WO2025054236A2 - Sars-cov-2 vaccine compositions and related methods - Google Patents
Sars-cov-2 vaccine compositions and related methods Download PDFInfo
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- WO2025054236A2 WO2025054236A2 PCT/US2024/045262 US2024045262W WO2025054236A2 WO 2025054236 A2 WO2025054236 A2 WO 2025054236A2 US 2024045262 W US2024045262 W US 2024045262W WO 2025054236 A2 WO2025054236 A2 WO 2025054236A2
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/53—DNA (RNA) vaccination
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Definitions
- SARS-CoV-2 spike proteins and polypeptides e.g., SARS- CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)
- polypeptides that comprise at least one non-naturally occurring glycosylation site (described herein) and nucleic acid molecules encoding the same.
- compositions comprising of the same (e.g., vaccine compositions, pharmaceutical compositions) and methods of making and utilizing the same.
- BACKGROUND Coronaviruses are a family of enveloped, positive-sense, single stranded RNA viruses that infect a wide variety of mammalian and avian species.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N- linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: Attorney Docket No.62801.28WO01 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N- linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic Attorney Docket No.62801.28
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389,
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389,
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions Attorney Docket No.62801.28WO01 set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set Attorney Docket No.62801.28WO01 forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, Attorney Docket No.62801.28WO01 a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position
- a cysteine at position 379 e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a Attorney Docket No.62801.28WO01 serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glut
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD).
- the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11-76.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76.
- the amino acid sequence of the encoded SARS-CoV-2 spike protein is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1).
- the encoded SARS-CoV-2 spike protein is an immunogen (or Attorney Docket No.62801.28WO01 an immunogenic fragment and/or immunogenic variant thereof).
- the nucleic acid molecule is RNA or DNA.
- the RNA is messenger ribonucleic acid (mRNA).
- the nucleic acid molecule comprises at least one modified nucleotide.
- the nucleic acid molecule comprises N1-methyl-pseudouridine, cytosine, adenine, and guanine.
- the nucleic acid molecule comprises a heterologous 5’- untranslated region (UTR), 3’-UTR, or both a 5’-UTR and 3’-UTR.
- the nucleic acid molecule comprises a poly(A) sequence.
- the nucleic acid molecule comprises a 5’cap structure.
- the nucleotide sequence of the nucleic acid molecule is codon optimized.
- the nucleic acid molecule further encodes a heterologous polypeptide or protein.
- the nucleic acid molecule is recombinant.
- the nucleic acid molecule is isolated.
- the nucleic acid molecule encodes a signal peptide. In some embodiments, the nucleic acid molecule encodes a homologous or heterologous signal peptide. In some embodiments, the nucleic acid molecule does not encode a signal peptide.
- vectors comprising a nucleic acid molecule described herein. In some embodiments, the vector is a non-viral vector (e.g., a plasmid) or a viral vector.
- the heterologous protein is a trimerization domain.
- the trimerization domain is operably connected to the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)) via a peptide linker.
- SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)
- compositions comprising at least one nucleic acid molecule described herein, a vector described herein, a conjugate described herein, or a fusion protein described herein.
- the composition is monovalent or polyvalent.
- the composition is polyvalent.
- the composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the nucleic acid molecule(s) are comprised in one or more vectors. In some embodiments, the nucleic acid molecule(s) or the one or more vectors are formulated in one or more carrier.
- the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. In some embodiments, the carrier is an LNP.
- the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. In some embodiments, the LNP has a mean particle size of between 80 nm and 160 nm. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or a vaccine composition.
- the composition comprises an adjuvant.
- SARS-CoV-2 spike proteins e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- the amino acid sequence of the SARS-CoV-2 spike protein e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1).
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-naturally occurring glycosylation motifs each comprising a non-naturally occurring glycosylation site, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the glycosylation motif is an N-linked glycosylation motif.
- the N-linked glycosylation motif is an NXT sequence, wherein X is any amino acid except for proline.
- the N-linked glycosylation motif is an NXS sequence, wherein X is any amino acid except for proline.
- the glycosylation site is an N-linked glycosylation site.
- the glycosylation motif is an O-linked glycosylation motif.
- the O-linked glycosylation motif is a serine or threonine.
- the glycosylation site is an O-linked glycosylation site.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-linked glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparag
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., Attorney Docket No.62801.28WO01 the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an aspara
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 428, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the glycosylation motif is an N-linked glycosylation motif.
- N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline.
- the glycosylation site is an N-linked glycosylation site.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 3
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N- glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the glycosylation motif is an N-linked glycosylation motif.
- the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline.
- the glycosylation site is an N-linked glycosylation site.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif Attorney Docket No.62801.28WO01 comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threon
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 4
- the amino acid sequence of the SARS-CoV-2 spike protein comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a naturally occurring glycosylation site) at one or more of amino acid positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation motif e.g., a naturally occurring glycosylation motif
- a glycosylation site e.g., a naturally occurring glycosylation site
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) relative the reference sequence (e.g., SEQ ID NO: 1).
- a reference sequence e.g., SEQ ID NO: 1
- the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) )) in a prefusion state relative the reference sequence (e.g., SEQ ID NO: 1).
- a reference sequence e.g., SEQ ID NO: 1
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)) comprises an inactive furin cleavage site.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 986 and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a Attorney Docket No.62801.28WO01 threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a Attorney Docket No.62801.28WO01 glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a Attorney Docket No.62801.28WO01 glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389,
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or se
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino Attorney Docket No.62801.28WO01 acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except pro
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 3
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 4
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a th
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 4
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428,
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set Attorney Docket No.62801.28WO01 forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 4
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429,
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., Attorney Docket No.62801.28WO01 the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at Attorney Docket No.62801.28WO01 position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline at position 987, amino acid numbering relative to the amino acid
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a proline at position 817 amino acid numbering relative
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at Attorney Docket No.62801.28WO01 position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442,
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD).
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises from about 10-15, 10- 20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10- 600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1190, 10-1300, 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1100, 100-1190, or 100-1300 Attorney Docket No.62801.28WO01 amino acids.
- the amino acid sequence of the SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76.
- the amino acid sequence of the SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11- 76].
- the amino acid sequence of the SARS-CoV-2 spike protein is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76.
- the amino acid sequence of the SARS-CoV-2 spike protein is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1).
- the SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and/or immunogenic variant thereof). In some embodiments, the SARS-CoV-2 spike protein is recombinant. In some embodiments, the SARS-CoV-2 spike protein is isolated. In some embodiments, the SARS-CoV-2 spike protein comprises a signal peptide. In some embodiments, the SARS-CoV-2 spike protein comprises a homologous or heterologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein does not comprise a signal peptide.
- the composition comprises a plurality of SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different.
- the SARS-CoV-2 spike protein(s) e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- carriers comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.
- the carrier is monovalent or polyvalent. In some embodiments, the carrier is polyvalent.
- the composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein Attorney Docket No.62801.28WO01 immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different.
- the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. In some embodiments, the carrier is an LNP.
- the vaccine composition is a boost vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition and a boost vaccine composition. In some embodiments, the vaccine composition can be utilized as a prime vaccine composition and/or a booster vaccine composition in a homologous or heterologous prime boost vaccine regimen. In some embodiments, the vaccine composition is monovalent or polyvalent. In some embodiments, the vaccine composition is polyvalent.
- the vaccine composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different.
- SARS-CoV-2 spike protein immunogens or immunogenic fragments and/or immunogenic variants thereof
- the vaccine composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein.
- the vaccine composition further comprises an adjuvant.
- vaccine compositions comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) that comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1, formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (a) the LNPs comprise
- compositions comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, or a vaccine composition described herein, and a pharmaceutically acceptable excipient.
- the pharmaceutical composition is monovalent or polyvalent. In some embodiments, the pharmaceutical composition is polyvalent.
- the pharmaceutical composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the pharmaceutical composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
- the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different.
- SARS-CoV-2 spike protein immunogens or immunogenic fragments and/or immunogenic variants thereof
- the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS- Co described herein V-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different.
- SARS-CoV-2 spike proteins e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)
- V-2 spike protein immunogens or immunogenic fragments and/or immunogenic variants thereof
- the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein.
- the pharmaceutical composition further comprises an adjuvant.
- host cells comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.
- kits comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein.
- the kit comprises instructions for use of the nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition.
- nucleic acid molecule in one aspect, provided herein are methods of delivering a nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, host cell, carrier, composition, vaccine composition, or pharmaceutical composition to a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or Attorney Docket No.62801.28WO01 immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby deliver the nucleic acid molecule, the vector, the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate
- provided herein are methods of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response in the subject.
- provided herein are methods of inducing or enhancing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject.
- provided herein are methods of preventing or decreasing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical Attorney Docket No.62801.28WO01 composition described herein, to thereby prevent or decrease an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject.
- provided herein are methods of inducing or enhancing an immune response against a plurality of strains of SARS-CoV-2 virus in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS- CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject.
- provided herein are methods of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby prevent, ameliorate, or treat the SARS-CoV-2 infection in the subject.
- provided herein are methods of preventing, ameliorating, or treating an infection by a plurality of SARS-CoV-2 strains in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby prevent, ameliorate, or treat an infection by a plurality of SARS-CoV-2 strains in the subject.
- kits for vaccinating a subject against SARS- CoV-2 comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described Attorney Docket No.62801.28WO01 herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby vaccinate the subject against SARS-CoV-2.
- mRNA molecule e.g., an mRNA molecule described herein
- a SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (or a conjugate or fusion protein thereof)
- a vector comprising the mRNA molecule
- a carrier comprising the mRNA molecule or the vector
- a vaccine composition comprising the mRNA molecule, the vector, or the carrier, or
- a pharmaceutical composition comprising the mRNA molecule, the vector, the carrier, or the vaccine composition
- provided herein are methods of vaccinating a subject against SARS- CoV-2, the method comprising administering to the subject a vaccine composition described herein or a pharmaceutical composition described herein, to thereby vaccinate the subject against SARS-CoV-2.
- kits for vaccinating a subject against a plurality of strains of SARS-CoV-2 comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby vaccinate the subject against a plurality of strains of SARS-CoV-2.
- the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered to the subject at least Attorney Docket No.62801.28WO01 twice.
- the subject is a human.
- the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a prime and/or a boost in a homologous or heterologous prime- boost regimen.
- the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.
- FIGS. 1A-1C depicts a sequence alignment of the amino acid sequence of a reference SARS-CoV-2 spike protein Wuhan-Hu-1 Spike protein sequence (SEQ ID NO: 1) and a variant thereof (SEQ ID NO: 9). 5.
- SARS-CoV-2 continues to evolve into new variants comprising a variety of amino acid variations, e.g., substitutions, deletions, and insertions. Many of the variations are found in the RBD of the spike protein, which is vital for SARS-CoV-2 entry into the cell. As most of the SARS- CoV-2 vaccines and current antibody therapies target the RBD of the spike protein, this creates the potential for the evolution of SARS-CoV-2 variants that evade vaccine-induced immunity, infection-induced immunity, and/or current antibody therapies.
- novel SARS-CoV-2 spike proteins e.g., SARS-CoV- 2 spike protein immunogens (and immunogenic fragments and/or immunogenic variants thereof)
- nucleic acid molecules e.g., mRNAs, encoding such SARS-CoV-2 spike proteins
- the current disclosure provides, inter alia, novel immunogens for use in pharmaceutical compositions and vaccines to induce a desired Attorney Docket No.62801.28WO01 immune response against one or more (e.g., multiple) variants of SARS-CoV-2.
- any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
- proteins and/or polypeptides are described herein, it is understood that polynucleotides (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) encoding the protein or polypeptide are also provided herein.
- proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are also provided herein.
- proteins, polypeptides, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, polypeptides, nucleic acid molecules, etc. are also provided herein.
- proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa.
- adjuvant refers to a substance that causes stimulation of the immune system of a subject when administered to the subject.
- administering refers to the physical introduction of an agent, e.g., a vaccine or therapeutic agent (or a precursor of the therapeutic agent that is metabolized or altered (e.g., translation of a nucleic acid molecule) within the body of the subject to produce the therapeutic agent in vivo) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. The term administering includes self- administration. As used herein, the term “agent” is used generically to describe any macro or micro molecule.
- moieties include, but are not limited to polypeptides, proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG).
- nucleic acid molecules e.g., DNA, RNA
- small molecules e.g., DNA, RNA
- carbohydrates e.g., lipids
- synthetic polymers e.g., polymers of PEG.
- the term “derived from,” with reference to a polynucleotide refers to a polynucleotide that has at least 70% sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof.
- derived from refers to a polypeptide or protein that comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein).
- the term “derived from” as used herein does not denote any specific process or method for obtaining the polynucleotide, polypeptide, or protein.
- the polynucleotide, polypeptide, or protein can be recombinant produced or chemically synthesized.
- the term “disease” refers to any abnormal condition that impairs physiological function.
- the term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology.
- disease includes infection (e.g., a viral (e.g., a SARS-Cov-2 infection), bacterial, fungal, protozoal infection).
- DNA and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose.
- the term “full-length” with reference to a SARS-CoV-2 spike protein refers to a SARS-CoV-2 spike protein, wherein the amino acid sequence of the SARS-CoV-2 spike protein comprises substantially the same number of amino acids as a reference SARS-CoV-2 spike Attorney Docket No.62801.28WO01 protein.
- the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a first polypeptide or protein to a second polypeptide or protein, wherein the first and second polypeptides or proteins are not naturally found operably connected together.
- the first and second polypeptides or proteins are derived from different proteins.
- fuse encompasses both a direct connection of the at least two polypeptides or proteins through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker).
- linker e.g., a peptide linker
- the term “glycosylation site” refers to the amino acid residue of a protein or peptide which is the attachment point for a glycan.
- the glycosylation site will be the asparagine residue to which the glycan is attached.
- the glycosylation site will be the serine or threonine residue to which the glycan is attached.
- non-naturally occurring glycosylation site within a protein or peptide refers to a glycosylation site that is not found in a specific protein or peptide in nature.
- the non-naturally occurring glycosylation site can be generated by creating a non-naturally occurring glycosylation motif, which does not necessarily require varying the amino acid of the glycosylation site.
- a non-naturally occurring glycosylation site would include a glycosylation site generated from the variation of a N-A-P amino acid sequence of a protein to a N-A-T amino acid sequence (creating an N-linked glycosylation motif) even though the amino acid that mediates attachment of the glycan (N) was not varied.
- glycosylation motif refers to the amino acid or sequence of amino acids that comprises the glycosylation site and facilities glycosylation.
- the canonical glycosylation motifs are NXT and NXS, wherein X is any amino acid except for proline and the asparagine (N) residue is the glycosylation site.
- N the asparagine residue
- the glycosylation motif is a S or T residue that also serves as the glycosylation site.
- fusion protein and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide operably connected to another polypeptide, wherein the first and second polypeptides are not naturally found operably connected together.
- the first and second polypeptides of the fusion protein are each derived from different proteins.
- the at least two polypeptides of the fusion protein can be directly operably connected Attorney Docket No.62801.28WO01 through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker).
- fusion polypeptide encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A – Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A – peptide linker – Polypeptide B).
- half-life extension protein or “half-life extension polypeptide” refers to a polypeptide or protein that when operably connected to another moiety (e.g., a subject moiety) (e.g., a protein), increases the half-life of the subject moiety (e.g., the subject protein) in vivo when administered to a subject (e.g., a human subject).
- a subject moiety e.g., a protein
- the pharmacokinetic properties of the protein can be evaluated utilizing in vivo models known in the art.
- heterologous when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described.
- a polypeptide comprising a “heterologous moiety” means a polypeptide that is joined to a moiety (e.g., small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the polypeptide in nature.
- a moiety e.g., small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.
- heterologous signal peptide refers to a signal peptide that is not operably connected to a subject polypeptide or protein in nature.
- heterologous prime boost refers to a prime boost vaccine regimen wherein the prime vaccine composition and the boost (or booster) vaccine composition are different (e.g., the immunogen is different, the form of the immunogen is different (e.g., a nucleic acid (e.g., mRNA) molecule-based vaccine versus a protein-based vaccine), the immunogen is expressed from a different vector (e.g., plasmid, viral vector), the method of delivering the immunogen to the subject is different, etc.).
- the prime vaccine composition and the boost (or booster) vaccine composition are different (e.g., the immunogen is different, the form of the immunogen is different (e.g., a nucleic acid (e.g., mRNA) molecule-based vaccine versus a protein-based vaccine), the immunogen is expressed from a different vector (e.g., plasmid, viral vector), the method of delivering the immunogen to the subject is different, etc.).
- homologous signal peptide refers to a signal peptide that is operably connected to a subject polypeptide or protein in nature.
- the human IL-2 signal peptide would constitute a homologous signal peptide.
- homologous prime boost refers to a prime boost vaccine regimen wherein the prime vaccine composition and the boost (or booster) vaccine composition are the same.
- the term “immunogen” refers to a substance that is capable of inducing an immune response (e.g., an adaptive immune response) in a subject (e.g., a human subject).
- the term “immunogenic fragment” refers to a fragment of a reference substance (e.g., a reference polypeptide or protein) that retains an immunogen.
- the term “immunogenic variant” refers to a variant of a reference substance (e.g., a reference polypeptide or protein) that retains an immunogen.
- the immunogenic variant is a polypeptide or protein that comprises at least one but no more than 25%, (e.g., no more than 20%, no more than 15%, no more than 12%, no more than 10%, no more than 8%) amino acid variation (e.g., substitutions, deletions, additions) compared to the amino acid sequence of a reference polypeptide or protein.
- the term “in combination with” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease.
- the treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap.
- the delivery of the two or more agents is simultaneous or concurrent and the agents may be co- formulated.
- the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen (e.g., a prime-boost vaccine regimen).
- administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disease is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other.
- the effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic).
- Sequential or substantially simultaneous administration of each agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues.
- the agents can be administered by the same route or by different routes.
- the term “isolated” with reference to an agent refers to the agent (e.g., the polypeptide, protein, or nucleic acid molecule) that is substantially free of other cellular components with which it is associated in the Attorney Docket No.62801.28WO01 natural state.
- modification in reference to a nucleic acid sequence refers to a nucleic acid molecule that comprises at least one nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and/or a modified internucleotide linkage, or any combination thereof.
- exemplary nucleotide modifications are provided herein, see, e.g., ⁇ 5.3 (e.g., ⁇ 5.3.2).
- nucleic acid molecule and “polynucleotide” are used interchangeably herein and refer to a polymer of DNA or RNA.
- the nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non- natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.
- Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means.
- recombinant means e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome
- synthetic means e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means.
- recombinant means e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and
- any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U).
- RNA e.g., mRNA
- T thymidine
- U uracil
- operably connected refers to the linkage of two moieties in a functional relationship.
- a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein).
- a transcription regulatory polynucleotide e.g., a promoter, enhancer, or other expression control element is operably connected to a polynucleotide that encodes a protein if it affects the transcription of the Attorney Docket No.62801.28WO01 polynucleotide that encodes the protein.
- the term “operably connected” can also refer to the conjugation of a moiety to e.g., a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein).
- the term “peptide” refers to a polymer of at least two amino acids linked by peptide bonds.
- peptide does not limit the length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2- 50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” are used interchangeably herein.
- the determination of “percent identity” between two sequences e.g., peptide or protein (amino acid sequences) or nucleic acid sequences) can be accomplished using a mathematical algorithm.
- Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety.
- PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
- the default parameters of the respective programs e.g., of XBLAST and NBLAST
- NCBI National Center for Biotechnology Information
- Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety.
- composition means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent.
- a “pharmaceutically acceptable carrier or diluent” means a substance for use in contact with the tissues of human beings and/or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit/risk ratio.
- the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more).
- poly(A) sequence refers to a sequence of adenosine nucleotides, typically located at the 3’-end of a linear RNA (or in a circular RNA), of up to about 1000 adenosine nucleotides.
- the poly(A) sequence is essentially homopolymeric, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides has essentially the length of 100 nucleotides.
- the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide).
- a poly(A) sequence of e.g., 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide).
- poly(A) sequence typically relates to RNA - however in the context of the invention, the term likewise relates to corresponding sequences in a DNA molecule (e.g. a “poly(T) sequence”).
- poly(T) sequence refers to a vaccine that contains a plurality of different immunogens.
- the polyvalent vaccine comprises a plurality of immunogens from more than one strain of a virus, different immunogens from the same strain of a virus, or any combination thereof.
- the polyvalent vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different Attorney Docket No.62801.28WO01 immunogens or nucleic acid molecules encoding the immunogens.
- the term “prime boost” refers to a vaccine regimen comprising at least an initial vaccine dose and one or more subsequent vaccine doses.
- the initial vaccine dose comprises the prime vaccine composition and the one or more subsequent vaccine doses are referred to as boost (or booster) vaccine compositions.
- Prime boost vaccine regimens can comprise more than one booster (e.g., 2, 3, 4, 5, 6, or more, etc.).
- a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
- protein refers to a one or more peptides folded into its three- dimensional structure.
- receptor binding domain or “RBD” in reference to the SARS-CoV-2 spike protein refers to a functional fragment of a SARS-CoV-2 spike protein that is capable of binding to ACE2.
- the amino acid sequence of an exemplary reference SARS-CoV-2 spike protein RBD is set forth in SEQ ID NO: 3.
- RNA and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose.
- RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule.
- SARS-CoV-2 spike protein refers to the SARS-CoV-2 protein that mediates binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2).
- ACE2 angiotensin-converting enzyme 2
- An exemplary amino acid sequence of a reference full-length SARS-CoV-2 spike protein is set forth in SEQ ID NO: 1.
- SARS-CoV-2 spike protein includes naturally occurring and engineered variants.
- the term “set” with reference to glycosylation sites does not require more than glycosylation site (e.g., non-naturally occurring glycosylation site).
- Table 2 herein describe “sets” glycosylation sites (e.g., non-naturally occurring glycosylation sites), some sets have only one glycosylation site (e.g., non- naturally occurring glycosylation site) and some sets have more than one glycosylation site (e.g., Attorney Docket No.62801.28WO01 non-naturally occurring glycosylation site).
- signal peptide or “signal sequence” refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export.
- the term encompasses both the signal peptide (the amino acid sequence of the signal peptide) and the nucleic acid sequence encoding the signal peptide.
- references to a signal peptide in the context of a nucleic acid molecule refers to the nucleic acid sequence encoding the signal peptide.
- subject includes any animal, such as a human or other animal.
- the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian).
- the subject is a human.
- the method subject is a non- human mammal.
- the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit).
- a non-human primate e.g., monkeys, apes
- ungulate e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys
- carnivore e.g., dog, cat
- the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots).
- avian taxa Galliformes e.g., chickens, turkeys, pheasants, quail
- Anseriformes e.g., ducks, geese
- Paleaognathae e.g., ostriches, emus
- Columbiformes e.g., pigeons, doves
- Psittaciformes e.g., par
- a “therapeutically effective amount” of an agent refers to any amount of the agent (e.g., the therapeutic agent, the vaccine) that, when used alone or in combination with another agent (e.g., a therapeutic agent, a vaccine), protects a subject against the onset of a disease (e.g., an infection), ameliorates the severity of a disease (e.g., an infection), and/or promotes disease (e.g., infection) regression evidenced by a decrease in severity of disease (e.g., infection) symptoms, an increase in frequency and duration of disease (e.g., infection) symptom-free periods, or a prevention of impairment or disability due to the disease (e.g., infection) affliction.
- translatable RNA refers to any RNA that encodes at least one polypeptide or protein and can be translated to produce the encoded polypeptide or protein in vitro, in vivo, in situ or ex vivo.
- a translatable RNA may be an mRNA or a circular RNA encoding a polypeptide or protein.
- the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease or infection and/or symptom(s) associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disease or infection does not require that the disease or infection, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and/or adverse symptom attributable to the disease or infection.
- the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or infection.
- the presently disclosed methods comprise administering a therapeutically effective amount of a compositions as described herein.
- the term “variant” or “variation” with reference to a nucleic acid molecule refers to a nucleic acid molecule that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid molecule.
- the term “variant” or “variation” with reference to a peptide or protein refers to a peptide or protein that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference peptide or protein.
- the term “5’-untranslated region” or “5’-UTR” refers to a part of a nucleic acid molecule located 5’ (i.e., “upstream”) of a coding sequence and which is not translated into protein. Typically, a 5’-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence.
- a 5’-UTR may comprise elements for controlling gene expression, also called regulatory elements.
- the 5’-UTR may be post-transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5’-cap structure.
- the term “3’-untranslated region” or “3’-UTR” refers to a part of a nucleic acid molecule located 3’ (i.e., downstream) of a coding sequence and which is not translated into protein.
- a 3’-UTR may located between a coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence.
- a 3'-UTR may comprise elements for controlling gene expression, also called regulatory elements.
- Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc.
- Glycoengineered SARS-CoV-2 Spike Proteins e.g., Immunogens
- Fragments Attorney Docket No.62801.28WO01 Thereof (e.g., RBDs)
- SARS-CoV-2 spike proteins and polypeptides e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof (e.g., receptor binding domains thereof)
- nucleic acid molecules encoding the same that comprise one or more non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site (e.g., a non-naturally occurring glycosylation site at one or more of positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ
- the SARS-CoV-2 spike glycoprotein mediates viral entry into host cells.
- the spike protein comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit).
- S1 subunit The SARS-CoV-2 spike protein is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently associated in the prefusion conformation.
- the distal S1 subunit comprises the RBD and contributes to stabilization of the prefusion state of the membrane anchored S2 subunit that contains the fusion machinery.
- the RBD mediates binding to the host cell receptor ACE2.
- the spike protein is cleaved by host proteases at the so-called S2' site located immediately upstream of the fusion peptide.
- the signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 1); the amino acid sequence of the corresponding mature first reference SARS- CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 2.
- the amino acid sequence of a RBD of the first reference SARS-CoV-2 spike protein is set forth in SEQ ID NO: 3 and SEQ ID NO: 4.
- the amino acid sequence of a second reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 5.
- the signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 5); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 6.
- the amino acid sequence Attorney Docket No.62801.28WO01 of the second reference SARS-CoV-2 spike protein comprises a glycine at position 614 and a proline at each of positions 817, 892, 899, 942, 986, and 987, amino acid numbering relative to SEQ ID NO: 1 (the Wuhan-Hu-1 Spike Numbering System described herein).
- the amino acid sequence of a second reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 7.
- the signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 7); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 8.
- the amino acid sequence of the second reference SARS-CoV-2 spike protein comprises a proline at each of positions 817, 892, 899, 942, 986, and 987, amino acid numbering relative to SEQ ID NO: 1.
- the amino acid sequence of a fourth reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 9.
- the signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 9); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 10.
- Table 1 Reference SARS-CoV-2 Spike Proteins and RBDs Thereof.
- amino acid positions in the amino acid sequence of a SARS-Cov-2 spike protein e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof) herein is relative to the amino acid sequence of SEQ ID NO: 1.
- a person of ordinary skill in the art can determine the corresponding amino acid for a Attorney Docket No.62801.28WO01 given amino acid position in any query spike protein by (i) aligning the amino acid sequence of the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) to the amino acid sequence of the query spike protein, (ii) locating the relevant amino acid position in the Wuhan-Hu-1 Spike reference sequence, and (iii) determining the amino acid located at the corresponding amino acid position in the query spike protein.
- FIGS. 1A-1C shows an alignment of the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) to the amino acid sequence of a query spike protein (SEQ ID NO: 9).
- the query spike protein comprises, inter alia, a deletion of four amino acids relative to the Wuhan- Hu-1 Spike reference sequence (amino acids L24, P25, P26, and Y125).
- the alignment keeps the corresponding amino acids in the query spike protein (SEQ ID NO: 9) correctly aligned to the Wuhan-Hu-1 Spike reference sequence.
- a query spike protein e.g., SEQ ID NO: 9
- one would generate the alignment in FIGS. 1A-1C locate amino acid position 440 in the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) and subsequently locate the corresponding amino acid in the query spike protein (SEQ ID NO: 9), in this example position 440 (according to Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) numbering) in the query spike protein (SEQ ID NO: 9) is lysine (K).
- Amino acid residue 440 (according to Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) numbering) in the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) is asparagine (N). This numbering system is indicated throughout the present disclosure by noting, “amino acid numbering relative to SEQ ID NO: 1” or the like.
- amino acid positions of the SARS-CoV-2 spike protein are typically defined herein relative to the full-length immature SARS-Cov-2 spike protein that contains the native signal sequence; it should be understood that this does not require the SARS-Cov-2 spike proteins (e.g., the SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein to include a native signal sequence.
- the SARS-CoV-2 spike protein or polypeptide is an immunogen (a SARS-CoV-2 spike protein or polypeptide immunogen).
- the SARS- Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide comprises an immunogen (a SARS-CoV-2 spike protein or polypeptide immunogen).
- the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein immunogen. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein immunogen.
- an immunogen refers to a substance that is capable of inducing an immune response (e.g., an adaptive immune response) in a subject (e.g., a human subject).
- the immunogenicity of a protein or polypeptide described herein can be determined through standard in silico, in vitro, ex vivo, and in vivo assays known in the art.
- the type of immune response measured can be determined by a person of ordinary skill in the art, e.g., in some embodiments, an antibody based immune response and/or a T cell based immune response is evaluated.
- a sample e.g., a blood, cell
- a subject e.g., an animal, human
- the protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide
- the sample can be utilized to evaluate indicators of an immune response.
- a blood (or serum) sample can be utilized to assess the level of antibodies that bind the antigen. Such binding can be assessed through standard ELISAs.
- the presence of neutralizing antibodies in the sample can also be assessed using standard assays, including, e.g., plaque reduction neutralization assays and microneutralization assays.
- the induction of a T-cell based immune response can be measured using standard assays known in the art, including, e.g., immunospot assays to assess cytokine production utilizing peptide stimulated peripheral blood mononuclear cells (PBMCs) from the subject.
- PBMCs peripheral blood mononuclear cells
- the SARS-CoV-2 spike protein or polypeptide is recombinant. In some embodiments, the SARS-CoV-2 spike protein or polypeptide is isolated. 5.2.1 Engineered Glycosylation Sites As described above, provided herein are, inter alia, SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof (e.g., receptor binding domains thereof))) and nucleic acid molecules encoding the same, that comprise one or more non-naturally occurring glycosylation sites (e.g., a non-naturally occurring glycosylation site at one or more of positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1).
- SARS-CoV-2 spike proteins and polypeptides e.g., SARS-CoV-2 spike protein and polypeptide immunogen
- the non-naturally occurring glycosylation site is an N-linked glycosylation site (an asparagine (N) residue within a N-linked glycosylation site (e.g., NXT or NXS (wherein X is any amino acid except for proline)).
- N an asparagine
- NXS N-linked glycosylation site
- O O-linked glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more non- naturally occurring glycosylation sites.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more non-naturally occurring glycosylation motifs each comprising a non- Attorney Docket No.62801.28WO01 naturally occurring glycosylation site.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1) (e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites).
- a reference SARS-CoV-2 spike protein e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- SEQ ID NO: 1 e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of amino acid positions 354, 369, 370, 383, 388, 394, 415, 428, 441, 447, 448, 460, 468, 476, 477, 494, 502, 517, 518, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- amino acid positions 354, 369, 370, 383, 388, 394, 415, 428, 441, 447, 448, 460, 468, 476, 4
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of amino acid positions 354, 369, 370, 388, 394, 415, 428, 447, 448, 460, 468, 476, 494, 502, 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- amino acid positions 354, 369, 370, 388, 394, 415, 428, 447, 448, 460, 468, 476, 494, 502, 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO:
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of amino acid positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- amino acid positions 369, 388, 447, 460, 476, and/or 529 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) further comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 383, 394, 415, 428, 441, 447, 448, 468, 494, 502, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) further Attorney Docket No.62801.28WO01 comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of amino acid positions 369, 447, 388, 428, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and optionally further comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and/or 9) of amino acid positions 354, 370, 388, 394, 448, 468, 476, 494, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of amino acid positions 354, 370, 388, 394, 415, 428, 460, 468, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- a glycosylation site e.g., a non-naturally occurring glycosylation site
- amino acid positions 354, 370, 388, 394, 415, 428, 460, 468, 476, and/or 529 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a Attorney Docket No.62801.28WO01 serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an Attorney Docket No.62801.28WO01 asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an aspara
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at Attorney Docket No.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosy
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N- glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threon
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449, amino acid
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447,
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449, amino acid numbering relative
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) Attorney Docket No.62801.28WO01 comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an Attorney Docket No.62801.28WO01 asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, amino acid numbering relative to the amino
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N- glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an as
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an Attorney Docket No.62801.28WO01 asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosy
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid at position 478 except for proline, and a threonine or a serine at position 479, amino acid numbering
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative Attorney Docket No.62801.28WO01 to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine at position 385, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid at position 478 except for proline, and a threonine at position 479, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at
- SARS-CoV-2 spike proteins and polypeptides e.g., SARS- CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)
- nucleic acids encoding the same that comprise any set (e.g., any singular or combinatorial use) of the non-naturally occurring glycosylation sites described herein.
- Exemplary sets of engineered glycosylation sites in the SARS-CoV-2 spike protein immunogens described herein are provided in Table 2. The amino acid numbering set forth in Table 2 is relative to SEQ ID NO: 1.
- Table 2 further provides exemplary glycosylation motifs that can be utilized to generate the exemplary glycosylation sites within each set.
- a number followed by an amino acid residue denotes the amino acid residue at the specified amino acid position (e.g., 494N denotes an asparagine at position 494 (amino acid numbering relative to the amino acid positions of SEQ ID NO: 1)); and Attorney Docket No.62801.28WO01 an “X” denotes any amino acid residue other than proline.
- Table 2 Engineered Glycosylation Sites of SARS-CoV-2 Spike Protein Immunogens.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more non- naturally glycosylation motifs set forth in Table 2.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more non-naturally glycosylation motifs set forth in Table 2 that comprises the corresponding non-naturally glycosylation motifs set forth in Table 2.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., one) set (e.g., set A, B, C, D, E, F, G, H, I, J, K) of non-naturally glycosylation sites set forth in Table 2.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more sets of non-naturally occurring glycosylation motifs, wherein each set comprises a set (e.g., set A, B, C, D, E, F, G, H, I, J, K) of non-naturally glycosylation sites set forth in Table 2.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid Attorney Docket No.62801.28WO01 numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N- glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino Attorney Docket No.62801.28WO01 acid except proline at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a th
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or Attorney Docket No.62801.28WO01 polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 3.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosy
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 8
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described Attorney Docket No.62801.28WO01 herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 3.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring Attorney Docket No.62801.28WO01 glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or Attorney Docket No.62801.28WO01 polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 11-32.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or Attorney Docket No.62801.28WO01 polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non- naturally glycosy
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any Attorney Docket No.62801.28WO01 one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 11-32.
- SARS-CoV-2 spike proteins e.g., immunogens
- exemplary SARS-CoV-2 spike proteins comprising at least one non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site set forth herein (see, e.g., Table 2) is provided in Table 4.
- Table 4 provides the amino acid sequence of the full-length spike protein (immature SEQ ID NOS: 33-43 and mature SEQ ID NOS: 44-54) of eleven exemplary SARS-CoV-2 immunogens (A-K) that comprise the corresponding set of engineered glycosylation sites set forth above in Table 2 (i.e., set A, B, C, D, E, F, G, H, I, J, or K) and a glycine at amino acid position 614 (amino acid numbering relative to SEQ ID NO: 1.).
- A-K SARS-CoV-2 immunogens
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Attorney Docket No.62801.28WO01 the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 4.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosy
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 8
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of one or more of the Attorney Docket No.62801.28WO01 following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 4.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 33-54.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising one or more (e.g., 1, 2, 3,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non- naturally glycosy
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation Attorney Docket No.62801.28WO01 motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or Attorney Docket No.62801.28WO01 polypeptide comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 33-54.
- SARS-CoV-2 spike proteins comprising at least one non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site set forth herein (see, e.g., Table 2) is provided in Table 5.
- Table 5 provides the amino acid sequence of the receptor binding domain of the spike protein (full receptor binding domain SEQ ID NOS: 55-65 and fragment of receptor binding domain SEQ ID NOS: 66-76) of eleven exemplary SARS-CoV-2 immunogens (A-K) that comprise the corresponding set of engineered glycosylation sites set forth above in Table 2 (i.e., set A, B, C, D, E, F, G, H, I, J, or K). Table 5.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, Attorney Docket No.62801.28WO01 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 5.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 8
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a Attorney Docket No.62801.28WO01 contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 5.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to Attorney Docket No.62801.28WO01 the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 55-76.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described here
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 55-76.
- SARS-CoV-2 Spike Proteins e.g., Immunogens
- the SARS-CoV-2 spike proteins and polypeptides e.g., the SARS-CoV-2 spike Attorney Docket No.62801.28WO01 protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein can be full-length proteins or fragments (e.g., receptor binding domains thereof).
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises or consists of from about 10-1300, 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10- 500, 10-400, 10-500, 10-400, 10-300, 10-250, 10-200, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-1300, 20-1300, 30-1300, 40-1300, 50-1300, 60-1300, 70-1300, 80-1300, 90-1300, 100-1300,10-250, 20-250, 30-250, 40-250, 50-250, 60-250, 70-250, 80-250, 90-250, or 100-250 amino acids.
- the SARS-CoV-2 spike protein or polypeptide comprises at least a portion of the RBD of the SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the SARS- CoV-2 spike protein or polypeptide comprises a full- length SARS-CoV-2 spike protein.
- the full-length SARS-CoV-2 spike protein comprises a homologous signal peptide.
- the full-length SARS- CoV-2 spike protein does not comprise a homologous signal peptide.
- the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises the ectodomain of the SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises the ectodomain of the SARS-CoV-2 spike protein and does not include the transmembrane domain or the cytoplasmic domain of the SARS-CoV-2 spike protein.
- the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a homologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) does not comprise a homologous signal peptide.
- SARS-CoV-2 Spike Proteins e.g., Immunogens
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- can exhibit additional structural and functional features Exemplary additional structural and functional features are provided below.
- the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1) (e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites).
- a reference SARS-CoV-2 spike protein e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- SARS-CoV-2 spike protein set forth in SEQ ID NO: 1 e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites.
- Standard methods known in the art can be utilized to evaluate the glycosylation of a protein or peptide, including e.g., the level of glycosylation, the sites of glycosylation, etc. See, e.g., Roth et al, Identification and Quantification of Protein Glycosylation (2012) International Journal of Carbohydrate Chemistry, Volume 201, Article ID 640923, https://doi.org/10.1155/2012/640923; Lebede et al, Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry, Anal. Chem.
- Standard methods include for example, the enzymatic or chemical release of glycans from the subject proteins followed by chromatography or mass spectrometry based structural analysis.
- SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- SARS-CoV-2 spike protein or polypeptide immunogen or immunogenic fragment and/or immunogenic variant thereof
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises at least one amino acid variation that stabilizes said SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) in a prefusion state.
- the amino acid sequence of the SARS- CoV-2 spike protein or polypeptide comprises at least one amino acid variation relative to the amino acid sequence set forth in any one of SEQ ID NO: 1 that stabilizes the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) in a prefusion state.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein Attorney Docket No.62801.28WO01 or polypeptide comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises a proline at amino acid position 986 and/or a proline at amino acid position 987, amino acid numbering amino acid numbering relative to SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises a proline at amino acid position 986 and a proline at amino acid position 987, amino acid numbering relative to SEQ ID NO: 1.
- the SARS-CoV-2 spike protein or polypeptide comprises an inactive furin cleavage site.
- the spike protein of SARS-Cov-2 has to be cleaved into the S1 and S2 subunit.
- the cleavage site in SARS-Cov-2 is a polybasic motif (RRAR (SEQ ID NO: 77)) (for example, amino acids 682-685 of SEQ ID NO: 1) that can be activated by furin-like proteases. Modifications to the furin cleavage site that inactivate it are known in the art.
- the amino acid sequence of the SARS- CoV-2 spike protein or polypeptide comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site.
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide e.g., the Attorney Docket No.62801.28WO01 SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide comprises a replacement of the RRAR cleavage site (SEQ ID NO: 77) with the amino acid sequence QQAQ (SEQ ID NO: 78).
- Multimerization e.g., Trimerization
- the SARS-CoV-2 spike protein or polypeptide comprises a multimerization (e.g., trimerization) domain.
- the multimerization domain (e.g., trimerization domain) facilities multimerization (e.g., trimerization) of the proteins upon expression in/from a cell.
- Suitable multimerization (e.g., trimerization) domains are known in the art, including e.g., a fibritin - based trimerization domain. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was previously described as an artificial natural trimerization domain. See, e.g., (Letarov et al., 1993) Biochemistry Moscow 64: 817- 823; S-Guthe et al., (2004) J. Mol. Biol. 337: 905-915).
- the amino acid sequence of the SARS-CoV-2 spike protein is derived from a circulating strain (i.e., naturally occurring variant) of SARS-CoV-2.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2.
- the amino acid sequence of the SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2 known to have previously circulated. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated. For example, in some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline Attorney Docket No.62801.28WO01 at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
- the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic
- nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.2).
- the nucleic acid molecule is RNA (e.g., mRNA or circular RNA) or DNA.
- the nucleic acid (e.g., RNA) molecule is a translatable RNA.
- the nucleic acid (e.g., RNA) molecule is a circular RNA.
- the nucleic acid (e.g., RNA) molecule is mRNA.
- the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) comprises Attorney Docket No.62801.28WO01 from about 30 to about 20000 nucleotides, about 50 to about 20000 nucleotides, about 500 to about 10000 nucleotides, about 1000 to about 10000 nucleotides, about 1000 to about 5000 nucleotides, or about 2000 to about 5000 nucleotides.
- the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide comprises at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides.
- the nucleic acid molecule is altered (e.g., compared to the sequence of a reference nucleic acid molecule, a naturally occurring nucleic acid molecule), e.g., to impart one or more of (a) improved resistance to in vivo degradation, (b) improved stability in vivo, (c) reduced secondary structures, and/or (d) improved translatability in vivo, compared to the reference nucleic acid sequence.
- Alterations include, without limitation, e.g., codon optimization, nucleotide modifications (see, e.g., described herein), etc.
- the sequence of the nucleic acid molecule is codon optimized, e.g., for expression in humans.
- Codon optimization may be used to match codon frequencies in target and host organisms to ensure proper folding; bias guanosine (G) and/or cytosine (C) content to increase nucleic acid stability; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation alteration sites in an encoded protein (e.g., glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of a protein to fold properly; and/or to reduce or eliminate secondary structures (e.g., problem secondary structures) within the nucleic acid molecule.
- G guanosine
- C cytosine
- the codon optimized nucleic acid sequence shows one or more of the above (compared to a reference nucleic acid sequence). In some embodiments, the codon optimized nucleic acid sequence shows one or more of improved resistance to in vivo degradation, improved stability in vivo, reduced secondary structures, and/or improved translatability in vivo, compared to a reference nucleic acid sequence. Codon optimization methods, tools, algorithms, and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.). In some Attorney Docket No.62801.28WO01 embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms (e.g., optimization algorithms known in the art).
- the nucleic acid sequence is modified to optimize the number of G and/or C nucleotides as compared to a reference nucleic acid sequence.
- An increase in the number of G and C nucleotides may be generated by substitution of codons containing adenosine (A) or thymidine (T) (or uracil (U)) nucleotides by codons containing G or C nucleotides.
- the nucleic acid molecule is recombinant.
- the nucleic acid molecule is isolated. 5.3.1 DNA Nucleic Acids
- the nucleic acid molecule is DNA.
- the DNA is a linear coding DNA construct.
- the DNA contained within a vector e.g., a non-viral vector (e.g., a plasmid) or a viral vector).
- the DNA is contained within a non-viral vector (e.g., a plasmid).
- the DNA is contained within a viral vector (e.g., described herein).
- a more detailed description of vectors for both RNA and DNA nucleic acids is provided in ⁇ 5.6.
- the coding DNA molecule may also comprise one or more heterologous nucleic acid elements to mediate expression of the coding region.
- promoter(s), enhancer(s), polyadenylation signal(s), synthetic introns, transcriptional termination signals, polyadenylation sequences, and other transcription regulatory elements include, e.g., promoter(s), enhancer(s), polyadenylation signal(s), synthetic introns, transcriptional termination signals, polyadenylation sequences, and other transcription regulatory elements.
- a person of ordinary skill in the art is familiar with the transcriptional regulatory elements needed for expression of coding DNA can optimize the expression construct (e.g., linear DNA, plasmid DNA, etc.) accordingly.
- a promoter is operably connected to the respective coding nucleic acid sequence.
- promoters from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter.
- SV40 simian virus 40
- MMTV mouse mammary tumor virus
- HV human immunodeficiency virus
- BIV bovine immunodeficiency virus
- LTR long terminal repeat
- Moloney virus promoter an avian leukosis virus (ALV) promoter
- AMV avian leukosis virus
- CMV cytomegalovirus
- EMV Epstein Barr virus
- the promoter can also be a promoter from a human gene, for example, from human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein.
- the Attorney Docket No.62801.28WO01 promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the entire contents of which is incorporated by reference herein for all purposes.
- Exemplary polyadenylation signals include, but are not limited, to the bovine growth hormone (BGH) polyadenylation site, SV40 polyadenylation signals, and LTR polyadenylation signals.
- BGH bovine growth hormone
- the nucleic acid molecule is an RNA molecule.
- the RNA molecule is a translatable RNA molecule.
- the RNA molecule is selected from an mRNA, a self-replicating RNA, a circular RNA, a viral RNA, or a replicon RNA.
- the RNA molecule a circular RNA.
- the RNA molecule is a mRNA.
- the basic components of an mRNA molecule typically include at least one coding region (herein a coding region encoding at least one peptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or variant thereof)) (e.g., described herein), a 5’-untranslated region (UTR), a 3’-UTR, a 5’ cap, and a poly-A tail.
- the RNA e.g., mRNA
- the UTRs may harbor regulatory sequence elements that determine the RNA (e.g., mRNA) turnover, stability, localization, and/or expression of operably connected coding sequence(s).
- the heterologous UTRs may be derived from a naturally occurring gene or may be synthetically engineered.
- the 5’-UTR comprises elements for controlling gene expression, e.g., ribosomal binding sites, miRNA binding sites.
- the 5’-UTR may be post- transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5’cap structure.
- the 3’-UTR comprises a polyadenylation signal.
- the RNA (e.g., mRNA) comprises at least one coding region encoding the polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) and 5’-UTR and/or a 3’-UTR.
- a SARS-CoV-2 protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- 5’-UTR and/or a 3’-UTR e.g., described herein
- the RNA comprises at least one coding sequence encoding a polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) operably connected to at least one heterologous 5’-UTR Attorney Docket No.62801.28WO01 and at least one 3’-UTR.
- the RNA comprises a poly(A) sequence.
- the poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides.
- poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides.
- the poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides, wherein the 3’ terminal nucleotide of said nucleic acid molecule is an adenosine.
- poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides, wherein the 3’ terminal nucleotide of said nucleic acid molecule is an adenosine.
- the RNA e.g., mRNA
- the RNA comprises a 5’-cap structure.
- the 5’-cap structure stabilizes the RNA (e.g., mRNA), enhances expression of the encoded polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) and/or reduces the stimulation of the innate immune system (e.g., after administration to a subject).
- a SARS-CoV-2 protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- reduces the stimulation of the innate immune system e.g., after administration to a subject.
- Exemplary 5’-cap structures include, but are not limited to, cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphorothioate modified ARCA), inosine, N1-methyi-guanosine, 2'-fluoro-guanosine, 7-deaza
- the 5’ cap structure comprises m7G, cap0, cap1, cap2, a modified capO, or a modified cap1 structure.
- the RNA e.g., mRNA
- the RNA comprises one or more modified nucleotide (e.g., nucleotide analogue, backbone modifications, sugar modifications, and/or base modifications).
- a backbone modification in the context of the present disclosure is a modification, Attorney Docket No.62801.28WO01 in which phosphates of the backbone of the nucleotides of the RNA (e.g., mRNA) are chemically modified.
- a sugar modification in the context of the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA (e.g., mRNA).
- a base modification in the context of the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA (e.g., mRNA).
- the RNA e.g., mRNA
- the RNA comprises at least one modified nucleotide.
- nucleotide analogues/modifications include, but are not limited to, 2- amino-6-chloropurineriboside-5’-triphosphate, 2-Aminopurine-riboside-5’-triphosphate; 2- aminoadenosine-5'-triphosphate, 2’-Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5'- triphosphate, 2-thiouridine-5’-triphosphate, 2’-Fluorothymidine-5’-triphosphate, 2’-O-Methyl- inosine-5’-triphosphate 4-thiouridine-5’-triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5- aminoallyluridine-5’-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’- triphosphate, 5-Bromo-2’-deoxycytidine-5'-triphosphate, 5-Bromo-2’-deoxyuridine
- nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5’-triphosphate, 7- deazaguanosine-5'-triphosphate, 5-bromocytidine-5’-triphosphate, and pseudouridine-5’- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-
- the RNA (e.g., mRNA) comprises pseudouridine, N1 - methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5- Attorney Docket No.62801.28WO01 methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and/or 2’-O-methyl uridine
- the RNA comprises one or more pseudouridine ( ⁇ ), N 1 -methylpseudouridine (m1 ⁇ ), 5-methylcytosine, and 5-methoxyuridine.
- RNA e.g., mRNA
- pseudouridine ⁇
- N 1 -methylpseudouridine m1 ⁇
- 5-methylcytosine 5-methoxyuridine.
- essentially all, e.g., essentially 100% of the uracil in the coding sequence of the RNA e.g., mRNA
- have a chemical modification preferably a chemical modification is in the 5- position of the uracil.
- Incorporating modified nucleotides such as e.g., pseudouridine ( ⁇ ), N1 - methylpseudouridine (m1 ⁇ ), 5-methylcytosine, and/or 5-methoxyuridine into the coding sequence may be advantageous as unwanted innate immune responses (upon administration of the coding RNA or the vaccine) may be adjusted or reduced (if required).
- modified nucleotides such as e.g., pseudouridine ( ⁇ ), N1 - methylpseudouridine (m1 ⁇ ), 5-methylcytosine, and/or 5-methoxyuridine into the coding sequence may be advantageous as unwanted innate immune responses (upon administration of the coding RNA or the vaccine) may be adjusted or reduced (if required).
- the RNA comprises: (i) a 5'-cap structure; (ii) a 5'- UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly- A region.
- RNA (e.g., mRNA) described herein can be generated by e.g., in vitro transcription. In vitro transcription is a method well known to those of ordinary skill in the art for the production of RNA (e.g., mRNA).
- the RNA is obtained by DNA-dependent in vitro transcription of an appropriate DNA template, e.g., a linearized plasmid DNA template or a PCR-amplified DNA template.
- the promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Examples of DNA-dependent RNA polymerases include the 17, T3, SP6, or Syn5 RNA polymerases.
- the DNA template is linearized with a suitable restriction enzyme before it is subjected to RNA in vitro transcription.
- Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g., T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to Attorney Docket No.62801.28WO01 degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgCh, which supplies Mg2+ ions as
- RNA (e.g., mRNA) products can be purified according to methods known in the art. For example, using PureMessenger® (CureVac, Tubingen, Germany; RP-HPLC according to W02008077592) and/or tangential flow filtration (as described in WO2016193206) and/or oligo d(T) purification (see WO2016180430); or using RP-HPLC, e.g., using Reversed- Phase High pressure liquid chromatography (RP-HPLC), the entire contents of each reference is incorporated by reference herein for all purposes.
- PureMessenger® CureVac, Tubingen, Germany
- RP-HPLC tangential flow filtration
- oligo d(T) purification see WO2016180430
- RP-HPLC Reversed- Phase High pressure liquid chromatography
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (see, e.g., ⁇ 5.2) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.3) is operably connected to a heterologous moiety (e.g., a heterologous polypeptide) forming a fusion protein or polypeptide or conjugate.
- a heterologous moiety e.g., a heterologous polypeptide
- the heterologous moiety imparts an additional function to the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), e.g., to promote or improve secretion of the encoded SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV- 2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), (e.g., via secretory signal peptides), promote or improve anchoring of the encoded SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), described herein in the plasma membrane (e.g., via transmembrane elements), promote or improve formation of antigen complexes (e.g., via multimerization domains or antigen clustering elements), or promote or promote or promote or
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS-CoV-2 spike protein or polypeptide Attorney Docket No.62801.28WO01 immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., the ectodomain of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof))
- the heterologous moiety is a half-life extension moiety.
- Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human immunoglobulin (hIg)), a fragment of an Ig (e.g., a hIg), an Ig (e.g., a hIg) constant region, a fragment of an Ig (e.g., a hIg) constant region, an Ig (e.g., a hIg) Fc region, human transferrin, human serum albumin (HSA), an HSA binding protein, and polyethylene glycol (PEG) (and polymers thereof).
- the heterologous polypeptide is a half-life extension polypeptide.
- Exemplary half-life extension polypeptides include, but are not limited to, an Ig (e.g., a hIg), a fragment of an Ig (e.g., a hIg), one or more Ig (e.g., a hIg) heavy chain constant region, a fragment of an Ig (e.g., a hIg) constant region, an Ig (e.g., a hIg) Fc region, human transferrin, human serum albumin (HSA), and an HSA binding protein.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) fused or conjugated to a half-life extending moiety (e.g., a half-life extension polypeptide)
- a half-life extending moiety e.g., a half-life extension polypeptide
- the heterologous moiety is a detectable agent (e.g., protein, e.g., a fluorescent protein). In some embodiments, the heterologous moiety is a heterologous signal peptide. In some embodiments, the heterologous moiety is a trimerization domain.
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was previously described as an artificial natural trimerization domain. See, e.g., (Letarov et al., 1993) Biochemistry Moscow 64: 817- 823; S-Guthe et al., (2004) J. Mol. Biol. 337: 905-915). See, also, Meier et al., J Mol Biol. Attorney Docket No.62801.28WO01 (2004) 344(4): 1051-69; WO 2018/081318; the entire contents of each of which are incorporated herein by reference for all purposes.
- heterologous moiety e.g., heterologous polypeptide
- SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)).
- the heterologous moiety is directly operably connected to the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)).
- SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the heterologous moiety is indirectly operably connected to the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)).
- SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- a person of ordinary skill in the art can evaluate and select a suitable linker for the fusion or conjugation of a specific heterologous moiety.
- a peptide linker may be employed.
- Peptide linkers are known in the art and can be selected based on specific properties, including e.g., length, flexibility, rigidity, cleavability, etc.
- the amino acid sequence of commonly employed peptide linkers comprises glycine amino acid residues, serine amino acid residues, glycine and serine amino acid residues, or glycine, serine, and proline amino acid residues.
- heterologous moiety e.g., heterologous polypeptide
- SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic Attorney Docket No.62801.28WO01 variant thereof))
- a SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (see, e.g., ⁇ 5.2) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.3) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition,
- compositions comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.2) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.3) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)).
- SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic
- the composition is a vaccine composition.
- the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen.
- the vaccine composition is a vaccine booster composition of a prime-boost vaccine regimen.
- the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen.
- the vaccine composition can be utilized as a prime and/or a booster (e.g., as described herein).
- the vaccine composition forms a single dose vaccine that does not require a booster.
- the vaccine composition is a polyvalent vaccine composition.
- the vaccine composition is a monovalent vaccine composition.
- a SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.2) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition, a vaccine booster composition, a vaccine prime and booster composition)).
- a vaccine composition e.g., a prime vaccine composition, a vaccine booster composition, a vaccine prime and booster composition
- compositions comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.2) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)).
- the composition is a vaccine composition.
- the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen.
- the vaccine composition is a vaccine boost composition of a prime-boost vaccine regimen.
- the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen.
- the prime boost composition can be utilized as a prime and/or a booster (e.g., as described herein).
- the vaccine composition forms a single dose vaccine that does not require a booster.
- the composition e.g., vaccine composition
- the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)).
- SARS-CoV-2 spike proteins or polypeptides e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)
- the plurality comprises or consists of from about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5- 60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30- 50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogenas,
- the plurality comprises at least 2 but no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)).
- the amino acid sequence of each of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different.
- the amino acid sequence of at least one of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is derived from a circulating strain of SARS-CoV- 2.
- the plurality comprises at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein.
- the plurality comprises at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, wherein the amino acid sequence of the at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., a set of) non-naturally occurring glycosylation site described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in ⁇ 5.2).
- SARS-CoV-2 spike protein or polypeptide e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- the amino acid sequence of the at least one SARS-CoV-2 spike protein or polypeptide e.g., immunogen (or immunogenic fragment
- the plurality comprises at least two SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the at least two SARS- CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) comprises a different combination of non-naturally occurring glycosylation sites described herein or a different combination of sets of non-naturally occurring glycosylation sites described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in ⁇ 5.2).
- SARS-CoV-2 spike proteins or polypeptides e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof) described herein, wherein the amino acid sequence of each of
- the composition comprises a multimer (e.g., trimer) of multiple Attorney Docket No.62801.28WO01 (e.g., 3) SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein.
- the composition comprises a trimer of three SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein.
- the composition comprises a homotrimer of three SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein.
- the amino acid sequence of the SARS-CoV-2 spike protein is derived from a circulating strain of SARS-CoV-2.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2.
- the amino acid sequence of the SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2 known to have previously circulated.
- the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated.
- the vaccine composition further comprises at least one immunogen (or immunogenic fragment and/or immunogenic variant thereof) from a non-SARS- CoV-2 virus, e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus.
- a non-SARS- CoV-2 virus e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus.
- Polypeptides and proteins may be produced by recombinant technology in host cells (e.g., Attorney Docket No.62801.28WO01 mammalian cells) that have been transfected or transduced with a nucleic acid expression vector (e.g., plasmid, viral vector (e.g., a baculoviral expression vector)) encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)).
- host cells e.g., Attorney Docket No.62801.28WO01 mammalian cells
- a nucleic acid expression vector e.g., plasmid, viral vector (e.g., a baculoviral expression vector)
- the cell is specifically selected to ensure the cell can mediate the desired glycosylation pattern (N-linked glycosylation, O-linked glycosylation, etc.) on a subject protein being produced.
- the cell is a eukaryotic cell.
- the cell is a mammalian cell.
- the cell is a murine cell.
- the cell is a human cell.
- the cell is a hamster cell.
- host cells containing the expression vector encoding the SARS-CoV-2 spike protein or polypeptide are cultured under conditions conducive to expression of the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)).
- Culture media is available from various vendors, and a suitable medium can be routinely chosen for a host cell to express a polypeptide or protein of interest, here the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)).
- the Attorney Docket No.62801.28WO01 peptide or protein may be concentrated, buffer exchanged by ultrafiltration, and the retentate from the ultrafiltration may be filtered through an appropriate filter, e.g., a 0.22 ⁇ m filter.
- an appropriate filter e.g., a 0.22 ⁇ m filter.
- McPherson et al. “Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant,” Chapter 4, in Sunil Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases, Methods in Molecular Biology, Springer, New York, 2016. See also U.S. Pat.5,762,939, the entire contents of each of which is incorporated by reference herein for all purposes.
- the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein (and fusion proteins, conjugates, etc. thereof) may also be produced synthetically.
- the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein may be produced by using an egg-based manufacturing method.
- the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the vaccine composition are formulated in one or more carrier (e.g., a carrier described herein (see, e.g., ⁇ 5.7)).
- the vaccine compositions are pharmaceutical compositions (e.g., described herein, e.g., see ⁇ 5.8).
- the vaccine compositions comprise an adjuvant (e.g., described herein, e.g., see ⁇ 5.9).
- a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., ⁇ 5.3) (or a fusion or conjugate thereof (see, e.g., ⁇ 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition, a booster composition, a vaccine prime and booster composition)).
- a vaccine composition e.g., a prime vaccine composition, a booster composition, a vaccine prime and booster composition
- the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine booster composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition can be utilized as a prime and/or a booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster.
- the composition (e.g., vaccine composition) comprises a plurality of nucleic acid molecules, each comprising a coding region encoding at least one SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein).
- each of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule.
- each of the nucleic acid molecules of the plurality are separate (i.e., not connected) nucleic acid molecules.
- At least two of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule. In some embodiments, at least two of the nucleic acid molecules of the plurality are separate (i.e., not connected) nucleic acid molecules. In some embodiments, at least two of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule; and at least one (e.g., at least 2, 3, 4, 5, etc.) of the nucleic acid molecules of the plurality is a separate (i.e., not connected) nucleic acid molecule.
- the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid molecules. In some embodiments, the plurality comprises or consists of from about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40- 90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100,
- the plurality comprises at least 2 but no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 nucleic acid molecules.
- Attorney Docket No.62801.28WO01 the amino acid sequence of each of the encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different.
- the amino acid sequence of at least one of the encoded SARS- CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is derived from a circulating strain of SARS-CoV- 2.
- the plurality comprises at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein.
- the plurality comprises at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, wherein the amino acid sequence of the at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., a set of) non-naturally occurring glycosylation site described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in ⁇ 5.2).
- the amino acid sequence of the at least one encoded SARS-CoV-2 spike protein or polypeptide e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the plurality comprises at least two encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the at least two encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) comprises a different combination of non- naturally occurring glycosylation sites described herein or a different combination of sets of non- naturally occurring glycosylation sites described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in ⁇ 5.2).
- the amino acid sequence of each of the at least two encoded SARS-CoV-2 spike proteins or polypeptides e.g., immunogens (or immunogenic fragments and/or immunogenic variants
- the amino acid sequence of the encoded SARS-CoV-2 spike protein is derived from a circulating strain of SARS-CoV-2. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2 Attorney Docket No.62801.28WO01 known to have previously circulated. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated.
- the vaccine composition further comprises at least one nucleic acid comprising a coding region encoding an immunogen (or immunogenic fragment and/or immunogenic variant thereof) from a non-SARS-CoV-2 virus, e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus.
- a non-SARS-CoV-2 virus e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus.
- vaccine composition comprises one or more nucleic acid molecules encoding an immunogen (or immunogenic fragment or immunogenic variant thereof) from an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, and/or an adenovirus (or any combination thereof).
- influenza virus e.g., influenza A, influenza B
- RSV respiratory syncytial virus
- rhinovirus e.g., a respiratory syncytial virus
- parvovirus e.g., a parvovirus
- parainfluenza virus e.g., parainfluenza virus, and/or an adenovirus (or any combination thereof).
- the encoded SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- ectodomain of the SARS-CoV-2 spike protein or polypeptide is modified to improve expression of the protein in host cells (e.g., insect cells, mammalian cells, eggs).
- the nucleic acid molecules are comprised within one or more vectors (e.g., vectors described herein (see, e.g., ⁇ 5.6)).
- the nucleic acid molecules or the vectors of the vaccine composition are formulated in one or more carrier (e.g., a carrier described herein (see, e.g., ⁇ 5.7)).
- the vaccine compositions are pharmaceutical compositions (e.g., described herein, e.g., see ⁇ 5.8).
- the vaccine compositions comprise an adjuvant (e.g., described herein, e.g., see ⁇ 5.9).
- Nucleic acid molecules can be generated using common methods known in the art and described above in ⁇ 5.3.
- nucleic acid molecules described herein e.g., DNA molecules, RNA molecules (e.g., mRNA molecules)
- a vector e.g., a non-viral vector, a viral vector.
- vectors e.g., viral vectors, non-viral vectors (e.g., plasmids, minicircles) comprising one or more nucleic acid molecule described herein (e.g., nucleic acid molecules encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)).
- nucleic acid molecule described herein e.g., nucleic acid molecules encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- the vector is a non-viral vector (e.g., a plasmid, minicircle).
- the vector is a plasmid.
- suitable plasmid DNA may be generated to allow efficient production of the encoded peptides or proteins (e.g., SARS-CoV-2 proteins or polypeptides (e.g., immunogens) in cell lines, e.g., in insect cell lines, for example using vectors as described in W02009150222A2 and as defined in PCT claims 1 to 33, the disclosure relating to claim 1 to 33 of W02009150222A2 the entire contents of which is incorporated by reference herein for all purposes.
- the vector is a viral vector.
- Viral vectors include both RNA and DNA based vectors.
- the vectors can be designed to meet a variety of specifications.
- viral vectors can be engineered to be capable or incapable of replication in prokaryotic and/or eukaryotic cells.
- the vector is replication deficient.
- the vector is replication competent.
- Viral vectors can be engineered or selected that either will (or will not) integrate in whole or in part into the genome of host cells, resulting (or not (e.g., episomal expression)) in stable host cells comprising the desired nucleic acid in their genome.
- Exemplary viral vectors include, but are not limited to, adenovirus vectors, adeno- associated virus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, parapoxivirus vectors, vaccinia virus vectors, fowlpox virus vectors, herpes virus vectors, adeno-associated virus vectors, alphavirus vectors, lentivirus vectors, rhabdovirus vectors, measles virus, Newcastle disease virus vectors, picornaviruses vectors, or lymphocytic choriomeningitis virus vectors.
- the viral vector is an adenovirus vector, adeno-associated virus vector, or a lentivirus vector.
- the vector is an adenovirus vector (e.g., a human adenoviral vector, e.g., HAdV or AdHu).
- the adenovirus vector has the E1 region deleted, rendering it replication-deficient in human cells. Other regions of the adenovirus such as Attorney Docket No.62801.28WO01 E3 and E4 may also be deleted.
- Exemplary adenovirus vectors include, but are not limited to, those described in e.g., W02005071093 or WQ2006048215, the entire contents of each of which is incorporated herein by reference for all purposes.
- the adenovirus-based vector used is a simian adenovirus, thereby avoiding dampening of the immune response after vaccination by pre-existing antibodies to common human entities such as AdHu5.
- simian adenovirus vectors include AdCh63 (see, e.g., W02005071093, the entire contents of which is incorporated herein by reference for all purposes) or AdCh68.
- Viral vectors can be generated through the use of a packaging/producer cell line (e.g., a mammalian cell line) using standard methods known to the person of ordinary skill in the art.
- a nucleic acid construct encoding the peptide or protein of interest (e.g., a peptide or protein described herein (e.g., SARS-CoV-2 peptide or protein described herein (e.g., a SARS-CoV-2 immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (along with additional elements e.g., a promoter, inverted terminal repeats (ITRs) flanking the transgene, a plasmid encoding e.g., viral replication and structural proteins, along with one or more helper plasmids a host cell (e.g., a host cell line) are transfected into a host cell line (i.e., the packing/producer cell line).
- a host cell e.g., a host cell line
- a host cell line i.e., the packing/producer cell line
- helper plasmid may also be needed that include helper genes from another virus (e.g., in the instance of adeno-associated viral vectors).
- Eukaryotic expression plasmids are commercially available from a variety of suppliers, for example the plasmid series: pcDNATM, pCR3.1 TM, pCMVTM, pFRTTM, pVAX1 TM, pCITM, NanoplasmidTM, and Pcaggs.
- the person of ordinary skill in the art is aware of numerous transfection methods and any suitable method of transfection may be employed (e.g., using a biochemical substance as carrier (e.g., lipofectamine), by mechanical means, by electroporation).
- the cells are cultured under conditions suitable and for a sufficient time for plasmid expression.
- the viral particles may be purified from the cell culture medium using standard methods known to the person of ordinary skill in the art. For example, by centrifugation followed by e.g., chromatography and/or ultrafiltration.
- a SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof) described herein (or a fusion or conjugate thereof)
- a nucleic acid Attorney Docket No.62801.28WO01 molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof)
- a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV- 2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)
- carriers comprising a SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, or a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein.
- a SARS-CoV-2 spike protein or polypeptide immunogen e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic
- Proteins, peptides, nucleic acid molecules e.g., RNA (e.g., mRNA), DNA
- vectors can be encapsulated within a carrier, chemically conjugated to a carrier, and/or associated with a carrier.
- the term “associated” refers to the essentially stable combination of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA, DNA), or vector with one or more molecules of a carrier (e.g., one or more lipids of a lipid-based carrier, e.g., a lipid nanoparticle (LNP), liposome, lipoplex, and/or nanoliposome) into larger complexes or assemblies without covalent binding.
- a carrier e.g., one or more lipids of a lipid-based carrier, e.g., a lipid nanoparticle (LNP), liposome, lipoplex, and/or nanoliposome
- the term “encapsulation” refers to the incorporation of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), or vector into a carrier (e.g., a lipid-based carrier, e.g., an LNP, liposome, lipoplex, and/or nanoliposome) wherein the protein, peptide, nucleic acid molecule, e.g., the RNA (e.g., mRNA, DNA), or vector is entirely contained within the interior space of the carrier (e.g., the lipid-based carrier, e.g., the LNP, liposome, lipoplex, and/or nanoliposome).
- a carrier e.g., a lipid-based carrier, e.g., an LNP, liposome, lipoplex, and/or nanoliposome
- Exemplary carriers includes, but are not limited to, lipid-based carriers (e.g., LNPs, liposomes, lipoplexes, and nanoliposomes).
- the carrier is a lipid-based carrier.
- the carrier is an LNP.
- the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid.
- Lipid based carriers are further Attorney Docket No.62801.28WO01 described below in ⁇ 5.7.1.
- a SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof) described herein (or a fusion or conjugate thereof), a nucleic acid molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), or a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV- 2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein is encapsulated or associated with one or
- any of the foregoing molecules e.g., proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.
- lipids e.g., cationic lipids and/or neutral lipids
- LNPs lipid nanoparticles
- liposomes lipoplexes
- nanoliposomes lipid nanoparticles
- the molecule e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) is associated with one or more lipids (e.g., cationic lipids and/or neutral lipids), thereby forming lipid-based carriers such as lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes.
- lipids e.g., cationic lipids and/or neutral lipids
- the molecule e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) is encapsulated in LNPs (e.g., as described herein).
- LNPs e.g., as described herein.
- the use of LNPs for mRNA delivery is further detailed in e.g., Hou X et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078-1094. doi: 10.1038/s41578- 021-00358-0. Epub 2021 Aug 10.
- the molecules e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.) described herein may be completely or partially located in the interior space of the LNPs, liposomes, lipoplexes, and/or nanoliposomes, within the lipid layer/membrane, or associated with the exterior surface of the lipid layer/membrane.
- RNA e.g., RNA (e.g., mRNA), DNA
- vector etc.
- a molecule e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) into LNPs, liposomes, lipoplexes, and/or nanoliposomes
- the molecule e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA
- an environment may contain enzymes or chemicals or conditions that degrade the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) from molecules or conditions that cause the rapid excretion of the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g.,
- incorporating the molecules into LNPs, liposomes, lipoplexes, and/or nanoliposomes may promote the uptake of the molecules (e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.), and hence, may enhance the therapeutic effect of the proteins or nucleic acid molecules (e.g., RNA, e.g., mRNA).
- a molecule e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.
- LNPs liposomes, lipoplexes, and/or nanoliposomes
- molecules e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.
- molecules e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.
- lipid-based carrier or lipid nanoformulation
- the lipid-based carrier is a liposome or a lipid nanoparticle (LNP).
- the lipid-based carrier is an LNP.
- the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid (e.g., an ionizable lipid), a non-cationic lipid (e.g., phospholipid), a structural lipid (e.g., cholesterol), and a PEG-modified lipid.
- the lipid-based carrier contains one or more molecules described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein), or a pharmaceutically acceptable salt thereof.
- suitable compounds to be used in the lipid-based carrier (or lipid nanoformulation) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.
- the lipid-based carrier may further include a second lipid.
- the second lipid is a cationic lipid, a non-cationic (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid.
- One or more naturally occurring and/or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation).
- the lipid-based carrier may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.
- cationic lipids may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof.
- Cationic Lipids Pulsitively Charged
- Ionizable Lipids In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more cationic lipids, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated.
- the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions.
- Exemplary cationic lipids include one or more amine group(s) which bear the positive charge.
- Examples of positively charged (cationic) lipids include, but are not limited to, N,N'- dimethyl-N,N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3 ⁇ -[N-(N',N'- dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), 1,2-dioleoyloxy-3-[trimethylammonio]- propane (DOTAP), 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2- dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), N,N-dioleyl-
- the lipid-based carrier comprises more than one cationic lipid.
- Attorney Docket No.62801.28WO01 the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0.
- the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid.
- cationic lipids that can be used in the lipid-based carrier (or lipid nanoformulation) include, for example those described in Table 4 of WO 2019/217941, the entire contents of which are incorporated by reference herein for all purposes.
- the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH).
- the lipid-based carrier (or lipid nanoformulation) may comprise one or more additional ionizable lipids, different than the ionizable lipids described herein.
- Exemplary ionizable lipids include, but are not limited to, , ,
- the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described by WO 2021/113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO 2021/113777), the entire contents of which are incorporated by reference herein for all purposes.
- the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078–1094 (2021).
- Examples of other ionizable lipids that can be used in lipid-based carrier (or lipid nanoformulation) include, without limitation, one or more of the following formulas: X of US 2016/0311759; I of US 20150376115 or in US 2016/0376224; Compound 5 or Compound 6 in US 2016/0376224; I, IA, or II of US 9,867,888; I, II or III of US 2016/0151284; I, IA, II, or IIA of US 2017/0210967; I-c of US 2015/0140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV, or V of US 2015/0239926; I of US 2017/0119904; I or II of WO 2017/117528; A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372;
- the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,l2Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-(((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca-9,l2-dienoate).
- biodegradable ionizable lipids for instance, (9Z,l2Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbon
- Non-Cationic Lipids e.g., Phospholipids
- the lipid-based carrier or lipid nanoformulation
- the non-cationic lipid is a phospholipid.
- the non-cationic lipid is a phospholipid substitute or replacement.
- the non-cationic lipid is a negatively charged (anionic) lipid.
- non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (
- acyl groups in these lipids are preferably acyl groups derived from fatty acids having C 10- C 24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl.
- Additional exemplary lipids include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, the entire contents of which are incorporated by reference herein for all purposes.
- lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS).
- the lipid-based carrier (or lipid nanoformulation) may comprise a combination of distearoylphosphatidylcholine/cholesterol, dipalmitoylphosphatidylcholine/cholesterol, dimyrystoylphosphatidylcholine/cholesterol, 1,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol, or egg sphingomyelin/cholesterol.
- DOPC 1,2- Dioleoyl-sn-glycero-3-phosphocholine
- non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like.
- nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl my
- the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipid that is oleic acid or a compound of Formula I, II, or IV of US Attorney Docket No.62801.28WO01 2018/0028664, the entire contents of which are incorporated by reference herein for all purposes.
- the non-cationic lipid content can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10- 15% (mol) of the total lipid components present.
- the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, and the molar ratio of an ionizable lipid to a neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1).
- the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids.
- the lipid-based carrier (or lipid nanoformulation) can further include one or more phospholipids, and optionally one or more additional molecules of similar molecular shape and dimensions having both a hydrophobic moiety and a hydrophilic moiety (e.g., cholesterol).
- the lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or more structural lipids.
- structural lipid refers to sterols (e.g., cholesterol) and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipid in the particle.
- Structural lipids can be selected from the group including but not limited to, cholesterol or cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof.
- the structural lipid is a sterol.
- the structural lipid is a steroid.
- the structural lipid is cholesterol.
- the structural lipid is an analog of cholesterol.
- the structural lipid is alpha-tocopherol.
- structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid).
- sterols when present, can include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009/127060 or US 2010/0130588, the entire contents of each of which are incorporated by reference herein for all purposes. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), Nano Attorney Docket No.62801.28WO01 Lett. 2020;20(6):4543-4549, the entire contents of which are incorporated by reference herein for all purposes.
- the structural lipid is a cholesterol derivative.
- Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53- coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof.
- the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-buty1 ether.
- the lipid-based carrier (or lipid nanoformulation) further comprises sterol in an amount of 0-50 mol% (e.g., 0-10 mol %, 10-20 mol %, 20-50 mol%, 20-30 mol %, 30-40 mol %, or 40-50 mol %) of the total lipid components.
- the lipid-based carrier may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PFAG) nanoparticles.
- the lipid-based carrier may include one or more polyethylene glycol (PEG) lipid.
- PEG-lipids examples include, but are not limited to, 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 350] (mPEG 350 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000] (mPEG 1000 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (mPEG 2000 PE); 1,2- Diacyl-sn-Glycero
- the PEG lipid is a polyethyleneglycol- diacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG- Attorney Docket No.62801.28WO01 DMB) conjugate.
- the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO 2019/217941, the entire contents of which are incorporated by reference herein for all purposes).
- the one or more conjugated lipids is formulated with one or more ionic lipids (e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid); and one or more sterols (e.g., cholesterol).
- one or more ionic lipids e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid
- one or more sterols e.g., cholesterol
- the PEG conjugate can comprise a PEG-dilaurylglycerol (C12), a PEG- dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG- dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18).
- a PEG-dilaurylglycerol C12
- PEG- dimyristylglycerol C14
- PEG-dipalmitoylglycerol C16
- PEG-disterylglycamide C18
- conjugated lipids when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn
- DAG P
- PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003/0077829, US 2003/0077829, US 2005/0175682, US 2008/0020058, US 2011/0117125, US 2010/0130588, US 2016/0376224, US 2017/0119904, US 2018/0028664, and WO 2017/099823, the entire contents of each of which are incorporated by reference herein for all purposes.
- the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III- b-1, III-b-2, or V of US 2018/0028664, which is incorporated herein by reference in its entirety.
- the PEG-lipid is of Formula II of US 2015/0376115 or US 2016/0376224, the entire contents of each of which are incorporated by reference herein for all purposes.
- the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl.
- the PEG-lipid includes one of the following: be used in place of PEG-lipid.
- polyoxazoline (POZ)-lipid conjugates polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid.
- exemplary conjugated lipids e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids, include those described in Table 2 of WO 2019/051289A9, the entire contents of which are incorporated by reference herein for all purposes.
- the conjugated lipid e.g., the PEGylated lipid
- the conjugated lipid can be present in an amount of 0-20 mol% of the total lipid components present in the lipid-based carrier (or lipid nanoformulation).
- the conjugated lipid (e.g., the PEGylated lipid) content is 0.5-10 mol% or 2-5 mol% of the total lipid components.
- the lipid-based carrier (or lipid nanoformulation) described herein may be coated with a polymer layer to enhance stability in vivo (e.g., sterically stabilized LNPs).
- Suitable polymers include, but are not limited to, poly(ethylene glycol), which may form a hydrophilic surface layer that improves the circulation half-life of liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, e.g., Working et al.
- the lipid-based carrier comprises one of more of the molecules described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.), optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and optionally conjugated lipid (e.g., a PEGylated lipid) that inhibits aggregation of particles.
- a protein e.g., a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.)
- a non-cationic lipid e.g., a phospholipid
- sterol e.g., a sterol
- neutral lipid e.g., a neutral lipid
- conjugated lipid e.g., a P
- the lipid-based carrier (or lipid nanoformulation) further comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein).
- a payload e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein).
- a payload e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein).
- RNA e.g., mRNA
- DNA e.g.
- the ionizable lipid including the lipid compounds described herein is present in an amount from about 20 mol% to about 100 mol% (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50-90 mol%) of the total lipid components; a non-cationic lipid (e.g., phospholipid) is present in an amount from about 0 mol% to about 50 mol% (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%) of the total lipid components, a conjugated lipid (e.g., a PEGylated lipid) in an amount from about 0.5 mol% to about 20 mol% (e.g., 1-10 mol% or 5-10%) of the total lipid components; a
- the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid.
- the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- the encapsulation efficiency of the payload may be at least 70%.
- the lipid-based carrier (or lipid nanoformulation) comprises about Attorney Docket No.62801.28WO01 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid.
- the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- the lipid nanoparticle comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid.
- the encapsulation efficiency of the payload may be at least 70%.
- the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid.
- the lipid-based carrier comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5
- the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- a payload e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- the lipid nanoparticle comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid.
- the encapsulation efficiency of the payload may be at least 70%.
- molar ratios of ionizable lipid/sterol/phospholipid (or another structural lipid)/PEG-lipid/additional components is varied in the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).
- the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- the lipid nanoparticle comprises molar ratios of ionizable lipid/sterol/phospholipid (or another structural lipid)/PEG-lipid/additional components in the following ranges: ionizable lipid (25-100%); Attorney Docket No.62801.28WO01 phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%).
- the encapsulation efficiency of the payload may be at least 70%.
- the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid).
- the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- the lipid nanoparticle comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid).
- the encapsulation efficiency of the payload may be at least 70%.
- the lipid-based carrier (or lipid nanoformulation) comprises (i) a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- a molecule described herein e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- a cationic lipid comprising from 50 mol% to 65 mol% of the total lipid present in the lipid-based carrier;
- a non-cationic lipid comprising a mixture of a phospholipid and a cholesterol derivative thereof, wherein the phospholipid comprises from 3 mol% to 15 mol% of the total lipid present in the lipid-based carrier and the cholesterol or derivative thereof comprises from 30 mol% to 40 mol% of the total lipid present in the lipid-based carrier; and
- a conjugated lipid comprising 0.5 mol% to 2 mol% of the total lipid present in the particle.
- the lipid-based carrier (or lipid nanoformulation) comprises (i) a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- a molecule described herein e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.
- a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the lipid-based carrier;
- a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the lipid-based carrier;
- a conjugated lipid comprising from 0.5 mol % to 2 mol % of the total lipid present in the lipid-based carrier.
- the phospholipid component in the mixture may be present from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15 Attorney Docket No.62801.28WO01 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol%, (or any fraction of these ranges) of the total lipid components.
- the lipid-based carrier or lipid nanoformulation
- the sterol component e.g.
- cholesterol or derivative) in the mixture may comprise from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 25 mol% to 35 mol%, from 25 mol% to 30 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 30 mol% to 35 mol%, from 35 mol% to 40 mol%, from 27 mol% to 37 mol%, or from 27 mol% to 35 mol% (or any fraction of these ranges) of the total lipid components.
- the non-ionizable lipid components in the lipid-based carrier may be present from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% (or any fraction of these ranges) of the total lipid components.
- the ratio of total lipid components to the payload e.g., an encapsulated therapeutic agent such as a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein) can be varied as desired.
- the total lipid components to the payload (mass or weight) ratio can be from about 10:1 to about 30:1.
- the total lipid components to the payload ratio can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1.
- the amounts of total lipid components and the payload can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher.
- the lipid-based carrier (or lipid nanoformulation’s) overall lipid content can range from about 5 mg/ml to about 30 mg/mL.
- Nitrogen:phosphate ratios (N:P ratio) is evaluated at values between 0.1 and 100.
- the efficiency of encapsulation of a payload such as a protein and/or nucleic acid describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation, relative to the initial amount provided.
- the encapsulation efficiency is desirably high (e.g., at least 70%. 80%. 90%. 95%, close to 100%).
- the encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents.
- An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in Attorney Docket No.62801.28WO01 a solution.
- Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution.
- the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.
- the encapsulation efficiency may be at least 70%.
- the encapsulation efficiency may be at least 80%.
- the encapsulation efficiency may be at least 90%.
- the encapsulation efficiency may be at least 95%.
- compositions comprising a SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (or a fusion or conjugate thereof), a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a fusion or conjugate thereof), a vector described herein (
- compositions described herein comprising providing a SARS-CoV-2 spike protein or polypeptide Attorney Docket No.62801.28WO01 (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein), a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike
- Acceptable excipients are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid or methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol;or m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine
- a pharmaceutical composition may be formulated for any route of administration to a subject.
- the skilled person knows the various possibilities to administer a pharmaceutical Attorney Docket No.62801.28WO01 composition described herein a in order to induce an immune response to the immunogens(s) and/or antigen(s) in the pharmaceutical composition.
- Non-limiting embodiments include parenteral administration, such as intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal administration, e.g., inhalation, intranasal, oral, and the like.
- the pharmaceutical composition is formulated for administration by intramuscular, intradermal, or subcutaneous injection.
- the pharmaceutical composition is formulated for administration by intramuscular injection.
- the pharmaceutical composition is formulated for administration by intradermal injection. In one embodiment, the pharmaceutical composition is formulated for administration by subcutaneous injection.
- injectables can be prepared in conventional forms, either as liquid solutions or suspensions.
- the injectables can contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol or ethanol.
- the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.
- auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins.
- the pharmaceutical composition is formulated in a single dose.
- the pharmaceutical compositions if formulated as a multi-dose.
- compositions described herein include for example, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents or other pharmaceutically acceptable substances.
- aqueous vehicles which can be incorporated in one or more of the formulations described herein, include sodium chloride injection, Ringer’s injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer’s injection.
- Nonaqueous parenteral vehicles which can be incorporated in one or more of the formulations described herein, include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil.
- Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to the parenteral preparations described herein and packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride or benzethonium chloride.
- Isotonic agents which can be incorporated in one or more of the formulations described herein, include sodium chloride or dextrose.
- Buffers Attorney Docket No.62801.28WO01 which can be incorporated in one or more of the formulations described herein, include phosphate or citrate.
- Antioxidants which can be incorporated in one or more of the formulations described herein, include sodium bisulfate.
- Local anesthetics which can be incorporated in one or more of the formulations described herein, include procaine hydrochloride.
- Suspending and dispersing agents which can be incorporated in one or more of the formulations described herein, include sodium carboxymethylcelluose, hydroxypropyl methylcellulose or polyvinylpyrrolidone.
- Emulsifying agents which can be incorporated in one or more of the formulations described herein, include Polysorbate 80 (TWEEN ® 80).
- a sequestering or chelating agent of metal ions which can be incorporated in one or more of the formulations described herein, is EDTA.
- Pharmaceutical carriers which can be incorporated in one or more of the formulations described herein, also include ethyl alcohol, polyethylene glycol or propylene glycol for water miscible vehicles; orsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment.
- dose to be employed in a pharmaceutical composition will also depend on the route of administration, and the seriousness of the condition caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances.
- effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), other medications administered, or whether therapy is prophylactic or therapeutic.
- Therapeutic dosages are preferably titrated to optimize safety and efficacy.
- Adjuvants Any of the foregoing, e.g., SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein (or a fusion or conjugate thereof), nucleic acid molecules described herein (e.g., nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (or a fusion or conjugate thereof), vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/
- Adjuvants are known in the art to further increase the immune response (e.g., to an immunogen).
- General categories of adjuvants include, but are not limited to, inorganic adjuvants, small molecule adjuvants, oil in water emulsions, lipids, polymers, peptides, peptidoglycans, carbohydrates, polysaccharides, RNA-based adjuvants, DNA-based adjuvants, viral particles, bacterial adjuvants, nanoparticles (e.g., inorganic nanoparticles), and multi-component adjuvants.
- adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO90/14837, the entire contents of which is incorporated herein by reference for all purposes), MF59, AS03, and Montanide; saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see, e.g., US5,057,540; WO90/03184, WO96/11711, WO2004/004762, WO2005/002620, the entire contents of each of which is incorporated herein by reference for all purposes); protamine or a protamine salt (e.g., protamine sulfate); calcium salt; bacterial or microbial derivatives, examples of which include monophosphoryl lipid A (MPL), 3-O
- coli heat labile enterotoxin LT cholera toxin CT, and the like
- eukaryotic proteins e.g., antibodies or fragments thereof (e.g., directed against the antigen itself or CD1a, CD3, CD7, CD80) and ligands to receptors (e.g., CD40L, GMCSF, GCSF, etc.).
- RNA-based adjuvants include, but are not limited to, Poly IC, Poly IC:LC, hairpin RNAs, e.g., with a 5’PPP containing sequence, viral sequences, polyU containing sequences, dsRNA, natural or synthetic immunostimulatory RNA sequences, nucleic acids analogs, optionally cyclic GMP-AMP or a cyclic dinucleotide such as cyclic di-GMP, and immunostimulatory base analogs, e.g., C8-substitued or an N7,C8-disubstituted guanine ribonucleotide.
- Exemplary DNA-based adjuvants include, but are not limited to, CpGs, dsDNA, Attorney Docket No.62801.28WO01 or natural or synthetic immunostimulatory DNA sequences.
- Exemplary bacteria-based adjuvants include, but are not limited, to bacterial adjuvant is flagellin, LPS, or a bacterial toxin, e.g., enterotoxins, heat-labile toxins, and Cholera toxins.
- Exemplary carbohydrate or polysaccharide adjuvants include, but are not limited to, dextran (branched microbial polysaccharide), dextran- sulfate, Lentinan, zymosan, Betaglucan, Deltin, Mannan, and Chitin.
- Exemplary small molecule adjuvants include, but are not limited to, imiquimod, resiquimod, and gardiquimod.
- Exemplary lipid or polymer adjuvants include, but are not limited to, polymeric nanoparticles (e.g., PLGA, PLG, PLA, PGA, or PHB), liposomes (e.g., Virosomes and CAF01), LNPs or a component thereof, lipopolysaccharide (LPS) (e.g., monophosphoryl lipid A (MPLA) or glucopyranosyl Lipid A (GLA)), lipopeptides (e.g., Pam2 (Pam2CSK4) or Pam3 (Pam3CSK4)), and glycolipid (e.g., trehalose dimycolate).
- polymeric nanoparticles e.g., PLGA, PLG, PLA, PGA, or PHB
- liposomes e.g., Virosomes and CAF
- Exemplary peptides or peptidoglycan include, but are not limited to, N- acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), flagellin-fusion protein, mannose-binding lectin (MBL), cytokines, and chemokine.
- Exemplary inorganic nanoparticle adjuvants include, but are not limited to, gold nanorods, silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)).
- Exemplary multicomponent adjuvants include, but are not limited to, AS01, AS03, AS04, Complete Freunds Adjuvant, and CAF01.
- SARS-CoV-2 spike proteins and polypeptides e.g., immunogens (and immunogenic fragments and/or immunogenic variants thereof) described herein (or fusions or conjugates thereof)
- the nucleic acid molecules described herein e.g., nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof))
- the vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the carriers described herein e.g., carriers comprising a nucleic acid molecule described herein (e.g., a nucleic
- the methods include administering one or more of the foregoing (e.g., protein (or a fusion or conjugate thereof), polypeptide (or a fusion or conjugate thereof), immunogen (or a fusion or conjugate thereof), nucleic acid molecule (or a fusion or conjugate thereof), vector, carrier, composition (e.g., vaccine composition), pharmaceutical composition) to a subject.
- exemplary subjects include mammals, e.g., humans, non-human mammals, e.g., non-human primates.
- the subject is a human.
- the subject is, elderly, pregnant, a newborn, immunocompromised, or immunosuppressed.
- the subject has one or more of the following cancer, heart disease, obesity, diabetes, asthma, chronic lung disease, and/or sickle cell disease.
- the subject has a weakened immune system or weakened immune response (e.g., a weakened immune response to a vaccine).
- the subject is immunocompromised or immunosuppressed.
- the subject is clinically vulnerable to the infection.
- the subject has cancer, has an autoimmune disease, has an immunodeficiency, received a bone marrow or organ transplant, is undergoing a therapy that depletes immune cells, is undergoing chemotherapy, has a chronic viral infection, post viral syndrome or post viral fatigue syndrome (e.g., HIV infection or AIDS; long Covid or persistent post-Covid syndrome), is using or has had prolonged use of an immunosuppressive medication, is currently a smoker or has a history of smoking, or is at least 50 (e.g., at least 55, 60, 65, 70, 75, 80, 85, 90, or 100) years of age. In some embodiments, the subject at least 50, 60, 65, 70, or 75 years of age.
- a chronic viral infection, post viral syndrome or post viral fatigue syndrome e.g., HIV infection or AIDS; long Covid or persistent post-Covid syndrome
- is using or has had prolonged use of an immunosuppressive medication is currently a smoker or has a history of smoking, or is at least 50 (e.g., at least 55
- the subject is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, or 120 years of age. In some embodiments, the subject is from about 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-120, 70-110, 70-100, 70-90, 70-80, 80-120, 80-110, 80-100, 80-90, 90-120, 90-110, or 90-100 years of age.
- the dosage of one or more of the foregoing e.g., protein, polypeptide, immunogen, nucleic acid molecule, vector, carrier, vaccine composition, pharmaceutical composition
- the dosage of one or more of the foregoing can be determined in accordance with standard techniques well known Attorney Docket No.62801.28WO01 to those of ordinary skill in the art, including the type (if any) adjuvant is used, the route of administration, and the age and weight of the subject.
- a single dose of any one of the foregoing is administered to a subject in need thereof.
- a series of doses of any one of the foregoing are administered to a subject in need thereof (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks) apart or within a range (e.g., 2-6 weeks apart)).
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule described herein, the vector described herein, the carrier described herein, the vaccine composition, or the pharmaceutical composition described herein is administered in a therapeutically effective amount.
- a dose of an mRNA molecule encoding a SARS-CoV-2 spike protein or polypeptide is between 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg.
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g.,
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition
- the nucleic acid molecule e.g., the RNA molecule, e.g., an mRNA molecule
- the vector, the carrier, the vaccine composition, or the pharmaceutical composition to the subject.
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, Attorney Docket No.62801.28WO01 e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof),
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule described herein (e.g., a nucleic acid molecule described herein (e.g., a nucleic acid
- the medicament further comprises an adjuvant (e.g., an adjuvant described herein).
- an adjuvant e.g., an adjuvant described herein.
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogen).
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response the subject.
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an m
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an m
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g.,
- a SARS-CoV-2 Attorney Docket No.62801.28WO01 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein)
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in the subject.
- the epitope is conserved in SARS-CoV-2 viruses.
- the epitope is in a functionally constrained region of the SARS-CoV-2 spike protein.
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject.
- An immune response in a subject can be measured by common methods known to those Attorney Docket No.62801.28WO01 of skill in the art.
- serological assays can be employed to detect a humoral response by measuring titers of anti-antigen (e.g., anti-SARS-CoV-2 spike protein, anti- SARS-CoV-2 spike protein RBD) IgG antibodies post administration.
- anti-antigen e.g., anti-SARS-CoV-2 spike protein, anti- SARS-CoV-2 spike protein RBD
- ELISA enzyme-linked immunosorbent assay
- blood is collected from a consenting subject, centrifuged, and the serum isolated according to standard techniques.
- the recombinant target antigen e.g., SARS-CoV-2 spike protein, SARS-CoV-2 spike protein RBD
- SARS-CoV-2 spike protein SARS-CoV-2 spike protein RBD
- the microplate is blocked by through the incubation with an irrelevant antigen (e.g., bovine serum albumin).
- an irrelevant antigen e.g., bovine serum albumin
- the serum sample from the subject is prepared and added to the blocked wells to allow for binding of an antigen specific antibodies to the immobilized antigen.
- the bound antibodies are detected using a secondary tagged antibody that binds to the previously bound antibodies (e.g., anti-human IgG antibodies). See, e.g., Front.
- Cell based assays can also be utilized to detect a cell based immune response (e.g., T cell immune response).
- antigen specific T cells e.g., CD4+ or CD8+ T cells
- ELISpot enzyme-linked immunospot
- ICS intracellular cytokine staining
- AIM activation induced marker assay
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-Co
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to prevent and/or an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen the subject.
- the epitope is not conserved in SARS-CoV-2 viruses.
- the epitope is not in a functionally constrained region of the SARS-CoV-2 spike protein.
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein
- the SARS-CoV-2 spike protein or polypeptide e.g., the SARS- CoV-2 protein or peptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to prevent, ameliorate, and/or treat the SARS-CoV-2 infection the subject.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject as a prophylactic treatment.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2 infection associated disease.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS- CoV-2, reduces the likelihood of developing an established infection after challenge with SARS- CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID- 19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof.
- Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.
- the SARS-CoV-2 spike proteins e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)
- the nucleic acid molecules e.g., RNA molecules, e.g., mRNA molecules
- the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein may be administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen.
- the boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like Attorney Docket No.62801.28WO01 particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- the prime vaccine composition contains the same immunogen as the booster vaccine.
- the primary vaccine contains a different immunogen as the booster vaccine.
- the SARS-CoV-2 spike proteins e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)
- the nucleic acid molecules e.g., RNA molecules, e.g., mRNA molecules
- the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen.
- the prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno- associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- the prime vaccine composition contains the same immunogen as the booster vaccine.
- the primary vaccine contains a different immunogen as the booster vaccine.
- a single dose of the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector e.g., the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition
- a series of doses of the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)).
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is Attorney Docket No.62801.28WO01 administered to the subject in a therapeutically effective amount.
- an mRNA molecule encoding a SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (e.g., a vaccine or pharmaceutical composition comprising the same) is administered to the subject
- the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an m
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), Attorney Docket No.62801.28WO01
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), Attorney Docket No.62801.28WO01
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., a nucleic acid molecule (e.g.,
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof),
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g.
- vaccinating subjects e.g., human subjects
- various methods of vaccinating subjects utilizing one or more of the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein.
- SARS-CoV-2 spike proteins e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)
- the nucleic acid molecules e.g., RNA molecules, e.g., mRNA molecules
- the vectors e.g., the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein.
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- a vector e.g., a vector
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to vaccinate the subject against SARS-CoV-2.
- the SARS-CoV-2 spike proteins e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)
- the nucleic acid molecules e.g., RNA molecules, e.g., mRNA molecules
- the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described Attorney Docket No.62801.28WO01 herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen.
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen.
- the boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- the prime vaccine composition contains the same immunogen as the booster vaccine.
- the primary vaccine contains a different immunogen as the booster vaccine.
- the SARS-CoV-2 spike proteins e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)
- the nucleic acid molecules e.g., RNA molecules, e.g., mRNA molecules
- the vectors, the carriers, the vaccine compositions, and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen.
- the prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- the prime vaccine composition contains the same immunogen as the booster vaccine.
- the primary vaccine contains a different immunogen as the booster vaccine.
- a single dose of the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector e.g., the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition
- a series of doses of the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)).
- the SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject in a therapeutically effective amount.
- an mRNA molecule encoding a SARS-CoV-2 spike protein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (e.g., a vaccine or pharmaceutical composition comprising the same) is administered to the subject
- the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject as a prophylactic treatment.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2 Attorney Docket No.62801.28WO01 infection associated disease.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., the RNA molecule, e.g., the mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS- CoV-2, reduces the likelihood of developing an established infection after challenge with SARS- CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID- 19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof.
- Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an m
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an m
- SARS-CoV-2 spike proteins or polypeptides described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein
- nucleic acid molecules described herein e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- vectors described herein e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g.,
- a SARS-CoV-2 spike protein or polypeptide described herein e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof)
- a nucleic acid molecule described herein e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g.
- mRNA molecule e.g., an mRNA molecule described herein
- a SARS-CoV-2 spike protein e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein (or a fusion or conjugate thereof)
- a vector comprising the mRNA molecule
- a carrier comprising the mRNA molecule or the vector
- a composition e.g., vaccine composition
- a pharmaceutical composition comprising the mRNA molecule, the vector, the carrier, or the vaccine Attorney Docket No.62801.28WO
- the mRNA molecule is formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1- methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the mRNA molecule the vector, the carrier, the composition (e.g.,
- the mRNA molecules, the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen.
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen.
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen.
- the mRNA molecule, the vector, the carrier, the v composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen.
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen.
- the boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- the prime vaccine composition contains the same immunogen as the Attorney Docket No.62801.28WO01 booster vaccine.
- the primary vaccine contains a different immunogen as the booster vaccine.
- the mRNA molecules, the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen.
- the prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like.
- viral vectors e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vector
- the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, a single dose of the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)).
- a set interval e.g., 2 weeks, 3 weeks apart
- a range e.g., 2-6 weeks apart
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in a therapeutically effective amount.
- the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg).
- the mRNA molecule, the vector, the carrier, or the pharmaceutical composition is administered to the subject as a prophylactic treatment.
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2 infection associated disease.
- the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection.
- the mRNA molecule, the vector, the carrier, the vaccine Attorney Docket No.62801.28WO01 composition, or the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS-CoV-2, reduces the likelihood of developing an established infection after challenge with SARS-CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID-19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof.
- Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.
- kits comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule) described herein, a vector described herein, a carrier described herein, a composition (e.g., vaccine composition) described herein, and/or a pharmaceutical composition described herein.
- the kit may comprise a liquid vehicle for solubilizing or diluting any one of the foregoing, and/or technical instructions.
- the technical instructions of the kit may contain information about administration and dosage and subjects (e.g., subject groups).
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector, the carrier, the composition e.g., vaccine composition
- the pharmaceutical composition described herein is provided in a separate part of the kit, wherein the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is optionally lyophilized
- the kit may further contain as a part a vehicle (e.g., buffer solution) for solubilizing the dried or lyophilized pharmaceutical composition.
- the kit comprises a single dose container of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition.
- a vehicle e.g., buffer solution
- the kit comprises a single dose container of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector,
- the kit comprises a multi- dose container for administration of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein and/or an administration device (e.g., an injector for intradermal injection or a syringe for intramuscular injection).
- an administration device e.g., an injector for intradermal injection or a syringe for intramuscular injection.
- the kit comprises an adjuvant in a separate container from the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein.
- the SARS-CoV-2 spike protein or polypeptide e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)
- the nucleic acid molecule e.g., RNA molecule, e.g., mRNA molecule
- the vector e.g., mRNA molecule
- the composition e.g., vaccine composition
- the kit may further contain technical instructions for mixing the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein and the adjuvant prior to administration or for co- administration.
- any of the kits described herein may be used in any of the methods described herein, e.g., in ⁇ 5.10.
- kits described herein may be used in a treatment or prophylaxis as defined herein (e.g., for the treatment, amelioration, and/or prophylaxis of SARS-CoV-2 infection).
- DNA constructs comprising SARS-CoV-2 proteins (e.g., immunogens) comprising at least one of the non-naturally glycosylation sites described herein are prepared and used for subsequent RNA in vitro transcription.
- Preparation of the DNA coding sequences can include codon optimization for stabilization and expression by introducing specific codons to generate a DNA coding sequence with an optimized G/C content (as discussed herein).
- Optimized coding sequences are introduced into a DNA plasmid comprising a 3’-UTR, a 5-UTR, and polyadenylation sequence.
- the obtained DNA plasmids are transformed and propagated in bacteria using common protocols known in the art.
- the DNA plasmids are subsequently extracted, purified, and used for RNA in vitro transcription. 6.1.2 RNA in vitro transcription
- the DNA plasmids are enzymatically linearized using a restriction enzyme used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and a 5’ cap (or analog) under suitable buffer conditions.
- the obtained RNA constructs are purified using a suitable method known in the art e.g., RP-HPLC.
- RNA in vitro transcription is performed in the presence of modified nucleotides for incorporation in the RNA e.g., pseudouridine or N1 -methylpseudouridine (m1 ⁇ ) instead of UTP.
- modified nucleotides for incorporation in the RNA e.g., pseudouridine or N1 -methylpseudouridine (m1 ⁇ ) instead of UTP.
- the 5’ cap is enzymatically added to the RNA after in vitro transcription using capping enzymes as commonly known in the art.
- LNPs are prepared using according to the general procedures known in the art using e.g., cationic lipids, structural lipids, a PEG-lipids, and cholesterol see, e.g., WO2015199952, WO2017004143 and WO2017075531, the full contents of each of which is incorporated by reference herein for al purposes.
- the lipid solution in ethanol
- RNA in aqueous solution
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Abstract
Provided herein are SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike proteins and polypeptide immunogens (and immunogenic fragments and immunogenic variants thereof)) comprising at least one non-naturally occurring glycosylation site, and nucleic acid molecules encoding the same. Further provided herein are compositions (e.g., pharmaceutical compositions) and vaccines comprising the same for use in e.g., the prevention, treatment, and/or amelioration of a SARS-CoV-2 infection.
Description
Attorney Docket No.62801.28WO01 SARS-CoV-2 VACCINE COMPOSITIONS AND RELATED METHODS RELATED APPLICATIONS This application claims priority to U.S. Serial No.: 63/580,865, filed September 6, 2023; and U.S. Serial No.: 63/604,285, filed November 30, 2023, the entire contents of each of which is incorporated herein by reference. 1. FIELD This disclosure relates to SARS-CoV-2 spike proteins and polypeptides (e.g., SARS- CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)), that comprise at least one non-naturally occurring glycosylation site (described herein) and nucleic acid molecules encoding the same. The disclosure further relates to compositions comprising of the same (e.g., vaccine compositions, pharmaceutical compositions) and methods of making and utilizing the same. 2. BACKGROUND Coronaviruses are a family of enveloped, positive-sense, single stranded RNA viruses that infect a wide variety of mammalian and avian species. The viral genome is packaged into a capsid that is comprised of the viral nucleocapsid protein and surrounded by a lipid envelope. Embedded in the lipid envelope are several proteins, including, the membrane protein, the envelope small membrane protein, hemagglutinin-esterase, and the spike protein. Human coronaviruses typically cause respiratory illnesses, and include, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus 1 (SARS- CoV-1), and Middle East respiratory syndrome (MERS-CoV). SARS-Cov-2 emerged in humans in 2019, spread rapidly, and led to a global pandemic. SARS-CoV-2 is the cause of the coronavirus disease 2019 (COVID-19). COVID-19 caused a continuing public health crisis, with millions of deaths and severe illness attributed to COVID-19 worldwide. Protection against COVID-19 is mediated in large part by an immune response directed against the SARS-CoV-2 spike protein, a main target of SARS-CoV-2 vaccines. The spike protein mediates binding and entry into host cells, through binding of the receptor binding domain (RBD) to the host cell receptor angiotensin-converting enzyme 2 (ACE2).
Attorney Docket No.62801.28WO01 3. SUMMARY Provided herein are, inter alia, SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)), nucleic acid molecules encoding the same, compositions (e.g., vaccine compositions, pharmaceutical compositions) comprising the SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)) or nucleic acids encoding the same, methods of manufacturing, and methods of use including, e.g., methods of preventing, reducing, or treating a SARS-CoV-2 infection, methods of vaccination against a SARS-CoV-2 infection, etc. In one aspect, provided herein are nucleic acid molecules comprising at least one coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper-glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-naturally occurring glycosylation motifs each comprising a non-naturally occurring glycosylation site, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT sequence, wherein X is any amino
Attorney Docket No.62801.28WO01 acid except for proline. In some embodiments, the N-linked glycosylation motif is an NXS sequence, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the glycosylation motif is an O-linked glycosylation motif. In some embodiments, the O-linked glycosylation motif is a serine or threonine. In some embodiments, the glycosylation site is an O-linked glycosylation site. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-linked glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N- glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N- glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or
Attorney Docket No.62801.28WO01 immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID
Attorney Docket No.62801.28WO01 NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N- glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 428, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid
Attorney Docket No.62801.28WO01 position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N- linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448,
Attorney Docket No.62801.28WO01 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N- linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-
Attorney Docket No.62801.28WO01 glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid
Attorney Docket No.62801.28WO01 numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine or a serine at position 479 and an N-glycosylation
Attorney Docket No.62801.28WO01 site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine or a serine at position 519 and an N- glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine or a serine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N- linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a naturally occurring glycosylation site) at one or more of amino acid positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) relative the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) )) in a prefusion state relative the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an inactive furin cleavage site. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or
Attorney Docket No.62801.28WO01 immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 986 and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N- linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO:
Attorney Docket No.62801.28WO01 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N- linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic
Attorney Docket No.62801.28WO01 acid at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions
Attorney Docket No.62801.28WO01 set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a
Attorney Docket No.62801.28WO01 threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions
Attorney Docket No.62801.28WO01 set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a
Attorney Docket No.62801.28WO01 serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions
Attorney Docket No.62801.28WO01 set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino
Attorney Docket No.62801.28WO01 acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at
Attorney Docket No.62801.28WO01 position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at
Attorney Docket No.62801.28WO01 position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set
Attorney Docket No.62801.28WO01 forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at
Attorney Docket No.62801.28WO01 position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position
Attorney Docket No.62801.28WO01 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering
Attorney Docket No.62801.28WO01 relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or
Attorney Docket No.62801.28WO01 immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429,
Attorney Docket No.62801.28WO01 a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a
Attorney Docket No.62801.28WO01 serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises from about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10- 100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10- 1200, 10-1300, 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100- 1000, 100-1100, 100-1200, or 100-1300 amino acids.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11-76. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1). In some embodiments, the encoded SARS-CoV-2 spike protein is an immunogen (or
Attorney Docket No.62801.28WO01 an immunogenic fragment and/or immunogenic variant thereof). In some embodiments, the nucleic acid molecule is RNA or DNA. In some embodiments, the RNA is messenger ribonucleic acid (mRNA). In some embodiments, the nucleic acid molecule comprises at least one modified nucleotide. In some embodiments, the nucleic acid molecule comprises N1-methyl-pseudouridine, cytosine, adenine, and guanine. In some embodiments, the nucleic acid molecule comprises a heterologous 5’- untranslated region (UTR), 3’-UTR, or both a 5’-UTR and 3’-UTR. In some embodiments, the nucleic acid molecule comprises a poly(A) sequence. In some embodiments, the nucleic acid molecule comprises a 5’cap structure. In some embodiments, the nucleotide sequence of the nucleic acid molecule is codon optimized. In some embodiments, the nucleic acid molecule further encodes a heterologous polypeptide or protein. In some embodiments, the nucleic acid molecule is recombinant. In some embodiments, the nucleic acid molecule is isolated. In some embodiments, the nucleic acid molecule encodes a signal peptide. In some embodiments, the nucleic acid molecule encodes a homologous or heterologous signal peptide. In some embodiments, the nucleic acid molecule does not encode a signal peptide. In one aspect, provided herein are vectors comprising a nucleic acid molecule described herein. In some embodiments, the vector is a non-viral vector (e.g., a plasmid) or a viral vector. In one aspect, provided herein are conjugates comprising a nucleic acid molecule described herein operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a heterologous polypeptide or protein). In one aspect, provided herein are fusion proteins comprising the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) encoded by a nucleic acid molecule described herein operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous protein. In some embodiments, the heterologous protein is a trimerization domain. In some embodiments, the trimerization domain is operably connected to the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) via a peptide linker. In one aspect, provided herein are compositions comprising at least one nucleic acid molecule described herein, a vector described herein, a conjugate described herein, or a fusion protein described herein. In some embodiments, the composition is monovalent or polyvalent. In some embodiments, the composition is polyvalent. In some embodiments, the composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the nucleic acid molecule(s) are comprised in one or more vectors. In some embodiments, the nucleic acid molecule(s) or the one or more vectors are formulated in one or more carrier. In some embodiments, the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. In some embodiments, the LNP has a mean particle size of between 80 nm and 160 nm. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or a vaccine composition. In some embodiments, the composition comprises an adjuvant. In one aspect, provided herein are SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-naturally occurring glycosylation motifs each comprising a non-naturally occurring glycosylation site, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT sequence, wherein X is any amino acid except for proline. In some embodiments, the N-linked glycosylation motif is an NXS sequence, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the glycosylation motif is an O-linked glycosylation motif. In some embodiments, the O-linked glycosylation motif is a serine or threonine. In some embodiments, the glycosylation site is an O-linked glycosylation site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-linked glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N- glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at
Attorney Docket No.62801.28WO01 position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N- linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N- linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g.,
Attorney Docket No.62801.28WO01 the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N- glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an
Attorney Docket No.62801.28WO01 N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 428, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth
Attorney Docket No.62801.28WO01 in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative
Attorney Docket No.62801.28WO01 to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N- linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid
Attorney Docket No.62801.28WO01 position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N- glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N- glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth
Attorney Docket No.62801.28WO01 in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine or a serine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine or a serine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine or a serine at position 520 and an N- glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-
Attorney Docket No.62801.28WO01 linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a naturally occurring glycosylation site) at one or more of amino acid positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) relative the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) )) in a prefusion state relative the reference sequence (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) comprises an inactive furin cleavage site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 986 and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a
Attorney Docket No.62801.28WO01 threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at
Attorney Docket No.62801.28WO01 position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N- linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising
Attorney Docket No.62801.28WO01 an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388,
Attorney Docket No.62801.28WO01 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N- linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino
Attorney Docket No.62801.28WO01 acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N- linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N- linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine
Attorney Docket No.62801.28WO01 at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising
Attorney Docket No.62801.28WO01 an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478
Attorney Docket No.62801.28WO01 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set
Attorney Docket No.62801.28WO01 forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising
Attorney Docket No.62801.28WO01 an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an
Attorney Docket No.62801.28WO01 N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g.,
Attorney Docket No.62801.28WO01 the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at
Attorney Docket No.62801.28WO01 position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at
Attorney Docket No.62801.28WO01 position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises from about 10-15, 10- 20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10- 600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1190, 10-1300, 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1100, 100-1190, or 100-1300
Attorney Docket No.62801.28WO01 amino acids. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11- 76]. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1).
Attorney Docket No.62801.28WO01 In some embodiments, the SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and/or immunogenic variant thereof). In some embodiments, the SARS-CoV-2 spike protein is recombinant. In some embodiments, the SARS-CoV-2 spike protein is isolated. In some embodiments, the SARS-CoV-2 spike protein comprises a signal peptide. In some embodiments, the SARS-CoV-2 spike protein comprises a homologous or heterologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein does not comprise a signal peptide. In one aspect, provided herein are conjugates comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a heterologous polypeptide or protein). In one aspect, provided herein are fusion proteins comprising a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous protein. In some embodiments, the heterologous protein is a trimerization domain. In some embodiments, the trimerization domain is operably connected to the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) via a peptide linker. In one aspect, provided herein are compositions comprising at least one SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, a conjugate described herein, or a fusion protein described herein. In some embodiments, the composition is monovalent or polyvalent. In some embodiments, the composition is polyvalent. In some embodiments, the composition comprises a plurality SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. In some embodiments, the composition comprises a plurality of SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. In some embodiments, the SARS-CoV-2 spike protein(s) (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) are formulated in one or more carrier. In some embodiments, the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. In some embodiments, the LNP has a mean particle size of between 80 nm and 160 nm. In some embodiments, the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or a vaccine composition. In some embodiments, the composition further comprises an adjuvant. In one aspect, provided herein are nucleic acid molecules encoding a SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein. In one aspect, provided herein are SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) encoded by a nucleic acid molecule described herein. In one aspect, provided herein are carriers comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein. In some embodiments, the carrier is monovalent or polyvalent. In some embodiments, the carrier is polyvalent. In some embodiments, the composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein
Attorney Docket No.62801.28WO01 immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. In some embodiments, the LNP has a mean particle size of between 80 nm and 160 nm. In one aspect, provided herein are vaccine compositions comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, or a pharmaceutical composition described herein. In some embodiments, the vaccine composition is a prime vaccine composition. In some embodiments, the vaccine composition is a boost vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition and a boost vaccine composition. In some embodiments, the vaccine composition can be utilized as a prime vaccine composition and/or a booster vaccine composition in a homologous or heterologous prime boost vaccine regimen. In some embodiments, the vaccine composition is monovalent or polyvalent. In some embodiments, the vaccine composition is polyvalent. In some embodiments, the vaccine composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g.,
Attorney Docket No.62801.28WO01 SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the vaccine composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS- CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the vaccine composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the vaccine composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the vaccine composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein. In some embodiments, the vaccine composition further comprises an adjuvant. In one aspect, provided herein are vaccine compositions comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) that comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1, formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region.
Attorney Docket No.62801.28WO01 In one aspect, provided herein are pharmaceutical compositions comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, or a vaccine composition described herein, and a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is monovalent or polyvalent. In some embodiments, the pharmaceutical composition is polyvalent. In some embodiments, the pharmaceutical composition comprises a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the pharmaceutical composition comprises a plurality of nucleic acid molecules described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. In some embodiments, the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS- Co described herein V-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. In some embodiments, the pharmaceutical composition comprises a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein. In some embodiments, the pharmaceutical composition further comprises an adjuvant.
Attorney Docket No.62801.28WO01 In one aspect, provided herein are pharmaceutical compositions comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV- 2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) that comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1, formulated in a lipid nanoparticle, the pharmaceutical composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region; and a pharmaceutically acceptable excipient. In one aspect, provided herein are host cells comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein. In one aspect, provided herein are kits comprising a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein. In some embodiments, the kit comprises instructions for use of the nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition. In one aspect, provided herein are methods of delivering a nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, host cell, carrier, composition, vaccine composition, or pharmaceutical composition to a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or
Attorney Docket No.62801.28WO01 immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby deliver the nucleic acid molecule, the vector, the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the host cell, the carrier, the composition, the vaccine composition, or the pharmaceutical composition to the subject. In one aspect, provided herein are methods of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response in the subject. In one aspect, provided herein are methods of inducing or enhancing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject. In one aspect, provided herein are methods of preventing or decreasing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical
Attorney Docket No.62801.28WO01 composition described herein, to thereby prevent or decrease an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject. In one aspect, provided herein are methods of inducing or enhancing an immune response against a plurality of strains of SARS-CoV-2 virus in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS- CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby induce or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject. In one aspect, provided herein are methods of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby prevent, ameliorate, or treat the SARS-CoV-2 infection in the subject. In one aspect, provided herein are methods of preventing, ameliorating, or treating an infection by a plurality of SARS-CoV-2 strains in a subject in need thereof, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby prevent, ameliorate, or treat an infection by a plurality of SARS-CoV-2 strains in the subject. In one aspect, provided herein are methods of vaccinating a subject against SARS- CoV-2, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described
Attorney Docket No.62801.28WO01 herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby vaccinate the subject against SARS-CoV-2. In one aspect, provided herein are methods of vaccinating a subject against SARS- CoV-2, the method comprising administering to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (or a conjugate or fusion protein thereof), (b) a vector comprising the mRNA molecule, (c) a carrier comprising the mRNA molecule or the vector, (d) a vaccine composition comprising the mRNA molecule, the vector, or the carrier, or (e) a pharmaceutical composition comprising the mRNA molecule, the vector, the carrier, or the vaccine composition, to thereby vaccinate the subject against SARS-CoV-2, to thereby vaccinate the subject against SARS-CoV-2. In one aspect, provided herein are methods of vaccinating a subject against SARS- CoV-2, the method comprising administering to the subject a vaccine composition described herein or a pharmaceutical composition described herein, to thereby vaccinate the subject against SARS-CoV-2. In one aspect, provided herein are methods of vaccinating a subject against a plurality of strains of SARS-CoV-2, the method comprising administering to the subject a nucleic acid molecule described herein, a SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) described herein, a vector described herein, a conjugate described herein, a fusion protein described herein, a host cell described herein, a carrier described herein, a composition described herein, a vaccine composition described herein, or a pharmaceutical composition described herein, to thereby vaccinate the subject against a plurality of strains of SARS-CoV-2. For the sake of clarity, it should be understood that the following embodiments relate to any of the above forementioned methods. In some embodiments, the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered to the subject at least
Attorney Docket No.62801.28WO01 twice. In some embodiments, the subject is a human. In some embodiments, the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a prime and/or a boost in a homologous or heterologous prime- boost regimen. In some embodiments, the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen. 4. BRIEF DESCRIPTION OF THE FIGURES FIGS. 1A-1C depicts a sequence alignment of the amino acid sequence of a reference SARS-CoV-2 spike protein Wuhan-Hu-1 Spike protein sequence (SEQ ID NO: 1) and a variant thereof (SEQ ID NO: 9). 5. DETAILED DESCRIPTION SARS-CoV-2 continues to evolve into new variants comprising a variety of amino acid variations, e.g., substitutions, deletions, and insertions. Many of the variations are found in the RBD of the spike protein, which is vital for SARS-CoV-2 entry into the cell. As most of the SARS- CoV-2 vaccines and current antibody therapies target the RBD of the spike protein, this creates the potential for the evolution of SARS-CoV-2 variants that evade vaccine-induced immunity, infection-induced immunity, and/or current antibody therapies. The inventors have identified novel engineered glycosylation sites within the SARS-CoV-2 spike protein (e.g., in the RBD) that shield select epitopes from immune recognition, while leaving target epitopes (e.g., evolutionary conserved epitopes) exposed. Accordingly, novel SARS-CoV-2 spike proteins (e.g., SARS-CoV- 2 spike protein immunogens (and immunogenic fragments and/or immunogenic variants thereof)) (or nucleic acid molecules, e.g., mRNAs, encoding such SARS-CoV-2 spike proteins) comprising at least one such non-naturally occurring glycosylation site are good candidates for variant-proof or variant-resistant pan-SARS-CoV-2 vaccines. As such, the current disclosure provides, inter alia, novel immunogens for use in pharmaceutical compositions and vaccines to induce a desired
Attorney Docket No.62801.28WO01 immune response against one or more (e.g., multiple) variants of SARS-CoV-2. TABLE OF CONTENTS 5.1 Definitions 5.2 Glycoengineered SARS-CoV-2 Spike Proteins (e.g., Immunogens) & Fragments Thereof (e.g., RBDs) 5.2.1 Engineered Glycosylation Sites 5.2.2 Engineered Glycosylation Motifs 5.2.3 Exemplary Sets of Engineered Glycosylation Sites & Motifs 5.2.4 Exemplary Glycoengineered SARS-CoV-2 Spike Proteins (e.g., Immunogens) & Fragments Thereof 5.2.5 Structure of SARS-CoV-2 Spike Proteins (e.g., Immunogens) 5.2.6 Exemplary Additional Features of SARS-CoV-2 Spike Proteins (e.g., Immunogens) 5.2.6.1 Hyperglycosylation 5.2.6.2 Stabilization 5.6.3.2 Multimerization (e.g., Trimerization) Domains 5.2.6.4 Variations from Different Strains of SARS-CoV-2 5.2.6.5 Conserved Structural Elements 5.3 Nucleic Acid Molecules 5.3.1 DNA Nucleic Acids 5.3.2 RNA Nucleic Acids 5.4 Fusions & Conjugates 5.5 Compositions (e.g., Vaccine Compositions) 5.5.1 Peptide and Protein-Based Vaccines 5.5.2 Nucleic Acid-Based Vaccines 5.6 Vectors 5.7 Carriers 5.7.1 Lipid Based Carriers/Lipid Nanoformulations 5.7.1.1 Cationic Lipids (Positively Charged) and Ionizable Lipids 5.7.1.2 Non-Cationic Lipids (e.g., Phospholipids) 5.7.1.3 Structural Lipids
Attorney Docket No.62801.28WO01 5.7.1.4 Polymers and Polyethylene Glycol (PEG) - Lipids 5.7.1.5 Percentages of Lipid Nanoformulation Components 5.8 Pharmaceutical Compositions 5.9 Adjuvants 5.10 Methods of Use 5.10.1 Methods of Delivery 5.10.2 Manufacture of a Medicament 5.10.3 Methods of Inducing or Enhancing an Immune Response 5.10.4 Methods of Preventing or Decreasing an Immune Response 5.10.5 Methods of Preventing, Ameliorating, and/or Treating Infection 5.10.6 Methods of Vaccination against SARS-CoV-2 5.10.6.1 Methods of Vaccinating a Subject against SARS-CoV-2 5.10.6.2 Methods of Vaccinating a Subject Utilizing a SARS-CoV-2 mRNA Vaccine 5.11 Kits 5.1 Definitions The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. For example, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of” and “consisting essentially of” are also provided.
Attorney Docket No.62801.28WO01 The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The terms “about” or “comprising essentially of” refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. When particular values or compositions are provided in the application and claims, unless otherwise stated, the meaning of “about” or “comprising essentially of” should be assumed to be within an acceptable error range for that particular value or composition. Where proteins and/or polypeptides are described herein, it is understood that polynucleotides (e.g., RNA (e.g., mRNA) or DNA nucleic acid molecules) encoding the protein or polypeptide are also provided herein. Where proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are described herein, it is understood that isolated forms of the proteins, polypeptides, nucleic acid molecules, vectors, carriers, etc. are also provided herein. Where proteins, polypeptides, nucleic acid molecules, etc. are described herein, it is understood that recombinant forms of the proteins, polypeptides, nucleic acid molecules, etc. are also provided herein. Where polypeptides or sets of polypeptides are described herein, it is understood that proteins comprising the polypeptides or sets of polypeptides folded into their three-dimensional structure (i.e., tertiary or quaternary structure) are also provided herein and vice versa. As used herein, the term “adjuvant” refers to a substance that causes stimulation of the immune system of a subject when administered to the subject.
Attorney Docket No.62801.28WO01 As used herein, the term “administering” refers to the physical introduction of an agent, e.g., a vaccine or therapeutic agent (or a precursor of the therapeutic agent that is metabolized or altered (e.g., translation of a nucleic acid molecule) within the body of the subject to produce the therapeutic agent in vivo) to a subject, using any of the various methods and delivery systems known to those skilled in the art. Administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. The term administering includes self- administration. As used herein, the term “agent” is used generically to describe any macro or micro molecule. Exemplary moieties include, but are not limited to polypeptides, proteins, peptides, nucleic acid molecules (e.g., DNA, RNA), small molecules, carbohydrates, lipids, synthetic polymers (e.g., polymers of PEG). As used herein, the term “derived from,” with reference to a polynucleotide refers to a polynucleotide that has at least 70% sequence identity to a reference polynucleotide (e.g., a naturally occurring polynucleotide) or a fragment thereof. The term “derived from,” with reference to a polypeptide or protein refers to a polypeptide or protein that comprises an amino acid sequence that has at least 70% sequence identity to the amino acid sequence of a reference polypeptide or protein (e.g., a naturally occurring polypeptide or protein). The term “derived from” as used herein does not denote any specific process or method for obtaining the polynucleotide, polypeptide, or protein. For example, the polynucleotide, polypeptide, or protein can be recombinant produced or chemically synthesized. As used herein, the term “disease” refers to any abnormal condition that impairs physiological function. The term is used broadly to encompass any disorder, illness, abnormality, pathology, sickness, condition, or syndrome in which physiological function is impaired, irrespective of the nature of the etiology. The term disease includes infection (e.g., a viral (e.g., a SARS-Cov-2 infection), bacterial, fungal, protozoal infection). The terms “DNA” and “polydeoxyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple deoxyribonucleotides that are polymerized via phosphodiester bonds. Deoxyribonucleotides are nucleotides in which the sugar is deoxyribose. As used herein, the term “full-length” with reference to a SARS-CoV-2 spike protein refers to a SARS-CoV-2 spike protein, wherein the amino acid sequence of the SARS-CoV-2 spike protein comprises substantially the same number of amino acids as a reference SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein. As used herein, the term “fuse” and grammatical equivalents thereof refer to the operable connection of at least a first polypeptide or protein to a second polypeptide or protein, wherein the first and second polypeptides or proteins are not naturally found operably connected together. For example, the first and second polypeptides or proteins are derived from different proteins. The term fuse encompasses both a direct connection of the at least two polypeptides or proteins through a peptide bond, and the indirect connection through a linker (e.g., a peptide linker). As used herein, the term “glycosylation site” refers to the amino acid residue of a protein or peptide which is the attachment point for a glycan. For example, in N-linked glycosylation, the glycosylation site will be the asparagine residue to which the glycan is attached. For further example, in O-linked glycosylation, the glycosylation site will be the serine or threonine residue to which the glycan is attached. The term “non-naturally occurring glycosylation site” within a protein or peptide refers to a glycosylation site that is not found in a specific protein or peptide in nature. The non-naturally occurring glycosylation site can be generated by creating a non-naturally occurring glycosylation motif, which does not necessarily require varying the amino acid of the glycosylation site. For example, a non-naturally occurring glycosylation site would include a glycosylation site generated from the variation of a N-A-P amino acid sequence of a protein to a N-A-T amino acid sequence (creating an N-linked glycosylation motif) even though the amino acid that mediates attachment of the glycan (N) was not varied. As used herein, the term “glycosylation motif” refers to the amino acid or sequence of amino acids that comprises the glycosylation site and facilities glycosylation. For example, in N- linked glycosylation, the canonical glycosylation motifs are NXT and NXS, wherein X is any amino acid except for proline and the asparagine (N) residue is the glycosylation site. For further example, in O-linked glycosylation, the glycosylation motif is a S or T residue that also serves as the glycosylation site. As used herein, the term “fusion protein” and grammatical equivalents thereof refers to a protein that comprises at least one polypeptide operably connected to another polypeptide, wherein the first and second polypeptides are not naturally found operably connected together. For example, the first and second polypeptides of the fusion protein are each derived from different proteins. The at least two polypeptides of the fusion protein can be directly operably connected
Attorney Docket No.62801.28WO01 through a peptide bond; or can be indirectly operably connected through a linker (e.g., a peptide linker). Therefore, for example, the term fusion polypeptide encompasses embodiments, wherein Polypeptide A is directly operably connected to Polypeptide B through a peptide bond (Polypeptide A – Polypeptide B), and embodiments, wherein Polypeptide A is operably connected to Polypeptide B through a peptide linker (Polypeptide A – peptide linker – Polypeptide B). As used herein, the term “half-life extension protein or “half-life extension polypeptide” refers to a polypeptide or protein that when operably connected to another moiety (e.g., a subject moiety) (e.g., a protein), increases the half-life of the subject moiety (e.g., the subject protein) in vivo when administered to a subject (e.g., a human subject). The pharmacokinetic properties of the protein can be evaluated utilizing in vivo models known in the art. As used herein, the term “heterologous”, when used to describe a first element in reference to a second element means that the first element and second element do not exist in nature disposed as described. For example, a polypeptide comprising a “heterologous moiety” means a polypeptide that is joined to a moiety (e.g., small molecule, polypeptide, polynucleotide, carbohydrate, lipid, synthetic polymer (e.g., polymers of PEG), etc.) that is not joined to the polypeptide in nature. As used, herein the term “heterologous signal peptide” refers to a signal peptide that is not operably connected to a subject polypeptide or protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL-12, the human IL-2 signal peptide would constitute a heterologous signal peptide. As used herein, the term “heterologous prime boost” refers to a prime boost vaccine regimen wherein the prime vaccine composition and the boost (or booster) vaccine composition are different (e.g., the immunogen is different, the form of the immunogen is different (e.g., a nucleic acid (e.g., mRNA) molecule-based vaccine versus a protein-based vaccine), the immunogen is expressed from a different vector (e.g., plasmid, viral vector), the method of delivering the immunogen to the subject is different, etc.). As used herein, the term “homologous signal peptide” refers to a signal peptide that is operably connected to a subject polypeptide or protein in nature. For example, in reference to a polypeptide comprising a signal peptide from human IL-2 operably connected to human IL-2, the human IL-2 signal peptide would constitute a homologous signal peptide.
Attorney Docket No.62801.28WO01 As used herein, the term “homologous prime boost” refers to a prime boost vaccine regimen wherein the prime vaccine composition and the boost (or booster) vaccine composition are the same. As used herein, the term “immunogen” refers to a substance that is capable of inducing an immune response (e.g., an adaptive immune response) in a subject (e.g., a human subject). As used herein, the term “immunogenic fragment” refers to a fragment of a reference substance (e.g., a reference polypeptide or protein) that retains an immunogen. As used herein, the term “immunogenic variant” refers to a variant of a reference substance (e.g., a reference polypeptide or protein) that retains an immunogen. In some embodiments, the immunogenic variant is a polypeptide or protein that comprises at least one but no more than 25%, (e.g., no more than 20%, no more than 15%, no more than 12%, no more than 10%, no more than 8%) amino acid variation (e.g., substitutions, deletions, additions) compared to the amino acid sequence of a reference polypeptide or protein. As used herein, the term “in combination with” means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease. The treatment regimen defines the doses and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of the two or more agents is simultaneous or concurrent and the agents may be co- formulated. In other embodiments, the two or more agents are not co-formulated and are administered in a sequential manner as part of a prescribed regimen (e.g., a prime-boost vaccine regimen). In some embodiments, administration of two or more agents or treatments in combination is such that the reduction in a symptom, or other parameter related to the disease is greater than what would be observed with one agent or treatment delivered alone or in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each agent can be effected by any appropriate route including, but not limited to, oral routes, intravenous routes, intramuscular routes, and direct absorption through mucous membrane tissues. The agents can be administered by the same route or by different routes. As used herein, the term “isolated” with reference to an agent (e.g., a polypeptide, protein, or nucleic acid molecule) refers to the agent (e.g., the polypeptide, protein, or nucleic acid molecule) that is substantially free of other cellular components with which it is associated in the
Attorney Docket No.62801.28WO01 natural state. As used herein, the term “modification” in reference to a nucleic acid sequence refers to a nucleic acid molecule that comprises at least one nucleotide comprising a chemical modification, e.g., a modified sugar moiety, a modified nucleobase, and/or a modified internucleotide linkage, or any combination thereof. Exemplary nucleotide modifications are provided herein, see, e.g., § 5.3 (e.g., § 5.3.2). In certain embodiments of the instant disclosure, inclusion of a deoxynucleotide - which is acknowledged as a naturally occurring form of nucleotide - if present within an RNA molecule (e.g., an mRNA molecule) is considered to constitute a modified nucleotide. The terms “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymer of DNA or RNA. The nucleic acid molecule can be single-stranded or double-stranded; contain natural, non-natural, or altered nucleotides; and contain a natural, non- natural, or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. Nucleic acid molecules include, but are not limited to, all nucleic acid molecules which are obtained by any means available in the art, including, without limitation, recombinant means, e.g., the cloning of nucleic acid molecules from a recombinant library or a cell genome, using ordinary cloning technology and polymerase chain reaction, and the like, and by synthetic means. The skilled artisan will appreciate that, except where otherwise noted, nucleic acid sequences set forth in the instant application will recite thymidine (T) in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the thymidines (Ts) would be substituted for uracils (Us). Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each thymidine (T) of the DNA sequence is substituted with uracil (U). As used herein, the term “operably connected” refers to the linkage of two moieties in a functional relationship. For example, a polypeptide is operably connected to another polypeptide when they are linked (either directly or indirectly via a peptide linker) in frame such that both polypeptides are functional (e.g., a fusion protein described herein). Or for example, a transcription regulatory polynucleotide e.g., a promoter, enhancer, or other expression control element is operably connected to a polynucleotide that encodes a protein if it affects the transcription of the
Attorney Docket No.62801.28WO01 polynucleotide that encodes the protein. The term “operably connected” can also refer to the conjugation of a moiety to e.g., a polynucleotide or polypeptide (e.g., the conjugation of a PEG polymer to a protein). As used herein, the term “peptide” refers to a polymer of at least two amino acids linked by peptide bonds. The term “peptide” does not limit the length of the polymer chain of amino acids. It is common in the art to refer to shorter polymers of amino acids (e.g., approximately 2- 50 amino acids) as peptides; and to refer to longer polymers of amino acids (e.g., approximately over 50 amino acids) as polypeptides. However, the terms “peptide” and “polypeptide” are used interchangeably herein. The determination of “percent identity” between two sequences (e.g., peptide or protein (amino acid sequences) or nucleic acid sequences)) can be accomplished using a mathematical algorithm. A specific, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin S & Altschul SF (1990) PNAS 87: 2264- 2268, modified as in Karlin S & Altschul SF (1993) PNAS 90: 5873-5877, each of which is herein incorporated by reference in its entirety. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul SF et al., (1990) J Mol Biol 215: 403, which is herein incorporated by reference in its entirety. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecule described herein. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul SF et al., (1997) Nuc Acids Res 25: 3389-3402, which is herein incorporated by reference in its entirety. Alternatively, PSI BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., National Center for Biotechnology Information (NCBI) on the worldwide web, ncbi.nlm.nih.gov). Another specific, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17, which is herein incorporated by reference in its entirety. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package.
Attorney Docket No.62801.28WO01 When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted. As used herein the term “pharmaceutical composition” means a composition that is suitable for administration to an animal, e.g., a human subject, and comprises a therapeutic agent and a pharmaceutically acceptable carrier or diluent. A “pharmaceutically acceptable carrier or diluent” means a substance for use in contact with the tissues of human beings and/or non-human animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable therapeutic benefit/risk ratio. As used herein, the term “plurality” means 2 or more (e.g., 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 9 or more, or 10 or more). As used herein, the term “poly(A) sequence,” refers to a sequence of adenosine nucleotides, typically located at the 3’-end of a linear RNA (or in a circular RNA), of up to about 1000 adenosine nucleotides. In some embodiments, the poly(A) sequence is essentially homopolymeric, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides has essentially the length of 100 nucleotides. In other embodiments, the poly(A) sequence may be interrupted by at least one nucleotide different from an adenosine nucleotide, e.g., a poly(A) sequence of e.g., 100 adenosine nucleotides may have a length of more than 100 nucleotides (comprising 100 adenosine nucleotides and in addition said at least one nucleotide - or a stretch of nucleotides - different from an adenosine nucleotide). It has to be understood that “poly(A) sequence” as defined herein typically relates to RNA - however in the context of the invention, the term likewise relates to corresponding sequences in a DNA molecule (e.g. a “poly(T) sequence”). As used herein, the term “polyvalent vaccine” refers to a vaccine that contains a plurality of different immunogens. In some embodiments, the polyvalent vaccine comprises a plurality of immunogens from more than one strain of a virus, different immunogens from the same strain of a virus, or any combination thereof. In some embodiments, the polyvalent vaccine comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more different
Attorney Docket No.62801.28WO01 immunogens or nucleic acid molecules encoding the immunogens. As used herein, the term “prime boost” refers to a vaccine regimen comprising at least an initial vaccine dose and one or more subsequent vaccine doses. The initial vaccine dose comprises the prime vaccine composition and the one or more subsequent vaccine doses are referred to as boost (or booster) vaccine compositions. Prime boost vaccine regimens can comprise more than one booster (e.g., 2, 3, 4, 5, 6, or more, etc.). A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology. As used herein, the term “protein” refers to a one or more peptides folded into its three- dimensional structure. As used herein, the term “receptor binding domain” or “RBD” in reference to the SARS-CoV-2 spike protein refers to a functional fragment of a SARS-CoV-2 spike protein that is capable of binding to ACE2. The amino acid sequence of an exemplary reference SARS-CoV-2 spike protein RBD is set forth in SEQ ID NO: 3. The terms “RNA” and “polyribonucleotide” are used interchangeably herein and refer to macromolecules that include multiple ribonucleotides that are polymerized via phosphodiester bonds. Ribonucleotides are nucleotides in which the sugar is ribose. RNA may contain modified nucleotides; and contain natural, non-natural, or altered internucleotide linkages, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified nucleic acid molecule. As used herein, the term “SARS-CoV-2 spike protein” refers to the SARS-CoV-2 protein that mediates binding to the host cell receptor angiotensin-converting enzyme 2 (ACE2). An exemplary amino acid sequence of a reference full-length SARS-CoV-2 spike protein is set forth in SEQ ID NO: 1. The term SARS-CoV-2 spike protein includes naturally occurring and engineered variants. As used herein, the term “set” with reference to glycosylation sites (e.g., non-naturally occurring glycosylation sites) does not require more than glycosylation site (e.g., non-naturally occurring glycosylation site). For example, Table 2 herein describe “sets” glycosylation sites (e.g., non-naturally occurring glycosylation sites), some sets have only one glycosylation site (e.g., non- naturally occurring glycosylation site) and some sets have more than one glycosylation site (e.g.,
Attorney Docket No.62801.28WO01 non-naturally occurring glycosylation site). As used herein, the term “signal peptide” or “signal sequence” refers to a sequence (e.g., an amino acid sequence) that can direct the transport or localization of a protein to a certain organelle, cell compartment, or extracellular export. The term encompasses both the signal peptide (the amino acid sequence of the signal peptide) and the nucleic acid sequence encoding the signal peptide. Thus, references to a signal peptide in the context of a nucleic acid molecule refers to the nucleic acid sequence encoding the signal peptide. As used herein, the term “subject” includes any animal, such as a human or other animal. In embodiments, the subject is a vertebrate animal (e.g., mammal, bird, fish, reptile, or amphibian). In embodiments, the subject is a human. In embodiments, the method subject is a non- human mammal. In embodiments, the subject is a non-human mammal is such as a non-human primate (e.g., monkeys, apes), ungulate (e.g., cattle, buffalo, sheep, goat, pig, camel, llama, alpaca, deer, horses, donkeys), carnivore (e.g., dog, cat), rodent (e.g., rat, mouse), or lagomorph (e.g., rabbit). In embodiments, the subject is a bird, such as a member of the avian taxa Galliformes (e.g., chickens, turkeys, pheasants, quail), Anseriformes (e.g., ducks, geese), Paleaognathae (e.g., ostriches, emus), Columbiformes (e.g., pigeons, doves), or Psittaciformes (e.g., parrots). A “therapeutically effective amount” of an agent (e.g., a therapeutic agent, a vaccine) refers to any amount of the agent (e.g., the therapeutic agent, the vaccine) that, when used alone or in combination with another agent (e.g., a therapeutic agent, a vaccine), protects a subject against the onset of a disease (e.g., an infection), ameliorates the severity of a disease (e.g., an infection), and/or promotes disease (e.g., infection) regression evidenced by a decrease in severity of disease (e.g., infection) symptoms, an increase in frequency and duration of disease (e.g., infection) symptom-free periods, or a prevention of impairment or disability due to the disease (e.g., infection) affliction. The ability of an agent (e.g., a therapeutic agent, a vaccine) to do any of the foregoing (or any combination thereof) can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays. As used herein, the term “translatable RNA” refers to any RNA that encodes at least one polypeptide or protein and can be translated to produce the encoded polypeptide or protein in vitro, in vivo, in situ or ex vivo. A translatable RNA may be an mRNA or a circular RNA encoding a polypeptide or protein.
Attorney Docket No.62801.28WO01 As used herein, the terms “treat,” treating,” “treatment,” and the like refer to reducing or ameliorating a disease or infection and/or symptom(s) associated therewith or obtaining a desired pharmacologic and/or physiologic effect. It will be appreciated that, although not precluded, treating a disease or infection does not require that the disease or infection, or symptom(s) associated therewith be completely eliminated. In some embodiments, the effect is therapeutic, i.e., without limitation, the effect partially or completely reduces, diminishes, abrogates, abates, alleviates, decreases the intensity of, or cures a disease and/or adverse symptom attributable to the disease or infection. In some embodiments, the effect is preventative, i.e., the effect protects or prevents an occurrence or reoccurrence of a disease or infection. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a compositions as described herein. As used herein, the term “variant” or “variation” with reference to a nucleic acid molecule, refers to a nucleic acid molecule that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid molecule. As used herein, the term “variant” or “variation” with reference to a peptide or protein refers to a peptide or protein that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference peptide or protein. As used herein, the term “5’-untranslated region” or “5’-UTR” refers to a part of a nucleic acid molecule located 5’ (i.e., “upstream”) of a coding sequence and which is not translated into protein. Typically, a 5’-UTR starts with the transcriptional start site and ends before the start codon of the coding sequence. A 5’-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. The 5’-UTR may be post-transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5’-cap structure. As used herein the term “3’-untranslated region” or “3’-UTR” refers to a part of a nucleic acid molecule located 3’ (i.e., downstream) of a coding sequence and which is not translated into protein. A 3’-UTR may located between a coding sequence and an (optional) terminal poly(A) sequence of a nucleic acid sequence. A 3'-UTR may comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements may be, e.g., ribosomal binding sites, miRNA binding sites etc. 5.2 Glycoengineered SARS-CoV-2 Spike Proteins (e.g., Immunogens) & Fragments
Attorney Docket No.62801.28WO01 Thereof (e.g., RBDs) Provided herein are, inter alia, SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof (e.g., receptor binding domains thereof))) and nucleic acid molecules encoding the same, that comprise one or more non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site (e.g., a non-naturally occurring glycosylation site at one or more of positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1). The SARS-CoV-2 spike glycoprotein mediates viral entry into host cells. The spike protein comprises two functional subunits responsible for binding to the host cell receptor (S1 subunit) and fusion of the viral and cellular membranes (S2 subunit). The SARS-CoV-2 spike protein is cleaved at the boundary between the S1 and S2 subunits, which remain non-covalently associated in the prefusion conformation. The distal S1 subunit comprises the RBD and contributes to stabilization of the prefusion state of the membrane anchored S2 subunit that contains the fusion machinery. The RBD mediates binding to the host cell receptor ACE2. The spike protein is cleaved by host proteases at the so-called S2' site located immediately upstream of the fusion peptide. This cleavage has been proposed to activate the protein for membrane fusion via extensive irreversible conformational changes. (See, e.g., Walls, Alexandra C et al. “Structure, Function, and Antigenicity of the SARS-CoV-2 Spike Glycoprotein.” Cell vol. 181,2 (2020): 281-292.e6. doi:10.1016/j.cell.2020.02.058, the entire contents of which is incorporated herein by reference for all purposes). The amino acid sequence of a first reference immature SARS-CoV-2 spike protein (Wuhan-Hu-1) is provided in SEQ ID NO: 1. The signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 1); the amino acid sequence of the corresponding mature first reference SARS- CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 2. The amino acid sequence of a RBD of the first reference SARS-CoV-2 spike protein is set forth in SEQ ID NO: 3 and SEQ ID NO: 4. The amino acid sequence of a second reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 5. The signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 5); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 6. The amino acid sequence
Attorney Docket No.62801.28WO01 of the second reference SARS-CoV-2 spike protein comprises a glycine at position 614 and a proline at each of positions 817, 892, 899, 942, 986, and 987, amino acid numbering relative to SEQ ID NO: 1 (the Wuhan-Hu-1 Spike Numbering System described herein). The amino acid sequence of a second reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 7. The signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 7); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 8. The amino acid sequence of the second reference SARS-CoV-2 spike protein comprises a proline at each of positions 817, 892, 899, 942, 986, and 987, amino acid numbering relative to SEQ ID NO: 1. The amino acid sequence of a fourth reference immature SARS-CoV-2 spike protein is provided in SEQ ID NO: 9. The signal peptide is underlined (amino acids 1-13 of SEQ ID NO: 9); the amino acid sequence of the corresponding mature second reference SARS-CoV-2 spike protein lacking the native signal peptide is provided in SEQ ID NO: 10. Table 1. Reference SARS-CoV-2 Spike Proteins and RBDs Thereof. Description Amino Acid Sequence SEQ ID NO
Attorney Docket No.62801.28WO01 ATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVT YVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQR NFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSF
Attorney Docket No.62801.28WO01 MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDK VFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNP
Attorney Docket No.62801.28WO01 PIKDFGGFNFSQILPDPSKPSKRSPIEDLLFNKVTLADAGF IKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYT SALLAGTITSGWTFGAGPALQIPFPMQMAYRFNGIGVTQNV
Attorney Docket No.62801.28WO01 peptide GFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWT AGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEV
Attorney Docket No.62801.28WO01 AEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKM SECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPA QEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYE
t s stan ar n t e art to num er t e am no ac pos t ons w t n a g ven - oV- 2 spike protein according to the corresponding amino acid position in the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1). As such, unless otherwise indicated, the designation of amino acid positions in the amino acid sequence of a SARS-Cov-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) herein is relative to the amino acid sequence of SEQ ID NO: 1. A person of ordinary skill in the art can determine the corresponding amino acid for a
Attorney Docket No.62801.28WO01 given amino acid position in any query spike protein by (i) aligning the amino acid sequence of the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) to the amino acid sequence of the query spike protein, (ii) locating the relevant amino acid position in the Wuhan-Hu-1 Spike reference sequence, and (iii) determining the amino acid located at the corresponding amino acid position in the query spike protein. As query spike proteins may have for example, insertions or deletions of amino acids relative to the Wuhan-Hu-1 Spike reference sequence, the amino acid corresponding to a particular position in the Wuhan-Hu-1 Spike reference sequence may not have the same amino acid number if one were to simply count the number of amino acids within the query spike protein. For example, FIGS. 1A-1C shows an alignment of the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) to the amino acid sequence of a query spike protein (SEQ ID NO: 9). The query spike protein comprises, inter alia, a deletion of four amino acids relative to the Wuhan- Hu-1 Spike reference sequence (amino acids L24, P25, P26, and Y125). However, the alignment keeps the corresponding amino acids in the query spike protein (SEQ ID NO: 9) correctly aligned to the Wuhan-Hu-1 Spike reference sequence. For example, if one wanted to locate amino acid position termed 440 in a query spike protein (e.g., SEQ ID NO: 9), one would generate the alignment in FIGS. 1A-1C, locate amino acid position 440 in the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) and subsequently locate the corresponding amino acid in the query spike protein (SEQ ID NO: 9), in this example position 440 (according to Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) numbering) in the query spike protein (SEQ ID NO: 9) is lysine (K). Amino acid residue 440 (according to Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) numbering) in the Wuhan-Hu-1 Spike reference sequence (SEQ ID NO: 1) is asparagine (N). This numbering system is indicated throughout the present disclosure by noting, “amino acid numbering relative to SEQ ID NO: 1” or the like. Although amino acid positions of the SARS-CoV-2 spike protein are typically defined herein relative to the full-length immature SARS-Cov-2 spike protein that contains the native signal sequence; it should be understood that this does not require the SARS-Cov-2 spike proteins (e.g., the SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein to include a native signal sequence. In some embodiments, the SARS-CoV-2 spike protein or polypeptide is an immunogen (a SARS-CoV-2 spike protein or polypeptide immunogen). In some embodiments, the SARS-
Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide comprises an immunogen (a SARS-CoV-2 spike protein or polypeptide immunogen). In some embodiments, the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic fragment of a SARS-CoV-2 spike protein immunogen. In some embodiments, the SARS-CoV-2 spike protein or polypeptide immunogen comprises or consists of an immunogenic variant of a SARS-CoV-2 spike protein immunogen. As defined herein, an immunogen refers to a substance that is capable of inducing an immune response (e.g., an adaptive immune response) in a subject (e.g., a human subject). The immunogenicity of a protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide) (or a nucleic acid molecule encoding the same) can be determined through standard in silico, in vitro, ex vivo, and in vivo assays known in the art. The type of immune response measured can be determined by a person of ordinary skill in the art, e.g., in some embodiments, an antibody based immune response and/or a T cell based immune response is evaluated. For example, a sample (e.g., a blood, cell) can be taken from a subject (e.g., an animal, human) post administration pf the protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide) (or a nucleic acid molecule encoding the same) to be evaluated. The sample can be utilized to evaluate indicators of an immune response. For example, a blood (or serum) sample can be utilized to assess the level of antibodies that bind the antigen. Such binding can be assessed through standard ELISAs. The presence of neutralizing antibodies in the sample can also be assessed using standard assays, including, e.g., plaque reduction neutralization assays and microneutralization assays. The induction of a T-cell based immune response can be measured using standard assays known in the art, including, e.g., immunospot assays to assess cytokine production utilizing peptide stimulated peripheral blood mononuclear cells (PBMCs) from the subject. See, e.g., Corominas J. et al, Safety and immunogenicity of the protein-based PHH-1V compared to BNT162b2 as a heterologous SARS-CoV-2 booster vaccine in adults vaccinated
Attorney Docket No.62801.28WO01 against COVID-19: a multicentre, randomized, double-blind, non-inferiority phase IIb trial, The Lancet Regional Health Europe, Vol. 28 (100613) May 2023 DOI:https://doi.org/10.1016/j.lanepe.2023.100613; Limin D et al, Growth, Antigenicity, and Immunogenicity of SARS-CoV-2 Spike Variants Revealed by a Live rVSV-SARS-CoV-2 Virus, Frontiers in Medicine, Vol 8 (2022) DOI=10.3389/fmed.2021.793437; Hana M El Sahly and others, Humoral Immunogenicity of the mRNA-1273 Vaccine in the Phase 3 Coronavirus Efficacy (COVE) Trial, The Journal of Infectious Diseases, Volume 226, Issue 10, 15 November 2022, Pages 1731–1742, https://doi.org/10.1093/infdis/jiac188; the entire contents of each of which is incorporated herein by reference for all purposes. In some embodiments, the SARS-CoV-2 spike protein or polypeptide is recombinant. In some embodiments, the SARS-CoV-2 spike protein or polypeptide is isolated. 5.2.1 Engineered Glycosylation Sites As described above, provided herein are, inter alia, SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof (e.g., receptor binding domains thereof))) and nucleic acid molecules encoding the same, that comprise one or more non-naturally occurring glycosylation sites (e.g., a non-naturally occurring glycosylation site at one or more of positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1). In some embodiments, the non-naturally occurring glycosylation site is an N-linked glycosylation site (an asparagine (N) residue within a N-linked glycosylation site (e.g., NXT or NXS (wherein X is any amino acid except for proline)). In some embodiments, the non-naturally occurring glycosylation site is an O-linked glycosylation site (e.g., a serine or threonine). In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more non- naturally occurring glycosylation sites. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more non-naturally occurring glycosylation motifs each comprising a non-
Attorney Docket No.62801.28WO01 naturally occurring glycosylation site. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1) (e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites). In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) of amino acid positions 354, 369, 370, 383, 388, 394, 415, 428, 441, 447, 448, 460, 468, 476, 477, 494, 502, 517, 518, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of amino acid positions 354, 369, 370, 388, 394, 415, 428, 447, 448, 460, 468, 476, 494, 502, 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of amino acid positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) further comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 383, 394, 415, 428, 441, 447, 448, 468, 494, 502, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) further
Attorney Docket No.62801.28WO01 comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) further comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) of amino acid positions 369, 447, 388, 428, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and optionally further comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and/or 9) of amino acid positions 354, 370, 388, 394, 448, 468, 476, 494, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 354, 370, 388, 394, 448, 468, 476, 494, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of amino acid positions 354, 370, 388, 394, 415, 428, 460, 468, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 354, 370, 388, 394, 415, 428, 460, 468, 476, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of amino acid positions 354, 370, 388, 394, 415, 428, 448, 460, 468, 476, 494, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 354, 370, 388, 394, 415, 428, 448, 460, 468, 476, 494, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 394, 448, 468, 476, 494, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 394, 448, 468, 476, 494, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, and/or 7) of amino acid positions 394, 415, 428, 460, 468, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 394, 415, 428, 460, 468, 476, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, and/or 9) of amino acid positions 394, 415, 428, 448, 460, 468, 476, 494, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 394, 415, 428, 448, 460, 468, 476, 494, and 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid
Attorney Docket No.62801.28WO01 positions 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more (e.g., 1 and/or 2) of amino acid positions 494 and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation site (e.g., a non-naturally occurring glycosylation site) at amino acid positions 494 and 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation motifs each comprising a naturally occurring glycosylation. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 5.2.2 Engineered Glycosylation Motifs As described above, provided herein are, inter alia, SARS-CoV-2 spike proteins and polypeptides (e.g., SARS-CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof (e.g., receptor binding domains thereof))) and nucleic acid molecules encoding the same, that comprise one or more non-naturally occurring
Attorney Docket No.62801.28WO01 glycosylation motif (e.g., comprising a non-naturally occurring glycosylation site (e.g., a non- naturally occurring glycosylation site at one or more of positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1)). Suitable glycosylation motifs are known in the art and can be identified by a person of ordinary still in the art, including, e.g., N-glycosylation motifs (e.g., canonical N-glycosylation motifs) and O-glycosylation motifs. In some embodiments, the glycosylation motif is an N-linked glycosylation motif. In some embodiments, the N-linked glycosylation motif is an NXT, wherein X is any amino acid except for proline. In some embodiments, the N-linked glycosylation motif is an NXS, wherein X is any amino acid except for proline. In some embodiments, the glycosylation site is an N-linked glycosylation site. In some embodiments, the glycosylation motif is an O-linked glycosylation motif. In some embodiments, the O-linked glycosylation motif is a serine. In some embodiments, the O- linked glycosylation motif is a threonine. In some embodiments, the corresponding glycosylation site is an O-linked glycosylation site. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions
Attorney Docket No.62801.28WO01 set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N- linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449, amino acid numbering relative the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a
Attorney Docket No.62801.28WO01 serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 461, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and
Attorney Docket No.62801.28WO01 an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO:
Attorney Docket No.62801.28WO01 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at
Attorney Docket No.62801.28WO01 position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N- glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except
Attorney Docket No.62801.28WO01 proline at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N- linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, a
Attorney Docket No.62801.28WO01 tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N- glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 394, a
Attorney Docket No.62801.28WO01 valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (l) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (m) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (n) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (o) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino
Attorney Docket No.62801.28WO01 acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449, amino acid numbering relative the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N-glycosylation
Attorney Docket No.62801.28WO01 site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 461, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N- glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an N- glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO:
Attorney Docket No.62801.28WO01 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to
Attorney Docket No.62801.28WO01 the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an
Attorney Docket No.62801.28WO01 N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N- glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N- linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid
Attorney Docket No.62801.28WO01 numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid at position 478 except for proline, and a threonine or a serine at position 479, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid at position 518 except for proline, and a threonine or a serine at position 519, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid at position 519 except for proline, and a threonine or a serine at position 520, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative
Attorney Docket No.62801.28WO01 to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine or a serine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine or a serine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine or a serine at position 520 and an N- glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine at position 385, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid at position 478 except for proline, and a threonine at position 479, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid at position 518 except for proline, and a threonine at position 519, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid at position 519 except for proline, and a threonine at position 520, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine at position 385 and an N-glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 5.2.3 Exemplary Sets of Engineered Glycosylation Sites & Motifs Further provided herein are SARS-CoV-2 spike proteins and polypeptides (e.g., SARS- CoV-2 spike protein and polypeptide immunogens (and immunogenic fragments and/or immunogenic variants thereof)) and nucleic acids encoding the same, that comprise any set (e.g., any singular or combinatorial use) of the non-naturally occurring glycosylation sites described herein. Exemplary sets of engineered glycosylation sites in the SARS-CoV-2 spike protein immunogens described herein are provided in Table 2. The amino acid numbering set forth in Table 2 is relative to SEQ ID NO: 1. Table 2 further provides exemplary glycosylation motifs that can be utilized to generate the exemplary glycosylation sites within each set. Within the exemplary glycosylation motifs provided in Table 2, a number followed by an amino acid residue denotes the amino acid residue at the specified amino acid position (e.g., 494N denotes an asparagine at position 494 (amino acid numbering relative to the amino acid positions of SEQ ID NO: 1)); and
Attorney Docket No.62801.28WO01 an “X” denotes any amino acid residue other than proline. Table 2. Engineered Glycosylation Sites of SARS-CoV-2 Spike Protein Immunogens. Set Glycosylation Site Exemplary Glycosylation Motif A 44 44 4 X4 T 44 4 Y4 T
Attorney Docket No.62801.28WO01 476 476N-477X-478T (e.g., 476N-477S-478T) 529 529N-530X-531T (e.g., 529N-530S-531T)
ike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more non-naturally glycosylation sites set forth in Table 2. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more non- naturally glycosylation motifs set forth in Table 2. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more non-naturally glycosylation motifs set forth in Table 2 that comprises the corresponding non-naturally glycosylation motifs set forth in Table 2. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., one) set (e.g., set A, B, C, D, E, F, G, H, I, J, K) of non-naturally glycosylation sites set forth in Table 2. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more sets of non-naturally occurring glycosylation motifs, wherein each set comprises a set (e.g., set A, B, C, D, E, F, G, H, I, J, K) of non-naturally glycosylation sites set forth in Table 2. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid
Attorney Docket No.62801.28WO01 numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N- glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, a threonine or a serine at position 496; and an N-glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N-glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at
Attorney Docket No.62801.28WO01 position 495, a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, a threonine at position 496 and an N-glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino
Attorney Docket No.62801.28WO01 acid except proline at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371, and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449, and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position
Attorney Docket No.62801.28WO01 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO:
Attorney Docket No.62801.28WO01 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in
Attorney Docket No.62801.28WO01 SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and
Attorney Docket No.62801.28WO01 a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and
Attorney Docket No.62801.28WO01 a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering
Attorney Docket No.62801.28WO01 relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except
Attorney Docket No.62801.28WO01 proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino
Attorney Docket No.62801.28WO01 acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an
Attorney Docket No.62801.28WO01 N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a
Attorney Docket No.62801.28WO01 serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and
Attorney Docket No.62801.28WO01 a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N- linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation
Attorney Docket No.62801.28WO01 site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at
Attorney Docket No.62801.28WO01 position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino
Attorney Docket No.62801.28WO01 acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position
Attorney Docket No.62801.28WO01 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the
Attorney Docket No.62801.28WO01 amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450,
Attorney Docket No.62801.28WO01 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476,
Attorney Docket No.62801.28WO01 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N- linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif
Attorney Docket No.62801.28WO01 comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid
Attorney Docket No.62801.28WO01 numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417,
Attorney Docket No.62801.28WO01 amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an
Attorney Docket No.62801.28WO01 asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID
Attorney Docket No.62801.28WO01 NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering
Attorney Docket No.62801.28WO01 relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any
Attorney Docket No.62801.28WO01 amino acid except proline at position 395, and a threonine or a serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and
Attorney Docket No.62801.28WO01 a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N- linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position
Attorney Docket No.62801.28WO01 429, and a threonine or a serine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N- linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at
Attorney Docket No.62801.28WO01 position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any
Attorney Docket No.62801.28WO01 amino acid except proline at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N- linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked
Attorney Docket No.62801.28WO01 glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N- linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises and (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 5.2.4 Exemplary Glycoengineered SARS-CoV-2 Spike Proteins (e.g., Immunogens) & Fragments Thereof The amino acid sequence of exemplary SARS-CoV-2 spike proteins (e.g., immunogens) comprising at least one non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site set forth herein (see, e.g., Table 2) is provided in Table 3. Table 3 provides the amino acid sequence of the full-length spike protein (immature SEQ ID NOS: 11-21 and mature SEQ ID NOS: 22-32) of eleven exemplary SARS-CoV-2 immunogens (A-K) that comprise the corresponding set of engineered glycosylation sites set forth above in Table 2 (i.e., set A, B, C, D, E, F, G, H, I, J, or K). Table 3. Amino Acid Sequence of SARS-CoV-2 Spike Protein Vaccine Immunogens. Desc Immature Full-Length Am SEQ ripti ino SEQ Mature Full-Length Amino Acid ID
Attorney Docket No.62801.28WO01 RKSNLKPFERDISTEIYQAGSTPC TEIYQAGSTPCNGVEGFNCYFPLQ NGVEGFNCYFPLQNYTFQPTNGVG NYTFQPTNGVGYQPYRVVVLSFEL YQPYRVVVLSFELLHAPATVCGPK LHAPATVCGPKKSTNLVKNKCVNF
Attorney Docket No.62801.28WO01 TKLNDLCFTNVYADSFVIRGDEVR DSFVIRGDEVRQIAPGQTGKIADY QIAPGQTGKIADYNYKLPDDFTGC NYKLPDDFTGCVIAWNSNNLDSKV VIAWNSNNLDSKVGGNYNYLYRLF GGNYNYLYRLFRKSNLKPFERDIS
Attorney Docket No.62801.28WO01 YQTSNFRVQPTESIVRFPNITNLC IVRFPNITNLCPFGEVFNATRFAS PFGEVFNATRFASVYAWNRKRISN VYAWNRKRISNCVADYSVLYNSAS CVADYSVLYNSASFSTFKCYGVSP FSTFKCYGVSPTKLNDLCFTNVYA
Attorney Docket No.62801.28WO01 LLALHRSYLTPGDSSSGWTAGAAA SSSGWTAGAAAYYVGYLQPRTFLL YYVGYLQPRTFLLKYNENGTITDA KYNENGTITDAVDCALDPLSETKC VDCALDPLSETKCTLKSFTVEKGI TLKSFTVEKGIYQTSNFRVQPTES
Attorney Docket No.62801.28WO01 EYVSQPFLMDLEGKQGNFKNLREF KQGNFKNLREFVFKNIDGYFKIYS VFKNIDGYFKIYSKHTPINLVRDL KHTPINLVRDLPQGFSALEPLVDL PQGFSALEPLVDLPIGINITRFQT PIGINITRFQTLLALHRSYLTPGD
Attorney Docket No.62801.28WO01 KSNIIRGWIFGTTLDSKTQSLLIV LDSKTQSLLIVNNATNVVIKVCEF NNATNVVIKVCEFQFCNDPFLGVY QFCNDPFLGVYYHKNNKSWMESEF YHKNNKSWMESEFRVYSSANNCTF RVYSSANNCTFEYVSQPFLMDLEG
Attorney Docket No.62801.28WO01 PPAYTNSFTRGVYYPDKVFRSSVL YPDKVFRSSVLHSTQDLFLPFFSN HSTQDLFLPFFSNVTWFHAIHVSG VTWFHAIHVSGTNGTKRFDNPVLP TNGTKRFDNPVLPFNDGVYFASTE FNDGVYFASTEKSNIIRGWIFGTT
Attorney Docket No.62801.28WO01 SCGSCCKFDEDDSEPVLKGVKLHY EPVLKGVKLHYT T MFVFLVLLPLVSSQCVNLTTRTQL QCVNLTTRTQLPPAYTNSFTRGVY
Attorney Docket No.62801.28WO01 VNIQKEIDRLNEVAKNLNESLIDL AKNLNESLIDLQELGKYEQYIKWP QELGKYEQYIKWPWYIWLGFIAGL WYIWLGFIAGLIAIVMVTIMLCCM IAIVMVTIMLCCMTSCCSCLKGCC TSCCSCLKGCCSCGSCCKFDEDDS
Attorney Docket No.62801.28WO01 TQRNFYEPQIITTDNTFVSGNCDV DNTFVSGNCDVVIGIVNNTVYDPL VIGIVNNTVYDPLQPELDSFKEEL QPELDSFKEELDKYFKNHTSPDVD DKYFKNHTSPDVDLGDISGINASV LGDISGINASVVNIQKEIDRLNEV
Attorney Docket No.62801.28WO01 VLGQSKRVDFCGKGYHLMSFPQSA GYHLMSFPQSAPHGVVFLHVTYVP PHGVVFLHVTYVPAQEKNFTTAPA AQEKNFTTAPAICHDGKAHFPREG ICHDGKAHFPREGVFVSNGTHWFV VFVSNGTHWFVTQRNFYEPQIITT
Attorney Docket No.62801.28WO01 TLVKQLSSNFGAISSVLNDILSRL SSVLNDILSRLDPPEAEVQIDRLI DPPEAEVQIDRLITGRLQSLQTYV TGRLQSLQTYVTQQLIRAAEIRAS TQQLIRAAEIRASANLAATKMSEC ANLAATKMSECVLGQSKRVDFCGK
or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or
Attorney Docket No.62801.28WO01 polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 3. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described
Attorney Docket No.62801.28WO01 herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 3 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 3. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 3. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring
Attorney Docket No.62801.28WO01 glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or
Attorney Docket No.62801.28WO01 polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 11-32. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or
Attorney Docket No.62801.28WO01 polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non- naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any
Attorney Docket No.62801.28WO01 one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 11-32 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 11-32.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 11-32. The amino acid sequence of exemplary SARS-CoV-2 spike proteins (e.g., immunogens) comprising at least one non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site set forth herein (see, e.g., Table 2) is provided in Table 4. Table 4 provides the amino acid sequence of the full-length spike protein (immature SEQ ID NOS: 33-43 and mature SEQ ID NOS: 44-54) of eleven exemplary SARS-CoV-2 immunogens (A-K) that comprise the corresponding set of engineered glycosylation sites set forth above in Table 2 (i.e., set A, B, C, D, E, F, G, H, I, J, or K) and a glycine at amino acid position 614 (amino acid numbering relative to SEQ ID NO: 1.). Table 4. Amino Acid Sequence of SARS-CoV-2 Spike Protein Vaccine Immunogens. Desc Immature Full-Length SEQ ripti Amino SEQ Mature Full-Length Amino Acid ID
Attorney Docket No.62801.28WO01 NGVEGFNCYFPLQNYTFQPTNGVG NYTFQPTNGVGYQPYRVVVLSFEL YQPYRVVVLSFELLHAPATVCGPK LHAPATVCGPKKSTNLVKNKCVNF KSTNLVKNKCVNFNFNGLTGTGVL NFNGLTGTGVLTESNKKFLPFQQF
Attorney Docket No.62801.28WO01 QIAPGQTGKIADYNYKLPDDFTGC NYKLPDDFTGCVIAWNSNNLDSKV VIAWNSNNLDSKVGGNYNYLYRLF GGNYNYLYRLFRKSNLKPFERDIS RKSNLKPFERDISTEIYQAGSTPC TEIYQAGSTPCNGVEGFNCYFPLQ
Attorney Docket No.62801.28WO01 PFGEVFNATRFASVYAWNRKRISN VYAWNRKRISNCVADYSVLYNSAS CVADYSVLYNSASFSTFKCYGVSP FSTFKCYGVSPTKLNDLCFTNVYA TKLNDLCFTNVYADSFVIRGDEVR DSFVIRGDEVRQIAPGQTGKIADY
Attorney Docket No.62801.28WO01 YYVGYLQPRTFLLKYNENGTITDA KYNENGTITDAVDCALDPLSETKC VDCALDPLSETKCTLKSFTVEKGI TLKSFTVEKGIYQTSNFRVQPTES YQTSNFRVQPTESIVRFPNITNLC IVRFPNITNLCPFGEVFNATRFAS
Attorney Docket No.62801.28WO01 VFKNIDGYFKIYSKHTPINLVRDL KHTPINLVRDLPQGFSALEPLVDL PQGFSALEPLVDLPIGINITRFQT PIGINITRFQTLLALHRSYLTPGD LLALHRSYLTPGDSSSGWTAGAAA SSSGWTAGAAAYYVGYLQPRTFLL
Attorney Docket No.62801.28WO01 NNATNVVIKVCEFQFCNDPFLGVY QFCNDPFLGVYYHKNNKSWMESEF YHKNNKSWMESEFRVYSSANNCTF RVYSSANNCTFEYVSQPFLMDLEG EYVSQPFLMDLEGKQGNFKNLREF KQGNFKNLREFVFKNIDGYFKIYS
Attorney Docket No.62801.28WO01 HSTQDLFLPFFSNVTWFHAIHVSG VTWFHAIHVSGTNGTKRFDNPVLP TNGTKRFDNPVLPFNDGVYFASTE FNDGVYFASTEKSNIIRGWIFGTT KSNIIRGWIFGTTLDSKTQSLLIV LDSKTQSLLIVNNATNVVIKVCEF
Attorney Docket No.62801.28WO01 T MFVFLVLLPLVSSQCVNLTTRTQL QCVNLTTRTQLPPAYTNSFTRGVY PPAYTNSFTRGVYYPDKVFRSSVL YPDKVFRSSVLHSTQDLFLPFFSN
Attorney Docket No.62801.28WO01 QELGKYEQYIKWPWYIWLGFIAGL WYIWLGFIAGLIAIVMVTIMLCCM IAIVMVTIMLCCMTSCCSCLKGCC TSCCSCLKGCCSCGSCCKFDEDDS SCGSCCKFDEDDSEPVLKGVKLHY EPVLKGVKLHYT
Attorney Docket No.62801.28WO01 VIGIVNNTVYDPLQPELDSFKEEL QPELDSFKEELDKYFKNHTSPDVD DKYFKNHTSPDVDLGDISGINASV LGDISGINASVVNIQKEIDRLNEV VNIQKEIDRLNEVAKNLNESLIDL AKNLNESLIDLQELGKYEQYIKWP
Attorney Docket No.62801.28WO01 PHGVVFLHVTYVPAQEKNFTTAPA AQEKNFTTAPAICHDGKAHFPREG ICHDGKAHFPREGVFVSNGTHWFV VFVSNGTHWFVTQRNFYEPQIITT TQRNFYEPQIITTDNTFVSGNCDV DNTFVSGNCDVVIGIVNNTVYDPL
Attorney Docket No.62801.28WO01 DPPEAEVQIDRLITGRLQSLQTYV TGRLQSLQTYVTQQLIRAAEIRAS TQQLIRAAEIRASANLAATKMSEC ANLAATKMSECVLGQSKRVDFCGK VLGQSKRVDFCGKGYHLMSFPQSA GYHLMSFPQSAPHGVVFLHVTYVP
or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
Attorney Docket No.62801.28WO01 the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 4. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2).
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 4 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 4. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the
Attorney Docket No.62801.28WO01 following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 4. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 33-54. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non- naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation
Attorney Docket No.62801.28WO01 motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 33-54 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 33-54. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or
Attorney Docket No.62801.28WO01 polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 33-54. The amino acid sequence of exemplary SARS-CoV-2 spike proteins (e.g., immunogens) comprising at least one non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site set forth herein (see, e.g., Table 2) is provided in Table 5. Table 5 provides the amino acid sequence of the receptor binding domain of the spike protein (full receptor binding domain SEQ ID NOS: 55-65 and fragment of receptor binding domain SEQ ID NOS: 66-76) of eleven exemplary SARS-CoV-2 immunogens (A-K) that comprise the corresponding set of engineered glycosylation sites set forth above in Table 2 (i.e., set A, B, C, D, E, F, G, H, I, J, or K). Table 5. Amino Acid Sequence of SARS-CoV-2 Vaccine Immunogens. Descripti Immunogenic Fragment – A SEQ Immunogenic Fragment – B SEQ on Full RBD ID NO Fragment RBD ID NO
Attorney Docket No.62801.28WO01 VFNATRFASVYAWNRKRISNCV RKRISNCVADYSVLYNSASFSTFK ADYSVLYNSASFSTFKCYGVSP CYGVSPTKLNDLCFTNVYADSFVI TKLNDLCFTNVYADSFVIRGDE RGDEVRQIAPGQTGKIADYNYKLP
Attorney Docket No.62801.28WO01 YTFQPTNGVGYQPYRVVVLSFE TVCGPKNST LLHAPATVCGPKNSTNLVKNKC VNF
In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90,
Attorney Docket No.62801.28WO01 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in Table 5. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a
Attorney Docket No.62801.28WO01 contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in Table 5 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 5. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in Table 5. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100 identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic
Attorney Docket No.62801.28WO01 fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of a SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to
Attorney Docket No.62801.28WO01 the amino acid sequence of a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS- CoV-2 protein set forth in any one of SEQ ID NOS: 55-76. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation sites described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs each comprising a non-naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%,
Attorney Docket No.62801.28WO01 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 19, 10, 11, 12, or more) non-naturally occurring glycosylation motifs described herein each comprising a non- naturally glycosylation site described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2).
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, or 1200 amino acids of any SARS-CoV-2 protein set forth in any one of SEQ ID NOS: 55-76 and comprises at least one set of non-naturally occurring glycosylation motifs described herein (e.g., set forth in Table 2) comprising a set non-naturally glycosylation sites described herein (e.g., set forth in Table 2). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, and other than the at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 (e.g., 1) of the sets of non-naturally occurring glycosylation motifs set forth in Table 2, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 55-76. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of one or more of the following sets of non-naturally occurring glycosylation motifs set forth in Table 2: A, B, C, D, E, F, G, H, I, J, and/or K; and other than the one or more set of non-naturally occurring glycosylation motifs is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence of any one of the SARS-Cov-2 proteins set forth in any one of SEQ ID NOS: 55-76. 5.2.5 Structure of SARS-CoV-2 Spike Proteins (e.g., Immunogens) The SARS-CoV-2 spike proteins and polypeptides (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein can be full-length proteins or fragments (e.g., receptor binding domains thereof). In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of at least about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of no more than about 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of from about 10-1300, 10-1200, 10-1100, 10-1000, 10-900, 10-800, 10-700, 10-600, 10- 500, 10-400, 10-500, 10-400, 10-300, 10-250, 10-200, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 10-1300, 20-1300, 30-1300, 40-1300, 50-1300, 60-1300, 70-1300, 80-1300, 90-1300, 100-1300,10-250, 20-250, 30-250, 40-250, 50-250, 60-250, 70-250, 80-250, 90-250, or 100-250 amino acids. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least a portion of the RBD of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises the RBD of the SARS-CoV-2 spike protein. In some embodiments the SARS- CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a full- length SARS-CoV-2 spike protein. In some embodiments, the full-length SARS-CoV-2 spike protein comprises a homologous signal peptide. In some embodiments, the full-length SARS- CoV-2 spike protein does not comprise a homologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-
Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises the ectodomain of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises the ectodomain of the SARS-CoV-2 spike protein and does not include the transmembrane domain or the cytoplasmic domain of the SARS-CoV-2 spike protein. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a homologous signal peptide. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) does not comprise a homologous signal peptide. 5.2.6 Exemplary Additional Features of SARS-CoV-2 Spike Proteins (e.g., Immunogens) The SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) can exhibit additional structural and functional features. Exemplary additional structural and functional features are provided below. 5.2.6.1 Hyperglycosylation In some embodiments, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper- glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1) (e.g., a SARS-CoV-2 spike protein that does not contain the one or more non-naturally occurring glycosylation sites). Standard methods known in the art can be utilized to evaluate the glycosylation of a protein or peptide, including e.g., the level of glycosylation, the sites of glycosylation, etc. See, e.g., Roth et al, Identification and Quantification of Protein Glycosylation (2012) International Journal of Carbohydrate Chemistry, Volume 201, Article ID 640923, https://doi.org/10.1155/2012/640923; Lebede et al, Exploring the Chemical Space of Protein Glycosylation in Noncovalent Protein Complexes: An Expedition along Different Structural Levels of Human Chorionic Gonadotropin by Employing Mass Spectrometry, Anal. Chem. 2021,
Attorney Docket No.62801.28WO01 93, 30, 10424–10434 (July 21, 2021) https://doi.org/10.1021/acs.analchem.1c02199; Leymarie et al., Effective Use of Mass Spectrometry for Glycan and Glycopeptide Structural Analysis, Anal. Chem. 2012, 84, 7, 3040–3048 (February 21, 2012) https://doi.org/10.1021/ac3000573the entire contents of each of which is incorporated by reference herein for all purposes. Standard methods include for example, the enzymatic or chemical release of glycans from the subject proteins followed by chromatography or mass spectrometry based structural analysis. Glycan site mapping is further standard in art through the use of enzymatic digestions and subsequent tandem mass spectrometry-based analysis. See, e.g., Roth et al, Identification and Quantification of Protein Glycosylation (2012) International Journal of Carbohydrate Chemistry, Volume 201, Article ID 640923, https://doi.org/10.1155/2012/640923, the entire contents of which is incorporated by reference herein for all purposes. 5.2.6.2 Stabilization In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is stabilized in a prefusion state. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation that stabilizes said SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) in a prefusion state. In some embodiments, the amino acid sequence of the SARS- CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation relative to the amino acid sequence set forth in any one of SEQ ID NO: 1 that stabilizes the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) in a prefusion state. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein
Attorney Docket No.62801.28WO01 or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at amino acid position 986 and/or a proline at amino acid position 987, amino acid numbering amino acid numbering relative to SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at amino acid position 986 and a proline at amino acid position 987, amino acid numbering relative to SEQ ID NO: 1. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises an inactive furin cleavage site. In order to be able to trigger fusion, the spike protein of SARS-Cov-2 has to be cleaved into the S1 and S2 subunit. The cleavage site in SARS-Cov-2 is a polybasic motif (RRAR (SEQ ID NO: 77)) (for example, amino acids 682-685 of SEQ ID NO: 1) that can be activated by furin-like proteases. Modifications to the furin cleavage site that inactivate it are known in the art. See, e.g., Amanat F, Strohmeier S, Rathnasinghe R, et al. Introduction of two prolines and removal of the polybasic cleavage site leads to optimal efficacy of a recombinant spike-based SARS-CoV-2 vaccine in the mouse model. Preprint. bioRxiv. 2020;2020.09.16.300970. Published 2020 Sep 17. doi:10.1101/2020.09.16.300970 (describes the replacement of the RRAR cleavage site (SEQ ID NO: 77) with a single alanine); and WO2022203963 (which describes the replacement of the RRAR cleavage site (SEQ ID NO: 77) with the amino acid sequence QQAQ (SEQ ID NO: 78)), the entire contents of each of which are incorporated by reference herein. In some embodiments, the amino acid sequence of the SARS- CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the
Attorney Docket No.62801.28WO01 SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a replacement of the RRAR cleavage site (SEQ ID NO: 77) with a single alanine. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a replacement of the RRAR cleavage site (SEQ ID NO: 77) with the amino acid sequence QQAQ (SEQ ID NO: 78). 5.2.6.3 Multimerization (e.g., Trimerization) Domains In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a multimerization (e.g., trimerization) domain. The multimerization domain (e.g., trimerization domain) facilities multimerization (e.g., trimerization) of the proteins upon expression in/from a cell. Suitable multimerization (e.g., trimerization) domains are known in the art, including e.g., a fibritin - based trimerization domain. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was previously described as an artificial natural trimerization domain. See, e.g., (Letarov et al., 1993) Biochemistry Moscow 64: 817- 823; S-Guthe et al., (2004) J. Mol. Biol. 337: 905-915). See, also, Meier et al., J Mol Biol. (2004) 344(4): 1051-69; WO 2018/081318; the entire contents of each of which are incorporated herein by reference for all purposes. 5.2.6.4 Variations from Different Strains of SARS-CoV-2 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein is derived from a circulating strain (i.e., naturally occurring variant) of SARS-CoV-2. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2 known to have previously circulated. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated. For example, in some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 5.2.6.5 Conserved Structural Elements
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline
Attorney Docket No.62801.28WO01 at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 5.3 Nucleic Acid Molecules In one aspect, provided herein are nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2). In some embodiments, the nucleic acid molecule is RNA (e.g., mRNA or circular RNA) or DNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is a translatable RNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is a circular RNA. In some embodiments, the nucleic acid (e.g., RNA) molecule is mRNA. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) comprises
Attorney Docket No.62801.28WO01 from about 30 to about 20000 nucleotides, about 50 to about 20000 nucleotides, about 500 to about 10000 nucleotides, about 1000 to about 10000 nucleotides, about 1000 to about 5000 nucleotides, or about 2000 to about 5000 nucleotides. In some embodiments, the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least 30 nucleotides, 50 nucleotides, 100 nucleotides, 200 nucleotides, 300 nucleotides, 400 nucleotides, 500 nucleotides, 1000 nucleotides, 2000 nucleotides, 3000 nucleotides, or 5000 nucleotides. In some embodiments, the nucleic acid molecule is altered (e.g., compared to the sequence of a reference nucleic acid molecule, a naturally occurring nucleic acid molecule), e.g., to impart one or more of (a) improved resistance to in vivo degradation, (b) improved stability in vivo, (c) reduced secondary structures, and/or (d) improved translatability in vivo, compared to the reference nucleic acid sequence. Alterations include, without limitation, e.g., codon optimization, nucleotide modifications (see, e.g., described herein), etc. In some embodiments, the sequence of the nucleic acid molecule is codon optimized, e.g., for expression in humans. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias guanosine (G) and/or cytosine (C) content to increase nucleic acid stability; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove/add post translation alteration sites in an encoded protein (e.g., glycosylation sites); add, remove, or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of a protein to fold properly; and/or to reduce or eliminate secondary structures (e.g., problem secondary structures) within the nucleic acid molecule. In some embodiments, the codon optimized nucleic acid sequence shows one or more of the above (compared to a reference nucleic acid sequence). In some embodiments, the codon optimized nucleic acid sequence shows one or more of improved resistance to in vivo degradation, improved stability in vivo, reduced secondary structures, and/or improved translatability in vivo, compared to a reference nucleic acid sequence. Codon optimization methods, tools, algorithms, and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies) and DNA2.0 (Menlo Park Calif.). In some
Attorney Docket No.62801.28WO01 embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms (e.g., optimization algorithms known in the art). In some embodiments, the nucleic acid sequence is modified to optimize the number of G and/or C nucleotides as compared to a reference nucleic acid sequence. An increase in the number of G and C nucleotides may be generated by substitution of codons containing adenosine (A) or thymidine (T) (or uracil (U)) nucleotides by codons containing G or C nucleotides. In some embodiments, the nucleic acid molecule is recombinant. In some embodiments, the nucleic acid molecule is isolated. 5.3.1 DNA Nucleic Acids In some embodiments, the nucleic acid molecule is DNA. In some embodiments, the DNA is a linear coding DNA construct. In some embodiments, the DNA contained within a vector (e.g., a non-viral vector (e.g., a plasmid) or a viral vector). In some embodiments, the DNA is contained within a non-viral vector (e.g., a plasmid). In some embodiments, the DNA is contained within a viral vector (e.g., described herein). A more detailed description of vectors for both RNA and DNA nucleic acids is provided in § 5.6. The coding DNA molecule may also comprise one or more heterologous nucleic acid elements to mediate expression of the coding region. These include, e.g., promoter(s), enhancer(s), polyadenylation signal(s), synthetic introns, transcriptional termination signals, polyadenylation sequences, and other transcription regulatory elements. A person of ordinary skill in the art is familiar with the transcriptional regulatory elements needed for expression of coding DNA can optimize the expression construct (e.g., linear DNA, plasmid DNA, etc.) accordingly. In some embodiments, a promoter is operably connected to the respective coding nucleic acid sequence. The person of ordinary skill in the art is aware of various promoters that can be employed, for example, a promoter from simian virus 40 (SV40), a mouse mammary tumor virus (MMTV) promoter, a human immunodeficiency virus (HIV) promoter, bovine immunodeficiency virus (BIV) long terminal repeat (LTR) promoter, a Moloney virus promoter, an avian leukosis virus (ALV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter, Epstein Barr virus (EBV) promoter, or a Rous sarcoma virus (RSV) promoter. The promoter can also be a promoter from a human gene, for example, from human actin, human myosin, human hemoglobin, human muscle creatine, or human metalothionein. The
Attorney Docket No.62801.28WO01 promoter can also be a tissue specific promoter, such as a muscle or skin specific promoter, natural or synthetic. Examples of such promoters are described in US Patent Application Publication No. US20040175727, the entire contents of which is incorporated by reference herein for all purposes. Exemplary polyadenylation signals, include, but are not limited, to the bovine growth hormone (BGH) polyadenylation site, SV40 polyadenylation signals, and LTR polyadenylation signals. 5.3.2 RNA Nucleic Acids In some embodiments, the nucleic acid molecule is an RNA molecule. In some embodiments, the RNA molecule is a translatable RNA molecule. In some embodiments, the RNA molecule is selected from an mRNA, a self-replicating RNA, a circular RNA, a viral RNA, or a replicon RNA. In some embodiments, the RNA molecule a circular RNA. In some embodiments, the RNA molecule is a mRNA. The basic components of an mRNA molecule typically include at least one coding region (herein a coding region encoding at least one peptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or variant thereof)) (e.g., described herein), a 5’-untranslated region (UTR), a 3’-UTR, a 5’ cap, and a poly-A tail. In some embodiments, the RNA (e.g., mRNA) comprises at least one heterologous UTR. The UTRs may harbor regulatory sequence elements that determine the RNA (e.g., mRNA) turnover, stability, localization, and/or expression of operably connected coding sequence(s). The heterologous UTRs may be derived from a naturally occurring gene or may be synthetically engineered. In some embodiments, the 5’-UTR comprises elements for controlling gene expression, e.g., ribosomal binding sites, miRNA binding sites. The 5’-UTR may be post- transcriptionally modified, e.g., by enzymatic or post-transcriptional addition of a 5’cap structure. In some embodiments, the 3’-UTR comprises a polyadenylation signal. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding region encoding the polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) and 5’-UTR and/or a 3’-UTR. In some embodiments, the RNA (e.g., mRNA) comprises at least one coding sequence encoding a polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) operably connected to at least one heterologous 5’-UTR
Attorney Docket No.62801.28WO01 and at least one 3’-UTR. In some embodiments, the RNA (e.g., mRNA) comprises a poly(A) sequence. The poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides. In some embodiments, poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides. The poly(A) sequence may comprise from about 10 to 500 adenosine nucleotides, 10 to 200 adenosine nucleotides, 20 to 200 adenosine nucleotides, 30 to 200 adenosine nucleotides, 40 to 200 adenosine nucleotides, or 50 to 200 adenosine nucleotides, wherein the 3’ terminal nucleotide of said nucleic acid molecule is an adenosine. In some embodiments, poly(A) sequence comprises at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or 500 adenosine nucleotides, wherein the 3’ terminal nucleotide of said nucleic acid molecule is an adenosine. In some embodiments, the RNA (e.g., mRNA) comprises a 5’-cap structure. In some embodiments, the 5’-cap structure stabilizes the RNA (e.g., mRNA), enhances expression of the encoded polypeptide or protein (e.g., a SARS-CoV-2 protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein) and/or reduces the stimulation of the innate immune system (e.g., after administration to a subject). Exemplary 5’-cap structures include, but are not limited to, cap0 (methylation of the first nucleobase, e.g., m7GpppN), cap1 (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA (anti-reverse cap analogue), modified ARCA (e.g., phosphorothioate modified ARCA), inosine, N1-methyi-guanosine, 2'-fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the 5’ cap structure comprises m7G, cap0, cap1, cap2, a modified capO, or a modified cap1 structure. In some embodiments, the RNA (e.g., mRNA) comprises one or more modified nucleotide (e.g., nucleotide analogue, backbone modifications, sugar modifications, and/or base modifications). A backbone modification in the context of the present disclosure is a modification,
Attorney Docket No.62801.28WO01 in which phosphates of the backbone of the nucleotides of the RNA (e.g., mRNA) are chemically modified. A sugar modification in the context of the present disclosure is a chemical modification of the sugar of the nucleotides of the RNA (e.g., mRNA). A base modification in the context of the present disclosure is a chemical modification of the base moiety of the nucleotides of the RNA (e.g., mRNA). In some embodiments, the RNA (e.g., mRNA) comprises at least one modified nucleotide. Exemplary nucleotide analogues/modifications include, but are not limited to, 2- amino-6-chloropurineriboside-5’-triphosphate, 2-Aminopurine-riboside-5’-triphosphate; 2- aminoadenosine-5'-triphosphate, 2’-Amino-2’-deoxycytidine-triphosphate, 2-thiocytidine-5'- triphosphate, 2-thiouridine-5’-triphosphate, 2’-Fluorothymidine-5’-triphosphate, 2’-O-Methyl- inosine-5’-triphosphate 4-thiouridine-5’-triphosphate, 5-aminoallylcytidine-5’-triphosphate, 5- aminoallyluridine-5’-triphosphate, 5-bromocytidine-5’-triphosphate, 5-bromouridine-5’- triphosphate, 5-Bromo-2’-deoxycytidine-5'-triphosphate, 5-Bromo-2’-deoxyuridine-5'- triphosphate, 5-iodocytidine-5’-triphosphate, 5-lodo-2’-deoxycytidine-5'-triphosphate, 5- iodouridine-5’ -triphosphate, 5-lodo-2’-deoxyuridine-5’-triphosphate, 5-methylcytidine-5’- triphosphate, 5-methyluridine-5’-triphosphate, 5-Propynyl-2’-deoxycytidine-5’-triphosphate, 5- Propynyl-2'-deoxyuridine-5’-triphosphate, 6-azacytidine-5’-triphosphate, 6-azauridine-5’- triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5’-triphosphate, 7- deazaguanosine-5’-triphosphate, 8-azaadenosine-5’-triphosphate, 8-azidoadenosine-5’- triphosphate, benzimidazole-riboside-5’-triphosphate, N1-methyladenosine-5’-triphosphate, N1- methylguanosine-5’-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5’- triphosphate, pseudouridine-5’-triphosphate, or puromycin-5’-triphosphate, xanthosine-5’- triphosphate. Particular preference is given to nucleotides for base modifications selected from the group of base-modified nucleotides consisting of 5-methylcytidine-5’-triphosphate, 7- deazaguanosine-5'-triphosphate, 5-bromocytidine-5’-triphosphate, and pseudouridine-5’- triphosphate, pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4- thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyl- uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine, 1- taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-1-methyl- pseudouridine, 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-
Attorney Docket No.62801.28WO01 methyl-1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2- thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl- cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1- methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2 -thiocytidine, 2-thio-5- methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1- deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5- methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy-1-methyl-pseudoisocytidine, 2- aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2- aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6- diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6-(cis- hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza- 8-aza-guanosine, 6-thio-guanosine, 6-thio-7 -deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 - methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl- 8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl- 6-thio-guanosine, 5’-O-(1-thiophosphate)-adenosine, 5’-O-(1-thiophosphate)-cytidine, 5’-O-(1- thiophosphate)-guanosine, 5’-O-(1-thiophosphatej-uridine, 5’-O-(1-thiophosphate)- pseudouridine, 6-aza-cytidine, 2-thio-cytidine, alpha-thio-cytidine, Pseudoiso-cytidine, 5- aminoallyl-uridine, 5-iodo-uridine, N1-methyl-pseudouridine, 5,6-dihydrouridine, alpha -thio- uridine, 4-thio-uridine, 6-aza-uridine, 5-hydroxy-uridine, deoxy-thymidine, 5-methyl-uridine, Pyrrolo-cytidine, inosine, alpha -thioguanosine, 6-methyl-guanosine, 5-methyl-cytdine, 8-oxo- guanosine, 7-deaza-guanosine, N1-methyl-adenosine, 2-amino-6-Chloro-purine, N6-methyl-2- amino-purine, Pseudo-iso-cytidine, 6-Chloro-purine, N6-methyl-adenosine, alpha -thioadenosine, 8-azido-adenosine, and 7-deaza-adenosine. In some embodiments, the RNA (e.g., mRNA) comprises pseudouridine, N1 - methylpseudouridine, N1-ethylpseudouridine, 2-thiouridine, 4’-thiouridine, 5-methylcytosine, 5-
Attorney Docket No.62801.28WO01 methyluridine, 2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine, 2-thio-5- aza-uridine, 2-thio-dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4- methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine, 4-thio- pseudouridine, 5-aza-uridine, dihydropseudouridine, 5-methoxyuridine, and/or 2’-O-methyl uridine. In some embodiments, the RNA (e.g., mRNA) comprises one or more pseudouridine (ψ), N 1 -methylpseudouridine (m1ψ), 5-methylcytosine, and 5-methoxyuridine. In some embodiments, essentially all, e.g., essentially 100% of the uracil in the coding sequence of the RNA (e.g., mRNA) have a chemical modification, preferably a chemical modification is in the 5- position of the uracil. Incorporating modified nucleotides such as e.g., pseudouridine (ψ), N1 - methylpseudouridine (m1ψ), 5-methylcytosine, and/or 5-methoxyuridine into the coding sequence may be advantageous as unwanted innate immune responses (upon administration of the coding RNA or the vaccine) may be adjusted or reduced (if required). In one embodiment, the RNA (e.g., mRNA) comprises: (i) a 5'-cap structure; (ii) a 5'- UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly- A region. RNA (e.g., mRNA) described herein can be generated by e.g., in vitro transcription. In vitro transcription is a method well known to those of ordinary skill in the art for the production of RNA (e.g., mRNA). Generally, the RNA is obtained by DNA-dependent in vitro transcription of an appropriate DNA template, e.g., a linearized plasmid DNA template or a PCR-amplified DNA template. The promoter for controlling RNA in vitro transcription can be any promoter for any DNA-dependent RNA polymerase. Examples of DNA-dependent RNA polymerases include the 17, T3, SP6, or Syn5 RNA polymerases. In some instances, the DNA template is linearized with a suitable restriction enzyme before it is subjected to RNA in vitro transcription. Reagents used in RNA in vitro transcription typically include: a DNA template (linearized plasmid DNA or PCR product) with a promoter sequence that has a high binding affinity for its respective RNA polymerase such as bacteriophage-encoded RNA polymerases (T7, T3, SP6, or Syn5); ribonucleotide triphosphates (NTPs) for the four bases (adenine, cytosine, guanine and uracil); a DNA-dependent RNA polymerase capable of binding to the promoter sequence within the DNA template (e.g., T7, T3, SP6, or Syn5 RNA polymerase); optionally, a ribonuclease (RNase) inhibitor to inactivate any potentially contaminating RNase; optionally, a pyrophosphatase to
Attorney Docket No.62801.28WO01 degrade pyrophosphate, which may inhibit RNA in vitro transcription; MgCh, which supplies Mg2+ ions as a co-factor for the polymerase; a buffer (TRIS or HEPES) to maintain a suitable pH value, which can also contain antioxidants (e.g., DTT), and/or polyamines such as spermidine at optimal concentrations, e.g., a buffer system comprising TRIS-Citrate as disclosed in W02017109161. The obtained RNA (e.g., mRNA) products can be purified according to methods known in the art. For example, using PureMessenger® (CureVac, Tubingen, Germany; RP-HPLC according to W02008077592) and/or tangential flow filtration (as described in WO2016193206) and/or oligo d(T) purification (see WO2016180430); or using RP-HPLC, e.g., using Reversed- Phase High pressure liquid chromatography (RP-HPLC), the entire contents of each reference is incorporated by reference herein for all purposes. 5.4 Fusions & Conjugates In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.3) is operably connected to a heterologous moiety (e.g., a heterologous polypeptide) forming a fusion protein or polypeptide or conjugate. In some embodiments, the heterologous moiety imparts an additional function to the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), e.g., to promote or improve secretion of the encoded SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV- 2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), (e.g., via secretory signal peptides), promote or improve anchoring of the encoded SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), described herein in the plasma membrane (e.g., via transmembrane elements), promote or improve formation of antigen complexes (e.g., via multimerization domains or antigen clustering elements), or promote or improve virus-like particle formation (VLP forming sequence). In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide
Attorney Docket No.62801.28WO01 immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., the ectodomain of the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof))) is modified to improve stability of the protein produced. In some embodiments, the heterologous moiety is a half-life extension moiety. Exemplary half-life extension moieties include, but are not limited to, an immunoglobulin (e.g., human immunoglobulin (hIg)), a fragment of an Ig (e.g., a hIg), an Ig (e.g., a hIg) constant region, a fragment of an Ig (e.g., a hIg) constant region, an Ig (e.g., a hIg) Fc region, human transferrin, human serum albumin (HSA), an HSA binding protein, and polyethylene glycol (PEG) (and polymers thereof). In some embodiments, the heterologous polypeptide is a half-life extension polypeptide. Exemplary half-life extension polypeptides include, but are not limited to, an Ig (e.g., a hIg), a fragment of an Ig (e.g., a hIg), one or more Ig (e.g., a hIg) heavy chain constant region, a fragment of an Ig (e.g., a hIg) constant region, an Ig (e.g., a hIg) Fc region, human transferrin, human serum albumin (HSA), and an HSA binding protein. The SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) fused or conjugated to a half-life extending moiety (e.g., a half-life extension polypeptide) can be evaluated for their pharmacokinetic properties utilizing standard in vivo methods known in the art. In some embodiments, the heterologous moiety is a detectable agent (e.g., protein, e.g., a fluorescent protein). In some embodiments, the heterologous moiety is a heterologous signal peptide. In some embodiments, the heterologous moiety is a trimerization domain. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises a multimerization (e.g., trimerization) domain. Suitable multimerization (e.g., trimerization) domains are known in the art, including e.g., a fibritin - based trimerization domain. This fibritin domain or ‘Foldon’ is derived from T4 fibritin and was previously described as an artificial natural trimerization domain. See, e.g., (Letarov et al., 1993) Biochemistry Moscow 64: 817- 823; S-Guthe et al., (2004) J. Mol. Biol. 337: 905-915). See, also, Meier et al., J Mol Biol.
Attorney Docket No.62801.28WO01 (2004) 344(4): 1051-69; WO 2018/081318; the entire contents of each of which are incorporated herein by reference for all purposes. The heterologous moiety (e.g., heterologous polypeptide) can be directly operably connected or indirectly operably connected to the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)). In some embodiments, the heterologous moiety is directly operably connected to the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)). In some embodiments, the heterologous moiety is indirectly operably connected to the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)). A person of ordinary skill in the art can evaluate and select a suitable linker for the fusion or conjugation of a specific heterologous moiety. For example, in embodiments, wherein the heterologous moiety comprises a heterologous polypeptide, a peptide linker may be employed. Peptide linkers are known in the art and can be selected based on specific properties, including e.g., length, flexibility, rigidity, cleavability, etc. The amino acid sequence of commonly employed peptide linkers comprises glycine amino acid residues, serine amino acid residues, glycine and serine amino acid residues, or glycine, serine, and proline amino acid residues. The heterologous moiety (e.g., heterologous polypeptide) and the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) or the nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic
Attorney Docket No.62801.28WO01 variant thereof)) can be arranged in orientation so long as each functional part of the fusion protein or polypeptide or conjugate maintains the ability to mediate its function. 5.5 Compositions (e.g., Vaccine Compositions) In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2) (or a fusion or conjugate thereof (see, e.g., §5.4)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition, a vaccine booster composition, a vaccine prime and booster composition)). Therefore, in one aspect, provided herein are compositions (e.g., vaccine compositions, (e.g., prime vaccine compositions, vaccine booster compositions, vaccine prime and booster compositions)) comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2) (or a fusion or conjugate thereof (see, e.g., § 5.4)) or a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)). In some embodiments, the composition is a vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine booster composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition can be utilized as a prime and/or a booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster. In some embodiments, the vaccine composition is a polyvalent vaccine composition. In some embodiments, the vaccine composition is a monovalent vaccine composition. 5.5.1 Peptide and Protein-Based Vaccines
Attorney Docket No.62801.28WO01 In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2) (or a fusion or conjugate thereof (see, e.g., § 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition, a vaccine booster composition, a vaccine prime and booster composition)). Therefore, in one aspect, provided herein are compositions (e.g., vaccine compositions (e.g., a prime vaccine composition, a vaccine booster composition, a vaccine prime and booster composition)) comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.2) (or a fusion or conjugate thereof (see, e.g., § 5.4)). In some embodiments, the composition is a vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine boost composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition can be utilized as a prime and/or a booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster. In some embodiments, the composition (e.g., vaccine composition) comprises a plurality of SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)). In some embodiments, the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)). In some embodiments, the plurality comprises or consists of from about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5- 60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30- 50, 30-40, 40-100, 40-90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants
Attorney Docket No.62801.28WO01 thereof)). In some embodiments, the plurality comprises at least 2 but no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)). In some embodiments, the amino acid sequence of each of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. In some embodiments, the amino acid sequence of at least one of the SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is derived from a circulating strain of SARS-CoV- 2. In some embodiments, the plurality comprises at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein. In some embodiments, the plurality comprises at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, wherein the amino acid sequence of the at least one SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., a set of) non-naturally occurring glycosylation site described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in § 5.2). In some embodiments, the plurality comprises at least two SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the at least two SARS- CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) comprises a different combination of non-naturally occurring glycosylation sites described herein or a different combination of sets of non-naturally occurring glycosylation sites described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in § 5.2). In some embodiments, the composition comprises a multimer (e.g., trimer) of multiple
Attorney Docket No.62801.28WO01 (e.g., 3) SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein. In some embodiments, the composition comprises a trimer of three SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein. In some embodiments, the composition comprises a homotrimer of three SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein is derived from a circulating strain of SARS-CoV-2. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2 known to have previously circulated. In some embodiments, the amino acid sequence of the SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated. In some embodiments, the vaccine composition further comprises at least one immunogen (or immunogenic fragment and/or immunogenic variant thereof) from a non-SARS- CoV-2 virus, e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus. In some embodiments, vaccine composition comprises one or more immunogen (or immunogenic fragment or immunogenic variant thereof) from an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, and/or an adenovirus (or any combination thereof). In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., ectodomain of the SARS-CoV-2 spike protein or polypeptide) is modified to improve expression of the protein in host cells (e.g., insect cells, mammalian cells, eggs) as described below. Polypeptides and proteins (e.g., immunogens) described herein, e.g., SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (and immunogenic fragments and/or immunogenic variants thereof)) may be produced by recombinant technology in host cells (e.g.,
Attorney Docket No.62801.28WO01 mammalian cells) that have been transfected or transduced with a nucleic acid expression vector (e.g., plasmid, viral vector (e.g., a baculoviral expression vector)) encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)). In some embodiments, the cell is specifically selected to ensure the cell can mediate the desired glycosylation pattern (N-linked glycosylation, O-linked glycosylation, etc.) on a subject protein being produced. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a murine cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is a hamster cell. Such general methods are common knowledge in the art. The expression vector typically contains an expression cassette that includes nucleic acid sequences capable of bringing about expression of the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)), such as promoter(s), enhancer(s), polyadenylation signals, and the like. The person of ordinary skill in the art is aware that various promoter and enhancer elements can be used to obtain expression of a nucleic acid molecule in a host cell. For example, promoters can be constitutive or regulated, and can be obtained from various sources, e.g., viruses, prokaryotic or eukaryotic sources, or artificially designed. Post transfection or transduction, host cells containing the expression vector encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) are cultured under conditions conducive to expression of the nucleic acid molecule encoding the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)). Culture media is available from various vendors, and a suitable medium can be routinely chosen for a host cell to express a polypeptide or protein of interest, here the SARS-CoV-2 spike protein or polypeptide (e.g., the immunogen (or the immunogenic fragment and/or immunogenic variant thereof)). Host cells can be adherent or suspension cultures, and a person of ordinary skill in the art can optimize culture methods for specific host cells selected. For example, suspension cells can be cultured in, for example, bioreactors in e.g., a batch process or a fed-batch process. The produced immunogenic peptide or protein may be isolated from the cell cultures, by, for example, column chromatography in either flow-flow through or bind-and-elute modes. Examples include, but are not limited to, ion exchange resins and affinity resins, such as lentil lectin Sepharose, and mixed mode cation exchange-hydrophobic interaction columns (CEX-HIC). The
Attorney Docket No.62801.28WO01 peptide or protein may be concentrated, buffer exchanged by ultrafiltration, and the retentate from the ultrafiltration may be filtered through an appropriate filter, e.g., a 0.22µm filter. See, e.g., McPherson et al., “Development of a SARS Coronavirus Vaccine from Recombinant Spike Protein Plus Delta Inulin Adjuvant,” Chapter 4, in Sunil Thomas (ed.), Vaccine Design: Methods and Protocols: Volume 1: Vaccines for Human Diseases, Methods in Molecular Biology, Springer, New York, 2016. See also U.S. Pat.5,762,939, the entire contents of each of which is incorporated by reference herein for all purposes. The SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein (and fusion proteins, conjugates, etc. thereof) may also be produced synthetically. The SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein may be produced by using an egg-based manufacturing method. In some embodiments, the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the vaccine composition are formulated in one or more carrier (e.g., a carrier described herein (see, e.g., § 5.7)). In some embodiments, the vaccine compositions are pharmaceutical compositions (e.g., described herein, e.g., see § 5.8). In some embodiments, the vaccine compositions comprise an adjuvant (e.g., described herein, e.g., see § 5.9). 5.5.2 Nucleic Acid-Based Vaccines In some embodiments, a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)) forms the basis for a composition (e.g., a vaccine composition (e.g., a prime vaccine composition, a booster composition, a vaccine prime and booster composition)). Therefore, in one aspect, provided herein are compositions (e.g., vaccine compositions (e.g., prime vaccine compositions, booster compositions, vaccine prime and booster compositions)) comprising a nucleic acid molecule comprising a coding region encoding at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof))
Attorney Docket No.62801.28WO01 described herein (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)). In some embodiments, the composition is a vaccine composition. In some embodiments, the vaccine composition is a prime vaccine composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine booster composition of a prime-boost vaccine regimen. In some embodiments, the vaccine composition is a vaccine prime and booster composition of a prime-boost vaccine regimen. In some embodiments, the prime boost composition can be utilized as a prime and/or a booster (e.g., as described herein). In some embodiments, the vaccine composition forms a single dose vaccine that does not require a booster. In some embodiments, the composition (e.g., vaccine composition) comprises a plurality of nucleic acid molecules, each comprising a coding region encoding at least one SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., described herein). In some embodiments, each of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule. In some embodiments, each of the nucleic acid molecules of the plurality are separate (i.e., not connected) nucleic acid molecules. In some embodiments, at least two of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule. In some embodiments, at least two of the nucleic acid molecules of the plurality are separate (i.e., not connected) nucleic acid molecules. In some embodiments, at least two of the nucleic acid molecules of the plurality are part of the same larger nucleic acid molecule; and at least one (e.g., at least 2, 3, 4, 5, etc.) of the nucleic acid molecules of the plurality is a separate (i.e., not connected) nucleic acid molecule. In some embodiments, the plurality comprises or consists of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more nucleic acid molecules. In some embodiments, the plurality comprises or consists of from about 2-100, 2-90, 2-80, 2-70, 2-60, 2-50, 2-40, 2-30, 2-20, 2-10, 2-5, 5-100, 5-90, 5-80, 5-70, 5-60, 5-50, 5-40, 5-30, 5-20, 5-10, 10-100, 10-90, 10-80, 10-70, 10-60, 10-50, 10-40, 10-30, 10-20, 20-100, 20-90, 20-80, 20-70, 20-60, 20-50, 20-40, 20-30, 30-100, 30-90, 30-80, 30-70, 30-60, 30-50, 30-40, 40-100, 40- 90, 40-80, 40-70, 40-60, 40-50, 50-100, 50-90, 50-80, 50-70, 50-60, 60-100, 60-90, 60-80, 60-70, 70-100, 70-90, 70-80, 80-100, 80-90, or 90-100 nucleic acid molecules. In some embodiments, the plurality comprises at least 2 but no more than 100, 90, 80, 70, 60, 50, 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3 nucleic acid molecules.
Attorney Docket No.62801.28WO01 In some embodiments, the amino acid sequence of each of the encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. In some embodiments, the amino acid sequence of at least one of the encoded SARS- CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is derived from a circulating strain of SARS-CoV- 2. In some embodiments, the plurality comprises at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein. In some embodiments, the plurality comprises at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, wherein the amino acid sequence of the at least one encoded SARS-CoV-2 spike protein or polypeptide (e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises one or more (e.g., a set of) non-naturally occurring glycosylation site described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in § 5.2). In some embodiments, the plurality comprises at least two encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, wherein the amino acid sequence of each of the at least two encoded SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) comprises a different combination of non- naturally occurring glycosylation sites described herein or a different combination of sets of non- naturally occurring glycosylation sites described herein (for example, any of such SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein, e.g., described in § 5.2). In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein is derived from a circulating strain of SARS-CoV-2. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more circulating variants of SARS-CoV-2. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein is derived from a strain of SARS-CoV-2
Attorney Docket No.62801.28WO01 known to have previously circulated. In some embodiments, the amino acid sequence of the encoded SARS-CoV-2 spike protein comprises one or more amino acid variation found in one or more SARS-CoV-2 variants known to have previously circulated. In some embodiments, the vaccine composition further comprises at least one nucleic acid comprising a coding region encoding an immunogen (or immunogenic fragment and/or immunogenic variant thereof) from a non-SARS-CoV-2 virus, e.g., an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, an adenovirus. In some embodiments, vaccine composition comprises one or more nucleic acid molecules encoding an immunogen (or immunogenic fragment or immunogenic variant thereof) from an influenza virus (e.g., influenza A, influenza B), a respiratory syncytial virus (RSV), a rhinovirus, a parvovirus, a parainfluenza virus, and/or an adenovirus (or any combination thereof). In some embodiments, the encoded SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (e.g., ectodomain of the SARS-CoV-2 spike protein or polypeptide) is modified to improve expression of the protein in host cells (e.g., insect cells, mammalian cells, eggs). In some embodiments, the nucleic acid molecules are comprised within one or more vectors (e.g., vectors described herein (see, e.g., § 5.6)). In some embodiments, the nucleic acid molecules or the vectors of the vaccine composition are formulated in one or more carrier (e.g., a carrier described herein (see, e.g., § 5.7)). In some embodiments, the vaccine compositions are pharmaceutical compositions (e.g., described herein, e.g., see § 5.8). In some embodiments, the vaccine compositions comprise an adjuvant (e.g., described herein, e.g., see § 5.9). Nucleic acid molecules can be generated using common methods known in the art and described above in § 5.3. 5.6 Vectors In some embodiments, the nucleic acid molecules described herein (e.g., DNA molecules, RNA molecules (e.g., mRNA molecules)) (see, e.g., § 5.3) (or a fusion or conjugate thereof (see, e.g., § 5.4)) are contained in a vector (e.g., a non-viral vector, a viral vector). Thus,
Attorney Docket No.62801.28WO01 in one aspect, also provided herein are vectors (e.g., viral vectors, non-viral vectors (e.g., plasmids, minicircles)) comprising one or more nucleic acid molecule described herein (e.g., nucleic acid molecules encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)). Such vectors can be easily manipulated by methods well known to the ordinary person of skill in the art. In some embodiments, the vector is a non-viral vector (e.g., a plasmid, minicircle). In some embodiments, the vector is a plasmid. A person of ordinary skill in the art is aware of suitable plasmids for expression of the DNA of interest. For example, Suitable plasmid DNA may be generated to allow efficient production of the encoded peptides or proteins (e.g., SARS-CoV-2 proteins or polypeptides (e.g., immunogens) in cell lines, e.g., in insect cell lines, for example using vectors as described in W02009150222A2 and as defined in PCT claims 1 to 33, the disclosure relating to claim 1 to 33 of W02009150222A2 the entire contents of which is incorporated by reference herein for all purposes. In some embodiments, the vector is a viral vector. Viral vectors include both RNA and DNA based vectors. The vectors can be designed to meet a variety of specifications. For example, viral vectors can be engineered to be capable or incapable of replication in prokaryotic and/or eukaryotic cells. In some embodiments, the vector is replication deficient. In some embodiments, the vector is replication competent. Viral vectors can be engineered or selected that either will (or will not) integrate in whole or in part into the genome of host cells, resulting (or not (e.g., episomal expression)) in stable host cells comprising the desired nucleic acid in their genome. Exemplary viral vectors include, but are not limited to, adenovirus vectors, adeno- associated virus vectors, lentivirus vectors, retrovirus vectors, poxvirus vectors, parapoxivirus vectors, vaccinia virus vectors, fowlpox virus vectors, herpes virus vectors, adeno-associated virus vectors, alphavirus vectors, lentivirus vectors, rhabdovirus vectors, measles virus, Newcastle disease virus vectors, picornaviruses vectors, or lymphocytic choriomeningitis virus vectors. In some embodiments, the viral vector is an adenovirus vector, adeno-associated virus vector, or a lentivirus vector. In some embodiments, the vector is an adenovirus vector (e.g., a human adenoviral vector, e.g., HAdV or AdHu). In some embodiments, the adenovirus vector has the E1 region deleted, rendering it replication-deficient in human cells. Other regions of the adenovirus such as
Attorney Docket No.62801.28WO01 E3 and E4 may also be deleted. Exemplary adenovirus vectors include, but are not limited to, those described in e.g., W02005071093 or WQ2006048215, the entire contents of each of which is incorporated herein by reference for all purposes. In some embodiments, the adenovirus-based vector used is a simian adenovirus, thereby avoiding dampening of the immune response after vaccination by pre-existing antibodies to common human entities such as AdHu5. Exemplary, simian adenovirus vectors include AdCh63 (see, e.g., W02005071093, the entire contents of which is incorporated herein by reference for all purposes) or AdCh68. Viral vectors can be generated through the use of a packaging/producer cell line (e.g., a mammalian cell line) using standard methods known to the person of ordinary skill in the art. Generally, a nucleic acid construct (e.g., a plasmid) encoding the peptide or protein of interest (e.g., a peptide or protein described herein (e.g., SARS-CoV-2 peptide or protein described herein (e.g., a SARS-CoV-2 immunogen (or immunogenic fragment and/or immunogenic variant thereof)) (along with additional elements e.g., a promoter, inverted terminal repeats (ITRs) flanking the transgene, a plasmid encoding e.g., viral replication and structural proteins, along with one or more helper plasmids a host cell (e.g., a host cell line) are transfected into a host cell line (i.e., the packing/producer cell line). In some instances, depending on the viral vector, a helper plasmid may also be needed that include helper genes from another virus (e.g., in the instance of adeno-associated viral vectors). Eukaryotic expression plasmids are commercially available from a variety of suppliers, for example the plasmid series: pcDNA™, pCR3.1 ™, pCMV™, pFRT™, pVAX1 ™, pCI™, Nanoplasmid™, and Pcaggs. The person of ordinary skill in the art is aware of numerous transfection methods and any suitable method of transfection may be employed (e.g., using a biochemical substance as carrier (e.g., lipofectamine), by mechanical means, by electroporation). The cells are cultured under conditions suitable and for a sufficient time for plasmid expression. The viral particles may be purified from the cell culture medium using standard methods known to the person of ordinary skill in the art. For example, by centrifugation followed by e.g., chromatography and/or ultrafiltration. 5.7 Carriers In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), a nucleic acid
Attorney Docket No.62801.28WO01 molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), or a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV- 2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein is formulated within one or more carrier. Therefore, further provided herein are carriers comprising a SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, or a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein. Proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), and vectors, can be encapsulated within a carrier, chemically conjugated to a carrier, and/or associated with a carrier. In this context, the term “associated” refers to the essentially stable combination of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA, DNA), or vector with one or more molecules of a carrier (e.g., one or more lipids of a lipid-based carrier, e.g., a lipid nanoparticle (LNP), liposome, lipoplex, and/or nanoliposome) into larger complexes or assemblies without covalent binding. In this context, the term “encapsulation” refers to the incorporation of a protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), or vector into a carrier (e.g., a lipid-based carrier, e.g., an LNP, liposome, lipoplex, and/or nanoliposome) wherein the protein, peptide, nucleic acid molecule, e.g., the RNA (e.g., mRNA, DNA), or vector is entirely contained within the interior space of the carrier (e.g., the lipid-based carrier, e.g., the LNP, liposome, lipoplex, and/or nanoliposome). Exemplary carriers includes, but are not limited to, lipid-based carriers (e.g., LNPs, liposomes, lipoplexes, and nanoliposomes). In some embodiments, the carrier is a lipid-based carrier. In some embodiments, the carrier is an LNP. In some embodiments, the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. Lipid based carriers are further
Attorney Docket No.62801.28WO01 described below in § 5.7.1. 5.7.1 Lipid Based Carriers/Lipid Nanoformulations In some embodiments, a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), a nucleic acid molecule comprising a coding region encoding the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), or a vector comprising a nucleic acid molecule comprising a coding region encoding the SARS-CoV- 2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein is encapsulated or associated with one or more lipids (e.g., cationic lipids and/or neutral lipids), thereby forming lipid-based carriers such as lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes. In some embodiments, any of the foregoing molecules (e.g., proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.) is encapsulated in one or more lipids (e.g., cationic lipids and/or neutral lipids), thereby forming lipid-based carriers such as lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes. In some embodiments, the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) is associated with one or more lipids (e.g., cationic lipids and/or neutral lipids), thereby forming lipid-based carriers such as lipid nanoparticles (LNPs), liposomes, lipoplexes, or nanoliposomes. In some embodiments, the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) is encapsulated in LNPs (e.g., as described herein). The use of LNPs for mRNA delivery is further detailed in e.g., Hou X et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater. 2021;6(12):1078-1094. doi: 10.1038/s41578- 021-00358-0. Epub 2021 Aug 10. PMID: 34394960; PMCID: PMC8353930, the entire contents of each of which are incorporated by reference herein for all purposes. The molecules (e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.) described herein may be completely or partially located in the interior space of the LNPs, liposomes, lipoplexes, and/or nanoliposomes, within the lipid layer/membrane, or associated with the exterior surface of the lipid layer/membrane. One purpose
Attorney Docket No.62801.28WO01 of incorporating the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) into LNPs, liposomes, lipoplexes, and/or nanoliposomes is to protect the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) from an environment which may contain enzymes or chemicals or conditions that degrade the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.) from molecules or conditions that cause the rapid excretion of the molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.). Moreover, incorporating the molecules (e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.) into LNPs, liposomes, lipoplexes, and/or nanoliposomes may promote the uptake of the molecules (e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.), and hence, may enhance the therapeutic effect of the proteins or nucleic acid molecules (e.g., RNA, e.g., mRNA). Accordingly, incorporating a molecule (e.g., the protein, peptide, nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), vector, etc.), into LNPs, liposomes, lipoplexes, and/or nanoliposomes may be particularly suitable for a pharmaceutical composition described herein, e.g., for intramuscular and/or intradermal administration. In some embodiments, molecules (e.g., the proteins, peptides, nucleic acid molecules (e.g., RNA (e.g., mRNA), DNA), vectors, etc.) described herein are formulated into a lipid-based carrier (or lipid nanoformulation). In some embodiments, the lipid-based carrier (or lipid nanoformulation) is a liposome or a lipid nanoparticle (LNP). In one embodiment, the lipid-based carrier is an LNP. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid (e.g., an ionizable lipid), a non-cationic lipid (e.g., phospholipid), a structural lipid (e.g., cholesterol), and a PEG-modified lipid. In some embodiments, the lipid-based carrier (or lipid nanoformulation) contains one or more molecules described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein), or a pharmaceutically acceptable salt thereof. As described herein, suitable compounds to be used in the lipid-based carrier (or lipid nanoformulation) include all the isomers and isotopes of the compounds described above, as well as all the pharmaceutically acceptable salts, solvates, or hydrates thereof, and all crystal forms, crystal form mixtures, and anhydrides or hydrates.
Attorney Docket No.62801.28WO01 In addition to one or more (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.) described herein, the lipid-based carrier (or lipid nanoformulation) may further include a second lipid. In some embodiments, the second lipid is a cationic lipid, a non-cationic (e.g., neutral, anionic, or zwitterionic) lipid, or an ionizable lipid. One or more naturally occurring and/or synthetic lipid compounds may be used in the preparation of the lipid-based carrier (or lipid nanoformulation). The lipid-based carrier (or lipid nanoformulation) may contain positively charged (cationic) lipids, neutral lipids, negatively charged (anionic) lipids, or a combination thereof. 5.7.1.1 Cationic Lipids (Positively Charged) and Ionizable Lipids In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one or more cationic lipids, e.g., a cationic lipid that can exist in a positively charged or neutral form depending on pH, or an amine-containing lipid that can be readily protonated. In some embodiments, the cationic lipid is a lipid capable of being positively charged, e.g., under physiological conditions. Exemplary cationic lipids include one or more amine group(s) which bear the positive charge. Examples of positively charged (cationic) lipids include, but are not limited to, N,N'- dimethyl-N,N'-dioctacyl ammonium bromide (DDAB) and chloride DDAC), N-(l-(2,3- dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), 3β-[N-(N',N'- dimethylaminoethyl)carbamoyl) cholesterol (DC-chol), 1,2-dioleoyloxy-3-[trimethylammonio]- propane (DOTAP), 1,2-dioctadecyloxy-3-[trimethylammonio]-propane (DSTAP), and 1,2- dioleoyloxypropyl-3-dimethyl-hydroxy ethyl ammonium chloride (DORI), N,N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-Dioleoyl-3-Dimethylammonium-propane (DODAP), 1,2-Dioleoylcarbamyl-3- Dimethylammonium-propane (DOCDAP), 1,2-Dilineoyl-3-Dimethylammonium-propane (DLINDAP), 3-Dimethylamino-2-(Cholest-5-en-3-beta-oxybutan-4-oxy)-1-(cis,cis-9,12- octadecadienoxy)propane (CLinDMA), 2-[5′-(cholest-5-en-3-beta-oxy)-3′-oxapentoxy)-3- dimethyl-1-(cis, cis-9′,12′-octadecadienoxy)propane (CpLin DMA), N,N-Dimethyl-3,4- dioleyloxybenzylamine (DMOBA), and the cationic lipids described in e.g. Martin et al., Current Pharmaceutical Design, pages 1-394, the entire contents of which are incorporated by reference herein for all purposes. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises more than one cationic lipid.
Attorney Docket No.62801.28WO01 In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises a cationic lipid having an effective pKa over 6.0. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a second cationic lipid having a different effective pKa (e.g., greater than the first effective pKa) than the first cationic lipid. In some embodiments, cationic lipids that can be used in the lipid-based carrier (or lipid nanoformulation) include, for example those described in Table 4 of WO 2019/217941, the entire contents of which are incorporated by reference herein for all purposes. In some embodiments, the cationic lipid is an ionizable lipid (e.g., a lipid that is protonated at low pH, but that remains neutral at physiological pH). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise one or more additional ionizable lipids, different than the ionizable lipids described herein. Exemplary ionizable lipids include, but are not limited to, , ,
Attorney Docket No.62801.28WO01 (Lipid A9), and MC3- , (see for all purposes).
In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more compounds described by WO 2021/113777 (e.g., a lipid of Formula (3) such as a lipid of Table 3 of WO 2021/113777), the entire contents of which are incorporated by reference herein for all purposes. In one embodiment, the ionizable lipid is a lipid disclosed in Hou, X., et al. Nat Rev Mater 6, 1078–1094 (2021). https://doi.org/10.1038/s41578-021-00358-0 (e.g., L319, C12-200, and DLin-MC3-DMA), (the entire contents of which are incorporated by reference herein for all purposes). Examples of other ionizable lipids that can be used in lipid-based carrier (or lipid nanoformulation) include, without limitation, one or more of the following formulas: X of US 2016/0311759; I of US 20150376115 or in US 2016/0376224; Compound 5 or Compound 6 in US 2016/0376224; I, IA, or II of US 9,867,888; I, II or III of US 2016/0151284; I, IA, II, or IIA of US 2017/0210967; I-c of US 2015/0140070; A of US 2013/0178541; I of US 2013/0303587 or US 2013/0123338; I of US 2015/0141678; II, III, IV, or V of US 2015/0239926; I of US 2017/0119904; I or II of WO 2017/117528; A of US 2012/0149894; A of US 2015/0057373; A of WO 2013/116126; A of US 2013/0090372; A of US 2013/0274523; A of US 2013/0274504; A of US 2013/0053572; A of WO 2013/016058; A of WO 2012/162210; I of US 2008/042973; I, II, III, or IV of US 2012/01287670; I or II of US 2014/0200257; I, II, or III of US 2015/0203446; I or III of US 2015/0005363; I, IA, IB, IC, ID, II, IIA, IIB, IIC, IID, or III-XXIV of US 2014/0308304; of US 2013/0338210; I, II, III, or IV of WO 2009/132131; A of US 2012/01011478; I or XXXV of US 2012/0027796; XIV or XVII of US 2012/0058144; of US 2013/0323269; I of US 2011/0117125; I, II, or III of US 2011/0256175; I, II, III, IV, V, VI, VII, VIII, IX, X, XI, XII of US 2012/0202871; I, II, III, IV, V, VI, VII, VIII, X, XII, XIII, XIV, XV, or XVI of US 2011/0076335; I or II of US 2006/008378; I of WO2015/074085 (e.g., ATX-002); I of US 2013/0123338; I or X-A-Y-Z of US 2015/0064242; XVI, XVII, or XVIII of US
Attorney Docket No.62801.28WO01 2013/0022649; I, II, or III of US 2013/0116307; I, II, or III of US 2013/0116307; I or II of US 2010/0062967; I-X of US 2013/0189351; I of US 2014/0039032; V of US 2018/0028664; I of US 2016/0317458; I of US 2013/0195920; 5, 6, or 10 of US 10,221,127; III-3 of WO 2018/081480; I-5 or I-8 of WO 2020/081938; I of WO 2015/199952 (e.g., compound 6 or 22) and Table 1 therein; 18 or 25 of US 9,867,888; A of US 2019/0136231; II of WO 2020/219876; 1 of US 2012/0027803; OF-02 of US 2019/0240349; 23 of US 10,086,013; cKK-E12/A6 of Miao et al (2020); C12-200 of WO 2010/053572; 7C1 of Dahlman et al (2017); 304-O13 or 503-O13 of Whitehead et al; TS- P4C2 of U S9,708,628; I of WO 2020/106946; I of WO 2020/106946; (1), (2), (3), or (4) of WO 2021/113777; and any one of Tables 1-16 of WO 2021/113777, the entire contents of each of which are incorporated by reference herein for all purposes. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further includes biodegradable ionizable lipids, for instance, (9Z,l2Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,l2-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,l2Z)-octadeca-9,l2-dienoate). See, e.g., lipids of WO 2019/067992, WO 2017/173054, WO 2015/095340, and WO 2014/136086, the entire contents of each of which are incorporated by reference herein for all purposes. 5.7.1.2 Non-Cationic Lipids (e.g., Phospholipids) In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is a phospholipid substitute or replacement. In some embodiments, the non-cationic lipid is a negatively charged (anionic) lipid. Exemplary non-cationic lipids include, but are not limited to, distearoyl-sn-glycero- phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE- mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (such as 16-O-monomethyl PE), dimethyl- phosphatidylethanolamine (such as 16-O-dimethyl PE), 18-l-
Attorney Docket No.62801.28WO01 trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoylphosphatidylglycerol (DSPG), dierucoylphosphatidylcholine (DEPC), palmitoyloleyolphosphatidylglycerol (POPG), dielaidoyl- phosphatidylethanolamine (DEPE), 1,2-dilauroyl- sn-glycero-3-phosphocholine (DLPC), Sodium 1,2- ditetradecanoyl-sn-glycero-3-phosphate (DMPA), phosphatidylcholine (lecithin), phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), phosphatidylethanolamine (cephalin), cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof. It is understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e.g., lauroyl, myristoyl, paimitoyl, stearoyl, or oleoyl. Additional exemplary lipids, in certain embodiments, include, without limitation, those described in Kim et al. (2020) dx.doi.org/10.1021/acs.nanolett.0c01386, the entire contents of which are incorporated by reference herein for all purposes. Such lipids include, in some embodiments, plant lipids found to improve liver transfection with mRNA (e.g., DGTS). In some embodiments, the lipid-based carrier (or lipid nanoformulation) may comprise a combination of distearoylphosphatidylcholine/cholesterol, dipalmitoylphosphatidylcholine/cholesterol, dimyrystoylphosphatidylcholine/cholesterol, 1,2- Dioleoyl-sn-glycero-3-phosphocholine (DOPC)/cholesterol, or egg sphingomyelin/cholesterol. Other examples of suitable non-cationic lipids include, without limitation, nonphosphorous lipids such as, e.g., stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyl dimethyl ammonium bromide, ceramide, sphingomyelin, and the like. Other non- cationic lipids are described in WO 2017/099823 or US 2018/0028664, the entire contents of each of which are incorporated by reference herein for all purposes. In one embodiment, the lipid-based carrier (or lipid nanoformulation) further comprises one or more non-cationic lipid that is oleic acid or a compound of Formula I, II, or IV of US
Attorney Docket No.62801.28WO01 2018/0028664, the entire contents of which are incorporated by reference herein for all purposes. The non-cationic lipid content can be, for example, 0-30% (mol) of the total lipid components present. In some embodiments, the non-cationic lipid content is 5-20% (mol) or 10- 15% (mol) of the total lipid components present. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a neutral lipid, and the molar ratio of an ionizable lipid to a neutral lipid ranges from about 2:1 to about 8:1 (e.g., about 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, or 8:1). In some embodiments, the lipid-based carrier (or lipid nanoformulation) does not include any phospholipids. In some embodiments, the lipid-based carrier (or lipid nanoformulation) can further include one or more phospholipids, and optionally one or more additional molecules of similar molecular shape and dimensions having both a hydrophobic moiety and a hydrophilic moiety (e.g., cholesterol). 5.7.1.3 Structural Lipids The lipid-based carrier (or lipid nanoformulation) described herein may further comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols (e.g., cholesterol) and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipid in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol or cholesterol derivative, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. In certain embodiments, the structural lipid is a steroid. In certain embodiments, the structural lipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol. In certain embodiments, the structural lipid is alpha-tocopherol. In some embodiments, structural lipids may be incorporated into the lipid-based carrier at molar ratios ranging from about 0.1 to 1.0 (cholesterol phospholipid). In some embodiments, sterols, when present, can include one or more of cholesterol or cholesterol derivatives, such as those described in WO 2009/127060 or US 2010/0130588, the entire contents of each of which are incorporated by reference herein for all purposes. Additional exemplary sterols include phytosterols, including those described in Eygeris et al. (2020), Nano
Attorney Docket No.62801.28WO01 Lett. 2020;20(6):4543-4549, the entire contents of which are incorporated by reference herein for all purposes. In some embodiments, the structural lipid is a cholesterol derivative. Non-limiting examples of cholesterol derivatives include polar analogues such as 5a-cholestanol, 53- coprostanol, cholesteryl-(2’-hydroxy)-ethyl ether, cholesteryl-(4'- hydroxy)-butyl ether, and 6- ketocholestanol; non-polar analogues such as 5a-cholestane, cholestenone, 5a-cholestanone, 5p- cholestanone, and cholesteryl decanoate; and mixtures thereof. In some embodiments, the cholesterol derivative is a polar analogue, e.g., cholesteryl-(4'-hydroxy)-buty1 ether. Exemplary cholesterol derivatives are described in WO 2009/127060 and US 2010/0130588, the entire contents of each of which are incorporated by reference herein for all purposes. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises sterol in an amount of 0-50 mol% (e.g., 0-10 mol %, 10-20 mol %, 20-50 mol%, 20-30 mol %, 30-40 mol %, or 40-50 mol %) of the total lipid components. 5.7.1.4 Polymers and Polyethylene Glycol (PEG) - Lipids In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polymers or co-polymers, e.g., poly(lactic-co-glycolic acid) (PFAG) nanoparticles. In some embodiments, the lipid-based carrier (or lipid nanoformulation) may include one or more polyethylene glycol (PEG) lipid. Examples of useful PEG-lipids include, but are not limited to, 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 350] (mPEG 350 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-550] (mPEG 550 PE); 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-750] (mPEG 750 PE); 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-1000] (mPEG 1000 PE); 1,2-Diacyl-sn- Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (mPEG 2000 PE); 1,2- Diacyl-sn-Glycero-3-Phosphoethanolamine-N- [Methoxy(Polyethylene glycol)-3000] (mPEG 3000 PE); 1,2-Diacyl-sn-Glycero-3- Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)- 5000] (mPEG 5000 PE); N-Acyl- Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol) 750] (mPEG 750 Ceramide); N-Acyl- Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol) 2000] (mPEG 2000 Ceramide); and N- Acyl-Sphingosine-1-[Succinyl(Methoxy Polyethylene Glycol) 5000] (mPEG 5000 Ceramide). In some embodiments, the PEG lipid is a polyethyleneglycol- diacylglycerol (i.e., polyethyleneglycol diacylglycerol (PEG-DAG), PEG-cholesterol, or PEG-
Attorney Docket No.62801.28WO01 DMB) conjugate. In some embodiments, the lipid-based carrier (or nanoformulation) includes one or more conjugated lipids (such as PEG-conjugated lipids or lipids conjugated to polymers described in Table 5 of WO 2019/217941, the entire contents of which are incorporated by reference herein for all purposes). In some embodiments, the one or more conjugated lipids is formulated with one or more ionic lipids (e.g., non-cationic lipid such as a neutral or anionic, or zwitterionic lipid); and one or more sterols (e.g., cholesterol). The PEG conjugate can comprise a PEG-dilaurylglycerol (C12), a PEG- dimyristylglycerol (C14), a PEG-dipalmitoylglycerol (C16), a PEG-disterylglycerol (C18), PEG- dilaurylglycamide (C12), PEG-dimyristylglycamide (C14), PEG-dipalmitoylglycamide (C16), and PEG-disterylglycamide (C18). In some embodiments, conjugated lipids, when present, can include one or more of PEG-diacylglycerol (DAG) (such as l-(monomethoxy-polyethyleneglycol)-2,3- dimyristoylglycerol (PEG-DMG)), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG- ceramide (Cer), a pegylated phosphatidylethanoloamine (PEG-PE), PEG succinate diacylglycerol (PEGS-DAG) (such as 4-0-(2',3'-di(tetradecanoyloxy)propyl-l-0-(w- methoxy(polyethoxy)ethyl) butanedioate (PEG-S-DMG)), PEG dialkoxypropylcarbam, N- (carbonyl-methoxypolyethylene glycol 2000)- 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine sodium salt, and those described in Table 2 of WO 2019/051289 (the entire contents of which are incorporated by reference herein for all purposes), and combinations of the foregoing. Additional exemplary PEG-lipid conjugates are described, for example, in US 5,885,613, US 6,287,591, US 2003/0077829, US 2003/0077829, US 2005/0175682, US 2008/0020058, US 2011/0117125, US 2010/0130588, US 2016/0376224, US 2017/0119904, US 2018/0028664, and WO 2017/099823, the entire contents of each of which are incorporated by reference herein for all purposes. In some embodiments, the PEG-lipid is a compound of Formula III, III-a-I, III-a-2, III- b-1, III-b-2, or V of US 2018/0028664, which is incorporated herein by reference in its entirety. In some embodiments, the PEG-lipid is of Formula II of US 2015/0376115 or US 2016/0376224, the entire contents of each of which are incorporated by reference herein for all purposes. In some embodiments, the PEG-DAA conjugate can be, for example, PEG-dilauryloxypropyl, PEG- dimyristyloxypropyl, PEG-dipalmityloxypropyl, or PEG-distearyloxypropyl. In some
Attorney Docket No.62801.28WO01 embodiments, the PEG-lipid includes one of the following: be
used in place of PEG-lipid. For example, polyoxazoline (POZ)-lipid conjugates, polyamide-lipid conjugates (such as ATTA-lipid conjugates), and cationic-polymer lipid (GPL) conjugates can be used in place of or in addition to the PEG-lipid. Exemplary conjugated lipids, e.g., PEG-lipids, (POZ)-lipid conjugates, ATTA-lipid conjugates and cationic polymer-lipids, include those described in Table 2 of WO 2019/051289A9, the entire contents of which are incorporated by reference herein for all purposes. In some embodiments, the conjugated lipid (e.g., the PEGylated lipid) can be present in an amount of 0-20 mol% of the total lipid components present in the lipid-based carrier (or lipid nanoformulation). In some embodiments, the conjugated lipid (e.g., the PEGylated lipid) content is 0.5-10 mol% or 2-5 mol% of the total lipid components. When needed, the lipid-based carrier (or lipid nanoformulation) described herein may be coated with a polymer layer to enhance stability in vivo (e.g., sterically stabilized LNPs). Examples of suitable polymers include, but are not limited to, poly(ethylene glycol), which may form a hydrophilic surface layer that improves the circulation half-life of liposomes and enhances the amount of lipid nanoformulations (e.g., liposomes or LNPs) that reach therapeutic targets. See, e.g., Working et al. J Pharmacol Exp Ther, 289: 1128-1133 (1999); Gabizon et al., J Controlled Release 53: 275-279 (1998); Adlakha Hutcheon et al., Nat Biotechnol 17: 775-779 (1999); and Koning et al., Biochim Biophys Acta 1420: 153-167 (1999), the entire
Attorney Docket No.62801.28WO01 contents of each of which are incorporated by reference herein for all purposes. 5.7.1.5 Percentages of Lipid Nanoformulation Components In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises one of more of the molecules described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc.), optionally a non-cationic lipid (e.g., a phospholipid), a sterol, a neutral lipid, and optionally conjugated lipid (e.g., a PEGylated lipid) that inhibits aggregation of particles. In some embodiments, the lipid-based carrier (or lipid nanoformulation) further comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein). The amounts of these components can be varied independently and to achieve desired properties. For example, in some embodiments, the ionizable lipid including the lipid compounds described herein is present in an amount from about 20 mol% to about 100 mol% (e.g., 20-90 mol%, 20-80 mol%, 20-70 mol%, 25-100 mol%, 30-70 mol%, 30-60 mol%, 30-40 mol%, 40-50 mol%, or 50-90 mol%) of the total lipid components; a non-cationic lipid (e.g., phospholipid) is present in an amount from about 0 mol% to about 50 mol% (e.g., 0-40 mol%, 0-30 mol%, 5-50 mol%, 5-40 mol%, 5-30 mol%, or 5-10 mol%) of the total lipid components, a conjugated lipid (e.g., a PEGylated lipid) in an amount from about 0.5 mol% to about 20 mol% (e.g., 1-10 mol% or 5-10%) of the total lipid components, and a sterol in an amount from about 0 mol % to about 60 mol% (e.g., 0-50 mol%, 10-60 mol%, 10-50 mol%, 15-60 mol%, 15-50 mol%, 20-50 mol%, 20-40 mol%) of the total lipid components, provided that the total mol% of the lipid component does not exceed 100%. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid. In some embodiments, the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein)) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein, about 0-50 mol% phospholipid, about 0-50 mol% sterol, and about 0-10 mol% PEGylated lipid. In some embodiments, the encapsulation efficiency of the payload may be at least 70%. In one embodiment, the lipid-based carrier (or lipid nanoformulation) comprises about
Attorney Docket No.62801.28WO01 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid. In some embodiments, the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein)) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 25-100 mol% of the ionizable lipid including the lipid compounds described herein; about 0-40 mol% phospholipid (e.g., DSPC), about 0-50 mol% sterol (e.g., cholesterol), and about 0-10 mol% PEGylated lipid. In some embodiments, the encapsulation efficiency of the payload may be at least 70%. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid. In some embodiments, the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein)) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises about 30-60 mol% (e.g., about 35-55 mol%, or about 40-50 mol%) of the ionizable lipid including the lipid compounds described herein, about 0-30 mol% (e.g., 5-25 mol%, or 10-20 mol%) phospholipid, about 15-50 mol% (e.g., 18.5-48.5 mol%, or 30-40 mol%) sterol, and about 0-10 mol% (e.g., 1-5 mol%, or 1.5-2.5 mol%) PEGylated lipid. In some embodiments, the encapsulation efficiency of the payload may be at least 70%. In some embodiments, molar ratios of ionizable lipid/sterol/phospholipid (or another structural lipid)/PEG-lipid/additional components is varied in the following ranges: ionizable lipid (25-100%); phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%). In some embodiments, the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein)) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises molar ratios of ionizable lipid/sterol/phospholipid (or another structural lipid)/PEG-lipid/additional components in the following ranges: ionizable lipid (25-100%);
Attorney Docket No.62801.28WO01 phospholipid (DSPC) (0-40%); sterol (0-50%); and PEG lipid (0-5%). In some embodiments, the encapsulation efficiency of the payload may be at least 70%. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid). In some embodiments, the lipid-based carrier comprises a payload (e.g., a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein)) that is formulated in a lipid nanoparticle, wherein the lipid nanoparticle comprises, by mol% or wt% of the total lipid components, 50-75% ionizable lipid (including the lipid compound as described herein), 20-40% sterol (e.g., cholesterol or derivative), 0 to 10% non-cationic-lipid, and 1-10% conjugated lipid (e.g., the PEGylated lipid). In some embodiments, the encapsulation efficiency of the payload may be at least 70%. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises (i) a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein); (ii) a cationic lipid comprising from 50 mol% to 65 mol% of the total lipid present in the lipid-based carrier; (iii) a non-cationic lipid comprising a mixture of a phospholipid and a cholesterol derivative thereof, wherein the phospholipid comprises from 3 mol% to 15 mol% of the total lipid present in the lipid-based carrier and the cholesterol or derivative thereof comprises from 30 mol% to 40 mol% of the total lipid present in the lipid-based carrier; and (iv) a conjugated lipid comprising 0.5 mol% to 2 mol% of the total lipid present in the particle. In some embodiments, the lipid-based carrier (or lipid nanoformulation) comprises (i) a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein); (ii) a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the lipid-based carrier; (iii) a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the lipid-based carrier; and (d) a conjugated lipid comprising from 0.5 mol % to 2 mol % of the total lipid present in the lipid-based carrier. In some embodiments, the phospholipid component in the mixture may be present from 2 mol% to 20 mol%, from 2 mol% to 15 mol%, from 2 mol% to 12 mol%, from 4 mol% to 15
Attorney Docket No.62801.28WO01 mol%, from 4 mol% to 10 mol%, from 5 mol% to 10 mol%, (or any fraction of these ranges) of the total lipid components. In some embodiments, the lipid-based carrier (or lipid nanoformulation) is phospholipid-free. In some embodiments, the sterol component (e.g. cholesterol or derivative) in the mixture may comprise from 25 mol% to 45 mol%, from 25 mol% to 40 mol%, from 25 mol% to 35 mol%, from 25 mol% to 30 mol%, from 30 mol% to 45 mol%, from 30 mol% to 40 mol%, from 30 mol% to 35 mol%, from 35 mol% to 40 mol%, from 27 mol% to 37 mol%, or from 27 mol% to 35 mol% (or any fraction of these ranges) of the total lipid components. In some embodiments, the non-ionizable lipid components in the lipid-based carrier (or lipid nanoformulation) may be present from 5 mol% to 90 mol%, from 10 mol% to 85 mol%, or from 20 mol% to 80 mol% (or any fraction of these ranges) of the total lipid components. The ratio of total lipid components to the payload (e.g., an encapsulated therapeutic agent such as a molecule described herein (e.g., a protein, a peptide, a nucleic acid molecule (e.g., RNA (e.g., mRNA), DNA), a vector, etc. described herein) can be varied as desired. For example, the total lipid components to the payload (mass or weight) ratio can be from about 10:1 to about 30:1. In some embodiments, the total lipid components to the payload ratio (mass/mass ratio; w/w ratio) can be in the range of from about 1:1 to about 25:1, from about 10:1 to about 14:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. The amounts of total lipid components and the payload can be adjusted to provide a desired N/P ratio, for example, N/P ratio of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or higher. Generally, the lipid-based carrier (or lipid nanoformulation’s) overall lipid content can range from about 5 mg/ml to about 30 mg/mL. Nitrogen:phosphate ratios (N:P ratio) is evaluated at values between 0.1 and 100. The efficiency of encapsulation of a payload such as a protein and/or nucleic acid, describes the amount of protein and/or nucleic acid that is encapsulated or otherwise associated with a lipid nanoformulation (e.g., liposome or LNP) after preparation, relative to the initial amount provided. The encapsulation efficiency is desirably high (e.g., at least 70%. 80%. 90%. 95%, close to 100%). The encapsulation efficiency may be measured, for example, by comparing the amount of protein or nucleic acid in a solution containing the liposome or LNP before and after breaking up the liposome or LNP with one or more organic solvents or detergents. An anion exchange resin may be used to measure the amount of free protein or nucleic acid (e.g., RNA) in
Attorney Docket No.62801.28WO01 a solution. Fluorescence may be used to measure the amount of free protein and/or nucleic acid (e.g., RNA) in a solution. For the lipid-based carrier (or lipid nanoformulation) described herein, the encapsulation efficiency of a protein and/or nucleic acid may be at least 50%, for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the encapsulation efficiency may be at least 70%. In some embodiments, the encapsulation efficiency may be at least 80%. In some embodiments, the encapsulation efficiency may be at least 90%. In some embodiments, the encapsulation efficiency may be at least 95%. 5.8 Pharmaceutical Compositions In one aspect, provided herein are pharmaceutical compositions comprising a SARS- CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (or a fusion or conjugate thereof), a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), a carrier described herein (e.g., a carrier comprising a SARS-CoV- 2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein or a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof))), and/or a composition described herein (e.g., a vaccine composition), and a pharmaceutically acceptable excipient (see, e.g., Remington’s Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA, the entire contents of which is incorporated by reference herein for all purposes). In one aspect, also provided herein are methods of making pharmaceutical compositions described herein comprising providing a SARS-CoV-2 spike protein or polypeptide
Attorney Docket No.62801.28WO01 (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein), a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)))), a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein (or polypeptide e.g., immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein or a nucleic acid molecule described herein (e.g., a nucleic acid molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof))), and/or a composition described herein (e.g., a vaccine composition), and formulating it into a pharmaceutically acceptable composition by the addition of one or more pharmaceutically acceptable excipient. Acceptable excipients (e.g., carriers and stabilizers) are preferably nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants including ascorbic acid or methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol;or m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, or other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG). A pharmaceutical composition may be formulated for any route of administration to a subject. The skilled person knows the various possibilities to administer a pharmaceutical
Attorney Docket No.62801.28WO01 composition described herein a in order to induce an immune response to the immunogens(s) and/or antigen(s) in the pharmaceutical composition. Non-limiting embodiments include parenteral administration, such as intramuscular, intradermal, subcutaneous, transcutaneous, or mucosal administration, e.g., inhalation, intranasal, oral, and the like. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular, intradermal, or subcutaneous injection. In one embodiment, the pharmaceutical composition is formulated for administration by intramuscular injection. In one embodiment, the pharmaceutical composition is formulated for administration by intradermal injection. In one embodiment, the pharmaceutical composition is formulated for administration by subcutaneous injection. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions. The injectables can contain one or more excipients. Exemplary excipients include, for example, water, saline, dextrose, glycerol or ethanol. In addition, if desired, the pharmaceutical compositions to be administered can also contain minor amounts of non-toxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, stabilizers, solubility enhancers, or other such agents, such as for example, sodium acetate, sorbitan monolaurate, triethanolamine oleate or cyclodextrins. In some embodiments, the pharmaceutical composition is formulated in a single dose. In some embodiments, the pharmaceutical compositions if formulated as a multi-dose. Pharmaceutically acceptable excipients used in the parenteral preparations described herein include for example, aqueous vehicles, nonaqueous vehicles, antimicrobial agents, isotonic agents, buffers, antioxidants, local anesthetics, suspending and dispersing agents, emulsifying agents, sequestering or chelating agents or other pharmaceutically acceptable substances. Examples of aqueous vehicles, which can be incorporated in one or more of the formulations described herein, include sodium chloride injection, Ringer’s injection, isotonic dextrose injection, sterile water injection, dextrose or lactated Ringer’s injection. Nonaqueous parenteral vehicles, which can be incorporated in one or more of the formulations described herein, include fixed oils of vegetable origin, cottonseed oil, corn oil, sesame oil or peanut oil. Antimicrobial agents in bacteriostatic or fungistatic concentrations can be added to the parenteral preparations described herein and packaged in multiple-dose containers, which include phenols or cresols, mercurials, benzyl alcohol, chlorobutanol, methyl and propyl p-hydroxybenzoic acid esters, thimerosal, benzalkonium chloride or benzethonium chloride. Isotonic agents, which can be incorporated in one or more of the formulations described herein, include sodium chloride or dextrose. Buffers,
Attorney Docket No.62801.28WO01 which can be incorporated in one or more of the formulations described herein, include phosphate or citrate. Antioxidants, which can be incorporated in one or more of the formulations described herein, include sodium bisulfate. Local anesthetics, which can be incorporated in one or more of the formulations described herein, include procaine hydrochloride. Suspending and dispersing agents, which can be incorporated in one or more of the formulations described herein, include sodium carboxymethylcelluose, hydroxypropyl methylcellulose or polyvinylpyrrolidone. Emulsifying agents, which can be incorporated in one or more of the formulations described herein, include Polysorbate 80 (TWEEN® 80). A sequestering or chelating agent of metal ions, which can be incorporated in one or more of the formulations described herein, is EDTA. Pharmaceutical carriers, which can be incorporated in one or more of the formulations described herein, also include ethyl alcohol, polyethylene glycol or propylene glycol for water miscible vehicles; orsodium hydroxide, hydrochloric acid, citric acid or lactic acid for pH adjustment. The precise dose to be employed in a pharmaceutical composition will also depend on the route of administration, and the seriousness of the condition caused by it, and should be decided according to the judgment of the practitioner and each subject’s circumstances. For example, effective doses may also vary depending upon means of administration, target site, physiological state of the subject (including age, body weight, and health), other medications administered, or whether therapy is prophylactic or therapeutic. Therapeutic dosages are preferably titrated to optimize safety and efficacy. 5.9 Adjuvants Any of the foregoing, e.g., SARS-CoV-2 spike proteins or polypeptides (e.g., immunogens (or immunogenic fragments and/or immunogenic variants thereof)) described herein (or a fusion or conjugate thereof), nucleic acid molecules described herein (e.g., nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (or a fusion or conjugate thereof), vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), carriers described herein (e.g., a carrier comprising a SARS-CoV-
Attorney Docket No.62801.28WO01 2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) described herein or a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or an immunogenic fragment and/or immunogenic variant thereof))), compositions described herein (e.g., a vaccine composition), and/or pharmaceutical compositions described herein may be co-formulated with and/or administered in combination with an adjuvant. Adjuvants are known in the art to further increase the immune response (e.g., to an immunogen). General categories of adjuvants include, but are not limited to, inorganic adjuvants, small molecule adjuvants, oil in water emulsions, lipids, polymers, peptides, peptidoglycans, carbohydrates, polysaccharides, RNA-based adjuvants, DNA-based adjuvants, viral particles, bacterial adjuvants, nanoparticles (e.g., inorganic nanoparticles), and multi-component adjuvants. Examples of adjuvants include, but are not limited to, aluminum salts such as aluminum hydroxide and/or aluminum phosphate; oil-emulsion compositions (or oil-in-water compositions), including squalene-water emulsions, such as MF59 (see, e.g., WO90/14837, the entire contents of which is incorporated herein by reference for all purposes), MF59, AS03, and Montanide; saponin formulations, such as for example QS21 and Immunostimulating Complexes (ISCOMS) (see, e.g., US5,057,540; WO90/03184, WO96/11711, WO2004/004762, WO2005/002620, the entire contents of each of which is incorporated herein by reference for all purposes); protamine or a protamine salt (e.g., protamine sulfate); calcium salt; bacterial or microbial derivatives, examples of which include monophosphoryl lipid A (MPL), 3-O-deacylated MPL (3dMPL), CpG-motif containing oligonucleotides, ADP-ribosylating bacterial toxins or mutants thereof, such as E. coli heat labile enterotoxin LT, cholera toxin CT, and the like; eukaryotic proteins (e.g., antibodies or fragments thereof (e.g., directed against the antigen itself or CD1a, CD3, CD7, CD80) and ligands to receptors (e.g., CD40L, GMCSF, GCSF, etc.). Exemplary RNA-based adjuvants include, but are not limited to, Poly IC, Poly IC:LC, hairpin RNAs, e.g., with a 5’PPP containing sequence, viral sequences, polyU containing sequences, dsRNA, natural or synthetic immunostimulatory RNA sequences, nucleic acids analogs, optionally cyclic GMP-AMP or a cyclic dinucleotide such as cyclic di-GMP, and immunostimulatory base analogs, e.g., C8-substitued or an N7,C8-disubstituted guanine ribonucleotide. Exemplary DNA-based adjuvants, include, but are not limited to, CpGs, dsDNA,
Attorney Docket No.62801.28WO01 or natural or synthetic immunostimulatory DNA sequences. Exemplary bacteria-based adjuvants include, but are not limited, to bacterial adjuvant is flagellin, LPS, or a bacterial toxin, e.g., enterotoxins, heat-labile toxins, and Cholera toxins. Exemplary carbohydrate or polysaccharide adjuvants include, but are not limited to, dextran (branched microbial polysaccharide), dextran- sulfate, Lentinan, zymosan, Betaglucan, Deltin, Mannan, and Chitin. Exemplary small molecule adjuvants, include, but are not limited to, imiquimod, resiquimod, and gardiquimod. Exemplary lipid or polymer adjuvants, include, but are not limited to, polymeric nanoparticles (e.g., PLGA, PLG, PLA, PGA, or PHB), liposomes (e.g., Virosomes and CAF01), LNPs or a component thereof, lipopolysaccharide (LPS) (e.g., monophosphoryl lipid A (MPLA) or glucopyranosyl Lipid A (GLA)), lipopeptides (e.g., Pam2 (Pam2CSK4) or Pam3 (Pam3CSK4)), and glycolipid (e.g., trehalose dimycolate). Exemplary peptides or peptidoglycan include, but are not limited to, N- acetyl-muramyl-L-alanyl-D-isoglutamine (MDP), flagellin-fusion protein, mannose-binding lectin (MBL), cytokines, and chemokine. Exemplary inorganic nanoparticle adjuvants, include, but are not limited to, gold nanorods, silica-based nanoparticles (e.g., mesoporous silica nanoparticles (MSN)). Exemplary multicomponent adjuvants include, but are not limited to, AS01, AS03, AS04, Complete Freunds Adjuvant, and CAF01. 5.10 Methods of Use Provided herein are various methods of utilizing the SARS-CoV-2 spike proteins and polypeptides (e.g., immunogens (and immunogenic fragments and/or immunogenic variants thereof)) described herein (or fusions or conjugates thereof), the nucleic acid molecules described herein (e.g., nucleic acid molecules comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof))) (or fusions or conjugates thereof), the vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a coding region encoding a SARS-CoV-2 spike protein e.g., immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the carriers described herein (e.g., carriers comprising a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) or a nucleic acid molecule described herein (e.g., a nucleic acid molecule comprising a encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-
Attorney Docket No.62801.28WO01 2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof), compositions (e.g., a vaccine composition (e.g., a vaccine composition comprising any of the foregoing)), and/or pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing). In some of the following aspects, the methods include administering one or more of the foregoing (e.g., protein (or a fusion or conjugate thereof), polypeptide (or a fusion or conjugate thereof), immunogen (or a fusion or conjugate thereof), nucleic acid molecule (or a fusion or conjugate thereof), vector, carrier, composition (e.g., vaccine composition), pharmaceutical composition) to a subject. Exemplary subjects include mammals, e.g., humans, non-human mammals, e.g., non-human primates. In some embodiments, the subject is a human. In some embodiments, the subject is, elderly, pregnant, a newborn, immunocompromised, or immunosuppressed. In some embodiments, the subject has one or more of the following cancer, heart disease, obesity, diabetes, asthma, chronic lung disease, and/or sickle cell disease. In some embodiments, the subject has a weakened immune system or weakened immune response (e.g., a weakened immune response to a vaccine). In some embodiments, the subject is immunocompromised or immunosuppressed. In some embodiments, the subject is clinically vulnerable to the infection. In some embodiments, the subject has cancer, has an autoimmune disease, has an immunodeficiency, received a bone marrow or organ transplant, is undergoing a therapy that depletes immune cells, is undergoing chemotherapy, has a chronic viral infection, post viral syndrome or post viral fatigue syndrome (e.g., HIV infection or AIDS; long Covid or persistent post-Covid syndrome), is using or has had prolonged use of an immunosuppressive medication, is currently a smoker or has a history of smoking, or is at least 50 (e.g., at least 55, 60, 65, 70, 75, 80, 85, 90, or 100) years of age. In some embodiments, the subject at least 50, 60, 65, 70, or 75 years of age. In some embodiments, the subject is at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 110, or 120 years of age. In some embodiments, the subject is from about 50-120, 50-110, 50-100, 50-90, 50-80, 50-70, 50-60, 60-120, 60-110, 60-100, 60-90, 60-80, 60-70, 70-120, 70-110, 70-100, 70-90, 70-80, 80-120, 80-110, 80-100, 80-90, 90-120, 90-110, or 90-100 years of age. The dosage of one or more of the foregoing (e.g., protein, polypeptide, immunogen, nucleic acid molecule, vector, carrier, vaccine composition, pharmaceutical composition) to be administered to a subject can be determined in accordance with standard techniques well known
Attorney Docket No.62801.28WO01 to those of ordinary skill in the art, including the type (if any) adjuvant is used, the route of administration, and the age and weight of the subject. In some embodiments, a single dose of any one of the foregoing is administered to a subject in need thereof. In some embodiments, a series of doses of any one of the foregoing are administered to a subject in need thereof (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks) apart or within a range (e.g., 2-6 weeks apart)). In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, the nucleic acid molecule described herein, the vector described herein, the carrier described herein, the vaccine composition, or the pharmaceutical composition described herein (e.g., any one of the foregoing) is administered in a therapeutically effective amount. In some embodiments, a dose of an mRNA molecule encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (e.g., a vaccine or pharmaceutical composition comprising the same) is between 30-200 mcg, e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg. 5.10.1 Methods of Delivery In one aspect, provided herein are methods of delivering (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or
Attorney Docket No.62801.28WO01 immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing) to a subject in need thereof, the method comprising administering to the subject (a) the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), (b) the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), (c) the vector, (d) the carrier, (e) the vaccine composition, or (f) the pharmaceutical composition to the subject, to thereby deliver the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition to the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to deliver the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., an mRNA molecule), the vector, the carrier, the vaccine composition, or the pharmaceutical composition to the subject. 5.10.2 Manufacture of a Medicament In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules,
Attorney Docket No.62801.28WO01 e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for the manufacture of a medicament. In some embodiments, the medicament further comprises an adjuvant (e.g., an adjuvant described herein). 5.10.3 Methods of Inducing or Enhancing an Immune Response In one aspect, provided herein are methods of inducing and/or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA
Attorney Docket No.62801.28WO01 molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby induce and/or enhance an immune response in the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response the subject. In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or
Attorney Docket No.62801.28WO01 polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in inducing or enhancing an immune response in a subject in need thereof. In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in a method of in inducing
Attorney Docket No.62801.28WO01 or enhancing an immune response in a subject in need thereof, the method comprising administering the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition to the subject. In one aspect, provided herein is the use of (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for the manufacture of a medicament for the induction or enhancement of an immune response in a subject in need thereof. In one aspect, provided herein are methods of inducing and/or enhancing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2
Attorney Docket No.62801.28WO01 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV- 2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby induce and/or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in the subject. In some embodiments, the epitope is conserved in SARS-CoV-2 viruses. In some embodiments, the epitope is in a functionally constrained region of the SARS-CoV-2 spike protein.
Attorney Docket No.62801.28WO01 In one aspect, provided herein are methods of inducing and/or enhancing an immune response against a plurality of strains of SARS-CoV-2 virus in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby induce and/or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to induce and/or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject. An immune response in a subject can be measured by common methods known to those
Attorney Docket No.62801.28WO01 of skill in the art. For example, serological assays can be employed to detect a humoral response by measuring titers of anti-antigen (e.g., anti-SARS-CoV-2 spike protein, anti- SARS-CoV-2 spike protein RBD) IgG antibodies post administration. For example, an enzyme-linked immunosorbent assay (ELISA) is a standard laboratory test for detecting and quantifying antibodies well known to the person of skill in the art. Generally, blood is collected from a consenting subject, centrifuged, and the serum isolated according to standard techniques. The recombinant target antigen (e.g., SARS-CoV-2 spike protein, SARS-CoV-2 spike protein RBD) is immobilized in microplate wells. The microplate is blocked by through the incubation with an irrelevant antigen (e.g., bovine serum albumin). The serum sample from the subject is prepared and added to the blocked wells to allow for binding of an antigen specific antibodies to the immobilized antigen. The bound antibodies are detected using a secondary tagged antibody that binds to the previously bound antibodies (e.g., anti-human IgG antibodies). See, e.g., Front. Immunol., Forgacs David et al., SARS-CoV-2 mRNA Vaccines Elicit Different Responses in Immunologically Naïve and Pre-Immune Humans; Vol 12 (27 September 2021) https://doi.org/10.3389/fimmu.2021.728021, the entire contents of which is incorporated by reference herein for all purposes. Cell based assays can also be utilized to detect a cell based immune response (e.g., T cell immune response). For example, antigen specific T cells (e.g., CD4+ or CD8+ T cells) can be measured using an enzyme-linked immunospot (ELISpot), an intracellular cytokine staining (ICS) assay, or an activation induced marker assay (AIM). Each of these assays is commonly used to detect cell based (e.g., T cell) immune responses to vaccines and well known to the person of ordinary skill in the art. See, e.g., Bowyer, Georgina et al. “Activation-induced Markers Detect Vaccine-Specific CD4⁺ T Cell Responses Not Measured by Assays Conventionally Used in Clinical Trials.” Vaccines vol. 6,3 50. 31 Jul. 2018, doi:10.3390/vaccines6030050, the entire contents of which is incorporated by reference herein for all purposes. 5.10.4 Methods of Preventing or Decreasing an Immune Response In one aspect, provided herein are methods of preventing or decreasing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or
Attorney Docket No.62801.28WO01 a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV- 2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby prevent and/or decrease an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to prevent and/or an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen the subject. In some embodiments, the epitope is not conserved in SARS-CoV-2 viruses. In some embodiments, the epitope is not in a functionally constrained region of the SARS-CoV-2 spike protein. 5.10.5 Methods of Preventing, Ameliorating, and/or Treating Infection
Attorney Docket No.62801.28WO01 In one aspect, provided herein are methods of preventing, ameliorating, and/or treating a SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby prevent, ameliorate, and/or treat the SARS-CoV-2 infection in the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS- CoV-2 protein or peptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to prevent, ameliorate, and/or treat the SARS-CoV-2 infection the subject. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-
Attorney Docket No.62801.28WO01 CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject as a prophylactic treatment. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2 infection associated disease. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS- CoV-2, reduces the likelihood of developing an established infection after challenge with SARS- CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID- 19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof. Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction.
Attorney Docket No.62801.28WO01 The SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein may be administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like
Attorney Docket No.62801.28WO01 particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno- associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, a single dose of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)). In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is
Attorney Docket No.62801.28WO01 administered to the subject in a therapeutically effective amount. In some embodiments, wherein an mRNA molecule encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (e.g., a vaccine or pharmaceutical composition comprising the same) is administered to the subject, the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg). In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof. In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof),
Attorney Docket No.62801.28WO01 (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in a method of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof, the method comprising administering the SARS-CoV-2 spike protein or polypeptide (e.g., the SARS-CoV-2 protein or peptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition to the subject . In one aspect, provided herein is the use of (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof),
Attorney Docket No.62801.28WO01 (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for the manufacture of a medicament for the prevention, amelioration, or treatment a SARS-CoV-2 infection in a subject in need thereof. In one aspect, provided herein are methods of preventing, ameliorating, and/or treating a SARS-CoV-2 an infection by a plurality of SARS-CoV-2 strains in a subject in need thereof, the method comprising administering to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or
Attorney Docket No.62801.28WO01 immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby prevent, ameliorate, and/or treat an infection by a plurality of SARS-CoV-2 strains in the subject. 5.10.6 Methods of Vaccination against SARS-CoV-2 Provided herein are, inter alia, various methods of vaccinating subjects (e.g., human subjects) against SARS-CoV-2. As such, provided below in §§ 5.10.6.1 and 5.10.6.2, are various methods of vaccinating subjects utilizing one or more of the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein. 5.10.6.1 Methods of Vaccinating a Subject against SARS-CoV-2 In one aspect, provided herein are methods of vaccinating a subject against SARS- CoV-2, the method comprising administering to the subject to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-
Attorney Docket No.62801.28WO01 2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), € a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby vaccinate the subject against SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to vaccinate the subject against SARS-CoV-2. In some embodiments, the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described
Attorney Docket No.62801.28WO01 herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen. In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, the SARS-CoV-2 spike proteins (e.g., immunogens (or immunogenic fragments or immunogenic variants thereof)), the nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules), the vectors, the carriers, the vaccine compositions, and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine.
Attorney Docket No.62801.28WO01 In some embodiments, a single dose of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)). In some embodiments, the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in a therapeutically effective amount. In some embodiments, wherein an mRNA molecule encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (e.g., a vaccine or pharmaceutical composition comprising the same) is administered to the subject, the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg). In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject as a prophylactic treatment. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2
Attorney Docket No.62801.28WO01 infection associated disease. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection. In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS- CoV-2, reduces the likelihood of developing an established infection after challenge with SARS- CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID- 19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof. Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction. In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region
Attorney Docket No.62801.28WO01 encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in vaccinating a subject against SARS-CoV-2. In one aspect, provided herein are (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein),
Attorney Docket No.62801.28WO01 (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for use in a method of vaccinating a subject against SARS-CoV-2, the method comprising administering the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., the RNA molecule, e.g., the mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition to the subject. In one aspect, provided herein is the use of (a) SARS-CoV-2 spike proteins or polypeptides described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogens (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) nucleic acid molecules described herein (e.g., nucleic acid molecules (e.g., RNA molecules, e.g., mRNA molecules) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) vectors described herein (e.g., vectors comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) carriers described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (e) compositions (e.g., vaccine compositions) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) pharmaceutical compositions described herein (e.g., a pharmaceutical composition comprising any of the foregoing) for the manufacture of a medicament for the vaccination a subject against SARS-CoV-2. In one aspect, provided herein are methods of vaccinating a subject against a plurality
Attorney Docket No.62801.28WO01 of strains of SARS-CoV-2, the method comprising administering to the subject to the subject (a) a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (b) a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) (or a fusion or conjugate thereof), (c) a vector described herein (e.g., a vector comprising a nucleic acid molecule described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), (d) a carrier described herein (e.g., a carrier comprising a SARS-CoV-2 spike protein or polypeptide described herein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein) or a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) described herein (e.g., a nucleic acid molecule (e.g., an RNA molecule, e.g., an mRNA molecule) comprising a coding region encoding a SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof) described herein), € a composition (e.g., vaccine composition) described herein (e.g., a vaccine composition comprising any of the foregoing), or (f) a pharmaceutical composition described herein (e.g., a pharmaceutical composition comprising any of the foregoing), to thereby vaccinate the subject against a plurality of strains of SARS-CoV-2. 5.10.6.2 Methods of Vaccinating a Subject Utilizing a SARS-CoV-2 mRNA Vaccine In one aspect, provided herein are methods of vaccinating in a subject against SARS- CoV-2, the method comprising administering to the subject to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding a SARS-CoV-2 spike protein (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein (or a fusion or conjugate thereof), (b) a vector comprising the mRNA molecule, (c) a carrier comprising the mRNA molecule or the vector, (d) a composition (e.g., vaccine composition) comprising the mRNA molecule, the vector, or the carrier, or (e) a pharmaceutical composition comprising the mRNA molecule, the vector, the carrier, or the vaccine
Attorney Docket No.62801.28WO01 composition, to thereby vaccinate the subject against SARS-CoV-2. In some embodiments, the mRNA molecule is formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1- methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in an amount and for a time sufficient to vaccinate the subject against SARS-CoV-2. In some embodiments, the mRNA molecules, the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a homologous or heterologous prime-boost regimen. In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a boost in a homologous or heterologous prime-boost regimen. In some embodiments, the mRNA molecule, the vector, the carrier, the v composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a vaccine prime and a vaccine boost in a homologous prime-boost regimen. In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is administered to the subject as a prime in a heterologous prime-boost regimen. The boost vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the
Attorney Docket No.62801.28WO01 booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, the mRNA molecules, the vectors, the carriers, the compositions (e.g., vaccine compositions), and the pharmaceutical compositions described herein are administered to a subject as a boost in a heterologous prime-boost regimen. The prime vaccine composition in the regimen may be a vaccine that is based on mRNAs, DNAs, viral vectors (e.g., adenoviral vectors, adeno-associated viral vectors, lentiviral vectors, vesicular stomatitis viral vectors, vaccinia viral vectors, or measles viral vectors), peptides or proteins, viral-like particles (VLP), capsid-like particles (CLP), live attenuated viruses, inactivated viruses (killed vaccines), and the like. In some embodiments, the prime vaccine composition contains the same immunogen as the booster vaccine. In some embodiments, the primary vaccine contains a different immunogen as the booster vaccine. In some embodiments, a single dose of the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject. In some embodiments, a series of doses of the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition are administered to the subject (e.g., two doses given at a set interval (e.g., 2 weeks, 3 weeks apart) or within a range (e.g., 2-6 weeks apart)). In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject in a therapeutically effective amount. In some embodiments, the mRNA molecule is administered at a dose from about 30-200 mcg (e.g., 30 mcg, 50 mcg, 75 mcg, 100 mcg, 150 mcg, or 200 mcg). In some embodiments, the mRNA molecule, the vector, the carrier, or the pharmaceutical composition is administered to the subject as a prophylactic treatment. In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered as a treatment after the onset of at least one symptom of a SARS-CoV-2 infection or a SARS-CoV-2 infection associated disease. In some embodiments, the mRNA molecule, the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition is administered to the subject after a determination that the subject does or does not have a SARS-CoV-2 infection. In some embodiments, the mRNA molecule, the vector, the carrier, the vaccine
Attorney Docket No.62801.28WO01 composition, or the pharmaceutical composition prevents infection with SARS-CoV-2, reduces the likelihood of infection with SARS-CoV-2, reduces the likelihood of developing an established infection after challenge with SARS-CoV-2, reduces the duration of a SARS-CoV-2 infection, prevents or delays onset one or more symptoms of COVID-19, reduces the frequency and/or severity one or more symptoms of COVID-19, and/or reduces the risk of hospitalization or death associated with COVID- 19, or any combination of thereof. Exemplary COVID-19 symptoms include, but are not limited to, shortness of breath, difficulty breathing, respiratory rate greater than or equal to 20 breaths per minutes, abnormal SpO2, clinical or radiological evidence of lower respiratory tract disease, radiological evidence of deep vein thrombosis, respiratory failure, evidence of shock, significant renal, hepatic, and neurological dysfunction. 5.11 Kits In a one aspect, provided herein are kits comprising at least one SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) described herein, a nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule) described herein, a vector described herein, a carrier described herein, a composition (e.g., vaccine composition) described herein, and/or a pharmaceutical composition described herein. In addition, the kit may comprise a liquid vehicle for solubilizing or diluting any one of the foregoing, and/or technical instructions. The technical instructions of the kit may contain information about administration and dosage and subjects (e.g., subject groups). In some embodiments, the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is provided in a separate part of the kit, wherein the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein is optionally lyophilized, spray-dried, or spray-
Attorney Docket No.62801.28WO01 freeze dried. The kit may further contain as a part a vehicle (e.g., buffer solution) for solubilizing the dried or lyophilized pharmaceutical composition. In some embodiments, the kit comprises a single dose container of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition. In some embodiments, the kit comprises a multi- dose container for administration of the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS- CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein and/or an administration device (e.g., an injector for intradermal injection or a syringe for intramuscular injection). In some embodiments, the kit comprises an adjuvant in a separate container from the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein. The kit may further contain technical instructions for mixing the SARS-CoV-2 spike protein or polypeptide (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the nucleic acid molecule (e.g., RNA molecule, e.g., mRNA molecule), the vector, the carrier, the composition (e.g., vaccine composition), or the pharmaceutical composition described herein and the adjuvant prior to administration or for co- administration. Any of the kits described herein may be used in any of the methods described herein, e.g., in § 5.10. Any of the kits described herein may be used in a treatment or prophylaxis as defined herein (e.g., for the treatment, amelioration, and/or prophylaxis of SARS-CoV-2 infection). 6. EXAMPLES 6.1 Example 1. SARS-CoV-2 RNA Vaccines.
Attorney Docket No.62801.28WO01 The following example provides an exemplary method of preparing an mRNA vaccine comprising an mRNA encoding any one or a plurality of the immunogens identified herein (e.g., an immunogen comprising one or more non-naturally glycosylation site described herein). 6.1.1 DNA Preparation DNA constructs comprising SARS-CoV-2 proteins (e.g., immunogens) comprising at least one of the non-naturally glycosylation sites described herein are prepared and used for subsequent RNA in vitro transcription. Preparation of the DNA coding sequences can include codon optimization for stabilization and expression by introducing specific codons to generate a DNA coding sequence with an optimized G/C content (as discussed herein). Optimized coding sequences are introduced into a DNA plasmid comprising a 3’-UTR, a 5-UTR, and polyadenylation sequence. The obtained DNA plasmids are transformed and propagated in bacteria using common protocols known in the art. The DNA plasmids are subsequently extracted, purified, and used for RNA in vitro transcription. 6.1.2 RNA in vitro transcription The DNA plasmids are enzymatically linearized using a restriction enzyme used for DNA dependent RNA in vitro transcription using T7 RNA polymerase in the presence of a nucleotide mixture (ATP/GTP/CTP/UTP) and a 5’ cap (or analog) under suitable buffer conditions. The obtained RNA constructs are purified using a suitable method known in the art e.g., RP-HPLC. The RNA in vitro transcription is performed in the presence of modified nucleotides for incorporation in the RNA e.g., pseudouridine or N1 -methylpseudouridine (m1ψ) instead of UTP. In some instances, the 5’ cap is enzymatically added to the RNA after in vitro transcription using capping enzymes as commonly known in the art. 6.1.3 Preparation of LNPs and Encapsulated RNAs LNPs are prepared using according to the general procedures known in the art using e.g., cationic lipids, structural lipids, a PEG-lipids, and cholesterol see, e.g., WO2015199952, WO2017004143 and WO2017075531, the full contents of each of which is incorporated by reference herein for al purposes. The lipid solution (in ethanol) is mixed with RNA (in aqueous solution) in a small T-piece to encapsulate the mRNA in the LNPs, see, e.g., Reichmuth, Andreas M et al. “mRNA vaccine delivery using lipid nanoparticles.” Therapeutic delivery vol.7,5 (2016):
Attorney Docket No.62801.28WO01 319-34. doi:10.4155/tde-2016-0006, the entire contents of which is incorporated by reference herein for all purposes. The LNP formulated mRNA is rebuffered as needed via dialysis and concentrated to a target concentration using ultracentrifugation. * * * The invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims. All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entireties and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.
Claims
Attorney Docket No.62801.28WO01 CLAIMS What is claimed is: 1. A nucleic acid molecule comprising at least one coding region encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a non-naturally occurring glycosylation motif comprising a non- naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369, 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 2. The nucleic acid molecule of claim 1, wherein the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper-glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1). 3. The nucleic acid molecule of claim 1 or 2, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-naturally occurring glycosylation motifs each comprising a non-naturally occurring glycosylation site, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 4. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation motif is an N-linked glycosylation motif. 5. The nucleic acid molecule of any one of the preceding claims, wherein the N-linked glycosylation motif is an NXT sequence, wherein X is any amino acid except for proline. 6. The nucleic acid molecule of any one of the preceding claims, wherein the N-linked glycosylation motif is an NXS sequence, wherein X is any amino acid except for proline. 7. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation site is an N-linked glycosylation site.
Attorney Docket No.62801.28WO01 8. The nucleic acid molecule of any one of claims 1-3, wherein the glycosylation motif is an O-linked glycosylation motif. 9. The nucleic acid molecule of claim 8, wherein the O-linked glycosylation motif is a serine or threonine. 10. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation site is an O-linked glycosylation site. 11. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-linked glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N- glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N- glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and an N- glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N- glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or
Attorney Docket No.62801.28WO01 (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N- glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 12. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino
Attorney Docket No.62801.28WO01 acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 13. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 14. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 15. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino
Attorney Docket No.62801.28WO01 acid positions 354, 370, 394, 415, 428, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 16. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation motif is an N-linked glycosylation motif. 17. The nucleic acid molecule of any one of the preceding claims, wherein the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. 18. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation site is an N-linked glycosylation site. 19. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N- glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N- glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N- glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N-
Attorney Docket No.62801.28WO01 glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N- glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N- glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N- glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N- glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 20. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N- glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 21. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein
Attorney Docket No.62801.28WO01 immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 22. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid
Attorney Docket No.62801.28WO01 position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 23. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 24. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation motif is an N-linked glycosylation motif. 25. The nucleic acid molecule of any one of the preceding claims, wherein the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. 26. The nucleic acid molecule of any one of the preceding claims, wherein the glycosylation site is an N-linked glycosylation site. 27. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following:
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N- glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N- glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine or a serine at position 479 and an N- glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine or a serine at position 519 and an N- glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine or a serine at position 520 and an N- glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 28. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N- glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at
Attorney Docket No.62801.28WO01 position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 29. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites. 30. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a naturally occurring glycosylation site) at one or more of amino acid positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 31. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 32. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) relative the reference sequence (e.g., SEQ ID NO: 1). 33. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) )) in a prefusion state relative the reference sequence (e.g., SEQ ID NO: 1). 34. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an inactive furin cleavage site. 35. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site. 36. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 37. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a
Attorney Docket No.62801.28WO01 proline at positions 986 and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 38. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 39. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 40. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 41. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 42. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position
Attorney Docket No.62801.28WO01 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 43. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 44. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 45. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 46. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 47. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 48. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 49. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 50. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 51. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 52. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 53. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 54. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 55. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 56. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 57. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 58. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 59. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 60. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and
Attorney Docket No.62801.28WO01 (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 61. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 62. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 63. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 64. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 65. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 66. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 67. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 68. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 69. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 70. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 71. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 72. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 73. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 74. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 75. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 76. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 77. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 78. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 79. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 80. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 81. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 82. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 83. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438,
Attorney Docket No.62801.28WO01 an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 84. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD). 85. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein. 86. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids. 87. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises from about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10-300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1200, 10-1300, 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100-1100, 100- 1200, or 100-1300 amino acids. 88. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76.
Attorney Docket No.62801.28WO01 89. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11-76]. 90. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76. 91. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, or 1300 amino acids set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1). 92. The nucleic acid molecule of any one of the preceding claims, wherein the amino acid sequence of the encoded SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1). 93. The nucleic acid molecule of any one of the preceding claims, wherein the encoded SARS-CoV-2 spike protein is an immunogen (or an immunogenic fragment and/or immunogenic variant thereof).
Attorney Docket No.62801.28WO01 94. The nucleic acid molecule of claim 93, wherein the nucleic acid molecule is RNA or DNA. 95. The nucleic acid molecule of any one of claim 93-94, wherein the RNA is messenger ribonucleic acid (mRNA). 96. The nucleic acid molecule of any one of claims 93-95, wherein the nucleic acid molecule comprises at least one modified nucleotide. 97. The nucleic acid molecule of any one of claims 93-96, wherein the nucleic acid molecule comprises N1-methyl-pseudouridine, cytosine, adenine, and guanine. 98. The nucleic acid molecule of any one of claims 93-97, wherein the nucleic acid molecule comprises a heterologous 5’-untranslated region (UTR), 3’-UTR, or both a 5’-UTR and 3’-UTR. 99. The nucleic acid molecule of any one of claims 93-98, wherein the nucleic acid molecule comprises a poly(A) sequence. 100. The nucleic acid molecule of any one of claims 93-99, wherein the nucleic acid molecule comprises a 5’cap structure. 101. The nucleic acid molecule of any one of claims 93-100, wherein the nucleotide sequence of the nucleic acid molecule is codon optimized. 102. The nucleic acid molecule of any one of claims 93-101, wherein the nucleic acid molecule further encodes a heterologous polypeptide or protein. 103. The nucleic acid molecule of any one of claims 93-102, wherein the nucleic acid molecule encodes a signal peptide. 104. The nucleic acid molecule of any one of claims 93-103, wherein the nucleic acid molecule encodes a homologous or heterologous signal peptide. 105. The nucleic acid molecule of any one of claims 93-104, wherein the nucleic acid molecule does not encode a signal peptide. 106. A vector comprising the nucleic acid molecule of any one of claims 1-105. 107. The vector of claim 106, wherein the vector is a non-viral vector (e.g., a plasmid) or a viral vector. 108. A conjugate comprising the nucleic acid molecule of any one of claims 1-105 operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a heterologous polypeptide or protein).
Attorney Docket No.62801.28WO01 109. A fusion protein comprising the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) encoded by the nucleic acid molecule of any one of claims 1-105 operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous protein. 110. The fusion protein of claim 109, wherein the heterologous protein is a trimerization domain. 111. The fusion protein of claim 110, wherein the trimerization domain is operably connected to the encoded SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) via a peptide linker. 112. A composition comprising at least one of the nucleic acid molecules set forth in any one of claims 1-105, the vector of any one of any one of claims 106-107, the conjugate of claim 108, or the fusion protein of any one of claims 109-111. 113. The composition of claim 112, wherein the composition is monovalent or polyvalent. 114. The composition of claim 112 or 113, wherein the composition is polyvalent. 115. The composition of any one of claims 112-114, comprising a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 116. The composition any one of claims 112-115, comprising a plurality of nucleic acid molecules set forth in any one of claims 1-105, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 117. The composition of any one of claims 112-116, wherein the nucleic acid molecule(s) are comprised in one or more vectors. 118. The composition of any one of claims 112-117, wherein the nucleic acid molecule(s) or the one or more vectors are formulated in one or more carrier. 119. The composition of claim 118, wherein the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. 120. The composition of claim 118 or 119, wherein the carrier is an LNP.
Attorney Docket No.62801.28WO01 121. The composition of any one of claims 118-120, wherein the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. 122. The composition of any one of claims 118-121, wherein the LNP has a mean particle size of between 80 nm and 160 nm. 123. The composition of any one of claims 112-122, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or a vaccine composition. 124. The composition of any one of claims 112-123, further comprising an adjuvant. 125. A SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 126. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of claim 125, wherein the SARS- CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) is hyper-glycosylated relative to a reference SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) (e.g., the SARS-CoV-2 spike protein set forth in SEQ ID NO: 1). 127. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of claim 125 or 126, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more non-naturally occurring glycosylation motifs each comprising a non-naturally occurring glycosylation site, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 128. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-127, wherein the glycosylation motif is an N-linked glycosylation motif. 129. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-128, wherein the N-linked glycosylation motif is an NXT sequence, wherein X is any amino acid except for proline. 130. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-129, wherein the N-linked glycosylation motif is an NXS sequence, wherein X is any amino acid except for proline. 131. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-130, wherein the glycosylation site is an N-linked glycosylation site. 132. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-131, wherein the glycosylation motif is an O-linked glycosylation motif. 133. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of claim 132, wherein the O-linked glycosylation motif is a serine or threonine. 134. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-133, wherein the glycosylation site is an O-linked glycosylation site. 135. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-134, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and an N-linked
Attorney Docket No.62801.28WO01 glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and an N- glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and an N- glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and an N- glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and an N- glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and an N- glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 136. The nucleic acid molecule of any one of claims 125-135, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 137. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-136, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and an N-glycosylation site at
Attorney Docket No.62801.28WO01 amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 138. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-137, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and an N-glycosylation site at amino acid position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and an N-glycosylation site at amino acid position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and an N-glycosylation site at amino acid position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and an N-glycosylation site at amino acid position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and an N-glycosylation site at amino acid position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and an N-glycosylation site at amino acid position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 139. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-138, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 354, 370, 394, 415, 428, 448, 468, 494, and/or 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 140. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-139, wherein the glycosylation motif is an N-linked glycosylation motif. 141. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-140, wherein the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline.
Attorney Docket No.62801.28WO01 142. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-141, wherein the glycosylation site is an N-linked glycosylation site. 143. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-142, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and an N- glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and an N- glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and an N- glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and an N- glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and an N- glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and an N-
Attorney Docket No.62801.28WO01 glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and an N- glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and an N- glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N- glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 144. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-143, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 504 and an N- glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 145. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-144, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and an N-glycosylation site at
Attorney Docket No.62801.28WO01 amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or
Attorney Docket No.62801.28WO01 (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 146. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-145, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and an N-glycosylation site at amino acid position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and an N-glycosylation site at amino acid position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and an N-glycosylation site at amino acid position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and an N-glycosylation site at amino acid position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and an N-glycosylation site at amino acid position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and an N-glycosylation site at amino acid position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (g) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and an N-glycosylation site at amino acid position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and an N-glycosylation site at amino acid position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (i) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and an N-glycosylation site at amino acid position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 147. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-146, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a non-naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a non-naturally occurring glycosylation site) at one or more of amino acid positions 383, 441, 477, 517, and/or 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 148. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-147, wherein the glycosylation motif is an N-linked glycosylation motif. 149. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-148, wherein the N-linked glycosylation motif is an NXT or NXS, wherein X is any amino acid except for proline. 150. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-149, wherein the glycosylation site is an N-linked glycosylation site. 151. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-150,
Attorney Docket No.62801.28WO01 wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N- glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine or a serine at position 443 and an N- glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine or a serine at position 479 and an N- glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine or a serine at position 519 and an N- glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine or a serine at position 520 and an N- glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 152. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-151, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more of the following: (a) an N-linked glycosylation motif comprising an asparagine at position 383, any amino acid except proline at position 384, and a threonine or a serine at position 385 and an N-
Attorney Docket No.62801.28WO01 glycosylation site at position 383, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 441, any amino acid except proline at position 442, and a threonine at position 443 and an N-glycosylation site at position 443, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 477, any amino acid except proline at position 478, and a threonine at position 479 and an N-glycosylation site at position 477, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 517, any amino acid except proline at position 518, and a threonine at position 519 and an N-glycosylation site at position 517, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (e) an N-linked glycosylation motif comprising an asparagine at position 518, any amino acid except proline at position 519, and a threonine at position 520 and an N-glycosylation site at position 518, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 153. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-152, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more naturally occurring glycosylation sites. 154. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-153, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycosylation motif (e.g., a naturally occurring glycosylation motif) comprising a glycosylation site (e.g., a naturally occurring glycosylation site) at one or more of amino acid
Attorney Docket No.62801.28WO01 positions 331 and/or 343, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 155. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-154, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 156. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-155, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) relative the reference sequence (e.g., SEQ ID NO: 1). 157. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-156, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises one or more amino acid variation relative to a reference sequence (e.g., SEQ ID NO: 1) that enhances the stability of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) )) in a prefusion state relative the reference sequence (e.g., SEQ ID NO: 1). 158. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-157, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an inactive furin cleavage site. 159. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-158,
Attorney Docket No.62801.28WO01 wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises at least one amino acid variation in the furin cleavage site that inactivates the furin cleavage site. 160. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-159, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at one or more of position 817, 892, 899, 986, and/or 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 161. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-160, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 986 and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 162. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-161, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a proline at positions 817, 892, 899, 986, and 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 163. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-162, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 164. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-163, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine or a serine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 165. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-164, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, any amino acid except proline at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 166. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-165, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 369, an asparagine at position 370, and a threonine at position 371 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 167. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-166, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 168. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-167, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine or a serine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 169. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-168, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, any amino acid except proline at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 170. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-170, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises an N-linked glycosylation motif comprising an asparagine at position 447, an asparagine at position 448, and a threonine at position 449 and a glycosylation site at position 447, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 171. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-170, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and
Attorney Docket No.62801.28WO01 (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 172. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-171, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 173. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-172, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 174. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-173, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 175. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-175, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 176. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-175, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 177. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-176, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 369, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 178. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-177, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (j) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 179. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-178, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine or a serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine or a serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine or a serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 180. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-179, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine or serine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine or serine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine or serine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and
Attorney Docket No.62801.28WO01 (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 181. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-180, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 354, any amino acid except proline at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, any amino acid except proline at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, any amino acid except proline at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (k) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 182. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-181, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 354, an arginine at position 355, and a threonine at position 356 and a glycosylation site at position 354, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 370, a serine at position 371, and a threonine at position 372 and a glycosylation site at position 370, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 388, an aspartic acid at position 389, and a threonine at position 390 and a glycosylation site at position 388, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (i) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (j) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (k) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (l) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 183. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-182, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and
Attorney Docket No.62801.28WO01 (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 184. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-183, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 185. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-184, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 186. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-185, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 187. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-186, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 188. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-187, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 189. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-188, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at
Attorney Docket No.62801.28WO01 position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 190. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-189, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (g) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 191. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-190, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine or a serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine or a serine at position 430 and a
Attorney Docket No.62801.28WO01 glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine or a serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine or a serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine or a serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine or a serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 192. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-191, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises
Attorney Docket No.62801.28WO01 (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine or serine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine or a serine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine or a serine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine or serine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine or a serine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine or serine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine or serine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine or serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine or serine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 193. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-192, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, any amino acid except proline at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, any amino acid except proline at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, any amino acid except proline at position 429, and a threonine at position 430 and a glycosylation site at position 428, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, any amino acid except proline at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, any amino acid except proline at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, any amino acid except proline at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (g) an N-linked glycosylation motif comprising an asparagine at position 476, any amino acid except proline at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (h) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, any amino acid except proline at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 194. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-194, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 394, a valine at position 395, and a threonine at position 396 and a glycosylation site at position 394, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) an N-linked glycosylation motif comprising an asparagine at position 415, a glycine at position 416, and a threonine at position 417 and a glycosylation site at position 415, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) an N-linked glycosylation motif comprising an asparagine at position 428, a phenylalanine at position 429, and a threonine at position 430 and a glycosylation site at position 429, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) an N-linked glycosylation motif comprising an asparagine at position 448, a tyrosine at position 449, and a threonine at position 450 and a glycosylation site at position 448, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) an N-linked glycosylation motif comprising an asparagine at position 460, a leucine at position 461, and a threonine at position 462 and a glycosylation site at position 460, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (f) an N-linked glycosylation motif comprising an asparagine at position 468, a serine at position 469, and a threonine at position 470 and a glycosylation site at position 468, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1;
Attorney Docket No.62801.28WO01 (g) an N-linked glycosylation motif comprising an asparagine at position 476, a serine at position 477, and a threonine at position 478 and a glycosylation site at position 476, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (h) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (i) an N-linked glycosylation motif comprising an asparagine at position 529, a serine at position 530, and a threonine at position 531 and a glycosylation site at position 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 195. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-194, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine or a serine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine or a serine at position 502 and a glycosylation site at position 504, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 196. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-195, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and
Attorney Docket No.62801.28WO01 (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 197. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-196, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, any amino acid except proline at position 495, and a threonine at position 496 and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, any amino acid except proline at position 503, and a threonine at position 504 and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 198. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-197, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) an N-linked glycosylation motif comprising an asparagine at position 494, a tyrosine at position 495, and a threonine at position 496, and a glycosylation site at position 494, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (b) an N-linked glycosylation motif comprising an asparagine at position 502, a valine at position 503, and a threonine at position 504, and a glycosylation site at position 502, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 199. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-198, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, or 6) of (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and/or (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 200. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-199, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises (a) a proline at position 817, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (b) a proline at position 892, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (c) a proline at position 899, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (d) a proline at position 942, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; (e) a proline at position 986, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1; and (f) a proline at position 987, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1.
Attorney Docket No.62801.28WO01 201. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-200, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a glycine at position 614, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 202. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-201, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, and/or 22) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431, an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 203. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-202, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, and/or 10) of a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and/or a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 204. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-203, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike
Attorney Docket No.62801.28WO01 protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, and a glycine at position 431, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 205. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-204, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises any one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and/or 12) of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 206. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-205, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises of an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and/or an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 207. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-206, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises a cysteine at position 379, a tyrosine at position 380, a proline at position 412, an asparagine at position 422, a tyrosine at position 423, a lysine at position 424, a leucine at position 425, a proline at position 426, a phenylalanine at position 429, a glycine at position 431,
Attorney Docket No.62801.28WO01 an alanine at position 435, a tryptophan at position 436, an asparagine at position 437, a serine at position 438, an aspartic acid at position 442, a serine at position 443, a glycine at position 447, a tyrosine at position 495, a phenylalanine at position 497, a glutamic acid at position 506, a proline at position 507, and an arginine at position 509, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1. 208. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-207, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of the SARS-CoV-2 spike protein receptor binding domain (RBD). 209. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-208, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises or consists of a full-length SARS-CoV-2 spike protein. 210. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-209, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids. 211. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-210, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) comprises from about 10-15, 10-20, 10-30, 10-40, 10-50, 10-60, 10-70, 10-80, 10-90, 10-100, 10-200, 10- 300, 10-400, 10-500, 10-600, 10-700, 10-800, 10-900, 10-1000, 10-1100, 10-1190, 10-1300, 100-200, 100-300, 100-400, 100-500, 100-600, 100-700, 100-800, 100-900, 100-1000, 100- 1100, 100-1190, or 100-1300 amino acids. 212. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-211,
Attorney Docket No.62801.28WO01 wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 11-76. 213. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-212, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 11-76. 214. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-213, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 55-76. 215. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-214, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous stretch of at least 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1190, or 1300 amino acids set forth in any one of SEQ ID NOS: 1-10 (e.g., SEQ ID NO: 1).
Attorney Docket No.62801.28WO01 216. The SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-215, wherein the amino acid sequence of the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)) is less than 100% identical to but at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence set forth in any one of SEQ ID NOS: 1- 10 (e.g., SEQ ID NO: 1). 217. A conjugate comprising the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-216 operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous moiety (e.g., a heterologous polypeptide or protein). 218. A fusion protein comprising the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-217 operably connected (e.g., directly or indirectly (e.g., via a linker)) to a heterologous protein. 219. The fusion protein of claim 218, wherein the heterologous protein is a trimerization domain. 220. The fusion protein of claim 219, wherein the trimerization domain is operably connected to the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) via a peptide linker. 221. A composition comprising at least one of the SARS-CoV-2 spike proteins (e.g., a SARS- CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) set forth in any one of claims 125-216, the conjugate of claim 217, or the fusion protein of any one of claims 218-220. 222. The composition of claim 221, wherein the composition is monovalent or polyvalent. 223. The composition of claim 221 or 222, wherein the composition is polyvalent. 224. The composition of any one of claims 221-223, comprising a plurality SARS-CoV-2 spike proteins (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)), wherein the amino acid sequence of each of the SARS-
Attorney Docket No.62801.28WO01 CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. 225. The composition any one of claims 221-224, comprising a plurality of SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-216, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) of the plurality is different. 226. The composition of any one of claims 221-225, wherein the SARS-CoV-2 spike protein(s) (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) are formulated in one or more carrier. 227. The composition of claim 226, wherein the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. 228. The composition of claim 226 or 227, wherein the carrier is an LNP. 229. The composition of claim 228, wherein the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid. 230. The composition of any one of claims 227-229, wherein the LNP has a mean particle size of between 80 nm and 160 nm. 231. The composition of any one of claims 221-230, wherein the composition is a pharmaceutical composition comprising a pharmaceutically acceptable excipient or a vaccine composition. 232. The composition of any one of claims 221-231, further comprising an adjuvant. 233. A nucleic acid molecule encoding the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-216. 234. A SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) encoded by the nucleic acid molecule of any one of claims 1-105. 235. A carrier comprising the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of
Attorney Docket No.62801.28WO01 claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109- 111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221- 232, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269. 236. The carrier of claim 235, wherein the carrier is monovalent or polyvalent. 237. The carrier of any one of claims 235-236, wherein the carrier is polyvalent. 238. The carrier of any one of claims 235-237, comprising a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 239. The carrier of any one of claims 235-238, comprising a plurality of nucleic acid molecules set forth in any one of claims 1-205 or 233, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 240. The carrier of any one of claims 235-239, comprising a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 241. The carrier of any one of claims 235-240, comprising a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 242. The carrier of any one of claims 235-241, wherein the carrier is a lipid nanoparticle (LNP), liposome, lipoplex, or nanoliposome. 243. The carrier of any one of claims 235-242, wherein the carrier is an LNP. 244. The carrier of claim 242 or 243, wherein the LNP comprises a cationic lipid, a neutral lipid, a cholesterol, and/or a PEG lipid.
Attorney Docket No.62801.28WO01 245. The carrier of any one of claims 242-244, wherein the LNP has a mean particle size of between 80 nm and 160 nm. 246. A vaccine composition comprising the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112- 124 or 221-232, the carrier of any one of claims 235-245, or the pharmaceutical composition of any one of claims 260-269. 247. The vaccine composition of claim 246, wherein the vaccine composition is a prime vaccine composition. 248. The vaccine composition of claim 246 or 247, wherein the vaccine composition is a boost vaccine composition. 249. The vaccine composition of any one of claims 246-248, wherein the vaccine composition is a prime vaccine composition and a boost vaccine composition. 250. The vaccine composition of any one of claims 246-249, wherein the vaccine composition can be utilized as a prime vaccine composition and/or a booster vaccine composition in a homologous or heterologous prime boost vaccine regimen. 251. The vaccine composition of any one of claims 246-250, wherein the vaccine composition is monovalent or polyvalent. 252. The vaccine composition of any one of claims 246-251, wherein the vaccine composition is polyvalent. 253. The vaccine composition of any one of claims 246-252, comprising a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 254. The vaccine composition of any one of claims 246-253, comprising a plurality of nucleic acid molecules set forth in any one of claims 1-105 or 233, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different.
Attorney Docket No.62801.28WO01 255. The vaccine composition of any one of claims 246-254, comprising a plurality of SARS- CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS- CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 256. The vaccine composition of any one of claims 246-255, comprising a plurality of SARS- CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 257. The vaccine composition of any one of claims 246-256, comprising a plurality of SARS- CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234. 258. A vaccine composition comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) that comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1, formulated in a lipid nanoparticle, the vaccine composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'-cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'- UTR; and (v) a poly-A region. 259. The vaccine composition of any one of claims 246-258, wherein the vaccine composition further comprises an adjuvant.
Attorney Docket No.62801.28WO01 260. A pharmaceutical composition comprising the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112- 124 or 221-232, the carrier of any one of claims 235-245, or the vaccine composition of any one of claims 246-259, or the vaccine composition of any one of claims 246-259, and a pharmaceutically acceptable excipient. 261. The pharmaceutical composition of claim 260, wherein the pharmaceutical composition is monovalent or polyvalent. 262. The pharmaceutical composition of any one of claims 260-261, wherein the pharmaceutical composition is polyvalent. 263. The pharmaceutical composition of any one of claims 260-262, comprising a plurality of nucleic acid molecules, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 264. The pharmaceutical composition of any one of claims 260-263, comprising a plurality of nucleic acid molecules set forth in any one of claims 1-105 or 223, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) encoded by each nucleic acid molecule of the plurality is different. 265. The pharmaceutical composition of any one of claims 260-264, comprising a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)), wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 266. The pharmaceutical composition of any one of claims 260-265, comprising a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234, wherein the amino acid sequence of each of the SARS-CoV-2 spike proteins (e.g., SARS-CoV-2
Attorney Docket No.62801.28WO01 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) is different. 267. The pharmaceutical composition of any one of claims 260-266, comprising a plurality of SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234, comprising a trimer of a SARS-CoV-2 spike proteins (e.g., SARS-CoV-2 spike protein immunogens (or immunogenic fragments and/or immunogenic variants thereof)) set forth in any one of claims 125-226 or 234. 268. A pharmaceutical composition comprising a messenger ribonucleic acid (mRNA) encoding a SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein immunogen (or an immunogenic fragment and/or immunogenic variant thereof)) that comprises a non-naturally occurring glycosylation motif comprising a non-naturally occurring glycosylation site at one or more (e.g., 1, 2, 3, 4, 5, and/or 6) of amino acid positions 369 , 388, 447, 460, 476, and/or 529, amino acid numbering relative to the amino acid positions set forth in SEQ ID NO: 1, formulated in a lipid nanoparticle, the pharmaceutical composition having the following characteristics: (a) the LNPs comprise a cationic lipid, a neutral lipid, a cholesterol, and a PEG lipid, (b) the LNPs have a mean particle size of between 80 nm and 160 nm, and (c) the mRNA comprises: (i) a 5'- cap structure; (ii) a 5'-UTR; (iii) N1-methyl-pseudouridine, cytosine, adenine, and guanine; (iv) a 3'-UTR; and (v) a poly-A region; and a pharmaceutically acceptable excipient. 269. The pharmaceutical composition of any one of claims 260-268, wherein the pharmaceutical composition further comprises an adjuvant. 270. A host cell comprising the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109- 111 or 218-220, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269. 271. A kit comprising the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of
Attorney Docket No.62801.28WO01 claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109- 111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221- 232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246- 259, or the pharmaceutical composition of any one of claims 260-269. 272. The kit of claim 271, comprising instructions for use of the nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, carrier, composition, vaccine composition, or pharmaceutical composition. 273. A method of delivering a nucleic acid molecule, vector, protein (or immunogenic fragment or immunogenic variant thereof), conjugate, fusion protein, host cell, carrier, composition, vaccine composition, or pharmaceutical composition to a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby deliver the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the host cell, the carrier, the composition, the vaccine composition, or the pharmaceutical composition to the subject. 274. A method of inducing or enhancing an immune response in a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112- 124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby induce or enhance an immune response in the subject.
Attorney Docket No.62801.28WO01 275. A method of inducing or enhancing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby induce or enhance an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject. 276. A method of preventing or decreasing an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby prevent or decrease an immune response to one or more predetermined epitopes of a SARS-CoV-2 spike protein antigen in a subject. 277. A method of inducing or enhancing an immune response against a plurality of strains of SARS-CoV-2 virus in a subject in need thereof, the method comprising administering to the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical
Attorney Docket No.62801.28WO01 composition of any one of claims 260-269, to thereby induce or enhance an immune response against a plurality of strains of SARS-CoV-2 virus in the subject. 278. A method of preventing, ameliorating, or treating a SARS-CoV-2 infection in a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby prevent, ameliorate, or treat the SARS-CoV-2 infection in the subject. 279. A method of preventing, ameliorating, or treating an infection by a plurality of SARS- CoV-2 strains in a subject in need thereof, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby prevent, ameliorate, or treat an infection by a plurality of SARS-CoV-2 strains in the subject. 280. A method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109- 111 or 218-220, the host cell of claim 270, the composition of any one of claims 112-124 or 221- 232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246- 259, or the pharmaceutical composition of any one of claims 260-269, to thereby vaccinate the subject against SARS-CoV-2.
Attorney Docket No.62801.28WO01 281. A method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject (a) an mRNA molecule (e.g., an mRNA molecule described herein) encoding the SARS-CoV-2 spike protein (e.g., a SARS-CoV-2 spike protein or polypeptide immunogen (or immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234 (or a conjugate or fusion protein thereof), (b) a vector comprising the mRNA molecule, (c) a carrier comprising the mRNA molecule or the vector, (d) a vaccine composition comprising the mRNA molecule, the vector, or the carrier, or (e) a pharmaceutical composition comprising the mRNA molecule, the vector, the carrier, or the vaccine composition, to thereby vaccinate the subject against SARS-CoV-2, to thereby vaccinate the subject against SARS-CoV-2. 282. A method of vaccinating a subject against SARS-CoV-2, the method comprising administering to the subject the vaccine composition of any one of claims 246-259 or the pharmaceutical composition of any one of claims 260-269, to thereby vaccinate the subject against SARS-CoV-2. 283. A method of vaccinating a subject against a plurality of strains of SARS-CoV-2, the method comprising administering to the subject the nucleic acid molecule of any one of claims 1-105 or 233, the protein (e.g., the SARS-CoV-2 spike protein immunogen (or the immunogenic fragment and/or immunogenic variant thereof)) of any one of claims 125-226 or 234, the vector of any one of claims 106-107, the conjugate of claim 108 or 217, the fusion protein of any one of claims 109-111 or 218-220, the host cell of claim 270, the composition of any one of claims 112- 124 or 221-232, the carrier of any one of claims 235-245, the vaccine composition of any one of claims 246-259, or the pharmaceutical composition of any one of claims 260-269, to thereby vaccinate the subject against a plurality of strains of SARS-CoV-2. 284. The method of any one of claims 273-283, wherein the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered to the subject at least twice. 285. The method of any one of claims 273-284, wherein the subject is a human. 286. The method of any one of claims 273-285, wherein the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or
Attorney Docket No.62801.28WO01 immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a prime and/or a boost in a homologous or heterologous prime-boost regimen. 287. The method of any one of claims 273-286, wherein the nucleic acid molecule, the vector, the SARS-CoV-2 spike protein (e.g., the SARS-CoV-2 spike protein immunogen (or immunogenic fragment and/or immunogenic variant thereof)), the conjugate, the fusion protein, the carrier, the composition, the vaccine composition, or the pharmaceutical composition is administered as a boost in a heterologous prime-boost regimen.
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