WO2025166067A1

WO2025166067A1 - Sars-cov-2 vaccine compositions and methods

Info

Publication number: WO2025166067A1
Application number: PCT/US2025/013877
Authority: WO
Inventors: Hannu Rajaniemi; Nikolai EROSHENKO; Marianna KEAVENEY; Everett WEBSTER; Nikhil Dhar; Arianna Lizeth Lopez SAUCEDA; Katherine Rowe; Justin Huang; Dario De Jesus Davila PASILLAS
Original assignee: Helix Nanotechnologies Inc
Current assignee: Helix Nanotechnologies Inc
Priority date: 2024-01-31
Filing date: 2025-01-30
Publication date: 2025-08-07
Anticipated expiration: 2026-07-31

Abstract

Disclosed herein are fusion polypeptides comprising: (i) a fragment antigen comprising an epitope of SARS-CoV-2; and (ii) a complement binding polypeptide. The disclosure also provides polynucleotides (e.g., mRNA) encoding the same. Also disclosed herein are methods of making and using the fusion polypeptides and fusion polynucleotides of the present disclosure.

Description

SARS-COV-2 VACCINE COMPOSITIONS AND METHODS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to US Provisional Patent Application No. 63/627,241, which was filed on January 31, 2024, and which is incorporated by reference herein in its entirety.

REFERENCE TO THE SEQUENCE LISTING

[0002] The application contains a Sequence Listing which has been submitted electronically in .XML format and is hereby incorporated by reference in its entirety. Said .XML copy, created on January 30, 2025, is named “01349-0001-00PCT. xml” and is 34,756 bytes in size. The sequence listing contained in this .XML file is part of the specification and is hereby incorporated by reference herein in its entirety.

BACKGROUND

[0003] In recent years, progress has been made in the development of vaccines for a variety of diseases and conditions, including CO VID 19, caused by the SARS-CoV-2 virus. However, these efforts have not been able to generate broadly neutralizing vaccines.

[0004] SARS-CoV-2 emerged in China in late 2019 and was declared a pandemic by the World Health Organization in March 2020. By March 2023, over 750 million cases and nearly 7 million deaths had been reported worldwide. The Spike glycoprotein of the SARS- CoV-2 virus binds to the ACE2 receptor on the surface of human cells, such as epithelial cells in the upper and lower airways, leading to infection, replication, and spread of the virus. The Spike protein is also the target of many neutralizing antibodies against the virus.

[0005] Several vaccines against CO VID 19 were approved for use in 2021, including mRNA vaccines developed by Pfizer and BioNTech, and by Modema, a viral vector vaccine developed by Johnson & Johnson, and a protein-based vaccine developed by Novavax, among others. All of these vaccines encode or comprise a full length Spike glycoprotein from one or more strains of SARS-CoV-2. Under selective pressure from the immunity acquired by prior infection and vaccination, new strains of SARS-CoV-2 containing mutations, for example, in the Spike glycoprotein, have emerged and spread globally. For example, by late 2021 the omicron variant of SARS-CoV-2 had emerged, and by early 2022, the BA.4 and BA.5 subvariant strains of omicron had emerged. In August 2022, the US Food and Drug Administration approved versions of the mRNA vaccines by Pfizer/BioNTech and Modema containing equal amounts of mRNA encoding the ancestral SARS-CoV-2 Spike protein and the Spike protein from the BA.4 and BA.5 strains of the omicron variant. In September 2023, the vaccines were further updated to encode the Spike protein from the XBB lineage of the omicron variant.

[0006] Further vaccine options against SARS-CoV-2 BA.4 and BA.5 strains and that protect against infection by those and future strains continue to be needed.

SUMMARY

[0007] The present disclosure provides compositions for stimulating an immune response against an antigen and/or for enhancing immunogenicity of an antigen. In some embodiments, compositions disclosed herein comprise immunogenic compositions comprising: (1) an antigen fragment or an antigen variant, fused to (2) an adjuvant comprising a complement C3d-binding region. In some embodiments, an immunogenic composition disclosed herein can enhance the titers of the resulting antibody response and/or result in a measurable T cell response. In some embodiments, the adjuvant is or comprises a complement C3d-binding region of a Sbi protein from Staphylococcus aureus (e.g., Sbi III and/or Sbi IV). Also provided herein are pharmaceutical compositions and methods of using said pharmaceutical compositions to stimulate an immune response against an antigen and/or to enhance immunogenicity of an antigen.

[0008] Embodiments herein include, for example, a single stranded RNA polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises: (a) a polypeptide comprising amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), or comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some cases, the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). In some cases, the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV- 2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). In some cases, the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some cases, the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some cases, the complement C3d-binding polypeptide comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain III (SEQ ID NO: 9) and/or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain IV (SEQ ID NO: 10), or wherein the complement C3d-binding polypeptide comprises or consists of one or both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10). In some cases, wherein the complement C3d-binding polypeptide comprises the Sbi domain III and Sbi domain IV, the Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10) are contiguous, or are separated by a linker. In some cases above, (i) (a) is disposed N-terminus of (b); or (ii) (a) is disposed C-terminus of (b); (iii) (a) and (b) are contiguous or separated by a linker; or the polypeptide has (iv) a combination of (i) and (iii) or (ii) and (iii). In some cases therefore, (a) is disposed N-terminus of (b), and (a) and (b) are separated by a linker. In some cases, where the polypeptide includes a linker, (i) the linker is a peptidyl linker; (ii) the linker is a peptidyl linker comprising at least 60% glycine and/or serine; or (iii) the linker is chosen from a Gly- Gly-Gly-Gly-Ser (Gly4-Ser) linker (SEQ ID NO: 18), optionally wherein the Gly4-Ser linker comprises the amino acid sequence of SEQ ID NO: 6, or a histidine linker. In some cases, the linker comprises the amino acid sequence of SEQ ID NO: 6 or is encoded by the nucleotide sequence of SEQ ID NO: 7. In some cases, the polypeptide further comprises a secretion peptide. For example, the secretion peptide in some cases may comprise an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, or is encoded by the nucleotide sequence of SEQ ID NO: 3. In some cases, the polynucleotide comprises a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1 wherein T is replaced by U. In some cases, the polynucleotide comprises a 3’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 12 wherein T is replaced by U. In some cases, the polynucleotide further comprises a poly-adenosine (poly- A) tail at the 3’ end of the polynucleotide, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110- 140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0009] The present disclosure also encompasses, for example, a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U: (i) a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1, (ii) a nucleotide sequence encoding a secretion peptide comprising a nucleotide sequence comprising SEQ ID NO: 3, or encoding an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, (iii) optionally, a nucleotide sequence encoding a linker sequence, such as Ala-Ala, (iv) a nucleotide sequence comprising SEQ ID NO: 5, or encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, (v) optionally, a nucleotide sequence comprising SEQ ID NO: 7, or encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18, and (vi) a nucleotide sequence comprising SEQ ID NO: 11, or encoding domain III and domain IV of the Sbi of Staphylococcus aureus comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0010] The present disclosure further encompasses, for example, a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U: (i) a 5’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 1, (ii) a nucleotide sequence encoding a secretion peptide comprising the amino acid sequence of SEQ ID NO: 2, (iii) optionally, a nucleotide sequence encoding a linker sequence, such as Ala- Ala, (iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) of SEQ ID NO: 4, (v) optionally, a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18, (vi) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, and (vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12, and optionally (viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50- 100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0011] The present disclosure additionally encompasses, for example, a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U: (i) a 5’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 1, (ii) a nucleotide sequence encoding a secretion peptide comprising the amino acid sequence of SEQ ID NO: 2, (iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala, (iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), (v) a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6, (vi) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, and (vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12, and optionally (viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0012] The disclosure also includes, for example a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13 wherein T is replaced by U, or is transcribed from the nucleotide sequence of SEQ ID NO: 13; or wherein the polynucleotide comprises the ribonucleotide sequence of SEQ ID NO: 19.

[0013] The disclosure yet further includes, for example, a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide encodes the following amino acid sequences, disposed from N-terminus to C-terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 and SEQ ID NO: 8, (b) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8, (c) SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or (d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; or encodes the amino acid sequence of SEQ ID NO: 20. [0014] In any of the above single stranded RNA polynucleotides, in some cases the polynucleotide comprises a 5’ cap. In some cases, the polynucleotide comprises at least one modified ribonucleotide, optionally comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof. In some cases, the at least one modified ribonucleotide comprises: a 5’ monophosphate; a 5’ diphosphate; or a 5’ triphosphate. In some cases, the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of: wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate. In some cases, the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of

In some cases, the polynucleotide comprises cytidine residues, and: (i) at least 5% of cytidine residues in the polynucleotide comprise N4-acetylcytidine; (ii) less than 100% of cytidine residues in the polynucleotide comprise N4-acetylcytidine; or (iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polynucleotide comprise N4-acetylcytidine. In some cases, the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate. In some cases, the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of

[0015] In some cases, the polynucleotide comprises uridine residues and: (i) at least 5% of uridine residues in the polynucleotide comprise 5-hydroxymethyluridine; (ii) less than 100% of uridine residues in the polynucleotide comprise 5-hydroxymethyluridine; (iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polynucleotide comprise 5-hydroxymethyluridine; or (iv) more than 60% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine.

[0016] In some cases, at least one modified ribonucleotide comprises: Nl- methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5- aza-uridine, 2-thio-uridine (s2U), 5-methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), or any combination thereof.

[0017] In some cases, the at least one modified ribonucleotide comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’-O-acetylated and the modified ribonucleotide has a structure of

(a) wherein X is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate; and (b) wherein R is a nucleobase chosen from: adenine or a modified version thereof, a guanine or a modified version thereof, a cytosine or a modified version thereof, or a uracil or a modified version thereof.

[0018] In some cases, the nucleobase is adenine, and the modified ribonucleotide has a 5’ triphosphate and a structure of

[0019] In some cases, the nucleobase is guanine, and the modified ribonucleotide has a 5’ triphosphate and a structure of

[0020] In some cases, the nucleobase is cytosine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0021] In some cases, the nucleobase is N4-acetylcytidine, and the modified ribonucleotide has a 5’ triphosphate and a structure of

[0022] In some cases, the nucleobase is uracil, and the modified ribonucleotide has a 5’ triphosphate and a structure of

[0023] In some cases, the nucleobase is 5-hydroxymethyluridine and the modified ribonucleotide has a 5’ triphosphate and a structure of: [0024] In some cases, the nucleobase is N1 -methylpseudouridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0025] The present disclosure also encompasses a single stranded RNA polynucleotide, wherein: (i) at least 5% of the ribose moi eties are acetylated (2’-O- acetylated), or (ii) about 5% to about 99% of the ribose moi eties are acetylated (2’-O- acetylated).

[0026] The disclosure further includes a single stranded RNA polynucleotide that comprises a cap structure and where the cap structure does not comprise a 2’-O-acetylated ribose. Alternatively, in some cases, the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose. In some embodiments herein, the polyribonucleotide further comprises one or more ribonucleotides that does not comprise a 2’-0 acetylated ribose.

[0027] The disclosure herein also includes a single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U: (i) a 5’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 1, (ii) a nucleotide sequence encoding a secretion peptide comprising the amino acid sequence of SEQ ID NO: 2, (iii) a nucleotide sequence encoding a linker sequence, such as Ala-Ala, (iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), (v) a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6, (vi) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, and (vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12, and optionally (viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues; and further wherein: (a) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of cytidine residues in the polynucleotide comprise N4-acetylcytidine, and (b) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polynucleotide comprise 5-hydroxymethyluridine, and optionally (c) wherein the polynucleotide comprises a 5’ cap.

[0028] The disclosure herein further encompasses a DNA polynucleotide encoding the single stranded RNA polynucleotide as described above or elsewhere herein. The disclosure also includes an expression vector comprising the DNA polynucleotide, as well as a host cell comprising the single stranded RNA polynucleotide or the DNA polynucleotide or the expression vector.

[0029] The disclosure herein also includes, for instance, a pharmaceutical composition comprising the single stranded RNA polynucleotide or the DNA polynucleotide and at least one carrier or excipient. In some cases, the carrier or excipient comprises liposome nanoparticles (LNP). The disclosure also includes a fusion polypeptide encoded by the single stranded RNA polynucleotide or the DNA polynucleotide or the expression vector. The disclosure further includes a method of making the single stranded RNA polynucleotide described above or elsewhere herein, comprising: recombinantly joining a first nucleotide sequence that encodes the polypeptide of (a) and a second nucleotide sequence that encodes the complement C3d-binding polypeptide from a immunoglobulin-binding protein (Sbi) of Staphylococcus aureus of (b) to form a polynucleotide sequence, optionally wherein the first and/or second nucleotide sequence comprises a further nucleotide sequence encoding a linker sequence, and optionally wherein the first nucleotide sequence comprises a further nucleotide sequence encoding a secretion peptide, and wherein the first and second nucleotide sequences comprise DNA, and transcribing the DNA to make single stranded RNA. The disclosure also includes a kit comprising the single stranded RNA polynucleotide or the DNA polynucleotide or the expression vector or the pharmaceutical composition described above or elsewhere herein, and optionally further comprising instructions for use.

[0030] The present disclosure also encompasses a method comprising administering to a subject in need of vaccination against SARS-CoV-2 at least one dose of the pharmaceutical composition or the single stranded RNA polynucleotide as described above or elsewhere herein. In some cases, the at least one dose is administered in an effective amount to: (i) induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject; (ii) stimulate B cells in the subject; or (iii) both (i) and (ii). The present disclosure also encompasses a method comprising administering to a subject: a first dose of the pharmaceutical composition or the single stranded RNA polynucleotide or the DNA polynucleotide described above or elsewhere herein; and a second dose of the pharmaceutical composition or the single stranded RNA polynucleotide or the DNA polynucleotide described above or elsewhere herein, optionally wherein the time between the first and second doses is from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks. In some such cases, the first dose and the second dose are in the same amounts. Alternatively, in some cases, the pharmaceutical composition, single stranded RNA polynucleotide, DNA polynucleotide, or fusion polypeptide is administered in a single dose to the subject. In some cases, the method comprises administering a single stranded RNA polynucleotide at a dose of 0.3-300 pg of RNA, such as 3-150 pg of RNA, 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10- 30 pg of RNA. In some cases, administration is by intramuscular injection.

[0031] The disclosure herein also includes a pharmaceutical composition or single stranded RNA polynucleotide as described above or elsewhere herein for use in vaccination of a subject against SARS-CoV-2. In some cases, the pharmaceutical composition or single stranded RNA polynucleotide is administered to the subject in an effective amount to: (i) induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject; (ii) stimulate B cells in the subject; or (iii) both (i) and (ii). In some cases, the pharmaceutical composition or single stranded RNA polynucleotide is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts. Alternatively, in some cases the pharmaceutical composition or polynucleotide is administered in a single dose to the subject. In some cases, the pharmaceutical composition or single stranded RNA polynucleotide is administered to the subject at a dose of 0.3-300 pg of RNA, such as 3-150 pg of RNA, 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA.

[0032] The disclosure herein also includes the use of the single stranded RNA polynucleotide or pharmaceutical composition described above or elsewhere herein in the preparation of a medicament for vaccination of a subject against SARS-CoV-2. In some cases, at least one dose of the single stranded RNA polynucleotide or composition is administered in an effective amount to: (i) induce an immune response against a SARS-CoV- 2 Spike RBD domain polypeptide in the subject; (ii) stimulate B cells in the subject; or (iii) both (i) and (ii). In some cases, the single stranded RNA polynucleotide or pharmaceutical composition is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts. In other cases, the composition or polynucleotide is administered in a single dose to the subject. In some cases, the composition or polynucleotide is administered to the subject at a dose of 0.3-300 pg of RNA, such as 3- 150 pg of RNA, 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3- 50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA.

[0033] In some cases of the methods, compositions or polynucleotides for use, or uses described above, the subject is immunosuppressed. In some such cases, the immunosuppressed subject has received, is receiving, or will be receiving one or more transplants. In some such cases, the one or more transplants is an organ transplant. In some such cases, the organ transplant comprises: a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a stomach transplant, an intestine transplant, or any combination thereof. In other cases, the one or more transplants is a cell transplant. In some such cases, the cell transplant is a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof. In some such cases, the cell transplant is a transplant of a population of bone marrow cells, blood cells, or any combination thereof. In some such cases, the cell transplant is a transplant of a population of engineered cells. In other such cases, the cell transplant is a transplant of a population of non-engineered cells. In some cases, the one or more transplants is a tissue transplant. In some such cases, the tissue transplant comprises skin tissue transplant, bone tissue transplant, cartilage tissue transplant, adrenal tissue transplant, corneal tissue transplant, or any combination thereof. In some such cases, the transplant is an allogeneic transplant. In some cases of treating an immunosuppressed subject, the subject is receiving or has received immunosuppressive therapy. In some cases, the immunosuppressive therapy comprises an organ transplant conditioning regimen, chemotherapy, radiation therapy, or a treatment for an autoimmune disease. In some cases, the immunosuppressive therapy comprises administration of one or more of a calcineurin inhibitor, an antiproliferative agent, a steroid, an mTOR inhibitor, or any combination thereof. In some cases, the immunosuppressive therapy comprises administration of one or more of tacrolimus, mycophenolate mofetil, or prednisone, or any combination thereof.

[0034] These, and other aspects encompassed by the present disclosure, are described in more detail below and in the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0035] FIG. 1 shows results of an anti-SARS-CoV-2 RBD IgG serology assay on D28 mouse sera at 1 : 1,000,000 dilution. A panel of SARS-CoV-2 variants were assayed.

[0036] FIG. 2 shows pseudovirus microneutralization titer for D28 mouse sera against SARS-CoV-2 Omicron BA.4/5 and ancestral (Wuhan) SARS-CoV-2 reporter virus particles.

[0037] FIG. 3 shows the changes in murine body weights post SARS-CoV-2 viral challenge, in a viral challenge model. Open circles show vehicle control, closed squares show a 1.2 pg dose, upward closed triangles show a 0.4 pg dose, downward closed triangles show a 0.12 pg dose, each given on day 0 and day 21, (Groups 1-4), while closed diamonds show a 0.4 pg dose given only on day 0 (Group 5).

[0038] FIG. 4 shows the survival rates of AC70 hACE2 mice post SARS-CoV-2 viral challenge in the viral challenge model. Symbols and groups are as in Fig. 3.

[0039] FIG. 5 (Figs. 5A-5B) shows the changes in body weights following drug induced immunosuppression and treatment with the vaccine for female (Fig. 5 A) and male (Fig. 5B) animals. In Fig. 5A, circles show Group 1 (Gl), upward triangles show Group 3 (G3), diamonds show Group 5 (G5), and squares show Group 7 (G7). In Fig. 5B, squares show Group 2 (G2), downward triangles show Group 4 (G4), circles show Group 6 (G6), and open upward triangles show Group 8 (G8).

[0040] FIG. 6 shows the immune cell count in female and male mice treated with TMP or saline, with or without vaccination (Day 17). Male mice results are shown in darker bars to the right of female mice results.

CERTAIN DEFINITIONS

[0041] About or approximately. As used herein, the terms “about” and “approximately,” when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” or “approximately” in that context. For example, in some embodiments, the term “about” or “approximately” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0042] Administering: As used herein, the term “administering” or “administration” typically refers to administration of a composition to a subject to achieve delivery of an agent that is, or is included in, the composition. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. For example, in some embodiments, administration may be ocular, oral, parenteral, topical, etc. In some particular embodiments, administration may be bronchial (e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal (e.g., by intratracheal instillation), vaginal, vitreal, etc. In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses. In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.

[0043] Adjuvant: The term “adjuvant,” as used herein, refers to an agent that modulates and/or enhances an immune response to an agent that elicits an immune response. In some embodiments, an adjuvant is administered before, concurrently with or after administration of an agent that elicits an immune response. In some embodiments, an adjuvant and an agent that elicits an immune response are in one composition. In some embodiments, an adjuvant and an agent that elicits an immune response are in different compositions. In some embodiments, an adjuvant is or comprises a nucleic acid, polypeptide, polysaccharide, or small molecule. In some embodiments, an adjuvant is or comprises a complement binding domain. In some embodiments, an adjuvant is or comprises a C3d binding domain. In some embodiments, an adjuvant is or comprises a domain III of Sbi immunoglobulin-binding protein of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, an adjuvant is or comprises a domain IV of Sbi immunoglobulin-binding protein of Staphylococcus aureus, or a functional fragment or variant thereof. In some embodiments, an adjuvant comprises both a domain III and a domain IV of the Sbi of Staphylococcus aureus, or a functional fragment or a variant thereof.

[0044] Antigen'. The term “antigen”, as used herein, refers to an agent that elicits an immune response; and/or (ii) an agent that binds to a T cell receptor (e.g., when presented by an MHC molecule) or to an antibody. In some embodiments, an antigen elicits a humoral response (e.g., including production of antigen-specific antibodies); in some embodiments, an antigen elicits a cellular response (e.g., involving T-cells whose receptors specifically interact with the antigen). In some embodiments, an antigen comprises at least one epitope of a target protein. In some embodiments, an epitope may be a linear epitope. In some embodiments, an epitope may be a conformational epitope. In some embodiments, an antigen binds to an antibody and may or may not induce a particular physiological response in an organism. In general, an antigen may be or include any chemical entity such as, for example, a small molecule, a nucleic acid, a polypeptide, a carbohydrate, a lipid, a polymer (in some embodiments other than a biologic polymer [e.g., other than a nucleic acid or amino acid polymer) etc. In some embodiments, an antigen is or comprises a polypeptide. In some embodiments, an antigen is or comprises a glycan. Those of ordinary skill in the art will appreciate that, in general, an antigen may be provided in isolated or pure form, or alternatively may be provided in crude form (e.g., together with other materials, for example in an extract such as a cellular extract or other relatively crude preparation of an antigencontaining source). In some embodiments, antigens utilized in accordance with the present invention are provided in a crude form. In some embodiments, an antigen is a recombinant antigen.

[0045] Antigen variant: As used herein, the term “antigen variant” refers to an antigen that shows significant structural identity with a target protein antigen but differs structurally from the target protein antigen in the presence or level of one or more chemical moieties as compared to the target protein antigen. In some embodiments, an antigen variant differs functionally from a target protein antigen. In some embodiments, an antigen variant does not differ functionally from a target protein antigen. In some embodiments, an antigen comprises an epitope of a target protein antigen. In some embodiments, an antigen variant differs from a target protein antigen as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.

[0046] Delivery/contacting'. As used interchangeably herein, the term “delivery,” “delivering,” or “contacting” refers to introduction of a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell. A target cell can be cultured in vitro or ex vivo or be present in a subject (in vivo). Methods of introducing a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) into a target cell can vary with in vitro, ex vivo, or in vivo applications. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a cell culture by in vitro transfection. In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell via delivery vehicles (e.g., nanoparticles, liposomes, and/or complexation with a cellpenetrating agent). In some embodiments, a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) can be introduced into a target cell in a subject by administering a fusion polynucleotide (e.g., as described herein) or a fusion polypeptide (e.g., as described herein) to a subject.

[0047] Functional: As used herein, the term “functional” is used to refer to a form or fragment of an entity that exhibits a particular property and/or activity.

[0048] Fragment: A “fragment” of a material or entity as described herein has a structure that includes a discrete portion of the whole, but lacks one or more moieties found in the whole. In some embodiments, a fragment consists of such a discrete portion. In some embodiments, a fragment consists of or comprises a characteristic structural element or moiety found in the whole. In some embodiments, a fragment comprises a polynucleotide fragment. In some embodiments, a fragment comprises a polypeptide fragment. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) as found in the whole polynucleotide or whole polypeptide. In some embodiments, a polynucleotide fragment or a polypeptide fragment comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the whole polynucleotide or whole polypeptide. The whole polypeptide or whole polynucleotide may in some embodiments be referred to as the “parent” of the polynucleotide fragment or polypeptide fragment.

[0049] Fragment antigen: A “fragment antigen” is used herein to refer to a fragment which comprises an epitope of a target protein antigen. In some embodiments, an epitope is or comprises an epitope presented by MHC Class I. In some embodiments, an epitope is or comprises an epitope presented by MHC Class II. In some embodiments, a fragment antigen is a polypeptide fragment antigen. In some embodiments, a fragment antigen is encoded by a polynucleotide encoding a fragment antigen. In some embodiments, a polypeptide fragment antigen comprises or consists of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, 300 or more monomeric units (e.g., residues) as found in a target protein antigen polypeptide. In some embodiments, a polypeptide fragment antigen comprises or consists of at least about 5%, 10%, 15%, 20%, 25%, 30%, 25%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in a target protein antigen polypeptide. In some embodiments, a polypeptide fragment antigen comprises or consists of no more than about 50%, 40%, 30%, 20%, 10%, or 5% of the monomeric units (e.g., residues) found in a target protein antigen polypeptide.

[0050] Fragment antigen variant: As used herein, the term “fragment antigen variant” refers to a fragment antigen that shows significant sequence and/or structural identity with a fragment antigen but differs in sequence and/or structure from the fragment antigen in the presence or level of one or more chemical moieties as compared to the fragment antigen. In some embodiments, a fragment antigen variant differs functionally from a fragment antigen. In some embodiments, a fragment antigen variant does not differ functionally from a fragment antigen. In some embodiments, a fragment antigen variant differs from a fragment antigen as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone.

[0051] Nucleic acid / Oligonucleotide / Polynucleotide'. As used herein, the terms

“nucleic acid” and “polynucleotide” and “oligonucleotide” are used interchangeably, and refer to a polymer of 3 nucleotides or more. In some embodiments, a nucleic acid comprises DNA. In some embodiments, a nucleic acid comprises RNA. In some embodiments, a nucleic acid comprises messenger RNA (mRNA). In some embodiments, a nucleic acid is single stranded. In some embodiments, a nucleic acid is double stranded. In some embodiments, a nucleic acid comprises both single and double stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N-phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a “peptide nucleic acid”. In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxy cytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises on or more, or all, non-natural residues. In some embodiments, a non-natural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5- iodouridine, C5-propynyl-uridine, C5 -propynyl-cytidine, C5-methylcytidine, 2- aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6- O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 1 10, 120, 130, 140, 150, 160, 170, 180, 190, 20, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long. When a number of nucleotides is used as an indication of size, e.g., of a fusion polynucleotide, a certain number of nucleotides refers to the number of nucleotides on a single strand, e.g, of a fusion polynucleotide. In some embodiments, a nucleic acid is linear, i.e., beginning at a 5’ end and ending at a 3’ end. In some embodiments, a nucleic acid is circular, such as a circular RNA molecule.

[0052] Polypeptide. The term “polypeptide”, as used herein, generally has its art- recognized meaning of a polymer of at least three amino acids or more. Those of ordinary skill in the art will appreciate that the term “polypeptide” is intended to be sufficiently general as to encompass not only polypeptides having a complete sequence recited herein, but also to encompass polypeptides that represent functional, biologically active, or characteristic fragments, portions or domains (e.g, fragments, portions, or domains retaining at least one activity) of such complete polypeptides. Polypeptides may contain L-amino acids, D-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, polypeptides may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof.

[0053] Fusion polypeptide. As used herein, a “fusion polypeptide” or “fusion protein” refers to a “polypeptide” comprising amino acid sequences derived from two or more different proteins, such as at least one antigen sequence or domain from SARS CoV-2 and an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus, for example. In a “fusion polypeptide,” an amino acid sequence from one protein may be disposed N-terminal to an amino acid sequence from a different protein, for instance, and the two amino acid sequences may either be directly fused together, or indirectly fused together via a peptide linker sequence.

[0054] Secretion peptide'. As used herein, a “secretion peptide” refers to a generally

N-terminal polypeptide sequence of typically about 10-30 amino acids that serves to direct a translated polypeptide for secretion. It may also be referred to, in some cases, as a “signal peptide” or a “leader peptide” or “leader sequence.” A secretion peptide may be from a SARS-CoV-2 protein or it may be from a heterologous protein.

[0055] Linker '. A “linker” or “linker sequence” herein, in the context of a fusion polypeptide, refers to an amino acid sequence that serves to link together two portions of the fusion polypeptide. Linkers may have a wide variety of amino acid sequences and lengths, and may, in some embodiments, be flexible (i.e. lacking in tertiary structure), or in other cases may possess tertiary structure.

[0056] RNA oligonucleotide'. As used herein, the term “RNA oligonucleotide” refers to an oligonucleotide of ribonucleotides. In some embodiments, an RNA oligonucleotide is single stranded. In some embodiments, an RNA oligonucleotide is double stranded. In some embodiments, an RNA oligonucleotide comprises both single and double stranded portions. In some embodiments, an RNA oligonucleotide can comprise a backbone structure as described in the definition oV Nucleic acid/ Oligonucleotide" above. An RNA oligonucleotide can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA) oligonucleotide. In some embodiments where an RNA oligonucleotide is a mRNA oligonucleotide, an RNA oligonucleotide typically comprises at its 3’ end a poly(A) region. In some embodiments where an RNA oligonucleotide is a mRNA oligonucleotide, an RNA oligonucleotide typically comprises at its 5’ end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, a polynucleotide (e.g., a fusion polynucleotide) comprises an RNA oligonucleotide. When a number of ribonucleotides is used as an indication of size, e.g., of a fusion polynucleotide, a certain number of nucleotides refers to the number of ribonucleotides on a single strand, e.g., of a fusion polynucleotide.

[0057] Percent (%) amino acid sequence identity: with respect to a reference polypeptide or polynucleotide sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical with the amino acid residues or nucleotides in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity for the purposes of the alignment. Alignment for purposes of determining percent sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, Clustal W, Megalign (DNASTAR) software or the FASTA program package. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0058] Variant'. As used herein, the term “variant” refers to an entity that shows significant structural identity with a reference entity but differs structurally from the reference entity in the presence or level of one or more chemical moieties as compared with the reference entity. In many embodiments, a variant also differs functionally from its reference entity. In general, whether a particular entity is properly considered to be a “variant” of a reference entity is based on its degree of structural identity with the reference entity. For example, a variant polypeptide may differ from a reference polypeptide as a result of one or more differences in amino acid sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, etc.) covalently attached to the polypeptide backbone. Alternatively or additionally, in some embodiments, a variant polypeptide does not share at least one characteristic sequence element with a reference polypeptide. In some embodiments, the reference polypeptide has one or more biological activities. In some embodiments, a variant polypeptide shares one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide lacks one or more of the biological activities of the reference polypeptide. In some embodiments, a variant polypeptide shows a reduced level of one or more biological activities as compared with the reference polypeptide.

[0059] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., mRNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)), which is incorporated herein by reference for any purpose.

[0060] Subject: As used herein, in the context of vaccination against SARS-CoV-2, the term “subject” refers to a human unless clearly stated otherwise (e.g., use of a murine model, etc., or vaccination of another mammalian subject). In some embodiments, a subject is suffering from Covid-19. In some embodiments, a subject is susceptible to Covid-19 or to SARS-CoV-2 infection (for instance, a subject that has been exposed to others who have been diagnosed with Covid- 19 or who have Covid- 19 symptoms, or is a subject located in an area undergoing or at risk of undergoing high rates of SARS-CoV-2 infection such that vaccination is desirable). In some embodiments, a subject displays one or more symptoms or characteristics of Covid- 19 or to SARS-CoV-2 infection. In some embodiments, a subject does not display any symptom or characteristic of Covid- 19 or to SARS-CoV-2 infection. In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered. In some embodiments, the subject is an immunosuppressed subject. In some embodiments, the subject is an immunocompromised subject.

[0061] Immunosuppressed subject. As used herein, the term “immunosuppressed subject” refers to a subject having an immune response that is reduced, e.g., suppressed or compromised, compared to a reference. In some embodiments, an immune response that is reduced, e.g., suppressed or compromised, is reduced by about 5% to about 100% compared to a reference. In some embodiments, a reference comprises an immune response of a subject prior to initiation of a therapy. In some embodiments, a reference comprises an immune response of a healthy subject. In some embodiments, a reference comprises an immune response of a population of comparable healthy subjects. In some embodiments, an immunosuppressed subject is an “immunocompromised subject.” As described herein, in some instances, an immunosuppressed subject is receiving an agent (e.g., drug) to suppress an immune response in the subject. In some situations, an immunosuppressed subject has a disease, disorder, or condition that suppresses the subject’s immune system, e.g., one or more genetic mutations that suppresses a subject’s immune system. Methods for determining whether a subject is immunosuppressed or diagnosing a subject as being immunosuppressed are known in the art. For example, in some embodiments, a subject may have a reduced number or percentage of certain immune cells (e.g., B cells, T cells, macrophages, NK cells, etc.). In some embodiments, a subject may have certain immune cells (e.g., B cells, T cells, macrophages, NK cells, etc.) that have reduced functionality. In some embodiments, immunosuppression is characterized by a frequency or intensity of diseases, disorders, symptoms or conditions experienced by a subject in a set time frame. In some embodiments, immunosuppression is characterized by a reduced immune response (e.g., in magnitude and/or timing) to one or more pathogens by a subject as compared to a reference, e.g., a healthy subject.

[0062] Standard techniques may be used for recombinant DNA, oligonucleotide synthesis, e.g., RNA synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques may be performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures may be generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), which is incorporated herein by reference for any purpose.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0063] Traditional vaccine adjuvants, such as alum and poly-I:C, typically work by activating one or more Toll like receptors (TLR) and thereby putting local tissue into an antiviral state. In contrast, achieving high levels of antigen production with RNA vaccines, e.g., mRNA vaccines, demands minimal immunogenicity, as localized innate immune responses can dramatically reduce expression levels. This disclosure provides an RNA-native adjuvant technology, e.g., mRNA-native adjuvant technology, that can provide improvements in the strength of the immune response, without creating a countervailing effect on antigen expression. This can be accomplished, e.g., by using protein-based fusions to make the RNA- encoded antigen, e.g., mRNA-encoded antigen, more immunogenic in a way that was decoupled from the immunogenicity of the vaccine itself. In one embodiment, the fusion protein would either directly stimulate B cells and/or drive uptake by B cells, while minimizing non-specific inflammation at the site of RNA, e.g., mRNA, expression. A large set of candidate fusion domains were screened, as described, for example, in International Patent Publication No. WO2022/187424, and it was observed that Sbi fragments improved antibody titers across a range of antigen expression levels.

[0064] Without wishing to be bound by theory, it is believed that antibody production is driven by B cells that both 1) bind the antigen via their B cell receptor (BCR); and 2) become activated by a second signal. The activation signal is most commonly provided by a helper T cell, which means that effective antigens need to both bind BCRs, and contain peptides that are efficiently presented on MHC and that bind T cell receptor (TCR)s. A complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus, in contrast, binds to complement fragments that can directly license B cells, bypassing the need to generate a strong response in helper T cells. This can extend the design space of effective antigens by enabling, e.g., the use of fragments that lack good T cell epitopes. In some embodiments, one of the applications of the Sbi-based fusion architecture is to make minimal antigens containing only a portion of the naturally occurring protein. As an example, this disclosure provides compositions and uses of Sbi-based fusion polypeptides and polynucleotides encoding them, wherein the fusion polypeptides comprise a portion of the SARS-CoV-2 Spike protein (e.g., RBD and sub-RBD portions) from the Omicron BA.4 and BA.5 strains as an exemplary antigen fused to a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus (Sbi fragment), such as an Sbi fragment comprising one or both of Sbi domains III and IV (Sbi- III-IV) of S. aureus. SARS-CoV-2 BA.4 and BA.5 antigens

[0065] The present application relates to fusion polypeptides and polynucleotides encoding fusion polypeptides, wherein the fusion polypeptides comprise a portion of a SARS-CoV-2 Omicron BA.4 and/or BA.5 strain protein fused to a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some embodiments, the SARS-CoV-2 Omicron BA.4/BA.5 antigen is a Spike glycoprotein, such as an RBD or sub-RBD fragment. In some cases, the SARS-CoV-2 Omicron BA.4/BA.5 antigen comprises or consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD), which is provided herein as SEQ ID NO: 4. In other cases, the SARS-CoV-2 Omicron BA.4/BA.5 antigen comprises or consists of an amino acid sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4.

[0066] In some cases, the SARS-CoV-2 Omicron BA.4/BA.5 antigen polynucleotide sequence encodes amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD), which is provided herein as SEQ ID NO: 4. For example, in some cases, the nucleic acid sequence that encodes the SARS-CoV-2 Omicron BA.4/BA.5 polypeptide comprises or consists of the nucleic acid sequence of SEQ ID NO: 5. In other cases, the nucleic acid sequence encoding the SARS-CoV-2 Omicron BA.4/BA.5 polypeptide sequence is, for example, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 5, yet retains the ability encode a SARS-CoV-2 Omicron BA.4/BA.5 RBD domain or RBD subdomain polypeptide or one that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to such a polypeptide. For example, due to degeneracy in the genetic code, those of ordinary skill in the art would understand that other DNA sequences (including codon- optimized sequences) could encode the polypeptide sequence of SEQ ID NO: 4 or a sequence at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 4. Thus, in some cases, the nucleic acid sequence that encodes the SARS- CoV-2 Omicron BA.4/BA.5 polypeptide sequence is not identical to SEQ ID NO: 5, but is nonetheless degenerate to SEQ ID NO: 5 in that it comprises nucleotide changes that do not affect the encoded amino acid sequence. For example, certain changes in a polynucleotide sequence may be helpful in improving expression of a polypeptide within a particular host cell or organism. Complement C3d-binding polypeptide from an immunoglobulin-binding protein

[0067] Fusion polypeptides herein may also comprise a complement binding polypeptide that comprises a complement C3d binding polypeptide. An exemplary C3d binding polypeptide is an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. For instance, S. aureus binder of immunoglobulin (Sbi) is an exemplary polypeptide which can bind complement C3d (as described in Clark et al. (2011) Mol Immunol. 48(4): 452-462, the entire contents of which is incorporated herein by reference). Sbi comprises two immunoglobulin binding domains (Domains I and II) and two complement C3d binding domains (Domains III and IV). Sbi domains III and IV can bind C3d (in native C3, iC3b and C3dg) and can result in fluid phase consumption of C3 via activation of the alternative pathway (see Clark et al 2011). It has also been shown that Sbi can be secreted and is involved in S. aureus immune evasion (Burman et al., 2008 J. Biol. Chem: 283:17579- 17593). Without wishing to be bound by theory, it is believed that in some embodiments, a complement C3d-binding polypeptide from Sbi of S. aureus can be used as an adjuvant to enhance and/or modulate an immune response from a SARS-CoV-2 antigen described herein.

[0068] In some embodiments, any of the fusion polypeptides comprises a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of S. aureus. In some embodiments, the complement C3d binding polypeptide comprises one or both of Sbi domain III and Sbi domain IV of S. aureus, or a functional fragment or a variant thereof. In some embodiments, the complement C3d-binding polypeptide comprises or consists of Sbi domain III of S. aureus. In some embodiments, the complement C3d-binding polypeptide comprises or consists of Sbi domain IV of S. aureus. In some embodiments, the C3d-binding polypeptide comprises or consists of both Sbi domain III and Sbi domain IV of S. aureus. In such cases, the domains III and IV may be directly linked or may be separated by a linker peptide sequence. In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 8, 9, or 10. In some embodiments, a Sbi complement C3d binding polypeptide comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, a Sbi complement C3d binding polypeptide comprises the amino acid sequence of SEQ ID NO: 8, 9, or 10. In some embodiments, a Sbi complement C3d binding polypeptide comprises the amino acid sequence of SEQ ID NO: 8.

[0069] In some embodiments, the complement C3d-binding polypeptide is encoded by a polynucleotide that encodes one or both of Sbi domain III and Sbi domain IV of S. aureus, or a functional fragment or a variant thereof. In some embodiments, the polynucleotide encodes a complement C3d-binding polypeptide comprising or consisting of Sbi domain III of S. aureus. In some embodiments, the polynucleotide encodes a complement C3d-binding polypeptide comprising or consisting of Sbi domain IV of S. aureus. In some embodiments, the polynucleotide encodes a C3d-binding polypeptide comprising or consisting of both Sbi domain III and Sbi domain IV of S. aureus. In such cases, the domains III and IV may be directly linked or may be separated by a linker peptide sequence. In some embodiments, the polynucleotide sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 8, 9, or 10. In some embodiments, the polynucleotide sequence encodes an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 100% identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 8, 9, or 10. In some embodiments, the polynucleotide sequence encodes the amino acid sequence of SEQ ID NO: 8. In some cases, the polynucleotide sequence comprises or consists of SEQ ID NO: 11 or a nucleic acid sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 11. In some cases, the polynucleotide sequence encoding the complement C3d-binding polypeptide comprises or consists of SEQ ID NO: 11.

Exemplary Fusion polypeptides

[0070] This disclosure provides, inter alia, fusion polypeptides comprising (a) a polypeptide comprising amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), or comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus as well as polynucleotides encoding them. In some cases, the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). In some cases, the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). Such sequences may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 5 or a nucleic acid sequence degenerate to that sequence, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 5.

[0071] In some cases, the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. .In some cases, the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some cases, the complement C3d-binding polypeptide comprises or consists of both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10). In some cases, the complement C3d- binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some cases, the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some cases, the Sbi domain III (e.g., SEQ ID NO: 9) and Sbi domain IV (e.g., SEQ ID NO: 10) are contiguous, or are separated by a linker. Such sequences may be encoded, for example, by a nucleic acid sequence comprising or consisting of SEQ ID NO: 11 or a nucleic acid sequence degenerate to that sequence, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 11.

[0072] In some cases, (a) is disposed N-terminus of (b), while in other cases (a) is disposed C-terminal of (b). In some embodiments, (a) and (b) are contiguous (i.e. fused directly) or separated by a linker. In some embodiments, the linker is a peptidyl linker. In some embodiments, the peptidyl linker comprises at least 60% glycine and/or serine. In some embodiments, the linker is chosen from a linker comprising one or more repeats of Gly-Gly- Gly-Gly-Ser (Gly4-Ser) (SEQ ID NO: 18), or a Histidine linker. In some embodiments, the linker is a Gly4-Ser linker (SEQ ID NO: 18). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the Gly4-Ser linker (SEQ ID NO: 21). In some embodiments, the linker comprises 3 repeats of the Gly4-Ser linker (SEQ ID NO: 6). In some embodiments, the linker comprises the sequence of SEQ ID NO: 6. The Gly4-Ser linker with 3 repeats (SEQ ID NO: 6) may be encoded, for example, by the nucleic acid sequence of SEQ ID NO: 7, for instance, or a sequence that is degenerate to that sequence.

[0073] In some embodiments, the polypeptide further comprises a secretion peptide. In some embodiments, the secretion peptide is 10-30, or 15-30 amino acids in length. In some embodiments, the secretion peptide is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. In some embodiments, the secretion peptide comprises an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or 100% identity to the amino acid sequence of SEQ ID NO: 2. In some embodiments, the secretion peptide is encoded by a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to the amino acid sequence of SEQ ID NO: 3.

[0074] In some embodiments, the fusion polypeptide comprises the following amino acid sequences, disposed from N-terminus to C-terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 and SEQ ID NO: 8, (b) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8, (c) SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or (d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8. In some embodiments, the polypeptide comprises the following amino acid sequences, disposed from N-terminus to C-terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and SEQ ID NO: 8 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 8, (b) SEQ ID NO: 2 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 2; SEQ ID NO: 4 or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and SEQ ID NO: 8 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 8, (c) SEQ ID NO: 4 or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 6 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 6; and SEQ ID NO: 8 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 8, or (d) SEQ ID NO: 2 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 2; SEQ ID NO: 4 or an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; SEQ ID NO: 6 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 6; and SEQ ID NO: 8 or an amino acid having at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 8. In some cases, the secretion peptide (e.g., of SEQ ID NO: 2 or an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97% identical to the amino acid sequence of SEQ ID NO: 2) is removed during production of the polypeptide.

[0075] In some embodiments, the fusion polypeptide comprises the amino acid sequence of SEQ ID NO: 20, or is encoded by a polynucleotide sequence that encodes the amino acid sequence of SEQ ID NO: 20. In some embodiments, the fusion polypeptide comprises an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 20, or is encoded by a polynucleotide sequence that encodes an amino acid sequence at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 20.

[0076] In some embodiments, the fusion polypeptide comprises an amino acid sequence encoded by a polynucleotide comprising the DNA sequence of SEQ ID NO: 13 or or encoded by a polynucleotide comprising the RNA sequence of SEQ ID NO: 19. In some embodiments, the fusion polypeptide comprises an amino acid sequence encoded by polynucleotide comprising a DNA sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the DNA sequence of SEQ ID NO: 13, or by a polynucleotide comprising an RNA sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the RNA sequence of SEQ ID NO: 19. In some embodiments, the polynucleotide comprises an RNA sequence at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identical to the RNA sequence of SEQ ID NO: 19, or an RNA sequence 100% identical to SEQ ID NO: 19, and further comprising a poly-A tail, such as comprising for example, from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues in length. In some embodiments, the RNA polynucleotide is single stranded. In some embodiments, the RNA polynucleotide comprises a 5’ cap.

Polynucleotides encoding Fusion Polypeptides

[0077] The disclosure also provides polynucleotides encoding fusion polypeptides comprising (a) a polypeptide comprising amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), or comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus. In some cases, the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). In some cases, the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4). In some cases, the polynucleotide comprises, for example, a nucleic acid sequence comprising or consisting of SEQ ID NO: 5 or a nucleic acid sequence degenerate to that sequence, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 5.

[0078] In some cases, the complement C3d-binding polypeptide of (b) comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8. In some cases, the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8. In some cases, the complement C3d-binding polypeptide comprises or consists of both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10). In some cases, the complement C3d- binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 9. In some cases, the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 10. In some cases, the Sbi domain III (e.g., SEQ ID NO: 9) and Sbi domain IV (e.g., SEQ ID NO: 10) are contiguous, or are separated by a linker. In some cases, the polynucleotide comprises, for example, a nucleic acid sequence comprising or consisting of SEQ ID NO: 11 or a nucleic acid sequence degenerate to that sequence, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 11.

[0079] In some embodiments, the polynucleotide comprises a nucleic acid coding sequence arranged such that, in the encoded polypeptide, (a) is disposed N-terminus of (b). In other embodiments, it is arranged such that, in the polypeptide, (a) is disposed C-terminus of (b).

[0080] In some embodiments, (a) and (b) are contiguous or separated by a nucleotide sequence encoding a linker. In some embodiments, the linker is a peptidyl linker. In some embodiments, the peptidyl linker comprises at least 60% glycine and/or serine. In some embodiments, the linker is chosen from a linker comprising one or more repeats of Gly-Gly- Gly-Gly-Ser (Gly4-Ser) (SEQ ID NO: 18), or a Histidine linker. In some embodiments, the linker is a Gly4-Ser linker (SEQ ID NO: 18). In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the Gly4-Ser linker (SEQ ID NO: 21). In some embodiments, the linker comprises 3 repeats of the Gly4-Ser linker (SEQ ID NO: 6). In some embodiments, the linker comprises the sequence of SEQ ID NO: 6. The Gly4-Ser linker with 3 repeats (SEQ ID NO: 6) may be encoded, for example, by the nucleic acid sequence of SEQ ID NO: 7, for instance, or a sequence that is degenerate to that sequence.

[0081] In some embodiments, the polynucleotide further comprises a nucleotide sequence encoding a secretion peptide, such as SEQ ID NO: 3 or a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 3.

[0082] In some embodiments, the polynucleotide comprises a 5’ untranslated region and/or a 3’ untranslated region. For instance, an exemplary 5’ untranslated region comprises a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1. An exemplary 3’ untranslated region comprises a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 12.

[0083] In some cases, the polynucleotide comprises a poly-A tail following the 3’ untranslated region, which may be, for example, from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues in length. The length of the poly-A tail is approximate, and thus a specific length represents the expected length of the poly-A tail based on the DNA sequence to be transcribed, or the expected RNA sequence derived from that DNA sequence. The actual length of the poly-A tail of the transcribed RNA may vary from the expected length, for example, due to enzymatic processing events. Thus, a length of, for instance, 120, is based on the expected length from the DNA sequence to be transcribed to RNA, while the actual length may vary due to such post-transcriptional events.

[0084] In some embodiments, the polynucleotide comprises the following elements, arranged 5’ to 3’ :

(i) a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1,

(ii) a nucleotide sequence encoding a secretion peptide comprising a nucleotide sequence comprising SEQ ID NO: 3, or encoding an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2,

(iii) optionally, a nucleotide sequence encoding a linker sequence, such as Ala-Ala,

(iv) a nucleotide sequence comprising SEQ ID NO: 5, or encoding amino acid residues 331- 527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 4,

(v) optionally, a nucleotide sequence comprising SEQ ID NO: 7, or encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18, and

(vi) a nucleotide sequence comprising SEQ ID NO: 11, or encoding domain III and domain IV of the Sbi of Staphylococcus aureus comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

In some cases, the polynucleotide comprises a 3’ untranslated region. In some cases, the nucleotide sequence also includes a 3’ untranslated region and a poly-A tail following the 3’ untranslated region, for example, the poly-A tail comprising from 50-200 adenosine residues, such as from 50-150 adenosine residues, such as from 100-150 adenosine residues.

[0085] In some cases, the nucleotide sequence comprises, from 5’ to 3’:

(i) a 5’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 1,

(ii) a nucleotide sequence encoding a secretion peptide comprising the amino acid sequence of SEQ ID NO: 2,

(iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) of SEQ ID NO: 4,

(v) optionally, a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18,

(vi) a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 8, and

(vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12.

In some cases, the nucleotide sequence also includes a 3’ untranslated region and a poly-A tail following the 3’ untranslated region, for example, the poly-A tail comprising from 50-200 adenosine residues, such as from 50-150 adenosine residues, such as 100-150 residues in length.

[0086] In some cases, the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’

(iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala,

(iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), (v) a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6,

In some cases, the nucleotide sequence also includes a 3’ untranslated region and a poly-A tail following the 3’ untranslated region, for example, the poly-A tail comprising from 50-200 adenosine residues, such as from 50-150 adenosine residues, such as 100-150 adenosine residues.

[0087] In some cases, the polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13. In sone cases, the polynucleotide comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 13. In some cases, the polynucleotide differs from SEQ ID NO: 13, but encodes the polypeptide encoded by SEQ ID NO: 13 (i.e., it is degenerate to SEQ ID NO: 13).

[0088] The polynucleotides herein may be composed of RNA or DNA, and may be included within various types of vectors, for example, to improve expression of the encoded fusion polypeptides. For example, vectors comprising polynucleotides herein include viral vectors, nonlimiting examples of which include a retrovirus vector, an adenovirus vector, an adeno-associated virus vector or a lentivirus vector or an RNA vector, among others. In some embodiments, a polynucleotide is or comprises RNA. In some embodiments, a polynucleotide is or comprises messenger RNA (mRNA). It is understood that an RNA polynucleotide comprising a specified nucleotide sequence herein comprises the version of that sequence in which T is replaced by U. The RNA may be single stranded in some cases, or it may be double stranded. In some embodiments, the RNA may be chemically modified, such as modified at the 5’ and/or 3’ end. In some embodiments, the RNA may be circular RNA rather than linear RNA. In some embodiments, a fusion polynucleotide is or comprises DNA. In some embodiments, the DNA may be chemically modified, such as modified at the 5’ and/or 3’ end. And as described further below, an RNA polynucleotide may also comprise a modified backbone or modified ribonucleobases, such as a modified U and/or C. In cases where a polynucleotide comprising a particular sequence herein also comprises a modified nucleotide, such as, for instance, a modified C or U, it is understood that when referring to the sequence by its SEQ ID NO, the wild type nucleotide, such as C or U, in the SEQ ID NO will be replaced by its modified version. [0089] In cases where the polynucleotide is RNA, the RNA may be single or double stranded, and may be encoded by one or more of the DNA sequences provided herein. For example, in some embodiments, a DNA polynucleotide may be prepared and used to transcribe an RNA polypeptide, which may then be administered to a subject in order to cause expression of the fusion polypeptide in the subject.

[0090] Accordingly, the disclosure also provides RNA polynucleotides encoding fusion polypeptides, which may be single or double stranded. In some embodiments, the RNA polynucleotide comprises a nucleic acid sequence encoding a Spike RBD domain comprising or consisting of SEQ ID NO: 5, or a nucleic acid sequence degenerate to SEQ ID NO: 5, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 5, wherein T in SEQ ID NO: 5 is replaced by U.

[0091] In some cases, the RNA polynucleotide comprises, for example, a nucleic acid sequence encoding domains III and IV of the Sbi of Staphylococcus aureus comprising or consisting of SEQ ID NO: 11 or a nucleic acid sequence degenerate to that sequence, or a nucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% identical to SEQ ID NO: 11, wherein T is replaced by U.

[0092] In some such embodiments, the polynucleotide comprises a nucleic acid coding sequence arranged such that, in the encoded polypeptide, the RBD domain is disposed N-terminus of the Sbi domain. In other embodiments, it is arranged such that, in the polypeptide, the RBD domain is disposed C-terminus of the Sbi domain.

[0093] In some embodiments, the RBD and Sbi domains encoded by the RNA polynucleotide are contiguous or separated by a linker. In some embodiments, the linker is a peptidyl linker. In some embodiments, the peptidyl linker comprises at least 60% glycine and/or serine. In some embodiments, the linker is chosen from a linker comprising one or more repeats of Gly-Gly-Gly-Gly-Ser (Gly4-Ser) (SEQ ID NO: 18), or a Histidine linker. In some embodiments, the linker is a Gly4-Ser linker. In some embodiments, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 repeats of the Gly4-Ser linker. In some embodiments, the linker comprises 3 repeats of the Gly4-Ser linker. In some embodiments, the linker comprises the sequence of SEQ ID NO: 6. The Gly4-Ser linker with 3 repeats may be encoded, for example, by the nucleic acid sequence of SEQ ID NO: 7, for instance, or a sequence that is degenerate to that sequence, wherein T is replaced by U.

[0094] In some embodiments, the RNA polynucleotide further comprises a nucleotide sequence encoding a secretion peptide, such as SEQ ID NO: 3 or a polynucleotide sequence having at least 80%, at least 85%, at least 90%, at least 95% identity to SEQ ID NO: 3, wherein T is replaced by U.

[0095] In some embodiments, the polynucleotide comprises a 5’ untranslated region and/or a 3’ untranslated region. For instance, an exemplary 5’ untranslated region comprises a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1, in which T is replaced by U. An exemplary 3’ untranslated region comprises a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 12, in which T is replaced by U.

[0096] In some cases, the polynucleotide comprises a poly-A tail following the 3’ untranslated region, which may be, for example, from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues in length.

[0097] In some embodiments, the RNA polynucleotide comprises the following elements, arranged 5’ to 3’, wherein U replaces T in the nucleotide sequences referred to below:

(iv) a nucleotide sequence comprising SEQ ID NO: 5, or encoding amino acid residues 331- 527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 4, (v) optionally, a nucleotide sequence comprising SEQ ID NO: 7, or encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18, and

[0098] In some cases, the RNA polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein U replaces T in the nucleotide sequences referred to below:

In some cases, the nucleotide sequence also includes a 3’ untranslated region and a poly-A tail following the 3’ untranslated region, for example, the poly-A tail comprising from 50-200 adenosine residues, such as from 50-150 adenosine residues, such as 100-150 residues in length. [0099] In some cases, the RNA polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein U replaces T in the nucleotide sequences referred to below:

(iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala,

(iv) a nucleotide sequence encoding amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4),

(v) a nucleotide sequence encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6,

[0100] In some cases herein, the polynucleotide is RNA, and comprises a 5’ cap. In some cases, the 5’ cap may comprise the structure of a m7G(5’)ppp(5’)(2’OMeA)pG, i.e. a 7- m ethyl guanosine triphosphate linked to a 2’O-methyl adenosine - guanine dinucleotide. A commercially available 5’ capping reagent, for example, includes CleanCap® Reagent AG (N-7113; TriLink Biotechnologies, a division of Maravai Life Sciences).

[0101] In some cases, the RNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 13, wherein T is replaced by U. In sone cases, the polynucleotide comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 13, wherein T is replaced by U. In some cases, the polynucleotide differs from SEQ ID NO: 13 (T replaced by U), but encodes the polypeptide encoded by SEQ ID NO: 13 (i.e., it is degenerate to SEQ ID NO: 13). In some cases, the RNA polynucleotide is transcribed from the nucleic acid sequence of SEQ ID NO: 13, or from a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 13, such as a sequence that differs from SEQ ID NO: 13 but is degenerate to it (i.e. encodes the same polypeptide).

[0102] In some cases, the RNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19. In some cases, the RNA polynucleotide comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 19. In some cases, the polynucleotide differs from SEQ ID NO: 19, but encodes the polypeptide encoded by SEQ ID NO: 19 (i.e., it is degenerate to SEQ ID NO: 19). In some cases, the RNA polynucleotide comprises the nucleic acid sequence of SEQ ID NO: 19 or comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 19, or a nucleotide sequence degenerate to SEQ ID NO: 19, and further comprises a poly-A tail, which may be, for example, from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues in length. In some cases, the RNA is single stranded. In some cases, the RNA polynucleotide also comprises a 5’ cap, which in some cases may comprise the structure of a m7G(5’)ppp(5’)(2’OMeA)pG, i.e. a 7-methyl guanosine triphosphate linked to a 2’O-methyl adenosine - guanine dinucleotide.

Polynucleotides comprising modified nucleotides

[0103] In certain embodiments herein, the DNA or RNA polynucleotides encoding fusion proteins herein comprise modified nucleotides. In some cases, an RNA polynucleotide comprises modified nucleotides. For example, a polyribonucleotide (i.e., an RNA polynucleotide) may further comprise one or more modified ribonucleotides comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0104] In some embodiments, the polynucleotide is an RNA comprising a modified cytidine. In some embodiments, at least 5% of cytidine residues in the polyribonucleotide are modified. In some embodiments, less than 100% of cytidine residues in the polyribonucleotide are modified. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polyribonucleotide are modified. [0105] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate.

[0106] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of

[0107] In some embodiments, at least 5% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine rather than cytidine. In some embodiments, less than 100% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine. In some embodiments, the N4-acetylcytidine is introduced by using N4-acetylcytidine ribonucleotides during transcription of a DNA sequence into RNA in place of cytidine ribonucleotides, such that at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine in the transcribed RNA. In some embodiments, more than 95% of cytidine residues in the polyribonucleotide comprise N4- acetylcy didine. In cases where the polynucleotide is stated to include such a modified cytidine of at least a particular percentage, the “C” in the sequence of the RNA will be understood to mean either cytidine or N4-acetylcytidine.

[0108] In some embodiments, the polynucleotide is RNA comprising modified uridine. In some embodiments, at least 5% of uridine residues in the polyribonucleotide are modified. In some embodiments, less than 100% of uridine residues in the polyribonucleotide are modified. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of uridine residues are modified.

[0109] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5- hydroxymethyluridine and has a structure of wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate. [0110] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5- hydroxymethyluridine and has a structure of

[oni] In some embodiments, the polyribonucleotide comprises modified uridine residues. In some embodiments, at least 5% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, less than 100% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polyribonucleotide comprise 5- hydroxymethyluridine. In some embodiments, more than 95% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine. In cases where the polynucleotide is stated to include such a modified uridine of at least a particular percentage, the “U” in the sequence of the RNA will be understood to mean either uridine or 5-hydroxymethyluridine.

[0112] In some embodiments, the polynucleotide is RNA comprising both modified cytidine and modified uridine. In some embodiments, the RNA comprises both N4- acetylcytidine and 5-hydroxymethyluridine, as described above. For example, the RNA may be transcribed from DNA in a process that uses N4-acetylcytidine and 5- hydroxymethyluridine ribonucleotides in place of cytidine and uridine ribonucleotides so that in some embodiments, at least 5% of both cytidine and uridine residues in the polyribonucleotide are modified. In some embodiments, less than 100% of each of cytidine and uridine residues in the polyribonucleotide are modified. In some embodiments, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of both cytidine and uridine residues are modified. In some cases, at least 95% of cytidine and uridine residues are modified.

[0113] In some embodiments, a polyribonucleotide disclosed herein comprises one or more modified ribonucleotides comprising: N1 -methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5- methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), or any combination thereof. Similarly, RNA may be transcribed from DNA in a process that includes modified ribonucleotides such that the modified ribonucleotides are incorporated in place of A, C, G and/or U ribonucleotides either completely, or at a desired percentage.

[0114] In some embodiments, one or more modified ribonucleotides comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’- O-acetylated and the modified ribonucleotide has a structure of:

(a) wherein X is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate; and

(b) wherein R is a nucleobase chosen from: adenine or a modified version thereof, a guanine or a modified version thereof, a cytosine or a modified version thereof, or a uracil or a modified version thereof. [0115] In some embodiments, the nucleobase is adenine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0116] In some embodiments, the nucleobase is guanine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0117] In some embodiments, the nucleobase is cytosine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0118] In some embodiments, the nucleobase is N4-acetylcytidine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0119] In some embodiments, the nucleobase is uracil, and the modified ribonucleotide has a 5’ triphosphate and a structure of

[0120] In some embodiments, the nucleobase is 5-hydroxymethyluridine and the modified ribonucleotide has a 5’ triphosphate and a structure of: [0121] In some embodiments, the nucleobase is N1 -methylpseudouridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0122] In some embodiments, at least 5% of the ribose moieties are acetylated (2’-O- acetylated).

[0123] In some embodiments, about 5% to about 99% of the ribose moieties are acetylated (2’-O-acetylated).

[0124] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure does not comprise a 2’-O-acetylated ribose.

[0125] In some embodiments, a polyribonucleotide disclosed herein comprises a cap structure and the cap structure comprises a 2’-O-acetylated ribose.

[0126] In some embodiments, a polyribonucleotide disclosed herein further comprises one or more ribonucleotides that does not comprise a 2’-0 acetylated ribose.

[0127] In some cases, the disclosure relates to an RNA polynucleotide that comprises the nucleic acid sequence of SEQ ID NO: 19. In sone cases, the RNA polynucleotide comprises a nucleic acid sequence at least 80%, at least 85%, at least 90%, or at least 95% identical to that of SEQ ID NO: 19. In some cases, the polynucleotide differs from SEQ ID NO: 19, but encodes the polypeptide encoded by SEQ ID NO: 19 (i.e., it is degenerate to SEQ ID NO: 19). In some cases, the RNA polypeptide comprises one or more modified nucleotides, for example, which may be introduced during in vitro transcription by using modified nucleotide precursors. For example, in some embodiments, the RNA polynucleotide comprises N4-acetylcytidine and 5-hydroxymethyluridine residues. For instance, in some cases, the RNA polynucleotide comprises at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% N4- acetylcytidine and 5-hydroxymethyluridine residues in place of C and U residues, such as due to use of modified nucleotide precursors during transcription.

Methods of making polynucleotides and fusion polypeptides

[0128] The present disclosure also concerns methods of making the above-described polynucleotides, for example, in some embodiments comprising recombinantly joining a first nucleotide sequence that encodes the polypeptide of (a) and a second nucleotide sequence that encodes the complement C3d-binding polypeptide from a immunoglobulin-binding protein (Sbi) of Staphylococcus aureus of (b) to form a polynucleotide sequence, optionally wherein the first and/or second nucleotide sequence comprises a further nucleotide sequence encoding a linker sequence, and optionally wherein the first nucleotide sequence comprises a further nucleotide sequence encoding a secretion peptide. In some cases, DNA polynucleotides can then be made in the form of a vector, which may then be used for expression of fusion polypeptides. For example, fusion polypeptides herein can also be made by expressing a polynucleotide herein or a vector comprising the polynucleotide in a host cell under conditions promoting expression of the fusion polypeptide, and optionally recovering the fusion polypeptide.

[0129] In other cases, DNA polynucleotides may be used directly in pharmaceutical compositions, or may be used for transcription to RNA polynucleotides, such as in vitro transcription. In some cases, transcribed polyribonucleotides may contain modifications comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof, as described above. For example, to incorporate such modifications, ribonucleotides comprising a modified ribose, modified backbone, or modified nucleobase may be used in place of one of the four natural ribonucleotides, either fully or partially, in order to obtain an RNA polynucleotide including such modifications. In some embodiments, the modifications may be as described above. For example, in order to obtain a modified C or modified U at a desired frequency in an RNA, for instance, one could use a particular percentage of unmodified and modified C or U ribonucleotide triphosphate starting materials so as to give the desired percentage of modified C or U in the final RNA transcribed in vitro. The same process may be used with other modified bases or backbones.

[0130] The following table provides polypeptide and DNA sequences disclosed herein:

Compositions and Kits Comprising Polynucleotides Encoding Fusion Polypeptides

[0131] Pharmaceutical compositions of the present disclosure may comprise a polypeptides disclosed herein (e.g., a fusion polypeptide), a polynucleotide disclosed herein, or an expression vector comprising a polynucleotide. In some embodiments, a pharmaceutical composition may comprise a pharmaceutically acceptable excipient, a diluent, or a combination thereof. In some embodiments, a pharmaceutical composition may comprise buffers such as neutral buffered saline, phosphate buffered saline and the like; carbohydrates such as glucose, mannose, sucrose, or dextrans; mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; and preservatives.

[0132] In some embodiments, a pharmaceutical composition is formulated for administration according to any of the routes of administration disclosed herein. In some embodiments, a pharmaceutical composition is formulated for intramuscular administration, intradermal administration, intravenous administration, or subcutaneous administration.

Lipid Nanoparticle (LNP) formulations

[0133] In some embodiments, a polynucleotide herein may be formulated in a lipid nanoparticle (LNP) formulation, or in another medium intended for use with pharmaceutical polynucleotide molecules. In some such embodiments, the polynucleotide is RNA. In other cases, the polynucleotide is DNA. In some cases, the polynucleotide is RNA comprising one or more modified nucleotides, as described above. The RNA may be single stranded, or it may be double stranded.

[0134] Such LNP formulations may be provided in a variety of pharmaceutically suitable media, such as aqueous buffers, and may optionally comprise further excipients or carriers.

[0135] For example, in some cases, the polynucleotide comprises an RNA, e.g., an mRNA, that is formulated in an LNP formulation. Accordingly, in some embodiments, the disclosure provides an LNP formulation comprising a polynucleotide comprising an RNA, e.g., mRNA. In some cases, the pharmaceutical compositions are for use in vaccination against SARS-CoV-2.

[0136] The disclosure herein also relates to kits comprising a polynucleotide herein, or a vector comprising the polynucleotide, a fusion polypeptide herein, and/or a pharmaceutical composition comprising the polynucleotide, vector, or fusion polypeptide. For example, a kit may be used, for example, for testing a potential pharmaceutical product, or for infecting a cell with the polynucleotide or vector, for example, to make copies of the polynucleotide or vector in order to produce pharmaceutical compositions or for administration to subjects. In some cases, a kit may further comprise instructions for use based on such purposes. Therapeutic Uses

[0137] In some embodiments, a pharmaceutical composition comprising a polynucleotide, vector, or fusion polypeptide, such as an LNP formulation comprising a polynucleotide comprising an RNA, e.g., mRNA, is administered to a subject, for example, to provide an immune response to the Spike RBD polypeptide of the fusion polypeptide. In some embodiments, such an immune response may help to protect a subject against one or more symptoms of Covid-19 and/or against infection with SARS-CoV-2, such as SARS- CoV-2 Omicron BA.4 or BA.5 strains or similar strains. Thus, the present disclosure also contemplates methods comprising administering such pharmaceutical compositions to subjects that are in need of vaccination against SARS-CoV-2 to enhance and/or modulate an immune response. In some embodiments, the immune response is elicited by an antigen comprised in the polynucleotide, e.g., the Spike RBD polypeptide sequence.

[0138] This disclosure provides a method comprising administering to a subject in need thereof at least one dose of a pharmaceutical composition comprising a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein. In some embodiments, the at least one dose is administered in an effective amount to induce an immune response against the fragment antigen in the subject. In some cases, methods comprise administering to a subject in need of vaccination against SARS-CoV-2 at least one dose of a pharmaceutical composition herein, or a polynucleotide or fusion polypeptide herein. In some embodiments, the dose is effective to induce an immune response against the SARS-CoV-2 antigen of the fusion polypeptide, and/or against other related SARS-CoV-2 antigens. In some cases, the dose is sufficient to induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject, and/or to stimulate B cells in the subject.

[0139] Disclosed herein is a method comprising administering to a subject: a first dose of a pharmaceutical composition disclosed herein; and a second dose of a pharmaceutical composition disclosed herein. In some embodiments, a pharmaceutical composition comprises a fusion polypeptide disclosed herein, a fusion polynucleotide disclosed herein, or an expression vector comprising a fusion polynucleotide disclosed herein.

[0140] In some cases, more than one dose of the pharmaceutical composition, polynucleotide, or fusion polypeptide is administered. For example, the time between the first and second doses may optionally be from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks. In some cases, a first dose and a second dose are administered at least 3 days, 4 days, 5 days, 6 days, 7 days, 2 weeks, 3 weeks, or 4 weeks apart. In some embodiments, a first dose and a second dose are in the same amount. In some embodiments, a first dose and a second dose are in different amounts.

[0141] In some embodiments, the pharmaceutical composition, polynucleotide, or polypeptide is administered in an amount effective for enhancing the immunogenicity of the SARS-CoV-2 antigen component of the fusion polypeptide. In some embodiments of any of the methods or uses disclosed herein, administration of the polynucleotide, fusion polypeptide or pharmaceutical composition results in a humoral response. In some embodiments, the humoral response is an antibody response. In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide or pharmaceutical composition results in an increased titer of an antibody response (e.g., titer in serum of antibodies binding to Spike (S) protein of SARS-CoV-2 or the receptor binding domain (RBD) of the S protein). In some embodiments, the increase in titer is an increase of about 10 fold to about 500 fold. In some embodiments of any of the methods or uses disclosed herein, administration of the fusion polynucleotide, fusion polypeptide or pharmaceutical composition results in an increased titer of neutralizing antibodies against SARS-CoV-2 strain Omicron BA.4 and/or BA.5. In some embodiments, the increased titer of the antibody response and/or the increase in neutralizing antibodies is compared to administration of an otherwise similar fusion polynucleotide that does not comprise an Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or a variant thereof. In some embodiments, the increased titer of the antibody response and/or the increase in neutralizing antibodies is compared to administration of an otherwise similar fusion polypeptide that does not comprise an Sbi domain III, or a fragment or variant thereof; and an Sbi domain IV, or a fragment or a variant thereof. In some embodiments, the increased titer of the antibody response and/or the increase in neutralizing antibodies is compared to administration of an otherwise similar pharmaceutical composition that does not comprise a nucleotide sequence encoding Sbi domain III, or a fragment or variant thereof; and Sbi domain IV, or a fragment or a variant thereof. In some embodiments, the subject shows a seroresponse, calculated based on serum antibody titer (e.g., of antibodies binding to Spike (S) protein of SARS-CoV-2 or the receptor binding domain (RBD) of the S protein), defined as at least a 4-fold increase from a pre-dose baseline, or shows a seroresponse, calculated based on increased neutralizing antibodies. In some embodiments, the increased titer of the antibody response, the increase in neutralizing antibodies, and/or the seroresponse lasts for at least 7, at least 30, at least 90, or at least 180 days post dosing (if a single dose is administered) or for at least 7, at least 30, at least 90, or at least 180 days post final dosing (if more than one dose is administered).

[0142] In some embodiments of any of the methods or uses disclosed herein, the composition is administered via any one of the following routes of administration: intramuscular, intravenous, subcutaneous, intrathecal, intradermal, ocular, intranasal, sublingual, or oral. In some embodiments, the formulation is administered intramuscularly.

[0143] In some cases, the subject to be vaccinated is 18-65 years of age. In some embodiments, the subject to be vaccinated is immunosuppressed. In some embodiments, the subject to be vaccinated is immunocompromised.

[0144] For example, in some cases, the subject is receiving immunosuppressants, has been diagnosed as having a weakened immune system naturally or because of a disease, disorder, or condition, and/or is a transplant recipient, such as an organ transplant recipient. For example, the present disclosure recognizes that immunosuppressed subjects often have the lowest vaccination responses (see Bin Lee et al., (2022) BMJ 2022;376: e068632). In some embodiments, the subject is an organ transplant recipient. In some embodiments, the subject has received or is receiving an immunosuppressive therapy. In some embodiments, an immunosuppressive therapy comprises: an organ transplant conditioning regimen, chemotherapy, radiation therapy, or a treatment for an autoimmune disease. In some embodiments, an organ transplant conditioning regimen comprises a calcineurin inhibitor, an antiproliferative agent, a steroid, an mTOR inhibitor, or any combination thereof. In some embodiments, a calcineurin inhibitor comprises tacrolimus. In some embodiments, an antiproliferative agent comprises mycophenolate mofetil. In some embodiments, a steroid comprises prednisone. In some embodiments, organ transplant immunosuppression induction and/or maintenance treatment regimen comprises tacrolimus, mycophenolate mofetil and prednisone, or any combination thereof. In some embodiments, where the subject has received an organ transplant, the organ transplant comprises: a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a stomach transplant, an intestine transplant, or any combination thereof. In other cases, the transplant is a cell transplant. In some embodiments, a cell transplant is a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof. In some embodiments, a cell transplant is a transplant of a population of bone marrow cells, blood cells, or any combination thereof. In some embodiments, a cell transplant is a transplant of a population of engineered cells. In some embodiments, a cell transplant is a transplant of a population of non-engineered cells. In yet other cases, the transplant is a tissue transplant. In some embodiments, a tissue transplant comprises skin tissue transplant, bone tissue transplant, cartilage tissue transplant, adrenal tissue transplant, corneal tissue transplant, or any combination thereof. In some cases, the transplant is an allogeneic transplant.

[0145] Further, immunosuppressed subjects as described herein can have, or be suspected of having, or be predisposed to one or more disorders, e.g., as described herein, and/or have received or will be receiving one or more treatments, e.g., as described herein. For example, an immunosuppressed subject can be a subject who: (1) has received, is receiving, or will be receiving a transplant, and (2) has one or more disorders disclosed herein. As another example, an immunosuppressed subject can be a subject who: (1) has a suppressed or compromised immune system due to a drug, e.g., an immunosuppressive therapy (e.g., an organ transplant conditioning regimen, a therapy that suppresses an immune system (e.g., an antibody therapy), a B-cell targeting therapy, a T-cell targeting therapy, a chemotherapy, a radiation therapy, a cancer therapy, a treatment for an inflammatory disease and/or a treatment for an autoimmune disease), and (2) has one or more disorders disclosed herein.

[0146] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving one or more transplants. In some embodiments, an immunosuppressed subject who has received, is receiving, or will be receiving one or more transplants can also be administered one or more therapeutic agents and/or be subjected to one or more procedures, e.g., dialysis.

[0147] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving one or more dialysis treatments. In some embodiments, a dialysis treatment is or comprises hemodialysis. In some embodiments, a dialysis treatment is or comprises peritoneal dialysis. [0148] In some embodiments, one or more transplants described herein is an organ transplant. In some embodiments, an organ transplant comprises: a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a stomach transplant, an intestine transplant, or any combination thereof.

[0149] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a kidney transplant.

[0150] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a liver transplant.

[0151] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a heart transplant.

[0152] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving lung transplant.

[0153] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a pancreas transplant.

[0154] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a stomach transplant.

[0155] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving an intestine transplant. In some embodiments, an immunosuppressed subject who has received, is receiving, or will be receiving one or more transplants can also be administered one or more therapeutic agents and/or be subjected to one or more procedures, e.g., dialysis. In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving one or more dialysis treatments. In some embodiments, a dialysis treatment is or comprises hemodialysis. In some embodiments, a dialysis treatment is or comprises peritoneal dialysis.

[0156] In some embodiments, one or more transplants described herein is a cell transplant. In some embodiments, a cell transplant is a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof. In some embodiments, a cell transplant is a transplant of a population of bone marrow cells, blood cells, or any combination thereof. [0157] In some embodiments, a cell transplant is a transplant of a population of engineered cells.

[0158] In some embodiments, a cell transplant is a transplant of a population of nonengineered cells.

[0159] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof.

[0160] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a transplant of a population of bone marrow cells, blood cells, or any combination thereof.

[0161] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a transplant of a population of engineered cells.

[0162] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a transplant of a population of non-engineered cells.

[0163] In some embodiments, one or more transplants described herein is a tissue transplant. In some embodiments, a tissue transplant comprises skin tissue transplant, bone tissue transplant, cartilage tissue transplant, adrenal tissue transplant, corneal tissue transplant, or any combination thereof.

[0164] In some embodiments, a transplant is an allogeneic transplant.

[0165] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a skin tissue transplant.

[0166] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a bone tissue transplant.

[0167] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a cartilage tissue transplant.

[0168] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving an adrenal tissue transplant. [0169] In some embodiments, an immunosuppressed subject has received, is receiving, or will be receiving a corneal tissue transplant.

[0170] In some embodiments, an immunosuppressed subject has or has been diagnosed with one or more diseases, e.g., as described herein. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for one or more diseases, e.g., as described herein.

[0171] In some embodiments, an immunosuppressed subject has or has been diagnosed with a rare disease, a lung disease, a liver disease, a kidney disease, a blood disorder, an autoimmune disease, an immunodeficiency disorder, a cardiovascular disorder, a cancer, or any combination thereof.

[0172] In some embodiments, an immunosuppressed subject has or has been diagnosed with a rare disease, e.g., cystic fibrosis. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a rare disease, e.g., cystic fibrosis. In some embodiments, treatments for cystic fibrosis include cystic fibrosis transmembrane conductance regulator (“CFTR”) modulator therapy (e.g., ivacaftor, lumacaftor-ivacaftor, tezacaftor-ivacaftor, ETI, etc.). See, for example, Southern, K. et al., “Standards for the care of people with cystic fibrosis; establishing and maintaining health,” Journal of Cystic Fibrosis, Volume 23, Issue 1, 12-28, the entire contents of which are hereby incorporated by reference in its entirety.

[0173] In some embodiments, an immunosuppressed subject has or has been diagnosed with a lung disease, e.g., chronic lung disease, idiopathic pulmonary fibrosis (“IPF”), pulmonary arterial hypertension (“PAH”), chronic obstructive pulmonary disease (“COPD”), or emphysema. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a lung disease. In some embodiments, treatments for lung disease include bronchodilators (e.g., short-acting betaagonists, long-acting beta-agonists), inhaled corticosteroids, and/or combinations thereof. See, for example, Nici L, et al. Pharmacologic management of chronic obstructive pulmonary disease: An official American Thoracic Society clinical practice guideline. American Journal of Respiratory and Critical Care Medicine. 2020; doi: 10.1164/rccm.202003-0625ST, the entire contents of which are hereby incorporated by reference in its entirety. [0174] In some embodiments, an immunosuppressed subject has or has been diagnosed with autoimmune disease, e.g., diabetes, systemic lupus erythematosus (“SLE”) or multiple sclerosis. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for an autoimmune disease. In some embodiments, a treatment for diabetes includes insulin therapy. See for example, American Diabetes Association Professional Practice Committee; 9. Pharmacologic Approaches to Glycemic Treatment: Standards of Care in Diabetes — 2024. Diabetes Care 1 January 2024; 47 (Supplement l): S 158— S 178, the entire contents of which are hereby incorporated by reference in its entirety. In some embodiments, exemplary treatments for SLE includes hydroxychloroquine, corticosteroids (e.g., prednisone), immunosuppressants (e.g., azathioprine, cyclophosphamide, mycophenolate, methotrexate), and/or combinations thereof. See, for example, Fanouriakis A, et al, “EULAR recommendations for the management of systemic lupus erythematosus: 2023 update,” Annals of the Rheumatic Diseases 2024;83:15-29, the entire contents of which are hereby incorporated by reference in its entirety. In some embodiments, exemplary treatments for multiple sclerosis include steroids, immunosuppressants (e.g., mitoxantrone, cyclophosphamide, methotrexate). See, for example, Hauser SL, Cree BAC. Treatment of Multiple Sclerosis: A Review. Am J Med. 2020 Dec; 133(12): 1380-1390.e2. doi: 10.1016/j.amjmed.2020.05.049. Epub 2020 Jul 17. PMID: 32682869, the entire contents of which are incorporated by reference in its entirety.

[0175] In some embodiments, an immunosuppressed subject has or has been diagnosed with liver disease, e.g., chronic liver disease (e.g., cirrhosis). In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a liver disease. In some embodiments, treatment for chronic liver disease includes anti-viral agents.

[0176] In some embodiments, an immunosuppressed subject has or has been diagnosed with kidney disease, e.g., end-stage renal disease, chronic kidney disease, or IgA nephropathy. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for kidney disease. In some embodiments, exemplary treatments for kidney diseases comprise angiotensin-converting-enzyme (“ACE”) inhibitors, angiotension receptor blockers (“ARBs”), empagliflozin, dialysis, and/or combinations thereof. See, for example, Breyer MD, Susztak K. Developing Treatments for Chronic Kidney Disease in the 21st Century. Semin Nephrol. 2016 Nov;36(6):436-447. doi: 10.1016, the entire contents of which are hereby incorporated by reference in its entirety.

[0177] In some embodiments, an immunosuppressed subject has or has been diagnosed with a cardiovascular disease. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a cardiovascular disease. In some embodiments, exemplary treatments for cardiovascular include anticoagulants, antiplatelet agents and dual antiplatelet therapy, ACE inhibitors, angiotensin II receptor blockers, angiotensin receptor-neprilysin inhibitors, beta blockers, calcium channel blockers, cholesterol-lowering medications, digitalis preparations, diuretics, vasodilators, or combinations thereof.

[0178] In some embodiments, an immunosuppressed subject has or has been diagnosed with a blood disorder. In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a blood disorder. In some embodiments, exemplary treatments for a blood disorder includes blood transfusions, platelet transfusions, anticoagulants, growth factor supplements, corticosteroids,

[0179] In some embodiments, an immunosuppressed subject has or has been diagnosed with a neurological disease, e.g., neuromyelitis optica (“NMO”), Guillain-Barre syndrome (“GBS”). In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for a neurological disease.

[0180] In some embodiments, an immunosuppressed subject has or has been diagnosed with an immunodeficiency disorder, e.g., human immunodeficiency virus (“HIV”) infection, acquired immune deficiency syndrome (“AIDS”), and other various primary immunodeficiency disorders (“PIDDs”). In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for an immunodeficiency disorder. In some embodiments, exemplary treatments for HIV include nucleoside reverse transcriptase inhibitors (“NRTIs”), non-nucleoside reverse transcriptase inhibitors (“NNRTIs”), protease inhibitors (“Pls”), fusion inhibitors, CCR5 antagonists, integrase strand transfer inhibitors (“INSTIs”), attachment inhibitors, post-attachment inhibitors, capsid inhibitors, combinations thereof. See, for example, Phanuphak N, Gulick RM. HIV treatment and prevention 2019: current standards of care. Curr Opin HIV AIDS. 2020 Jan; 15, the entire contents of which are hereby incorporated by reference in its entirety. [0181] In some embodiments, an immunosuppressed subject has or has been diagnosed with a cancer, e.g., a blood cancer or a solid cancer. In some embodiments, a blood cancer is a leukemia, a lymphoma (e.g., Hodgkin’s lymphoma, non-Hodgkin’s lymphoma), or a myeloma (e.g., multiple myeloma). In some embodiments, an immunosuppressed subject has received, is receiving or will receive one or more treatments for cancer, e.g., chemotherapy and/or radiation therapy.

[0182] In some embodiments, an immunosuppressed subject is more than 65 years old. In some embodiments, an immunosuppressed subject is about 65 years to about 100 years old.

[0183] In some embodiments, an immunosuppressed subject has received or is receiving an immunosuppressive therapy. In some embodiments, an immunosuppressive therapy comprises: an organ transplant conditioning regimen, a therapy that suppresses an immune system, a B-cell targeting therapy, a T-cell targeting therapy, a chemotherapy, a radiation therapy, a cancer therapy, a treatment for an inflammatory disease and/or a treatment for an autoimmune disease.

[0184] In some embodiments, an immunosuppressed subject has received or is receiving a B cell targeting therapy (e.g., rituximab, or belimumab).

[0185] In some embodiments, an organ transplant conditioning regimen comprises a calcineurin inhibitor, an antiproliferative agent, a steroid, an mTOR inhibitor or any combination thereof. In some embodiments, a calcineurin inhibitor comprises tacrolimus. In some embodiments, an antiproliferative agent comprises mycophenolate mofetil. In some embodiments, a steroid comprises prednisone. In some embodiments, an organ transplant conditioning regimen comprises tacrolimus, mycophenolate mofetil and prednisone, or any combination thereof.

[0186] In some embodiments, the formulation comprises an LNP formulation of an RNA polynucleotide, such as a single stranded RNA polynucleotide. In some embodiments the formulation is administered in a single dose, while in other embodiments, the formulation is administered in two doses, such as at least one week, at least two weeks, at least three weeks, at least four weeks, at least six weeks, or at least two months apart. In some embodiments, the subject to be vaccinated is an immunosuppressed subject, as described above. In some such cases, the subject lacks adequate helper T cell functionality to activate B cells. For example, in some cases a subject has a relatively low number of helper T cells, while in other cases a subject has helper T cells with relatively low ability to activate B cells. In some cases, the LNP formulation is administered intramuscularly (i.e., via IM injection) at a dose of from 0.3 to 300 pg of RNA, such as from 0.3 to 150 pg of RNA, such as from 3 to 300 pg of RNA, such as from 3 to 150 pg of RNA, such as from 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA. For example, in some cases an immunocompromised individual might require a higher dose than a normal individual. In some cases, the LNP formulation may be administered in a volume of 0.2-1 mL, such as 0.2- 0.5 mL, or 0.5-1 mL, or 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1 mL. In some cases the volume of administration is 0.5 mL. In some such cases, the LNP formulation is diluted from a concentrated solution in 0.9% NaCl for injection. In some cases, the subject to be vaccinated with the LNP formulation is 18-65 years of age.

[0187] Exemplary embodiments of the disclosure include the following, as well as those listed in the embodiments elsewhere herein:

[0188] 1. A polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises:

(a) a polypeptide comprising amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), or comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and

(b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

[0189] 2 The polynucleotide of embodiment 1, wherein the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

[0190] 3. The polynucleotide of embodiment 1, wherein the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

[0191] 4. The polynucleotide of any one of embodiments 1-3, wherein the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0192] 5 The polynucleotide of any one of embodiments 1-3, wherein the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0193] 6. The polynucleotide of any one of embodiments 1-3, wherein the complement C3d-binding polypeptide comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain III (SEQ ID NO: 9) and/or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain IV (SEQ ID NO: 10), or wherein the complement C3d-binding polypeptide comprises or consists of one or both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10).

[0194] 7 The polynucleotide of any one of embodiments 4-6, wherein the Sbi domain

III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10) are contiguous, or are separated by a linker.

[0195] 8. The polynucleotide of any one of embodiments 4-7, wherein:

(i) (a) is disposed N-terminus of (b); or

(ii) (a) is disposed C-terminus of (b);

(iii) (a) and (b) are contiguous or separated by a linker; or

(iv) a combination thereof.

[0196] 9. The polynucleotide of embodiment 8, wherein (a) is disposed N-terminus of

(b), and wherein (a) and (b) are separated by a linker.

[0197] 10. The polynucleotide of embodiment 8 or 9, wherein:

(i) the linker is a peptidyl linker;

(ii) the linker is a peptidyl linker comprising at least 60% glycine and/or serine; or (iii) the linker is chosen from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) linker (SEQ ID NO: 18), optionally wherein the Gly4-Ser linker comprises the amino acid sequence of SEQ ID NO: 6, or a histidine linker.

[0198] 11. The polynucleotide of embodiment 8, 9, or 10, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6 or is encoded by the nucleotide sequence of SEQ ID NO: 7.

[0199] 12. The polynucleotide of any one of embodiments 1-11, wherein the polypeptide further comprises a secretion peptide.

[0200] 13. The polynucleotide of embodiment 12, wherein the secretion peptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, or is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0201] 14. The polynucleotide of any one of embodiments 1-13, wherein the polynucleotide comprises a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1.

[0202] 15. The polynucleotide of any one of embodiments 1-14, wherein the polynucleotide comprises a 3’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 12.

[0203] 16. The polynucleotide of any one of embodiments 1-15, wherein the polynucleotide further comprises a poly-adenosine (poly-A) tail at the 3’ end of the polynucleotide, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0204] 17. A polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’,

(i) a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1, (ii) a nucleotide sequence encoding a secretion peptide comprising a nucleotide sequence comprising SEQ ID NO: 3, or encoding an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2,

[0205] 18. A polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’,

(vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12, and optionally (viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0206] 19. A polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’,

(iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala,

(vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12, and optionally

(viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0207] 20. A polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13, or is an RNA transcribed from the nucleotide sequence of SEQ ID NO: 13.

[0208] 21. A polynucleotide encoding a fusion polypeptide, wherein the polynucleotide encodes the following amino acid sequences, disposed from N-terminus to C- terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 and SEQ ID NO: 8, (b) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8, (c) SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or (d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; or wherein the polynucleotide encodes the amino acid sequence of SEQ ID NO: 20.

[0209] 22. The polynucleotide of any one of embodiments 1-21, wherein the polynucleotide is DNA.

[0210] 23. The polynucleotide of any one of embodiments 1-21, wherein the polynucleotide is RNA.

[0211] 24. The polynucleotide of embodiment 23, wherein the RNA is single stranded, optionally wherein the RNA comprises a 5’ cap.

[0212] 25. The polynucleotide of embodiment 22, 23 or 24, wherein the polynucleotide comprises at least one modified ribonucleotide, optionally comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

[0213] 26. The polynucleotide of embodiment 25, wherein the at least one modified ribonucleotide comprises: a 5’ monophosphate; a 5’ diphosphate; or a 5’ triphosphate

[0214] 27. The polynucleotide of embodiment 25 or 26, wherein the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of: wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate.

[0215] 28. The polynucleotide of any one of embodiments 25-27, wherein the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of:

[0216] 29. The polynucleotide of embodiment 28, wherein the polyribonucleotide comprises cytidine residues, and:

(i) at least 5% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine;

(ii) less than 100% of cytidine residues in the polyribonucleotide comprise N4- acetylcytidine; or

(iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polyribonucleotide comprise N4- acetylcytidine.

[0217] 30. The polynucleotide of any one of embodiments 25-29, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate.

[0218] 31. The polynucleotide of 30, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of

[0219] 32. The polynucleotide of embodiment 31, wherein the polyribonucleotide comprises uridine residues and:

(i) at least 5% of uridine residues in the polyribonucleotide comprise 5- hydroxymethyluridine;

(ii) less than 100% of uridine residues in the polyribonucleotide comprise 5- hydroxymethyluridine;

(iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polyribonucleotide comprise 5- hydroxymethyluridine; or

(iv) more than 60% of uridine residues in the polyribonucleotide comprise 5- hydroxymethyluridine.

[0220] 33. The polynucleotide of any one of embodiments 25-32, wherein the at least one modified ribonucleotide comprises: N1 -methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5- methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), or any combination thereof.

[0221] 34. The polynucleotide of any one embodiments 25-33, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’-O-acetylated and the modified ribonucleotide has a structure of:

(b) wherein R is a nucleobase chosen from: adenine or a modified version thereof, a guanine or a modified version thereof, a cytosine or a modified version thereof, or a uracil or a modified version thereof.

[0222] 35. The polynucleotide of embodiment 34, wherein the nucleobase is adenine, and the modified ribonucleotide has a 5’ triphosphate and a structure of: [0223] 36. The polynucleotide of embodiment 34, wherein the nucleobase is guanine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0224] 37. The polynucleotide of embodiment 34, wherein the nucleobase is cytosine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0225] 38. The polynucleotide of embodiment 34, wherein the nucleobase is N4- acetylcytidine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0226] 39. The polynucleotide of embodiment 34, wherein the nucleobase is uracil, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0227] 40. The polynucleotide of embodiment 34, wherein the nucleobase is 5- hydroxymethyluridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0228] 41. The polynucleotide of embodiment 34, wherein the nucleobase is Nl- methylpseudouridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

[0229] 42. The polynucleotide of any one of embodiments 34-41, wherein:

(i) at least 5% of the ribose moi eties are acetylated (2’-O-acetylated), or

(ii) about 5% to about 99% of the ribose moi eties are acetylated (2’-O-acetylated).

[0230] 43. The polynucleotide of any one of embodiments 34-42, wherein the polyribonucleotide comprises a cap structure and the cap structure does not comprise a 2’-O- acetylated ribose.

[0231] 44. The polynucleotide of any one of embodiments 34-43, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O- acetylated ribose.

[0232] 45. The polynucleotide of any one of embodiments 34-44, wherein the polyribonucleotide further comprises one or more ribonucleotides that does not comprise a 2’-0 acetylated ribose.

[0233] 46. A single-stranded RNA polynucleotide comprising a sequence at least

90%, at least 95%, at least 97%, at least 98%, at least 99%, or 100% identical to the sequence of SEQ ID NO: 19, optionally wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polyribonucleotide comprise N4-acetylcytidine, and/or wherein at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of uridine residues in the polyribonucleotide comprise 5-hydroxymethyluridine.

[0234] 47. The polynucleotide of any one of claims 25-46, wherein the polynucleotide further comprises a 5’ cap and/or further comprises a poly-A tail, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100- 200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

[0235] 48. An expression vector comprising the polynucleotide of any one of embodiments 1-24.

[0236] 49. The expression vector of embodiment 46, wherein the expression vector comprises a viral vector chosen from: a retrovirus vector, an adenovirus vector, an adeno- associated virus vector or a lentivirus vector or an RNA vector.

[0237] 50. A host cell comprising the polynucleotide of any one of embodiments 1-

24, or the expression vector of embodiment 48 or 49.

[0238] 51. A pharmaceutical composition comprising the polynucleotide of any one of embodiments 1-49, and at least one carrier or excipient.

[0239] 52. The pharmaceutical composition of embodiment 51, wherein the carrier or excipient comprises liposome nanoparticles (LNP).

[0240] 53. The pharmaceutical composition of embodiment 51 or 52, wherein the polynucleotide is single stranded RNA comprising a 5’ cap.

[0241] 54. A fusion polypeptide encoded by the polynucleotide of any one of embodiments 1-47 or the expression vector of embodiment 48 or 49.

[0242] 55. A fusion polypeptide comprising:

(a) a polypeptide comprising amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4), or comprising an amino acid sequence at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of SEQ ID NO: 4; and (b) a complement C3d-binding polypeptide from an immunoglobulin-binding protein (Sbi) of Staphylococcus aureus.

[0243] 56. The fusion polypeptide of embodiment 55, wherein the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

[0244] 57. The fusion polypeptide of embodiment 55, wherein the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

[0245] 58. The fusion polypeptide of any one of embodiments 55-57, wherein the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0246] 59. The fusion polypeptide of any one of embodiments 55-57, wherein the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8.

[0247] 60. The fusion polypeptide of any one of embodiments 55-57, wherein the complement C3d-binding polypeptide comprises or consists of both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10).

[0248] 61. The fusion polypeptide of embodiment 60, wherein the Sbi domain III

(SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10) are contiguous, or are separated by a linker.

[0249] 62. The fusion polypeptide of any one of embodiments 55-61, wherein:

(i) (a) is disposed N-terminus of (b); or

(ii) (a) is disposed C-terminus of (b);

(iii) (a) and (b) are contiguous or separated by a linker; or

(iv) a combination thereof.

[0250] 63. The fusion polypeptide of embodiment 62, wherein (a) is disposed N- terminus of (b), and wherein (a) and (b) are separated by a linker.

[0251] 64. The fusion polypeptide of embodiment 62 or 63, wherein: (i) the linker is a peptidyl linker;

(ii) the linker is a peptidyl linker comprising at least 60% glycine and/or serine; or

(iii) the linker is chosen from a Gly-Gly-Gly-Gly-Ser (Gly4-Ser) linker (SEQ ID NO: 18), optionally wherein the Gly4-Ser linker comprises the amino acid sequence of SEQ ID NO: 6, or a Histidine linker.

[0252] 65. The fusion polypeptide of embodiment 62, 63, or 64, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6, or is encoded by the nucleotide sequence of SEQ ID NO: 7.

[0253] 66. The fusion polypeptide of any one of embodiments 55-65, wherein the polypeptide further comprises a secretion peptide.

[0254] 67. The fusion polypeptide of embodiment 66, wherein the secretion peptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, or is encoded by the nucleotide sequence of SEQ ID NO: 3.

[0255] 68. A fusion polypeptide, which is encoded by a polynucleotide comprising a nucleotide sequence comprising, from 5’ to 3’,

(v) optionally, a nucleotide sequence comprising SEQ ID NO: 7, or encoding a linker sequence, such as a glycine-serine linker, such as comprising the amino acid sequence of SEQ ID NO: 6 or 18, and (vi) a nucleotide sequence comprising SEQ ID NO: 11, or encoding domain III and domain IV of the Sbi of Staphylococcus aureus comprising an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

[0256] 69. A fusion polypeptide, which is encoded by a polynucleotide comprising a nucleotide sequence comprising, from 5’ to 3’,

[0257] 70. A fusion polypeptide, which is encoded by a polynucleotide comprising a nucleotide sequence comprising, from 5’ to 3’,

(iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala,

(vii) a 3’ untranslated region comprising the nucleotide sequence of SEQ ID NO: 12. [0258] 71. A fusion polypeptide, comprising the following amino acid sequences, disposed from N-terminus to C-terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 and SEQ ID NO: 8, (b) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8, (c) SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or (d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; or comprising the amino acid sequence of SEQ ID NO: 20.

[0259] 72. A fusion polypeptide encoded by the DNA sequence of SEQ ID NO: 13, or encoded by the RNA sequence of SEQ ID NO: 19.

[0260] 73. A pharmaceutical composition comprising the polynucleotide or expression vector of any one of embodiments 1-49, or the fusion polypeptide of any one of embodiments 54-72, and optionally further comprising at least one carrier or excipient.

[0261] 74. The pharmaceutical composition of embodiment 73, wherein the pharmaceutical composition comprises the polynucleotide or expression vector of any one of embodiments 1-49, and optionally further comprises at least one carrier or excipient.

[0262] 75. The pharmaceutical composition of embodiment 74, wherein the composition comprises at least one carrier or excipient which comprises a lipid nanoparticle (LNP).

[0263] 76. A method of making the polynucleotide of any one of embodiments 1-47 or the expression vector of embodiment 48 or 49, comprising: recombinantly joining a first nucleotide sequence that encodes the polypeptide of (a) and a second nucleotide sequence that encodes the complement C3d-binding polypeptide from a immunoglobulin-binding protein (Sbi) of Staphylococcus aureus of (b) to form a polynucleotide sequence, optionally wherein the first and/or second nucleotide sequence comprises a further nucleotide sequence encoding a linker sequence, and optionally wherein the first nucleotide sequence comprises a further nucleotide sequence encoding a secretion peptide.

[0264] 77. A host cell comprising the fusion polypeptide of any one of embodiments

54-72.

[0265] 78. A kit comprising the polynucleotide of any one of embodiments 1-47, or the expression vector of embodiment 48 or 49, the fusion polypeptide of any one of embodiments 54-72, or the pharmaceutical composition of embodiment 73-75, and optionally further comprising instructions for use.

[0266] 79. A method comprising administering to a subject in need of vaccination against SARS-CoV-2 at least one dose of the pharmaceutical composition of embodiment 73- 75 or the polynucleotide of any one of embodiments 1-47, or the fusion polypeptide of any one of embodiments 54-72.

[0267] 80. The method of embodiment 79, wherein the at least one dose is administered in an effective amount to:

(i) induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject;

(ii) stimulate B cells in the subject; or iii) both (i) and (ii).

[0268] 81. A method comprising administering to a subject: a first dose of the pharmaceutical composition of embodiment 73-75 or the polynucleotide of any one of embodiments 1-47, or the fusion polypeptide of any one of embodiments 54-72; and a second dose of the pharmaceutical composition of embodiment 73-75 or the polynucleotide of any one of embodiments 1-47, or the fusion polypeptide of any one of embodiments 54-72, optionally wherein the time between the first and second doses is from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks.

[0269] 82. The method of embodiment 81, wherein the first dose and the second dose are in the same amounts.

[0270] 83. The method of embodiment 79 or 80, wherein the pharmaceutical composition, polynucleotide, or fusion polypeptide is administered in a single dose to the subject.

[0271] 84. The method of any one of embodiments 79-83, wherein the method comprises administering a polynucleotide at a dose of 0.3-300 pg, such as 3-150 pg, 3-100 pg, 3-50 pg, 3-10 pg, 3-30 pg, 0.3-50 pg, 0.3-10 pg, 3-6 pg, 6-10 pg, or 10-30 pg. [0272] 85. The method of any one of embodiments 79-84, wherein administration is by intramuscular injection.

[0273] 86. The pharmaceutical composition of embodiment 73-75 or the polynucleotide of any one of embodiments 1-47, or the fusion polypeptide of any one of embodiments 54-72, for use in vaccination of a subject against SARS-CoV-2.

[0274] 87. The pharmaceutical composition, polynucleotide, or fusion polypeptide for use of embodiment 86, wherein the composition, polynucleotide, or polypeptide is administered to the subject in an effective amount to:

(ii) stimulate B cells in the subject; or

(iii) both (i) and (ii).

[0275] 88. The pharmaceutical composition, polynucleotide, or fusion polypeptide for use of embodiment 86 or 87, wherein the composition is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts.

[0276] 89. The pharmaceutical composition of embodiment 86 of 87, wherein the pharmaceutical composition, polynucleotide, or fusion polypeptide is administered in a single dose to the subject.

[0277] 90. The pharmaceutical composition of any one of embodiments 86-89, wherein the pharmaceutical composition is a polynucleotide, which is administered to the subject at a dose of 0.3-300 pg, such as 3-150 pg, 3-100 pg, 3-50 pg, 3-10 pg, 3-30 pg, 0.3- 50 pg, 0.3-10 pg, 3-6 pg, 6-10 pg, or 10-30 pg.

[0278] 91. Use of the pharmaceutical composition of embodiment 73-75 or the polynucleotide of any one of embodiments 1-47, or the fusion polypeptide of any one of embodiments 54-72 in the preparation of a medicament for vaccination of a subject against SARS-CoV-2.

[0279] 92. The use of embodiment 91, wherein at least one dose of the composition is administered in an effective amount to: (i) induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject;

(ii) stimulate B cells in the subject; or

(iii) both (i) and (ii).

[0280] 93. The use of embodiment 91 or 92, wherein the composition is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts.

[0281] 94. The use of embodiment 91 or 92, wherein the composition is administered in a single dose to the subject.

[0282] 95. The use of any one of embodiments 91-94, wherein the pharmaceutical composition is a polynucleotide, which is administered to the subject at a dose of 0.3-300 pg of the polynucleotide, such as 3-150 pg, 3-100 pg, 3-50 pg, 3-10 pg, 3-30 pg, 0.3-50 pg, 0.3- 10 pg, 3-6 pg, 6-10 pg, or 10-30 pg.

[0283] 96. The method, pharmaceutical composition, or use of any one of embodiments 79-95, wherein the subject is immunosuppressed or immunocompromised.

[0284] 97. The method, pharmaceutical composition, or use of embodiment 96, wherein the immunosuppressed or immunocompromised subject has received, is receiving, or will be receiving one or more transplants.

[0285] 98. The method, pharmaceutical composition, or use of embodiment 97, wherein the one or more transplants is an organ transplant.

[0286] 99. The method, pharmaceutical composition, or use of embodiment 98, wherein the organ transplant comprises: a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a stomach transplant, an intestine transplant, or any combination thereof.

[0287] 100. The method, pharmaceutical composition, or use of embodiment 96, wherein the one or more transplants is a cell transplant.

[0288] 101. The method, pharmaceutical composition, or use of embodiment 100, wherein the cell transplant is a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof.

[0289] 102. The method, pharmaceutical composition, or use of embodiment 100, wherein the cell transplant is a transplant of a population of bone marrow cells, blood cells, or any combination thereof.

[0290] 103. The method, pharmaceutical composition, or use of embodiment 100, wherein the cell transplant is a transplant of a population of engineered cells.

[0291] 104. The method, pharmaceutical composition, or use of embodiment 100, wherein the cell transplant is a transplant of a population of non-engineered cells.

[0292] 105. The method, pharmaceutical composition, or use of embodiment 96, wherein the one or more transplants is a tissue transplant.

[0293] 106. The method, pharmaceutical composition, or use of embodiment 96, wherein the tissue transplant comprises skin tissue transplant, bone tissue transplant, cartilage tissue transplant, adrenal tissue transplant, corneal tissue transplant, or any combination thereof.

[0294] 107. The method, pharmaceutical composition, or use of any one of embodiments 96-106, wherein the transplant is an allogeneic transplant.

[0295] 108. The method, pharmaceutical composition, or use of embodiment 95, wherein the subject is receiving or has received immunosuppressive therapy.

[0296] 109. The method, pharmaceutical composition, or use of embodiment 108, wherein the immunosuppressive therapy comprises an organ transplant conditioning regimen, chemotherapy, radiation therapy, or a treatment for an autoimmune disease.

[0297] 110. The method, pharmaceutical composition, or use of embodiment 106, wherein the immunosuppressive therapy comprises administration of one or more of a calcineurin inhibitor, an antiproliferative agent, a steroid, an mTOR inhibitor, or any combination thereof.

[0298] 111. The method, pharmaceutical composition, or use of embodiment 110, wherein the immunosuppressive therapy comprises administration of one or more of tacrolimus, mycophenolate mofetil, or prednisone, or any combination thereof. [0299] 112. The method, pharmaceutical composition, or use of any one of embodiments 96-111, wherein the immunosuppressed or immunocompromised subject has received, is receiving or will be receiving one or more dialysis treatments.

[0300] 113. The method, pharmaceutical composition, or use of any one of embodiments 96-112, wherein the immunosuppressed or immunocompromised subject has one or more disorders, wherein the one or more disorders optionally comprises a rare disease, lung disease, an autoimmune disease, liver disease, kidney disease, a cardiovascular disease, a blood disorder, a neurologic disease, an immunodeficiency disorder, a cancer, or any combination thereof.

[0301] 114. The method, pharmaceutical composition, or use of any one of embodiments 79-113, wherein the method, pharmaceutical composition, or use results in a seroresponse in the subject after at least 8, at least 30, at least 90, or at least 180 days post dosage (if a single dose) or post final dosage (if two or more doses are administered).

[0302] 115. The method, pharmaceutical composition, or use of any one of embodiments 79-114, wherein the method, pharmaceutical composition, or use results in increased titer of neutralizing antibodies against SARS-CoV-2 strain Omicron BA.4 and/or BA.5 in the subject after at least 8, at least 30, at least 90, or at least 180 days post dosage (if a single dose) or post final dosage (if two or more doses are administered).

[0303] 116. The method, pharmaceutical composition, or use of any one of embodiments 79-115, wherein the method, pharmaceutical composition, or use results in increased titer of serum antibodies against SARS-CoV-2 S protein or S protein RBD domain in the subject after at least 8, at least 30, at least 90, or at least 180 days post dosage (if a single dose) or post final dosage (if two or more doses are administered).

[0304] Other features of the invention will become apparent in the course of the following descriptions of exemplary embodiments, which are given for illustration of the invention and are not intended to be limiting thereof.

EXEMPLIFICATION

Example 1: Preparation of mRNA-LNP Formulation

[0305] DNA constructs: Transcription templates for mRNA vaccines comprising SEQ ID NO: 13 were synthesized as gBlock dsDNA fragments (Integrated DNA Technologies). The mRNA vaccine was prepared as a synthetic, purified, single-stranded, 5'- capped (CleanCap™ AG (TriLink Biotech)) messenger RNA optimized for expression of a fusion protein of SARS-CoV-2 (strain BA. 4/5) linked to Sbi lll-IV.

[0306] Each gBlock template was amplified with T7-AGG_fwd

(gaattTAATACGACTCACTATAAGGcttgttctttttgcagaagc; SEQ ID NO: 14) and 120pA_rev

_{TTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTaga} atgtgaagaaactttctttttattag) (SEQ ID NO: 15) using Herculase® II polymerase (Agilent) with an annealing temperature of 50°C.

[0307] mRNA synthesis: The PCR products were cleaned up using a 0.8x ratio of SPRISelect™ beads (Beckman Coulter) to PCR reaction volume. 19.9 pL transcription mixes consisting of lx HiScribe™ T7 High Yield buffer (NEB), 7.5 mM of each NTP, 7.5 mM CleanCap™ AG (TriLink Biotech), 2M betaine (ThermoSci), 20 mM MgC12, and 0.1 pL/pL HiScribe™ T7 Polymerase Mix were added to 2.1 pL DNA solution consisting of 200 ng T7 template in nuclease free H2O. Transcription was carried out for 1 hr at 50°C. Natural A and G NTPs and chemically modified ac4C and 5hmU NTPs were used for the transcription, in order to produce an mRNA with modified C and U nucleotides but natural A and G nucleotides. The transcribed RNAs were purified using the 500 pg capacity Monarch RNA Cleanup Kit (NEB), treated with DNAse I, and purified again using 500 pg capacity Monarch columns. mRNAs were then treated with Alkaline Phosphatase (Millipore) for 10 min at 37°C and purified using 500 pg Monarch columns. Concentrations were determined using a NanoDrop® spectrophotometer (ThermoSci).

[0308] LNP formulation: Formulations of mRNA in lipid nanoparticles (mRNA- LNPs) were prepared using the NanoAssemir Ignite® microfluidic mixer (Precision Nanosystems). GenVoy-ILM lipid mixture (Precision Nanosystems) was diluted to 12.5 mM in anhydrous ethanol and combined with an aqueous solution of mRNA (0.14 mg/mL) in PNI buffer (Precision Nanosystems), using the manufacturer-recommended formulation parameters. Formulations were immediately diluted 30: 1 in phosphate-buffered saline (pH 7.4) and concentrated using Amicon® centrifugation filters (Millipore Sigma UFC901008). LNP mRNA payload concentration (pg/mL) was quantified using a Stunner® instrument (Unchained Labs). Formulations were then diluted with PBS to the appropriate concentration to allow for a dose of 2 pg mRNA in 50 pL per animal. Formulations were stored at 4°C and used for in vivo studies within 21 days of production. Example 2: Murine immunization in vivo methods

[0309] The mRNA-LNP formulation described in Example 1 was used in the experiments of Example 2 (including parts 2A and 2B). All animal experiments were carried out in accordance with the guidelines set forth by Charles River Accelerator Development Lab (CRADL, Cambridge, MA, USA) and were approved by the CRADL Institutional Animal Care and Use (IACUC) committee. Female BALB/c mice (aged 7-9 weeks) were purchased from Charles River Laboratories (Wilmington, MA, USA) and housed at CRADL. Mice were provided standard feed and water ad libitum. Mice were acclimated for at least 2 days before the initiation of study.

[0310] Immunization: Each treatment group consisted of n = 5 mice. Mice were immunized via intramuscular injection in the quadriceps with 50 pL mRNA-LNP formulation as described in Example 1 (2 pg mRNA dose per animal) at study Day 0 and again at Day 21. The same formulation was administered for both immunization timepoints. A control group consisting of n = 2 mice were dosed with 50 pL PBS in the same manner and at the same timepoints.

[0311] Termination and sample collection: On study Day 28, 7 days after the second immunization, mice were euthanized via CO2 inhalation. Whole blood was collected via intracardiac (IC) venipuncture. Whole blood was collected into MiniCollect™ serum separator tubes (GreinerBio-One 450472) and allowed to sit at room temperature for 30 minutes to promote clotting before serum was separated by centrifugation at 4°C, 1200 x g, for 10 minutes. Serum was stored frozen at -80°C for immunogenicity readouts.

[0312] Anti-RBD mouse IgG serology assay: Antigen-specific serum IgG antibody response against Omicron subvariant BA.4/5 RBD was assessed using Meso Scale Discovery (MSD) V-PLEX SARS-CoV-2 Serology panel 28 kit. Briefly, plates were blocked with 150 pL / well of MSD Blocker A at room temperature, shaking at 700 rpm on an orbital plate shaker for 1 hour (Heidolph Instuments, Titramax 1000). Plates were then washed 3 times with 150 pL / well IX MSD Wash buffer using an automated plate washer (BioTek, 405-TS) and were thoroughly tapped dry. Serum thawed from -80C storage was prepared at 1 : 100,000 and 1 : 1,000,000 dilutions in MSD Diluent-100 and added to MSD plates in duplicates at 50 pL / well. 1 hour incubation and plate wash step were performed as previously described. Secondary detection antibody, anti-mouse IgG MSD GOLD SULFO-TAG (Fisher Scientific), was prepared according to kit protocol as a 1 :200 dilution in MSD Diluent-100 and was added 50 pL / well. 1 hour incubation and plate wash step were performed as previously described. To read plates, 150 pL I well of MSD GOLD Read Buffer B was added to plates and signal was detected using the MESO QuickPlex™ SQ 120MM instrument (Meso Scale Discovery). Results were reported as the mean detected signal in relative light units (RLU). Increased signal indicated a larger quantity of antigen specific IgG antibody in a given sample. Signal was averaged across n = 5 samples [mice] per group, or n = 2 samples [mice] for PBS control group. Error bars represented the standard deviation across the same number of samples.

[0313] Pseudovirus Microneutralization Titer (MNT) assay: Pseudovirus neutralizing antibody titers were measured in an MNT assay. Briefly, human 293T-hsACE2 cells were cultured and plated in 96 well plates, then incubated with pseudotyped lentiviral reporter virus particles (RVP, Integral Molecular) for 48 hours, in the presence or absence of serum sample. The RVPs bore a GFP reporter gene and were pseudotyped with a SARS- CoV-2 Spike protein from a specific lineage/strain (in this case Omicron BA.4/5 RVP-774 or Wuhan RVP-701). Each serum sample was run in duplicate 3-fold dilution series covering 1 :50 to 1 :328,050. Dilution preparation was automated using a Hamilton Star instrument (Hamilton Robotics). After 48 hours, GFP+ single cell counts (focus forming units; FFU) were quantified for each well on a Cytation 5™ plate reader. Pseudovirus neutralization 50% inhibitory dilution (ID50) titer was then calculated for each serum sample as the inverse of the dilution required to reduce the infected GFP+ FFU count per well by 50% compared to the virus control wells. The data was reported as the geometric mean ID50 for the indicated RVP (BA.4/5 or Wuhan) across n =5 mice for the vaccinated group and n=2 mice for the PBS Control group. Error bars represented the geometric standard deviation across the same number of samples.

Example 2A: Anti-RBD Serology Assay Results

[0314] To demonstrate the immunogenicity of the vaccine, a multiplexed serology assay (V-PLEX SARS-CoV-2 Panel 28 Mouse IgG Kit, Meso Scale Discovery) measuring serum IgG antibodies against the receptor binding domain (RBD) of various SARS-CoV-2 virus strains was performed.

[0315] The multiplexed assay captured antigen proteins including the RBD of SARS- CoV-2 sub-variant Omicron BA.4/5 (which the vaccine sequence is based on), as well as RBDs from 5 additional sub-variants from the Omicron lineage (BA.2.12.1, BA.2, BA.2+L452R, BA.2+L452M, BA.3) and 4 earlier SARS-CoV-2 lineages (Wuhan, Alpha, Beta, Delta). RBD proteins were pre-conjugated to the kit plate (CAT# K15618U-2). Each bar in the Figure 1 graph represents the average serum IgG antibody response against the indicated RBD antigen across n=5 mice as extrapolated from relative light units (RLUs) (with error bars representing the standard deviation of the response across the mice within a given treatment group) (FIG. 1).

[0316] The anti-RBD serology assay results depicted in FIG. 1 indicated that the vaccine strongly induces serum IgG antibodies against Omicron BA.4/5 RBD, and that these antibodies are able to bind a wide breadth of RBDs from SARS-CoV-2 variants and subvariants (FIG. 1). Because this assay was performed at serum dilution of 1 : 1,000,000 and the signal for all vaccinated mice was significantly elevated compared to the negative (PBS) control mouse serums, the anti-RBD serum IgG titers for the vaccinated mice serums could be considered to be > 1 : 1,000,000. As expected, the signal across the RBDs varied in strength, corresponding to RBD sequence differences between distinct SARS-CoV-2 lineages and sub-variants, but a significant signal was seen for each. This result demonstrated that in addition to inducing a robust response against the targeted BA.4/5 variant, the vaccine may also induce anti-RBD antibodies capable of recognizing new and emerging SARS-CoV-2 variants.

Example 2B: Pseudovirus Microneutralization Titer (MNT) Assay Results

[0317] To demonstrate that the anti-RBD antibodies induced by the vaccine are functional, we performed (via CRO PPD) a pseudovirus microneutralization titer (MNT) assay on D28 serums from vaccinated and control mice. This assay serves as a model of SARS-CoV-2 infection and quantifies the ability of antibodies to inhibit viral infection in cultured human cells.

[0318] In this case, the D28 mouse serum samples were run in two separate experiments, using different reporter virus particles (RVPs). Both were lentiviral RVPs bearing a GFP reporter gene. The first was pseudotyped with SARS-CoV-2 Omicron BA.4/5 Spike protein, and the second with ancestral (Wuhan) SARS-CoV-2 Spike protein. In this assay, 293T-hsACE2 cells were plated in 96 well plates and incubated with RVP for 48 hours, in the presence or absence of serum sample. Each serum sample was run in duplicate 3-fold dilution series covering 1 :50 to 1 :328,050. After 48 hours, GFP+ single cell counts (focus forming units; FFU) were quantified for each well on a Cytation 5™ plate reader. A titer, or in this case more specifically a 50% inhibitory dilution (ID50), was then calculated for each serum sample as the inverse of the dilution required to reduce the infected GFP+ FFU count per well by 50% compared to the virus control wells. The bars in the FIG. 2 graph represent the geometric mean ID50 for the indicated RVP (BA.4/5 or Wuhan) across n =5 mice (FIG. 2).

[0319] These MNT assay results (FIG. 2) indicated that the vaccine induces high ID50 titers (>19,000) of functional, neutralizing antibodies against Omicron BA.4/5, as well as low but detectable (potentially still protective) neutralizing ID50 titers (-112) against a distant (ancestral Wuhan) strain. For comparison, Moderna has reported BA.4/5 pseudovirus neutralizing titers (ID50) of 2,372 (for subjects with no previous SARS-CoV-2 infection) and 7,676 (for subjects with previous SARS-CoV-2 infection) for human serums from subjects immunized with their updated Omicron BA.4/5 booster, mRNA-1273.214 (Chalkias et al., N Engl J Med. 2022, 387(14), 1279-1291). Although there is still not an established correlation of protection for SARS-CoV-2 vaccines, human clinical studies provide strong evidence that high serum neutralizing antibody titers are inversely correlated with occurrence of COVID- 19 cases (and therefore positively correlated to vaccine efficacy). In fact, ID50 titers of at least 100 corresponded to vaccine efficacy of 90.7%, while ID50 titers of at least 1000 corresponded to vaccine efficacy of 96.1% in humans (Gilbert et al., Science 2022, 375, 43- 50).

Example 3: Murine SARS-CoV-2 Challenge

[0320] The vaccine was next tested in a SARS-CoV-2 challenge model in mice. The mRNA vaccine was prepared as a synthetic, purified, single-stranded, 5'-capped (CleanCap™ AG (TriLink Biotech)) messenger RNA optimized for expression of a fusion protein of SARS-CoV-2 (strain BA. 4/5) linked to Sbi lll-IV. The mRNA was produced from a synthetic DNA template comprising SEQ ID NO: 13 via in vitro transcription with natural A and G ribonucleotides and chemically modified ribonucleotides ac4C and 5hmU in order to produce an mRNA with modified C and U nucleotides, and with CleanCap™ AG (TriLink Biotech) in order to provide the 5’ cap. It was purified via OligodT column (ThermoSci) purification and formulated as an LNP formulation.

[0321] The efficacy of the vaccine against infection and/or severe disease in animals challenged with SARS-CoV-2 was evaluated in female AC70 hACE-2 mice. The mice were randomized to 5 groups with 12 F/group, with 4 of the groups being dosed by IM injection with either vehicle, 0.12, 0.4 or 1.2 pg vaccine on Study Days 0 and 21. The 5^th group was dosed with 0.4 pg only on Day 0. On Study Day 35 blood was collected. On Study Day 42, all the animals were challenged with a potentially lethal dose (2.2 x 10^A4 TCIDso/dose) of SARS-CoV-2 (BA.4) via the intranasal route. Oral swabs were taken from animals on Study Days 41, 44, and 46 to assess viral load. On Study Day 47, 6 animals/group were euthanized, and the lungs were collected for histopathological evaluation. The remaining animals were euthanized on Study Day 57. During the In-life phase of the study animals were monitored for viability, clinical signs, body weights and injection sites were examined. Pre-challenge antigen-specific serum IgG titers and serum pseudovirus neutralizing titers, in addition to survival and viral load were assessed.

[0322] A total of 3 control (Group 1) animals were euthanized due to adverse clinical signs on study days 50, 51, and 52 (8, 9, and 10 days following the SARS CoV-2 challenge), respectively. A total of 3 animals in Group 4 animals (treated 0.12 pg on study days 0 and 21) were euthanized on study day 51 and 2 animals in Group 5 (treated 0.4 pg on study day 0) were euthanized on study days 51 and 52 due to adverse clinical signs. Following SARS CoV-2 challenge, body weights in general tended to increase out to 15 days post-challenge. The mean percent body weight change for Group 4 animals declined by about -8% by 8 days post challenge, reaching a NADIR of approximately -10% at 10 days, then rapidly recovered by 15 days post challenge. The mean percent body weight change for control animals declined at the same rate reaching a NADIR of about -12% by 10 days post challenge, then rising to almost 0% change by 15 days post challenge (Fig. 3).

[0323] The most common clinical observations noted post challenge were mild ruffled coat and hair loss. Other observations included hunched posture, ruffled coat, lethargy, body weight loss >20%, and ocular effects (Discharge/Opacity/Squint/ Abnormal Cornea or Sclera).

[0324] Survival was 100% by 15 days post challenge in animals treated with 1.2 g of the vaccine on study days 0 and 21 (Group 2) and -80% by day 9 post challenge, -65% by days 9-15 post challenge in animals treated 0.4 g only on study day 0 (Group 5). Survival of Group 4 animals (0.12 g on study days 0 and 21) was -50% by 10 days post challenge and remained that way by 15 days post challenge. Survival of the vehicle control animals (Group 1) declined to -80%, then -65%, then -50% by 8-, 9-, and 10-15-days post challenge (FIG. 4). [0325] Draize scores for both edema and erythema were either grade 0 or 1 for all dose groups with no clear dose-response relationship. Regardless of the dose group, all the animals that survived to 15 days post challenge had mild disease (COVID scores of <2). The animals that died post-challenge all had severe disease (CO VID scores of <7).

Example 4: Tests in Drug-Induced Immunocompromised Murine Model

[0326] The vaccine as described in Example 3 was next tested in a drug-induced immunocompromised mouse model in BALB/c mice, in order to further characterize the immune response to the vaccine in an immunocompromised setting. In this study, a total of 12 mice (6/sex/treatment group) were subjected to daily immunosuppressive treatment with a drug cocktail containing tacrolimus, mycophenolate mofetil, and prednisone via oral gavage throughout the duration of the study. Of these mice, 3/sex were immunized with 50 pL of the vaccine, 3 and 24 days following immunosuppression. Clinical signs, body weight, and immunophenotyping via FACS were performed throughout the study on Day 3, Day 17, and Day 38 (end of study). Serum was also collected throughout the study for the determination of antigen-specific serum IgG, serum pseudovirus neutralization, and serum cytokine levels. Cellular and humoral immune responses to the vaccine were also analyzed.

[0327] With the exception of a few individual observations in the mice (which randomly occurred across treatment groups), administration of the immunosuppression regimen as well as the vaccine was well tolerated. Food consumption remained generally consistent throughout the study. Immunosuppression resulted in a body weight loss in the range of 3 to 10% for both male and female mice that occurred over the first seven days of treatment but then remained stable over the remainder of the study. However, immunization with the vaccine did not negatively impact bodyweights for either males or females, regardless of their immunocompetent status (FIG. 5).

[0328] Upon evaluation of the local injection site (via Draize scoring), there was no erythema or edema observed at any time post vaccination for any of the groups.

[0329] The quantity and cell cycle phases of CD4+ T and CD8+ T cells of immunosuppressed or saline-treated mice, with or without vaccination, were evaluated in samples collected on Day 17. Immunosuppression alone resulted in a decrease in the total number of T cells and CD4+ T cells (in female mice). Vaccination with the vaccine increased total T cells, CD4+ T cells, CD8+ T cell, and B cells of saline-treated male but not female mice, nor immunosuppressed mice, regardless of sex. Immunosuppression and vaccination did not affect counts of NK cells and neutrophils (FIG. 6). By Day 38, immunosuppressed mice showed trends for decreased immune cell counts, regardless of vaccination status.

[0330] Following splenocyte peptide stimulation (JPT #PM-SARS-RBDMUT10-l), spleen weights, splenocyte counts, and the quantity of IFN-y and IL-5 T-cells following immunosuppression and/or vaccination with the vaccine were evaluated by ELISpot analysis. Spleen weights and splenocyte counts were reduced in the immunosuppressed groups supporting the conclusion that the mice had drug-induced immunosuppression. The ELISpot analysis revealed that even with immunosuppression, vaccinated animals elicited sufficient IFN- y and IL-5 T-cells responses.

Example 5: Phase I Dose Escalation Study in Human Subjects

[0331] The vaccine as described in Example 3 is tested in a human Phase I study.

The vaccine is a synthetic, purified, single-stranded, 5'-capped messenger RNA optimized for expression of a fusion protein of SARS-CoV-2 (strain BA. 4/5) linked to Sbi lll-IV. The synthetic mRNA was produced from a synthetic DNA template (not plasmid) comprising SEQ ID NO: 13 via in vitro transcription and was synthesized with chemically modified nucleotides ac4C and 5hmU and natural nucleotides used for adenosine and guanosine. It was purified via OligodT column (ThermoSci) purification. The vaccine antigen encoded by the mRNA is the RBD portion of the viral S protein of SARS-CoV-2 strain BA.4/5. An immunostimulatory protein domain, Sbi lll-IV, was co-encoded on the mRNA as a fusion to the RBD via a flexible linker. The native “SSP” signal peptide of SARS-CoV-2 S protein was encoded on the N terminus of the RBD, to promote secretion of the RBD-Sbi_III-IV protein from transfected producer cells. The drug product was a preservative-free sterile dispersion of mRNA in lipid nanoparticles (LNPs) in an aqueous sucrose-based cryoprotectant buffer. The drug product is intended for IM administration.

[0332] The safety, reactogenicity, and immunogenicity of the vaccine is evaluated in previously vaccinated (SARS-CoV-2) healthy adults that are 18-65 years of age (inclusive) to assess the safety and immunogenicity of ascending doses of vaccine. Inclusion criteria include having received at least one vaccination against COVID-19 previously with the most recent dose greater than 180 days prior to the start of the trial, being negative for SARS-CoV- 2 infection as determined by a PCR test at the screening visit and by rapid antigen test on day 1 of the trial, having certain laboratory test values deemed not clinically significant by the Investigator, and a prothrombin time, fibrinogen, and activated partial thromboplastin time in the reference range. Females must not be pregnant or trying to become pregnant, while male participants that have not had a vasectomy at least 6 months prior to day 1 must agree to use a barrier method of contraception from the time of vaccination until at least 90 days after vaccination and must not donate sperm until after 90 days post-vaccination. Exclusion criteria include any of the following at screening: concomitant disease, condition, or treatment that could interfere with conduct of the study or pose an unacceptable risk to the participant in the study or interfere with interpretation of study data; level of serum IgG antibodies against recombinant purified S protein and/or S protein RBD derived from SARS- CoV-2 Omicron BA.4/BA.5 that exceeds a predetermined threshold; acute illness with or without fever within 48 hours of vaccination; history of hypersensitivity or severe allergic reaction to any previous licensed or unlicensed vaccines; receipt of immunoglobulins and/or blood or blood products within 6 months of vaccination; blood dyscrasias or significant coagulation disorder; abnormality or permanent body art (e.g. tattoo) that would interfere with the ability to observe local reactions at the injection site (i.e. deltoid region); received or plans to receive any licensed vaccine within 4 weeks before or after vaccination; receipt of any other SARS-CoV-2 or other experimental COVID vaccine within 180 days before vaccination or any planned receipt of such vaccine during the study duration.

[0333] The study is conducted on 5 cohorts.

[0334] Cohort 1 : Open-label, single-arm, single dose of 3 pg vaccine (n=10; 2 sentinel + 8 ROC)

[0335] Cohort 2: Open-label, single-arm, single dose of 10 pg vaccine (n=10; 2 sentinel + 8 ROC)

[0336] Cohort 3: Double-blind, randomized, single dose of 50 pg vaccine or 50 pg SPIKEVAX® XBB1.5 (elasomeran, Modema) COVID-19 Vaccine (n=20; 4 sentinel randomized 1 : 1 + 16 ROC randomized 1 :1)

[0337] Cohort 4: Open-label, single-arm, single dose of 100 pg vaccine (n=10; 2 sentinel + 8 ROC)

[0338] Cohort 5: Open-label, single-arm, single dose of 150 pg vaccine (n=18; 2 sentinel + 16 ROC) [0339] The commencement of each cohort is overseen by a Safety Review Committee

(SRC).

[0340] The vaccine is provided as a frozen, concentrated solution at 0.60 mg/mL (concentration of mRNA drug substance). To prepare the final solution for injection, the drug product is thawed and diluted with isotonic sodium chloride solution (0.9% NaCl, saline).

[0341] The vaccine is administered as a single 0.5 mL IM injection into the deltoid muscle on Day 1 of each cohort dose level. The concentration of the vaccine (0.6 mg/mL) is diluted in 0.9% NaCl for injection, USP to obtain 3, 10, 50, 100, and 150 pg in 0.5 mL dosages.

[0342] A ‘sentinel approach’ is employed for dosing of each study cohort, where two sentinel participants in each cohort are dosed and monitored for 48 hours post vaccination, prior to dosing the remaining participants in that cohort. The remaining participants in that cohort are only dosed if the adverse event (AE) profile in the sentinel participants is considered acceptable by the Principal Investigator (PI) and the Medical Monitors (MM) for the clinical trial and the halting criteria provided below are not met. After all participants in a cohort complete at least 7 days of follow-up (Day 8), the SRC will review the safety data. If no safety concerns or pausing criteria are met, then the next ascending dose level cohort will be initiated using the same sentinel approach as implemented for the first cohort. Participants will have follow-up visits at Days 2, 3, 8, 15, 29, 90, and 180 for safety and early efficacy assessments. The overall duration of study participation is to be approximately 208 days.

[0343] Primary endpoints of the trial include frequency and grade of solicited local and systemic reactogenicity adverse events (AEs) during a 7-day follow-up period post vaccination (days 1-8), and frequency and grade of unsolicited local and systemic AEs during the 28-day follow-up period post vaccination (days 1-29), as well as frequency and grade of any serious adverse events (SAEs), medically attended adverse events (MAAEs) and adverse events of special interest (AESIs) throughout the duration of the study. Secondary objectives of the trial include evaluation of serum neutralizing antibodies (nAbs) against spike (S) protein from SARS-CoV-2 Omicron BA.4/BA.5 up to day 180 and evaluation of serum IgG antibodies against a recombinant purified S protein and/or S protein receptor binding domain (RBD domain) from SARS-CoV-2 Omicron BA.4/BA.5 up to day 180. Secondary endpoints include geometric mean of nAb levels at baseline to day 180, geometric mean fold rise of nAb levels from baseline through to day 180, geometric mean of antigen-specific serum IgG antibodies at baseline through day 180, and geometric mean fold rise of serum IgG levels from baseline through day 180. The trial also evaluates vaccine-elicited cell-mediated immunity in healthy adults, using peripheral blood mononuclear cells, and the breadth of serum nAb and antigen-specific serum antibody responses against a variety of SARS-CoV-2 strains, such as by determining change in cell-mediated immune response markers from baseline to day 29, and serum nAb against S protein and/or S protein RBD domain from SARS-CoV-2 strains of interest from day 1 to day 180.

Example 6: Updated Phase I Dose Escalation Study in Healthy Human Subjects

[0344] The vaccine as described in Example 3 is being tested in a human Phase I study, to assess safety and tolerability of a single dose administration in healthy adults. As described below, the study is being conducted in six cohorts and is currently in progress. Currently, subjects in all cohorts have been dosed, and certain results for three of the cohorts are available and are provided below.

[0345] The vaccine was a synthetic, purified, single-stranded, 5'-capped messenger RNA optimized for expression of a fusion protein of SARS-CoV-2 antigen protein (strain BA. 4/5) linked to Sbi_III-IV. The synthetic mRNA was produced from a synthetic DNA template (not plasmid) comprising SEQ ID NO: 13 via in vitro transcription and was synthesized with chemically modified nucleotides ac4C and 5hmU and natural nucleotides used for adenosine and guanosine. It was purified via OligodT column (ThermoSci) purification. The vaccine antigen encoded by the mRNA is the RBD portion of the viral S protein of SARS- CoV-2 strain BA.4/5. An immunostimulatory protein domain, Sbi lll-IV, was co-encoded on the mRNA as a fusion to the RBD via a flexible linker. The native “SSP” signal peptide of SARS-CoV-2 S protein was encoded on the N terminus of the RBD, to promote secretion of the RBD-Sbi_III-IV protein from transfected producer cells. The drug product was a preservative-free sterile dispersion of mRNA in lipid nanoparticles (LNPs) in an aqueous sucrose-based cryoprotectant buffer, intended for IM administration.

[0346] The study is being conducted on 6 cohorts.

Cohort 1 : Open-label, single-arm, single dose of 3 pg vaccine (n=10; 2 sentinel + 8 ROC)

Cohort 2: Open-label, single-arm, single dose of 10 pg vaccine (n=10; 2 sentinel + 8 ROC)

Cohort 3: Open-label, single-arm, single dose of 30 pg vaccine (n=18; 2 sentinel + 8 ROC + 8 additional ROC (as 4+4)) Cohort 4: Open-label, single-arm, single dose of 50 pg vaccine (n=10; 2 sentinel + 8 ROC)

Cohort 5: Open-label, single-arm, single dose of 6 pg vaccine (n=10)

Cohort 6: Open-label, single-arm, single dose of 30 pg COMIRNATY (Pfizer) comparator vaccine (n=10)

[0347] The commencement of Cohorts 1-4 was overseen by a Safety Review Committee (SRC). Participants in all cohorts have been dosed and representative data for Cohorts 1-3 are provided below.

[0348] The participants were healthy adults that are 18-65 years of age (inclusive) at the time of screening and are determined to be healthy by the Investigator based on medical history, clinical laboratory results, vital signs and electrocardiogram measurements, and physical examination at screening. Inclusion criteria included having received at least one vaccination against COVID-19 previously with the most recent dose greater than 180 days prior to the start of the study, being negative for SARS-CoV-2 infection as determined by a PCR test at the screening visit and by rapid antigen test on Day 1 of the study, having certain laboratory test values deemed not clinically significant by the Investigator, and a prothrombin time, fibrinogen, and activated partial thromboplastin time in the reference range. Females must not have been pregnant lactating or trying to become pregnant, and those of childbearing potential must have used an effective contraception method, while male participants that have not had a vasectomy at least 6 months prior to Day 1 must have agreed to use a barrier method of contraception from the time of vaccination until at least 90 days after vaccination and must not have donated sperm until after 90 days post-vaccination.

[0349] Exclusion criteria included any of the following at screening: concomitant disease, condition, or treatment that could interfere with conduct of the study or pose an unacceptable risk to the participant in the study or interfere with interpretation of study data; confirmed SARS-CoV-2 infection within 6 months before vaccine administration; acute illness with or without fever within 48 hours of vaccination; history of hypersensitivity or severe allergic reaction to any previous licensed or unlicensed vaccines or a component of the tested vaccine; receipt of immunoglobulins and/or blood or blood products within 6 months of vaccination; blood dyscrasias or significant coagulation disorder; abnormality or permanent body art (e.g. tattoo) that would interfere with the ability to observe local reactions at the injection site (i.e. deltoid region); received or plans to receive any licensed vaccine within 4 weeks before or after vaccination; receipt of any other SARS-CoV-2 or other experimental COVID vaccine within 180 days before vaccination or any planned receipt of such vaccine during the study duration.

[0350] The vaccine was provided to the clinical site as a suspension at 0.6 mg/mL (concentration of mRNA drug substance), and was administered by IM injection (0.5 mL). The concentrated vaccine (0.6 mg/mL) was diluted in 0.9% NaCl for injection, USP, to obtain the 3, 6, 10, 30, and 50 pg in 0.5 mL dosages. The COMIRNATY vaccine was provided as a suspension at 0.1 mg/mL and administered by IM injection (0.3 mL), without dilution.

[0351] Participants were screened on Day -28 to Day -1, and certain tests were conducted. Dosing occurred on Day 1 of the study. On Day 1 of the study, participants also underwent pre-dose and post-dose assessments. Participants have had or will have follow-up screening on Days 2, 3, 8 (+/- 1), 15 (+/- 1), 29 (+/-2), 90 (+/-3), and 180 (+/- 7), and thus are or were followed for 180 days.

[0352] A ‘sentinel approach’ was employed for dosing of Cohorts 1-4, where two sentinel participants in each cohort were dosed and monitored for 48 hours post vaccination, prior to dosing the remaining participants in that cohort. The remaining participants in that cohort were only dosed if the adverse event (AE) profile in the sentinel participants was considered acceptable by the Principal Investigator (PI) and the Medical Monitors (MM) for the clinical study and the study halting criteria were not met. Non-sentinel participants were monitored for 30 minutes following vaccine administration and discharged if stable, unless required to stay longer by the Investigator for further monitoring. After all participants in a cohort completed at least 7 days of follow-up (Day 8), the SRC reviewed the safety data. If no safety concerns or pausing criteria were met, then the next ascending dose level cohort was initiated using the same sentinel approach as implemented for the first cohort. For Cohort 3, safety data were additionally reviewed for all dosed participants after the first 8 ROC participants, and again after the first additional 4 ROC participants reached 7 days of followup (Day 8), to allow dosing of the second additional 4 ROC participants.

[0353] Primary endpoints of the study include frequency and grade of solicited local and systemic reactogenicity adverse events (AEs) during a 7-day follow-up period post vaccination (Days 1-8), and frequency and grade of unsolicited local and systemic AEs during the 28-day follow-up period post vaccination (Days 1-29), as well as frequency and grade of any serious adverse events (SAEs), medically attended adverse events (MAAEs) and adverse events of special interest (AESIs) throughout the duration of the study.

[0354] Secondary objectives of the study include evaluation of geometric mean titer (GMT) and geometric mean fold rise (GMFR) of serum neutralizing antibodies (nAbs) against spike (S) protein or S protein receptor binding domain (RBD domain) from SARS- CoV-2 from baseline (Day 1) up to Day 180; evaluation of GMT and GMFR of antigenspecific serum IgG antibodies against a recombinant purified S protein and/or S protein RBD domain from SARS-CoV-2 up to Day 180; and the geometric mean ratio (GMR) of the postvaccination antibody levels compared to COMIRNATY; as well as serum antibody seroresponse rate (SRR) from baseline to Day 180.

[0355] Further exploratory endpoints include evaluating vaccine-elicited cell mediated immunity in participants using peripheral blood mononuclear cells (PBMCs) by determining change in PBMC cell-type markers and/or antigen-induced cytokines from baseline (Day 1) to Day 29; evaluating vaccine-elicited complement response by determining change in plasma complement response markers from baseline (Day 1) to 4 hours post dose, Day 2, Day 8, and Day 29; and evaluating the breadth of vaccine-elicited serum nAbs and antigen-specific serum antibody responses against a variety of SARS-CoV-2 strains, and/or serum antibody responses against components of the LNP delivery system, including polyethylene glycol (PEG). This last set of exploratory endpoints can be assessed by determining serum nAbs against S protein and/or S protein RBD domain from SARS-CoV-2 strains of interest at baseline (Day 1) up to Day 180, and by determining serum antigenspecific antibody levels against S protein and/or S protein RBD domain from SARS-CoV-2 strains of interest and/or against components of the LNP delivery system at baseline (Day 1) up to Day 180.

[0356] Data for participants in Cohorts 1-3 (3, 10, and 30 pg doses, respectively) have been obtained, with those in Cohorts 1-2 assessed for the complete 180 day period, while those in Cohort 3 assessed up to day 29. These 38 participants comprise 21 males and 17 females with a mean age of 34.3 years old (from 18 to 60).

[0357] Tolerability of the vaccine in these cohorts was found to be similar to that of currently approved SARS-CoV-2 vaccines. For example, the following table provides a summary of solicited local and systemic adverse events during Days 1-7 of the study. Cohort 1 Cohort 2 Cohorts Total

(3 ug) (10 ug) (30 ug) (N=38)

Day 1 To 7

Any Reactogenicity Event

Mild (Gradel) 6(60.0%) 5(50.0%) 9(50.0%) 20(52.6%)

Moderate (Grade 2) 1 (10.0%) 3(30.0%) 9(50.0%) 13(34.2%)

Local

Pain at the injection site

Mild (Gradel) 2(20.0%) 5(50.0%) 12(66.7%) 19(50.0%)

Moderate (Grade 2) 0 1 (10.0%) 1 (5.6%) 2(5.3%)

Tenderness at the injection site

Mild (Gradel) 5(50.0%) 4(40.0%) 8(44.4%) 17(44.7%)

Moderate (Grade 2) 0 3(30.0%) 8(44.4%) 11 (28.9%)

Redness at the injection site

Below Gradel 1 (10.0%) 1 (10.0%) 1 (5.6%) 3(7.9%)

Warmth at the injection site

Mild (Gradel) 3(30.0%) 2(20.0%) 2(11.1%) 7(18.4%)

Itching at the injection site

Mild (Gradel) 0 1 (10.0%) 0 1 (2.6%)

Bruising at the injection site

Mild (Gradel) 0 0 2 (11.1%) 2(5.3%)

Arm motion limitations

Mild (Gradel) 0 1 (10.0%) 4(22.2%) 5(13.2%)

Moderate (Grade 2) 0 1 (10.0%) 2(11.1%) 3(7.9%)

Systemic

Nausea

Mild (Gradel) 1 (10.0%) 1 (10.0%) 0 2(5.3%)

Moderate (Grade 2) 0 0 1 ( 5.6%) 1 ( 2.6%)

Diarrhea

Below Gradel 1 (10.0%) 1 (10.0%) 0 2(5.3%)

Mild (Gradel) 1 (10.0%) 0 1 (5.6%) 2(5.3%)

Headache

Mild (Gradel) 1 (10.0%) 2(20.0%) 8(44.4%) 11 (28.9%)

Moderate (Grade 2) 1 (10.0%) 0 1 (5.6%) 2(5.3%)

Fatigue

Mild (Gradel) 2(20.0%) 3(30.0%) 4(22.2%) 9(23.7%)

Moderate (Grade 2) 0 0 5(27.8%) 5(13.2%)

Muscle Pain

Mild (Gradel) 2(20.0%) 2(20.0%) 5(27.8%) 9(23.7%)

Moderate (Grade 2) 0 0 2(11.1%) 2(5.3%)

Joint Pain

Mild (Gradel) 0 1 (10.0%) 1 (5.6%) 2(5.3%)

Moderate (Grade 2) 0 0 1 ( 5.6%) 1 ( 2.6%)

Chills

Mild (Gradel) 1 (10.0%) 0 2(11.1%) 3(7.9%)

Moderate (Grade 2) 0 0 1 ( 5.6%) 1 ( 2.6%)

For all corresponding percentages, the denominator is the respective numbers of exposed participants, i.e., participants who received a vaccination and were still on-study for that time point or time interval, irrespective of whether a Diary was present or not. [0358] Safety of the vaccine in these cohorts was also found to be similar to that of currently approved SARS-CoV-2 vaccines. The following table provides a summary of unsolicited treatment related adverse events for participants in Cohorts 1-3 across all body systems. _o . Cohort 1 Cohort 2 Cohort 3

System Orga" C ass (SOC) _{(3 ug) (10 u}g) _{(30 ug) Tota}|

Preferred Term (PT) (N=10) (N=10) (N=18) (N=38) n (%) M n (%) M n (%) M n (%) M

All Body Systems 1 (10.0%) 1 0 0 3 (16.7%) 11 4 (10.5%) 12

Mild (Grade 1) 0 0 0 0 0 4 0 4

Moderate (Grade 2) 1 (10.0%) 1 0 0 1 ( 5.6%) 5 2 ( 5.3%) 6

Severe (Grade 3) _{0 0 0} 0 2 (11.1 %) 2 2 ( 5.3%) 2

Adverse events were coded to System Organ Class (SOC) and Preferred Term (PT) using the Medical Dictionary for Regulatory Activities (MedDRA) Version 26.1.

A treatment-emergent adverse event (TEAE) is defined as an adverse event (AE) that commences on, or after the first administration of study drug.

If a participant has multiple occurrences of an AE, the participant is presented only once in the n (%) column at the maximum severity for a given SOC and preferred term. Occurrences are counted each time in the M column.

Percentages based on the number of participants in the relevant population.

[0359] Secondary endpoint data are provided in the following tables, and are at least comparable to those of approved SARS-CoV-2 vaccines.

[0360] The geometric mean titer (GMT) (arbitrary units per mL; AU/mL) of an antibody against the SARS-CoV-2 BA.5 strain S protein in participant serum (antigenspecific serum IgG) was assessed by ELISA and results were as follows in the table below. (Data for Cohort 3 at Days 90 and 180 have not yet been obtained.) GMT was calculated as the mean of the assay results after logarithmic transformation and then exponentiating the mean to express results on the original scale. Two-sided 95% confidence intervals (CI) were obtained by taking natural log transforms of concentrations or titers, calculating the 95% CI with reference to the t-distribution, and exponentiating the confidence limits.

Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic _ (N=10) _ (N=10) _ (N=18) _

Baseline n 10 10 18

GMT (95% 61 ,684.73 (37,739.67 - 68,448.65 (55,256.58 - 58,047.21 (37,401 .14 -

Cl) 100,822.43) 84,790.21) 90,090.29) Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

(Min - Max) (15,445.33 - 230,630.96) (36,221.30 - 107,167.37) (4,193.71 - 347,620.75)

Day 8 n 10 10 18

GMT (95% 102,744.13 (72,872.68 - 200,169.01 (157,963.20 - 214,259.42 (165,644.20 - Cl) 144,860.27) 253,651.70) 277,142.82)

(Min - Max) (36,815.88 - 272,009.24) (122,258.92 - 432,097.81) (57,854.22 - 674,978.56)

Day 15 n 10 9 18

GMT (95% 180,832.47 (139,184.15 - 375,202.58 (296,393.81 - 343,386.90 (271 ,327.89 -

Cl) 234,943.29) 474,965.99) 434,583.29)

(Min - Max) (100,622.60 - 361 ,251 .71) (228,627.97 - 671 ,506.19) (144,044.28 - 880,000.00)

Day 29 n 10 9 18

GMT (95% 197,255.89 (153,523.54 - 370,955.67 (289,862.43 - 293,581.09 (227,248.45 -

Cl) 253,445.73) 474,735.92) 379,275.89)

(Min - Max) (105,585.39 - 410,446.80) (198,219.16 - 794,108.32) (109,273.93 - 855,025.62)

Day 90 n 9 8

GMT (95% 107,729.84 (83,245.63 - 264,717.61 (194,558.07 -

Cl) 139,415.35) 360,177.37)

(Min - Max) (53,770.24 - 197,970.13) (144,938.90 - 648,830.04)

Day 180 n 8 8

GMT (95% 79,262.12 (56,801.61 - 179,919.16 (149,118.29 -

Cl) 110,603.97) 217,082.04)

(Min - Max) (36,580.03 - 193,165.39) (120,918.77 - 273,007.30)

CI: Confidence Interval; GMT: Geometric Mean Titer; Max: Maximum; Min: Minimum.

The 95% CI is calculated using the t-distribution with n-1 degrees of freedom.

[0361] The geometric mean fold rise (GMFR) of antigen-specific serum IgG in participants from baseline (Day 1) to Day 180 (in Cohorts 1-2) and Day 29 (in Cohort 3) was as shown in the table below. GMFR was calculated as the mean of the difference of logarithmically transformed results (later time point - earlier time point) and exponentiating the mean. GMFR was determined by Analysis of Covariance (ANCOVA) with baseline titer as covariate.

Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

Day 8 n 10 10 18

GMFR (95% Cl) 1.66 (1.29 - 2.15) 2.93 (2.33 - 3.67) 3.69 (2.45 - 5.57)

Day 15 n 10 9 18 Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

GMFR (95% Cl) 2.93 (1.97-4.37) 5.71 (4.22-7.72) 5.92 (3.94-8.89)

Day 29 n 10 9 18

GMFR (95% Cl) 3.20 (2.23-4.58) 5.64 (4.05-7.85) 5.06 (3.42-7.48)

Day 90 n 9 8

GMFR (95% Cl) 1.69 (1.17-2.42) 3.97 (2.56-6.15)

Day 180 n 8 8

GMFR (95% Cl) 1.20 (0.84- 1.71) 2.70(1.91 -3.80)

CI: Confidence Interval; GMFR: Geometric Mean Fold Rise.

The 95% CI is calculated using the t-distribution with n-1 degrees of freedom.

[0362] The seroresponse rate (SRR) of antigen-specific serum IgGs (in %) was as shown in the table below. A positive seroresponse for an individual participant was defined as a change from less than the lower limit of quantification (LLOQ) to greater than or equal to 4 X LLOQ, in other words, at least a 4-fold increase from baseline (Day 1) if the baseline level is greater than or equal to LLOQ. The SRR is defined as the percentage of participants with a positive seroresponse, presented with the 95% CI calculated using the Clopper Pearson method. As shown in the table below, most participants in Cohort 2 retained a seroresponse through to Day 180.

Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

Day 8 n/nn 1 /10 3/10 4/18

SRR (95% Cl) 10.0 (0.25 - 44.50) 30.0 (6.67 - 65.25) 22.2 (6.41 - 47.64)

Day15 n/nn 3/10 6/9 12/18

SRR (95% Cl) 30.0 (6.67-65.25) 66.7 (29.93-92.51) 66.7 (40.99-86.66)

Day29 n/nn 5/10 6/9 12/18

SRR (95% Cl) 50.0 (18.71 -81.29) 66.7 (29.93-92.51) 66.7 (40.99-86.66)

Day 90 n / nn 4/9 6/8

SRR (95% Cl) 44.4 (13.70 - 78.80) 75.0 (34.91 - 96.81)

Day 180 n/nn 3/8 6/8

SRR (95% Cl) 37.5 (8.52 - 75.51) 75.0 (34.91 - 96.81)

CI: Confidence Interval; SRR: Seroresponse Rate. n: number of participants with a seroresponse at given timepoint; nn: number of analyzable participants at given timepoint.

The 95% CI is calculated using the Clopper-Pearson method. [0363] The GMT for titers of neutralizing antibodies in serum against a SARS-CoV-2

BA.5 strain were as follows in Cohorts 1-3. GMT and CI were calculated as described above.

Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

Baseline n 10 10 18

GMT (95% Cl) 196.98 (128.70- 196.98 (134.55- 296.28 (187.77-

301.50) 288.39) 467.49)

(Min -Max) (40-320) (80-640) (20- 1,280)

Day 8 n 10 10 18

GMT (95% Cl) 452.55 (366.18- 970.06 (788.29- 1,551.78 (1,161.98-

559.29) 1,193.74) 2,072.33)

(Min -Max) (320-640) (640- 1,280) (320-5,120)

Day 15 n 10 9 18

GMT (95% Cl) 735.17 (535.47- 2,194.54 (1,372.19- 2,194.54 (1,575.53-

1,009.33) 3,509.73) 3,056.76)

(Min -Max) (320- 1,280) (640-5,120) (320- 10,240)

Day 29 n 10 9 18

GMT (95% Cl) 970.06(629.82- 2,194.54(1,309.44- 2,111.64(1,416.00-

1,494.11) 3,677.91) 3,149.03)

(Min -Max) (160-2,560) (640-10,240) (320-10,240)

Day 90 n 9 8

GMT (95% Cl) 1,280.00 (791.76- 2,791.70 (1,894.94-

2,069.32) 4,112.84)

(Min -Max) (640-5,120) (1,280-5,120)

Day 180 n 8 8

GMT (95% Cl) 697.92 (473.73- 1,974.03 (1,206.37-

CI: Confidence Interval; GMT: Geometric Mean Titer; Max: Maximum; Min: Minimum.

The 95% CI is calculated using the t-distribution with n-1 degrees of freedom.

[0364] The geometric mean fold rise (GMFR) of neutralizing antibodies was as follows in the table below. GMFR was calculated as described above. Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

Day 8 n 10 10 18

GMFR (95% Cl) 2.30 (1.40-3.76) 4.92 (3.54-6.86) 5.24 (3.54-7.75)

Day 15 n 10 9 18

GMFR (95% Cl) 3.73 (2.08 - 6.68) 12.70 (8.75 - 18.42) 7.41 (4.69 - 11.69)

Day 29 n 10 9 18

GMFR (95% Cl) 4.92 (2.98-8.14) 12.70 (7.86-20.53) 7.13 (4.33- 11.74)

Day 90 n 9 8

GMFR (95% Cl) 6.35 (3.75- 10.75) 14.67 (8.70-24.75)

Day 180 n 8 8

GMFR (95% Cl) 3.67 (2.06 - 6.54) 10.37 (5.40 - 19.95)

CI: Confidence Interval; GMFR: Geometric Mean Fold Rise.

The 95% CI is calculated using the t-distribution with n-1 degrees of freedom.

[0365] The seroresponse rate (SRR) (in %) for neutralizing antibody titer was as follows in the table below. SRR was calculated as described above.

Cohort 1 Cohort 2 Cohort 3

(3 ug) (10 ug) (30 ug)

Visit Statistic (N=10) (N=10) (N=18)

Day 8 n/nn 3/10 9/10 14/18

SRR (95% Cl) 30.0 (6.67-65.25) 90.0 (55.50-99.75) 77.8 (52.36-93.59)

Day15 n/nn 5/10 9/9 16/18

SRR (95% Cl) 50.0 (18.71 -81.29) 100.0 (66.37- 100.0) 88.9 (65.29-98.62)

Day29 n/nn 8/10 9/9 16/18

SRR (95% Cl) 80.0 (44.39-97.48) 100.0 (66.37- 100.0) 88.9 (65.29-98.62)

Day 90 n / nn 8/9 8/8

SRR (95% Cl) 88.9 (51.75-99.72) 100.0 (63.06- 100.0)

Day 180 n/nn 7/8 8/8

SRR (95% Cl) 87.5 (47.35 - 99.68) 100.0 (63.06 - 100.0)

The 95% CI is calculated using the Clopper-Pearson method. [0366] As shown in the table above, all or nearly all participants in Cohort 2 had a seroresponse by Day 8.

EQUIVALENTS

[0367] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim dependent on the same base claim (or, as relevant, any other claim) unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise. Further, it should also be understood that any embodiment or aspect of the invention can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the claims that follow.

Claims

1. A single stranded RNA polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide, wherein the fusion polypeptide comprises:

2. The single stranded RNA polynucleotide of claim 1, wherein the polypeptide of (a) comprises amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

3. The single stranded RNA polynucleotide of claim 1, wherein the polypeptide of (a) consists of amino acid residues 331-527 of SARS-CoV-2 Omicron BA.4/BA.5 Spike receptor binding domain (RBD) (SEQ ID NO: 4).

4. The single stranded RNA polynucleotide of any one of claims 1-3, wherein the complement C3d-binding polypeptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identical to the amino acid sequence of SEQ ID NO: 8.

5. The single stranded RNA polynucleotide of any one of claims 1-3, wherein the complement C3d-binding polypeptide comprises or consists of the amino acid sequence of SEQ ID NO: 8.

6. The single stranded RNA polynucleotide of any one of claims 1-3, wherein the complement C3d-binding polypeptide comprises an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain III (SEQ ID NO: 9) and/or an amino acid sequence with at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, or 100% identity to Sbi domain IV (SEQ ID NO: 10), or wherein the complement C3d-binding polypeptide comprises or consists of one or both Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10).

7. The single stranded RNA polynucleotide of any one of claims 4-6, wherein the Sbi domain III (SEQ ID NO: 9) and Sbi domain IV (SEQ ID NO: 10) are contiguous, or are separated by a linker.

8. The single stranded RNA polynucleotide of any one of claims 4-7, wherein:

(i) (a) is disposed N-terminus of (b), or

(ii) (a) is disposed C-terminus of (b); or

(iii) (a) and (b) are contiguous or separated by a linker; or

(iv) a combination of (i) and (iii) or of (ii) and (iii).

9. The single stranded RNA polynucleotide of claim 8, wherein (a) is disposed N-terminus of (b), and wherein (a) and (b) are separated by a linker.

10. The single stranded RNA polynucleotide of claim 8 or 9, wherein:

(i) the linker is a peptidyl linker;

11. The single stranded RNA polynucleotide of claim 8, 9, or 10, wherein the linker comprises the amino acid sequence of SEQ ID NO: 6 or is encoded by the nucleotide sequence of SEQ ID NO: 7.

12. The single stranded RNA polynucleotide of any one of claims 1-11, wherein the polypeptide further comprises a secretion peptide.

13. The single stranded RNA polynucleotide of claim 12, wherein the secretion peptide comprises an amino acid sequence at least 80%, at least 85%, at least 90%, at least 95%, or 100% identical to the amino acid sequence of SEQ ID NO: 2, or is encoded by the nucleotide sequence of SEQ ID NO: 3.

14. The single stranded RNA polynucleotide of any one of claims 1-13, wherein the polynucleotide comprises a 5’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 1 wherein T is replaced by U.

15. The single stranded RNA polynucleotide of any one of claims 1-14, wherein the polynucleotide comprises a 3’ untranslated region comprising a nucleotide sequence at least 90%, at least 95%, at least 97%, or 100% identical to the sequence of SEQ ID NO: 12 wherein T is replaced by U.

16. The single stranded RNA polynucleotide of any one of claims 1-15, wherein the polynucleotide further comprises a poly-adenosine (poly-A) tail at the 3’ end of the polynucleotide, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues.

17. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U:

18. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U:

19. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U:

(ii) a nucleotide sequence encoding a secretion peptide comprising the amino acid sequence of SEQ ID NO: 2, (iii) a nucleotide sequence encoding a linker sequence, such as Ala- Ala,

20. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 13 wherein T is replaced by U; or is transcribed from the nucleotide sequence of SEQ ID NO: 13; or wherein the polynucleotide comprises the ribonucleotide sequence of SEQ ID NO: 19.

21. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide encodes the following amino acid sequences, disposed from N-terminus to C- terminus, wherein each of the following amino acid sequences is optionally separated by a linker: (a) SEQ ID NO: 4 and SEQ ID NO: 8, (b) SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 8, (c) SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8, or (d) SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, and SEQ ID NO: 8; or encodes the amino acid sequence of SEQ ID NO: 20.

22. The single stranded RNA polynucleotide of any one of claims 1-21, wherein the polynucleotide comprises a 5’ cap.

23. The single stranded RNA polynucleotide of any one of claims 1-22, wherein the polynucleotide comprises at least one modified ribonucleotide, optionally comprising: a modified nucleobase, a modified ribose, a modified backbone, or any combination thereof.

24. The single stranded RNA polynucleotide of claim 23, wherein the at least one modified ribonucleotide comprises: a 5’ monophosphate; a 5’ diphosphate; or a 5’ triphosphate.

25. The single stranded RNA polynucleotide of claim 23 or 24, wherein the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of: wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate.

26. The single stranded RNA polynucleotide of any one of claims 23-25, wherein the at least one modified ribonucleotide comprises a nucleoside comprising an acetyl group, wherein the nucleoside is N4-acetylcytidine and the modified ribonucleotide has a structure of:

27. The single stranded RNA polynucleotide of claim 26, wherein the polynucleotide comprises cytidine residues, and:

(i) at least 5% of cytidine residues in the polynucleotide comprise N4-acetylcytidine; (ii) less than 100% of cytidine residues in the polynucleotide comprise N4- acetylcytidine; or

(iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% of cytidine residues in the polynucleotide comprise N4-acetylcytidine.

28. The single stranded RNA polynucleotide of any one of claims 23-27, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of wherein R is a 5’ monophosphate, a 5’ diphosphate, or a 5’ triphosphate.

29. The single stranded RNA polynucleotide of 28, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a hydroxymethyl group, wherein the nucleoside is 5-hydroxymethyluridine and has a structure of

30. The single stranded RNA polynucleotide of claim 29, wherein the polynucleotide comprises uridine residues and:

(i) at least 5% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine;

(ii) less than 100% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine;

(iii) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine; or

(iv) more than 60% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine.

31. The single stranded RNA polynucleotide of any one of claims 23-30, wherein the at least one modified ribonucleotide comprises: N1 -methylpseudouridine, pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 5- methyl cytidine (m5C), 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 2-amino-purine, 2, 6- diaminopurine, 2-amino-6-halo-purine, 6-halo-purine, inosine (I), 1-methyl-inosine (ml I), wyosine (imG), methylwyosine (mimG), or any combination thereof.

32. The single stranded RNA polynucleotide of any one of claims 23-31, wherein the at least one modified ribonucleotide comprises a nucleoside comprising a ribose moiety comprising an acetyl group, wherein the ribose is 2’-O-acetylated and the modified ribonucleotide has a structure of:

33. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is adenine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

34. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is guanine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

35. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is cytosine, and the modified ribonucleotide has a 5’ triphosphate and a structure of:

36. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is N4- acetylcytidine, and the modified ribonucleotide has a 5’ triphosphate and a structure of

37. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is uracil, and the modified ribonucleotide has a 5’ triphosphate and a structure of

38. The single stranded RNA polynucleotide of claim 32, wherein the nucleobase is 5- hydroxymethyluridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

39. The polynucleotide of claim 32, wherein the nucleobase is N1 -methylpseudouridine and the modified ribonucleotide has a 5’ triphosphate and a structure of:

40. The single stranded RNA polynucleotide of any one of claims 32-39, wherein:

(i) at least 5% of the ribose moi eties are acetylated (2’-O-acetylated), or

41. The single stranded RNA polynucleotide of any one of claims 32-40, wherein the polynucleotide comprises a cap structure and the cap structure does not comprise a 2’-O- acetylated ribose.

42. The single stranded RNA polynucleotide of any one of claims 32-41, wherein the polyribonucleotide comprises a cap structure and the cap structure comprises a 2’-O- acetylated ribose.

43. The single stranded RNA polynucleotide of any one of claims 32-42, wherein the polyribonucleotide further comprises one or more ribonucleotides that does not comprise a 2’-0 acetylated ribose.

44. A single stranded RNA polynucleotide encoding a fusion polypeptide, wherein the polynucleotide comprises a nucleotide sequence comprising, from 5’ to 3’, wherein T is replaced by U:

(viii) a poly-A tail following the 3’ untranslated region, optionally wherein the poly-A tail comprises from 50 to 200 adenosine residues in length, such as 100-200, 150-200, 100-150, 100-140, 110-140, 50-100 75-100, 75-150, or 100, 110, 115, 120, 125, 130, 135, 140, 150, 160, 170, 180, or 200 adenosine residues; and further wherein:

(a) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of cytidine residues in the polynucleotide comprise N4- acetylcytidine, and

(b) at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% of uridine residues in the polynucleotide comprise 5- hydroxymethyluridine, and optionally

(c) wherein the polynucleotide comprises a 5’ cap.

45. A DNA polynucleotide encoding the single stranded RNA polynucleotide of any one of claims 1-44.

46. An expression vector comprising the DNA polynucleotide of claim 45.

47. A host cell comprising the polynucleotide of any one of claims 1-45, or the expression vector of claim 46.

48. A pharmaceutical composition comprising the polynucleotide of any one of claims 1-45, and at least one carrier or excipient.

49. The pharmaceutical composition of claim 48, wherein the carrier or excipient comprises liposome nanoparticles (LNP).

50. A fusion polypeptide encoded by the polynucleotide of any one of claims 1-45 or the expression vector of claim 46.

51. A method of making the single stranded RNA polynucleotide of any one of claims 1-44, comprising: recombinantly joining a first nucleotide sequence that encodes the polypeptide of (a) and a second nucleotide sequence that encodes the complement C3d-binding polypeptide from a immunoglobulin-binding protein (Sbi) of Staphylococcus aureus of (b) to form a polynucleotide sequence, optionally wherein the first and/or second nucleotide sequence comprises a further nucleotide sequence encoding a linker sequence, and optionally wherein the first nucleotide sequence comprises a further nucleotide sequence encoding a secretion peptide, and wherein the first and second nucleotide sequences comprise DNA, and transcribing the DNA to make single stranded RNA.

52. A kit comprising the single stranded RNA polynucleotide of any one of claims 1-44, or the DNA polynucleotide of claim 45, or the expression vector of claim 46, or the pharmaceutical composition of claim 48 or 49, and optionally further comprising instructions for use.

53. A method comprising administering to a subject in need of vaccination against SARS- CoV-2 at least one dose of the pharmaceutical composition of claim 48 or 49 or the single stranded RNA polynucleotide of any one of claims 1-44.

54. The method of claim 53, wherein the at least one dose is administered in an effective amount to: (i) induce an immune response against a SARS-CoV-2 Spike RBD domain polypeptide in the subject;

(ii) stimulate B cells in the subject; or

(iii) both (i) and (ii).

55. A method comprising administering to a subject: a first dose of the pharmaceutical composition of claim 48 or 49 or the single stranded RNA polynucleotide of any one of claims 1-44; and a second dose of the pharmaceutical composition of claim 48 or 49 or the polynucleotide of any one of claims 1-44, optionally wherein the time between the first and second doses is from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks.

56. The method of claim 55, wherein the first dose and the second dose are in the same amounts.

57. The method of claim 53 or 54, wherein the pharmaceutical composition or polynucleotide is administered in a single dose to the subject.

58. The method of any one of claims 53-57, wherein the method comprises administering the pharmaceutical composition or the single stranded RNA polynucleotide at a dose of 0.3-300 pg of RNA, such as 3-150 pg of RNA, 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA.

59. The method of any one of claims 53-58, wherein administration is by intramuscular injection.

60. The pharmaceutical composition of claim 48 or 49 or the single stranded RNA polynucleotide of any one of claims 1-44 for use in vaccination of a subject against SARS- CoV-2.

61. The pharmaceutical composition or single stranded RNA polynucleotide for use of claim

60, wherein the composition or single stranded RNA polynucleotide is administered to the subject in an effective amount to:

(ii) stimulate B cells in the subject; or

(iii) both (i) and (ii).

62. The pharmaceutical composition or single stranded RNA polynucleotide for use of claim 60 or 61, wherein the composition or polynucleotide is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts.

63. The pharmaceutical composition or single-stranded RNA polynucleotide of claim 60 or

61, wherein the pharmaceutical composition or polynucleotide is administered in a single dose to the subject.

64. The pharmaceutical composition or single stranded RNA polynucleotide of any one of claims 60-63, wherein the pharmaceutical composition or polynucleotide is administered to the subject at a dose of 0.3-300 pg of RNA, such as 3-150 pg of RNA, 3-100 pg of RNA, 3- 50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA.

65. Use of the single stranded RNA polynucleotide of any one of claims 1-44 or the pharmaceutical composition of claim 48 or 49 in the preparation of a medicament for vaccination of a subject against SARS-CoV-2.

66. The use of claim 65, wherein at least one dose of the polynucleotide or composition is administered in an effective amount to:

(ii) stimulate B cells in the subject; or (iii) both (i) and (ii).

67. The use of claim 65 or 66, wherein the polynucleotide or composition is administered to the subject as a first dose followed by a second dose after a period of from one week to six weeks, such as from two weeks to six weeks, such as from three weeks to six weeks, such as from three weeks to four weeks, optionally wherein the first dose and the second dose are in the same amounts.

68. The use of claim 65 or 66, wherein the polynucleotide or composition is administered in a single dose to the subject.

69. The use of any one of claims 65-68, wherein the polynucleotide or composition is administered to the subject at a dose of 0.3-300 pg of RNA, such as 3-150 pg of RNA, 3-100 pg of RNA, 3-50 pg of RNA, 3-10 pg of RNA, 3-30 pg of RNA, 0.3-50 pg of RNA, 0.3-10 pg of RNA, 3-6 pg of RNA, 6-10 pg of RNA, or 10-30 pg of RNA.

70. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of any one of claims 53-69, wherein the subject is immunosuppressed.

71. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 70, wherein the immunosuppressed subject has received, is receiving, or will be receiving one or more transplants.

72. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 71, wherein the one or more transplants is an organ transplant.

73. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 72, wherein the organ transplant comprises: a kidney transplant, a liver transplant, a heart transplant, a lung transplant, a pancreas transplant, a stomach transplant, an intestine transplant, or any combination thereof.

74. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 71, wherein the one or more transplants is a cell transplant.

75. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 74, wherein the cell transplant is a transplant of a population of stem cells (e.g., hematopoietic stem cells, induced pluripotent stem cells, or embryonic stem cells), immune cells, or any combination thereof.

76. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 74, wherein the cell transplant is a transplant of a population of bone marrow cells, blood cells, or any combination thereof.

77. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 74, wherein the cell transplant is a transplant of a population of engineered cells.

78. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 74, wherein the cell transplant is a transplant of a population of non-engineered cells.

79. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 71, wherein the one or more transplants is a tissue transplant.

80. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 79, wherein the tissue transplant comprises skin tissue transplant, bone tissue transplant, cartilage tissue transplant, adrenal tissue transplant, corneal tissue transplant, or any combination thereof.

81. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of any one of claims 71-80, wherein the transplant is an allogeneic transplant.

82. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of any one of claims 70-81, wherein the subject is receiving or has received immunosuppressive therapy.

83. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 82, wherein the immunosuppressive therapy comprises an organ transplant conditioning regimen, chemotherapy, radiation therapy, or a treatment for an autoimmune disease.

84. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 82, wherein the immunosuppressive therapy comprises administration of one or more of a calcineurin inhibitor, an antiproliferative agent, a steroid, an mTOR inhibitor, or any combination thereof.

85. The method, pharmaceutical composition or single stranded RNA polynucleotide, or use of claim 84, wherein the immunosuppressive therapy comprises administration of one or more of tacrolimus, mycophenolate mofetil, or prednisone, or any combination thereof.