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EP4436595A1 - Reducing risk of antigen mimicry in immunogenic medicaments - Google Patents

Reducing risk of antigen mimicry in immunogenic medicaments

Info

Publication number
EP4436595A1
EP4436595A1 EP22814525.6A EP22814525A EP4436595A1 EP 4436595 A1 EP4436595 A1 EP 4436595A1 EP 22814525 A EP22814525 A EP 22814525A EP 4436595 A1 EP4436595 A1 EP 4436595A1
Authority
EP
European Patent Office
Prior art keywords
protein
antigenic polypeptide
cross
antigenic
host
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP22814525.6A
Other languages
German (de)
French (fr)
Inventor
Michael John Agostino
Seda ARAT
Patrick Michel Caubel
Mark Matthew GOSINK
Kanwar Nasir Mahmood KHAN
Petra Helen KOZA-TAYLOR
Matthew Thomas MARTIN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Pfizer Corp Belgium
Pfizer Corp SRL
Pfizer Inc
Original Assignee
Pfizer Corp Belgium
Pfizer Corp SRL
Pfizer Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Pfizer Corp Belgium, Pfizer Corp SRL, Pfizer Inc filed Critical Pfizer Corp Belgium
Publication of EP4436595A1 publication Critical patent/EP4436595A1/en
Withdrawn legal-status Critical Current

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/575—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 humoral response
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • This disclosure relates to the field of immunology, specifically to immunogenic compositions, methods for improving safety and/or reducing side effects of immunogenic compositions, and immunogenic compositions having improved safety and/or side effect profiles.
  • Adverse reactions following administration of vaccines are not limited to only certain vaccines against only a certain pathogenic organism, as side effects have been observed for a broad range of vaccines against a variety of pathogens including, for example, coronavirus, influenza, hepatitis, human papilloma virus, smallpox, anthrax, and typhoid.
  • pathogens including, for example, coronavirus, influenza, hepatitis, human papilloma virus, smallpox, anthrax, and typhoid.
  • pathogens including, for example, coronavirus, influenza, hepatitis, human papilloma virus, smallpox, anthrax, and typhoid.
  • pathogens including, for example, coronavirus, influenza, hepatitis, human papilloma virus, smallpox, anthrax, and typhoid.
  • Vaccines (Basel), Oct 15 ;9( 10): 1186, 2021 (stating that post-vaccine myocarditis has been reported for live vaccines (e.g., smallpox, anthrax and some typhoid vaccines).
  • the present disclosure provides methods for reducing the side effects of immunogenic compositions and for improving side effect profdes of immunogenic compositions. Reducing the side effects of immunogenic compositions by reducing and/or preventing antigen mimicry and immune cross-reactivity associated with immunogenic compositions is a desirable outcome in the safety and efficacy of vaccines.
  • the present disclosure is based, at least in part, on the discovery that antigenic polypeptide constructs comprised in immunogenic compositions can be analyzed for antigenic peptide sequences having high homology with peptides comprised in host proteins that are predicted to be antigenic after immunization. Without wishing to be bound by theory, subsequent modification of the antigenic peptide sequences results in immunogenic compositions capable of eliciting an immune response but having attenuated cross-reactivity with potentially antigenic host proteins.
  • an immunogenic composition for administration to a host comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct differs from an unmodified antigenic polypeptide or viral spike protein construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide
  • the antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 9 or more consecutive amino acids having at least about 75% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity with the cross-reactive host peptide.
  • the antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 15 consecutive amino acids having less than about 50% sequence identity with the cross-reactive host peptide.
  • an immunogenic composition for administration to a host, the composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct differs from an unmodified antigenic polypeptide or viral spike protein construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptid
  • an immunogenic composition for administration to a host comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 3-127); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c.
  • an unmodified antigenic polypeptide construct such as, for example, SARS-CoV-2 spike protein
  • antigenic peptide sequences such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 3-127
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide
  • an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 3-127) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the cross-reactive host peptide sequence set forth in SEQ ID NOs: 128-137) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 5 for SEQ ID Nos: 128-137); and d.
  • the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • an immunogenic composition for administration to a host comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c.
  • an unmodified antigenic polypeptide construct such as, for example, SARS-CoV-2 spike protein
  • antigenic peptide sequences such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide
  • an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 185-194) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in SEQ ID NOs: 175-184) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 3 for SEQ ID Nos: 175-184); and d.
  • a cross-reactive host peptide such as, for example, any one of the respective cross-reactive host peptide sequence set forth in SEQ ID Nos: 175-184
  • a host tissue such as, for example, the respective polypeptide named in Table 3 for SEQ ID Nos: 175-184
  • modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in SEQ ID NOs: 175-184).
  • an immunogenic composition for administration to a host comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c.
  • an unmodified antigenic polypeptide construct such as, for example, SARS-CoV-2 spike protein
  • antigenic peptide sequences such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide
  • an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in even numbered SEQ ID Nos: 140-174) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in odd numbered SEQ ID NOs: 139-173) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 3 for odd numbered SEQ ID Nos: 139-173); and d.
  • a cross-reactive host peptide such as, for example, any one of the respective cross-reactive host peptide sequence set forth in odd numbered SEQ ID Nos: 139-173
  • a host tissue such as, for example, the respective polypeptide named in Table 3 for odd numbered SEQ ID
  • modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in odd numbered SEQ ID NOs: 139-173).
  • an immunogenic composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide or viral spike protein construct and/or a nucleic acid encoding the modified antigenic polypeptide or
  • modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified antigenic polypeptide or viral spike protein construct having an unmodified antigenic peptide sequence.
  • the immunogenic composition comprising the modified antigenic polypeptide or viral spike protein construct elicits an immune response.
  • an immunogenic composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide or viral spike protein construct and/or a nucleic acid encoding the
  • a method for reducing or preventing crossreactivity of (1) an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, and/or (2) a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic peptide sequence and/
  • a method for reducing or preventing cross-reactivity of (1) an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, and/or (2) a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic
  • the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
  • the viral protein is a viral spike protein.
  • the bacterial protein is a protein from Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium. In some embodiments, the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
  • the parasite protein is a protein from Leishmania, Plasmodium, or Schistosoma. In some embodiments, the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
  • the fungal protein is a protein from Candida, Cryptococcus, or Aspergillus .
  • the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
  • the nucleic acid is RNA.
  • the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), selfamplifying RNA (saRNA), and/or modified RNA (modRNA).
  • the RNA is comprised in an expression vector.
  • the immunogenic composition is an RNA vaccine.
  • the nucleic acid is DNA.
  • the DNA is double stranded, single stranded, and/or antisense single stranded.
  • the DNA is comprised in an expression vector.
  • the immunogenic composition is a DNA vaccine.
  • the immunogenic composition comprises a polypeptide. In some instances, the immunogenic composition comprises a polypeptide vaccine.
  • the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
  • the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. In specific embodiments, the cross-reactive host polypeptide is expressed in the heart tissue. In some embodiments, the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • At least one cross-reactive host polypeptide comprises and/or is located within NEBL. In specific embodiments, at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In some preferred embodiments, the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. In specific embodiments, the cross-reactive host polypeptide is expressed in the heart tissue.
  • the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain l(ANKRDl), synaptopodin 2-like protein (SYNPO2L), myosin 7(MYH7), ankyrin repeat and SOCS box containing 15(ASB15), cysteine and glycine rich protein 3(CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase -coupling factor 6, mitochondrial(ATP5PF), and/or pericentriolar material 1 protein (PCM1).
  • at least one cross-reactive host polypeptide is located within MYH6.
  • At least one cross-reactive host polypeptide is located within MYH6 and has, for example, the amino acid sequence SEQ ID NO: 128. In specific embodiments, at least one cross-reactive host polypeptide is located within PCM1. In specific embodiments, at least one cross-reactive host polypeptide is located within PCM1 and comprises, for example, the amino acid sequence SEQ ID NO: 137. In specific embodiments, at least one cross-reactive host polypeptide comprises the amino acid sequence selected from any one of SEQ ID NO: 3-25. [0022] In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct after the ranking strategy is preferably selected from any one of the peptides of SEQ ID NO: 3-138.
  • the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 3-138. In some embodiments, the antigenic peptide sequence ofthe unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 3-25. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 26- 36. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 37- 60.
  • the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 61- 70. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 71- 94. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 95- 104. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 105- 127.
  • the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 128- 137. Such preferred peptides may be used for further analyses such as antigenicity predictions.
  • an immunogenic composition produced according to any method disclosed herein.
  • an immunogenic composition comprising any antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct disclosed herein.
  • the immunogenic composition elicits an immune response comprising an antibody response.
  • the immunogenic composition elicits an immune response comprising a T cell response.
  • an immunogenic composition for administration to a host comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • an immunogenic composition for administration to a host comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross- reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • E2 The immunogenic composition of El, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • E3 The immunogenic composition of E2, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E4 The immunogenic composition of E2 or E3, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
  • E5. The immunogenic composition of any one of E2-E4, wherein the viral protein is a viral spike protein.
  • E6 The immunogenic composition of E2, wherein the bacterial protein is a protein from Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium.
  • E7 The immunogenic composition of clause 2, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
  • E8 The immunogenic composition of E2, wherein the parasite protein is a protein from Leishmania, Plasmodium, or Schistosoma.
  • E9 The immunogenic composition of E2, wherein the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
  • E10 The immunogenic composition of E2, wherein the fungal protein is a protein from Candida, Cryptococcus, or Aspergillus .
  • El l The immunogenic composition of E2, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
  • E12. The immunogenic composition of any one of El-El 1, wherein nucleic acid is RNA.
  • El 3 The immunogenic composition of El 2, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E14 The immunogenic composition of E12 or E13, wherein the RNA is comprised in an expression vector.
  • El 5 The immunogenic composition of any one of E12-E14, wherein the immunogenic composition is an RNA vaccine.
  • E16 The immunogenic composition of any one of clauses El-El 1, wherein the nucleic acid is DNA.
  • El 7 The immunogenic composition of El 6, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E19 The immunogenic composition of any one of E16-E18, wherein the immunogenic composition is a DNA vaccine.
  • E20 The immunogenic composition of any one of El-El l, wherein the immunogenic composition comprises a polypeptide.
  • E21 The immunogenic composition of any one of E1-E20, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • E22 The immunogenic composition of any one of E1-E21, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
  • E23 The immunogenic composition of any one of E1-E22, wherein the cross- reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue.
  • E24 The immunogenic composition of any one of E1-E23, wherein the cross- reactive host polypeptide is expressed in the heart tissue.
  • E25 The immunogenic composition of any one of E1-E24, wherein the cross- reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 kelch like family member 41
  • MYH6 myosin
  • myosin 6 MYH6
  • ANKRDl synaptopodin 2-like protein
  • MYH7 myosin 7
  • SOCS box containing 15 ASB15
  • cysteine and glycine rich protein 3 CSRP3
  • DSP desmoplakin
  • E26 The immunogenic composition of E25, wherein at least one cross- reactive host polypeptide comprises and/or is located within NEBL. In some embodiments, the immunogenic composition of E25, wherein at least one cross-reactive host polypeptide is comprised located withins MYH6.
  • E27 The immunogenic composition of E25, wherein at least one cross- reactive host polypeptide comprises and/or is located within RYR2. In some embodiments, the immunogenic composition of E25, wherein at least one cross-reactive host polypeptide is located within PCM1.
  • an immunogenic composition for administration to a host comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct differs from an unmodified viral spike protein construct at one or more antigenic peptide sequences, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, wherein an antigenic peptide sequence of the unmodified viral spike protein construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified viral spike protein construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • an immunogenic composition for administration to a host comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct differs from an unmodified viral spike protein construct at one or more antigenic peptide sequences, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, wherein an antigenic peptide sequence of the unmodified viral spike protein construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15consecutive amino acids having less than about 50% sequence identity with the cross-reactive host peptide.
  • E29 The immunogenic composition of E28, wherein the viral spike protein is a spike protein from arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral spike protein is a spike protein from arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E30 The immunogenic composition of E28 or E29, wherein nucleic acid is
  • E31 The immunogenic composition of E30, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E32 The immunogenic composition of E30 or 31, wherein the RNA is comprised in an expression vector.
  • E33 The immunogenic composition of any one of E30-E32, wherein the immunogenic composition is an RNA vaccine.
  • E34 The immunogenic composition of E28 or E29, wherein the nucleic acid is DNA.
  • E35 The immunogenic composition of E34, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E36 The immunogenic composition of E34 or E35, wherein the DNA is comprised in an expression vector.
  • E37 The immunogenic composition of any one of E34-E36, wherein the immunogenic composition is a DNA vaccine.
  • E38 The immunogenic composition of E28 or E29, wherein the immunogenic composition comprises a polypeptide.
  • E39 The immunogenic composition of any one of E28-E38, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • E40 The immunogenic composition of any one of E28-E39, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified viral spike protein construct.
  • E41 The immunogenic composition of any one of E28-E40, wherein the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue.
  • E42 The immunogenic composition of any one of E28-E41, wherein the cross-reactive host polypeptide is expressed in the heart tissue.
  • E43 The immunogenic composition of any one of E28-E42, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 myosin heavy chain 6
  • MYH7 myosin heavy
  • myosin 6 MYH6
  • ANKRDl synaptopodin 2-like protein
  • SYNPO2L synaptopodin 2-like protein
  • MYH7 myosin 7
  • ankyrin repeat and SOCS box containing 15 ASB15
  • E44 The immunogenic composition of E43, wherein at least one cross- reactive host polypeptide comprises and/or is located within NEBL. In some embodiments, the immunogenic composition of E43, wherein at least one cross-reactive host polypeptide is located within MYH6.
  • E45 The immunogenic composition of E43, wherein at least one cross- reactive host polypeptide comprises and/or is located within RYR2. In some embodiments, the immunogenic composition of E43, wherein at least one cross-reactive host polypeptide is located within PCM1
  • a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in the host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
  • a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in the host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct
  • E47 The method of E46, wherein modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified antigenic polypeptide construct having an unmodified antigenic peptide sequence.
  • E48 The method of E46 or E47, wherein the immunogenic composition comprising the modified antigenic polypeptide construct elicits an immune response.
  • E49 The method of any one of E46-E48, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • E50 The method of E49, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E51 The method of E49 or E50, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
  • E52 The method of any one of E49-E51, wherein the viral protein is a viral spike protein.
  • E53 The method of E49, wherein the bacterial protein is a protein from
  • Streptococcus Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium .
  • E54 The method of E49, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
  • E55 The method of E49, wherein the parasite protein is a protein from
  • E56 The method of E49, wherein the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
  • E57 The method of E49, wherein the fungal protein is a protein from
  • Candida Cryptococcus, or Aspergillus .
  • E58 The method of E49, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
  • E59 The method of any one of E46-E58, wherein nucleic acid is RNA.
  • E60 The method of E59, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E62 The method of any one of E59-E61, wherein the immunogenic composition is an RNA vaccine.
  • E63 The method of any one of E46-E58, wherein the nucleic acid is DNA.
  • E64 The method of E63, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E65 The method of E63 or E64, wherein the DNA is comprised in an expression vector.
  • E66 The method of any one of E63-E65, wherein the immunogenic composition is a DNA vaccine.
  • E67 The method of any one of E46-E58, wherein the immunogenic composition comprises a polypeptide.
  • E68 The method of any one of E46-E67, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • E69 The method of any one of E46-E68, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
  • E70 The method of any one of E46-E69, wherein the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue.
  • E71 The method of any one of E46-E70, wherein the cross-reactive host polypeptide is expressed in the heart tissue.
  • E72 The method of any one of E46-E71, wherein the cross-reactive host polypeptide comprises and/or is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 myosin heavy chain 6
  • MYH7 my
  • E72 wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6.
  • E73 The method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within PCM1.
  • E74 The method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2.
  • a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross- reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • E76 The method of E75, wherein modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified viral spike protein construct having an unmodified antigenic peptide sequence.
  • E77 The method of E75 or E76, wherein the immunogenic composition comprising the modified viral spike protein construct elicits an immune response.
  • E78 The method of any one of E75-E77, wherein the viral spike protein is a spike protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral spike protein is a spike protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E79 The method of any one of E75-E78, wherein nucleic acid is RNA.
  • E80 The method of E79, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E81 The method of E79 or E80, wherein the RNA is comprised in an expression vector.
  • E82 The method of any one of E79-E81, wherein the immunogenic composition is an RNA vaccine.
  • E83 The method of any one of E75-E78, wherein the nucleic acid is DNA.
  • E84 The method of E83, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E85 The method of E83 or E84, wherein the DNA is comprised in an expression vector.
  • E87 The method of any one of E75-E78, wherein the immunogenic composition comprises a polypeptide.
  • E88 The method of any one of E75-E87, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • E89 The method of any one of E75-E88, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified viral spike protein construct.
  • E90 The method of any one of E75-E89, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
  • E91 The method of any one of E75-E90, wherein the cross-reactive host polypeptide is expressed in heart tissue.
  • E92 The method of any one of E75-E91, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 kelch like family member 41
  • MYH6 myosin heavy
  • myosin 6 MYH6
  • ANKRD1 ankyrin repeat domain 1
  • SYNPO2L synaptopodin 2- like protein
  • MYH7 myosin 7
  • SOCS box containing 15 ASB15
  • cysteine and glycine rich protein 3 CS
  • E93 The method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6. [0119] E94. The method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In another embodiment, the method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within PCM1
  • an immunogenic composition produced according to the method of any one of E46-E74 or E66-E82.
  • a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence, wherein the modified antigenic
  • a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence, wherein the modified
  • E97 The method of E96, wherein the modified antigenic polypeptide construct elicits an immune response.
  • E98 The method of E96 or E97, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • E99 The method of E98, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E100 The method of E98 or E99, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
  • E101 The method of any one of E98-E100, wherein the viral protein is a viral spike protein.
  • E102 The method of E98, wherein the bacterial protein is a protein from
  • Streptococcus Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium .
  • E103 The method of E98, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
  • E104 The method of E98, wherein the parasite protein is a protein from
  • E105 The method of E98, wherein the parasite protein is aparasite cell surface protein and/or a parasite secreted protein.
  • E106 The method of E98, wherein the fungal protein is a protein from
  • Candida Cryptococcus, or Aspergillus .
  • E107 The method of E98, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
  • E108 The method of any one of E96-E 107, wherein nucleic acid is RNA.
  • E109 The method of E108, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E110 The method of E108 or E109, wherein the RNA is comprised in an expression vector.
  • El 12. The method of El 11, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • El 14 The method of any one of E96-E113, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • El 15. The method of any one of E96-E114, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
  • El 16. The method of any one of E96-E115, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
  • El 17. The method of any one of E96-E116, wherein the cross-reactive host polypeptide is expressed in heart tissue.
  • El 18 The method of any one of E96-E117, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • myosin 6 MYH6
  • ANKRD1 ankyrin repeat domain 1
  • SYNPO2L synaptopodin 2-like protein
  • MYH7 myosin 7
  • SOCS box containing 15 ASB15
  • cysteine and glycine rich protein 3 CSRP3
  • El 19 The method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6.
  • E120 The method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In another embodiment, the method of El 18, wherein at least one cross-reactive host polypeptide is located within comprises PCM1. [0146] E121. According to another aspect of the present disclosure, there is provided an immunogenic composition comprising the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct any one of E96-E120.
  • E122 The immunogenic composition of E121, wherein nucleic acid is RNA.
  • El 23 The immunogenic composition of El 22, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E124 The immunogenic composition of E122 or E123, wherein the RNA is comprised in an expression vector.
  • El 25 The immunogenic composition of any one of E122-E124, wherein the immunogenic composition is an RNA vaccine.
  • E126 The immunogenic composition E121, wherein the nucleic acid is DNA.
  • El 27 The immunogenic composition of El 26, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E128 The immunogenic composition of E126 or E127, wherein the DNA is comprised in an expression vector.
  • E129 The immunogenic composition of any one of E126-E128, wherein the immunogenic composition is a DNA vaccine.
  • El 30 The immunogenic composition of E121, wherein the immunogenic composition comprises a polypeptide.
  • a method for reducing or preventing cross-reactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct comprising: identifying at least one antigenic peptide sequence of an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross- reactive host peptide to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the un
  • a method for reducing or preventing crossreactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct comprising: identifying at least one antigenic peptide sequence of an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with
  • E131 wherein the modified viral protein construct elicits an immune response.
  • E133 The method of E131 or E132, wherein the viral spike protein is a spike protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
  • E134 The method of E131 or E133, wherein nucleic acid is RNA.
  • RNA double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E136 The method of E134 or E135, wherein the RNA is comprised in an expression vector.
  • E137 The method of E131 or E133, wherein the nucleic acid is DNA.
  • E138 The method of El 37, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E139 The method of E137 or E138, wherein the DNA is comprised in an expression vector.
  • E140 The method of any one of E131-E139, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
  • E 141 The method of any one of E 131 -E 140, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about
  • El 42 The method of any one of E131-E141, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
  • El 43 The method of any one of E131-E142, wherein the cross-reactive host polypeptide is expressed in heart tissue.
  • El 44 The method of any one of E131-E143, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 myosin heavy chain 6
  • MYH7 myosin heavy
  • myosin 6 MYH6
  • ANKRD1 ankyrin repeat domain 1
  • SYNPO2L synaptopodin 2-like protein
  • MYH7 myosin 7
  • SOCS box containing 15 ASB15
  • cysteine and glycine rich protein 3 CSRP3
  • El 45 The method of El 44, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL.
  • an immunogenic composition comprising the viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct any one of E131-146.
  • E148 The immunogenic composition of E147, wherein nucleic acid is RNA.
  • E149 The immunogenic composition of E148, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA
  • modRNA modified RNA
  • E150 The immunogenic composition of E148 or E149, wherein the RNA is comprised in an expression vector.
  • E151 The immunogenic composition of any one of E148-E150, wherein the immunogenic composition is an RNA vaccine.
  • E152 The immunogenic composition E147, wherein the nucleic acid is DNA.
  • E153 The immunogenic composition of E152, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
  • E154 The immunogenic composition of E152 or E153, wherein the DNA is comprised in an expression vector.
  • E155 The immunogenic composition of any one of E152-E154, wherein the immunogenic composition is a DNA vaccine.
  • El 56 The immunogenic composition of El 47, wherein the immunogenic composition comprises a polypeptide.
  • E157 According to another aspect of the present disclosure, there is provided a method of vaccinating a subject, comprising administering to the subject in need thereof an effective amount of the immunogenic composition of any of any one of E1-E45, E95, E121- E130, or E147-E156.
  • E158 A method for treating or preventing an infectious disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of E1-E45, E95, E121-E130, or E147-E156.
  • E159 The method of E158, wherein the immunogenic composition elicits an immune response comprising an antibody response.
  • El 62 A method identifying a potential toxicity of an immunogenic composition in a biological host system, the immunogenic composition comprising an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, the method comprising: a. identifying one or more proteins of the host that have a degree of similarity to the antigenic polypeptide construct; b. identifying properties of the one or more proteins, the properties comprising cellular location, gene- and protein-tissue expression profdes, and/or toxic state associations; c. ranking or sorting the one or more proteins using one or more of the properties; d. identifying immunogenicity of the one or more proteins; e. comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence; f. predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence.
  • E165 The method of any one of clauses E162-E164, wherein the immunogenic composition is a vaccine.
  • El 66 The method of any one of clauses E162-E165, wherein the method comprises searching a human proteome with a spike protein peptide and identifying a protein from the human proteome with homology to the spike protein peptide.
  • E168 The method any one of clauses E162-E167, wherein ranking or sorting the one or more proteins using one or more of the properties comprises ranking cellular locations that are extracellular or on a surface of a cell as more likely to cause the potential toxicity than intracellular locations, ranking gene- and protein-tissue expression profiles that overlap in tissue location of the potential toxicity as more likely to cause the potential toxicity than other tissue locations, and/or ranking toxic state associations that overlap or are related to the potential toxicity as more likely to cause the potential toxicity than other toxic state associations.
  • identifying immunogenicity of the one or more proteins comprises identifying a ranking or sorting of degree of immunogenicity and/or degree of potential immunogenicity.
  • comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence comprises comparing the rank of likely to cause the potential toxicity and degree of immunogenicity and/or degree of potential immunogenicity and ranking: more likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity, and either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than less likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity.
  • E172 The method any one of clauses E162- E171, wherein each of the steps are performed on a computational model.
  • FIG. 1 illustrates a strategy for analyzing antigenic polypeptide constructs for antigenic peptide sequences having high homology with peptides comprised in host proteins to predict an immune response to the antigenic polypeptide construct that will have crossreactivity to the host protein.
  • FIG. 2 illustrates a strategy for a viral spike protein-to-human protein peptide homology search.
  • FIG. 3 illustrates the concept of molecular mimicry in the context of an mRNA vaccine against a viral spike protein.
  • the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value.
  • the term “about” may encompass a range ofvalues that are 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 measurement or quantitation.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C.
  • “and/or” operates as an inclusive or.
  • essentially all is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.
  • compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification.
  • the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
  • inhibiting or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result
  • promote or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.
  • the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest.
  • a reference, standard, or control may be tested and/or determined substantially simultaneously and/or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and/or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.
  • the term “antigen mimicry,” “molecular mimicry,” and any variation of these terms refers to a phenomenon in which an antigenic peptide or protein sequence used in an immunogenic composition cross-reacts with endogenously-expressed host proteins.
  • the endogenously-expressed human proteins act as an “antigen mimic” or “molecular mimic” of the antigenic peptide or protein sequence.
  • immune responses raised against the antigenic peptide or protein sequence can cross-react with endogenously-expressed human proteins that share similar or identical peptide sequences (or structures) with the antigenic peptide or protein sequence.
  • the concept of molecular mimicry in the context of an mRNA vaccine against a viral spike protein is illustrated in FIG. 3
  • the cross-reactive host protein or polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
  • the cross-reactive host polypeptide is located withing the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca 2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP).
  • NEBL nebulette
  • RYR2 ryanodine receptor 2
  • TTN titin
  • KLHL41 kelch like family
  • the cross-reactive host protein or polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
  • the cross-reactive host polypeptide is located withing the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
  • MYH6 myosin 6
  • ANKRD1 ankyrin repeat domain 1
  • SYNPO2L synaptopodin 2-like protein
  • MYH7 myosin 7
  • SOCS box containing 15 ASB15
  • RNA means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and/or uracil (U)).
  • ribonucleotide means a nucleotide with a hydroxyl group at the 2’ position of a [3-D-ribofuranosyl group.
  • RNA can be messenger RNA (mRNA) that relates to a RNA transcript which encodes a peptide or protein.
  • mRNA generally contains a 5' untranslated region (5'- UTR), a polypeptide coding region, and a 3' untranslated region (3'-UTR).
  • RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, and modified RNA (modRNA).
  • RNA is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid.
  • a “modified RNA” or “modRNA” refers to an RNA molecule having at least one addition, deletion, substitution, and/or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and/or 3' end(s) of RNA.
  • such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide.
  • a modified nucleotide can replace one or more uridine and/or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and/or cytidine in the RNA sequence, or can occur for only select uridine and/or cytidine nucleotides.
  • Such alterations to the standard nucleotides in RNA can include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides.
  • at least one uridine nucleotide can be replaced with 1 -methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art.
  • RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides.
  • the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA).
  • DNA means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and/or thymine (T)).
  • DNA can contain all, or a majority of, deoxyribonucleotide residues.
  • deoxyribonucleotide means a nucleotide lacking a hydroxyl group at the 2’ position of a [3-D-ribofiiranosyl group.
  • DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.
  • a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues.
  • wild-type refers to the endogenous version of a molecule that occurs naturally in an organism.
  • wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response.
  • a “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide.
  • a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity.
  • a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein.
  • the protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods.
  • nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof).
  • a polypeptide e.g., an antigen or fragment thereof.
  • recombinant may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
  • RNA, DNA, and/or peptides or polypeptides can be used as a modality to treat and/or prevent a number of diseases and/or conditions in mammals, including humans.
  • Methods described herein comprise administration of the RNA, DNA, and/or peptides or polypeptides described herein to a mammal, such as a human.
  • RNA, DNA, and/or peptides or polypeptides include an antigen-coding RNA or DNA vaccine or a peptide or polypeptide vaccine to induce robust neutralizing antibodies and accompanying/concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses.
  • RNA or DNA can be used to encode at least one antigen intended to generate an immune response in said mammal.
  • pathogenic antigenic polypeptide constructs are peptide or protein antigens derived from a pathogen associated with infectious disease, including viruses, bacteria, parasites, and fungi.
  • the antigenic polypeptide constructs can comprise one or more antigenic peptide sequences that elicit an immune response.
  • the antigenic polypeptide constructs are viral spike protein constructs, and the viral spike protein constructs can comprise one or more antigenic peptide sequences that elicit an immune response.
  • Viral antigenic polypeptide constructs can be from a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus, for example.
  • the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
  • the viral protein is a viral spike protein.
  • the viral antigenic polypeptide constructs can be selected from antigens derived from viral pathogens including an arenavirus (such as Lassa virus, or lymphocytic choriomeningitis virus (LCMV)); an astrovirus; a bunyavirus (such as a Hantavirus); a calicivirus; a coronavirus (such as a severe acute respiratory syndrome virus (SARS) - e.g.
  • an arenavirus such as Lassa virus, or lymphocytic choriomeningitis virus (LCMV)
  • an astrovirus such as a Hantavirus
  • a bunyavirus such as a Hantavirus
  • a calicivirus such as a severe acute respiratory syndrome virus (SARS) - e.g.
  • SARS severe acute respiratory syndrome virus
  • SARS-CoV-2 SARS-CoV-2, or a middle east respiratory syndrome (MERS) virus
  • a filovirus such as Ebola virus or Marburg virus
  • a flavivirus such as Yellow Fever virus, West Nile virus, or Hepatitis C virus (HCV)
  • HCV Hepatitis C virus
  • a hepadnavirus a hepevirus
  • an orthomyxovirus such as Influenza A virus, Influenza B virus, or Influenza C virus
  • a paramyxovirus such as Rubeola virus, or Rubulavirus
  • a picomavirus such as Poliovirus, Hepatitis A virus, or Rhinovirus
  • a reovirus such as Rotavirus
  • a retrovirus such as Human Immunodeficiency Virus (HIV), or Human T-lymphotropic virus (HTLV)
  • a rhabdovirus such as Rabies virus or Rabies lyssavirus
  • a togavirus such as Sindbis
  • antigenic polypeptide constructs can be from a protein from bacterial pathogens including Staphylococcus, Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium.
  • the bacterial antigen is a bacterial protein, and in some embodiments, the bacterial protein is a bacterial membrane protein and/or a bacterial cell wall protein.
  • Antigens derived from Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRSA) that are contemplated for use include virulence regulators, such as the Agr system, Sar and Sae, the Ari system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP.
  • MRSA Methicillin-resistant Staphylococcus aureus
  • Staphylococcus proteins that may serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay).
  • the genomes for two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007).
  • Staphylococcus proteins for use as antigens may also be identified in other public databases such as GenBank®, Swiss-Prot®, and TrEMBL®.
  • Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline -binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC).
  • Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as an antigen in some embodiments (see, e.g., Zysk et al., 2000).
  • antigenic polypeptide constructs examples include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B.
  • influenzae type b outer membrane protein Helicobacter polypeptides, Klebsiella polypeptides, L-form bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S.
  • pneumoniae polypeptides Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., S. pyogenes M proteins), group B streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis Fl and V antigens).
  • group A streptococcus polypeptides e.g., S. pyogenes M proteins
  • group B streptococcus (S. agalactiae) polypeptides e.g., Treponema polypeptides
  • Yersinia polypeptides e.
  • one or more of the bacterial pathogens and/or one or more bacterial antigens in the above lists may be excluded.
  • the antigenic polypeptide constructs can be from a protein from parasitic pathogens including Leishmania, Plasmodium, or Schistosoma.
  • the parasite antigen is a parasite protein, and in some embodiments, the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
  • parasite antigens that can be used for antigenic polypeptide constructs include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostom
  • PfCSP falciparum circumsporozoite
  • PfSSP2 sporozoite surface protein 2
  • PfLSAl c- term carboxyl terminus of liver state antigen 1
  • PfExp-1 exported protein 1
  • Pneumocystis polypeptides Sarcocystis polypeptides
  • Schistosoma polypeptides Theileria polypeptides, Toxoplasma polypeptides, Trypanosoma polypeptides, and polypeptides (including antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kiss
  • antigenic polypeptide constructs can be from a protein from fungal pathogens including Candida, Cryptococcus, or Aspergillus .
  • the fungal antigen is a fungal protein, and in some embodiments, the fungal protein is a fungal surface protein and/or a fungal cell wall protein.
  • fungal antigens that can be used for antigenic polypeptide constructs include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptide
  • antigenic polypeptide constructs can be from a protein from a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
  • Exemplary tumor-/cancer-associated antigens and tumor neoantigens that can be used for antigenic polypeptide constructs include EBNA, CD123, HER1, HER2, CA-125, CA 19-9, CA 72-4, CA 15-3 ⁇ CA 27.29 ⁇ BCAA, CA-195, CA- 242, CA-50, CA LX, MN-CA IX, TRAIL/DR4, CD2, CD5, CD7, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD47, CD56, CD68/P1, CD70, CD97, CD99, CD123, CD 171, CD 179, CD200, CD319 (CS1), HLA-G, carcinoembryonic antigen, alphafetoprotein,
  • ART-4 CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, MUC16, MUC18, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, P16, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, [3-catenin, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, interferon regulatory factor 4 (
  • sequences for antigens are known in the art, for example, in the GenBank® database: CD 19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD 123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No.
  • Tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancers, as examples.
  • Exemplary tumor- associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO 99/40188); PRAME; BAGE; RAGE, Lü (also known as NY ESO 1); SAGE; and HAGE or GAGE.
  • tumor antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, e.g., U.S. Patent No. 6,544,518.
  • Prostate cancer tumor-associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate-carcinoma tumor antigen- 1 (PCTA-1), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).
  • one or more of the tumor-/cancer-associated antigens in the above list may be excluded.
  • antigenic polypeptide constructs can be from a protein from Acinetobacter baumannii, Anciplcisma genus, Anciplcisma phagocy tophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei
  • viruses include, but are not limited to, those caused and/or impacted by viral infection.
  • viruses include, but are not limited to, an arenavirus (such as Lassa virus, or lymphocytic choriomeningitis virus (LCMV)); an astrovirus; a bunyavirus (such as a Hantavirus); a calicivirus; a coronavirus (such as a severe acute respiratory syndrome virus (SARS) - e.
  • SARS-CoV-2 SARS-CoV-2, or a middle east respiratory syndrome (MERS) virus
  • a filovirus such as Ebola virus or Marburg virus
  • a flavivirus such as Yellow Fever virus, West Nile virus, or Hepatitis C virus (HCV)
  • HCV Hepatitis C virus
  • a hepadnavirus a hepevirus
  • an orthomyxovirus such as Influenza A virus, Influenza B virus, or Influenza C virus
  • a paramyxovirus such as Rubeola virus, or Rubulavirus
  • a picomavirus such as Poliovirus, Hepatitis A virus, or Rhinovirus
  • a reovirus such as Rotavirus
  • a retrovirus such as Human Immunodeficiency Virus (HIV), or Human T-lymphotropic virus (HTLV)
  • a rhabdovirus such as Rabies virus or Rabies lyssavirus
  • a togavirus such as Sindbis
  • Conditions and/or diseases that can be treated with the RNA, DNA, and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by bacteria, fungi, parasites, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
  • an antigen intended to generate an immune response is encoded by a nucleic acid.
  • the nucleic acid encodes one or more peptide or protein antigen(s) derived from a pathogen associated with one or more infectious disease(s), including virus(es), bacteria, parasite(s), and fungi.
  • the nucleic acids encoding the antigenic polypeptide constructs can comprise one or more nucleotide sequences corresponding to one or more antigenic peptide sequences that elicit an immune response.
  • Nucleic acid sequences can exist in a variety of instances such as: isolated segments; recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described herein.
  • Nucleic acids may encode an antigen or epitope to which antibodies may bind.
  • Nucleic acids encoding fusion proteins that include antigens or epitopes are also provided.
  • the nucleic acids can be singlestranded or double-stranded and can comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
  • polynucleotide refers to a nucleic acid molecule that can be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences.
  • Polynucleotides may be singlestranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
  • the term “gene” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar polypeptide.
  • polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters).
  • the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
  • nucleic acid segments regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably.
  • the nucleic acids can be any length.
  • nucleotides in length can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector.
  • a nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol.
  • a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic and/or prophylatic benefits such as targeting or efficacy.
  • a tag or other heterologous polypeptide may be added to the modified polypeptide -encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
  • the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof encoded by a nucleic acid elicit an immune response.
  • the immune response may be against the antigenic polypeptide constructs and/or antigenic peptide sequences and/or a homologous endogenously-expressed (e.g. cross-reactive) host protein.
  • the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously-expressed (e.g. cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%,
  • the nucleic acids may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
  • the one or more antigenic polypeptide constructs and/or antigenic peptide sequence thereof encoded by a nucleic acid are modified such that the antigenic polypeptide constructs and/or antigenic peptide sequences thereof continue to elicit an immune response but homology with a homologous endogenously-expressed (e.g., cross- reactive) host protein is reduced to reduce or prevent cross-reactivity of the immune response to the host protein after immunization with a nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences thereof.
  • a homologous endogenously-expressed host protein e.g., cross- reactive
  • the homology between the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed (e.g., cross-reactive) host protein may be reduced by modifying the nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences thereof such that the nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences and the nucleic acid, e.g., genomic DNA, mRNA, or cDNA, encoding the homologous endogenously-expressed (e.g., cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 2
  • the nucleic acids of the disclosure encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations in the sequence encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide compared to nucleic acids, e.g., genomic DNA, mRNA, cDNA, encoding unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences
  • the portions of the nucleic acids of the disclosure encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
  • Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. In some instances, however it is made, a mutant polypeptide can be expressed and screened for a desired property. [0017] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes.
  • one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g. , Romain Studer et al., Biochem. J. 449:581-594 (2013).
  • the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.
  • the RNA molecule described herein is a coding RNA molecule.
  • Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide.
  • the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide.
  • the coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs), which may be a sequence of codons that is translatable into a polypeptide or protein of interest.
  • the coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule, or self-amplifying RNA molecule (saRNA, also referred to as a replicon).
  • mRNA messenger RNA
  • saRNA self-amplifying RNA molecule
  • the RNA molecule is an mRNA.
  • the RNA molecule is a saRNA.
  • the saRNA molecule may be a coding RNA molecule.
  • the RNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides.
  • one RNA molecule may also encode more than one polypeptide of interest or more, such as an antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens.
  • the sequence of the RNA molecule may be codon optimized or deoptimized for expression in a desired host, such as a human cell.
  • the RNA molecule includes equal to any one of, at least any one of, at most any one of, or between any two of from about 20 to about 100,000 nucleotides (e.g., equal to any one of, at least any one of, at most any one of, or between any two of from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to
  • the RNA molecule includes at least 100 nucleotides.
  • the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; between 7,000 and 25,000 nucleotides.
  • one or more of the nucleotide size ranges in the list may be excluded.
  • the sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression or replication of the RNA, or to provide additional stability or resistance to degradation.
  • the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA.
  • the RNA molecules may include one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
  • a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides.
  • the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”.
  • the same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”.
  • the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.
  • the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. Naturally occurring nucleotide modifications are known in the art. [0025] In some embodiments, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA may include a 5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.
  • Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include, for example, m5C (5- methylcytidine), m5U (5 -methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1 -methyladenosine); m2A (2- methyladenosine); Am (2-1-0- methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis), methyl
  • Additional exemplary modified nucleotides include any one of N-l- methylpseudouridine; pseudouridine, N6-methyladenosine, 5 -methylcytidine, and 5- methyluridine.
  • the modified nucleotide is N-l -methylpseudouridine.
  • the RNA molecule may include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages.
  • the RNA molecule includes a modified nucleotide selected from any one of pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2- thiouridine, 4 '-thiouridine, 5-methylcytosine, 5 -methyluridine, 2-thio-l -methyl- 1 -deazapseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl -pseudouridine
  • the UTR is derived from an mRNA that is naturally abundant in a specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted.
  • the UTR increases protein synthesis.
  • the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and/or the rate at which ribosomes initiate translation on the message (message translation efficiency).
  • the UTR sequence may prolong protein synthesis in a tissue-specific manner.
  • the 5' UTR and the 3' UTR sequences are computationally derived.
  • the 5' UTR and the 3' UTRs are derived from a naturally abundant mRNA in a tissue.
  • the tissue may be, for example, liver, a stem cell, or lymphoid tissue.
  • the lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T- lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte.
  • the 5' UTR and the 3' UTR are derived from an alphavirus.
  • the 5' UTR and the 3' UTR are from a wild-type alphavirus. Examples of alphaviruses are described below. [0031]
  • the RNA molecule includes a 5 ' UTR and the 3 ' UTR derived from a naturally abundant mRNA in a tissue.
  • the RNA molecule includes a 5' UTR and the 3' UTR derived from an alphavirus.
  • the RNA molecule includes a 5 ' UTR and the 3 ' UTR from a wild-type alphavirus .
  • the RNA molecule includes a 5' cap.
  • the 5' and 3' UTRs may be operably linked to an ORF, which may be a sequence of codons that is capable of being translated into a polypeptide of interest.
  • ORF an open reading frame
  • the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).
  • the ORF encodes a modified antigenic polypeptide construct.
  • the ORF further includes one or more subgenomic promoters.
  • the RNA molecule includes a subgenomic promoter operably linked to the ORF.
  • the subgenomic promoter comprises a cisacting regulatory element.
  • the cis-acting regulatory element is an AU- rich element.
  • the AU-rich element is au, auaaaagau, auaaaagau, auag, auauauauau, auauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug.
  • a RNA molecule may include (i) an ORF encoding a replicase which may transcribe RNA from the RNA molecule and (ii) an ORF encoding at least one modified antigenic polypeptide construct.
  • the polymerase may be an alphavirus replicase, e.g., including any one of the non-structural alphavirus proteins nsPl, nsP2, nsP3 and nsP4, or a combination thereof.
  • the RNA molecule includes alphavirus nonstructural protein nsPl.
  • the RNA molecule includes alphavirus nonstructural protein nsP2.
  • the RNA molecule includes alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsPl, nsP2, and nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsPl, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsPl, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.
  • the RNA molecule does not encode alphavirus structural proteins.
  • the RNA molecule may have one or more additional (e.g., downstream) open reading frames, e.g., to encode further antigen(s) or to encode accessory polypeptides.
  • a first RNA molecule does not include an ORF encoding a modified antigenic polypeptide or protein
  • a second RNA or a saRNA molecule includes an ORF encoding the modified antiogenic polypeptide or protein.
  • the first RNA molecule does not include a subgenomic promoter.
  • the first RNA molecule includes an ORF for a nonstructural protein derived from an alphavirus.
  • the ORF encoding a nonstructural protein in the first RNA molecule and in the second RNA or the saRNA molecule are identical.
  • the second RNA or the saRNA molecule further includes an ORF encoding a modified antiogenic polypeptide or protein.
  • the second RNA or the saRNA molecule may lead to a production of genomic RNA copies of itself in a cell, but not to the production of RNA- containing virions.
  • the second RNA or the saRNA molecule cannot perpetuate itself in infectious form.
  • the genes encoding alphavirus structural proteins, which are necessary for perpetuation in wild-type alphaviruses are absent from the second RNA or saRNA molecules of the present disclosure, and their place is taken by a gene(s) encoding the polypeptide of interest, such that the subgenomic transcript encodes the modified antiogenic polypeptide or protein, rather than the structural alphavirus virion proteins.
  • the RNA molecule described herein may include a gene of interest.
  • the gene of interest can encode a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.
  • a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptide
  • the sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g. , GenBank.
  • the RNA molecule includes a coding region for an antigen derived from a pathogen associated with infectious disease or cancer, as described elsewhere herein.
  • the RNA molecule described herein includes a 5' cap.
  • the 5 '-cap moiety is a natural 5 '-cap.
  • a “natural 5 '-cap” is defined as a cap that includes 7-methylguanosine connected to the 5' end of an mRNA molecule through a 5' to 5' triphosphate linkage.
  • the 5 '-cap moiety is a 5'- cap analog.
  • the 5' end of the RNA is capped with a modified ribonucleotide, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping).
  • the 5' end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription.
  • capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule.
  • an exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE)
  • the 5' terminal cap includes a cap analog, for example, a 5' terminal cap may include a guanine analog.
  • Exemplary guanine analogs include, but are not limited to, inosine, N1 -methyl -guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine.
  • the capping region may include a single cap or a series of nucleotides forming the cap.
  • the capping region may be equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides in length.
  • the cap is absent.
  • a first and/or second operational region of the RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 to 40, e.g. , 5-30, 10-20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
  • poly A tail refers to a stretch of consecutive adenine residues, which may be attached to the 3' end of the RNA molecule.
  • the poly-A tail may increase the half-life of the RNA molecule.
  • Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation.
  • Exemplary designs include a poly-A tails of about 40 adenine residues to about 80 adenine residues.
  • the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly A tail sequence.
  • the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end.
  • a poly-A polymerase recognition sequence e.g., AAUAAA
  • a “full length” RNA molecule is one that includes a 5 '-cap and a poly A tail.
  • the poly A tail includes 5-400 nucleotides in length.
  • the poly A tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two of 5, 6, 7, 8, 9, 10, 15, 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, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400.
  • the RNA molecule includes a poly A tail that includes about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides.
  • the RNA molecule includes a poly A tail includes a sequence of greater than 30 adenosine nucleotides (“As”).
  • the RNA molecule includes a poly A tail that includes about 40 As. In some embodiments, the RNA molecule includes a poly A tail that includes about 80 As. As used herein, regarding poly A tail length, the term “about” refers to a deviation of ⁇ 10% of the value(s) to which it is attached.
  • the 3' poly-A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some embodiments, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 80 consecutive adenosine residues.
  • the RNA molecule is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/ diafiltration .
  • the RNA molecule is a saRNA.
  • saRNA self-amplifying RNA
  • replicon refer to RNA with the ability to replicate itself.
  • Self-amplifying RNA molecules may be produced by using replication elements derived from a virus or viruses, e.g. , alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest.
  • a self-amplifying RNA molecule is typically a positivestrand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA.
  • the delivered RNA leads to the production of multiple daughter RNAs.
  • These daughter RNAs, as well as collinear subgenomic transcripts may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein of interest, e.g. , an antigen.
  • the overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g. , a viral antigen, can become a major polypeptide product of the cells.
  • the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases, and other nonstructural viral proteins.
  • the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest).
  • a subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA.
  • the heterologous sequence e.g. , an antigen of interest
  • IRS internal ribosome entry site
  • the self-amplifying RNA molecule is not encapsulated in a virus-like particle.
  • Self-amplifying RNA molecules described herein may be designed so that the self-amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA.
  • an alphavirus such as Sinbis virus (SIN), Semliki forest virus, and Venezuelan equine encephalitis virus (VEE)
  • one or more genes encoding viral structural proteins, such as capsid and/or envelope glycoproteins may be omitted.
  • a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen.
  • the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein nsPl, nsP2, nsP3, nsP4, and any combination thereof.
  • the selfamplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5 -methyluridine). In some embodiments, the self-amplifying RNA molecules does not include a modified nucleotide (e.g. , pseudouridine, N6-methyladenosine, 5- methylcytidine, 5 -methyluridine).
  • modified nucleotides e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5 -methyluridine.
  • the saRNA construct may encode at least one non-structural protein (NSP), disposed 5' or 3' of the sequence encoding at least one peptide or polypeptide of interest.
  • the sequence encoding at least one NSP is disposed 5' of the sequences encoding the peptide or polypeptide of interest.
  • the sequence encoding at least one NSP may be disposed at the 5' end of the RNA construct.
  • at least one non- structural protein encoded by the RNA construct may be the RNA polymerase nsP4.
  • the saRNA construct encodes nsPl, nsP2, nsP3 and, nsP4.
  • nsPl is the viral capping enzyme and membrane anchor of the replication complex (RC).
  • nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing.
  • nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP- ribosylation.
  • nsP4 is the core viral RNA-dependent RNA polymerase.
  • the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsPl, nsP2, nsP3, and nsP4.
  • the self-amplifying RNA molecules do not encode alphavirus structural proteins.
  • the selfamplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the selfamplifying RNA molecule cannot perpetuate itself in infectious form.
  • the alphavirus structural proteins which are necessary for perpetuation in wild-type viruses can be absent from selfamplifying RNAs of the present disclosure and their place can be taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.
  • the self-amplifying RNA molecule may have two open reading frames.
  • the first (5') open reading frame can encode a replicase; the second (3') open reading frame can encode a polypeptide comprising an antigen of interest.
  • the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides.
  • the saRNA molecule further includes (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence.
  • the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.
  • self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.
  • the saRNA molecule is alphavirus-based.
  • Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family.
  • Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), and Venezuelan equine encephalitis virus (VEEV).
  • Sindbis virus SINV
  • SFV Semliki Forest virus
  • RRV Ross River virus
  • VEEV Venezuelan equine encephalitis virus
  • the self-amplifying RNA described herein may incorporate an RNA replicase derived from any one of SFV, SINV, VEEV, RRV, or other viruses belonging to the alphavirus family.
  • the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wild-type virus sequence, e.g., the attenuated TC83 mutant of
  • Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase).
  • the replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA.
  • These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell.
  • Suitable alphavirus saRNAs may use a replicase from a SINV, a SFV, an eastern equine encephalitis virus, a VEEV, or mutant variants thereof.
  • the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a positive-stranded RNA virus, and in particular a picomavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.
  • a virus other than an alphavirus such as a positive-stranded RNA virus, and in particular a picomavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus.
  • Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md.
  • alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR
  • the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., mRNA).
  • the self-amplifying RNA molecules described herein include at least about 4 kb.
  • the self-amplifying RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb.
  • the self-amplifying RNA may include at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb.
  • the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb
  • the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g. , linked in series) when expressed as an amino acid sequence.
  • the polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
  • the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some embodiments, the self- amplifying RNA described herein may encode epitopes capable of eliciting either a helper T- cell response or a cytotoxic T-cell response or both.
  • the antigenic polypeptide constructs can comprise one or more antigenic peptide sequences that elicit an immune response.
  • pathogenic antigenic polypeptide constructs are peptide or protein antigens derived from a pathogen associated with infectious disease, including viruses, bacteria, parasites, and fungi.
  • the antigenic polypeptide constructs are peptide or protein antigens derived from cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
  • peptides or proteins can exist in a variety of instances such as: fragments or functional derivatives, recombinant polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, peptides or proteins sufficient for use as hybridization probes, peptides or proteins for inhibiting expression of a polynucleotide, and complementary amino acid sequences of the foregoing described herein.
  • Peptides or proteins may be an epitope to which antibodies may bind.
  • the peptides or proteins can comprise RNA and/or DNA nucleotides (e.g., peptide nucleic acids).
  • the size of a protein or peptide or derivative of a corresponding amino sequence described or referenced herein can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97
  • proteins or peptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or peptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.).
  • domain refers to any distinct functional or structural unit of a protein or peptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
  • the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof elicit an immune response.
  • the immune response may be against the antigenic polypeptide constructs and/or antigenic peptide sequences and/or a homologous endogenously-expressed (e.g. cross-reactive) host protein.
  • the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed e.g.
  • cross-reactive host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous.
  • the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
  • the one or more antigenic polypeptide constructs and/or antigenic peptide sequence thereof are modified such that the antigenic polypeptide constructs and/or antigenic peptide sequences thereof continue to elicit an immune response but homology with a homologous endogenously-expressed (e.g., cross-reactive) host protein is reduced to reduce or prevent cross-reactivity after immunization with the antigenic polypeptide constructs and/or antigenic peptide sequences thereof.
  • a homologous endogenously-expressed host protein is reduced to reduce or prevent cross-reactivity after immunization with the antigenic polypeptide constructs and/or antigenic peptide sequences thereof.
  • the homology between the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously-expressed (e.g., cross-reactive) host protein may be reduced by modifying the antigenic polypeptide constructs and/or antigenic peptide sequences thereof such that the modified antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed (e.g., cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
  • the modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more variant amino acids (e.g. , amino acid substitutions, insertions, and/or deletions) compared to unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences thereof.
  • variant amino acids e.g. , amino acid substitutions, insertions, and/or deletions
  • modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
  • Nucleotide as well as protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • antigenic polypeptide constructs and/or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof of the present disclosure may be modified, such that they are substantially identical to the antigenic polypeptide constructs or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof comprised in immunogenic compositions described herein.
  • the modified antigenic polypeptide constructs and/or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof continue to be bound by antibodies to elicit an immune response but produce attenuated immune cross-reactivity after immunization.
  • Polypeptide and/or nucleic acid sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.
  • the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the disclosure may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more variant amino acids (e.g., amino acid substitutions, insertions, and/or deletions) or be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
  • variant amino acids e.g., amino acid substitutions, insertions, and/or deletions
  • the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
  • the modified antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the “wildtype” or standard protein or peptide or “modified” or “variant” protein or peptide. This can be accomplished to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids.
  • the term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine.
  • neutral substitutions or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids.
  • one of skill in the art may wish to introduce a mutation into a polynucleotide to reduce cross-reactivity of the protein encoded by the polynucleotide with endogenously-expressed host proteins while not disturbing the ability of that polynucleotide to encode a protein that is bound by an antibody and that elicits an immune response.
  • Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that retain the ability to be bound by an antibody and elicit an immune response and/or proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that have reduced homology with endogenously-expressed host proteins, such that cross-reactivity of the proteins is diminished, for example.
  • a polynucleotide may be (and encode) a biological functional equivalent with significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like.
  • Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge.
  • Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine.
  • Non-conservative changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein’s ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention.
  • substitutions may be non-conservative such that a function or activity of the polypeptide is affected.
  • Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa.
  • Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
  • alteration of the function of a polypeptide is intended by introducing one or more substitutions.
  • certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
  • Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. For example, it is contemplated that peptides may be mutated by truncation, or deletion of a number of contiguous amino acids, rendering them shorter than their corresponding endogenous form.
  • Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein. For example, it is contemplated that peptides might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g. , for targeting or localization, for enhanced activity, for purification purposes, etc.).
  • polypeptides of the disclosure may be chemically modified. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861).
  • amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned.
  • the addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region.
  • Modified antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the disclosure can be substitutional, insertional, or deletion variants, for example.
  • Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and/or the like.
  • An analysis of the size, shape and/or type of the amino acid side-chain substituents reveals that arginine, lysine and/or histidine are all positively charged residues; that alanine, glycine and/or serine are all a similar size; and/or that phenylalanine, tryptophan and/or tyrosine all have a generally similar shape.
  • arginine, lysine and/or histidine; alanine, glycine and/or serine; and/or phenylalanine, tryptophan and/or tyrosine; are defined herein as biologically functional equivalents.
  • the hydropathic index of amino acids may be considered.
  • the hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics.
  • the importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982).
  • the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ⁇ 2 is included. In some aspects of the disclosure, those that are within ⁇ 1 are included, and in other aspects of the disclosure, those within ⁇ 0.5 are included.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • the substitution of amino acids whose hydrophilicity values are within ⁇ 2 are included, in other embodiments, those which are within ⁇ 1 are included, and in still other embodiments, those within ⁇ 0.5 are included.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue.
  • single or multiple amino acid substitutions may be made in the naturally occurring sequence. Substitutions can be made in that portion of the protein that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the primary, secondary, or tertiary structure that characterizes the native protein).
  • amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
  • an “amino molecule” refers to any amino acid, amino acid derivative, or amino acid mimic as would be known to one of ordinary skill in the art.
  • the residues of the peptide or protein are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues.
  • the sequence may comprise one or more non-amino molecule moieties.
  • the sequence of residues of the proteinaceous molecule may be interrupted by one or more non- amino molecule moieties.
  • Peptides and proteins include the twenty “natural” amino acids, and post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified and/or unusual amino acids.
  • protein encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown in the Table below.
  • peptide mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993).
  • the underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen.
  • a peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
  • peptide mimetic concept Some successful applications of the peptide mimetic concept have focused on mimetics of [3-tums within proteins, which are known to be highly antigenic. Likely [3-tum structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
  • Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids.
  • Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties.
  • alpha-helix mimetics are disclosed in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
  • Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures.
  • Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Patents 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Patents 5,672,681 and 5,674,976.
  • nucleic acid molecules encoding peptides of interest, e.g. , antigens. These nucleic acids may be generated by methods known in the art.
  • the nucleic acid molecules may be used to express large quantities of the polypeptide of interest, such as an antigen.
  • RNA or DNA molecules comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g. , a fragment containing one or more polypeptides, or antigens).
  • RNA or DNA molecules comprising nucleic acid molecules may encode antigens, fusion proteins, modified antibodies, antibody fragments, and probes thereof.
  • the RNA or DNA molecules may contain nucleic acid sequences that serve other functions as well.
  • isolated means altered or removed from the natural state through human intervention.
  • a DNA or RNA naturally present in a living animal is not “isolated,” but a synthetic DNA or RNA, or a DNA or RNA partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated DNA or RNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the DNA or RNA has been delivered.
  • the DNA or RNA molecule is an analog and may include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and/or improve binding specificity.
  • modifications particularly modifications that increase nuclease resistance, improve binding affinity, and/or improve binding specificity.
  • the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar.
  • other substitutions such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true species. All such compounds are considered to be analogs.
  • reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids.
  • reference to inter-sugar linkages shall be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.
  • Modified oligonucleotides and oligonucleotide analogs may exhibit increased chemical and/or enzymatic stability relative to their naturally occurring counterparts.
  • Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds.
  • the lack of a negatively charged backbone may facilitate cellular penetration.
  • the modified intemucleoside linkages are intended to replace naturally-occurring phosphodiester-5'-methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound.
  • Modifications may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA or RNA synthesizer using methodology commonly known to those skilled in DNA or RNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5' to 3' sense, an “upstream” synthon such as structure H is modified at its terminal 3' site, while a “downstream” synthon such as structure Hl is modified at its terminal 5' site.
  • Oligonucleosides linked by hydrazines, hydroxylamines, and other linking groups can be protected by a dimethoxytrityl group at the 5 '-hydroxyl and activated for coupling at the 3'-hydroxyl with cyanoethyldiisopropyl-phosphite moieties. These compounds can be inserted into any desired sequence by standard, solid phase, automated DNA or RNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing a uniform backbone linkage can be synthesized by use of CPG- solid support and standard nucleic acid synthesizing machines such as Applied Biosystems Inc.
  • the initial nucleotide (number 1 at the 3 '-terminus) is attached to a solid support such as controlled pore glass. In sequence specific order, each new nucleotide is attached either by manual manipulation or by the automated synthesizer system.
  • Free amino groups can be alkylated with, for example, acetone and sodium cyanoboro hydride in acetic acid.
  • the alkylation step can be used to introduce other, useful, functional molecules on the macromolecule.
  • useful functional molecules include but are not limited to reporter molecules, RNA cleaving groups, groups for improving the pharmacokinetic properties of an oligonucleotide, and groups for improving the pharmacodynamic properties of an oligonucleotide.
  • Such molecules can be attached to or conjugated to the macromolecule via attachment to the nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to pendent groups extending from a hydroxyl group of the sugar moiety of one or more of the nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is herein incorporated by reference, and in other of the above-referenced patent applications.
  • Solid supports may include any of those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel Support — an aminopolyethyleneglycol derivatized support or Poros — a copolymer of polystyrene/divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides can be effected via standard procedures. As used herein, the term solid support further includes any linkers (e.g., long chain alkyl amines and succinyl residues) used to bind a growing oligonucleoside to a stationary phase such as CPG.
  • CPG controlled pore glass
  • TentaGel Support an aminopolyethyleneglycol derivatized support
  • Poros a copolymer of polystyrene/divinylbenzene. Attachment and cleavage of nucleotides and oli
  • the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or a 5 '(E)-vinyl -phosphonate (VP) modification.
  • the oligonucleotides have one or more phosphorothioated DNA or RNA bases.
  • RNA or DNA molecules discussed above.
  • Prokaryote- and/or eukaryote-based systems or cell free systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides.
  • Commercially and widely available systems include but are not limited to bacterial, mammalian, yeast, insect cell, and cell free systems.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins.
  • Appropriate cell lines, host systems, or expression systems can be chosen to ensure the correct modification and processing of the nucleic acid or polypeptide(s) expressed.
  • Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.
  • antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the present disclosure are achieved by operably linking a nucleic acid encoding the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs to a promoter, and incorporating the construct into an expression vector, which is taken up and expressed by cells.
  • the vectors can be suitable for replication and, in some cases, integration in eukaryotes. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence.
  • the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid.
  • Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.
  • a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
  • the expression vector may be provided to a cell in the form of a viral vector.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
  • Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including selfinactivating lentivirus vectors).
  • retroviruses include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including selfinactivating lentivirus vectors).
  • retroviruses include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including selfinactivating lentivirus vectors).
  • adenoviruses provide a convenient platform for gene delivery systems.
  • a selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art.
  • the recombinant virus can then be isolated and delivered to cells of the subject either in viv
  • the nucleic acid encoding antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the present disclosure is introduced into cells using a recombinant vector such as a viral vector including, for example, a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, or an adenovirus.
  • a viral vector including, for example, a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, or an adenovirus.
  • Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells.
  • Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
  • Such components also might include markers, such as detectable and/or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • markers such as detectable and/or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector.
  • Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities.
  • a large variety of such vectors are known in the art and are generally available.
  • the vector When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell’s nucleus or cytoplasm.
  • Eukaryotic expression cassettes included in the vectors particularly contain (in a 5'- to-3' direction) regulatory elements including a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, a transcriptional termination/polyadenylation sequence, post-transcriptional regulatory elements, and origins of replication.
  • regulatory elements including a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, a transcriptional termination/polyadenylation sequence, post-transcriptional regulatory elements, and origins of replication.
  • Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a particular nucleic acid (e.g., RNA, mRNA, saRNA, DNA) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods may include, but are not limited to, direct delivery of nucleic acids such as by injection (U.S.
  • One illustrative delivery vehicle is a lipid and/or a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid.
  • the nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid.
  • Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution.
  • Lipids are fatty substances which may be naturally occurring or synthetic lipids.
  • lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
  • a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome.
  • Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991).
  • liposomes used may vary upon the nature of the liposome as well as the cell used, for example, about 5 to about 20 ⁇ g vector DNA per 1 to 10 million of cells may be contemplated.
  • Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987).
  • the feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
  • a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda etal., 1989).
  • a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991).
  • HMG-1 nuclear non-histone chromosomal proteins
  • a liposome may be complexed or employed in conjunction with both HVJ and HMG- 1.
  • a delivery vehicle may comprise a ligand and a liposome.
  • lipids suitable for use can be obtained from commercial sources.
  • lipofectamine can be obtained from Thermo Fisher Scientific, Waltham, Mass.
  • dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.
  • dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.)
  • cholesterol (“Choi”) can be obtained from Calbiochem- Behring
  • dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.).
  • Liposome is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution.
  • compositions that have different structures in solution than the normal vesicular structure are also encompassed.
  • the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules.
  • lipofectamine-nucleic acid complexes are also contemplated.
  • a nucleic acid is introduced into a cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. Recipient cells can be made more susceptible to transformation by mechanical wounding. Also the amount of vectors used may vary upon the nature of the cells used, for example, about 5 to about 20 pg vector DNA per 1 to 10 million of cells may be contemplated.
  • a nucleic acid is introduced to the cells using calcium phosphate precipitation.
  • Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique.
  • mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
  • a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol.
  • reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
  • RNA or DNA can be transfected into cells according to a variety of methods known in the art. RNA or DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some RNA or DNA may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells.
  • the polypeptide of interest expression construct or RNA or DNA replicase can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NF AT- 1 or NF-KB, both of which are transcription factors that can be activated upon T-cell activation.
  • Control of expression allows T cells, such as tumortargeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells.
  • T cells such as tumortargeting T cells
  • cytokine signaling both in the T cells themselves and in surrounding endogenous immune cells.
  • RNA molecule such as a saRNA, or DNA.
  • techniques and conditions that would allow large-scale production of RNA or DNA molecules, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides.
  • RNA or DNA and transfection technique For transfection of mammalian cells, it is known, depending upon the RNA or DNA and transfection technique used, only a small fraction of cells may integrate the foreign RNA or DNA into their cells. Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to identify and select these integrants, a variety of assays may be performed.
  • Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR
  • biochemical assays such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
  • cells containing an exogenous nucleic acid may be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector.
  • a selection marker may be one that confers a property that allows for selection.
  • a positive selection marker may be one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection.
  • An example of a positive selection marker is a drug resistance marker.
  • markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions are also contemplated.
  • screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized.
  • tk herpes simplex virus thymidine kinase
  • CAT chloramphenicol acetyltransferase
  • One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.
  • Selectable markers may include a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell.
  • Selectable markers are often antibiotic resistance genes; cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those cells that can grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include: the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin.
  • a screenable marker may comprise a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells. Certain embodiments of the present invention utilize reporter genes to indicate specific cell lineages.
  • the reporter gene can be located within expression elements and under the control of the ventricular- or atrial-selective regulatory elements normally associated with the coding region of a ventricular- or atrial- selective gene for simultaneous expression.
  • a reporter allows the cells of a specific lineage to be isolated without placing them under drug or other selective pressures or otherwise risking cell viability.
  • reporter examples include genes encoding cell surface proteins (e.g. , CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., [3-galactosidase).
  • cell surface proteins e.g. , CD4, HA epitope
  • fluorescent proteins e.g., antigenic determinants
  • enzymes e.g., [3-galactosidase.
  • the vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme.
  • the reporter gene is a fluorescent protein.
  • a broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.
  • the cells of the disclosure may be specifically formulated and/or they may be cultured in a particular medium.
  • the cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.
  • the medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined.
  • a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham
  • the medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s).
  • the serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).
  • the medium may contain or may not contain any alternatives to serum.
  • the alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto.
  • the alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience.
  • the commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).
  • the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and/or T3 (triodo-I-thyronine). In specific embodiments, one or more of these may be explicitly excluded.
  • Vitamins such as biotin; DL Alpha Tocop
  • the medium further comprises vitamins.
  • the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof.
  • the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B 12.
  • the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof.
  • the medium further comprises proteins.
  • the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof.
  • the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof.
  • the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof.
  • the medium comprises or further comprises amino acids, monosaccharides, inorganic ions.
  • the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof.
  • the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof.
  • the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof.
  • the medium comprises or consists essentially of one or more vitamins discussed herein and/or one or more proteins discussed herein, and/or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and/or phosphorus) or salts thereof, and/or molyb
  • the medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and/or inorganic salts. . In specific embodiments, one or more of these may be explicitly excluded.
  • One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/mL, pg/mL, mg/mL, or any range derivable therein.
  • the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration.
  • the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cry opreservation agents, such as DMSO (for example, in 5% DMSO).
  • the cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin.
  • the cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour.
  • the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.
  • nucleic acid molecules disclosed herein may be obtained from any source that produces nucleic acids. Methods of isolating mRNA are well known in the art. See e.g., Sambrook et al., supra.
  • the disclosure relates to an immunogenic composition for administration to a host.
  • the host is a human. In other embodiments, the host is a non-human.
  • the composition comprises a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
  • the modified antigenic polypeptide construct can differ from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences.
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
  • an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • the composition comprises a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
  • the modified antigenic polypeptide construct can differ from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences.
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
  • an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • the composition comprises a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
  • the modified viral spike protein construct can differ from an unmodified viral spike protein construct at one or more antigenic peptide sequences.
  • the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • an antigenic peptide sequence of the unmodified viral spike protein construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified viral spike protein construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • the composition comprises a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
  • the modified viral spike protein construct can differ from an unmodified viral spike protein construct at one or more antigenic peptide sequences.
  • the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • an antigenic peptide sequence of the unmodified viral spike protein construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
  • the compositions described herein are immunogenic compositions.
  • the compositions described herein include at least one isolated nucleic acid or polypeptide molecule as described herein.
  • the compositions described herein are vaccines.
  • the immunogenic compositions comprise RNA, and the immunogenic compositions are RNA vaccines.
  • the immunogenic compositions comprise DNA, and vaccines are DNA vaccines.
  • the immunogenic compositions comprise a polypeptide, and vaccines are polypeptide vaccines.
  • Conditions and/or diseases that can be treated with the RNA, DNA, and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
  • the composition is substantially free of one or more impurities or contaminants and, for instance, includes nucleic acid or polypeptide molecules that are equal to any one of, at least any one of, at most any one of, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure.
  • the composition further includes a lipid-based delivery system, which delivers a nucleic acid molecule to the interior of a cell, where it can then replicate, inhibit protein expression of interest, and/or express the encoded polypeptide of interest.
  • the delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen.
  • the composition further includes neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG), and forms nanoparticles that encompass, or encapsulate, the nucleic acid molecules.
  • the composition further includes any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water- in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion.
  • the nucleic acid molecule is encapsulated in, bound to or adsorbed on any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune -stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion, or a combination thereof.
  • encapsulate As used herein, “encapsulate,” “encapsulated,” “encapsulation,” and grammatically comparable variants thereof mean that at least a portion of a substance is enclosed or surrounded by another material or another substance in a composition.
  • a substance such as a nucleic acid
  • a composition such as a lipid.
  • compositions further comprise one or more stabilizing agents and one or more buffers.
  • a nucleic acid molecule e.g. , a naked or encapsulated nucleic acid, or a polypeptide as disclosed herein may be comprised in a solution comprising the one or more stabilizing agents and one or more buffers.
  • the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrolidone, glycine, or a combination thereof.
  • the stabilizing agent is a disaccharide, or sugar.
  • the stabilizing agent is sucrose.
  • the stabilizing agent is trehalose.
  • the stabilizing agent is a combination of sucrose and trehalose.
  • the total concentration of the stabilizing agent(s) in the composition is about 5% to about 10% w/v.
  • the total concentration of the stabilizing agent can be equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v or any range or value derivable therein.
  • the total concentration of the stabilizing agent(s) in the composition is 10% w/v.
  • the amino acid concentration is 5% w/v.
  • buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g.,
  • the buffer is a HEPES buffer, a Tris buffer, or a PBS buffer. In one embodiment, the buffer is Tris buffer. In another embodiment, the buffer is a HEPES buffer. In a further embodiment, the buffer is a PBS buffer. In some embodiments, the concentration of the buffer in the composition is about 10 mM.
  • the buffer concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein.
  • the buffer concentration is 10 mM.
  • the buffer can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6.
  • the buffer can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,
  • the buffer is at pH 7.4.
  • compositions may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents, and/or excipients, in some cases.
  • the composition further includes a pharmaceutically acceptable vehicle.
  • each of a buffer, stabilizing agent, salt, surfactant, preservative, and excipient are included in the compositions.
  • any one or more of a buffer, stabilizing agent, salt, surfactant, preservative, excipient, carrier, diluent, or vehicle may be excluded from compositions.
  • salts include but not limited to sodium salts and/or potassium salts.
  • the sodium salt comprises sodium chloride.
  • the potassium salt comprises potassium chloride.
  • the concentration of the salts in the composition can be about 70 mM to about 140 mM.
  • the salt concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein.
  • the salt concentration is 70 mM. In specific embodiments, the salt concentration is 140 mM.
  • the salt can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6.
  • the salt can be at a pH equal to any one of, at least any one of, at most any one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or
  • excipients which refer to ingredients in the compositions that are not active ingredients, include but are not limited to carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants.
  • Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal.
  • “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art.
  • aqueous solvents e.g.
  • Diluents include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, com rice, and potato; and celluloses such as microcrystalline cellulose.
  • the amount of diluent in the composition can range from about 10% to about 90% by weight of the total composition, about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%.
  • compositions described herein can be carried out via any of the accepted modes of administration of agents for serving similar utilities.
  • Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal.
  • parenteral includes subcutaneous injections, intravenous, intramuscular, intradermal, intrastemal injection, or infusion techniques.
  • Pharmaceutical compositions described herein are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient.
  • Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound in aerosol form may hold a plurality of dosage units.
  • composition to be administered will, in any event, contain a therapeutically and/or prophylactically effective amount of a compound within the scope of this disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings described herein.
  • a pharmaceutical composition within the scope of this disclosure may be in the form of a solid or liquid.
  • the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form.
  • the carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalator administration.
  • the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid.
  • the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form.
  • Such a solid composition will typically contain one or more inert diluents or edible carriers.
  • one or more of the following may be present or exclude: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose, or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate, or orange flavoring; and a coloring agent.
  • binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin
  • excipients such as starch, lactose, or dextrins
  • disintegrating agents such as al
  • the pharmaceutical composition When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.
  • the pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension.
  • the liquid may be for oral administration or for delivery by injection, as two examples.
  • preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant, and flavor enhancer.
  • a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included or exclude.
  • a liquid pharmaceutical composition may include or exclude one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose
  • a liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a compound such that a suitable dosage will be obtained.
  • compositions may be prepared by methodology well known in the pharmaceutical art.
  • a pharmaceutical composition intended to be administered by injection can be prepared by combining the nucleic acid or polypeptide with sterile, distilled water or other carrier so as to form a solution.
  • a surfactant may be added to facilitate the formation of a homogeneous solution or suspension.
  • Surfactants are compounds that non- covalently interact with a compound consistent with the teachings herein so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
  • compositions within the scope of the disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and length of action of the therapeutic and/or prophylactic agent; the age, body weight, general health, gender, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • the disclosure relates to a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
  • the method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
  • the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
  • the disclosure also relates to a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
  • the method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence.
  • the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence.
  • modifying the antigenic peptide sequence reduces or prevents crossreactivity of the modified antigenic polypeptide construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified antigenic polypeptide construct comprising an unmodified antigenic peptide sequence.
  • the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
  • the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
  • antigenic peptide sequences of antigenic polypeptide constructs to be modified to reduce or prevent cross-reactivity with host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue can be identified using one or more of peptide sharing analysis (e.g., protein alignment), mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue, rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, and immune function), and algorithmic immunogenicity prediction tools (e.g., http://tools.iedb.org/main/tcell).
  • peptide sharing analysis e.g., protein alignment
  • mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association
  • the cross-reactivity of the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the cross-reactivity of the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3- fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or
  • the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity equivalent to or higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof.
  • the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40- fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-
  • the disclosure relates to a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
  • the method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
  • the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
  • the disclosure also relates to a method for reducing or preventing cross-reactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct.
  • the method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence.
  • the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence.
  • modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified viral spike protein construct comprising an unmodified antigenic peptide sequence.
  • the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue .
  • the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
  • antigenic peptide sequences of viral spike protein constructs to be modified to reduce or prevent cross-reactivity with host peptides comprised in a cross- reactive host polypeptides expressed in a host tissue can be identified using one or more of peptide sharing analysis (e.g., protein alignment), mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue, rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, and immune function), and algorithmic immunogenicity prediction tools (e.g., http://tools.iedb.org/main/tcell).
  • peptide sharing analysis e.g., protein alignment
  • mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic link
  • the cross-reactivity of the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue.
  • the cross-reactivity of the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3- fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or
  • the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity equivalent to or higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof.
  • the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40- fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-fold or more, about 400
  • the disclosure relates to a method of inducing an immune response in a subject.
  • the method includes administering to the mammalian cell an effective amount of a composition as described herein.
  • the disclosure relates to a method of vaccinating a subject.
  • the method includes administering to the subject in need thereof an effective amount of a composition described herein.
  • the disclosure relates to a method of treating or preventing an infectious disease.
  • the method includes administering to the subject an effective amount of a composition as described herein.
  • disclosure relates to a method of treating or preventing an infectious disease in a subject by, for example, inducing an immune response to an infectious disease in the subject.
  • the method includes administering a priming composition that includes an effective amount of a composition described herein, and administering a booster composition including an effective amount of an adenoviral vector encoding an antigen.
  • the method includes administering a priming composition including an effective amount of an adenoviral vector encoding an antigen, and administering a booster composition that includes an effective amount of a composition described herein.
  • the composition elicits an immune response including an antibody response.
  • the composition elicits an immune response including a T cell response.
  • Computational toxicology concerns the use of computational tools to support integrative approaches to toxicological research and chemical safety assessments via predictive modeling, analyses of complex, multifaceted data sets, and extrapolation and translation among evidence streams, particularly new approach methodologies that rely upon alternatives to animal testing. Understanding which biological pathway targets are related to different types of organ level toxicities may be useful to design new in vitro assays and to develop new predictive computational methods and/or interpret their results.
  • the disclosure relates to a method of identifying a potential toxicity of an immunogenic composition in a biological host system.
  • the immunogenic composition may include an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
  • the method includes a. identifying one or more proteins of the host that have a degree of similarity to the antigenic polypeptide construct; b. identifying properties of the one or more proteins, the properties comprising cellular location, gene- and protein-tissue expression profiles, and/or toxic state associations; c. ranking or sorting the one or more proteins using one or more of the properties; d. identifying immunogenicity of the one or more proteins; e.
  • step c. comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence; and f. predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence.
  • One or more of the steps may be performed on a computer. In some embodiments all of the steps above are performed on a computer. In some embodiments one or more of the steps are performed on a different computer than one or more of the other steps. Performing one or more of the steps on a computer may include performing the steps in a single or in a combination of computational models.
  • identifying a potential toxicity may include identifying a potential toxicity from a list or database of toxicities that the immunogenic composition is likely to increase a risk in a biological host system, if the immunogenic composition is administered to the biological host.
  • identifying a potential toxicity may include identifying an immunogenic composition or a portion of an immunogenic composition, such as a portion of the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct that is likely to increase the risk of a specific toxicity in a biological host system if the immunogenic composition is administered to the biological host.
  • identifying a potential toxicity includes identifying a potential toxicity from a list or database of toxicities that the immunogenic composition is likely to increase a risk of in a biological host system if the immunogenic composition is administered to the biological host and includes identifying an immunogenic composition or a portion of an immunogenic composition, such as a portion of the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct that is likely to increase the risk of a specific toxicity in a biological host system if the immunogenic composition is administered to the biological host.
  • the potential toxicity can be any toxicity of the biological host.
  • the potential toxicity is a toxicity associated with administration to the biological host of the immunogenic composition or a portion of an immunogenic composition, such as the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
  • the potential toxicity includes myocarditis or pericarditis.
  • the potential toxicity includes myocarditis or pericarditis and the antigenic polypeptide construct is a viral spike protein.
  • the antigenic polypeptide is a prefusion spike protein, such as any of the spike proteins comprising a SARS-CoV-2 S ectodomain trimer comprising protomers, which comprise an amino acid sequence at least 95% identical to residues 16-1208 of SEQ ID NO: 2 and comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 2 that stabilize the S ectodomain trimer in a prefusion conformation, as described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021 by The United States of America, as represented by The Secretary, Department of Health and Human Services.
  • WO2021163365 PCT/US2021/017709
  • sequence of SEQ ID NO: 2 from WO2021163365, filed Feburary 11, 2021, is: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTK
  • stabilization of the perfusion conformation is obtained by introducing two consecutive proline substitutions at residues K986 and V987 in the spike protein (see SEQ ID NO: 2 described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021).
  • the pre-fusion stabilized spike protein comprises at least one pre fusion stabilizing mutation, wherein the at least one pre-fusion stabilizing mutation comprises the following amino acid substitutions: K986P and V987P (see SEQ ID NO: 2 described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021).
  • the stabilized spike protein has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGE
  • the immunogenic composition can be a vaccine.
  • the immunogenic composition can be a treatment or preventative treatment for an infection, cancer, rare diseases, and/or other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
  • the host can be a plant or an animal.
  • the host can be a mammal.
  • the host can be a human.
  • the method includes searching a human proteome with a spike protein peptide and identifying a protein from the human proteome with homology to the spike protein peptide. In some instances, the method includes identifying whether the protein from the human proteome with homology to the spike protein peptide is present in a target organ.
  • ranking or sorting the one or more proteins using one or more of the properties includes ranking cellular locations that are extracellular or on a surface of a cell as more likely to cause the potential toxicity than intracellular locations, ranking gene- and protein-tissue expression profdes that overlap in tissue location of the potential toxicity as more likely to cause the potential toxicity than other tissue locations, and/or ranking toxic state associations that overlap or are related to the potential toxicity as more likely to cause the potential toxicity than other toxic state associations.
  • the ranking or sorting can be from greatest to least or can assign a numerical degree of likelihood to cause the potential toxicity. In some instances, all of cellular locations, gene- and protein-tissue expression profiles, and toxic state associations are used in this method.
  • Cellular locations, gene- and protein-tissue expression profiles, and toxic state associations can be gathered from one or more premade lists or databases. In some instances, cellular locations, gene- and protein-tissue expression profiles, and toxic state associations can be predicted by computational methods and the predicted properties can be used in the methods herein.
  • identifying immunogenicity of the one or more proteins comprises identifying a ranking or sorting of degree of immunogenicity and/or degree of potential immunogenicity.
  • the ranking or sorting can be from greatest to least or can assign a numerical degree of immunogenicity and/or potential immunogenicity.
  • Immunogenicity can be the ability of the one or more proteins to provoke an immune response in the biological host.
  • the immunogenicity of the one or more proteins can be calculated or gathered from one or more premade lists or databases. In some instances, immunogenicity can be predicted by computational methods and the predicted immunogenicity can be used in the methods herein. [0198] Comparing the ranking or sorting in step c.
  • step d to produce a degree of correspondence can include comparing the rank or degree of likelihood to cause the potential toxicity and the degree of immunogenicity and/or degree of potential immunogenicity and ranking: more likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity, and either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than less likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity.
  • the degree of correspondence can be from greatest to least or can assign a numerical degree of correspondence.
  • Predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence can include predicting a greater degree of correspondence as more likely to cause a potential toxicity.
  • the method can further include modifying the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct to produce a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct that is less likely to cause the potential toxicity when compared to the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
  • the modification process can include, but is not limited to, modifying the nucleic acid or protein sequence, changing one or more of the naturally occurring nucleotides or amino acids to analogs or non-naturally occurring nucleotides or amino acid, removing one or more of the nucleotides or amino acids, adding additional nucleotides or amino acids, or combinations thereof.
  • FIG. 1 A strategy employed for identifying antigen mimicry occurring following immunization with the BNT162b2 mRNA vaccine against COVID-19 is illustrated in FIG. 1.
  • peptide sharing analysis between the BNT162b2 spike protein sequence and human proteins was conducted to identify human proteins with similarity to the COVID-19 spike protein.
  • FIG. 2 in a preferred analysis, hundreds of protein “hits” corresponding to similarity between an antigenic peptide sequence comprising 9 and 15 consecutive amino acids from the BNT162b2 spike protein sequence and an endogenously- expressed human protein were obtained.
  • mRNA and protein expression profiles in various tissues were also assessed to determine whether the human proteins identified as similar to the BNT162b2 spike protein sequence are enriched in the heart. Out of the proteins that comprise of at least one of the 2236 peptides, only 103 were determined to be predominantly expressed in the heart. The results were further narrowed using a rank-based approach to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, immune function, known epitopes) supporting a role for the human protein in pericarditis and/or myocarditis, as well as algorithmic immunogenicity predictions (http:// tools.iedb.org/main/tcell).
  • additional evidence e.g., disease association, genetic linkage, immune function, known epitopes
  • % identity means the percentage of human peptide sequence that is similar to the vaccine sequence (including antigen and non-coding regions) or spike protein sequence as referenced in the table. 100% means vaccine peptide sequence perfectly matches with a human peptide sequence.
  • RYR2 (ryanodine receptor 2) and NEBL (nebulette) were the top candidates identified due to the fact that they are plasma membrane proteins and they have heart-specific protein expression profiles.
  • the RYR2 protein profile is similar in humans compared to preclinical species, and RYR2 has been associated with cardiomyopathies (considered to be a rare genetic variant).
  • RYR2 is important in fetal heart development, and auto-antibodies against RYR2 have been found in myasthenia gravis patients. Additionally, RYR2 is important in mechanical stretch and TGFb-1 signaling during exertion.
  • the NEBL protein profile is distinct in humans compared to preclinical species.
  • NEBL binds actin and interacts with thin filaments and Z-line associated proteins in striated muscle.
  • NEBL may be involved in cardiac myofibril assembly and is a component of extracellular vesicular exosomes; I bands; stress fibers; and Z discs in the heart. Additionally, NEBL is known to play a role in familial isolated dilated cardiomyopathy.
  • Kelch like family member 41 was identified as a top hit using the rank-based strategy. KLHL41 is involved in skeletal muscle development and differentiation, and it regulates proliferation and differentiation of myoblasts and plays a role in myofibril assembly by promoting lateral fusion of adjacent thin fibrils into mature, wide myofibrils.
  • KLHL41 is also associated with an autosomal recessive form of nemaline myopathy.
  • Nemaline myopathies are muscular disorders characterized by muscle weakness of varying severity and onset, and abnormal thread-like or rod-shaped structures in muscle fibers on histologic examination.
  • data are generated to demonstrate cross-reactivity of host peptide sequences with antibodies (e.g., spike protein antibodies).
  • specific regions of the spike protein are conserved in MHC presentation and are important for cellular immune responses.
  • the antigenic sequence responsible for antibody production against the spike protein and the host peptide sequences is determined. Additionally, or alternatively, in some such embodiments, the region of the spike protein comprising the peptide sequences is determined.
  • overlapping region(s) of the antigenic peptide sequence e.g. , spike protein sequence
  • host peptide sequences e.g., NEBL and RYR2
  • SARS-CoV-2 virus is a member of a large family of viruses called coronaviruses that causes a respiratory disease COVID-19. While there are short-term effects of SARS-CoV- 2 infection such as fever, fatigue, headache and loss of taste/smell, the long-term effects can be very serious, including organ damage. In some people, lasting health effects may include, heart complications, chronic kidney impairment, stroke, and Guillain-Barre syndrome. This study focused on determining if these long-term effects/multi-organ failures that have been observed after infection are occurring due to molecular mimicry. Initially, we identified -2500 human peptides (9 and 15 amino acids) with at least 50% similarity within the SARS-CoV-2 spike protein sequences.
  • the proteins that these peptides are a part of were triaged to 26 human endogenous proteins highly expressed in heart, kidney and brain tissues that could potentially lead to immune-mediated adverse events.
  • the present example will describe our computational approach that can be used by infectious disease, vaccine and drug safety scientists for molecular mimicry risk assessment.
  • Step B Peptide Homology Search a. Blasting spike protein sequences against human proteome b. 9-aa with >75% similarity (for MHC I predictions) c. 15-aa with >50% similarity (for MHC II predictions)
  • Step C) Antigenicity Predictions Determining binding capability of human peptides to MHC Class I and II molecules using Immune epitope database (IEDB)
  • the method includes steps in the following order: (B) Peptide homology search followed by (A) Ranking strategy. In some embodiments, the method includes steps in the following order: (B) Peptide homology search followed by (C) Antigenicity predictions. In some embodiments, the method further includes in the following order the step of (C) Antigenicity predictions followed by (A) Ranking strategy.

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Abstract

The present disclosure provides methods of reducing risk of antigen mimicry in immunogenic medicaments. The present disclosure also provides immunogenic compositions comprising antigenic polypeptide constructs having a reduced risk of antigen mimicry.

Description

REDUCING RISK OF ANTIGEN MIMICRY IN IMMUNOGENIC MEDICAMENTS
BACKGROUND
I. Technical Field
[0001] This disclosure relates to the field of immunology, specifically to immunogenic compositions, methods for improving safety and/or reducing side effects of immunogenic compositions, and immunogenic compositions having improved safety and/or side effect profiles.
II. Background
[0002] Since at least the 1800s, vaccines have successfully been used to prevent lethal infections. However, adverse events have been observed following vaccinations. These adverse reactions may potentially be due to interactions between susceptibility of the vaccinated subject and various vaccine components. Among the potentially implicated mechanisms for these reactions is molecular mimicry, which refers to potentially significant similarity between certain vaccine elements and specific endogenously-expressed human proteins. The similarity of viral and bacterial antigenic proteins comprised in vaccines to the human proteome has been researched. For example, Kanduc described an extensive overlap of the human proteome with both viral and bacterial proteomes, with up to 90% of viral pentapeptides and 99.7% of bacterial heptapeptides shared by the human proteome. (Kanduc D. ResearchGate , 8: 65-76, 2009; Trost B et al. SelfNonself 1: 328-334, 2010). The mimicry between antigenic components in vaccines and host proteins may lead to immune crossreactivity, wherein the reaction of the immune system toward vaccine elements is extended to similar human proteins.
[0003] While it is clear that immunogenic compositions like vaccines can have powerful and beneficial prophylactic effects, a potential challenge to their use involves immune crossreactivity due to antigen mimicry, wherein side effects including redness or swelling at the site of vaccination, fever, fatigue, headache, muscle pain, stomach pain, chest pain, joint pain, chills, nausea and vomiting, shortness of breath, and/or swollen lymph nodes may be observed. A strategy to explore the rationale that, following immunization, immune responses raised against the antigen of the immunogenic composition cross-react with host proteins that share peptide sequences (or structures) with the antigen sequence, in this way leading to harmful pathologies.
[0004] Adverse reactions following administration of vaccines are not limited to only certain vaccines against only a certain pathogenic organism, as side effects have been observed for a broad range of vaccines against a variety of pathogens including, for example, coronavirus, influenza, hepatitis, human papilloma virus, smallpox, anthrax, and typhoid. See, e.g., Witberg G. et al. NEJM, 2021, and Sulemankhil I. et al. Cardiovasc Revasc Med. 2021 Aug 16 (describing incidence of myocarditis in patients receiving at least two different COVID-19 vaccines); Segal Y. & Shoenfeld Y. Cell Mol Immunol, 15: 586-594, 2018 (describing examples of H1N1, HBV, and HPV vaccines associated with immunological crossreaction); Giannotta G. & Giannotta N. Clin Case Rep Rev, 5: 1-12, 2019 (case report of neuroinflammation following receipt of an HPV vaccine); and Watanabe S. et al. European Respiratory Journal, 41: 474-477, 2013 (case reports of interstitial lung disease following receipt of an influenza vaccine); and Hajjo R. et al. Vaccines (Basel), Oct 15 ;9( 10): 1186, 2021 (stating that post-vaccine myocarditis has been reported for live vaccines (e.g., smallpox, anthrax and some typhoid vaccines).
[0005] Overcoming these antigen mimicry and immune cross-reactivity issues may yield improved immunogenic compositions. Discovering ways to reduce or prevent antigen mimicry and immune cross-reactivity to immunogenic compositions is currently an unmet challenge where solutions would potentially have a large benefit towards the safety and efficacy of immunogenic compositions. Furthermore, the discovery of methods that reduce side effects would enable research groups world-wide to evaluate and develop improved immunogenic compositions.
SUMMARY
[0006] The present disclosure provides methods for reducing the side effects of immunogenic compositions and for improving side effect profdes of immunogenic compositions. Reducing the side effects of immunogenic compositions by reducing and/or preventing antigen mimicry and immune cross-reactivity associated with immunogenic compositions is a desirable outcome in the safety and efficacy of vaccines. The present disclosure is based, at least in part, on the discovery that antigenic polypeptide constructs comprised in immunogenic compositions can be analyzed for antigenic peptide sequences having high homology with peptides comprised in host proteins that are predicted to be antigenic after immunization. Without wishing to be bound by theory, subsequent modification of the antigenic peptide sequences results in immunogenic compositions capable of eliciting an immune response but having attenuated cross-reactivity with potentially antigenic host proteins.
[0007] Disclosed herein, in some embodiments, is an immunogenic composition for administration to a host, the composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct differs from an unmodified antigenic polypeptide or viral spike protein construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide. In a preferred embodiment, the antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 9 or more consecutive amino acids having at least about 75% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity with the cross-reactive host peptide. In a preferred embodiment, the antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 15 consecutive amino acids having less than about 50% sequence identity with the cross-reactive host peptide. In another embodiment, disclosed herein is an immunogenic composition for administration to a host, the composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct differs from an unmodified antigenic polypeptide or viral spike protein construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct or viral spike protein comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide or viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
[0008] Also disclosed herein, in some aspects, an immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 3-127); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c. wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 3-127) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the cross-reactive host peptide sequence set forth in SEQ ID NOs: 128-137) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 5 for SEQ ID Nos: 128-137); and d. wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
[0009] Also disclosed herein, in some aspects, an immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c. wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 185-194) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in SEQ ID NOs: 175-184) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 3 for SEQ ID Nos: 175-184); and d. wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in SEQ ID NOs: 175-184). [0010] Also disclosed herein, in some aspects, an immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a) wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct (such as, for example, SARS-CoV-2 spike protein) at one or more antigenic peptide sequences (such as, for example, any one of the antigenic peptide sequences set forth in SEQ ID Nos: 1-2); b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c. wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct (such as, for example, any one of the antigenic peptide sequences set forth in even numbered SEQ ID Nos: 140-174) comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in odd numbered SEQ ID NOs: 139-173) comprised in a cross-reactive host polypeptide expressed in a host tissue (such as, for example, the respective polypeptide named in Table 3 for odd numbered SEQ ID Nos: 139-173); and d. wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide (such as, for example, any one of the respective cross-reactive host peptide sequence set forth in odd numbered SEQ ID NOs: 139-173).
[0011] Also disclosed herein, in some aspects, is a method for producing an immunogenic composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide or viral spike protein construct and/or a nucleic acid encoding the modified antigenic polypeptide or viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct. In some embodiments, modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified antigenic polypeptide or viral spike protein construct having an unmodified antigenic peptide sequence. In some embodiments, the immunogenic composition comprising the modified antigenic polypeptide or viral spike protein construct elicits an immune response. Also disclosed herein, in some aspects, is a method for producing an immunogenic composition comprising (1) a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, and/or (2) a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide or viral spike protein construct and/or a nucleic acid encoding the modified antigenic polypeptide or viral spike protein construct, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct.
[0012] Also disclosed herein, in some aspects, is a method for reducing or preventing crossreactivity of (1) an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, and/or (2) a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified antigenic polypeptide or viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified antigenic polypeptide or viral spike protein construct comprising an unmodified antigenic peptide sequence. Also disclosed herein, in some aspects, is a method for reducing or preventing cross-reactivity of (1) an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, and/or (2) a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide or viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide or viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide or viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified antigenic polypeptide or viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide or viral spike protein construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified antigenic polypeptide or viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified antigenic polypeptide or viral spike protein construct comprising an unmodified antigenic peptide sequence.
[0013] In some embodiments, the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition. In some embodiments, the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus. In some embodiments, the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein. In specific embodiments, the viral protein is a viral spike protein.
[0014] In some embodiments, the bacterial protein is a protein from Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium. In some embodiments, the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
[0015] In some embodiments, the parasite protein is a protein from Leishmania, Plasmodium, or Schistosoma. In some embodiments, the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
[0016] In some embodiments, the fungal protein is a protein from Candida, Cryptococcus, or Aspergillus . In some embodiments, the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein. [0017] In some embodiments, the nucleic acid is RNA. In some embodiments, the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), selfamplifying RNA (saRNA), and/or modified RNA (modRNA). In some embodiments, the RNA is comprised in an expression vector. In specific embodiments, the immunogenic composition is an RNA vaccine.
[0018] In some embodiments, the nucleic acid is DNA. In some embodiments, the DNA is double stranded, single stranded, and/or antisense single stranded. In some embodiments, the DNA is comprised in an expression vector. In specific embodiments the immunogenic composition is a DNA vaccine.
[0019] In some embodiments, the immunogenic composition comprises a polypeptide. In some instances, the immunogenic composition comprises a polypeptide vaccine.
[0020] In some embodiments, the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 65%, about 60%, about 55%, about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide. In some embodiments, the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
[0021] In some embodiments, the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. In specific embodiments, the cross-reactive host polypeptide is expressed in the heart tissue. In some embodiments, the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In specific embodiments, at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In specific embodiments, at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In some preferred embodiments, the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. In specific embodiments, the cross-reactive host polypeptide is expressed in the heart tissue. In some embodiments, the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain l(ANKRDl), synaptopodin 2-like protein (SYNPO2L), myosin 7(MYH7), ankyrin repeat and SOCS box containing 15(ASB15), cysteine and glycine rich protein 3(CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase -coupling factor 6, mitochondrial(ATP5PF), and/or pericentriolar material 1 protein (PCM1). In specific embodiments, at least one cross-reactive host polypeptide is located within MYH6. In specific embodiments, at least one cross-reactive host polypeptide is located within MYH6 and has, for example, the amino acid sequence SEQ ID NO: 128. In specific embodiments, at least one cross-reactive host polypeptide is located within PCM1. In specific embodiments, at least one cross-reactive host polypeptide is located within PCM1 and comprises, for example, the amino acid sequence SEQ ID NO: 137. In specific embodiments, at least one cross-reactive host polypeptide comprises the amino acid sequence selected from any one of SEQ ID NO: 3-25. [0022] In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct after the ranking strategy is preferably selected from any one of the peptides of SEQ ID NO: 3-138. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 3-138. In some embodiments, the antigenic peptide sequence ofthe unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 3-25. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 26- 36. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 37- 60. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 61- 70. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 71- 94. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 95- 104. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 105- 127. In some embodiments, the antigenic peptide sequence of the unmodified antigenic polypeptide construct has the amino acid sequence selected from any one of SEQ ID NO: 128- 137. Such preferred peptides may be used for further analyses such as antigenicity predictions. [0023] Disclosed herein, in some aspects, is an immunogenic composition produced according to any method disclosed herein. Disclosed herein, in some aspects, is an immunogenic composition comprising any antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct disclosed herein.
[0024] Also disclosed herein, in some aspects, are methods of: (1) vaccinating a subject, comprising administering to the subject in need thereof an effective amount of the immunogenic composition disclosed herein; and (2) treating or preventing an infectious disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of disclosed herein. In some embodiments, the immunogenic composition elicits an immune response comprising an antibody response. In some embodiments, the immunogenic composition elicits an immune response comprising a T cell response.
[0025] Also disclosed are the following aspects or embodiments (E) 1 to 173 of the present disclosure.
[0026] El. According to a first aspect of the present disclosure, there is provided an immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide. In some embodiments, there is provided an immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross- reactive host polypeptide expressed in a host tissue, and wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
[0027] E2. The immunogenic composition of El, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
[0028] E3. The immunogenic composition of E2, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0029] E4. The immunogenic composition of E2 or E3, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
[0030] E5. The immunogenic composition of any one of E2-E4, wherein the viral protein is a viral spike protein.
[0031] E6. The immunogenic composition of E2, wherein the bacterial protein is a protein from Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium. [0032] E7. The immunogenic composition of clause 2, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
[0033] E8. The immunogenic composition of E2, wherein the parasite protein is a protein from Leishmania, Plasmodium, or Schistosoma.
[0034] E9. The immunogenic composition of E2, wherein the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
[0035] E10. The immunogenic composition of E2, wherein the fungal protein is a protein from Candida, Cryptococcus, or Aspergillus .
[0036] El l. The immunogenic composition of E2, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein. [0037] E12. The immunogenic composition of any one of El-El 1, wherein nucleic acid is RNA.
[0038] El 3. The immunogenic composition of El 2, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0039] E14. The immunogenic composition of E12 or E13, wherein the RNA is comprised in an expression vector.
[0040] El 5. The immunogenic composition of any one of E12-E14, wherein the immunogenic composition is an RNA vaccine.
[0041] E16. The immunogenic composition of any one of clauses El-El 1, wherein the nucleic acid is DNA.
[0042] El 7. The immunogenic composition of El 6, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0043] El 8. The immunogenic composition of E16 or E17, wherein the DNA is comprised in an expression vector.
[0044] E19. The immunogenic composition of any one of E16-E18, wherein the immunogenic composition is a DNA vaccine.
[0045] E20. The immunogenic composition of any one of El-El l, wherein the immunogenic composition comprises a polypeptide.
[0046] E21. The immunogenic composition of any one of E1-E20, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 50%, about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0047] E22. The immunogenic composition of any one of E1-E21, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
[0048] E23. The immunogenic composition of any one of E1-E22, wherein the cross- reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. [0049] E24. The immunogenic composition of any one of E1-E23, wherein the cross- reactive host polypeptide is expressed in the heart tissue.
[0050] E25. The immunogenic composition of any one of E1-E24, wherein the cross- reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In some embodiments, the immunogenic composition of any one of E1-E24, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain l(ANKRDl), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase -coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0051] E26. The immunogenic composition of E25, wherein at least one cross- reactive host polypeptide comprises and/or is located within NEBL. In some embodiments, the immunogenic composition of E25, wherein at least one cross-reactive host polypeptide is comprised located withins MYH6.
[0052] E27. The immunogenic composition of E25, wherein at least one cross- reactive host polypeptide comprises and/or is located within RYR2. In some embodiments, the immunogenic composition of E25, wherein at least one cross-reactive host polypeptide is located within PCM1.
[0053] E28. According to another aspect of the present disclosure, there is provided an immunogenic composition for administration to a host, the composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct differs from an unmodified viral spike protein construct at one or more antigenic peptide sequences, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, wherein an antigenic peptide sequence of the unmodified viral spike protein construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified viral spike protein construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide. In some embodiments, there is provided an immunogenic composition for administration to a host, the composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct differs from an unmodified viral spike protein construct at one or more antigenic peptide sequences, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, wherein an antigenic peptide sequence of the unmodified viral spike protein construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and wherein the modified viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15consecutive amino acids having less than about 50% sequence identity with the cross-reactive host peptide. [0054] E29. The immunogenic composition of E28, wherein the viral spike protein is a spike protein from arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0055] E30. The immunogenic composition of E28 or E29, wherein nucleic acid is
RNA.
[0056] E31. The immunogenic composition of E30, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0057] E32. The immunogenic composition of E30 or 31, wherein the RNA is comprised in an expression vector.
[0058] E33. The immunogenic composition of any one of E30-E32, wherein the immunogenic composition is an RNA vaccine.
[0059] E34. The immunogenic composition of E28 or E29, wherein the nucleic acid is DNA.
[0060] E35. The immunogenic composition of E34, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0061] E36. The immunogenic composition of E34 or E35, wherein the DNA is comprised in an expression vector.
[0062] E37. The immunogenic composition of any one of E34-E36, wherein the immunogenic composition is a DNA vaccine. [0063] E38. The immunogenic composition of E28 or E29, wherein the immunogenic composition comprises a polypeptide.
[0064] E39. The immunogenic composition of any one of E28-E38, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0065] E40. The immunogenic composition of any one of E28-E39, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified viral spike protein construct.
[0066] E41. The immunogenic composition of any one of E28-E40, wherein the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue. [0067] E42. The immunogenic composition of any one of E28-E41, wherein the cross-reactive host polypeptide is expressed in the heart tissue.
[0068] E43. The immunogenic composition of any one of E28-E42, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In some embodiments, The immunogenic composition of any one of E28-E42, wherein the cross- reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain l(ANKRDl), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0069] E44. The immunogenic composition of E43, wherein at least one cross- reactive host polypeptide comprises and/or is located within NEBL. In some embodiments, the immunogenic composition of E43, wherein at least one cross-reactive host polypeptide is located within MYH6.
[0070] E45. The immunogenic composition of E43, wherein at least one cross- reactive host polypeptide comprises and/or is located within RYR2. In some embodiments, the immunogenic composition of E43, wherein at least one cross-reactive host polypeptide is located within PCM1
[0071] E46. According to another aspect of the present disclosure, there is provided a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in the host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct. According to another aspect of the present disclosure, there is provided a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in the host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct
[0072] E47. The method of E46, wherein modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified antigenic polypeptide construct having an unmodified antigenic peptide sequence.
[0073] E48. The method of E46 or E47, wherein the immunogenic composition comprising the modified antigenic polypeptide construct elicits an immune response.
[0074] E49. The method of any one of E46-E48, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
[0075] E50. The method of E49, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0076] E51. The method of E49 or E50, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
[0077] E52. The method of any one of E49-E51, wherein the viral protein is a viral spike protein.
[0078] E53. The method of E49, wherein the bacterial protein is a protein from
Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium .
[0079] E54. The method of E49, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
[0080] E55. The method of E49, wherein the parasite protein is a protein from
Leishmania, Plasmodium, or Schistosoma.
[0081] E56. The method of E49, wherein the parasite protein is a parasite cell surface protein and/or a parasite secreted protein.
[0082] E57. The method of E49, wherein the fungal protein is a protein from
Candida, Cryptococcus, or Aspergillus .
[0083] E58. The method of E49, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
[0084] E59. The method of any one of E46-E58, wherein nucleic acid is RNA.
[0085] E60. The method of E59, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA). [0086] E61. The method of E59 or E60, wherein the RNA is comprised in an expression vector.
[0087] E62. The method of any one of E59-E61, wherein the immunogenic composition is an RNA vaccine.
[0088] E63. The method of any one of E46-E58, wherein the nucleic acid is DNA.
[0089] E64. The method of E63, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0090] E65. The method of E63 or E64, wherein the DNA is comprised in an expression vector.
[0091] E66. The method of any one of E63-E65, wherein the immunogenic composition is a DNA vaccine.
[0092] E67. The method of any one of E46-E58, wherein the immunogenic composition comprises a polypeptide.
[0093] E68. The method of any one of E46-E67, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0094] E69. The method of any one of E46-E68, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
[0095] E70. The method of any one of E46-E69, wherein the cross-reactive host polypeptide is expressed in the heart, brain, kidney, liver, and/or lung tissue.
[0096] E71. The method of any one of E46-E70, wherein the cross-reactive host polypeptide is expressed in the heart tissue.
[0097] E72. The method of any one of E46-E71, wherein the cross-reactive host polypeptide comprises and/or is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In another embodiment, the method of any one of E46-E71, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM 1). E73. The method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6. [0098] E73. The method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within PCM1.
[0099] E74. The method of E72, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2.
[0100] E75. According to another aspect of the present disclosure, there is provided a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct. According to another aspect of the present disclosure, there is provided a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross- reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with the cross-reactive host peptide to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
[0101] E76. The method of E75, wherein modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified viral spike protein construct having an unmodified antigenic peptide sequence.
[0102] E77. The method of E75 or E76, wherein the immunogenic composition comprising the modified viral spike protein construct elicits an immune response.
[0103] E78. The method of any one of E75-E77, wherein the viral spike protein is a spike protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0104] E79. The method of any one of E75-E78, wherein nucleic acid is RNA.
[0105] E80. The method of E79, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0106] E81. The method of E79 or E80, wherein the RNA is comprised in an expression vector.
[0107] E82. The method of any one of E79-E81, wherein the immunogenic composition is an RNA vaccine.
[0108] E83. The method of any one of E75-E78, wherein the nucleic acid is DNA.
[0109] E84. The method of E83, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0110] E85. The method of E83 or E84, wherein the DNA is comprised in an expression vector.
[oni] E86. The method of any one of E83-E85, wherein the immunogenic composition is a DNA vaccine.
[0112] E87. The method of any one of E75-E78, wherein the immunogenic composition comprises a polypeptide. [0113] E88. The method of any one of E75-E87, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0114] E89. The method of any one of E75-E88, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified viral spike protein construct.
[0115] E90. The method of any one of E75-E89, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
[0116] E91. The method of any one of E75-E90, wherein the cross-reactive host polypeptide is expressed in heart tissue.
[0117] E92. The method of any one of E75-E91, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In another embodiment, the method of any one of E75-E91, wherein the cross-reactive host polypeptide comprises and/or is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2- like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0118] E93. The method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6. [0119] E94. The method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In another embodiment, the method of E92, wherein at least one cross-reactive host polypeptide comprises and/or is located within PCM1
[0120] E95. According to another aspect of the present disclosure, there is provided an immunogenic composition produced according to the method of any one of E46-E74 or E66-E82.
[0121] E96. According to another aspect of the present disclosure, there is provided a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified antigenic polypeptide construct to the cross-reactive host peptide compared to the crossreactivity of an unmodified antigenic polypeptide construct comprising an unmodified antigenic peptide sequence. According to another aspect of the present disclosure, there is provided a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, the method comprising: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified antigenic polypeptide construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified antigenic polypeptide construct comprising an unmodified antigenic peptide sequence.
[0122] E97. The method of E96, wherein the modified antigenic polypeptide construct elicits an immune response.
[0123] E98. The method of E96 or E97, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
[0124] E99. The method of E98, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0125] E100. The method of E98 or E99, wherein the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein.
[0126] E101. The method of any one of E98-E100, wherein the viral protein is a viral spike protein.
[0127] E102. The method of E98, wherein the bacterial protein is a protein from
Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium .
[0128] E103. The method of E98, wherein the bacterial protein is a bacterial cell surface protein and/or a bacterial cell wall protein.
[0129] E104. The method of E98, wherein the parasite protein is a protein from
Leishmania, Plasmodium, or Schistosoma.
[0130] E105. The method of E98, wherein the parasite protein is aparasite cell surface protein and/or a parasite secreted protein.
[0131] E106. The method of E98, wherein the fungal protein is a protein from
Candida, Cryptococcus, or Aspergillus .
[0132] E107. The method of E98, wherein the fungal protein is a fungal cell surface protein and/or a fungal cell wall protein.
[0133] E108. The method of any one of E96-E 107, wherein nucleic acid is RNA. [0134] E109. The method of E108, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0135] E110. The method of E108 or E109, wherein the RNA is comprised in an expression vector.
[0136] El 11. The method of any one of E96-E107, wherein the nucleic acid is DNA.
[0137] El 12. The method of El 11, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0138] El 13. The method of El 11 or El 12, wherein the DNA is comprised in an expression vector.
[0139] El 14. The method of any one of E96-E113, wherein the antigenic peptide sequence of the modified antigenic polypeptide construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0140] El 15. The method of any one of E96-E114, wherein the modified antigenic polypeptide construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about 64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified antigenic polypeptide construct.
[0141] El 16. The method of any one of E96-E115, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
[0142] El 17. The method of any one of E96-E116, wherein the cross-reactive host polypeptide is expressed in heart tissue.
[0143] El 18. The method of any one of E96-E117, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In another embodiment, the method of any one of E96-E117, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0144] El 19. The method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. In another embodiment, the method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6.
[0145] E120. The method of El 18, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2. In another embodiment, the method of El 18, wherein at least one cross-reactive host polypeptide is located within comprises PCM1. [0146] E121. According to another aspect of the present disclosure, there is provided an immunogenic composition comprising the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct any one of E96-E120.
[0147] E122. The immunogenic composition of E121, wherein nucleic acid is RNA.
[0148] El 23. The immunogenic composition of El 22, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0149] E124. The immunogenic composition of E122 or E123, wherein the RNA is comprised in an expression vector.
[0150] El 25. The immunogenic composition of any one of E122-E124, wherein the immunogenic composition is an RNA vaccine.
[0151] E126. The immunogenic composition E121, wherein the nucleic acid is DNA.
[0152] El 27. The immunogenic composition of El 26, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0153] E128. The immunogenic composition of E126 or E127, wherein the DNA is comprised in an expression vector.
[0154] E129. The immunogenic composition of any one of E126-E128, wherein the immunogenic composition is a DNA vaccine.
[0155] El 30. The immunogenic composition of E121, wherein the immunogenic composition comprises a polypeptide.
[0156] E131. According to another aspect of the present disclosure, there is provided a method for reducing or preventing cross-reactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence of an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross- reactive host peptide to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified viral spike protein construct comprising an unmodified antigenic peptide sequence. According to another aspect of the present disclosure, there is provided a method for reducing or preventing crossreactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct, the method comprising: identifying at least one antigenic peptide sequence of an unmodified viral spike protein construct, wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with the cross-reactive host peptide to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence, wherein the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct, and wherein modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified viral spike protein construct comprising an unmodified antigenic peptide sequence.
[0157] El 32. The method of E131, wherein the modified viral protein construct elicits an immune response. [0158] E133. The method of E131 or E132, wherein the viral spike protein is a spike protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
[0159] E134. The method of E131 or E133, wherein nucleic acid is RNA.
[0160] El 35. The method of El 34, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0161] E136. The method of E134 or E135, wherein the RNA is comprised in an expression vector.
[0162] E137. The method of E131 or E133, wherein the nucleic acid is DNA.
[0163] E138. The method of El 37, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0164] E139. The method of E137 or E138, wherein the DNA is comprised in an expression vector.
[0165] E140. The method of any one of E131-E139, wherein the antigenic peptide sequence of the modified viral spike protein construct has less than about 45%, about 40%, about 35%, about 30%, about 25%, about 20%, about 15%, or about 10% identity with the cross-reactive host peptide.
[0166] E 141. The method of any one of E 131 -E 140, wherein the modified viral spike protein construct has at least about 51%, about 52%, about 53%, about 54%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, about 61%, about 62%, about 63%, about
64%, about 65%, about 66%, about 67%, about 68%, about 69%, about 70%, about 71%, about 72%, about 73%, about 74%, about 75%, about 76%, about 77%, about 78%, about 79%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% sequence identity with the unmodified viral spike protein construct.
[0167] El 42. The method of any one of E131-E141, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue.
[0168] El 43. The method of any one of E131-E142, wherein the cross-reactive host polypeptide is expressed in heart tissue.
[0169] El 44. The method of any one of E131-E143, wherein the cross-reactive host polypeptide is located within the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In another embodiment, the method of any one of E131-E143, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0170] El 45. The method of El 44, wherein at least one cross-reactive host polypeptide comprises and/or is located within NEBL. The method of E 144, wherein at least one cross-reactive host polypeptide comprises and/or is located within MYH6.
[0171] El 46. The method of El 44, wherein at least one cross-reactive host polypeptide comprises and/or is located within RYR2. The method of El 44, wherein at least one cross-reactive host polypeptide comprises and/or is is located within PCM1
[0172] El 47. According to another aspect of the present disclosure, there is provided an immunogenic composition comprising the viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct any one of E131-146.
[0173] E148. The immunogenic composition of E147, wherein nucleic acid is RNA.
[0174] E149. The immunogenic composition of E148, wherein the RNA is double stranded, single stranded, antisense single stranded, messenger RNA (mRNA), self-amplifying RNA (saRNA), and/or modified RNA (modRNA).
[0175] E150. The immunogenic composition of E148 or E149, wherein the RNA is comprised in an expression vector.
[0176] E151. The immunogenic composition of any one of E148-E150, wherein the immunogenic composition is an RNA vaccine.
[0177] E152. The immunogenic composition E147, wherein the nucleic acid is DNA.
[0178] E153. The immunogenic composition of E152, wherein the DNA is double stranded, single stranded, and/or antisense single stranded.
[0179] E154. The immunogenic composition of E152 or E153, wherein the DNA is comprised in an expression vector.
[0180] E155. The immunogenic composition of any one of E152-E154, wherein the immunogenic composition is a DNA vaccine. [0181] El 56. The immunogenic composition of El 47, wherein the immunogenic composition comprises a polypeptide.
[0182] E157. According to another aspect of the present disclosure, there is provided a method of vaccinating a subject, comprising administering to the subject in need thereof an effective amount of the immunogenic composition of any of any one of E1-E45, E95, E121- E130, or E147-E156.
[0183] E158. A method for treating or preventing an infectious disease, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of E1-E45, E95, E121-E130, or E147-E156.
[0184] E159. The method of E158, wherein the immunogenic composition elicits an immune response comprising an antibody response.
[0185] El 60. The method according of El 58 or El 59, wherein the immunogenic composition elicits an immune response comprising a T cell response.
[0186] E161. A method for treating or preventing a disease or condition, comprising administering to a subject in need thereof an effective amount of the immunogenic composition of any one of E1-E45, E95, E121-E130, or E147-E156.
[0187] El 62. A method identifying a potential toxicity of an immunogenic composition in a biological host system, the immunogenic composition comprising an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, the method comprising: a. identifying one or more proteins of the host that have a degree of similarity to the antigenic polypeptide construct; b. identifying properties of the one or more proteins, the properties comprising cellular location, gene- and protein-tissue expression profdes, and/or toxic state associations; c. ranking or sorting the one or more proteins using one or more of the properties; d. identifying immunogenicity of the one or more proteins; e. comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence; f. predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence.
[0188] E 163. The method of E 162, wherein one or more of the steps are performed by a computational model. [0189] E164. The method of any one of E162 or E163, wherein the potential toxicity is myocarditis or pericarditis.
[0190] E165. The method of any one of clauses E162-E164, wherein the immunogenic composition is a vaccine.
[0191] El 66. The method of any one of clauses E162-E165, wherein the method comprises searching a human proteome with a spike protein peptide and identifying a protein from the human proteome with homology to the spike protein peptide.
[0192] El 67. The method of any one of clauses E162-E166, further comprising identifying whether the protein from the human proteome with homology to the spike protein peptide is present in a target organ.
[0193] E168. The method any one of clauses E162-E167, wherein ranking or sorting the one or more proteins using one or more of the properties comprises ranking cellular locations that are extracellular or on a surface of a cell as more likely to cause the potential toxicity than intracellular locations, ranking gene- and protein-tissue expression profiles that overlap in tissue location of the potential toxicity as more likely to cause the potential toxicity than other tissue locations, and/or ranking toxic state associations that overlap or are related to the potential toxicity as more likely to cause the potential toxicity than other toxic state associations.
[0194] E169. The method any one of clauses E162-E168, wherein identifying immunogenicity of the one or more proteins comprises identifying a ranking or sorting of degree of immunogenicity and/or degree of potential immunogenicity.
[0195] El 70. The method of clause El 69, wherein comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence comprises comparing the rank of likely to cause the potential toxicity and degree of immunogenicity and/or degree of potential immunogenicity and ranking: more likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity, and either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than less likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity.
[0196] E171. The method clause El 70, wherein predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence comprises predicting a greater degree of correspondence as more likely to cause a potential toxicity.
[0197] E172. The method any one of clauses E162- E171, wherein each of the steps are performed on a computational model.
[0198] E173. The method of any one of clauses E162- E172, further comprising modifying the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct to produce a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct that is less likely to cause the potential toxicity when compared to the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
[0199] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions of the disclosure can be used to achieve methods of the disclosure. [0200] Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0201] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The disclosure may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
[0202] FIG. 1 illustrates a strategy for analyzing antigenic polypeptide constructs for antigenic peptide sequences having high homology with peptides comprised in host proteins to predict an immune response to the antigenic polypeptide construct that will have crossreactivity to the host protein.
[0203] FIG. 2 illustrates a strategy for a viral spike protein-to-human protein peptide homology search.
[0204] FIG. 3 illustrates the concept of molecular mimicry in the context of an mRNA vaccine against a viral spike protein.
DETAILED DESCRIPTION
I. Examples of Definitions
[0205] Throughout this application, the term “about” is used according to its plain and ordinary meaning in the area of cell and molecular biology to indicate that a value includes the inherent variation or standard deviation of error for the measurement or quantitation method being employed to determine the value. For example, in some embodiments, the term “about” may encompass a range ofvalues that are 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 measurement or quantitation.
[0206] The use of the word “a” or “an” when used in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.”
[0207] The phrase “and/or” means “and” or “or”. To illustrate, A, B, and/or C includes: A alone, B alone, C alone, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B, and C. In other words, “and/or” operates as an inclusive or.
[0208] The phrase “essentially all” is defined as “at least 95%”; if essentially all members of a group have a certain property, then at least 95% of members of the group have that property. In some instances, essentially all means equal to any one of, at least any one of, or between any two of 95, 96, 97, 98, 99, or 100 % of members of the group have that property.
[0209] The compositions and methods for their use can “comprise,” “consist essentially of,” or “consist of’ any of the ingredients or steps disclosed throughout the specification. Throughout this specification, unless the context requires otherwise, the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. It is contemplated that embodiments described herein in the context of the term “comprising” may also be implemented in the context of the term “consisting of’ or “consisting essentially of.” Compositions and methods “consisting essentially of’ any of the ingredients or steps disclosed limits the scope of the claim to the specified materials or steps which do not materially affect the basic and novel characteristic of the claimed disclosure. The words “consisting of’ (and any form of consisting of, such as “consist of’ and “consists of’) means including, and limited to, whatever follows the phrase “consisting of.” Thus, the phrase “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present.
[0210] Reference throughout this specification to “one embodiment,” “an embodiment,” “a particular embodiment,” “a related embodiment,” “a certain embodiment,” “an additional embodiment,” or “a further embodiment” or combinations thereof means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment of the present disclosure. Thus, the appearances of the foregoing phrases in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.
[0211] The terms “inhibiting” or “reducing” or any variation of these terms includes any measurable decrease or complete inhibition to achieve a desired result The terms “promote” or “increase” or any variation of these terms includes any measurable increase to achieve a desired result or production of a protein or molecule.
[0212] As used herein, the terms “reference,” “standard,” or “control” describe a value relative to which a comparison is performed. For example, an agent, subject, population, sample, or value of interest is compared with a reference, standard, or control agent, subject, population, sample, or value of interest. A reference, standard, or control may be tested and/or determined substantially simultaneously and/or with the testing or determination of interest for an agent, subject, population, sample, or value of interest and/or may be determined or characterized under comparable conditions or circumstances to the agent, subject, population, sample, or value of interest under assessment.
[0213] The term “antigen mimicry,” “molecular mimicry,” and any variation of these terms refers to a phenomenon in which an antigenic peptide or protein sequence used in an immunogenic composition cross-reacts with endogenously-expressed host proteins. The endogenously-expressed human proteins act as an “antigen mimic” or “molecular mimic” of the antigenic peptide or protein sequence. Following immunization with the immunogenic composition, immune responses raised against the antigenic peptide or protein sequence can cross-react with endogenously-expressed human proteins that share similar or identical peptide sequences (or structures) with the antigenic peptide or protein sequence. The concept of molecular mimicry in the context of an mRNA vaccine against a viral spike protein is illustrated in FIG. 3
[0214] In some embodiments, the cross-reactive host protein or polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue. In some embodiments, the cross-reactive host polypeptide is located withing the following proteins: nebulette (NEBL), ryanodine receptor 2 (RYR2), ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2 (ATP2A2), titin (TTN), kelch like family member 41 (KLHL41), myosin heavy chain 6 (MYH6), myosin heavy chain 7 (MYH7), otogelin (OTOG), nicotinamide nucleotide transhydrogenase (NNT), and/or nebulin related anchoring protein (NRAP). In some embodiments, the cross-reactive host protein or polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue. In some embodiments, the cross-reactive host polypeptide is located withing the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1).
[0215] The term “RNA,” as used herein, means a nucleic acid molecule that includes ribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and/or uracil (U)). For example, RNA can contain all, or a majority of, ribonucleotide residues. As used herein, the term “ribonucleotide” means a nucleotide with a hydroxyl group at the 2’ position of a [3-D-ribofuranosyl group. In one aspect, RNA can be messenger RNA (mRNA) that relates to a RNA transcript which encodes a peptide or protein. As known to those of skill in the art, mRNA generally contains a 5' untranslated region (5'- UTR), a polypeptide coding region, and a 3' untranslated region (3'-UTR). Without any limitation, RNA can encompass double stranded RNA, antisense RNA, single stranded RNA, isolated RNA, synthetic RNA, RNA that is recombinantly produced, and modified RNA (modRNA).
[0216] An “isolated RNA” is defined as an RNA molecule that can be recombinant or has been isolated from total genomic nucleic acid. [0217] A “modified RNA” or “modRNA” refers to an RNA molecule having at least one addition, deletion, substitution, and/or alteration of one or more nucleotides as compared to naturally occurring RNA. Such alterations can refer to the addition of non-nucleotide material to internal RNA nucleotides, or to the 5' and/or 3' end(s) of RNA. In one aspect, such modRNA contains at least one modified nucleotide, such as an alteration to the base of the nucleotide. For example, a modified nucleotide can replace one or more uridine and/or cytidine nucleotides. For example, these replacements can occur for every instance of uridine and/or cytidine in the RNA sequence, or can occur for only select uridine and/or cytidine nucleotides. Such alterations to the standard nucleotides in RNA can include non-standard nucleotides, such as chemically synthesized nucleotides or deoxynucleotides. For example, at least one uridine nucleotide can be replaced with 1 -methylpseudouridine in an RNA sequence. Other such altered nucleotides are known to those of skill in the art. Such altered RNAs are considered analogs of naturally-occurring RNA. In some aspects, the RNA is produced by in vitro transcription using a DNA template, where DNA refers to a nucleic acid that contains deoxyribonucleotides. In some aspects, the RNA can be replicon RNA (replicon), in particular self-replicating RNA, or self-amplifying RNA (saRNA).
[0218] The term “DNA,” as used herein, means a nucleic acid molecule that includes deoxyribonucleotide residues (such as containing the nucleotide base(s) adenine (A), cytosine (C), guanine (G) and/or thymine (T)). For example, DNA can contain all, or a majority of, deoxyribonucleotide residues. As used herein, the term “deoxyribonucleotide” means a nucleotide lacking a hydroxyl group at the 2’ position of a [3-D-ribofiiranosyl group. Without any limitation, DNA can encompass double stranded DNA, antisense DNA, single stranded DNA, isolated DNA, synthetic DNA, DNA that is recombinantly produced, and modified DNA.
[0219] As used herein, a “protein,” “polypeptide,” or “peptide” refers to a molecule comprising at least two amino acid residues. As used herein, the term “wild-type” refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, wild-type versions of a protein or polypeptide are employed, however, in many embodiments of the disclosure, a modified protein or polypeptide is employed to generate an immune response. The terms described above may be used interchangeably. A “modified protein” or “modified polypeptide” or a “variant” refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, is altered with respect to the wild-type protein or polypeptide. In some embodiments, a modified/variant protein or polypeptide has at least one modified activity or function (recognizing that proteins or polypeptides may have multiple activities or functions). It is specifically contemplated that a modified/variant protein or polypeptide may be altered with respect to one activity or function yet retain a wild-type activity or function in other respects, such as immunogenicity. Where a protein is specifically mentioned herein, it is in general a reference to a native (wild-type) or recombinant (modified) protein. The protein may be isolated directly from the organism of which it is native, produced by recombinant DNA/exogenous expression methods, produced by solid-phase peptide synthesis (SPPS), or other in vitro methods. In particular embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences that encode a polypeptide (e.g., an antigen or fragment thereof). The term “recombinant” may be used in conjunction with a polypeptide or the name of a specific polypeptide, and this generally refers to a polypeptide produced from a nucleic acid molecule that has been manipulated in vitro or that is a replication product of such a molecule.
II. RNA, DNA, and Peptide Modalities
[0220] As contemplated herein, without any limitations, RNA, DNA, and/or peptides or polypeptides can be used as a modality to treat and/or prevent a number of diseases and/or conditions in mammals, including humans. Methods described herein comprise administration of the RNA, DNA, and/or peptides or polypeptides described herein to a mammal, such as a human. For example, in one aspect such methods of use for RNA, DNA, and/or peptides or polypeptides include an antigen-coding RNA or DNA vaccine or a peptide or polypeptide vaccine to induce robust neutralizing antibodies and accompanying/concomitant T-cell response to achieve protective immunization with preferably minimal vaccine doses.
[0221] For example, such RNA or DNA can be used to encode at least one antigen intended to generate an immune response in said mammal. These pathogenic antigenic polypeptide constructs are peptide or protein antigens derived from a pathogen associated with infectious disease, including viruses, bacteria, parasites, and fungi. The antigenic polypeptide constructs can comprise one or more antigenic peptide sequences that elicit an immune response. In specific embodiments, the antigenic polypeptide constructs are viral spike protein constructs, and the viral spike protein constructs can comprise one or more antigenic peptide sequences that elicit an immune response.
[0222] Viral antigenic polypeptide constructs can be from a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picomavirus, reovirus, retrovirus, rhabdovirus, or togavirus, for example. In some embodiments, the viral protein is a viral membrane protein, a viral capsid protein, a viral envelope protein, and/or a viral non-structural protein. In some embodiments, the viral protein is a viral spike protein. The viral antigenic polypeptide constructs can be selected from antigens derived from viral pathogens including an arenavirus (such as Lassa virus, or lymphocytic choriomeningitis virus (LCMV)); an astrovirus; a bunyavirus (such as a Hantavirus); a calicivirus; a coronavirus (such as a severe acute respiratory syndrome virus (SARS) - e.g. SARS-CoV-2, or a middle east respiratory syndrome (MERS) virus); a filovirus (such as Ebola virus or Marburg virus); a flavivirus (such as Yellow Fever virus, West Nile virus, or Hepatitis C virus (HCV)); a hepadnavirus; a hepevirus; an orthomyxovirus (such as Influenza A virus, Influenza B virus, or Influenza C virus); a paramyxovirus (such as Rubeola virus, or Rubulavirus); a picomavirus (such as Poliovirus, Hepatitis A virus, or Rhinovirus); a reovirus (such as Rotavirus); a retrovirus (such as Human Immunodeficiency Virus (HIV), or Human T-lymphotropic virus (HTLV)); a rhabdovirus (such as Rabies virus or Rabies lyssavirus); a togavirus (such as Sindbis virus (SINV), Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV); and/or or Rubella virus.
[0223] In some instances, antigenic polypeptide constructs can be from a protein from bacterial pathogens including Staphylococcus, Streptococcus, Neisseria, Salmonella, Vibrio, Clostridium, Bacillus, or Mycobacterium. In some embodiments, the bacterial antigen is a bacterial protein, and in some embodiments, the bacterial protein is a bacterial membrane protein and/or a bacterial cell wall protein.
[0224] Antigens derived from Staphylococcus species including Methicillin-resistant Staphylococcus aureus (MRSA) that are contemplated for use include virulence regulators, such as the Agr system, Sar and Sae, the Ari system, Sar homologues (Rot, MgrA, SarS, SarR, SarT, SarU, SarV, SarX, SarZ and TcaR), the Srr system and TRAP. Other Staphylococcus proteins that may serve as antigens include Clp proteins, HtrA, MsrR, aconitase, CcpA, SvrA, Msa, CfvA and CfvB (see, e.g., Staphylococcus: Molecular Genetics, 2008 Caister Academic Press, Ed. Jodi Lindsay). The genomes for two species of Staphylococcus aureus (N315 and Mu50) have been sequenced and are publicly available, for example at PATRIC (PATRIC: The VBI PathoSystems Resource Integration Center, Snyder et al., 2007). As would be understood by the skilled person, Staphylococcus proteins for use as antigens may also be identified in other public databases such as GenBank®, Swiss-Prot®, and TrEMBL®.
[0225] Antigens derived from Streptococcus pneumoniae that are contemplated for use in certain embodiments described herein include pneumolysin, PspA, choline -binding protein A (CbpA), NanA, NanB, SpnHL, PavA, LytA, Pht, and pilin proteins (RrgA; RrgB; RrgC). Antigenic proteins of Streptococcus pneumoniae are also known in the art and may be used as an antigen in some embodiments (see, e.g., Zysk et al., 2000). The complete genome sequence of a virulent strain of Streptococcus pneumoniae has been sequenced and, as would be understood by the skilled person, .S', pneumoniae proteins for use herein may also be identified in other public databases such as GENBANK®, SWISS-PROT®, and TREMBL®. Proteins of particular interest for antigens according to the present disclosure include virulence factors and proteins predicted to be exposed at the surface of the pneumococci (see, e.g., Frolet et al., 2010).
[0226] Examples of bacterial antigens that may be used as for antigenic polypeptide constructs include, but are not limited to, Actinomyces polypeptides, Bacillus polypeptides, Bacteroides polypeptides, Bordetella polypeptides, Bartonella polypeptides, Borrelia polypeptides (e.g., B. burgdorferi OspA), Brucella polypeptides, Campylobacter polypeptides, Capnocytophaga polypeptides, Chlamydia polypeptides, Corynebacterium polypeptides, Coxiella polypeptides, Dermatophilus polypeptides, Enterococcus polypeptides, Ehrlichia polypeptides, Escherichia polypeptides, Francisella polypeptides, Fusobacterium polypeptides, Haemobartonella polypeptides, Haemophilus polypeptides (e.g., H. influenzae type b outer membrane protein), Helicobacter polypeptides, Klebsiella polypeptides, L-form bacteria polypeptides, Leptospira polypeptides, Listeria polypeptides, Mycobacteria polypeptides, Mycoplasma polypeptides, Neisseria polypeptides, Neorickettsia polypeptides, Nocardia polypeptides, Pasteurella polypeptides, Peptococcus polypeptides, Peptostreptococcus polypeptides, Pneumococcus polypeptides (i.e., S. pneumoniae polypeptides), Proteus polypeptides, Pseudomonas polypeptides, Rickettsia polypeptides, Rochalimaea polypeptides, Salmonella polypeptides, Shigella polypeptides, Staphylococcus polypeptides, group A streptococcus polypeptides (e.g., S. pyogenes M proteins), group B streptococcus (S. agalactiae) polypeptides, Treponema polypeptides, and Yersinia polypeptides (e.g., Y. pestis Fl and V antigens).
[0227] In some aspects, one or more of the bacterial pathogens and/or one or more bacterial antigens in the above lists may be excluded.
[0228] In some instances, the antigenic polypeptide constructs can be from a protein from parasitic pathogens including Leishmania, Plasmodium, or Schistosoma. In some embodiments, the parasite antigen is a parasite protein, and in some embodiments, the parasite protein is a parasite cell surface protein and/or a parasite secreted protein. Examples of parasite antigens that can be used for antigenic polypeptide constructs include, but are not limited to, Babesia polypeptides, Balantidium polypeptides, Besnoitia polypeptides, Cryptosporidium polypeptides, Eimeria polypeptides, Encephalitozoon polypeptides, Entamoeba polypeptides, Giardia polypeptides, Hammondia polypeptides, Hepatozoon polypeptides, Isospora polypeptides, Leishmania polypeptides, Microsporidia polypeptides, Neospora polypeptides, Nosema polypeptides, Pentatrichomonas polypeptides, Plasmodium polypeptides, Acanthocheilonema polypeptides, Aelurostrongylus polypeptides, Ancylostoma polypeptides, Angiostrongylus polypeptides, Ascaris polypeptides, Brugia polypeptides, Bunostomum polypeptides, Capillaria polypeptides, Chabertia polypeptides, Cooperia polypeptides, Crenosoma polypeptides, Dictyocaulus polypeptides, Dioctophyme polypeptides, Dipetalonema polypeptides, Diphyllobothrium polypeptides, Diplydium polypeptides, Dirofilaria polypeptides, Dracunculus polypeptides, Enterobius polypeptides, Filaroides polypeptides, Haemonchus polypeptides, Lagochilascaris polypeptides, Loa polypeptides, Mansonella polypeptides, Muellerius polypeptides, Nanophyetus polypeptides, Necator polypeptides, Nematodirus polypeptides, Oesophagostomum polypeptides, Onchocerca polypeptides, Opisthorchis polypeptides, Ostertagia polypeptides, Parafilaria polypeptides, Paragonimus polypeptides, Parascaris polypeptides, Physaloptera polypeptides, Protostrongylus polypeptides, Setaria polypeptides, Spirocerca polypeptides Spirometra polypeptides, Stephanofilaria polypeptides, Strongyloides polypeptides, Strongylus polypeptides, Thelazia polypeptides, Toxascaris polypeptides, Toxocara polypeptides, Trichinella polypeptides, Trichostrongylus polypeptides, Trichuris polypeptides, Uncinaria polypeptides, and Wuchereria polypeptides, (e.g., P. falciparum circumsporozoite (PfCSP)), sporozoite surface protein 2 (PfSSP2), carboxyl terminus of liver state antigen 1 (PfLSAl c- term), and exported protein 1 (PfExp-1), Pneumocystis polypeptides, Sarcocystis polypeptides, Schistosoma polypeptides, Theileria polypeptides, Toxoplasma polypeptides, Trypanosoma polypeptides, and polypeptides (including antigens as well as allergens) from fleas; ticks, including hard ticks and soft ticks; flies, such as midges, mosquitoes, sand flies, black flies, horse flies, horn flies, deer flies, tsetse flies, stable flies, myiasis-causing flies and biting gnats; ants; spiders, lice; mites; and true bugs, such as bed bugs and kissing bugs. In some aspects, one or more of the parasite pathogens and/or one or more parasite antigens in the list may be excluded.
[0229] In some instances, antigenic polypeptide constructs can be from a protein from fungal pathogens including Candida, Cryptococcus, or Aspergillus . In some embodiments, the fungal antigen is a fungal protein, and in some embodiments, the fungal protein is a fungal surface protein and/or a fungal cell wall protein. Examples of fungal antigens that can be used for antigenic polypeptide constructs include, but are not limited to, Absidia polypeptides, Acremonium polypeptides, Alternaria polypeptides, Aspergillus polypeptides, Basidiobolus polypeptides, Bipolaris polypeptides, Blastomyces polypeptides, Candida polypeptides, Coccidioides polypeptides, Conidiobolus polypeptides, Cryptococcus polypeptides, Curvalaria polypeptides, Epidermophyton polypeptides, Exophiala polypeptides, Geotrichum polypeptides, Histoplasma polypeptides, Madurella polypeptides, Malassezia polypeptides, Microsporum polypeptides, Moniliella polypeptides, Mortierella polypeptides, Mucor polypeptides, Paecilomyces polypeptides, Penicillium polypeptides, Phialemonium polypeptides, Phialophora polypeptides, Prototheca polypeptides, Pseudallescheria polypeptides, Pseudomicrodochium polypeptides, Pythium polypeptides, Rhinosporidium polypeptides, Rhizopus polypeptides, Scolecobasidium polypeptides, Sporothrix polypeptides, Stemphylium polypeptides, Trichophyton polypeptides, Trichosporon polypeptides, and Xylohypha polypeptides. In some aspects, one or more of the fungal pathogens and/or one or more fungal antigens in the above list may be excluded.
[0230] In some instances, antigenic polypeptide constructs can be from a protein from a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition. Exemplary tumor-/cancer-associated antigens and tumor neoantigens (Linnemann et al., 2015) that can be used for antigenic polypeptide constructs include EBNA, CD123, HER1, HER2, CA-125, CA 19-9, CA 72-4, CA 15-3\CA 27.29\BCAA, CA-195, CA- 242, CA-50, CA LX, MN-CA IX, TRAIL/DR4, CD2, CD5, CD7, CD19, CD20, CD22, CD23, CD24, CD30, CD33, CD38, CD44v6, CD47, CD56, CD68/P1, CD70, CD97, CD99, CD123, CD 171, CD 179, CD200, CD319 (CS1), HLA-G, carcinoembryonic antigen, alphafetoprotein, b-human chorionic gonadotropin, AKT, Her3, epithelial tumor antigen, R0R1, folate binding protein, folate receptor, HIV-1 envelope glycoprotein gpl20, HIV-1 envelope glycoprotein gp41, HERV-K, 11-6, IL-l lRa, IL-13Ra, kappa chain, lambda chain, CSPG4, CLL-1, U5snRNP200, BAFF-R, BCMA, p53, mutated p53, Ras, mutated ras, c-Myc, cytoplasmic serine/threonine kinases (e.g., A-Raf, B-Raf, and C-Raf, cyclin-dependent kinases), MAGE- Al, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A10, MAGE-A12, MART-1, glioma-associated antigen, melanoma-associated antigen, BAGE, DAM-6, DAM- 10, GAGE-
1, GAGE-2, GAGE-8, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7B, pi 5, NA88-A, MC1R, mda-7, gp75, GplOO, PSA, PSM, Tyrosinase, tyrosinase-related protein, TRP-1, TRP-
2, ART-4, CAMEL, CEA, Cyp-B, hTERT, hTRT, iCE, MUC1, MUC2, MUC16, MUC18, Phosphoinositide 3-kinases (PI3Ks), TRK receptors, PRAME, P15, P16, RU1, RU2, SART-1, SART-3, Wilms’ tumor antigen (WT1), AFP, [3-catenin, Caspase-8/m, CDK-4/m, ELF2M, GnT-V, G250, HAGE, HSP70-2M, HST-2, KIAA0205, MUM-1, MUM-2, MUM-3, Myosin/m, RAGE, SART-2, TRP-2/INT2, 707-AP, Annexin II, CDC27/m, TPI/mbcr-abl, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, interferon regulatory factor 4 (IRF4), ETV6/AML, LDLR/FUT, Pml/RAR, Tumor-associated calcium signal transducer 1 (TACSTD1) TACSTD2, receptor tyrosine kinases (e.g., Epidermal Growth Factor receptor (EGFR) (in particular, EGFRvIII), platelet derived growth factor receptor (PDGFR), vascular endothelial growth factor receptor (VEGFR)), VEGFR2, cytoplasmic tyrosine kinases (e.g., src-family, syk-ZAP70 family), integrin-linked kinase (ILK), signal transducers and activators of transcription STAT3, STATS, and STATE, hypoxia inducible factors (e.g, HIF-1 and HIF- 2), Nuclear Factor-Kappa B (NF-B), Notch receptors (e.g., Notch 1-4), NY ESO 1, pl85erbB2, pl80erbB-3, c-Met, nm-23Hl, beta-HCG, BCA225, BTAA, CAM 17.1, CAM43, LI CAM, NCAM, NuMa, 43-9F, 791Tgp72, CO-029, FGF-5, HTgp-175, M344, MA-50, MG7-Ag, M0V18, NB/70K, NY-CO-1 , RCASI, SDCCAG1 6, TA- 90\Mac-2 binding protein\cyclophilm C-associated protein, TAAL6, TAG72, TLP, TPS, GPC3, EBMA-1, BARF-1, CS-1, ADRB3, thyroglobulin, EVT6-AML, TGS5, plysialic acid, neutrophil elastase, intestinal carboxyl esterase, prostase, prostein, lewisY, LY6K, PAP, OR51 E2, PANX3, SSEA-4, TARP, CXORF61, Flt3, TEM1, TEM7R, TSHR, UPK2, mammalian targets of rapamycin (mTOR), WNT, extracellular signal-regulated kinases (ERKs), and their regulatory subunits, k-ras, PMSA, PR-3, MDM2, Mesothelin, renal cell carcinoma-5T4, SM22-alpha, carbonic anhydrases I (CAI) and IX (CAIX) (also known as G250), STEAD, TEL/AML1, GD2, proteinase3, hTERT, sarcoma translocation breakpoints, EphA2, EphnnB2, ML-IAP, EpCAM, ERG (TMPRSS2 ETS fusion gene), NA 17, PAX3, ALK, androgen receptor, insulin growth factor (IGF)-I, IGFII, IGF-I receptor, cyclin Bl, polysialic acid, M-CSF, MYCN, RhoC, GD3, fucosyl GM1, mesothelian, PSCA, sLe, PLAC1, GM3, GPRC5D, GPR20, BORIS, Tn, GLoboH, NY-BR-1, RGsS, SAGE, SART3, STn, PAX5, OY-TES1, sperm protein 17, LCK, HMWMAA, HAVCR1, AKAP-4, SSX2, XAGE 1, B7H3, B7H6, Kit, legumain, TN Ag, TIE2, Page4, MAD-CT-1, FAP, MAD-CT-2, fos related antigen 1, CBX2, CLDN6, SPANX, TPTE, ACTL8, ANKRD30A, CDKN2A, MAD2L1, CTAG1B, SUNCI, TSP- 180, and LRRN 1. Examples of sequences for antigens are known in the art, for example, in the GenBank® database: CD 19 (Accession No. NG_007275.1), EBNA (Accession No. NG_002392.2), WT1 (Accession No. NG_009272.1), CD 123 (Accession No. NC_000023.11), NY-ESO (Accession No. NC_000023.11), EGFRvIII (Accession No. NG_007726.3), MUC1 (Accession No. NG_029383.1), HER2 (Accession No. NG_007503.1), CA-125 (Accession No. NG_055257.1), WT1 (Accession No. NG_009272.1), Mage-A3 (Accession No. NG_013244.1), Mage-A4 (Accession No. NG_013245.1), Mage-AlO (Accession No. NC_000023.11), TRAIL/DR4 (Accession No. NC_000003.12), and/or CEA (Accession No. NC_000019.10).
[0001] Tumor-associated antigens may be derived from prostate, breast, colorectal, lung, pancreatic, renal, mesothelioma, ovarian, liver, brain, bone, stomach, spleen, testicular, cervical, anal, gall bladder, thyroid, or melanoma cancers, as examples. Exemplary tumor- associated antigens or tumor cell-derived antigens include MAGE 1, 3, and MAGE 4 (or other MAGE antigens such as those disclosed in International Patent Publication No. WO 99/40188); PRAME; BAGE; RAGE, Lage (also known as NY ESO 1); SAGE; and HAGE or GAGE. These non-limiting examples of tumor antigens are expressed in a wide range of tumor types such as melanoma, lung carcinoma, sarcoma, and bladder carcinoma. See, e.g., U.S. Patent No. 6,544,518. Prostate cancer tumor-associated antigens include, for example, prostate specific membrane antigen (PSMA), prostate-specific antigen (PSA), prostate-carcinoma tumor antigen- 1 (PCTA-1), prostatic acid phosphates, NKX3.1, and six-transmembrane epithelial antigen of the prostate (STEAP).
[0002] In some aspects, one or more of the tumor-/cancer-associated antigens in the above list may be excluded.
[0003] In some instances, antigenic polypeptide constructs can be from a protein from Acinetobacter baumannii, Anciplcisma genus, Anciplcisma phagocy tophilum, Ancylostoma braziliense, Ancylostoma duodenale, Arcanobacterium haemolyticum, Ascaris lumbricoides, Aspergillus genus, Astroviridae, Babesia genus, Bacillus anthracis, Bacillus cereus, Bartonella henselae, BK virus, Blastocystis hominis, Blastomyces dermatitidis, Bordetella pertussis, Borrelia burgdorferi, Borrelia genus, Borrelia spp, Brucella genus, Brugia malayi, Bunyaviridae family, Burkholderia cepacia and other Burkholderia species, Burkholderia mallei, Burkholderia pseudomallei, Caliciviridae family, Campylobacter genus, Candida albicans, Candida spp, Chlamydia trachomatis, Chlamydophila pneumoniae, Chlamydophila psittaci, CJD prion, Clonorchis sinensis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium perfringens, Clostridium spp, Clostridium tetani, Coccidioides spp, coronaviruses, Corynebacterium diphtheriae, Coxiella burnetii, Crimean- Congo hemorrhagic fever virus, Cryptococcus neoformans, Cryptosporidium genus, Cytomegalovirus (CMV), Dengue viruses (DEN-1 , DEN-2, DEN-3 and DEN-4), Dientamoeba fragilis, Ebolavirus (EBOV), Echinococcus genus, Ehrlichia chaffeensis, Ehrlichia ewingii, Ehrlichia genus, Entamoeba histolytica, Enterococcus genus, Enterovirus genus, Enteroviruses, mainly Coxsackie A virus and Enterovirus 71 (EV71 ), Epidermophyton spp, Epstein-Barr Virus (EBV), Escherichia coli 01 57:H7, 01 1 1 and O104:H4, Fasciola hepatica and Fasciola gigantica, FFI prion, Filarioidea superfamily, Flavivimses, Francisella tularensis, Fusohacterium genus, Geotrichum candidum, Giardia intestinalis, Gnathostoma spp, GSS prion, Guanarito virus, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Henipavirus (Hendra virus Nipah virus)' , Hepatitis A Virus, Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), Hepatitis D Virus, Hepatitis E Virus, Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), Histoplasma capsulatum, HIV (Human immunodeficiency virus), Hortaea werneckii, Human bocavirus (HBoV), Human herpesvirus 6 (HHV-6) and Human herpesvirus 7 (HHV- 7), Human metapneumovirus (hMPV), Human papillomavirus (HPV), Human parainfluenza viruses (HPIV), Japanese encephalitis virus, JC virus, Junin virus, Kingella kingae, Klebsiella granulomatis, Kuru prion, Lassa virus, Legionella pneumophila, Leishmania genus, Leptospira genus, Listeria monocytogenes, Lymphocytic choriomeningitis virus (LCMV), Machupo virus, Malassezia spp, Marburg virus, Measles virus, Metagonimus yokagawai, Microsporidia phylum, Molluscum contagiosum virus (MCV), Mumps virus, Mycobacterium leprae and Mycobacterium lepromatosis, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Naegleria fowled, Necator americanus, Neisseria gonorrhoeae, Neisseria meningitidis, Nocardia asteroides, Nocardia spp, Onchocerca volvulus, Orientia tsutsugamushi, Orthomyxoviridae family (Influenza), Paracoccidioides brasiliensis, Paragonimus spp, Paragonimus westermani, Parvovirus Bl 9, Pasteurella genus, Plasmodium genus, Pneumocystis jirovecii, Poliovirus, Rabies virus, Respiratory syncytial virus (RSV), Rhinovirus, rhinoviruses, Rickettsia akari, Rickettsia genus, Rickettsia prowazekii, Rickettsia rickettsii, Rickettsia typhi, Rift Valley fever virus, Rotavirus, Rubella virus, Sabia virus, Salmonella genus, Sarcoptes scabiei, SARS coronavirus, Schistosoma genus, Shigella genus, Sin Nombre virus, Hantavirus, Sporothrix schenckii, Staphylococcus genus, Staphylococcus genus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Strongyloides stercoralis, Taenia genus, Taenia solium, Tick-borne encephalitis virus (TBEV), Toxocara canis or Toxocara cati, Toxoplasma gondii, Treponema pallidum, Trichinella spiralis, Trichomonas vaginalis, Trichophyton spp, Trichuris trichiura, Trypanosoma brucei, Trypanosoma cruzi, Ureaplasma urealyticum, Varicella zoster virus (VZV), Varicella zoster virus (VZV), Variola major or Variola minor, vCJD prion, Venezuelan equine encephalitis virus, Vibrio cholerae, West Nile virus, Western equine encephalitis virus, Wuchereria bancrofti, Yellow fever virus, Yersinia enterocolitica, Yersinia pestis, and/or Yersinia pseudotuberculosis . [0004] Conditions and/or diseases that can be treated and/or prevented with such RNA, DNA, and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by viral infection. Such viruses include, but are not limited to, an arenavirus (such as Lassa virus, or lymphocytic choriomeningitis virus (LCMV)); an astrovirus; a bunyavirus (such as a Hantavirus); a calicivirus; a coronavirus (such as a severe acute respiratory syndrome virus (SARS) - e.g. SARS-CoV-2, or a middle east respiratory syndrome (MERS) virus); a filovirus (such as Ebola virus or Marburg virus); a flavivirus (such as Yellow Fever virus, West Nile virus, or Hepatitis C virus (HCV)); a hepadnavirus; a hepevirus; an orthomyxovirus (such as Influenza A virus, Influenza B virus, or Influenza C virus); a paramyxovirus (such as Rubeola virus, or Rubulavirus); a picomavirus (such as Poliovirus, Hepatitis A virus, or Rhinovirus); a reovirus (such as Rotavirus); a retrovirus (such as Human Immunodeficiency Virus (HIV), or Human T-lymphotropic virus (HTLV)); a rhabdovirus (such as Rabies virus or Rabies lyssavirus); a togavirus (such as Sindbis virus (SINV), Eastern Equine Encephalitis virus (EEEV), Western Equine Encephalitis virus (WEEV), and/or Rubella virus.
[0005] Conditions and/or diseases that can be treated with the RNA, DNA, and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by bacteria, fungi, parasites, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
III. Nucleic Acids
[0006] In certain embodiments, an antigen intended to generate an immune response is encoded by a nucleic acid. In some embodiments, the nucleic acid encodes one or more peptide or protein antigen(s) derived from a pathogen associated with one or more infectious disease(s), including virus(es), bacteria, parasite(s), and fungi. The nucleic acids encoding the antigenic polypeptide constructs can comprise one or more nucleotide sequences corresponding to one or more antigenic peptide sequences that elicit an immune response.
[0007] Nucleic acid sequences can exist in a variety of instances such as: isolated segments; recombinant vectors of incorporated sequences or recombinant polynucleotides encoding polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof; polynucleotides sufficient for use as hybridization probes, PCR primers, or sequencing primers for identifying, analyzing, mutating or amplifying a polynucleotide; anti-sense nucleic acids for inhibiting expression of a polynucleotide; mRNA; saRNA; and complementary sequences of the foregoing described herein. Nucleic acids may encode an antigen or epitope to which antibodies may bind. Nucleic acids encoding fusion proteins that include antigens or epitopes are also provided. The nucleic acids can be singlestranded or double-stranded and can comprise RNA and/or DNA nucleotides and artificial variants thereof (e.g., peptide nucleic acids).
[0008] The term “polynucleotide” refers to a nucleic acid molecule that can be recombinant or has been isolated from total genomic nucleic acid. Included within the term “polynucleotide” are oligonucleotides (nucleic acids 100 residues or less in length), recombinant vectors, including, for example, plasmids, cosmids, phage, viruses, and the like. Polynucleotides include, in certain aspects, regulatory sequences, isolated substantially away from their naturally occurring genes or protein encoding sequences. Polynucleotides may be singlestranded (coding or antisense) or double- stranded, and may be RNA, DNA (genomic, cDNA or synthetic), analogs thereof, or a combination thereof. Additional coding or non-coding sequences may, but need not, be present within a polynucleotide.
[0009] In this respect, the term “gene” is used to refer to a nucleic acid that encodes a protein, polypeptide, or peptide (including any sequences required for proper transcription, post-translational modification, or localization). As will be understood by those in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences, and smaller engineered nucleic acid segments that express, or may be adapted to express, proteins, polypeptides, domains, peptides, fusion proteins, and mutants. A nucleic acid encoding all or part of a polypeptide may contain a contiguous nucleic acid sequence encoding all or a portion of such a polypeptide. It also is contemplated that a particular polypeptide may be encoded by nucleic acids containing variations having slightly different nucleic acid sequences but, nonetheless, encode the same or substantially similar polypeptide.
[0010] In certain embodiments, there are polynucleotide variants having substantial identity to the sequences disclosed herein; those comprising equal to any one of, at least any one of, at most any one of, or between any two of 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% or higher sequence identity, compared to a polynucleotide sequence provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 90% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide. In some embodiments, the isolated polynucleotide will comprise a nucleotide sequence encoding a polypeptide that has at least 95% identity to an amino acid sequence described herein, over the entire length of the sequence; or a nucleotide sequence complementary to said isolated polynucleotide.
[0011] The nucleic acid segments, regardless of the length of the coding sequence itself, may be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, and the like, such that their overall length may vary considerably. The nucleic acids can be any length. They can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 or more nucleotides in length, and/or can comprise one or more additional sequences, for example, regulatory sequences, and/or be a part of a larger nucleic acid, for example, a vector. It is therefore contemplated that a nucleic acid fragment of almost any length may be employed, with the total length being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, a nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow for purification of the polypeptide, transport, secretion, post-translational modification, or for therapeutic and/or prophylatic benefits such as targeting or efficacy. As discussed above, a tag or other heterologous polypeptide may be added to the modified polypeptide -encoding sequence, wherein “heterologous” refers to a polypeptide that is not the same as the modified polypeptide.
[0012] In some embodiments, the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof encoded by a nucleic acid elicit an immune response. The immune response may be against the antigenic polypeptide constructs and/or antigenic peptide sequences and/or a homologous endogenously-expressed (e.g. cross-reactive) host protein. The antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously-expressed (e.g. cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%,
57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%,
73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous.
[0013] In some embodiments, the nucleic acids may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,
41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65,
66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90,
91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 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 contiguous nucleotides that are equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with a homologous nucleic acid encoding a homologous endogenously-expressed (e.g. cross- reactive) host protein or a cDNA encoding a homologous endogenously-expressed (e.g. cross- reactive) host protein.
[0014] In some embodiments, the one or more antigenic polypeptide constructs and/or antigenic peptide sequence thereof encoded by a nucleic acid are modified such that the antigenic polypeptide constructs and/or antigenic peptide sequences thereof continue to elicit an immune response but homology with a homologous endogenously-expressed (e.g., cross- reactive) host protein is reduced to reduce or prevent cross-reactivity of the immune response to the host protein after immunization with a nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences thereof. The homology between the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed (e.g., cross-reactive) host protein may be reduced by modifying the nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences thereof such that the nucleic acid encoding the antigenic polypeptide constructs and/or antigenic peptide sequences and the nucleic acid, e.g., genomic DNA, mRNA, or cDNA, encoding the homologous endogenously-expressed (e.g., cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%,
39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,
55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,
71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,
87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% similar, identical, or homologous. [0015] The nucleic acids of the disclosure encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more mutations in the sequence encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide compared to nucleic acids, e.g., genomic DNA, mRNA, cDNA, encoding unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences thereof. The portions of the nucleic acids of the disclosure encoding the modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%,
30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%,
46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any derivable range therein) similar, identical, or homologous with nucleic acids encoding unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences thereof.
A. Mutation
[0016] Changes can be introduced by mutation into a nucleic acid, thereby leading to changes in the amino acid sequence of a polypeptide (e.g., an antigen or antibody or antibody derivative) that it encodes. Mutations can be introduced using any technique known in the art. In one embodiment, one or more particular amino acid residues are changed using, for example, a site-directed mutagenesis protocol. In another embodiment, one or more randomly selected residues are changed using, for example, a random mutagenesis protocol. In some instances, however it is made, a mutant polypeptide can be expressed and screened for a desired property. [0017] Mutations can be introduced into a nucleic acid without significantly altering the biological activity of a polypeptide that it encodes. For example, one can make nucleotide substitutions leading to amino acid substitutions at non-essential amino acid residues. Alternatively, one or more mutations can be introduced into a nucleic acid that selectively changes the biological activity of a polypeptide that it encodes. See, e.g. , Romain Studer et al., Biochem. J. 449:581-594 (2013). For example, the mutation can quantitatively or qualitatively change the biological activity. Examples of quantitative changes include increasing, reducing or eliminating the activity. Examples of qualitative changes include altering the antigen specificity of an antibody.
B. RNA Molecules
[0018] In some embodiments, the RNA molecule described herein is a coding RNA molecule. Coding RNA includes a functional RNA molecule that may be translated into a peptide or polypeptide. In some embodiments, the coding RNA molecule includes at least one open reading frame coding for at least one peptide or polypeptide. The coding RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs), which may be a sequence of codons that is translatable into a polypeptide or protein of interest. The coding RNA molecule may be a messenger RNA (mRNA) molecule, viral RNA molecule, or self-amplifying RNA molecule (saRNA, also referred to as a replicon). In some embodiments, the RNA molecule is an mRNA. In some embodiments, the RNA molecule is a saRNA. In some embodiments, the saRNA molecule may be a coding RNA molecule.
[0019] The RNA molecule may encode one polypeptide of interest or more, such as an antigen or more than one antigen, e.g., two, three, four, five, six, seven, eight, nine, ten, or more polypeptides. Alternatively, or in addition, one RNA molecule may also encode more than one polypeptide of interest or more, such as an antigen, e.g., a bicistronic, or tricistronic RNA molecule that encodes different or identical antigens.
[0020] The sequence of the RNA molecule may be codon optimized or deoptimized for expression in a desired host, such as a human cell.
[0021] In some embodiments, the RNA molecule includes equal to any one of, at least any one of, at most any one of, or between any two of from about 20 to about 100,000 nucleotides (e.g., equal to any one of, at least any one of, at most any one of, or between any two of from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000). In some embodiments, the RNA molecule includes at least 100 nucleotides. For example, in some embodiments, the RNA has a length between 100 and 15,000 nucleotides; between 7,000 and 16,000 nucleotides; between 8,000 and 15,000 nucleotides; between 9,000 and 12,500 nucleotides; between 11,000 and 15,000 nucleotides; between 13,000 and 16,000 nucleotides; between 7,000 and 25,000 nucleotides. In some aspects, one or more of the nucleotide size ranges in the list may be excluded.
[0022] The sequence of the RNA molecule may be modified if desired, for example to increase the efficacy of expression or replication of the RNA, or to provide additional stability or resistance to degradation. For example, the RNA sequence may be modified with respect to its codon usage, for example, to increase translation efficacy and half-life of the RNA.
[0023] In some embodiments, the RNA molecules may include one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced. As used herein, a “structural” feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in an RNA molecule without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide “ATCG” may be chemically modified to “AT-5meC-G”. The same polynucleotide may be structurally modified from “ATCG” to “ATCCCG”. Here, the dinucleotide “CC” has been inserted, resulting in a structural modification to the polynucleotide.
[0024] In some embodiments, the RNA molecule may include one or more modified nucleotides in addition to any 5' cap structure. Naturally occurring nucleotide modifications are known in the art. [0025] In some embodiments, the RNA molecule does not include modified nucleotides, e.g., does not include modified nucleobases, and all of the nucleotides in the RNA molecule are conventional standard ribonucleotides A, U, G, and C, with the exception of an optional 5' cap that may include, for example, 7-methylguanosine, which is further described below. In some embodiments, the RNA may include a 5' cap comprising a 7'-methylguanosine, and the first 1, 2 or 3 5' ribonucleotides may be methylated at the 2' position of the ribose.
1. Modified Nucleobases
[0026] Modified nucleobases which may be incorporated into modified nucleosides and nucleotides and be present in the RNA molecules include, for example, m5C (5- methylcytidine), m5U (5 -methyluridine), m6A (N6-methyladenosine), s2U (2- thiouridine), Um (2'-0-methyluridine), mlA (1 -methyladenosine); m2A (2- methyladenosine); Am (2-1-0- methyladenosine); ms2m6A (2-methylthio-N6- methyladenosine); i6A (N6- isopentenyladenosine); ms2i6A (2-methylthio- N6isopentenyladenosine); io6A (N6-(cis- hydroxyisopentenyl)adenosine); ms2io6A (2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine); g6A (N6- glycinylcarbamoyladenosine); t6A (N6-threonyl carbamoyladenosine); ms2t6A (2- methylthio-N6-threonyl carbamoyladenosine); m6t6A (N6-methyl-N6- threonylcarbamoyladenosine); hn6A(N6-hydroxynorvalylcarbamoyl adenosine); ms2hn6A (2- methylthio-N6-hydroxynorvalyl carbamoyladenosine); Ar(p) (2'-0- ribosyladenosine (phosphate)); I (inosine); mil (1 -methylinosine); m'lm (l,2'-0- dimethylinosine); m3C (3- methylcytidine); Cm (2T-0-methylcytidine); s2C (2- thiocytidine); ac4C (N4-acetylcytidine); £5C (5-fonnylcytidine); m5Cm (5,2-0- dimethylcytidine); ac4Cm (N4acetyl2TOmethylcytidine); k2C (lysidine); mlG (1- methylguanosine); m2G (N2- methylguanosine); m7G (7-methylguanosine); Gm (2'-0- methylguanosine); m22G (N2,N2- dimethylguanosine); m2Gm (N2,2'-0- dimethylguanosine); m22Gm (N2,N2,2'-0- trimethylguanosine); Gr(p) (2'-0- ribosylguanosine (phosphate)); yW (wybutosine); o2yW (peroxywybutosine); OHyW (hydroxywybutosine); OHyW* (undermodified hydroxywybutosine); imG (wyosine); mimG (methylguanosine); Q (queuosine); oQ (epoxyqueuosine); galQ (galtactosyl- queuosine); manQ (mannosyl-queuosine); preQo (7- cyano-7-deazaguanosine); preQi (7- aminomethyl-7-deazaguanosine); G* (archaeosine); D (dihydrouridine); m5Um (5,2'-0- dimethyluridine); s4U (4-thiouridine); m5s2U (5-methyl-2- thiouridine); s2Um (2-thio-2'- O-methyluridine); acp3U (3-(3-amino-3- carboxypropyl)uridine); ho5U (5- hydroxyuridine); mo5U (5 -methoxyuridine); cmo5U (uridine 5-oxyacetic acid); mcmo5U (uridine 5-oxyacetic acid methyl ester); chm5U (5- (carboxyhydroxymethyl)uridine)); mchm5U (5-(carboxyhydroxymethyl)uridine methyl ester); mcm5U (5 -methoxycarbonyl methyluridine); mcm5Um (S-methoxycarbonylmethyl-2-O- methyluridine); mcm5s2U (5- methoxycarbonylmethyl-2-thiouridine); nm5s2U (5- aminomethyl-2-thiouridine); mnm5U (5 -methylaminomethyluridine); mnm5s2U (5- methylaminomethyl-2 -thiouridine); mnm5se2U (5-methylaminomethyl-2-selenouridine); ncm5U (5 -carbamoylmethyl uridine); ncm5Um (5-carbamoylmethyl-2'-0-methyluridine); cmnm5U (5- carboxymethylaminomethyluridine); cnmm5Um (5-carboxymethy 1 aminomethyl-2-L- Omethyluridine); cmnm5s2U (5-carboxymethylaminomethyl-2- thiouridine); m62A (N6,N6-dimethyladenosine); Tm (2'-0-methylinosine); m4C (N4- methylcytidine); m4Cm (N4,2-0-dimethylcytidine); hm5C (5-hydroxymethylcytidine); m3U (3 -methyluridine); cm5U (5-carboxymethyluridine); m6Am (N6,T-0-dimethyladenosine); m62Am (N6,N6,0-2-trimethyladenosine); m2'7G (N2,7-dimethylguanosine); m2'2'7G (N2,N2,7- trimethylguanosine); m3Um (3,2T-0-dimethyluridine); m5D (5- methyldihydrouridine); f5Cm (5-formyl-2'-0-methylcytidine); mlGm (l,2'-0- dimethylguanosine); m'Am (1,2-0- dimethyl adenosine) irinomethyluridine); tm5s2U (S- taurinomethyl-2-thiouridine)); imG- 14 (4-demethyl guanosine); imG2 (isoguanosine); ac6A (N6-acetyladenosine), hypoxanthine, inosine, 8-oxo-adenine, 7-substituted derivatives thereof, dihydrouracil, pseudouracil, 2-thiouracil, 4-thiouracil, 5 -aminouracil, 5-(Ci-C6)-alkyluracil, 5- methyluracil, 5-(C2-Ce)-alkenyluracil, 5-(C2-Ce)-alkynyluracil, 5-(hydroxymethyl)uracil, 5- chlorouracil, 5 -fluorouracil, 5 -bromouracil, 5-hydroxycytosine, 5-(Ci-C6 )- alkylcytosine, 5- methylcytosine, 5-(C2-C6)-alkenylcytosine, 5-(C2-C6)-alkynylcytosine, 5- chlorocytosine, 5- fluorocytosine, 5 -bromocytosine, N2-dimethylguanine, 7-deazaguanine, 8-azaguanine, 7- deaza-7-substituted guanine, 7-deaza-7-(C2-C6)alkynylguanine, 7-deaza- 8-substituted guanine, 8-hydroxyguanine, 6-thioguanine, 8-oxoguanine, 2-aminopurine, 2-amino-6- chloropurine, 2,4-diaminopurine, 2,6-diaminopurine, 8-azapurine, substituted 7-deazapurine, 7-deaza-7-substituted purine, 7-deaza-8-substituted purine, hydrogen (abasic residue), m5C, m5U, m6A, s2U, W, or 2'-O-methyl-U. In some aspects, one or more of the modified nucleosides in the list may be excluded.
[0027] Additional exemplary modified nucleotides include any one of N-l- methylpseudouridine; pseudouridine, N6-methyladenosine, 5 -methylcytidine, and 5- methyluridine. In some embodiments, the modified nucleotide is N-l -methylpseudouridine.
[0028] In some embodiments, the RNA molecule may include phosphoramidate, phosphorothioate, and/or methylphosphonate linkages. [0029] In some embodiments, the RNA molecule includes a modified nucleotide selected from any one of pseudouridine, N1 -methylpseudouridine, N1 -ethylpseudouridine, 2- thiouridine, 4 '-thiouridine, 5-methylcytosine, 5 -methyluridine, 2-thio-l -methyl- 1 -deazapseudouridine, 2-thio-l-methyl-pseudouridine, 2-thio-5-aza-uridine, 2-thio- dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-pseudouridine, 4-methoxy-2-thio- pseudouridine, 4-methoxy-pseudouridine, 4-thio-l -methyl -pseudouridine, 4-thio- pseudouridine, 5 -aza-uridine, dihydropseudouridine, 5 -methoxyuridine, and 2'-O-methyl uridine.
2. UTRs
[0030] The 5' untranslated regions (UTR) is a regulatory region of DNA situated at the 5' end of a protein coding sequence that is transcribed into mRNA but not translated into protein. 5' UTRs may contain various regulatory elements, e.g., 5' cap structure, stem-loop structure, and an internal ribosome entry site (IRES), which may play a role in the control of translation initiation. The 3' UTR, situated downstream of a protein coding sequence, may be involved in regulatory processes including transcript cleavage, stability and polyadenylation, translation, and mRNA localization. In some embodiments, the UTR is derived from an mRNA that is naturally abundant in a specific tissue (e.g., lymphoid tissue), to which the mRNA expression is targeted. In some embodiments, the UTR increases protein synthesis. Without being bound by mechanism or theory, the UTR may increase protein synthesis by increasing the time that the mRNA remains in translating polysomes (message stability) and/or the rate at which ribosomes initiate translation on the message (message translation efficiency). According, the UTR sequence may prolong protein synthesis in a tissue-specific manner. In some embodiments, the 5' UTR and the 3' UTR sequences are computationally derived. In some embodiments, the 5' UTR and the 3' UTRs are derived from a naturally abundant mRNA in a tissue. The tissue may be, for example, liver, a stem cell, or lymphoid tissue. The lymphoid tissue may include, for example, any one of a lymphocyte (e.g., a B-lymphocyte, a helper T- lymphocyte, a cytotoxic T-lymphocyte, a regulatory T-lymphocyte, or a natural killer cell), a macrophage, a monocyte, a dendritic cell, a neutrophil, an eosinophil and a reticulocyte. In some embodiments, the 5' UTR and the 3' UTR are derived from an alphavirus. In some embodiments, the 5' UTR and the 3' UTR are from a wild-type alphavirus. Examples of alphaviruses are described below. [0031] In some embodiments, the RNA molecule includes a 5 ' UTR and the 3 ' UTR derived from a naturally abundant mRNA in a tissue. In some embodiments, the RNA molecule includes a 5' UTR and the 3' UTR derived from an alphavirus. In some embodiments, the RNA molecule includes a 5 ' UTR and the 3 ' UTR from a wild-type alphavirus . In some embodiments, the RNA molecule includes a 5' cap.
3. Open Reading Frame (ORF)
[0032] The 5' and 3' UTRs may be operably linked to an ORF, which may be a sequence of codons that is capable of being translated into a polypeptide of interest. As stated above, the RNA molecule may include one (monocistronic), two (bicistronic) or more (multicistronic) open reading frames (ORFs).
[0033] In some embodiments, the ORF encodes a modified antigenic polypeptide construct. In some embodiments, the ORF further includes one or more subgenomic promoters. In some embodiments, the RNA molecule includes a subgenomic promoter operably linked to the ORF. In some embodiments, the subgenomic promoter comprises a cisacting regulatory element. In some embodiments, the cis-acting regulatory element is an AU- rich element. In some embodiments, the AU-rich element is au, auaaaagau, auaaaaagau, auag, auauauauau, auauauau, auauauauauau, augaugaugau, augau, auaaaagaua, or auaaaagaug.
[0034] In some embodiments, a RNA molecule may include (i) an ORF encoding a replicase which may transcribe RNA from the RNA molecule and (ii) an ORF encoding at least one modified antigenic polypeptide construct. The polymerase may be an alphavirus replicase, e.g., including any one of the non-structural alphavirus proteins nsPl, nsP2, nsP3 and nsP4, or a combination thereof. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsPl. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP2. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural protein nsP4. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsPl, nsP2, and nsP3. In some embodiments, the RNA molecule includes alphavirus nonstructural proteins nsPl, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule includes any combination of nsPl, nsP2, nsP3, and nsP4. In some embodiments, the RNA molecule does not include nsP4.
[0035] In some embodiments, the RNA molecule does not encode alphavirus structural proteins. In some embodiments, the RNA molecule may have one or more additional (e.g., downstream) open reading frames, e.g., to encode further antigen(s) or to encode accessory polypeptides.
[0036] In some embodiments, a first RNA molecule does not include an ORF encoding a modified antigenic polypeptide or protein, whereas a second RNA or a saRNA molecule includes an ORF encoding the modified antiogenic polypeptide or protein. In some embodiments, the first RNA molecule does not include a subgenomic promoter.
[0037] In some embodiments, the first RNA molecule includes an ORF for a nonstructural protein derived from an alphavirus. In some embodiments, the ORF encoding a nonstructural protein in the first RNA molecule and in the second RNA or the saRNA molecule are identical. In some embodiments, the second RNA or the saRNA molecule further includes an ORF encoding a modified antiogenic polypeptide or protein.
[0038] In some embodiments, the second RNA or the saRNA molecule may lead to a production of genomic RNA copies of itself in a cell, but not to the production of RNA- containing virions. In some embodiments, the second RNA or the saRNA molecule cannot perpetuate itself in infectious form. For example, the genes encoding alphavirus structural proteins, which are necessary for perpetuation in wild-type alphaviruses, are absent from the second RNA or saRNA molecules of the present disclosure, and their place is taken by a gene(s) encoding the polypeptide of interest, such that the subgenomic transcript encodes the modified antiogenic polypeptide or protein, rather than the structural alphavirus virion proteins.
4. Genes of Interest
[0039] The RNA molecule described herein may include a gene of interest. The gene of interest can encode a polypeptide of interest selected from, e.g., biologies, antibodies, vaccines, therapeutic polypeptides or peptides, cell penetrating peptides, secreted polypeptides, plasma membrane polypeptides, cytoplasmic or cytoskeletal polypeptides, intracellular membrane bound polypeptides, nuclear polypeptides, polypeptides associated with human disease, targeting moieties or those polypeptides encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery. In some instances, the sequence for a particular gene of interest is readily identified by one of skill in the art using public and private databases, e.g. , GenBank. In some embodiments, the RNA molecule includes a coding region for an antigen derived from a pathogen associated with infectious disease or cancer, as described elsewhere herein.
5. 5' Cap [0040] In some embodiments, the RNA molecule described herein includes a 5' cap. In some embodiments, the 5 '-cap moiety is a natural 5 '-cap. A “natural 5 '-cap” is defined as a cap that includes 7-methylguanosine connected to the 5' end of an mRNA molecule through a 5' to 5' triphosphate linkage. In some embodiments, the 5 '-cap moiety is a 5'- cap analog. In some embodiments, the 5' end of the RNA is capped with a modified ribonucleotide, which may be incorporated during RNA synthesis (e.g., co-transcriptional capping) or may be enzymatically engineered after RNA transcription (e.g., post-transcriptional capping). In some embodiments, the 5' end of the RNA molecule is capped with a modified ribonucleotide via an enzymatic reaction after RNA transcription. In some embodiments, capping is performed after purification, e.g., tangential flow filtration, of the RNA molecule. An exemplary enzymatic reaction for capping may include use of Vaccinia Virus Capping Enzyme (VCE) In some embodiments, the 5' terminal cap includes a cap analog, for example, a 5' terminal cap may include a guanine analog. Exemplary guanine analogs include, but are not limited to, inosine, N1 -methyl -guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino- guanosine, LNA-guanosine, and 2-azido-guanosine. In some embodiments, the capping region may include a single cap or a series of nucleotides forming the cap. In some embodiments the capping region may be equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.
[0041] In some embodiments, a first and/or second operational region of the RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 to 40, e.g. , 5-30, 10-20, 15, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
6. Poly-A Tail
[0042] As used herein, “poly A tail” refers to a stretch of consecutive adenine residues, which may be attached to the 3' end of the RNA molecule. The poly-A tail may increase the half-life of the RNA molecule. Poly-A tails may play key regulatory roles in enhancing translation efficiency and regulating the efficiency of mRNA quality control and degradation. Short sequences or hyperpolyadenylation may signal for RNA degradation. Exemplary designs include a poly-A tails of about 40 adenine residues to about 80 adenine residues. In some embodiments, the RNA molecule further includes an endonuclease recognition site sequence immediately downstream of the poly A tail sequence. In some embodiments, such as for the second RNA or the saRNA molecule, the RNA molecule further includes a poly-A polymerase recognition sequence (e.g., AAUAAA) near its 3' end. A “full length” RNA molecule is one that includes a 5 '-cap and a poly A tail.
[0043] In some embodiments, the poly A tail includes 5-400 nucleotides in length. The poly A tail nucleotide length may be equal to any one of, at least any one of, at most any one of, or between any two of 5, 6, 7, 8, 9, 10, 15, 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, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, and 400. In some embodiments, the RNA molecule includes a poly A tail that includes about 25 to about 400 adenosine nucleotides, a sequence of about 50 to about 400 adenosine nucleotides, a sequence of about 50 to about 300 adenosine nucleotides, a sequence of about 50 to about 250 adenosine nucleotides, a sequence of about 60 to about 250 adenosine nucleotides, or a sequence of about 40 to about 100 adenosine nucleotides. In some embodiments, the RNA molecule includes a poly A tail includes a sequence of greater than 30 adenosine nucleotides (“As”). In some embodiments, the RNA molecule includes a poly A tail that includes about 40 As. In some embodiments, the RNA molecule includes a poly A tail that includes about 80 As. As used herein, regarding poly A tail length, the term “about” refers to a deviation of ±10% of the value(s) to which it is attached. In some embodiments, the 3' poly-A tail has a stretch of at least 10 consecutive adenosine residues and at most 300 consecutive adenosine residues. In some embodiments, the RNA molecule includes at least 20 consecutive adenosine residues and at most 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 40 consecutive adenosine residues. In some embodiments, the RNA molecule includes about 80 consecutive adenosine residues.
[0044] In some embodiments, the RNA molecule is purified, e.g., such as by filtration that may occur via, e.g., ultrafiltration, diafiltration, or, e.g., tangential flow ultrafiltration/ diafiltration .
7. Self-Amplifying RNA (saRNA)
[0045] In some embodiments, the RNA molecule is a saRNA. “saRNA,” “self-amplifying RNA,” and “replicon” refer to RNA with the ability to replicate itself. Self-amplifying RNA molecules may be produced by using replication elements derived from a virus or viruses, e.g. , alphaviruses, and substituting the structural viral polypeptides with a nucleotide sequence encoding a polypeptide of interest. A self-amplifying RNA molecule is typically a positivestrand molecule that may be directly translated after delivery to a cell, and this translation provides an RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. The delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded gene of interest, e.g., a viral antigen, or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the protein of interest, e.g. , an antigen. The overall result of this sequence of transcriptions is an amplification in the number of the introduced saRNAs and so the encoded gene of interest, e.g. , a viral antigen, can become a major polypeptide product of the cells.
[0046] In some embodiments, the self-amplifying RNA includes at least one or more genes selected from any one of viral replicases, viral proteases, viral helicases, and other nonstructural viral proteins. In some embodiments, the self-amplifying RNA may also include 5'- and 3 '-end tractive replication sequences, and optionally a heterologous sequence that encodes a desired amino acid sequence (e.g., an antigen of interest). A subgenomic promoter that directs expression of the heterologous sequence may be included in the self-amplifying RNA. Optionally, the heterologous sequence (e.g. , an antigen of interest) may be fused in frame to other coding regions in the self-amplifying RNA and/or may be under the control of an internal ribosome entry site (IRES).
[0047] In some embodiments, the self-amplifying RNA molecule is not encapsulated in a virus-like particle. Self-amplifying RNA molecules described herein may be designed so that the self-amplifying RNA molecule cannot induce production of infectious viral particles. This may be achieved, for example, by omitting one or more viral genes encoding structural proteins that are necessary to produce viral particles in the self-amplifying RNA. For example, when the self-amplifying RNA molecule is based on an alphavirus, such as Sinbis virus (SIN), Semliki forest virus, and Venezuelan equine encephalitis virus (VEE), one or more genes encoding viral structural proteins, such as capsid and/or envelope glycoproteins, may be omitted.
[0048] In some embodiments, a self-amplifying RNA molecule described herein encodes (i) an RNA- dependent RNA polymerase that may transcribe RNA from the self-amplifying RNA molecule and (ii) a polypeptide of interest, e.g., a viral antigen. In some embodiments, the polymerase may be an alphavirus replicase, e.g., including any one of alphavirus protein nsPl, nsP2, nsP3, nsP4, and any combination thereof. In some embodiments, the selfamplifying RNA molecules described herein may include one or more modified nucleotides (e.g., pseudouridine, N6-methyladenosine, 5- methylcytidine, 5 -methyluridine). In some embodiments, the self-amplifying RNA molecules does not include a modified nucleotide (e.g. , pseudouridine, N6-methyladenosine, 5- methylcytidine, 5 -methyluridine).
[0049] The saRNA construct may encode at least one non-structural protein (NSP), disposed 5' or 3' of the sequence encoding at least one peptide or polypeptide of interest. In some embodiments, the sequence encoding at least one NSP is disposed 5' of the sequences encoding the peptide or polypeptide of interest. Thus, the sequence encoding at least one NSP may be disposed at the 5' end of the RNA construct. In some embodiments, at least one non- structural protein encoded by the RNA construct may be the RNA polymerase nsP4. In some embodiments, the saRNA construct encodes nsPl, nsP2, nsP3 and, nsP4. As is known in the art, nsPl is the viral capping enzyme and membrane anchor of the replication complex (RC). nsP2 is an RNA helicase and the protease responsible for the ns polyprotein processing. nsP3 interacts with several host proteins and may modulate protein poly- and mono-ADP- ribosylation. nsP4 is the core viral RNA-dependent RNA polymerase. In some embodiments, the polymerase may be an alphavirus replicase, e.g., comprising one or more of alphavirus proteins nsPl, nsP2, nsP3, and nsP4.
[0050] Whereas natural alphavirus genomes encode structural virion proteins in addition to the non- structural replicase polypeptide, in some embodiments, the self-amplifying RNA molecules do not encode alphavirus structural proteins. In some embodiments, the selfamplifying RNA may lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA that includes virions. Without being bound by theory or mechanism, the inability to produce these virions means that, unlike a wild-type alphavirus, the selfamplifying RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild-type viruses can be absent from selfamplifying RNAs of the present disclosure and their place can be taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins.
[0051] In some embodiments, the self-amplifying RNA molecule may have two open reading frames. The first (5') open reading frame can encode a replicase; the second (3') open reading frame can encode a polypeptide comprising an antigen of interest. In some embodiments the RNA may have additional (e.g., downstream) open reading frames, e.g., to encode further antigens or to encode accessory polypeptides. [0052] In some embodiments, the saRNA molecule further includes (1) an alphavirus 5' replication recognition sequence, and (2) an alphavirus 3' replication recognition sequence. In some embodiments, the 5' sequence of the self-amplifying RNA molecule is selected to ensure compatibility with the encoded replicase.
[0053] Optionally, self-amplifying RNA molecules described herein may also be designed to induce production of infectious viral particles that are attenuated or virulent, or to produce viral particles that are capable of a single round of subsequent infection.
[0054] In some embodiments, the saRNA molecule is alphavirus-based. Alphaviruses include a set of genetically, structurally, and serologically related arthropod-borne viruses of the Togaviridae family. Exemplary viruses and virus subtypes within the alphavirus genus include Sindbis virus (SINV), Semliki Forest virus (SFV), Ross River virus (RRV), and Venezuelan equine encephalitis virus (VEEV). As such, the self-amplifying RNA described herein may incorporate an RNA replicase derived from any one of SFV, SINV, VEEV, RRV, or other viruses belonging to the alphavirus family. In some embodiments, the self-amplifying RNA described herein may incorporate sequences derived from a mutant or wild-type virus sequence, e.g., the attenuated TC83 mutant of VEEV has been used in saRNAs.
[0055] Alphavirus-based saRNAs are (+)-stranded saRNAs that may be translated after delivery to a cell, which leads to translation of a replicase (or replicase- transcriptase). The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic (-)-strand copies of the (+)-strand delivered RNA. These (-)-strand transcripts may themselves be transcribed to give further copies of the (+)-stranded parent RNA and also to give a subgenomic transcript which encodes the desired gene product. Translation of the subgenomic transcript thus leads to in situ expression of the desired gene product by the infected cell. Suitable alphavirus saRNAs may use a replicase from a SINV, a SFV, an eastern equine encephalitis virus, a VEEV, or mutant variants thereof.
[0056] In some embodiments, the self-amplifying RNA molecule is derived from or based on a virus other than an alphavirus, such as a positive-stranded RNA virus, and in particular a picomavirus, flavivirus, rubivirus, pestivirus, hepacivirus, calicivirus, or coronavirus. Suitable wild-type alphavirus sequences are well-known and are available from sequence depositories, such as the American Type Culture Collection, Rockville, Md. Representative examples of suitable alphaviruses include Aura (ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou (ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Eastern equine encephalomyelitis virus (ATCC VR-65, ATCC VR-1242), Fort Morgan (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagach (ATCC VR-927), Mayaro (ATCC VR- 66), Mayaro virus (ATCC VR-1277), Middleburg (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumu (ATCC VR-371), Pixuna virus (ATCC VR- 372, ATCC VR-1245), Ross River virus (ATCC VR-373, ATCC VR-1246), Semliki Forest (ATCC VR-67, ATCC VR-1247), Sindbis virus (ATCC VR-68, ATCC VR- 1248), Tonate (ATCC VR-925), Triniti (ATCC VR-469), Una (ATCC VR-374), Venezuelan equine encephalomyelitis (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCC VR- 1249, ATCC VR-532), Western equine encephalomyelitis (ATCC VR-70, ATCC VR- 1251, ATCC VR-622, ATCC VR-1252), Whataroa (ATCC VR-926), and Y-62-33 (ATCC VR-375). In some aspects, one or more of the alphaviruses in the list may be excluded.
[0057] In some embodiments, the self-amplifying RNA molecules described herein are larger than other types of RNA (e.g., mRNA). Typically, the self-amplifying RNA molecules described herein include at least about 4 kb. For example, the self-amplifying RNA may be equal to any one of, at least any one of, at most any one of, or between any two of 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb, 10 kb, 11 kb, 12 kb, 13 kb, 14 kb, 15 kb, 16 kb. In some instances the self-amplifying RNA may include at least about 5 kb, at least about 6 kb, at least about 7 kb, at least about 8 kb, at least about 9 kb, at least about 10 kb, at least about 11 kb, at least about 12 kb, or more than 12 kb. In certain examples, the self-amplifying RNA is about 4 kb to about 12 kb, about 5 kb to about 12 kb, about 6 kb to about 12 kb, about 7 kb to about 12 kb, about 8 kb to about 12 kb, about 9 kb to about 12 kb, about 10 kb to about 12 kb, about 11 kb to about 12 kb, about 5 kb to about 11 kb, about 5 kb to about 10 kb, about 5 kb to about 9 kb, about 5 kb to about 8 kb, about 5 kb to about 7 kb, about 5 kb to about 6 kb, about 6 kb to about 12 kb, about 6 kb to about 11 kb, about 6 kb to about 10 kb, about 6 kb to about 9 kb, about 6 kb to about 8 kb, about 6 kb to about 7 kb, about 7 kb to about 11 kb, about 7 kb to about 10 kb, about 7 kb to about 9 kb, about 7 kb to about 8 kb, about 8 kb to about 11 kb, about 8 kb to about 10 kb, about 8 kb to about 9 kb, about 9 kb to about 11 kb, about 9 kb to about 10 kb, or about 10 kb to about 11 kb.
[0058] In some embodiments, the self-amplifying RNA molecule may encode a single polypeptide antigen or, optionally, two or more of polypeptide antigens linked together in a way that each of the sequences retains its identity (e.g. , linked in series) when expressed as an amino acid sequence. The polypeptides generated from the self-amplifying RNA may then be produced as a fusion polypeptide or engineered in such a manner to result in separate polypeptide or peptide sequences.
[0059] In some embodiments, the self-amplifying RNA described herein may encode one or more polypeptide antigens that include a range of epitopes. In some embodiments, the self- amplifying RNA described herein may encode epitopes capable of eliciting either a helper T- cell response or a cytotoxic T-cell response or both.
IV. Polypeptides
[0060] The antigenic polypeptide constructs can comprise one or more antigenic peptide sequences that elicit an immune response. In certain embodiments, pathogenic antigenic polypeptide constructs are peptide or protein antigens derived from a pathogen associated with infectious disease, including viruses, bacteria, parasites, and fungi. In certain embodiments, the antigenic polypeptide constructs are peptide or protein antigens derived from cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
[0061] In certain embodiments, peptides or proteins can exist in a variety of instances such as: fragments or functional derivatives, recombinant polypeptides, such as antigens or one or both chains of an antibody, or a fragment, derivative, mutein, or variant thereof, peptides or proteins sufficient for use as hybridization probes, peptides or proteins for inhibiting expression of a polynucleotide, and complementary amino acid sequences of the foregoing described herein. Peptides or proteins may be an epitope to which antibodies may bind. The peptides or proteins can comprise RNA and/or DNA nucleotides (e.g., peptide nucleic acids).
[0062] In certain embodiments the size of a protein or peptide or derivative of a corresponding amino sequence described or referenced herein can be, for example, equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 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, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500, 3000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, 15000 amino acid residues or greater. It is contemplated that proteins or peptides may be mutated by truncation, rendering them shorter than their corresponding wild-type form, also, they might be altered by fusing or conjugating a heterologous protein or peptide sequence with a particular function (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term “domain” refers to any distinct functional or structural unit of a protein or peptide, and generally refers to a sequence of amino acids with a structure or function recognizable by one skilled in the art.
[0063] In some embodiments, the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof elicit an immune response. The immune response may be against the antigenic polypeptide constructs and/or antigenic peptide sequences and/or a homologous endogenously-expressed (e.g. cross-reactive) host protein. The antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed (e.g. cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% similar, identical, or homologous. [0064] In some embodiments, the one or more antigenic polypeptide constructs and/or antigenic peptide sequences thereof may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,
95, 96, 97, 98, 99, 100, 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 contiguous amino acids that are equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%,
52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%,
68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,
84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% (or any derivable range therein) similar, identical, or homologous with a homologous endogenously-expressed (e.g. cross-reactive) host protein.
[0065] In some embodiments, the one or more antigenic polypeptide constructs and/or antigenic peptide sequence thereof are modified such that the antigenic polypeptide constructs and/or antigenic peptide sequences thereof continue to elicit an immune response but homology with a homologous endogenously-expressed (e.g., cross-reactive) host protein is reduced to reduce or prevent cross-reactivity after immunization with the antigenic polypeptide constructs and/or antigenic peptide sequences thereof. The homology between the antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously-expressed (e.g., cross-reactive) host protein may be reduced by modifying the antigenic polypeptide constructs and/or antigenic peptide sequences thereof such that the modified antigenic polypeptide constructs and/or antigenic peptide sequences and the homologous endogenously- expressed (e.g., cross-reactive) host protein may be equal to any one of, at least any one of, at most any one of, or between any two of 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,
27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%,
43%, 44%. 45%. 46%. 47%, 48%. 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%,
59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%,
75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,
91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% similar, identical, or homologous.
[0066] The modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more variant amino acids (e.g. , amino acid substitutions, insertions, and/or deletions) compared to unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences thereof. The modified antigenic polypeptide constructs and/or modified antigenic peptide sequences thereof may equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%,
32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%,
48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,
64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,
80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,
96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% (or any derivable range therein) similar, identical, or homologous with unmodified antigenic polypeptide constructs and/or unmodified antigenic peptide sequences thereof.
[0067] Nucleotide as well as protein, polypeptide, and peptide sequences for various genes have been previously disclosed, and may be found in the recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information’s Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov) and The Universal Protein Resource (UniProt; on the World Wide Web at uniprot.org). The coding regions for these genes may be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
V. Antigenic Peptide Sequence Modifications
[0068] The antigenic polypeptide constructs and/or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof of the present disclosure may be modified, such that they are substantially identical to the antigenic polypeptide constructs or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof comprised in immunogenic compositions described herein. In some embodiments, the modified antigenic polypeptide constructs and/or antigenic peptide sequences thereof and/or nucleic acids encoding the antigenic polypeptide constructs or antigenic peptide sequences thereof continue to be bound by antibodies to elicit an immune response but produce attenuated immune cross-reactivity after immunization. In particular embodiments, it is the antigenic peptide sequences and/or nucleic acids encoding the antigenic peptide sequences comprised in the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs that are modified.
[0069] Polypeptide and/or nucleic acid sequences are “substantially identical” when optimally aligned using such programs as Clustal Omega, IGBLAST, GAP or BESTFIT using default gap weights, they share at least 80% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, or at least 99% sequence identity or any range therein.
[0070] The antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the disclosure may include equal to any one of, at least any one of, at most any one of, or between any two of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more variant amino acids (e.g., amino acid substitutions, insertions, and/or deletions) or be equal to any one of, at least any one of, at most any one of, or between any two of 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%,
62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,
78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,
94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% similar, identical, or homologous with equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45,
46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70,
71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95,
96, 97, 98, 99, 100, 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 contiguous amino acids of unmodified antigenic polypeptide constructs and/or nucleic acids encoding the unmodified antigenic polypeptide constructs of the present disclosure and/or of homologous peptides or proteins.
[0071] In some embodiments, the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs may comprise equal to any one of, at least any one of, at most any one of, or between any two of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,
42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66,
67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 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 contiguous amino acids that are equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%,
35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%,
51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,
67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,
83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99%, or 100% (or any derivable range therein) similar, identical, or homologous with unmodified antigenic polypeptide constructs and/or nucleic acids encoding the unmodified antigenic polypeptide constructs of the present disclosure and/or of homologous peptides or proteins.
[0072] As modifications and/or changes may be made in the sequence and/or structure of polynucleotides and/or proteins according to the present disclosure, while obtaining molecules having similar or improved characteristics (e.g., maintenance of antibody binding and immune response and attenuation of cross-reactivity with endogenously-expressed host proteins), such biologically functional equivalents of the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs are also encompassed within the present invention.
[0073] The modified antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs may comprise a polynucleotide that has been engineered to contain distinct sequences while at the same time retaining the capacity to encode the “wildtype” or standard protein or peptide or “modified” or “variant” protein or peptide. This can be accomplished to the degeneracy of the genetic code, i.e., the presence of multiple codons, which encode for the same amino acids. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six different codons for arginine. Also considered are “neutral substitutions” or “neutral mutations” which refers to a change in the codon or codons that encode biologically equivalent amino acids. In one example, one of skill in the art may wish to introduce a mutation into a polynucleotide to reduce cross-reactivity of the protein encoded by the polynucleotide with endogenously-expressed host proteins while not disturbing the ability of that polynucleotide to encode a protein that is bound by an antibody and that elicits an immune response.
[0074] In terms of functional equivalents, it is well understood by the skilled artisan that, inherent in the definition of a “biologically functional equivalent” protein and/or polynucleotide, is the concept that there is a limit to the number of changes that may be made within a defined portion of the molecule while retaining a molecule with an acceptable level of equivalent biological activity. Biologically functional equivalents are thus defined herein as those proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that retain the ability to be bound by an antibody and elicit an immune response and/or proteins (and polynucleotides) having substitutions or mutations in selected amino acids (or codons) that have reduced homology with endogenously-expressed host proteins, such that cross-reactivity of the proteins is diminished, for example.
[0075] In one example, a polynucleotide may be (and encode) a biological functional equivalent with significant changes. Certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies, binding sites on substrate molecules, receptors, and such like.
[0076] Substitutional variants typically contain the exchange of one amino acid for another at one or more sites within the protein, and may be designed to modulate one or more properties of the polypeptide, with or without the loss of other functions or properties. Substitutions may be conservative, that is, one amino acid is replaced with one of similar shape and charge. Conservative substitutions are well known in the art and include, for example, the changes of: alanine to serine; arginine to lysine; asparagine to glutamine or histidine; aspartate to glutamate; cysteine to serine; glutamine to asparagine; glutamate to aspartate; glycine to proline; histidine to asparagine or glutamine; isoleucine to leucine or valine; leucine to valine or isoleucine; lysine to arginine; methionine to leucine or isoleucine; phenylalanine to tyrosine, leucine or methionine; serine to threonine; threonine to serine; tryptophan to tyrosine; tyrosine to tryptophan or phenylalanine; and valine to isoleucine or leucine. So-called “conservative” changes do not disrupt the biological activity of the protein, as the structural change is not one that impinges of the protein’s ability to carry out its designed function. It is thus contemplated by the inventors that various changes may be made in the sequence of genes and proteins disclosed herein, while still fulfilling the goals of the present invention. Alternatively, substitutions may be non-conservative such that a function or activity of the polypeptide is affected. Non-conservative changes typically involve substituting a residue with one that is chemically dissimilar, such as a polar or charged amino acid for a nonpolar or uncharged amino acid, and vice versa. Non-conservative substitutions may involve the exchange of a member of one of the amino acid classes for a member from another class.
[0077] In other embodiments, alteration of the function of a polypeptide is intended by introducing one or more substitutions. For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity. Structures such as, for example, an enzymatic catalytic domain or interaction components may have amino acid substituted to maintain such function. Since it is the interactive capacity and nature of a protein that defines that protein’s biological functional activity, certain amino acid substitutions can be made in a protein sequence, and in its underlying DNA coding sequence, and nevertheless produce a protein with like properties. It is thus contemplated by the inventors that various changes may be made in the DNA sequences of genes without appreciable loss of their biological utility or activity.
[0078] Deletion variants typically lack one or more residues of the native or wild-type protein. Individual residues can be deleted or a number of contiguous amino acids can be deleted. A stop codon may be introduced (by substitution or insertion) into an encoding nucleic acid sequence to generate a truncated protein. For example, it is contemplated that peptides may be mutated by truncation, or deletion of a number of contiguous amino acids, rendering them shorter than their corresponding endogenous form.
[0079] Insertional mutants typically involve the addition of amino acid residues at a nonterminal point in the polypeptide. This may include the insertion of one or more amino acid residues. Terminal additions may also be generated and can include fusion proteins which are multimers or concatemers of one or more peptides or polypeptides described or referenced herein. For example, it is contemplated that peptides might be altered by fusing or conjugating a heterologous protein or polypeptide sequence with a particular function (e.g. , for targeting or localization, for enhanced activity, for purification purposes, etc.).
[0080] Additionally, the polypeptides of the disclosure may be chemically modified. Glycosylation of the polypeptides can be altered, for example, by modifying one or more sites of glycosylation within the polypeptide sequence to increase the affinity of the polypeptide for antigen (U.S. Pat. Nos. 5,714,350 and 6,350,861).
[0081] It also will be understood that amino acid and nucleic acid sequences may include additional residues, such as additional N- or C-terminal amino acids, or 5' or 3' sequences, respectively, and yet still be essentially as set forth in one of the sequences disclosed herein, so long as the sequence meets the criteria set forth above, including the maintenance of biological protein activity where protein expression is concerned. The addition of terminal sequences particularly applies to nucleic acid sequences that may, for example, include various noncoding sequences flanking either of the 5' or 3' portions of the coding region.
[0082] Modified antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the disclosure can be substitutional, insertional, or deletion variants, for example.
[0083] Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and/or the like. An analysis of the size, shape and/or type of the amino acid side-chain substituents reveals that arginine, lysine and/or histidine are all positively charged residues; that alanine, glycine and/or serine are all a similar size; and/or that phenylalanine, tryptophan and/or tyrosine all have a generally similar shape. Therefore, based upon these considerations, arginine, lysine and/or histidine; alanine, glycine and/or serine; and/or phenylalanine, tryptophan and/or tyrosine; are defined herein as biologically functional equivalents.
[0084] In making such changes to produce biologically functional equivalents, the hydropathic index of amino acids may be considered. The hydropathy profile of a protein is calculated by assigning each amino acid a numerical value (“hydropathy index”) and then repetitively averaging these values along the peptide chain. Each amino acid has been assigned a value based on its hydrophobicity and charge characteristics. They are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cysteine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn defines the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like. It is also known that certain amino acids may be substituted for other amino acids having a similar hydropathy index or score, and still retain a similar biological activity. In making changes based upon the hydropathy index, in certain embodiments, the substitution of amino acids whose hydropathy indices are within ±2 is included. In some aspects of the disclosure, those that are within ±1 are included, and in other aspects of the disclosure, those within ±0.5 are included.
[0085] It also is understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. U.S. Patent No. 4,554,101, specifically incorporated by reference herein in its entirety, states that the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. In certain embodiments, the greatest local average hydrophilicity of a protein, as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0+1); glutamate (+3.0+1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5+1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4). In making changes based upon similar hydrophilicity values, in certain embodiments, the substitution of amino acids whose hydrophilicity values are within ±2 are included, in other embodiments, those which are within ±1 are included, and in still other embodiments, those within ±0.5 are included. In some instances, one may also identify epitopes from primary amino acid sequences based on hydrophilicity. It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still produce a biologically equivalent and immunologically equivalent protein.
[0086] Additionally, one skilled in the art can review structure-function studies identifying residues in similar polypeptides or proteins that are important for activity or structure. In view of such a comparison, one can predict the importance of amino acid residues in a protein that correspond to amino acid residues important for activity or structure in similar proteins. One skilled in the art may opt for chemically similar amino acid substitutions for such predicted important amino acid residues.
[0087] One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar proteins or polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues with respect to its three-dimensional structure. One skilled in the art may choose not to make changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each desired amino acid residue. These variants can then be screened using standard assays for binding and/or activity, thus yielding information gathered from such routine experiments, which may allow one skilled in the art to determine the amino acid positions where further substitutions should be avoided either alone or in combination with other mutations. Various tools available to determine secondary structure can be found on the World Wide Web at expasy.org/proteomics/protein_structure.
[0088] In some embodiments of the disclosure, single or multiple amino acid substitutions (in certain embodiments, conservative amino acid substitutions) may be made in the naturally occurring sequence. Substitutions can be made in that portion of the protein that lies outside the domain(s) forming intermolecular contacts. In such embodiments, conservative amino acid substitutions can be used that do not substantially change the structural characteristics of the protein or polypeptide (e.g., one or more replacement amino acids that do not disrupt the primary, secondary, or tertiary structure that characterizes the native protein).
[0089] As outlined above, amino acid substitutions generally are based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions that take into consideration the various foregoing characteristics are well known and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.
A. Altered Amino Acids
[0090] As used herein, an “amino molecule” refers to any amino acid, amino acid derivative, or amino acid mimic as would be known to one of ordinary skill in the art. In certain embodiments, the residues of the peptide or protein are sequential, without any non-amino molecule interrupting the sequence of amino molecule residues. In other embodiments, the sequence may comprise one or more non-amino molecule moieties. In particular embodiments, the sequence of residues of the proteinaceous molecule may be interrupted by one or more non- amino molecule moieties. Peptides and proteins include the twenty “natural” amino acids, and post-translational modifications thereof. However, in vitro peptide synthesis permits the use of modified and/or unusual amino acids.
[0091] Accordingly, the term “protein,” “peptide,” or “polypeptide” encompasses amino molecule sequences comprising at least one of the 20 common amino acids in naturally synthesized proteins, or at least one modified or unusual amino acid, including but not limited to those shown in the Table below.
B. Mimetics
[0092] In addition to the modifications discussed above, structurally similar compounds may be formulated to mimic the key portions of peptide or polypeptides of the present disclosure. Such compounds, which may be termed peptidomimetics, may be used in the same manner as the peptides of the disclosure.
[0093] Certain mimetics that mimic elements of protein secondary and tertiary structure are described in Johnson et al. (1993). The underlying rationale behind the use of peptide mimetics is that the peptide backbone of proteins exists chiefly to orient amino acid side chains in such a way as to facilitate molecular interactions, such as those of antibody and/or antigen. A peptide mimetic is thus designed to permit molecular interactions similar to the natural molecule.
[0094] Some successful applications of the peptide mimetic concept have focused on mimetics of [3-tums within proteins, which are known to be highly antigenic. Likely [3-tum structure within a polypeptide can be predicted by computer-based algorithms, as discussed herein. Once the component amino acids of the turn are determined, mimetics can be constructed to achieve a similar spatial orientation of the essential elements of the amino acid side chains.
[0095] Other approaches have focused on the use of small, multidisulfide-containing proteins as attractive structural templates for producing biologically active conformations that mimic the binding sites of large proteins. Vita e/ al. (1998). A structural motif that appears to be evolutionarily conserved in certain toxins is small (30-40 amino acids), stable, and highly permissive for mutation. This motif is composed of a beta sheet and an alpha helix bridged in the interior core by three disulfides.
[0096] Beta II turns have been mimicked successfully using cyclic L-pentapeptides and those with D-amino acids. Weisshoff et al. (1999). Also, Johannesson et al. (1999) report on bicyclic tripeptides with reverse turn inducing properties.
[0097] Methods for generating specific structures have been disclosed in the art. For example, alpha-helix mimetics are disclosed in U.S. Patents 5,446,128; 5,710,245; 5,840,833; and 5,859,184. These structures render the peptide or protein more thermally stable, also increase resistance to proteolytic degradation. Six, seven, eleven, twelve, thirteen and fourteen membered ring structures are disclosed.
[0098] Methods for generating conformationally restricted beta turns and beta bulges are described, for example, in U.S. Patents 5,440,013; 5,618,914; and 5,670,155. Beta-turns permit changed side substituents without having changes in corresponding backbone conformation, and have appropriate termini for incorporation into peptides by standard synthesis procedures. Other types of mimetic turns include reverse and gamma turns. Reverse turn mimetics are disclosed in U.S. Patents 5,475,085 and 5,929,237, and gamma turn mimetics are described in U.S. Patents 5,672,681 and 5,674,976.
VI. Obtaining Encoded Polypeptide Embodiments
[0099] In some aspects, there are nucleic acid molecules encoding peptides of interest, e.g. , antigens. These nucleic acids may be generated by methods known in the art.
A. Expression
[0100] The nucleic acid molecules may be used to express large quantities of the polypeptide of interest, such as an antigen.
1. RNA or DNA molecule synthesis
[0101] In some aspects, contemplated are isolated RNA or DNA molecules comprising a nucleic acid molecule encoding a polypeptide of the desired sequence or a portion thereof (e.g. , a fragment containing one or more polypeptides, or antigens). In some aspects, RNA or DNA molecules comprising nucleic acid molecules may encode antigens, fusion proteins, modified antibodies, antibody fragments, and probes thereof. In addition to control sequences that govern transcription and translation, the RNA or DNA molecules may contain nucleic acid sequences that serve other functions as well.
[0102] As used herein, “isolated” means altered or removed from the natural state through human intervention. For example, a DNA or RNA naturally present in a living animal is not “isolated,” but a synthetic DNA or RNA, or a DNA or RNA partially or completely separated from the coexisting materials of its natural state is “isolated.” An isolated DNA or RNA can exist in substantially purified form, or can exist in a non-native environment such as, for example, a cell into which the DNA or RNA has been delivered.
[0103] In some embodiments, the DNA or RNA molecule is an analog and may include modifications, particularly modifications that increase nuclease resistance, improve binding affinity, and/or improve binding specificity. For example, when the sugar portion of a nucleoside or nucleotide is replaced by a carbocyclic moiety, it is no longer a sugar. Moreover, when other substitutions, such a substitution for the inter-sugar phosphodiester linkage are made, the resulting material is no longer a true species. All such compounds are considered to be analogs. Throughout this specification, reference to the sugar portion of a nucleic acid species shall be understood to refer to either a true sugar or to a species taking the structural place of the sugar of wild type nucleic acids. Moreover, reference to inter-sugar linkages shall be taken to include moieties serving to join the sugar or sugar analog portions in the fashion of wild type nucleic acids.
[0104] Modified oligonucleotides and oligonucleotide analogs may exhibit increased chemical and/or enzymatic stability relative to their naturally occurring counterparts. Extracellular and intracellular nucleases generally do not recognize and therefore do not bind to the backbone-modified compounds. When present as the protonated acid form, the lack of a negatively charged backbone may facilitate cellular penetration.
[0105] The modified intemucleoside linkages are intended to replace naturally-occurring phosphodiester-5'-methylene linkages with four atom linking groups to confer nuclease resistance and enhanced cellular uptake to the resulting compound.
[0106] Modifications may be achieved using solid supports which may be manually manipulated or used in conjunction with a DNA or RNA synthesizer using methodology commonly known to those skilled in DNA or RNA synthesizer art. Generally, the procedure involves functionalizing the sugar moieties of two nucleosides which will be adjacent to one another in the selected sequence. In a 5' to 3' sense, an “upstream” synthon such as structure H is modified at its terminal 3' site, while a “downstream” synthon such as structure Hl is modified at its terminal 5' site.
[0107] Oligonucleosides linked by hydrazines, hydroxylamines, and other linking groups can be protected by a dimethoxytrityl group at the 5 '-hydroxyl and activated for coupling at the 3'-hydroxyl with cyanoethyldiisopropyl-phosphite moieties. These compounds can be inserted into any desired sequence by standard, solid phase, automated DNA or RNA synthesis techniques. One of the most popular processes is the phosphoramidite technique. Oligonucleotides containing a uniform backbone linkage can be synthesized by use of CPG- solid support and standard nucleic acid synthesizing machines such as Applied Biosystems Inc. 380B and 394 and Milligen/Biosearch 7500 and 8800s. The initial nucleotide (number 1 at the 3 '-terminus) is attached to a solid support such as controlled pore glass. In sequence specific order, each new nucleotide is attached either by manual manipulation or by the automated synthesizer system.
[0108] Free amino groups can be alkylated with, for example, acetone and sodium cyanoboro hydride in acetic acid. The alkylation step can be used to introduce other, useful, functional molecules on the macromolecule. Such useful functional molecules include but are not limited to reporter molecules, RNA cleaving groups, groups for improving the pharmacokinetic properties of an oligonucleotide, and groups for improving the pharmacodynamic properties of an oligonucleotide. Such molecules can be attached to or conjugated to the macromolecule via attachment to the nitrogen atom in the backbone linkage. Alternatively, such molecules can be attached to pendent groups extending from a hydroxyl group of the sugar moiety of one or more of the nucleotides. Examples of such other useful functional groups are provided by WO1993007883, which is herein incorporated by reference, and in other of the above-referenced patent applications.
[0109] Solid supports may include any of those known in the art for polynucleotide synthesis, including controlled pore glass (CPG), oxalyl controlled pore glass, TentaGel Support — an aminopolyethyleneglycol derivatized support or Poros — a copolymer of polystyrene/divinylbenzene. Attachment and cleavage of nucleotides and oligonucleotides can be effected via standard procedures. As used herein, the term solid support further includes any linkers (e.g., long chain alkyl amines and succinyl residues) used to bind a growing oligonucleoside to a stationary phase such as CPG. In some embodiments, the oligonucleotide may be further defined as having one or more locked nucleotides, ethylene bridged nucleotides, peptide nucleic acids, or a 5 '(E)-vinyl -phosphonate (VP) modification. In some embodiments, the oligonucleotides have one or more phosphorothioated DNA or RNA bases.
2. Expression Systems
[0110] Numerous expression systems exist that comprise at least a part or all of the RNA or DNA molecules discussed above. Prokaryote- and/or eukaryote-based systems or cell free systems can be employed for use with an embodiment to produce nucleic acid sequences, or their cognate polypeptides, proteins and peptides. Commercially and widely available systems include but are not limited to bacterial, mammalian, yeast, insect cell, and cell free systems. Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins. Appropriate cell lines, host systems, or expression systems can be chosen to ensure the correct modification and processing of the nucleic acid or polypeptide(s) expressed. Those skilled in the art are able to express a vector to produce a nucleic acid sequence or its cognate polypeptide, protein, or peptide using an appropriate expression system.
[oni] In some embodiments, antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the present disclosure are achieved by operably linking a nucleic acid encoding the antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs to a promoter, and incorporating the construct into an expression vector, which is taken up and expressed by cells. The vectors can be suitable for replication and, in some cases, integration in eukaryotes. Typical vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the desired nucleic acid sequence. For example, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers (see, e.g., WO 01/96584; WO 01/29058; and U.S. Pat. No. 6,326,193).
[0112] In certain embodiments the expression vector may be provided to a cell in the form of a viral vector. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and in other virology and molecular biology manuals.
[0113] A number of viral based systems have been developed for gene transfer into mammalian cells. Viruses that are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses (including selfinactivating lentivirus vectors). For example, adenoviruses provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. Thus, in some embodiments, the nucleic acid encoding antigenic polypeptide constructs and/or nucleic acids encoding the antigenic polypeptide constructs of the present disclosure is introduced into cells using a recombinant vector such as a viral vector including, for example, a lentivirus, a retrovirus, gamma-retroviruses, an adeno-associated virus (AAV), a herpesvirus, or an adenovirus.
[0114] One of skill in the art would be well equipped to construct an vector comprising one or more polynucleotide sequences of interest through standard recombinant techniques (see, for example, Maniatis et al., 1988 and Ausubel et al., 1994, both specifically incorporated by reference herein in their entirety).
[0115] Vectors can also comprise other components or functionalities that further modulate gene delivery and/or gene expression, or that otherwise provide beneficial properties to the targeted cells. Such other components include, for example, components that influence binding or targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide.
[0116] Such components also might include markers, such as detectable and/or selection markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. A large variety of such vectors are known in the art and are generally available. When a vector is maintained in a host cell, the vector can either be stably replicated by the cells during mitosis as an autonomous structure, incorporated within the genome of the host cell, or maintained in the host cell’s nucleus or cytoplasm.
[0117] Eukaryotic expression cassettes included in the vectors particularly contain (in a 5'- to-3' direction) regulatory elements including a eukaryotic transcriptional promoter operably linked to a protein-coding sequence, splice signals including intervening sequences, a transcriptional termination/polyadenylation sequence, post-transcriptional regulatory elements, and origins of replication.
3. Methods of Nucleic Acid Delivery
[0118] Suitable methods for nucleic acid delivery to effect expression of compositions are anticipated to include virtually any method by which a particular nucleic acid (e.g., RNA, mRNA, saRNA, DNA) can be introduced into a cell, a tissue or an organism, as described herein or as would be known to one of ordinary skill in the art. Such methods may include, but are not limited to, direct delivery of nucleic acids such as by injection (U.S. Patents 5,994,624,5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference), including microinjection (Harland and Weintraub, 1985; U.S. Patent 5,789,215, incorporated herein by reference); by electroporation (U.S. Patent No. 5,384,253, incorporated herein by reference); by calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990); by using DEAE dextran followed by polyethylene glycol (Gopal, 1985); by direct sonic loading (Fechheimer et al., 1987); by liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991); by microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Patents 5,610,042; 5,322,783, 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference); by agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Patents 5,302,523 and 5,464,765, each incorporated herein by reference); by Agrobacierium-mediated transformation (U.S. Patents 5,591,616 and 5,563,055, each incorporated herein by reference); or by PEG mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Patents 4,684,611 and 4,952,500, each incorporated herein by reference); by desiccation/inhibition mediated DNA uptake (Potrykus et al., 1985). Other methods include viral transduction, such as gene transfer by lentiviral or retroviral transduction. [0119] One illustrative delivery vehicle is a lipid and/or a liposome. The use of lipid formulations is contemplated for the introduction of the nucleic acids into a host cell (in vitro, ex vivo, or in vivo). In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/DNA or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly forming aggregates that are not uniform in size or shape. Lipids are fatty substances which may be naturally occurring or synthetic lipids. For example, lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class of compounds which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.
[0120] In a certain embodiment, a nucleic acid may be entrapped in a lipid complex such as, for example, a liposome. Liposomes are vesicular structures characterized by a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh and Bachhawat, 1991). The amount of liposomes used may vary upon the nature of the liposome as well as the cell used, for example, about 5 to about 20 μg vector DNA per 1 to 10 million of cells may be contemplated. [0121] Liposome-mediated nucleic acid delivery and expression of foreign DNA in vitro has been very successful (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987). The feasibility of liposome-mediated delivery and expression of foreign DNA in cultured chick embryo, HeLa and hepatoma cells has also been demonstrated (Wong et al., 1980).
[0122] In certain embodiments, a liposome may be complexed with a hemagglutinating virus (HVJ). This has been shown to facilitate fusion with the cell membrane and promote cell entry of liposome-encapsulated DNA (Kaneda etal., 1989). In other embodiments, a liposome may be complexed or employed in conjunction with nuclear non-histone chromosomal proteins (HMG-1) (Kato et al., 1991). In yet further embodiments, a liposome may be complexed or employed in conjunction with both HVJ and HMG- 1. In other embodiments, a delivery vehicle may comprise a ligand and a liposome.
[0123] In various embodiments lipids suitable for use can be obtained from commercial sources. For example, lipofectamine can be obtained from Thermo Fisher Scientific, Waltham, Mass.; dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem- Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc. (Birmingham, Ala.). Stock solutions of lipids in chloroform or chloroform/methanol can be stored at about -20°C. Chloroform can be used as the only solvent since it is more readily evaporated than methanol. “Liposome” is a generic term encompassing a variety of single and multilamellar lipid vehicles formed by the generation of enclosed lipid bilayers or aggregates. Liposomes can be characterized as having vesicular structures with a phospholipid bilayer membrane and an inner aqueous medium. Multilamellar liposomes have multiple lipid layers separated by aqueous medium. They form spontaneously when phospholipids are suspended in an excess of aqueous solution. The lipid components undergo self-rearrangement before the formation of closed structures and entrap water and dissolved solutes between the lipid bilayers (Ghosh et al. (1991) Glycobiology 5: 505-510). However, compositions that have different structures in solution than the normal vesicular structure are also encompassed. For example, the lipids may assume a micellar structure or merely exist as nonuniform aggregates of lipid molecules. Also contemplated are lipofectamine-nucleic acid complexes.
[0124] In certain embodiments, a nucleic acid is introduced into a cell via electroporation. Electroporation involves the exposure of a suspension of cells and DNA to a high-voltage electric discharge. Recipient cells can be made more susceptible to transformation by mechanical wounding. Also the amount of vectors used may vary upon the nature of the cells used, for example, about 5 to about 20 pg vector DNA per 1 to 10 million of cells may be contemplated.
[0125] Transfection of eukaryotic cells using electroporation has been quite successful. Mouse pre-B lymphocytes have been transfected with human kappa-immunoglobulin genes (Potter et al., 1984), and rat hepatocytes have been transfected with the chloramphenicol acetyltransferase gene (Tur-Kaspa et al., 1986) in this manner.
[0126] In other embodiments, a nucleic acid is introduced to the cells using calcium phosphate precipitation. Human KB cells have been transfected with adenovirus 5 DNA (Graham and Van Der Eb, 1973) using this technique. Also in this manner, mouse L(A9), mouse C127, CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected with a neomycin marker gene (Chen and Okayama, 1987), and rat hepatocytes were transfected with a variety of marker genes (Rippe et al., 1990).
[0127] In another embodiment, a nucleic acid is delivered into a cell using DEAE-dextran followed by polyethylene glycol. In this manner, reporter plasmids were introduced into mouse myeloma and erythroleukemia cells (Gopal, 1985).
4. Host Cells
[0128] In another aspect, contemplated are the use of host cells into which a RNA or DNA molecule has been introduced. RNA or DNA can be transfected into cells according to a variety of methods known in the art. RNA or DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. Some RNA or DNA may employ control sequences that allow it to be replicated and/or expressed in both prokaryotic and eukaryotic cells. In certain aspects, the polypeptide of interest expression construct or RNA or DNA replicase can be placed under control of a promoter that is linked to T-cell activation, such as one that is controlled by NF AT- 1 or NF-KB, both of which are transcription factors that can be activated upon T-cell activation. Control of expression allows T cells, such as tumortargeting T cells, to sense their surroundings and perform real-time modulation of cytokine signaling, both in the T cells themselves and in surrounding endogenous immune cells. One of skill in the art would understand the conditions under which to incubate host cells to maintain them and to permit replication of a RNA molecule, such as a saRNA, or DNA. Also understood and known are techniques and conditions that would allow large-scale production of RNA or DNA molecules, as well as production of the nucleic acids encoded by vectors and their cognate polypeptides, proteins, or peptides. [0129] For transfection of mammalian cells, it is known, depending upon the RNA or DNA and transfection technique used, only a small fraction of cells may integrate the foreign RNA or DNA into their cells. Regardless of the method used to introduce exogenous nucleic acids into a host cell, in order to identify and select these integrants, a variety of assays may be performed. Such assays include, for example, “molecular biological” assays well known to those of skill in the art, such as Southern and Northern blotting, RT-PCR and PCR; “biochemical” assays, such as detecting the presence or absence of a particular peptide, e.g., by immunological means (ELISAs and Western blots) or by assays described herein to identify agents falling within the scope of the disclosure.
[0130] In certain embodiments, cells containing an exogenous nucleic acid may be identified in vitro or in vivo by including a marker in the expression vector or the exogenous nucleic acid. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression vector. Generally, a selection marker may be one that confers a property that allows for selection. A positive selection marker may be one in which the presence of the marker allows for its selection, while a negative selection marker is one in which its presence prevents its selection. An example of a positive selection marker is a drug resistance marker.
[0131] In addition to markers conferring a phenotype that allows for the discrimination of transformants based on the implementation of conditions, other types of markers including screenable markers such as GFP, whose basis is colorimetric analysis, are also contemplated. Alternatively, screenable enzymes as negative selection markers such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be utilized. One of skill in the art would also know how to employ immunologic markers, possibly in conjunction with FACS analysis. The marker used is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selection and screenable markers are well known to one of skill in the art.
[0132] Selectable markers may include a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes; cells that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those cells that can grow have successfully taken up and expressed the introduced genetic material. Examples of selectable markers include: the Abicr gene or Neo gene from Tn5, which confers antibiotic resistance to geneticin. [0133] A screenable marker may comprise a reporter gene, which allows the researcher to distinguish between wanted and unwanted cells. Certain embodiments of the present invention utilize reporter genes to indicate specific cell lineages. For example, the reporter gene can be located within expression elements and under the control of the ventricular- or atrial-selective regulatory elements normally associated with the coding region of a ventricular- or atrial- selective gene for simultaneous expression. A reporter allows the cells of a specific lineage to be isolated without placing them under drug or other selective pressures or otherwise risking cell viability.
[0134] Examples of such reporters include genes encoding cell surface proteins (e.g. , CD4, HA epitope), fluorescent proteins, antigenic determinants and enzymes (e.g., [3-galactosidase). The vector containing cells may be isolated, e.g., by FACS using fluorescently-tagged antibodies to the cell surface protein or substrates that can be converted to fluorescent products by a vector encoded enzyme.
[0135] In specific embodiments, the reporter gene is a fluorescent protein. A broad range of fluorescent protein genetic variants have been developed that feature fluorescence emission spectral profiles spanning almost the entire visible light spectrum. Mutagenesis efforts in the original Aequorea victoria jellyfish green fluorescent protein have resulted in new fluorescent probes that range in color from blue to yellow, and are some of the most widely used in vivo reporter molecules in biological research. Longer wavelength fluorescent proteins, emitting in the orange and red spectral regions, have been developed from the marine anemone, Discosoma striata, and reef corals belonging to the class Anthozoa. Still other species have been mined to produce similar proteins having cyan, green, yellow, orange, and deep red fluorescence emission. Developmental research efforts are ongoing to improve the brightness and stability of fluorescent proteins, thus improving their overall usefulness.
[0136] In particular embodiments, the cells of the disclosure may be specifically formulated and/or they may be cultured in a particular medium. The cells may be formulated in such a manner as to be suitable for delivery to a recipient without deleterious effects.
[0137] The medium in certain aspects can be prepared using a medium used for culturing animal cells as their basal medium, such as any of AIM V, X-VIVO-15, NeuroBasal, EGM2, TeSR, BME, BGJb, CMRL 1066, Glasgow MEM, Improved MEM Zinc Option, IMDM, Medium 199, Eagle MEM, aMEM, DMEM, Ham, RPMI-1640, and Fischer's media, as well as any combinations thereof, but the medium may not be particularly limited thereto as far as it can be used for culturing animal cells. Particularly, the medium may be xeno-free or chemically defined. [0138] The medium can be a serum-containing or serum-free medium, or xeno-free medium. From the aspect of preventing contamination with heterogeneous animal-derived components, serum can be derived from the same animal as that of the stem cell(s). The serum- free medium refers to medium with no unprocessed or unpurified serum and accordingly, can include medium with purified blood-derived components or animal tissue-derived components (such as growth factors).
[0139] The medium may contain or may not contain any alternatives to serum. The alternatives to serum can include materials which appropriately contain albumin (such as lipid- rich albumin, bovine albumin, albumin substitutes such as recombinant albumin or a humanized albumin, plant starch, dextrans and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'- thiolgiycerol, or equivalents thereto. The alternatives to serum can be prepared by the method disclosed in International Publication No. 98/30679, for example (incorporated herein in its entirety). Alternatively, any commercially available materials can be used for more convenience. The commercially available materials include knockout Serum Replacement (KSR), Chemically-defined Lipid concentrated (Gibco), and Glutamax (Gibco).
[0140] In certain embodiments, the medium may comprise one, two, three, four, five, six, seven, eight, nine, ten, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 ormore of the following: Vitamins such as biotin; DL Alpha Tocopherol Acetate; DL Alpha-Tocopherol; Vitamin A (acetate); proteins such as BSA (bovine serum albumin) or human albumin, fatty acid free Fraction V; Catalase; Human Recombinant Insulin; Human Transferrin; Superoxide Dismutase; Other Components such as Corticosterone; D-Galactose; Ethanolamine HC1; Glutathione (reduced); L-Carnitine HC1; Linoleic Acid; Linolenic Acid; Progesterone; Putrescine 2HC1; Sodium Selenite; and/or T3 (triodo-I-thyronine). In specific embodiments, one or more of these may be explicitly excluded.
[0141] In some embodiments, the medium further comprises vitamins. In some embodiments, the medium comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of the following (and any range derivable therein): biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, vitamin B12, or the medium includes combinations thereof or salts thereof. In some embodiments, the medium comprises or consists essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, choline chloride, calcium pantothenate, pantothenic acid, folic acid nicotinamide, pyridoxine, riboflavin, thiamine, inositol, and vitamin B 12. In some embodiments, the vitamins include or consist essentially of biotin, DL alpha tocopherol acetate, DL alpha-tocopherol, vitamin A, or combinations or salts thereof. In some embodiments, the medium further comprises proteins. In some embodiments, the proteins comprise albumin or bovine serum albumin, a fraction of BSA, catalase, insulin, transferrin, superoxide dismutase, or combinations thereof. In some embodiments, the medium further comprises one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, or combinations thereof. In some embodiments, the medium comprises one or more of the following: a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, or combinations thereof. In some embodiments, the medium comprises or further comprises amino acids, monosaccharides, inorganic ions. In some embodiments, the amino acids comprise arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine, or combinations thereof. In some embodiments, the inorganic ions comprise sodium, potassium, calcium, magnesium, nitrogen, or phosphorus, or combinations or salts thereof. In some embodiments, the medium further comprises one or more of the following: molybdenum, vanadium, iron, zinc, selenium, copper, or manganese, or combinations thereof. In certain embodiments, the medium comprises or consists essentially of one or more vitamins discussed herein and/or one or more proteins discussed herein, and/or one or more of the following: corticosterone, D-Galactose, ethanolamine, glutathione, L-camitine, linoleic acid, linolenic acid, progesterone, putrescine, sodium selenite, or triodo-I-thyronine, a B-27® supplement, xeno-free B-27® supplement, GS21TM supplement, an amino acid (such as arginine, cystine, isoleucine, leucine, lysine, methionine, glutamine, phenylalanine, threonine, tryptophan, histidine, tyrosine, or valine), monosaccharide, inorganic ion (such as sodium, potassium, calcium, magnesium, nitrogen, and/or phosphorus) or salts thereof, and/or molybdenum, vanadium, iron, zinc, selenium, copper, or manganese. In specific embodiments, one or more of these may be explicitly excluded.
[0142] The medium can also contain one or more externally added fatty acids or lipids, amino acids (such as non-essential amino acids), vitamin(s), growth factors, cytokines, antioxidant substances, 2-mercaptoethanol, pyruvic acid, buffering agents, and/or inorganic salts. . In specific embodiments, one or more of these may be explicitly excluded.
[0143] One or more of the medium components may be added at a concentration of at least, at most, or about 0.1, 0.5, 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 180, 200, 250 ng/L, ng/mL, pg/mL, mg/mL, or any range derivable therein. [0144] In specific embodiments, the cells of the disclosure are specifically formulated. They may or may not be formulated as a cell suspension. In specific cases they are formulated in a single dose form. They may be formulated for systemic or local administration. In some cases the cells are formulated for storage prior to use, and the cell formulation may comprise one or more cry opreservation agents, such as DMSO (for example, in 5% DMSO). The cell formulation may comprise albumin, including human albumin, with a specific formulation comprising 2.5% human albumin. The cells may be formulated specifically for intravenous administration; for example, they are formulated for intravenous administration over less than one hour. In particular embodiments the cells are in a formulated cell suspension that is stable at room temperature for 1, 2, 3, or 4 hours or more from time of thawing.
B. Isolation
[0145] The nucleic acid molecules disclosed herein may be obtained from any source that produces nucleic acids. Methods of isolating mRNA are well known in the art. See e.g., Sambrook et al., supra.
VII. Compositions
[0146] In one aspect, the disclosure relates to an immunogenic composition for administration to a host. In some embodiments, the host is a human. In other embodiments, the host is a non-human.
[0147] In some embodiments, the composition comprises a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct. The modified antigenic polypeptide construct can differ from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences. In some embodiments, the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct. In some embodiments, an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide. In some embodiments, the composition comprises a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct. The modified antigenic polypeptide construct can differ from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences. In some embodiments, the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct. In some embodiments, an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
[0148] In some embodiments, the composition comprises a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct. The modified viral spike protein construct can differ from an unmodified viral spike protein construct at one or more antigenic peptide sequences. In some embodiments, the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct. In some embodiments, an antigenic peptide sequence of the unmodified viral spike protein construct comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified viral spike protein construct comprises an antigenic peptide sequence of 5 or more consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide. In some embodiments, the composition comprises a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct. The modified viral spike protein construct can differ from an unmodified viral spike protein construct at one or more antigenic peptide sequences. In some embodiments, the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct. In some embodiments, an antigenic peptide sequence of the unmodified viral spike protein construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified viral spike protein construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
[0149] In some instances, the compositions described herein are immunogenic compositions. In some instances, the compositions described herein include at least one isolated nucleic acid or polypeptide molecule as described herein. In some instances, the compositions described herein are vaccines. In specific embodiments, the immunogenic compositions comprise RNA, and the immunogenic compositions are RNA vaccines. In other embodiments, the immunogenic compositions comprise DNA, and vaccines are DNA vaccines. In yet other embodiments, the immunogenic compositions comprise a polypeptide, and vaccines are polypeptide vaccines. Conditions and/or diseases that can be treated with the RNA, DNA, and/or peptide or polypeptide compositions include, but are not limited to, those caused and/or impacted by infection, cancer, rare diseases, and other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA.
[0150] In some embodiments, the composition is substantially free of one or more impurities or contaminants and, for instance, includes nucleic acid or polypeptide molecules that are equal to any one of, at least any one of, at most any one of, or between any two of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure; at least 98% pure, or at least 99% pure.
[0151] In some embodiments, the composition further includes a lipid-based delivery system, which delivers a nucleic acid molecule to the interior of a cell, where it can then replicate, inhibit protein expression of interest, and/or express the encoded polypeptide of interest. The delivery system may have adjuvant effects which enhance the immunogenicity of an encoded antigen. In some embodiments, the composition further includes neutral lipids, cationic lipids, cholesterol, and polyethylene glycol (PEG), and forms nanoparticles that encompass, or encapsulate, the nucleic acid molecules. In some embodiments, the composition further includes any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune-stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water- in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion. In some embodiments, the nucleic acid molecule is encapsulated in, bound to or adsorbed on any one of a cationic lipid, a liposome, a lipid nanoparticle, a polyplex, a cochleate, a virosome, an immune -stimulating complex, a microparticle, a microsphere, a nanosphere, a unilamellar vesicle, a multilamellar vesicle, an oil-in-water emulsion, a water-in-oil emulsion, an emulsome, a polycationic peptide, and a cationic nanoemulsion, or a combination thereof. As used herein, “encapsulate,” “encapsulated,” “encapsulation,” and grammatically comparable variants thereof mean that at least a portion of a substance is enclosed or surrounded by another material or another substance in a composition. In some embodiments, a substance, such as a nucleic acid, can be fully enclosed or surrounded by another material or another substance in a composition, such as a lipid.
[0152] In some embodiments, the compositions further comprise one or more stabilizing agents and one or more buffers. A nucleic acid molecule, e.g. , a naked or encapsulated nucleic acid, or a polypeptide as disclosed herein may be comprised in a solution comprising the one or more stabilizing agents and one or more buffers. In some embodiments, the stabilizing agent comprises sucrose, mannose, sorbitol, raffinose, trehalose, mannitol, inositol, sodium chloride, arginine, lactose, hydroxyethyl starch, dextran, polyvinylpyrolidone, glycine, or a combination thereof. In some embodiments, the stabilizing agent is a disaccharide, or sugar. In one embodiment, the stabilizing agent is sucrose. In another embodiment, the stabilizing agent is trehalose. In a further embodiment, the stabilizing agent is a combination of sucrose and trehalose. In some embodiments, the total concentration of the stabilizing agent(s) in the composition is about 5% to about 10% w/v. For example, the total concentration of the stabilizing agent can be equal to any one of, at least any one of, at most any one of, or between any two of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% w/v or any range or value derivable therein. In specific embodiments, the total concentration of the stabilizing agent(s) in the composition is 10% w/v. In specific embodiments, the amino acid concentration is 5% w/v.
[0153] Examples of buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, calcium lactobionate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, amino-sulfonate buffers (e.g., HEPES), magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer’s solution, ethyl alcohol, and/or combinations thereof. In some embodiments, the buffer is a HEPES buffer, a Tris buffer, or a PBS buffer. In one embodiment, the buffer is Tris buffer. In another embodiment, the buffer is a HEPES buffer. In a further embodiment, the buffer is a PBS buffer. In some embodiments, the concentration of the buffer in the composition is about 10 mM. For example, the buffer concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 11 mM, 12 mM, 13 mM, 14 mM, 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, or 20 mM, or any range or value derivable therein. In specific embodiments, the buffer concentration is 10 mM. The buffer can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the buffer can be at pH 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3,
7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or 8.5, or any range or value derivable therein. In specific embodiments, the buffer is at pH 7.4.
[0154] The compositions may further include one or more salts and/or one or more pharmaceutically acceptable surfactants, preservatives, carriers, diluents, and/or excipients, in some cases. In some embodiments, the composition further includes a pharmaceutically acceptable vehicle. In some embodiments, each of a buffer, stabilizing agent, salt, surfactant, preservative, and excipient are included in the compositions. In other embodiments, any one or more of a buffer, stabilizing agent, salt, surfactant, preservative, excipient, carrier, diluent, or vehicle may be excluded from compositions.
[0155] Examples of salts include but not limited to sodium salts and/or potassium salts. In some embodiments, the sodium salt comprises sodium chloride. In some embodiments, the potassium salt comprises potassium chloride. The concentration of the salts in the composition can be about 70 mM to about 140 mM. For example, the salt concentration can be equal to any one of, at least any one of, at most any one of, or between any two of 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM, or any range or value derivable therein. In specific embodiments, the salt concentration is 70 mM. In specific embodiments, the salt concentration is 140 mM. The salt can be at a neutral pH, pH 6.5 to 8.5, pH 7.0 to pH 8.0, or pH 7.2 to pH 7.6. For example, the salt can be at a pH equal to any one of, at least any one of, at most any one of, or between any two of 6.5, 6.6, 6.7, 6.8, 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, or
8.5, or any range or value derivable therein.
[0156] Examples of excipients, which refer to ingredients in the compositions that are not active ingredients, include but are not limited to carriers, binders, diluents, lubricants, thickeners, surface active agents, preservatives, stabilizers, emulsifiers, buffers, flavoring agents, disintegrants, coatings, plasticizers, compression agents, wet granulation agents, or colorants. Preservatives for use in the compositions disclosed herein include but are not limited to benzalkonium chloride, chlorobutanol, paraben and thimerosal. As used herein, “pharmaceutically acceptable carrier” includes any and all aqueous solvents (e.g., water, alcoholic/aqueous solutions, saline solutions, parenteral vehicles, such as sodium chloride, Ringer’s dextrose, etc.), non-aqueous solvents (e.g., propylene glycol, polyethylene glycol, vegetable oil, and injectable organic esters, such as ethyloleate), dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial or antifungal agents, anti-oxidants, chelating agents, and inert gases), isotonic agents, absorption delaying agents, salts, drugs, drug stabilizers, gels, binders, excipients, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, fluid and nutrient replenishers, such like materials and combinations thereof, as would be known to one of ordinary skill in the art. Diluents, or diluting or thinning agents, include but are not limited to ethanol, glycerol, water, sugars such as lactose, sucrose, mannitol, and sorbitol, and starches derived from wheat, com rice, and potato; and celluloses such as microcrystalline cellulose. The amount of diluent in the composition can range from about 10% to about 90% by weight of the total composition, about 25% to about 75%, about 30% to about 60% by weight, or about 12% to about 60%.
[0157] The pH and exact concentration of the various components in a pharmaceutical composition are adjusted according to well-known parameters. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredients, its use in immunogenic, prophylactic and/or therapeutic compositions is contemplated.
[0158] Administration of the compositions described herein can be carried out via any of the accepted modes of administration of agents for serving similar utilities. Pharmaceutical compositions may be formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalants, gels, microspheres, and aerosols. Typical routes of administering such pharmaceutical compositions include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intradermal, intrastemal injection, or infusion techniques. Pharmaceutical compositions described herein are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a compound in aerosol form may hold a plurality of dosage units. The composition to be administered will, in any event, contain a therapeutically and/or prophylactically effective amount of a compound within the scope of this disclosure, or a pharmaceutically acceptable salt thereof, for treatment of a disease or condition of interest in accordance with the teachings described herein.
[0159] A pharmaceutical composition within the scope of this disclosure may be in the form of a solid or liquid. In one aspect, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral syrup, injectable liquid, or an aerosol, which is useful in, for example, inhalator administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi-solid, semi-liquid, suspension, and gel forms are included within the forms considered herein as either solid or liquid. As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present or exclude: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth, or gelatin; excipients such as starch, lactose, or dextrins; disintegrating agents such as alginic acid, sodium alginate, Primogel, com starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate, or orange flavoring; and a coloring agent. When the pharmaceutical composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil. The pharmaceutical composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred composition contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant, and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer, and isotonic agent may be included or exclude.
[0160] A liquid pharmaceutical composition, whether they be solutions, suspensions, or other like form, may include or exclude one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose; agents to act as cryoprotectants such as sucrose or trehalose. The parenteral preparation can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.
[0161] A liquid pharmaceutical composition intended for either parenteral or oral administration should contain an amount of a compound such that a suitable dosage will be obtained.
[0162] The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a pharmaceutical composition intended to be administered by injection can be prepared by combining the nucleic acid or polypeptide with sterile, distilled water or other carrier so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non- covalently interact with a compound consistent with the teachings herein so as to facilitate dissolution or homogeneous suspension of the compound in the aqueous delivery system.
[0163] The compositions within the scope of the disclosure are administered in a therapeutically and/or prophylactically effective amount, which will vary depending upon a variety of factors including the activity of the specific therapeutic and/or prophylactic agent employed; the metabolic stability and length of action of the therapeutic and/or prophylactic agent; the age, body weight, general health, gender, and diet of the patient; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
VIII. Methods of Use
[0164] In one aspect, the disclosure relates to a method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct. The method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct. In some embodiments, the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct.
[0165] The disclosure also relates to a method for reducing or preventing cross-reactivity of an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct. The method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence. In some embodiments, the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, and modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified antigenic polypeptide construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified antigenic polypeptide construct comprising the modified antigenic peptide sequence. In some embodiments, modifying the antigenic peptide sequence reduces or prevents crossreactivity of the modified antigenic polypeptide construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified antigenic polypeptide construct comprising an unmodified antigenic peptide sequence. [0166] In some embodiments, the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct. In some embodiments, the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct.
[0167] In some embodiments, antigenic peptide sequences of antigenic polypeptide constructs to be modified to reduce or prevent cross-reactivity with host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue can be identified using one or more of peptide sharing analysis (e.g., protein alignment), mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue, rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, and immune function), and algorithmic immunogenicity prediction tools (e.g., http://tools.iedb.org/main/tcell).
[0168] In some embodiments, the cross-reactivity of the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue.
[0169] In some embodiments, the cross-reactivity of the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3- fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40-fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-fold or more, about 400-fold or more, about 500- fold or more, about 1000-fold or more, about 2000-fold or more, about 3000-fold or more, about 4000-fold or more, about 5000-fold or more, or about 10000-fold or more.
[0170] In some embodiments, the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity equivalent to or higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof.
[0171] In some embodiments, the modified antigenic polypeptide construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified antigenic peptide sequence and/or an unmodified antigenic polypeptide construct and/or compositions thereof by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40- fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-fold or more, about 400-fold or more, about 500-fold or more, about 1000-fold or more, about 2000-fold or more, about 3000-fold or more, about 4000-fold or more, about 5000-fold or more, or about 10000-fold or more.
[0172] In another aspect, the disclosure relates to a method for producing an immunogenic composition comprising a modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct. The method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct. In some embodiments, the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to have less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to form the modified viral spike protein construct and/or a nucleic acid encoding the modified viral spike protein construct.
[0173] The disclosure also relates to a method for reducing or preventing cross-reactivity of a viral spike protein construct and/or a nucleic acid encoding the viral spike protein construct. The method can comprise one or both of the following steps: identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence. In some embodiments, the method comprises both of the steps of identifying at least one antigenic peptide sequence comprised in an unmodified viral spike protein construct, and modifying the antigenic peptide sequence of the unmodified viral spike protein construct to produce a modified antigenic peptide sequence having less than about 50% amino acid sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue to produce a modified viral spike protein construct comprising the modified antigenic peptide sequence and/or a nucleic acid encoding the modified viral spike protein construct comprising the modified antigenic peptide sequence. In some embodiments, modifying the antigenic peptide sequence reduces or prevents cross-reactivity of the modified viral spike protein construct to the cross-reactive host peptide compared to the cross-reactivity of an unmodified viral spike protein construct comprising an unmodified antigenic peptide sequence.
[0174] In some embodiments, the antigenic peptide sequence comprises 5 or more consecutive amino acids having at least about 50% amino acid sequence identity with a cross- reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue. In some embodiments, the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct. In some embodiments, the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue . In some embodiments, the modified viral spike protein construct has at least about 50% amino acid sequence identity with the unmodified viral spike protein construct.
[0175] In some embodiments, antigenic peptide sequences of viral spike protein constructs to be modified to reduce or prevent cross-reactivity with host peptides comprised in a cross- reactive host polypeptides expressed in a host tissue can be identified using one or more of peptide sharing analysis (e.g., protein alignment), mRNA and protein expression profiles of cross-reactive host peptides comprised in a cross-reactive host polypeptides expressed in a host tissue, rank-based approaches to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, and immune function), and algorithmic immunogenicity prediction tools (e.g., http://tools.iedb.org/main/tcell).
[0176] In some embodiments, the cross-reactivity of the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue.
[0177] In some embodiments, the cross-reactivity of the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue is reduced or prevented compared to the cross-reactivity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof with a host peptide sequence comprised in a cross-reactive host polypeptide expressed in a host tissue by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3- fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40-fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-fold or more, about 400-fold or more, about 500- fold or more, about 1000-fold or more, about 2000-fold or more, about 3000-fold or more, about 4000-fold or more, about 5000-fold or more, or about 10000-fold or more.
[0178] In some embodiments, the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity equivalent to or higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof.
[0179] In some embodiments, the modified viral spike protein construct and/or compositions thereof produced by the methods of the present disclosure have an efficacy, intracellular delivery, and/or immunogenicity higher than the efficacy, intracellular delivery, and/or immunogenicity of an unmodified viral spike protein sequence and/or an unmodified viral spike protein construct and/or compositions thereof by equal to any one of, at least any one of, at most any one of, or between any two of about 5% or higher, about 10% or more, about 15% or more, about 20% or more, about 30% or more, about 40% or more, about 50% or more, about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 1-fold or more, about 2-fold or more, about 3-fold or more, about 4-fold or more, about 5-fold or more, about 10-fold or more, about 20-fold or more, about 30-fold or more, about 40- fold or more, about 50-fold or more, about 100-fold or more, about 200-fold or more, about 300-fold or more, about 400-fold or more, about 500-fold or more, about 1000-fold or more, about 2000-fold or more, about 3000-fold or more, about 4000-fold or more, about 5000-fold or more, or about 10000-fold or more.
[0180] In another aspect, the disclosure relates to a method of inducing an immune response in a subject. The method includes administering to the mammalian cell an effective amount of a composition as described herein.
[0181] In another aspect, the disclosure relates to a method of vaccinating a subject. The method includes administering to the subject in need thereof an effective amount of a composition described herein.
[0182] In another aspect, the disclosure relates to a method of treating or preventing an infectious disease. The method includes administering to the subject an effective amount of a composition as described herein.
[0183] In another aspect, disclosure relates to a method of treating or preventing an infectious disease in a subject by, for example, inducing an immune response to an infectious disease in the subject. In some embodiments, the method includes administering a priming composition that includes an effective amount of a composition described herein, and administering a booster composition including an effective amount of an adenoviral vector encoding an antigen. In another embodiment, the method includes administering a priming composition including an effective amount of an adenoviral vector encoding an antigen, and administering a booster composition that includes an effective amount of a composition described herein. In some embodiments, the composition elicits an immune response including an antibody response. In some embodiments, the composition elicits an immune response including a T cell response.
IX. Method of Identifying a potential toxicity
[0184] Computational toxicology concerns the use of computational tools to support integrative approaches to toxicological research and chemical safety assessments via predictive modeling, analyses of complex, multifaceted data sets, and extrapolation and translation among evidence streams, particularly new approach methodologies that rely upon alternatives to animal testing. Understanding which biological pathway targets are related to different types of organ level toxicities may be useful to design new in vitro assays and to develop new predictive computational methods and/or interpret their results.
[0185] Conventional risk and safety assessment approaches have included experimental methods and calculation methods, where the experimental methods can be further divided into in vitro methods and in vivo methods. There are advantages and disadvantages for both in vitro experiments and in vivo experiments. In vivo experiments have been developed and applied to various toxicological tests and screening methods, but are currently impacted by principles to refine, reduce, and replace animal use to address the costs and ethical issues from in vivo animal testing. In vitro experiments cannot fully mimic the complicated mechanisms of distribution, absorption, metabolism, excretion process and other factors in animals, and the accuracy of results obtained there from is not always comparable to that obtained from in vivo tests.
[0186] Further, both in vitro and in vivo methods are time-consuming, labor intensive and may have limited ability to address fully or identify potential toxicities.
[0187] Therefore, there is an urgent need to develop a computational toxicology method for screening compositions for potential reduction or elimination of adverse events.
[0188] In one aspect, the disclosure relates to a method of identifying a potential toxicity of an immunogenic composition in a biological host system. The immunogenic composition may include an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct. In some embodiments, the method includes a. identifying one or more proteins of the host that have a degree of similarity to the antigenic polypeptide construct; b. identifying properties of the one or more proteins, the properties comprising cellular location, gene- and protein-tissue expression profiles, and/or toxic state associations; c. ranking or sorting the one or more proteins using one or more of the properties; d. identifying immunogenicity of the one or more proteins; e. comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence; and f. predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence.
[0189] One or more of the steps may be performed on a computer. In some embodiments all of the steps above are performed on a computer. In some embodiments one or more of the steps are performed on a different computer than one or more of the other steps. Performing one or more of the steps on a computer may include performing the steps in a single or in a combination of computational models.
[0190] In some embodiments identifying a potential toxicity may include identifying a potential toxicity from a list or database of toxicities that the immunogenic composition is likely to increase a risk in a biological host system, if the immunogenic composition is administered to the biological host. In some embodiments identifying a potential toxicity may include identifying an immunogenic composition or a portion of an immunogenic composition, such as a portion of the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct that is likely to increase the risk of a specific toxicity in a biological host system if the immunogenic composition is administered to the biological host. In some embodiments identifying a potential toxicity includes identifying a potential toxicity from a list or database of toxicities that the immunogenic composition is likely to increase a risk of in a biological host system if the immunogenic composition is administered to the biological host and includes identifying an immunogenic composition or a portion of an immunogenic composition, such as a portion of the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct that is likely to increase the risk of a specific toxicity in a biological host system if the immunogenic composition is administered to the biological host.
[0191] The potential toxicity can be any toxicity of the biological host. In some instances, the potential toxicity is a toxicity associated with administration to the biological host of the immunogenic composition or a portion of an immunogenic composition, such as the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct. In some instances, the potential toxicity includes myocarditis or pericarditis. In some instances, the potential toxicity includes myocarditis or pericarditis and the antigenic polypeptide construct is a viral spike protein.
[0192] In some preferred embodiments, the antigenic polypeptide is a prefusion spike protein, such as any of the spike proteins comprising a SARS-CoV-2 S ectodomain trimer comprising protomers, which comprise an amino acid sequence at least 95% identical to residues 16-1208 of SEQ ID NO: 2 and comprising proline substitutions at positions 986 and 987 of SEQ ID NO: 2 that stabilize the S ectodomain trimer in a prefusion conformation, as described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021 by The United States of America, as represented by The Secretary, Department of Health and Human Services. The sequence of SEQ ID NO: 2 from WO2021163365, filed Feburary 11, 2021, is: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY ECD I P IGAGI CAS YQTQTNS PRRARS VASQS 1 I AYTMSLGAENSVAYSNNS I Al PTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDP LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQGGYIPEAPRDGQAYVRKDGEWVLLSTF ( SEQ ID NO : 2 ) .
Preferably, stabilization of the perfusion conformation is obtained by introducing two consecutive proline substitutions at residues K986 and V987 in the spike protein (see SEQ ID NO: 2 described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021).
Accordingly, in preferred embodiments, the pre-fusion stabilized spike protein (S_stab) comprises at least one pre fusion stabilizing mutation, wherein the at least one pre-fusion stabilizing mutation comprises the following amino acid substitutions: K986P and V987P (see SEQ ID NO: 2 described in WO2021163365 (PCT/US2021/017709), filed Feburary 11, 2021).
[0193] In some embodiments, the stabilized spike protein has an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% identity to the amino acid sequence of: MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNI IRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLE GKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQT LLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETK CTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIAD YNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPC NGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVN FNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITP GTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSY ECD I P IGAGI CAS YQTQTNS PRRARS VASQS I I AYTMSLGAENSVAYSNNS I Al PTNFTI SVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQE VFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDC LGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAM QMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALN TLVKQLSSNFGAISSVLNDILSRLDPPEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRA SANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPA ICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQI ITTDNTFVSGNCDWIGIVNNTVYDP LQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDL QELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDD SEPVLKGVKLHYT ( SEQ ID NO : 1 ) .
[0194] The immunogenic composition can be a vaccine. The immunogenic composition can be a treatment or preventative treatment for an infection, cancer, rare diseases, and/or other diseases or conditions caused by overproduction, underproduction, or improper production of proteins, DNA, or RNA, such as mRNA or siRNA. The host can be a plant or an animal. The host can be a mammal. The host can be a human.
[0195] In some instances, the method includes searching a human proteome with a spike protein peptide and identifying a protein from the human proteome with homology to the spike protein peptide. In some instances, the method includes identifying whether the protein from the human proteome with homology to the spike protein peptide is present in a target organ. [0196] In some instances, ranking or sorting the one or more proteins using one or more of the properties includes ranking cellular locations that are extracellular or on a surface of a cell as more likely to cause the potential toxicity than intracellular locations, ranking gene- and protein-tissue expression profdes that overlap in tissue location of the potential toxicity as more likely to cause the potential toxicity than other tissue locations, and/or ranking toxic state associations that overlap or are related to the potential toxicity as more likely to cause the potential toxicity than other toxic state associations. The ranking or sorting can be from greatest to least or can assign a numerical degree of likelihood to cause the potential toxicity. In some instances, all of cellular locations, gene- and protein-tissue expression profiles, and toxic state associations are used in this method. In some instances, other properties are also used in this method. Cellular locations, gene- and protein-tissue expression profiles, and toxic state associations can be gathered from one or more premade lists or databases. In some instances, cellular locations, gene- and protein-tissue expression profiles, and toxic state associations can be predicted by computational methods and the predicted properties can be used in the methods herein.
[0197] In some instances, identifying immunogenicity of the one or more proteins comprises identifying a ranking or sorting of degree of immunogenicity and/or degree of potential immunogenicity. The ranking or sorting can be from greatest to least or can assign a numerical degree of immunogenicity and/or potential immunogenicity. Immunogenicity can be the ability of the one or more proteins to provoke an immune response in the biological host. The immunogenicity of the one or more proteins can be calculated or gathered from one or more premade lists or databases. In some instances, immunogenicity can be predicted by computational methods and the predicted immunogenicity can be used in the methods herein. [0198] Comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence can include comparing the rank or degree of likelihood to cause the potential toxicity and the degree of immunogenicity and/or degree of potential immunogenicity and ranking: more likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity, and either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than less likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity.
The degree of correspondence can be from greatest to least or can assign a numerical degree of correspondence.
[0199] Predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence can include predicting a greater degree of correspondence as more likely to cause a potential toxicity.
[0200] The method can further include modifying the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct to produce a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct that is less likely to cause the potential toxicity when compared to the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct. The modification process can include, but is not limited to, modifying the nucleic acid or protein sequence, changing one or more of the naturally occurring nucleotides or amino acids to analogs or non-naturally occurring nucleotides or amino acid, removing one or more of the nucleotides or amino acids, adding additional nucleotides or amino acids, or combinations thereof.
EXAMPLES
[0201] The following examples are included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the disclosure, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the disclosure.
Example 1
[0202] A strategy employed for identifying antigen mimicry occurring following immunization with the BNT162b2 mRNA vaccine against COVID-19 is illustrated in FIG. 1. In particular, peptide sharing analysis between the BNT162b2 spike protein sequence and human proteins was conducted to identify human proteins with similarity to the COVID-19 spike protein. As shown in FIG. 2, in a preferred analysis, hundreds of protein “hits” corresponding to similarity between an antigenic peptide sequence comprising 9 and 15 consecutive amino acids from the BNT162b2 spike protein sequence and an endogenously- expressed human protein were obtained. 2236 unique human peptide sequences with 9 consecutive amino acids having 75-100% sequence identity and 15 consecutive amino acids having 50-100% sequence identity from the BNT162b2 spike protein sequence, and there is no human protein contained peptide sequences having 100% amino acid sequence identity with the 9 or 15 consecutive amino acid sequence from the BNT162b2 spike protein sequence. In a previous analysis, hundreds of protein “hits” corresponding to similarity between an antigenic peptide sequence comprising 5-8 consecutive amino acids from the BNT162b2 spike protein sequence and an endogenously-expressed human protein were obtained. Nearly 2500 unique human proteins contained peptide sequences having 50-100% amino acid sequence identity with the 5-8 consecutive amino acid sequence from the BNT162b2 spike protein sequence, and hundreds of unique human proteins contained peptide sequences having 100% amino acid sequence identity with the 5-8 consecutive amino acid sequence from the BNT162b2 spike protein sequence.
[0203] Next, as shown in FIG. 1, mRNA and protein expression profiles in various tissues were also assessed to determine whether the human proteins identified as similar to the BNT162b2 spike protein sequence are enriched in the heart. Out of the proteins that comprise of at least one of the 2236 peptides, only 103 were determined to be predominantly expressed in the heart. The results were further narrowed using a rank-based approach to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, immune function, known epitopes) supporting a role for the human protein in pericarditis and/or myocarditis, as well as algorithmic immunogenicity predictions (http:// tools.iedb.org/main/tcell). This allowed narrowing of the 2236 unique human peptides identified as having 75-100% amino acid sequence identity with the 9 consecutive amino acid sequences and 50-100% amino acid sequence identity with 15 consecutive amino acid sequences from the BNT162b2 spike protein sequence to ten human proteins with the potential to be antigenic mimics of the BNT162b2 spike protein. These ten proteins are described in the table below. The results of the analysis are shown in Table 2 and Table 3.
[0204] Unless stated otherwise, as used in the Tables of the Examples herein, % identity means the percentage of human peptide sequence that is similar to the vaccine sequence (including antigen and non-coding regions) or spike protein sequence as referenced in the table. 100% means vaccine peptide sequence perfectly matches with a human peptide sequence.
[0205] In a previous analysis, out of 1689 proteins with 75-100% homology, only 45 were determined to be predominantly expressed in the heart; 14 proteins were found to have 100% homology with 5-8 consecutive amino acid sequence from the BNT162b2 spike protein sequence (see Table 5). The results were further narrowed using a rank-based approach to account for the cellular location of the proteins and additional evidence (e.g., disease association, genetic linkage, and immune function) supporting a role for the human protein in pericarditis and/or myocarditis, as well as algorithmic immunogenicity predictions (http://tools.iedb.org/main/tcell). This allowed narrowing of the nearly 2500 unique human proteins identified as having 50-100% amino acid sequence identity with the 5-8 consecutive amino acid sequence from the BNT162b2 spike protein sequence to ten human proteins with the potential to be antigenic mimics of the BNT162b2 spike protein. These ten proteins are described in the table below (Table 4).
Table 4
RYR2 (ryanodine receptor 2) and NEBL (nebulette) were the top candidates identified due to the fact that they are plasma membrane proteins and they have heart-specific protein expression profiles. The RYR2 protein profile is similar in humans compared to preclinical species, and RYR2 has been associated with cardiomyopathies (considered to be a rare genetic variant). RYR2 is important in fetal heart development, and auto-antibodies against RYR2 have been found in myasthenia gravis patients. Additionally, RYR2 is important in mechanical stretch and TGFb-1 signaling during exertion. The NEBL protein profile is distinct in humans compared to preclinical species. NEBL binds actin and interacts with thin filaments and Z-line associated proteins in striated muscle. NEBL may be involved in cardiac myofibril assembly and is a component of extracellular vesicular exosomes; I bands; stress fibers; and Z discs in the heart. Additionally, NEBL is known to play a role in familial isolated dilated cardiomyopathy. Kelch like family member 41 (KLHL41) was identified as a top hit using the rank-based strategy. KLHL41 is involved in skeletal muscle development and differentiation, and it regulates proliferation and differentiation of myoblasts and plays a role in myofibril assembly by promoting lateral fusion of adjacent thin fibrils into mature, wide myofibrils. KLHL41 is also associated with an autosomal recessive form of nemaline myopathy. Nemaline myopathies are muscular disorders characterized by muscle weakness of varying severity and onset, and abnormal thread-like or rod-shaped structures in muscle fibers on histologic examination. In some embodiments, data are generated to demonstrate cross-reactivity of host peptide sequences with antibodies (e.g., spike protein antibodies). In some embodiments, specific regions of the spike protein are conserved in MHC presentation and are important for cellular immune responses. In some such embodiments, the antigenic sequence responsible for antibody production against the spike protein and the host peptide sequences is determined. Additionally, or alternatively, in some such embodiments, the region of the spike protein comprising the peptide sequences is determined. In some embodiments, overlapping region(s) of the antigenic peptide sequence (e.g. , spike protein sequence) that is associated with the host peptide sequences (e.g., NEBL and RYR2) are determined and aligned within the 3-D structures of the proteins.
Table 5
[0206] Example 2 A Computational Approach to Molecular Mimicry: SARS-CoV-2 Analysis for Immune- Mediated Adverse Events
[0207] SARS-CoV-2 virus is a member of a large family of viruses called coronaviruses that causes a respiratory disease COVID-19. While there are short-term effects of SARS-CoV- 2 infection such as fever, fatigue, headache and loss of taste/smell, the long-term effects can be very serious, including organ damage. In some people, lasting health effects may include, heart complications, chronic kidney impairment, stroke, and Guillain-Barre syndrome. This study focused on determining if these long-term effects/multi-organ failures that have been observed after infection are occurring due to molecular mimicry. Initially, we identified -2500 human peptides (9 and 15 amino acids) with at least 50% similarity within the SARS-CoV-2 spike protein sequences. The proteins that these peptides are a part of were triaged to 26 human endogenous proteins highly expressed in heart, kidney and brain tissues that could potentially lead to immune-mediated adverse events. The present example will describe our computational approach that can be used by infectious disease, vaccine and drug safety scientists for molecular mimicry risk assessment.
[0208] The methods used include:
[0209] Step A) Ranking Strategy: Overlaying gene and protein expression in tissues of interest, cellular location, peptide homology
[0210] Step B) Peptide Homology Search a. Blasting spike protein sequences against human proteome b. 9-aa with >75% similarity (for MHC I predictions) c. 15-aa with >50% similarity (for MHC II predictions)
[0211] Step C) Antigenicity Predictions: Determining binding capability of human peptides to MHC Class I and II molecules using Immune epitope database (IEDB)
[0212] In some embodiments, the method includes steps in the following order: (B) Peptide homology search followed by (A) Ranking strategy. In some embodiments, the method includes steps in the following order: (B) Peptide homology search followed by (C) Antigenicity predictions. In some embodiments, the method further includes in the following order the step of (C) Antigenicity predictions followed by (A) Ranking strategy.
[0213] The Table below lists the potential mimics for COVID-19-infection associated multi-organ failures. Group of proteins in bold text were further considered.
Table 6
[0214] We investigated molecular mimicry as potential pathogenesis of multi organ failures after SARS-CoV-2 infection. Based on our bottom-up approach, we prioritized potential “mimics” proteins that may play a role in cross-reactive adaptive immunity. 2470 shared human and viral peptides 26 “common” proteins with higher tissue expression and MHC hits 4 groups of proteins identified for hypothesis generation DVPIGAIIC (SEQ ID NO: 138) peptide from DLAT protein may be a potential mimic.
[0215] The table below shows exemplary peptide sequences obtained through the methods described in Examples 1 and 2:
Tab e 7
[0216] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.

Claims

CLAIMS What is claimed is:
1. An immunogenic composition for administration to a host, the composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, a. wherein the modified antigenic polypeptide construct differs from an unmodified antigenic polypeptide construct at one or more antigenic peptide sequences, b. wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; c. wherein an antigenic peptide sequence of the unmodified antigenic polypeptide construct comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue, and d. wherein the modified antigenic polypeptide construct comprises an antigenic peptide sequence of 9 consecutive amino acids having less than about 75% sequence identity or 15 consecutive amino acids having less than about 50% amino acid sequence identity with the cross-reactive host peptide.
2. The immunogenic composition of claim 1, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition.
3. The immunogenic composition of claim 2, wherein the viral protein is a protein from an arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, hepevirus, orthomyxovirus, paramyxovirus, picornavirus, reovirus, retrovirus, rhabdovirus, or togavirus.
4. The immunogenic composition of any one of claims 2-3, wherein the viral protein is a viral spike protein.
5. The immunogenic composition of any one of claims 1-4, wherein the cross-reactive host polypeptide is expressed in heart, brain, kidney, liver, and/or lung tissue. The immunogenic composition of any one of claims 1-5, wherein the cross-reactive host polypeptide is expressed in heart tissue. The immunogenic composition of any one of claims 1-6, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1). The immunogenic composition of claim 7, wherein at least one cross-reactive host polypeptide is located within MYH6. The immunogenic composition of claim 8, wherein at least one cross-reactive host polypeptide is located within PCM1. The immunogenic composition of any one of claims 1-6, wherein the cross-reactive host polypeptide has the amino acid sequence according to any one of SEQ ID NO: 3-138. A method for producing an immunogenic composition comprising a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, the method comprising: a. identifying at least one antigenic peptide sequence comprised in an unmodified antigenic polypeptide construct, i. wherein the antigenic peptide sequence comprises 9 consecutive amino acids having at least about 75% sequence identity or 15 consecutive amino acids having at least about 50% sequence identity with a cross-reactive host peptide comprised in a cross-reactive host polypeptide expressed in a host tissue; and b. modifying the antigenic peptide sequence of the unmodified antigenic polypeptide construct to have less than about 50% amino acid sequence identity with the cross- reactive host peptide to form the modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct, wherein the modified antigenic polypeptide construct has at least about 50% amino acid sequence identity with the unmodified antigenic polypeptide construct; and wherein modifying the antigenic peptide sequence to have less than about 50% identity with the cross-reactive host peptide reduces or prevents cross-reactivity of the immunogenic composition to the cross-reactive host peptide compared to the cross-reactivity of an immunogenic composition comprising an unmodified antigenic polypeptide construct having an unmodified antigenic peptide sequence. The method of claim 10, wherein the antigenic polypeptide construct is a viral protein, a bacterial protein, a protein of a host parasite, a fungal protein, a cancer protein, or a protein wherein reduction of the concentration of the protein treats a disease or condition. The method of any one of claims 10-12, wherein the cross-reactive host polypeptide is located within the following proteins: myosin 6 (MYH6), ankyrin repeat domain 1 (ANKRD1), synaptopodin 2-like protein (SYNPO2L), myosin 7 (MYH7), ankyrin repeat and SOCS box containing 15 (ASB15), cysteine and glycine rich protein 3 (CSRP3), desmoplakin (DSP), oxoglutarate dehydrogenase (OGDH), ATP synthase-coupling factor 6, mitochondrial (ATP5PF), and/or pericentriolar material 1 protein (PCM1). The method of claim 13, wherein at least one cross-reactive host polypeptide is located within MYH6. The method of claim 14, wherein at least one cross-reactive host polypeptide is located within PCM1. A method identifying a potential toxicity of an immunogenic composition in a biological host system, the immunogenic composition comprising an antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct, the method comprising: a. identifing one or more proteins of the host that have a degree of similarity to the antigenic polypeptide construct; b. identifying properties of the one or more proteins, the properties comprising cellular location, gene- and protein-tissue expression profiles, and/or toxic state associations; c. ranking or sorting the one or more proteins using one or more of the properties; d. identifying immunogenicity of the one or more proteins; e. comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence; f. predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence; wherein one or more of the steps are performed by a computational model. The method of claim 16, wherein the potential toxicity is myocarditis or pericarditis. The method of any one of claims 16-17, wherein the method comprises searching a human proteome with a spike protein peptide and identifying a protein from the human proteome with homology to the spike protein peptide. The method of any one of claims 16-18, further comprising identifying whether the protein from the human proteome with homology to the spike protein peptide is present in a target organ. The method any one of claims 16-19, wherein ranking or sorting the one or more proteins using one or more of the properties comprises ranking cellular locations that are extracellular or on a surface of a cell as more likely to cause the potential toxicity than intracellular locations, ranking gene- and protein-tissue expression profiles that overlap in tissue location of the potential toxicity as more likely to cause the potential toxicity than other tissue locations, and/or ranking toxic state associations that overlap or are related to the potential toxicity as more likely to cause the potential toxicity than other toxic state associations. The method any one of claims 16-20, wherein identifying immunogenicity of the one or more proteins comprises identifying a ranking or sorting of degree of immunogenicity and/or degree of potential immunogenicity. The method of claim 21, wherein comparing the ranking or sorting in step c. with the immunogenicity of the one or more proteins in step d to produce a degree of correspondence comprises comparing the rank of likely to cause the potential toxicity and degree of immunogenicity and/or degree of potential immunogenicity and ranking: more likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity, and either of less likely to cause the potential toxicity and greater degree of immunogenicity and/or degree of potential immunogenicity or more likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity as a greater degree of correspondence than less likely to cause the potential toxicity and less degree of immunogenicity and/or degree of potential immunogenicity. The method claim 22, wherein predicting which of the one or more proteins are the most likely to cause a potential toxicity based on the degree of correspondence comprises predicting a greater degree of correspondence as more likely to cause a potential toxicity. The method any one of claims 16-23, wherein each of the steps are performed on a computational model. The method of any one of claims 16-24, further comprising modifying the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct to produce a modified antigenic polypeptide construct and/or a nucleic acid encoding the modified antigenic polypeptide construct that is less likely to cause the potential toxicity when compared to the antigenic polypeptide construct and/or a nucleic acid encoding the antigenic polypeptide construct.
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