WO2026030253A1 - Vaccins contre le papillomavirus humain (vph) - Google Patents

Vaccins contre le papillomavirus humain (vph)

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Publication number
WO2026030253A1
WO2026030253A1 PCT/US2025/039540 US2025039540W WO2026030253A1 WO 2026030253 A1 WO2026030253 A1 WO 2026030253A1 US 2025039540 W US2025039540 W US 2025039540W WO 2026030253 A1 WO2026030253 A1 WO 2026030253A1
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WIPO (PCT)
Prior art keywords
polypeptide
hpv
sequence
derived
set forth
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.)
Pending
Application number
PCT/US2025/039540
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English (en)
Inventor
Jeffrey TEIGLER
Ciaran Daniel SCALLAN
Amy Rachel Rappaport
Karin Jooss
Italo Faria DO VALLE
Joshua KLEIN
Ankur DHANIK
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.)
Gritstone Bio Inc
Original Assignee
Gritstone Bio Inc
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Publication date
Application filed by Gritstone Bio Inc filed Critical Gritstone Bio Inc
Publication of WO2026030253A1 publication Critical patent/WO2026030253A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA 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
    • A61K39/12—Viral antigens
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • 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
    • A61K2039/572—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 cytotoxic response
    • 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
    • A61K2039/70—Multivalent vaccine
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20022—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
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/20011—Papillomaviridae
    • C12N2710/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • 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
    • C12N2770/36011—Togaviridae
    • C12N2770/36111—Alphavirus, e.g. Sindbis virus, VEE, EEE, WEE, Semliki
    • C12N2770/36122—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
    • C12N2820/00—Vectors comprising a special origin of replication system
    • C12N2820/60—Vectors comprising a special origin of replication system from viruses

Definitions

  • HPV human papillomavirus
  • HPV human papillomavirus
  • a human papillomavirus (HPV) vaccine comprising at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide, wherein the encoded immunogenic polypeptide comprises: - at least one MHC class I epitope comprising a polypeptide sequence as set forth in any one of Tables C1-C8; or
  • HPV-derived E6/E7 fusion protein comprising an HPV E6 derived polypeptide and an HPV E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the polypeptide sequences shown in Table A; or
  • compositions for delivery of an antigen expression system comprising (a) one or more vectors, the one or more vectors comprising a vector backbone, wherein the vector backbone comprises (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence; and (b) an antigen cassette, optionally wherein the antigen cassette is inserted into the vector backbone when present, and wherein the antigen cassette comprises:
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C8; or - any one of the polypeptide sequences of the cassette sequences set forth in Table D; or
  • HPV-derived E6/E7 fusion protein comprising an HPV E6 derived polypeptide and an HPV E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the polypeptide sequences shown in Table A; or
  • each of the HPV derived nucleic acid sequences comprises; (I) optionally, a 5’ linker sequence, and (II) optionally, a 3’ linker sequence; (ii) optionally, a second promoter nucleotide sequence operably linked to one or more of the three HPV derived nucleic acid sequences; and (iii) optionally, at least one MHC class II epitope-encoding nucleic acid sequence; (iv) optionally, at least one nucleic acid sequence encoding a GPGPG amino acid linker sequence (SEQ ID NO:56); and (v) optionally, at least one second poly(A) sequence, wherein the second poly(A) sequence is a native poly(A) sequence or an exogenous poly(A) sequence to the vector backbone optionally wherein the exogenous poly(A) sequence comprises an SV40 poIy(A) signal sequence or a Bovine Growth Hormone (BGH) poly(A) signal sequence.
  • BGH Bovine Growth Hormone
  • compositions for delivery of an antigen expression system comprising (a) one or more vectors, the one or more vectors comprising a vector backbone, wherein the vector backbone comprises a chimpanzee adenovirus vector, optionally wherein the chimpanzee adenovirus vector is a ChAdV68 vector, or an alphavirus vector, optionally wherein the alphavirus vector is a Venezuelan equine encephalitis virus vector, and wherein the vector backbone comprises (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone such that the antigen cassette is operably linked to the at least one promoter nucleotide sequence, and wherein the antigen cassette comprises:
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C2; or - at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C3, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C3; or
  • HPV-derived E6/E7 fusion protein comprising an HPV E6 derived polypeptide and an HPV E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the polypeptide sequences shown in Table A; or
  • compositions for delivery of an antigen expression system comprising: the antigen expression system, wherein the antigen expression system comprises: (a) a vector backbone, wherein the vector backbone comprises an alphavirus vector, optionally wherein the alphavirus vector is a Venezuelan equine encephalitis virus vector, and wherein the vector backbone comprises: (i) a subgenomic promoter nucleotide sequence, and (ii) a polyadenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone such that the antigen cassette is operably linked to the subgenomic promoter nucleotide sequence, and wherein the antigen cassette comprises, in order from 5’ to 3’: (i) a nucleotide sequence encoding a first HPV-derived nucleic acid sequence; (ii) a 2A ribosome skipping sequence element; and (iii) a nucleo
  • compositions for delivery of an antigen expression system comprising the antigen expression system, wherein the antigen expression system comprises (a) a vector backbone, wherein the vector backbone comprises an alphavirus vector, optionally wherein the alphavirus vector is a Venezuelan equine encephalitis virus vector, and wherein the vector backbone comprises (i) a first subgenomic promoter nucleotide sequence, and (ii) a polyadenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone such that the antigen cassette is operably linked to the subgenomic promoter nucleotide sequence, and wherein the antigen cassette comprises, in order from 5’ to 3’: (i) a nucleotide sequence encoding a first HPV-derived nucleic acid sequence; (ii) a second subgenomic promoter nucleotide sequence; and (iii) a nucle
  • the at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide comprises:
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables Cl, and an HPV16-derived E6/E7 fusion protein comprising an HPV 16 E6 derived polypeptide and an HPV 16 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV16-derived polypeptide sequences shown in Table A; or - at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C2, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C2, and an HPV18-derived E6/E7 fusion protein comprising an HPV18 E6 derived polypeptide and an HPV18 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV18-derived polypeptide sequences shown in Table
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C3, and an HPV31 -derived E6/E7 fusion protein comprising an HPV31 E6 derived polypeptide and an HPV31 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV31 -derived polypeptide sequences shown in Table A; or
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C4, and an HPV33-derived E6/E7 fusion protein comprising an HPV33 E6 derived polypeptide and an HPV33 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV33-derived polypeptide sequences shown in Table A; or
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C5, and an HPV35-derived E6/E7 fusion protein comprising an HPV35 E6 derived polypeptide and an HPV35 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV35-derived polypeptide sequences shown in Table A; or
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C6, and an HPV45-derived E6/E7 fusion protein comprising an HPV45 E6 derived polypeptide and an HPV45 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV45-derived polypeptide sequences shown in Table A; or
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C7, and an HPV52-derived E6/E7 fusion protein comprising an HPV52 E6 derived polypeptide and an HPV52 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV52-derived polypeptide sequences shown in Table A; or
  • the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C8, and an HPV58-derived E6/E7 fusion protein comprising an HPV58 E6 derived polypeptide and an HPV58 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV58-derived polypeptide sequences shown in Table A; or
  • the at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide comprises at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables Cl, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables Cl, and an HPV16-derived E6/E7 fusion protein comprising an HPV16 E6 derived polypeptide and an HPV 16 E7 derived polypeptide, optionally wherein the HPV-derived fusion protein comprises any one of the HPV16-derived polypeptide sequences shown in Table A; and at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C2, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C2, and an HPV18-derived E6/E7 fusion protein comprising an HPV18 E6 derived polypeptide and an HPV18 E7 derived polypeptide, optionally where
  • the at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables Cl, and an HPV16-derived E6/E7 fusion protein comprising an HPV16 E6 derived polypeptide and an HPV16 E7 derived polypeptide, wherein the HPV-derived fusion protein comprises any one of the HPV16-derived polypeptide sequences shown in Table A; and each of the polypeptide sequences as set forth in Tables C2, and an HPV 18-derived E6/E7 fusion protein comprising an HPV18 E6 derived polypeptide and an HPV18 E7 derived polypeptide, wherein the HPV-derived fusion protein comprises any one of the HPV18-derived polypeptide sequences shown in Table A.
  • the at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide comprises at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables Cl, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables Cl; and at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C2, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C2.
  • the at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide comprises: at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables Cl, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables Cl; and at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C2, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C2; and at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C5, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C5; and at least one MHC class I epitope comprising a polypeptide sequence as set forth in Tables C6, optionally wherein the immunogenic polypeptide comprises each of the polypeptide sequences as set forth in Tables C6.
  • a HPV vaccine comprising: (A) one or more vectors, wherein the one or more vectors comprises: (a) an RNA alphavirus backbone, wherein the RNA alphavirus backbone comprises: (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone, and wherein the antigen cassette comprises: (i) one or more of the HPV derived nucleic acid sequences provided herein, wherein the immunogenic polypeptide optionally comprises a N-terminal linker and/or a C-terminal linker; (ii) optionally, a second promoter nucleotide sequence operably linked to the HPV derived nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-
  • the composition for delivery of the self-amplifying alphavirus-based expression system comprises at least lOpg or 20 pg of each of the one or more vectors. In some aspects, the composition for delivery of the self- amplifying alphavirus-based expression system comprises between 10 pg- 20 pg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises 20pg or less of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises between 10-30pg or between 10-100pg of each of the one or more vectors.
  • the composition comprises at least two HPV-derived nucleic acid sequences, wherein a first HPV-derived nucleic acid sequence encodes an immunogenic polypeptide derived from a first HPV strain and a second HPV-derived nucleic acid sequence encodes an immunogenic polypeptide derived from a second HPV strain.
  • the first HPV strain and the second HPV strain are each selected from the group consisting of: HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58.
  • the first HPV strain and the second HPV strain are HPV16 and HPV18.
  • the immunogenic polypeptide is derived from an HPV early (E) protein selected from the group consisting of: HPV El, HPV E2, HPV E5, HPV E6, HPV E7, and combinations thereof.
  • the composition comprises the HPV derived nucleic acid sequence encodes at least one immunogenic polypeptide derived from each of HPV El, HPV E2, HPV E5, HPV E6, and HPV E7.
  • the composition further comprises a non-HPV derived nucleic acid sequence encoding at least one immunogenic polypeptide derived from an infectious disease other than HPV, optionally wherein the infectious disease is selected from the group consisting of: a pathogen, a virus, a bacteria, a fungus, and a parasite.
  • the RNA alphavirus backbone comprises one or more elements obtained from the sequence of SEQ ID NO: 3 or SEQ ID NO:5, optionally wherein the one or more elements are selected from the group consisting of the sequences necessary for nonstructural protein-mediated amplification, the 26S promoter nucleotide sequence, the poly (A) sequence, and the nsPl-4 genes of the sequence set forth in SEQ ID NO: 3 or SEQ ID NO:5, optionally the RNA alphavirus backbone comprises the sequence set forth in the sequences selected from the group consisting of SEQ ID NOs:6-9.
  • a method for stimulating an immune response in a subject comprising administering to the subject the composition for delivery of the selfamplifying alphavirus-based expression systems provided herein.
  • the method comprises administering at least two doses of the composition for delivery of the self-amplifying alphavirus-based expression system.
  • the at least two doses comprises a priming dose and at least one boosting dose.
  • the at least two doses are administered on days 1 and day 28 or later.
  • the at least two doses are administered on days 1 and on or after week 4.
  • the at least two doses comprise the same antigen cassette.
  • the method further comprises administration of a chimpanzee adenovirus (ChAdV)-based expression system, wherein the composition for delivery of the ChAdV-based expression system comprises the ChAdV-based expression system, wherein the ChAdV-based expression system comprises a viral particle comprising a ChAdV vector, wherein the ChAdV vector comprises (a) a ChAdV backbone, wherein the ChAdV backbone comprises: (i) at least one promoter nucleotide sequence, and (ii) at least one poly adenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone, and wherein the antigen cassette comprises at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide.
  • ChAdV chimpanzee adenovirus
  • the ChAdV-based expression system is administered as a priming dose.
  • the antigen cassette of the ChAdV-based expression system is the same as the antigen cassette of the self-amplifying alphavirus-based expression system.
  • a HPV vaccine comprising a chimpanzee adenovirus (ChAdV)-based expression system
  • the composition for delivery of the ChAdV-based expression system comprises: (a) a ChAdV backbone, wherein the ChAdV backbone comprises (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence; and (b) an antigen cassette, wherein the antigen cassette is inserted into the vector backbone, and wherein the antigen cassette comprises (i) one or more of the HPV derived nucleic acid sequences provided herein, wherein the immunogenic polypeptide optionally comprises a N-terminal linker and/or a C-terminal linker; (ii) optionally, a second promoter nucleotide sequence operably linked to the HPV derived nucleic acid sequence; and (iii) optionally, at least one MHC class II epitope-en
  • the composition for delivery of the ChAdV-based expression system comprises at least IxlO 11 of the viral particles. In some aspects, the composition for delivery of the ChAdV-based expression system comprises between IxlO 11 and IxlO 12 . In some aspects, the composition for delivery of the ChAdV-based expression system comprises IxlO 11 , 3xl0 n , or vp/mL.
  • the ChAdV backbone comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1, wherein the nucleotides 2 to 36,518 lack: (1) nucleotides 577 to 3403 of the sequence shown in SEQ ID NO: 1 corresponding to an El deletion; (2) nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO:1 corresponding to an E3 deletion; and (3) optionally nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1 corresponding to a partial E4 deletion; optionally wherein the antigen cassette is inserted within the El deletion.
  • Also provided herein is a method for stimulating an immune response in a subject, the method comprising administering to the subject the composition for delivery of the ChAdV- based expression systems provided herein.
  • the ChAdV-based expression system is administered as a priming dose.
  • the method further comprises administration of a composition for delivery of a self-amplifying alphavirus-based expression system, wherein the composition for delivery of the self-amplifying alphavirus-based expression system comprises (A) the selfamplifying alphavirus-based expression system, wherein the self-amplifying alphavirus-based expression system comprises one or more vectors, wherein the one or more vectors comprises
  • RNA alphavirus backbone comprising (i) at least one promoter nucleotide sequence, and (ii) at least one polyadenylation (poly(A)) sequence;
  • an antigen cassette wherein the antigen cassette is inserted into the vector backbone, and wherein the antigen cassette comprises at least one HPV derived nucleic acid sequence encoding an immunogenic polypeptide.
  • the antigen cassette of the ChAdV-based expression system is the same as the antigen cassette of the self- amplifying alphavirus-based expression system.
  • composition for delivery of the expression system is formulated in a pharmaceutical composition comprising a pharmaceutically acceptable carrier.
  • kits comprising the composition for delivery of the expression systems provided herein.
  • Also provided herein is a method for stimulating an immune response in a subject, the method comprising administering to the subject any one of the HPV vaccines provided herein.
  • a method for stimulating an immune response in a subject comprising administering to the subject any one of the HPV vaccines provided herein.
  • the subject is at risk for one or more of cervical cancer, anal cancer, oropharyngeal cancer, oral cancer, penile cancer, vaginal cancer, and/or vulvar cancer.
  • the subject is diagnosed as HPV positive.
  • the subject is diagnosed as having cervical intraepithelial neoplasia (CIN1).
  • a method for stimulating an immune response in a subject comprising administering to the subject a composition for delivery of a selfamplifying alphavirus-based expression system and administering to the subject a composition for delivery of a chimpanzee adenovirus (ChAdV)-based expression system, and wherein either: a. the composition for delivery of the ChAdV-based expression system comprises the ChAdV- based expression system, wherein the ChAdV-based expression system comprises a viral particle comprising a ChAdV vector, and wherein the composition comprises IxlO 12 or less of the viral particles, b.
  • the composition for delivery of the ChAdV-based expression system comprises the ChAdV- based expression system
  • the ChAdV-based expression system comprises a viral particle comprising a ChAdV vector
  • the composition comprises IxlO 12 or less of the viral particles
  • composition for delivery of the self-amplifying alphavirusbased expression system comprises the self-amplifying alphavirus-based expression system, wherein the self-amplifying alphavirus-based expression system comprises one or more vectors, and wherein the composition comprises at least lOpg of each of the one or more vectors, or c.
  • the composition for delivery of the ChAdV-based expression system comprises the ChAdV- based expression system, wherein the ChAdV-based expression system comprises a viral particle comprising a ChAdV vector, and wherein the composition comprises IxlO 12 or less of the viral particles and wherein the composition for delivery of the self-amplifying alphavirusbased expression system comprises the self-amplifying alphavirus-based expression system, wherein the self-amplifying alphavirus-based expression system comprises one or more vectors, and wherein the composition comprises at least lOpg of each of the one or more vectors.
  • composition for delivery of the ChAdV-based expression system is administered as a priming dose and the composition for delivery of the self-amplifying alphavirus-based expression system is administered as one or more boosting doses.
  • the composition for delivery of the self-amplifying alphavirus-based expression system comprises at least 3 pg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises at least lOpg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises at least 30pg of each of the one or more vectors. In some aspects, the composition for delivery of the selfamplifying alphavirus-based expression system comprises at least lOOpg of each of the one or more vectors.
  • the composition for delivery of the self-amplifying alphavirusbased expression system comprises at least 300pg of each of the one or more vectors. In some aspects, the composition for delivery of the self- amplifying alphavirus-based expression system comprises at least 400 pg, at least 500pg, at least 600pg, at least 700pg, at least 800pg, at least 900(ig, at least lOOOpg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises between 10-30pg, 10-lOOpg, 10-300pg, 30-100pg, 30-300
  • the composition for delivery of the self-amplifying alphavirus-based expression system comprises between 3-lOpg, 3-30pg, 3- lOOpg, 3-300pg, 10-30pg, or 100-300pg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises between 10-500pg, lO-lOOOpg, 30-500pg, 30- lOOOpg, or 500-1000pg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises lOpg, 30pg, lOOpg, or 300pg of each of the one or more vectors.
  • the composition for delivery of the self-amplifying alphavirus-based expression system comprises 400pg, 500 pg. 600pg, 700pg, 800pg, 900pg, or lOOOpg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises less than or equal to 300pg of each of the one or more vectors. In some aspects, the composition for delivery of the self-amplifying alphavirus-based expression system comprises less than or equal to lOOpg of each of the one or more vectors. In some aspects, the composition for delivery of the self- amplifying alphavirus-based expression system comprises less than or equal to 30pg of each of the one or more vectors.
  • a dose of can represent the total content of RNA/samRNA administered.
  • a dose of can represent the total content of RNA/samRNA administered and include only a single distinct samRNA construct.
  • an ordered sequence of one or more of the HPV derived nucleic acid sequences encoding the immunogenic polypeptide is described in the formula, from 5’ to 3’, comprising:
  • the composition further comprises a nanoparticulate delivery vehicle.
  • the nanoparticulate delivery vehicle is a lipid nanoparticle (LNP).
  • the LNP comprises ionizable amino lipids.
  • the ionizable amino lipids comprise MC3-like (dilinoleylmethyl-4-dimethylaminobutyrate) molecules.
  • the nanoparticulate delivery vehicle encapsulates the antigen expression system.
  • the antigen cassette is integrated between the at least one promoter nucleotide sequence and the at least one poly (A) sequence.
  • the at least one promoter nucleotide sequence is operably linked to the HPV derived nucleic acid sequence.
  • the one or more vectors comprise one or more +-stranded RNA
  • the one or more +-stranded RNA vectors comprise a 5’ 7-methylguanosine (m7g) cap.
  • the one or more +-stranded RNA vectors are produced by in vitro transcription.
  • the one or more vectors are self-replicating within a mammalian cell.
  • the backbone comprises at least one nucleotide sequence of an Aura virus, a Fort Morgan virus, a Venezuelan equine encephalitis virus, a Ross River virus, a Semliki Forest virus, a Sindbis virus, or a Mayaro virus. In some aspects, the backbone comprises at least one nucleotide sequence of a Venezuelan equine encephalitis virus.
  • the backbone comprises at least sequences for nonstructural protein-mediated amplification, a 26S promoter sequence, a poly(A) sequence, a nonstructural protein 1 (nsPl) gene, a nsP2 gene, a nsP3 gene, and a nsP4 gene encoded by the nucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus.
  • nsPl nonstructural protein 1
  • the backbone comprises at least sequences for nonstructural protein-mediated amplification, a 26S promoter sequence, and a poly(A) sequence encoded by the nucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus.
  • sequences for nonstructural protein- mediated amplification are selected from the group consisting of: an alphavirus 5’ UTR, a 51-nt CSE, a 24-nt CSE, a 26S subgenomic promoter sequence, a 19-nt CSE, an alphavirus 3’ UTR, or combinations thereof.
  • the backbone does not encode structural virion proteins capsid, E2 and El.
  • the antigen cassette is inserted in place of structural virion proteins within the nucleotide sequence of the Aura virus, the Fort Morgan virus, the Venezuelan equine encephalitis virus, the Ross River virus, the Semliki Forest virus, the Sindbis virus, or the Mayaro virus.
  • the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5.
  • the Venezuelan equine encephalitis virus comprises the sequence of SEQ ID NO:3 or SEQ ID NO:5 further comprising a deletion between base pair 7544 and 11176.
  • the backbone comprises the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.
  • the antigen cassette is inserted at position 7544 to replace the deletion between base pairs 7544 and 11176 as set forth in the sequence of SEQ ID NO:3 or SEQ ID NO:5.
  • the insertion of the antigen cassette provides for transcription of a polycistronic RNA comprising the nsPl-4 genes and the at least one HPV derived nucleic acid sequence, wherein the nsPl-4 genes and the at least one HPV derived nucleic acid sequence are in separate open reading frames.
  • the at least one promoter nucleotide sequence is the native 26S promoter nucleotide sequence encoded by the backbone.
  • the backbone comprises at least one nucleotide sequence of a chimpanzee adenovirus vector, optionally wherein the chimpanzee adenovirus vector is a ChAdV68 vector.
  • the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO:1.
  • the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO: 1, except that the sequence is fully deleted or functionally deleted in at least one gene selected from the group consisting of the chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO: 1, optionally wherein the sequence is fully deleted or functionally deleted in: (1) E1A and E1B; (2) El A, E1B, and E3; or (3) El A, E1B, E3, and E4 of the sequence set forth in SEQ ID NO:1.
  • the ChAdV68 vector backbone comprises a gene or regulatory sequence obtained from the sequence of SEQ ID NO: 1, optionally wherein the gene is selected from the group consisting of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO:1.
  • the ChAdV68 vector backbone comprises a partially deleted E4 gene comprising a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region.
  • the ChAdV68 vector backbone comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1 and further comprising: (1) an El deletion of at least nucleotides 577 to 3403 of the sequence shown in SEQ ID NO:1, (2) an E3 deletion of at least nucleotides 27,125 to 31,825 of the sequence shown in SEQ ID NO: 1, and (3) an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1; optionally wherein the antigen cassette is inserted within the El deletion.
  • the ChAdV68 vector backbone comprises the sequence set forth in SEQ ID NO:57 (“chAd68-Empty-E4deleted”), optionally wherein the antigen cassette is inserted within the El deletion.
  • the ChAdV68 vector backbone comprises one or more deletions between base pair number 577 and 3403 or between base pair 456 and 3014, and optionally wherein the vector further comprises one or more deletions between base pair 27,125 and 31,825 or between base pair 27,816 and 31,333 of the sequence set forth in SEQ ID NO:1.
  • the ChAdV68 vector backbone comprises one or more deletions between base pair number 3957 and 10346, base pair number 21787 and 23370, and base pair number 33486 and 36193 of the sequence set forth in SEQ ID NO:1.
  • the wherein the cassette is inserted in the ChAdV backbone at the El region, E3 region, and/or any deleted AdV region that allows incorporation of the cassette.
  • the ChAdV backbone is generated from one of a first generation, a second generation, or a helper-dependent adenoviral vector
  • the at least one promoter nucleotide sequence is selected from the group consisting of: a CMV, a SV40, an EF-1, a RSV, a PGK, a HSA, a MCK, and a EBV promoter sequence.
  • the at least one promoter nucleotide sequence is a CMV promoter sequence.
  • the at least one promoter nucleotide sequence is an exogenous RNA promoter.
  • the second promoter nucleotide sequence is a 26S promoter nucleotide sequence or a CMV promoter nucleotide sequence.
  • the second promoter nucleotide sequence comprises multiple 26S promoter nucleotide sequences or multiple CMV promoter nucleotide sequences, wherein each 26S promoter nucleotide sequence or CMV promoter nucleotide sequence provides for transcription of one or more of the separate open reading frames.
  • one or more of the cassettes are at least 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides in length. In some aspects, one or more of the cassettes are at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, or 10000 nucleotides in length. In some aspects, the one or more vectors are capable of driving expression of a cassette that is at least 3500 nucleotides in length. In some aspects, the one or more vectors are capable of driving expression of a cassette that is at least 6000 nucleotides in length.
  • At least one of the at least one HPV derived nucleic acid sequences encodes a polypeptide sequence or portion thereof that is presented by MHC class I. In some aspects, at least one of the at least one HPV derived nucleic acid sequences encodes a polypeptide sequence or portion thereof that is presented by MHC class II. In some aspects, at least one of the at least one HPV derived nucleic acid sequences encodes a polypeptide sequence or portion thereof capable of stimulating a B cell response, optionally wherein the polypeptide sequence or portion thereof capable of stimulating a B cell response comprises a full-length protein, a protein domain, a protein subunit, or an antigenic fragment predicted or known to be capable of being bound by an antibody.
  • the linker links
  • the linker is selected from the group consisting of: (1) consecutive glycine residues, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (2) consecutive alanine residues, at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 residues in length; (3) two arginine residues (RR); (4) alanine, alanine, tyrosine (AAY); (5) a consensus sequence at least 2, 3, 4, 5, 6, 7, 8 , 9, or 10 amino acid residues in length that is processed efficiently by a mammalian proteasome; (6) one or more native sequences flanking the antigen derived from the cognate protein of origin and that is at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 2-20 amino acid residues in length; and (7) a furin or TEV cleavage sequence.
  • the linker links two MHC class II sequences or an MHC class II sequence to an MHC class I sequence.
  • At least one sequence of the at least one HPV derived nucleic acid sequences is linked, operably or directly, to a separate or contiguous sequence that enhances the expression, stability, cell trafficking, processing and presentation, and/or immunogenicity of the at least one HPV derived nucleic acid sequences.
  • the separate or contiguous sequence comprises at least one of: a ubiquitin sequence, a ubiquitin sequence modified to increase proteasome targeting (e.g., the ubiquitin sequence contains a Gly to Ala substitution at position 76), an immunoglobulin signal sequence (e.g., IgK), a major histocompatibility class I sequence, lysosomal-associated membrane protein (LAMP)-l, human dendritic cell lysosomal- associated membrane protein, and a major histocompatibility class II sequence; optionally wherein the ubiquitin sequence modified to increase proteasome targeting is A76.
  • a ubiquitin sequence e.g., the ubiquitin sequence contains a Gly to Ala substitution at position 76
  • an immunoglobulin signal sequence e.g., IgK
  • a major histocompatibility class I sequence e.g., lysosomal-associated membrane protein (LAMP)-l, human dendritic cell ly
  • At least one of the at least one HPV derived nucleic acid sequences encodes two or more distinct polypeptides predicted or validated to be capable of presentation by at least one HLA allele.
  • each of the at least one HPV derived nucleic acid sequences encodes a polypeptide sequence or portion thereof that is less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 45%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, or less than 33% of the translated, corresponding full-length HPV protein.
  • each of the at least one HPV derived nucleic acid sequences encodes a polypeptide sequence or portion thereof that does not encode a functional protein, functional protein domain, functional protein subunit, or functional protein fragment of the translated, corresponding HPV protein.
  • two or more of the at least one HPV derived nucleic acid sequences are derived from the same HPV gene.
  • the two or more HPV derived nucleic acid sequences derived from the same HPV gene are ordered such that a first nucleic acid sequence cannot be immediately followed by or linked to a second nucleic acid sequence if the second nucleic acid sequence follows first nucleic acid sequence in the corresponding HPV gene.
  • the at least one HPV derived nucleic acid sequence comprises at least 2-10, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleic acid sequences. In some aspects, the at least one HPV derived nucleic acid sequence comprises at least 11-20, 15-20, 11-100, 11-200, 11-300, 11-400, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or up to 400 nucleic acid sequences. In some aspects, the at least one HPV derived nucleic acid sequence comprises at least 2-400 nucleic acid sequences and wherein at least two of the HPV derived nucleic acid sequences encode polypeptide sequences or portions thereof that are (1) presented by MHC class I, (2) presented by MHC class II, and/or (3) capable of stimulating a B cell response. In some aspects, at least two of the HPV derived nucleic acid sequences encode polypeptide sequences or portions thereof that are (1) presented by MHC class I, (2) presented by MHC class II, and/or (3) capable of stimulating a B cell response class.
  • At least one of the antigens encoded by the at least one HPV derived nucleic acid sequence are presented on antigen presenting cells resulting in an immune response targeting at least one of the antigens on a HPV infected cell surface.
  • at least one of the antigens encoded by the at least one HPV derived nucleic acid sequence results in an antibody response targeting at least one of the antigens on a HPV virus.
  • the at least one HPV derived nucleic acid sequences when administered to the subject and translated, at least one of the MHC class I or class II antigens are presented on antigen presenting cells resulting in an immune response targeting at least one of the antigens on a HPV infected cell surface, and optionally wherein the expression of each of the at least one HPV derived nucleic acid sequences is driven by the at least one promoter nucleotide sequence.
  • each MHC class I epitope-encoding HPV derived nucleic acid sequence encodes a polypeptide sequence between 8 and 35 amino acids in length, optionally 9- 17, 9-25, 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 or 35 amino acids in length.
  • the at least one MHC class II epitopeencoding nucleic acid sequence is present.
  • the at least one MHC class II epitope-encoding nucleic acid sequence is present and comprises at least one MHC class II HPV derived nucleic acid sequence.
  • the at least one MHC class II epitope-encoding nucleic acid sequence is 12-20, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 20-40 amino acids in length. In some aspects, the at least one MHC class II epitope-encoding nucleic acid sequence is present and comprises at least one universal MHC class II epitope-encoding nucleic acid sequence, optionally wherein the at least one universal sequence comprises at least one of Tetanus toxoid and PADRE, and/or at least one MHC class II HPV derived epitope-encoding nucleic acid sequence.
  • the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is inducible. In some aspects, the at least one promoter nucleotide sequence or the second promoter nucleotide sequence is non- inducible. In some aspects, the at least one poly(A) sequence comprises a poly(A) sequence native to the backbone. In some aspects, the at least one poly(A) sequence comprises a poly(A) sequence exogenous to the backbone.
  • the at least one poly(A) sequence is operably linked to at least one of the at least one HPV derived nucleic acid sequences.
  • the at least one poly(A) sequence is at least 20 , at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, or at least 90 consecutive A nucleotides.
  • the at least one poly(A) sequence is at least 80 consecutive A nucleotides.
  • the at least one second poly(A) sequence is present.
  • the at least one second poly(A) sequence comprises an SV40 poly(A) signal sequence or a Bovine Growth Hormone (BGH) poly(A) signal sequence, or a combination of two more SV40 poly (A) signal sequences or BGH poly (A) signal sequence.
  • the at least one second poly(A) sequence comprises two or more second poly(A) sequences, optionally wherein the two or more second poly(A) sequences comprises two or more SV40 poly(A) signal sequences two or more BGH poly(A) signal sequences, or a combination of SV40 poly(A) signal sequences and BGH poly(A) signal sequences.
  • the antigen cassette further comprises at least one of: an intron sequence, an exogenous intron sequence, a Constitutive Transport Element (CTE), a RNA Transport Element (RTE), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence, an internal ribosome entry sequence (IRES) sequence, a nucleotide sequence encoding a 2A self cleaving peptide sequence, a nucleotide sequence encoding a Furin cleavage site, or a sequence in the 5 ’ or 3 ’ non-coding region known to enhance the nuclear export, stability, or translation efficiency of mRNA that is operably linked to at least one of the at least one HPV derived nucleic acid sequences.
  • CTE Constitutive Transport Element
  • RTE RNA Transport Element
  • WPRE woodchuck hepatitis virus posttranscriptional regulatory element
  • IVS internal ribosome entry sequence
  • the antigen cassette further comprises a reporter gene, including but not limited to, green fluorescent protein (GFP), a GFP variant, secreted alkaline phosphatase, luciferase, a luciferase variant, or a detectable peptide or epitope.
  • GFP green fluorescent protein
  • the detectable peptide or epitope is selected from the group consisting of an HA tag, a Flag tag, a His-tag, or a V5 tag.
  • the one or more vectors further comprises one or more nucleic acid sequences encoding at least one immune modulator.
  • the immune modulator is an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD-1 antibody or an antigen-binding fragment thereof, an anti-PD-Ll antibody or an antigen-binding fragment thereof, an anti-4- IBB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof.
  • the antibody or antigenbinding fragment thereof is a Fab fragment, a Fab’ fragment, a single chain Fv (scFv), a single domain antibody (sdAb) either as single specific or multiple specificities linked together (e.g., camelid antibody domains), or full-length single-chain antibody (e.g., full-length IgG with heavy and light chains linked by a flexible linker).
  • the heavy and light chain sequences of the antibody are a contiguous sequence separated by either a self-cleaving sequence such as 2A or IRES ; or the heavy and light chain sequences of the antibody are linked by a flexible linker such as consecutive glycine residues.
  • the immune modulator is a cytokine.
  • the cytokine is at least one of IL-2, IL-7, IL-12, IL-15, or IL-21 or variants thereof of each.
  • a MHC class I or MHC class II epitope-encoding HPV derived nucleic acid sequence is selected by performing the steps of: (a) obtaining at least one of exome, transcriptome, or whole genome HPV nucleotide sequencing data from a HPV virus or HPV infected cell, wherein the HPV nucleotide sequencing data is used to obtain data representing peptide sequences of each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on a HPV infected cell surface, the set of numerical likelihoods having been identified at least based on received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens which are used to generate the MHC class I or MHC class II epitope-encoding HP
  • each MHC class I or MHC class II epitope-encoding HPV derived nucleic acid sequences is selected by performing the steps of: (a) obtaining at least one of exome, transcriptome, or whole genome HPV nucleotide sequencing data from a HPV virus or HPV infected cell, wherein the HPV nucleotide sequencing data is used to obtain data representing peptide sequences of each of a set of antigens; (b) inputting the peptide sequence of each antigen into a presentation model to generate a set of numerical likelihoods that each of the antigens is presented by one or more of the MHC alleles on a HPV infected cell surface, the set of numerical likelihoods having been identified at least based on received mass spectrometry data; and (c) selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens which are used to generate the at least 18 HPV derived nucleic acid sequences.
  • a number of the set of selected antigens is 2-20.
  • the presentation model represents dependence between: (a) presence of a pair of a particular one of the MHC alleles and a particular amino acid at a particular position of a peptide sequence; and (b) likelihood of presentation on a HPV infected cell surface, by the particular one of the MHC alleles of the pair, of such a peptide sequence comprising the particular amino acid at the particular position.
  • selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being presented on a HPV infected cell surface relative to unselected antigens based on the presentation model, optionally wherein the selected antigens have been validated as being presented by one or more specific HLA alleles. In some aspects, selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being capable of inducing a HPV specific immune response in the subject relative to unselected antigens based on the presentation model.
  • selecting the set of selected antigens comprises selecting antigens that have an increased likelihood of being capable of being presented to naive T cells by professional antigen presenting cells (APCs) relative to unselected antigens based on the presentation model, optionally wherein the APC is a dendritic cell (DC).
  • selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of being subject to inhibition via central or peripheral tolerance relative to unselected antigens based on the presentation model.
  • selecting the set of selected antigens comprises selecting antigens that have a decreased likelihood of being capable of inducing an autoimmune response to normal tissue in the subject relative to unselected antigens based on the presentation model.
  • exome or transcriptome HPV nucleotide sequencing data is obtained by performing sequencing on a HPV virus or HPV infected tissue or cell.
  • the sequencing is next generation sequencing (NGS) or any massively parallel sequencing approach.
  • the antigen cassette comprises junctional epitope sequences formed by adjacent sequences in the antigen cassette.
  • at least one or each junctional epitope sequence has an affinity of greater than 500 nM for MHC.
  • each junctional epitope sequence is non- self.
  • each of the MHC class I and/or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele present in at least 5% of a population. In some aspects, each of the MHC class I and/or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, wherein each antigen/HLA pair has an antigen/HLA prevalence of at least 0.01% in a population.
  • each of the MHC class I and/or MHC class II epitopes is predicted or validated to be capable of presentation by at least one HLA allele, wherein each antigen/HLA pair has an antigen/HLA prevalence of at least 0.1 % in a population.
  • the antigen cassette does not encode a non-therapeutic MHC class I or class II epitope nucleic acid sequence comprising a translated, wild-type nucleic acid sequence, wherein the non-therapeutic epitope is predicted to be displayed on an MHC allele of the subject.
  • the non-therapeutic predicted MHC class I or class II epitope sequence is a junctional epitope sequence formed by adjacent sequences in the antigen cassette.
  • the prediction is based on presentation likelihoods generated by inputting sequences of the non-therapeutic epitopes into a presentation model.
  • an order of the at least one HPV derived nucleic acid sequences in the antigen cassette is determined by a series of steps comprising: (a) generating a set of candidate antigen cassette sequences corresponding to different orders of the at least one HPV derived nucleic acid sequences; (b) determining, for each candidate antigen cassette sequence, a presentation score based on presentation of non-therapeutic epitopes in the candidate antigen cassette sequence; and (c) selecting a candidate cassette sequence associated with a presentation score below a predetermined threshold as the antigen cassette sequence for an antigen vaccine.
  • compositions any of the compositions provided herein and a pharmaceutically acceptable carrier.
  • the composition further comprises an adjuvant.
  • the composition further comprises an immune modulator.
  • the immune modulator is an anti-CTLA4 antibody or an antigenbinding fragment thereof, an anti-PD- 1 antibody or an antigen-binding fragment thereof, an anti- PD-L1 antibody or an antigen-binding fragment thereof, an anti-4- IBB antibody or an antigenbinding fragment thereof, or an anti-OX-40 antibody or an antigen-binding fragment thereof.
  • an isolated nucleotide sequence or set of isolated nucleotide sequences comprising the antigen cassette of any of the compositions described herein and one or more elements obtained from the sequence of SEQ ID NO: 3 or SEQ ID NO:5, optionally wherein the one or more elements are selected from the group consisting of the sequences necessary for nonstructural protein-mediated amplification, the 26S promoter nucleotide sequence, the poly(A) sequence, and the nsPl-4 genes of the sequence set forth in SEQ ID NO:3 or SEQ ID NO: 5, and optionally wherein the nucleotide sequence is cDNA.
  • the sequence or set of isolated nucleotide sequences comprises the antigen cassette of any of the above composition claims inserted at position 7544 of the sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.
  • the isolated sequence further comprises: a T7 or SP6 RNA polymerase promoter nucleotide sequence 5’ of the one or more elements obtained from the sequence of SEQ ID NO:3 or SEQ ID NO:5; and optionally, one or more restriction sites 3’ of the poly(A) sequence.
  • the antigen cassette of any of the compositions provided herein is inserted at position 7563 of SEQ ID NO:8 or SEQ ID NO:9.
  • an isolated nucleotide sequence or set of isolated nucleotide sequences comprising the antigen cassette of any of the compositions provided herein and one or more elements obtained from the sequence of SEQ ID NO:1 or SEQ ID NO:57 (“chAd68- Empty-E4deleted”), optionally wherein the one or more elements are selected from the group consisting of the chimpanzee adenovirus inverted terminal repeat (ITR), E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4, and L5 genes of the sequence set forth in SEQ ID NO:1, and optionally wherein the nucleotide sequence is cDNA.
  • ITR chimpanzee adenovirus inverted terminal repeat
  • the sequence or set of isolated nucleotide sequences comprises the antigen cassette of any of the compositions provided herein inserted within the El deletion of the sequence set forth in SEQ ID NO:57 (“chAd68-Empty- E4deleted”).
  • the isolated sequence further comprises: a T7 or SP6 RNA polymerase promoter nucleotide sequence 5’ of the one or more elements obtained from the sequence of SEQ ID NO:1 or SEQ ID NO:57 (“chAd68-Empty-E4deleted”); and optionally, one or more restriction sites 3 ’ of the poly( A) sequence.
  • vector or set of vectors comprising any of the isolated nucleotide sequences or set of isolated nucleotide sequences provided herein.
  • an isolated cell comprising any of the isolated nucleotide sequences or set of isolated nucleotide sequences provided herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AEl-2a cell.
  • kits comprising any of the compositions provided herein and instructions for use.
  • any of the methods described herein comprises a homologous prime/boost strategy.
  • any of the methods described herein comprises a heterologous prime/boost strategy.
  • the heterologous prime/boost strategy comprises an identical antigen cassette encoded by different vaccine platforms.
  • the heterologous prime/boost strategy comprises different antigen cassettes encoded by the same vaccine platform. In some aspects, the heterologous prime/boost strategy comprises different antigen cassettes encoded by different vaccine platforms. In some aspects, the different antigen cassettes comprise a E6/E7 fusion protein-encoding cassette and a separate T cell epitope encoding cassette. In some aspects, the different antigen cassettes comprise cassettes encoding distinct epitopes and/or antigens derived from different strains of HPV.
  • a method for inducing an immune response in a subject comprising administering to the subject any of the compositions or pharmaceutical compositions provided herein.
  • the subject expresses at least one HLA allele predicted or known to present a MHC class I or MHC class II epitope encoded by the at least one HPV derived nucleic acid sequence.
  • the subject expresses at least one HLA allele predicted or known to present a MHC class I epitope encoded by the at least one HPV derived nucleic acid sequence, and wherein the MHC class I epitope comprises at least one MHC class I epitope comprising a polypeptide sequence as set forth in Table A.
  • the subject express at least one HLA allele predicted or known to present a MHC class II epitope encoded by the at least one HPV derived nucleic acid sequence, and wherein the MHC class II epitope comprises at least one MHC class II epitope comprising a polypeptide sequence as set forth in Table B.
  • the composition is administered intramuscularly (IM), intradermally (ID), subcutaneously (SC), or intravenously (IV). In some aspects, the composition is administered intramuscularly
  • the method further comprises administration of one or more immune modulators, optionally wherein the immune modulator is administered before, concurrently with, or after administration of the composition or pharmaceutical composition.
  • the one or more immune modulators are selected from the group consisting of: an anti-CTLA4 antibody or an antigen-binding fragment thereof, an anti-PD- 1 antibody or an antigen-binding fragment thereof, an anti-PD-Ll antibody or an antigen-binding fragment thereof, an anti-4-lBB antibody or an antigen-binding fragment thereof, or an anti-OX-40 antibody or an antigenbinding fragment thereof.
  • the immune modulator is administered intravenously (IV), intramuscularly (IM), intradermally (ID), or subcutaneously (SC).
  • the subcutaneous administration is near the site of the composition or pharmaceutical composition administration or in close proximity to one or more vector or composition draining lymph nodes.
  • the method further comprises administering to the subject a second vaccine composition.
  • the second vaccine composition is administered prior to the administration of the first composition or pharmaceutical composition.
  • the second vaccine composition is administered subsequent to the administration of any of the compositions or pharmaceutical compositions provided herein.
  • the second vaccine composition is the same as the first composition or pharmaceutical composition administered.
  • the second vaccine composition is different from the first composition or pharmaceutical composition administered.
  • the second vaccine composition comprises a chimpanzee adenovirus vector encoding at least one HPV derived nucleic acid sequence.
  • the at least one HPV derived nucleic acid sequence encoded by the chimpanzee adenovirus vector is the same as the at least one HPV derived nucleic acid sequence of any of the compositions provided herein.
  • a method of manufacturing the one or more vectors of any of the above composition claims comprising: (a) obtaining a linearized DNA sequence comprising the backbone and the antigen cassette; (b) in vitro transcribing the linearized DNA sequence by addition of the linearized DNA sequence to an in vitro transcription reaction containing all the necessary components to transcribe the linearized DNA sequence into RNA, optionally further comprising in vitro addition of the m7g cap to the resulting RNA; and (c) isolating the one or more vectors from the in vitro transcription reaction.
  • the linearized DNA sequence is generated by linearizing a DNA plasmid sequence or by amplification using PCR.
  • the DNA plasmid sequence is generated using one of bacterial recombination or full genome DNA synthesis or full genome DNA synthesis with amplification of synthesized DNA in bacterial cells.
  • isolating the one or more vectors from the in vitro transcription reaction involves one or more of phenol chloroform extraction, silica column based purification, or similar RNA purification methods.
  • compositions of any of the above composition claims for delivery of the antigen expression system comprising: (a) providing components for the nanoparticulate delivery vehicle; (b) providing the antigen expression system; and (c) providing conditions sufficient for the nanoparticulate delivery vehicle and the antigen expression system to produce the composition for delivery of the antigen expression system.
  • the conditions are provided by microfluidic mixing.
  • Also provided herein is a method of manufacturing an adenovirus vector disclosed herein, the method comprising: obtaining a plasmid sequence comprising the at least one promoter sequence and the antigen cassette; transfecting the plasmid sequence into one or more host cells; and isolating the adenovirus vector from the one or more host cells.
  • isolating comprises: lysing the host cell to obtain a cell lysate comprising the adenovirus vector; and purifying the adenovirus vector from the cell lysate.
  • the plasmid sequence is generated using one of bacterial recombination or full genome DNA synthesis or full genome DNA synthesis with amplification of synthesized DNA in bacterial cells.
  • the one or more host cells are at least one of CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, and AEl-2a cells.
  • purifying the adenovirus vector from the cell lysate involves one or more of chromatographic separation, centrifugation, virus precipitation, and filtration.
  • any of the above compositions further comprise a nanoparticulate delivery vehicle.
  • the nanoparticulate delivery vehicle may be a lipid nanoparticle (LNP).
  • the LNP comprises ionizable amino lipids.
  • the ionizable amino lipids comprise MC3-like (dilinoleylmethyl- 4- dimethylaminobutyrate ) molecules.
  • the nanoparticulate delivery vehicle encapsulates the antigen expression system.
  • any of the above compositions further comprise a plurality of LNPs, wherein the LNPs comprise: the antigen expression system; a cationic lipid; a non-cationic lipid; and a conjugated lipid that inhibits aggregation of the LNPs, wherein at least about 95% of the LNPs in the plurality of LNPs either: have a non-lamellar morphology; or are electron-dense.
  • the non-cationic lipid is a mixture of (1) a phospholipid and (2) cholesterol or a cholesterol derivative.
  • the conjugated lipid that inhibits aggregation of the LNPs is a polyethyleneglycol (PEG)-lipid conjugate.
  • the PEG-lipid conjugate is selected from the group consisting of: a PEG-diacylglycerol (PEG-DAG) conjugate, a PEG dialkyloxypropyl (PEG-DAA) conjugate, a PEG-phospholipid conjugate, a PEG-ceramide (PEG-Cer) conjugate, and a mixture thereof.
  • the PEG-DAA conjugate is a member selected from the group consisting of: a PEG-didecyloxypropyl (Cio) conjugate, a PEG- dilauryloxypropyl (C12) conjugate, a PEG-dimyristyloxypropyl (C14) conjugate, a PEG- dipalmityloxypropyl (Cie) conjugate, a PEG-distearyloxypropyl (Cis) conjugate, and a mixture thereof.
  • the antigen expression system is fully encapsulated in the LNPs.
  • the non-lamellar morphology of the LNPs comprises an inverse hexagonal (I I//) or cubic phase structure.
  • the cationic lipid comprises from about 10 mol % to about 50 mol % of the total lipid present in the LNPs. In some aspects, the cationic lipid comprises from about 20 mol % to about 50 mol % of the total lipid present in the LNPs. In some aspects, the cationic lipid comprises from about 20 mol % to about 40 mol % of the total lipid present in the LNPs. [0092] In some aspects, the non-cationic lipid comprises from about 10 mol % to about 60 mol % of the total lipid present in the LNPs.
  • the non-cationic lipid comprises from about 20 mol % to about 55 mol % of the total lipid present in the LNPs. In some aspects, the non-cationic lipid comprises from about 25 mol % to about 50 mol % of the total lipid present in the LNPs.
  • the conjugated lipid comprises from about 0.5 mol % to about 20 mol % of the total lipid present in the LNPs. In some aspects, the conjugated lipid comprises from about 2 mol % to about 20 mol % of the total lipid present in the LNPs. In some aspects, the conjugated lipid comprises from about 1.5 mol % to about 18 mol % of the total lipid present in the LNPs.
  • greater than 95% of the LNPs have a non-lamellar morphology. In some aspects, greater than 95% of the LNPs are electron dense.
  • any of the above compositions further comprise a plurality of LNPs, wherein the LNPs comprise: a cationic lipid comprising from 50 mol % to 65 mol % of the total lipid present in the LNPs; a conjugated lipid that inhibits aggregation of LNPs comprising from 0.5 mol % to 2 mol % of the total lipid present in the LNPs; and a non-cationic lipid comprising either: a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises from 4 mol % to 10 mol % of the total lipid present in the LNPs and the cholesterol or derivative thereof comprises from 30 mol % to 40 mol % of the total lipid present in the LNPs; a mixture of a phospholipid and cholesterol or a derivative thereof, wherein the phospholipid comprises from 3 mol % to 15 mol % of the total lipid present in the LNPs and the cholesterol or derivative thereof comprises
  • any of the above compositions further comprise a plurality of LNPs, wherein the LNPs comprise: a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the LNPs; a conjugated lipid that inhibits aggregation of LNPs comprising from 0.5 mol % to 2 mol % of the total lipid present in the LNPs; and a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in the LNPs.
  • the LNPs comprise: a cationic lipid comprising from 50 mol % to 85 mol % of the total lipid present in the LNPs; a conjugated lipid that inhibits aggregation of LNPs comprising from 0.5 mol % to 2 mol % of the total lipid present in the LNPs; and a non-cationic lipid comprising from 13 mol % to 49.5 mol % of the total lipid present in
  • the phospholipid comprises dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), or a mixture thereof.
  • DPPC dipalmitoylphosphatidylcholine
  • DSPC distearoylphosphatidylcholine
  • the conjugated lipid comprises a polyethyleneglycol (PEG)-lipid conjugate.
  • the PEG-lipid conjugate comprises a PEG-diacylglycerol (PEGDAG) conjugate, a PEG-dialkyloxypropyl (PEG-DAA) conjugate, or a mixture thereof.
  • the PEG-DAA conjugate comprises a PEG-dimyristyloxypropyl (PEG-DMA) conjugate, a PEG-distearyloxypropyl (PEG-DSA) conjugate, or a mixture thereof.
  • the PEG portion of the conjugate has an average molecular weight of about 2,000 daltons.
  • the conjugated lipid comprises from 1 mol % to 2 mol % of the total lipid present in the LNPs.
  • the LNP comprises a compound having a structure of: or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein:
  • G is
  • the LNP comprises a compound having a structure of Formula II:
  • R la and R lb are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R la is H or Ci- C12 alkyl, and R lb together with the carbon atom to which it is bound is taken together with an adjacent R lb and the carbon atom to which it is bound to form a carbon-carbon double bond
  • R 2a and R 2b are, at each occurrence, independently either (a) H or C1-C12 alkyl, or (b) R 2a is H or Ci-C 12 alkyl, and R 2b together with the carbon atom to which it is bound is taken together with an adjacent R 2b and the carbon atom to which it is bound to form a carbon-carbon double bond
  • R 3a and R 3b are
  • any of the above compositions further comprise one or more excipients comprising a neutral lipid, a steroid, and a polymer conjugated lipid.
  • the neutral lipid comprises at least one of l,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), l,2-Dipalmiloyl-.S7?-glycero-3-phosphocholine (DPPC), l,2-Dimyristoyl-5n-glycero-3- phosphocholine (DMPC), l-Palmitoyl-2-oleoyl-.yn-glycero-3 -phosphocholine (POPC), 1,2- dioleoyl-OT-gdycero-3-phosphocholine (DOPC), and l,2-Dioleoyl-.vn-glycero-3- phosphoethanolamine (DOPE).
  • the neutral lipid is DSPC.
  • the molar ratio of the compound to the neutral lipid ranges from about 2:1 to about 8: 1.
  • the steroid is cholesterol. In some aspects, the molar ratio of the compound to cholesterol ranges from about 2:1 to 1:1.
  • the polymer conjugated lipid is a pegylated lipid.
  • the molar ratio of the compound to the pegylated lipid ranges from about 100: 1 to about 25: 1.
  • the pegylated lipid is PEG-DAG, a PEG polyethylene (PEG-PE), a PEG- succinoyl-diacylglycerol (PEG-S-DAG), PEG-cer or a PEG dialkyoxypropylcarbamate.
  • the pegylated lipid has the following structure III:
  • R 10 and R 11 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and z has a mean value ranging from 30 to 60.
  • R 10 and R 11 are each independently straight, saturated alkyl chains having 12 to 16 carbon atoms.
  • the average z is about 45. start here
  • the LNP self- ssembles into non-bilayer structures when mixed with polyanionic nucleic acid.
  • the non-bilayer structures have a diameter between 60nm and 120nm.
  • the non-bilayer structures have a diameter of about 70nm, about 80nm, about 90nm, or about lOOnm.
  • wherein the nanoparticulate delivery vehicle has a diameter of about lOOnm.
  • vector or set of vectors comprising any of the nucleotide sequence described herein. Also disclosed herein is a vector comprising an isolated nucleotide sequence disclosed herein.
  • an isolated cell comprising any of the nucleotide sequences or set of isolated nucleotide sequences described herein, optionally wherein the cell is a BHK-21, CHO, HEK293 or variants thereof, 911, HeLa, A549, LP-293, PER.C6, or AEl-2a cell.
  • kits comprising any of the compositions described herein and instructions for use. Also disclosed herein is a kit comprising a vector or a composition disclosed herein and instructions for use.
  • Also provided for herein is a method for treating a subject having one or more of cervical cancer, anal cancer, oropharyngeal cancer, oral cancer, penile cancer, vaginal cancer, and/or vulvar cancer, the method comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein.
  • Also provided for herein is a method for treating a subject infected with or at risk for infection by HPV, the method comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein.
  • Also provided for herein is a method for stimulating an immune response in a subject, the method comprising administering to the subject any of the compositions or any of the pharmaceutical compositions described herein.
  • Also disclosed herein is a method for treating a subject, the method comprising administering to the subject a vector disclosed herein or a pharmaceutical composition disclosed herein.
  • Also disclosed herein is a method of manufacturing the one or more vectors of any of the above compositions.
  • FIGS. 1A-C present architectures of samRNA vaccine constructs for individual HPV strains based on either double promoter systems (FIG. 1A and FIG. IB) or an internal ribosomal skip site (FIG. 1C) to facilitate the combination of E6E7 and T Cell Epitope Cassette (TCE) inclusion on a single nucleic acid backbone.
  • Filled black boxes indicate an IgG expression leader sequence and unfilled white boxes indicate an internal furin cleavage site.
  • FIG. 2 shows immunogenicity results as assessed by ELISpot in C57/B16 mice for antigens HPV16 and HPV 18 on individual samRNA vaccine constructs according to architectures found in FIG. 1A and FIG. IB, respectively.
  • FIGS. 3A-B present architectures of ChAd vaccine constructs for individual HPV strains based on an internal ribosomal skip site with E6E7 in either the first (FIG. 3A) or second (FIG. 3B) position to facilitate the combination of E6E7 and T Cell Epitope Cassette (TCE) inclusion on a single nucleic acid backbone.
  • Filled black boxes indicate an IgG expression leader sequence and unfilled white boxes indicate an internal furin cleavage site.
  • FIG. 4 shows immunogenicity results as assessed by ELISpot in C57/B16 mice for antigens HP VI 6 or HPV 18 E6E7 and T Cell Epitope cassettes on single ChAd vectors utilizing architectures from FIG. 3A.
  • FIGS. 5A-C present architectures for inclusion of HPV16 and HPV18 E6E7 and T Cell Epitope cassettes on single samRNA vectors. These architectures utilize a dual promoter system to either include single E6E7 and TCE’ s with internal ribosomal skip sites on each promoter (FIG. 4A) or a fusion of the two TCE’s expressed on either the first (FIG. 4B) or second (FIG. 4C) promoter. Filled black boxes indicate an IgG expression leader sequence and unfilled white boxes indicate an internal furin cleavage site.
  • FIG. 6 shows immunogenicity results as assessed by ELISpot in C57/B16 mice for antigens HPV16 or HPV 18 E6E7 and T Cell Epitope for either the multi-strain samRNA vaccine construct for HPV 16 and HPV18 (format architecture of FIG. 5 A) or as a combination of separate single-strain samRNA vaccine constructs for each of HPV16 and HPV18 (Blended). Note: Data compiled from separate experiments.
  • FIGS. 7A-B present architectures for inclusion of HPV 16 & HPV 18 E6E7 and T Cell Epitope cassettes on single ChAd vectors. These architectures utilize fused E6E7 proteins from both HPV16 & HPV18 as well as fused T Cell Epitope cassettes from both viruses, with the fused E6E7’s as the first (FIG. 7A) or second (FIG. 7B) component of an internal ribosomal skip site containing sequence. Filled black boxes indicate an IgG expression leader sequence and unfdled white boxes indicate an internal furin cleavage site.
  • FIG. 8 shows immunogenicity results as assessed by ELISpot in C57/B16 mice for antigens HP VI 6 or HPV 18 E6E7 and T Cell Epitope for either a multi-strain ChAd vaccine construct for HPV 16 and HPV 18 (format architecture of FIG. 7A) or as a combination of separate single-strain ChAd vaccine constructs for each of HPV16 and HPV18 (Blended).
  • FIG. 9 shows immunogenicity results as assessed by ELISpot in C57/B16 mice for the indicated prime/boost experiments: Mice primed with either samRNA (left), or ChAd (2 nd from left) vectors encoding antigens for both HPV 16 and HPV 18 were subsequently boosted at d28 with either samRNA (middle, right) at day 28 or ChAd (2 nd from right) at 4 months.
  • FIG. 10 shows immunogenicity results as assessed by ELISpot against included E6/E7 and T Cell Epitope antigens in C57/B16 and FVB mice for samRNA (left) and ChAd (right) based blended vaccines encoding the antigens for HPV35 or HPV45.
  • FIG. 11 shows immunogenicity results as assessed by ELISpot against included E6/E7 and T Cell Epitope antigens in C57/B16 and FVB mice for samRNA (left) and ChAd (right) based blended vaccines encoding the antigens for HPV31 or HPV33.
  • FIG. 12 shows immunogenicity results as assessed by ELISpot against included E6/E7 and T Cell Epitope antigens in C57/B16 and FVB mice for samRNA (left) and ChAd (right) based blended vaccines encoding the antigens for HPV52 or HPV58.
  • FIG. 13 shows a Phase lb study design in females age 25-55 who are HPV 16/18 positive with CIN1.
  • FIG. 14 shows a Phase lb study design in females age 25-55 who are HPV 16/18 positive.
  • FIG. 15A shows an illustration of an algorithm performing several ranking iterations used to construct a concatenated T-cell epitope cassette.
  • FIG. 15A shows an illustration of calculating coverage provided by a given epitope by the frequencies of haplotypes in a reference population that cover at least one allele associated with the epitope used to construct a concatenated T-cell epitope cassette.
  • FIG. 16 shows the performance of performance of epitope prediction and inclusion in an exemplary HPV16 TCE cassette design, as assessed using mass-spectrometry verification of presentation of predicted epitopes in HLA monoallelic lines.
  • FIG. 17 shows performance of population coverage for an exemplary HPV 16 TCE cassette design.
  • an antigen is a substance that stimulates an immune response.
  • An antigen can be a neoantigen.
  • An antigen can be a “shared antigen” that is an antigen found among a specific population, e.g., a specific population of HPV patients with or at risk of infection for an infectious disease.
  • the term “antigen-based vaccine” is a vaccine composition based on one or more antigens, e.g., a plurality of antigens.
  • the vaccines can be nucleotide-based (e.g., virally based, RNA based, or DNA based), protein-based (e.g., peptide based), or a combination thereof.
  • cancer antigen is a mutation or other aberration giving rise to a sequence that may represent an antigen.
  • coding region is the portion(s) of a gene that encode protein.
  • coding mutation is a mutation occurring in a coding region.
  • ORF means open reading frame
  • missense mutation is a mutation causing a substitution from one amino acid to another.
  • nonsense mutation is a mutation causing a substitution from an amino acid to a stop codon or causing removal of a canonical start codon.
  • frameshift mutation is a mutation causing a change in the frame of the protein.
  • the term “indel” is an insertion or deletion of one or more nucleic acids.
  • the term percent "identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection.
  • the percent “identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared.
  • sequence comparison typically one sequence acts as a reference sequence to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.
  • sequence similarity or dissimilarity can be established by the combined presence or absence of particular nucleotides, or, for translated sequences, amino acids at selected sequence positions (e.g., sequence motifs).
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTF1T, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra).
  • BLAST algorithm One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information.
  • non-stop or read-through is a mutation causing the removal of the natural stop codon.
  • epitopope is the specific portion of an antigen typically bound by an antibody or T cell receptor.
  • immunogenic is the ability to stimulate an immune response, e.g., via T cells, B cells, or both.
  • HLA binding affinity means affinity of binding between a specific antigen and a specific MHC allele.
  • the term “bait” is a nucleic acid probe used to enrich a specific sequence of DNA or RNA from a sample.
  • variant is a difference between a subject’ s nucleic acids and the reference human genome used as a control.
  • variant call is an algorithmic determination of the presence of a variant, typically from sequencing.
  • polymorphism is a germline variant, i.e., a variant found in all DNA-bearing cells of an individual.
  • somatic variant is a variant arising in non-germline cells of an individual.
  • allele is a version of a gene or a version of a genetic sequence or a version of a protein.
  • HLA type is the complement of HLA gene alleles.
  • nonsense-mediated decay or “NMD” is a degradation of an mRNA by a cell due to a premature stop codon.
  • exome is a subset of the genome that codes for proteins.
  • An exome can be the collective exons of a genome.
  • logistic regression is a regression model for binary data from statistics where the logit of the probability that the dependent variable is equal to one is modeled as a linear function of the dependent variables.
  • neural network is a machine learning model for classification or regression consisting of multiple layers of linear transformations followed by element-wise nonlinearities typically trained via stochastic gradient descent and back- propagation.
  • proteome is the set of all proteins expressed and/or translated by a cell, group of cells, or individual.
  • peptidome is the set of all peptides presented by MHC-I or MHC-II on the cell surface.
  • the peptidome may refer to a property of a cell or a collection of cells (e.g., the infectious disease peptidome, meaning the union of the peptidomes of all cells that are infected by the infectious disease).
  • the term “ELIS POT” means Enzyme-linked immunosorbent spot assay - which is a common method for monitoring immune responses in humans and animals.
  • the term “dextramers” is a dextran-based peptide-MHC multimers used for antigen-specific T-cell staining in flow cytometry.
  • tolerance or immune tolerance is a state of immune nonresponsiveness to one or more antigens, e.g. self-antigens.
  • central tolerance is a tolerance affected in the thymus, either by deleting self-reactive T-cell clones or by promoting self-reactive T-cell clones to differentiate into immunosuppressive regulatory T-cells (Tregs).
  • peripheral tolerance is a tolerance affected in the periphery by downregulating or anergizing self-reactive T-cells that survive central tolerance or promoting these T cells to differentiate into Tregs.
  • sample can include a single cell or multiple cells or fragments of cells or an aliquot of body fluid, taken from a subject, by means including venipuncture, excretion, ejaculation, massage, biopsy, needle aspirate, lavage sample, scraping, surgical incision, or intervention or other means known in the art.
  • subject encompasses a cell, tissue, or organism, human or non-human, whether in vivo, ex vivo, or in vitro, male or female.
  • subject is inclusive of mammals including humans.
  • mammal encompasses both humans and non-humans and includes but is not limited to humans, non-human primates, canines, felines, murines, bovines, equines, and porcines.
  • Clinical factor refers to a measure of a condition of a subject, e.g., disease activity or severity.
  • “Clinical factor” encompasses all markers of a subject’s health status, including non-sample markers, and/or other characteristics of a subject, such as, without limitation, age and gender.
  • a clinical factor can be a score, a value, or a set of values that can be obtained from evaluation of a sample (or population of samples) from a subject or a subject under a determined condition.
  • a clinical factor can also be predicted by markers and/or other parameters such as gene expression surrogates.
  • Clinical factors can include infection type (e.g., human papillomavirus species), infection sub-type (e.g., HPV strain), and medical history.
  • nucleic acid sequences derived from an infection refers to nucleic acid sequences obtained from infected cells or an infectious disease organism, e.g. via RT-PCR; or sequence data obtained by sequencing the infected cell or infectious disease organism and then synthesizing the nucleic acid sequences using the sequencing data, e.g., via various synthetic or PCR-based methods known in the art.
  • Derived sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and/or otherwise optimized for expression), that encode the same polypeptide sequence as the corresponding native infectious disease organism nucleic acid sequence.
  • Derived sequences can include nucleic acid sequence variants that encode a modified infectious disease organism polypeptide sequence having one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to a native infectious disease organism polypeptide sequence.
  • a modified polypeptide sequence can have one or more missense mutations relative to the native polypeptide sequence of an infectious disease organism protein.
  • HPV nucleic acid sequence encoding an immunogenic polypeptide refers to nucleic acid sequences obtained from a HPV virus, e.g.
  • Derived sequences can include nucleic acid sequence variants, such as sequence-optimized nucleic acid sequence variants (e.g., codon-optimized and/or otherwise optimized for expression), that encode the same polypeptide sequence as the corresponding native HPV nucleic acid sequence.
  • Derived sequences can include nucleic acid sequence variants that encode a modified HPV polypeptide sequence having one or more (e.g., 1, 2, 3, 4, or 5) mutations relative to a native HPV polypeptide sequence.
  • a modified HPV polypeptide sequence can have one or more mutations, such as one or more deletions that render proteins inactive.
  • alphavirus refers to members of the family Togaviridae, and are positivesense single-stranded RNA viruses. Alphaviruses are typically classified as either Old World, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World, such as eastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equine encephalitis and its derivative strain TC-83. Alphaviruses are typically self-replicating RNA viruses.
  • alphavirus backbone refers to minimal sequence(s) of an alphavirus that allow for self-replication of the viral genome.
  • Minimal sequences can include conserved sequences for nonstructural protein- mediated amplification, a nonstructural protein 1 (nsPl) gene, a nsP2 gene, a nsP3 gene, a nsP4 gene, and a polyA sequence, as well as sequences for expression of subgenomic viral RNA including a subgenomic promoter (e.g., a 26S promoter element).
  • a subgenomic promoter e.g., a 26S promoter element
  • sequences for nonstructural protein-mediated amplification includes alphavirus conserved sequence elements (CSE) well known to those in the art.
  • CSEs include, but are not limited to, an alphavirus 5’ UTR, a 51-nt CSE, a 24-nt CSE, a subgenomic promoter sequence (e.g. , a 26S subgenomic promoter sequence), a 19-nt CSE, and an alphavirus 3’ UTR.
  • RNA polymerase includes polymerases that catalyze the production of RNA polynucleotides from a DNA template. RNA polymerases include, but are not limited to, bacteriophage derived polymerases including T3, T7, and SP6.
  • lipid includes hydrophobic and/or amphiphilic molecules.
  • Lipids can be cationic, anionic, or neutral.
  • Lipids can be synthetic or naturally derived, and in some instances biodegradable.
  • Lipids can include cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, fats, and fat-soluble vitamins.
  • PEG polyethyleneglycol
  • Lipids can also include dilinoleylmethyl- 4- dimethylaminobutyrate (MC3) and MC3-like molecules.
  • lipid nanoparticle includes vesicle like structures formed using a lipid containing membrane surrounding an aqueous interior, also referred to as liposomes.
  • Lipid nanoparticles includes lipid-based compositions with a solid lipid core stabilized by a surfactant.
  • the core lipids can be fatty acids, acylglycerols, waxes, and mixtures of these surfactants.
  • Biological membrane lipids such as phospholipids, sphingomyelins, bile salts (sodium taurocholate), and sterols (cholesterol) can be utilized as stabilizers.
  • Lipid nanoparticles can be formed using defined ratios of different lipid molecules, including, but not limited to, defined ratios of one or more cationic, anionic, or neutral lipids.
  • Lipid nanoparticles can encapsulate molecules within an outer-membrane shell and subsequently can be contacted with target cells to deliver the encapsulated molecules to the host cell cytosol.
  • Lipid nanoparticles can be modified or functionalized with non- lipid molecules, including on their surface.
  • Lipid nanoparticles can be single-layered (unilamellar) or multi-layered (multilamellar).
  • Lipid nanoparticles can be complexed with nucleic acid.
  • Unilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior.
  • Multilamellar lipid nanoparticles can be complexed with nucleic acid, wherein the nucleic acid is in the aqueous interior, or to form or sandwiched between
  • MHC major histocompatibility complex
  • HLA human leukocyte antigen, or the human MHC gene locus
  • NGS next-generation sequencing
  • PPV positive predictive value
  • TSNA tumor-specific neoantigen
  • FFPE formalin-fixed, paraffin-embedded
  • NMD nonsense-mediated decay
  • NSCLC non-small-cell lung cancer
  • DC dendritic cell
  • Methods for identifying antigens include identifying antigens that are likely to be presented on a cell surface e.g., presented by MHC on an infected cell or an immune cell, including professional antigen presenting cells such as dendritic cells), and/or are likely to be immunogenic.
  • one such method may comprise the steps of: obtaining at least one of exome, transcriptome or whole genome nucleotide sequencing and/or expression data from an infected cell or an infectious disease organism (e.g., HPV), wherein the nucleotide sequencing data and/or expression data is used to obtain data representing peptide sequences of each of a set of antigens (e.g.
  • antigens derived from the infectious disease organism inputting the peptide sequence of each antigen into one or more presentation models to generate a set of numerical likelihoods that each of the antigens is presented by one or more MHC alleles on a cell surface, such as an infected cell of the subject, the set of numerical likelihoods having been identified at least based on received mass spectrometry data; and selecting a subset of the set of antigens based on the set of numerical likelihoods to generate a set of selected antigens.
  • Antigens can include nucleotides or polypeptides.
  • an antigen can be an RNA sequence that encodes for a polypeptide sequence.
  • Antigens useful in vaccines can therefore include nucleotide sequences or polypeptide sequences.
  • peptides and nucleic acid sequences encoding immunogenic peptides derived from any polypeptide associated with HPV, a HPV infection in a subject, or a HPV infected cell of a subject.
  • Antigens can be derived from nucleotide sequences or polypeptide sequences of a HPV virus.
  • Immunogenic polypeptide sequences of HPV include, but are not limited to, MHC class I epitopes shown in Tables C1-C9, which can include polypeptides sequences in Tables C1-C9 that contain one or more predicted HPV MHC class I epitopes and/or one or more HPV MHC class I epitopes validated in the literature.
  • Immunogenic polypeptide sequences of HPV include HPV MHC class I epitopes validated by mass- spectrometry to be presented by one or more HL A alleles, including one or more HL A alleles of a target vaccine demographic.
  • Immunogenic polypeptide sequences of HPV include, but are not limited to, multiple MHC class I epitopes (or multiple HPV polypeptides that contain one or more predicted HPV MHC class I epitopes and/or one or more HPV MHC class I epitopes) that are concatenated into a single polypeptide, e.g. in a cassette. Illustrative non-limitating examples of concatenated HPV MHC class I epitopes are shown in Table D.
  • HPV MHC class I epitopes can include epitopes from any protein encoded by HPV, including but not limited to any early (E) region encoding proteins E1-E7 or late (L) region encoding structural proteins L1-L2.
  • HPV MHC class I epitopes can include epitopes from one or more of HPV proteins E1-E7.
  • HPV MHC class I epitopes can include epitopes only from one or more of HPV proteins E1-E7.
  • HPV MHC class I epitopes can include epitopes from one or more of HPV proteins El, E2, E5, E6, and E7.
  • HPV MHC class 1 epitopes can include epitopes from at least each of HPV proteins El, E2, E5, E6, and E7.
  • HPV MHC class I epitopes can include epitopes from at least each of and limited to HPV proteins El, E2, E5, E6, and E7.
  • Immunogenic polypeptide sequences of HPV include, but are not limited to, HPV proteins (e.g., full-length HPV proteins), including HPV proteins that contain one or more predicted HPV MHC class I epitopes and/or one or more HPV MHC class I epitopes validated in the literature.
  • Full-length HPV proteins can include any early (E) region encoding proteins E1-E7 or late (L) region encoding structural proteins L1-L2.
  • Full-length HPV proteins can include one or more of any early (E) region encoding proteins E1-E7.
  • Full-length HPV proteins can include only one or more of early (E) region encoding proteins E1-E7.
  • Full-length HPV proteins can include E6.
  • Full-length HPV proteins can include E7.
  • Full-length HPV proteins can include E6 and E7.
  • Full-length HPV proteins can include only E6 and E7.
  • Full-length HPV proteins can be modified from their naturally occurring sequences, for example as shown in the non-limiing illustrative modifications shown in Table B. Modifications can include, but are not limited to, deletions or other substitutions that inactivate a protein or otherwise make a protein non-functional or reduce the function of a protein, e.g., to improve the safety of a vaccine.
  • HPV protein modifications can include one or more of the modifications shown in Table B.
  • HPV protein modifications can include each of the modifications shown in Table B for a respective HPV protein.
  • Full-length HPV proteins can include a fusion protein (e.g., two or more proteins modified to be co-expressed as a single polypeptide, including through use of a peptide linker) of one or more full-length HPV proteins.
  • a HPV fusion protein can include one or more full- length HPV proteins E1-E7.
  • a HPV fusion protein can include HPV proteins E6 and E7 modified to be co-expressed as a single polypeptide, such as in the illustrative examples shown in Table A.
  • Immunogenic polypeptide sequences can include full-length HPV proteins in combination with multiple MHC class I epitopes concatenated into a single polypeptide.
  • immunogenic polypeptide sequences can include full-length HPV proteins E6 and E7 (e.g., a HPV fusion protein can including HPV proteins E6 and E7 modified to be co-expressed as a single polypeptide, such as in the illustrative examples shown in Table A) in combination with multiple MHC class I epitopes concatenated into a single polypeptide, such as one or more of the polypeptides sequences in Tables C1-C9 or each of the polypeptides sequences shown in Tables C1-C9 (e.g., any one of the polypeptides sequences shown in Table D).
  • immunogenic polypeptide sequences include full-length HPV proteins in combination with multiple MHC class I epitopes concatenated into a single polypeptide
  • the selection of the multiple MHC class I epitopes concatenated into the single polypeptide can exclude those peptides encoded in the full-length HPV proteins.
  • multiple MHC class I epitopes concatenated into a single polypeptide can include only immunogenic polypeptide sequences derived from HPV proteins other than E6 and E7.
  • Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide corresponding to a polypeptide encoded by a HPV strain the subject is infected with or at risk for infection by, such as for use in prophylactic vaccines for a specific demographic or geographic population at risk for infection by the specific HPV strain.
  • Specific HPV strains include, but are not limited to, HPV strains HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58.
  • HPV strains for a specific demographic or geographic population can include HPV strains generally found in women from low middle income countries (LMIC) with persistent HPV infection, such as HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58.
  • LMIC low middle income countries
  • Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide corresponding to a polypeptide encoded by at least two or more HPV strains.
  • Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide corresponding to two or more HPV strains selected from HPV16, HPV18, HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58.
  • Vaccines can be designed to encode at least one HPV- derived immunogenic polypeptide derived from each of HPV16 and HPV18. Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide derived from each of HPV16, HPV18, HPV35, and HPV45. Vaccines can be designed to encode at least one HPV- derived immunogenic polypeptide derived from each of HPV35 and HPV45. Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide derived from at least each of HPV35 and HPV45.
  • Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide derived from each of HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58. Vaccines can be designed to encode at least one HPV-derived immunogenic polypeptide derived from at least each of HPV31, HPV33, HPV35, HPV45, HPV52, and HPV58.
  • Vaccine strategies can also include use of multiple separate vaccines used in combination to separately express multiple HPV-derived immunogenic polypeptides. Such a strategy can be called a “blended” vaccination strategy. Vaccine strategies can also include use of multiple separate vaccines used in combination to separately express multiple HPV-derived immunogenic polypeptides, where the separately expressed HPV-derived immunogenic polypeptides are derived from distinct HPV strains, such as in the non-limting illustrative example of a first vaccine that encodes HPV-derived immunogenic polypeptides derived from HPV 16 and a second vaccine that encodes HPV-derived immunogenic polypeptides derived from HPV18.
  • Vaccine strategies can also include use of multiple separate vaccines used in combination to separately express multiple HPV-derived immunogenic polypeptides, where the separately expressed HPV-derived immunogenic polypeptides are derived from distinct HPV proteins, such as in the non-limting illustrative example of a first vaccine that encodes HPV- derived immunogenic polypeptides derived from HPV E6 and/or E7 and a second vaccine that encodes HPV-derived immunogenic polypeptides derived from El, E2, and E5 (e.g., concatenated into a single polypeptide, such as in a cassette).
  • Vaccine strategies can also include use of multiple separate vaccines used in combination to separately express multiple HPV- derived immunogenic polypeptides, where the separately expressed HPV-derived immunogenic polypeptides are derived from distinct HPV proteins that include distinct proteins from multiple HPV strains, such as in the non-limting illustrative example of a first vaccine that encodes HPV- derived immunogenic polypeptides derived from HPV E6 and/or E7 from both HPV 16 and HPV 18 and a second vaccine that encodes HPV-derived immunogenic polypeptides derived from El, E2, and E5 from both HPV16 and HPV18 (e.g., concatenated into a single polypeptide for HPV 16 and concatenated into a single polypeptide for HPV 18).
  • a first vaccine that encodes HPV- derived immunogenic polypeptides derived from HPV E6 and/or E7 from both HPV 16 and HPV 18
  • a second vaccine that encodes HPV-derived immunogenic polypeptides
  • Vaccines can be designed to encode at least one immunogenic polypeptide corresponding to a polypeptide encoded by HPV and at least one immunogenic polypeptide corresponding to a polypeptide encoded by a an infectious disease other than HPV, e.g., polypeptide sequences of an infectious disease organism that can include, but are not limited to, a pathogen-derived peptide, a virus-derived peptide, a bacteria-derived peptide, a fungus -derived peptide, and/or a parasite-derived peptide.
  • Infectious disease organism include, but are not limited to, Severe acute respiratory syndrome-related coronavirus (SARS), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Ebola, HIV, Hepatitis B virus (HBV), influenza, Hepatitis C virus (HCV), Cytomegalovirus (CMV), Chikungunya virus, Respiratory syncytial virus (RSV), Dengue virus, a orthymyxoviridae family virus, and tuberculosis.
  • SARS Severe acute respiratory syndrome-related coronavirus
  • SARS-CoV-2 severe acute respiratory syndrome coronavirus 2
  • Ebola HIV
  • HBV Hepatitis B virus
  • HCV Hepatitis C virus
  • CMV Cytomegalovirus
  • RSV Respiratory syncytial virus
  • Dengue virus a orthymyxoviridae family virus
  • tuberculosis tuberculosis.
  • Antigens can be selected that are predicted to be presented on the cell surface of a cell, such as an infected cell or an immune cell, including professional antigen presenting cells such as dendritic cells. Antigens can be selected that are predicted to be immunogenic. Exemplary antigens predicted using the methods described herein to be presented on the cell surface by an MHC include predicted and/or previously validated MHC class I epitopes shown in Tables C1-C9..
  • Antigens can be selected that have been validated to be presented by a specific HLA and/or stimulate an immune response, such as previously reported/validated in the literature (for example, as in Nelde et al. [Nature Immunology volume 22, pages 74-85 2021], Tarke et al. 2021, or Schelien et al. [bioRxiv 2020.08.13.249433]).
  • the magnitude of stimulation of an immune response can be used to guide epitope/antigen selection, such as to select epitopes that stimulate as robust an immune response as possible, including when cassettes have a size constraint.
  • a cassette can be constructed to encode one or more validated epitopes and/or at least 4, 5, 6, or 7 predicted epitopes, wherein at least 85%, 90%, or 95% of a population carries at least one HLA validated to present at least one of the one or more validated epitopes and/or at least one HLA predicted to present each of the at least 4, 5, 6, or 7 predicted epitopes.
  • a cassette can be constructed to encode one or more validated epitopes and at least 4, 5, 6, or 7 predicted epitopes, wherein at least 85%, 90%, or 95% of a population carries at least one HLA validated to present at least one of the one or more validated epitopes or at least one HLA predicted to present each of the at least 4, 5, 6, or 7 predicted epitopes.
  • One or more polypeptides encoded by an antigen nucleotide sequence can comprise at least one of: a binding affinity with MHC with an IC50 value of less than lOOOnM, for MHC Class I peptides a length of 8-15, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids, presence of sequence motifs within or near the peptide promoting proteasome cleavage, and presence or sequence motifs promoting TAP transport.
  • MHC Class II peptides a length 6-30, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, presence of sequence motifs within or near the peptide promoting cleavage by extracellular or lysosomal proteases (e.g., cathepsins) or HLA-DM catalyzed HLA binding.
  • extracellular or lysosomal proteases e.g., cathepsins
  • HLA-DM catalyzed HLA binding e.g., HLA-DM catalyzed HLA binding.
  • One or more antigens can be presented on the surface of an infected cell (e.g. , a HPV infected cell).
  • One or more antigens can be immunogenic in a subject having or suspected to have an infection (e.g., a HPV infection, such as a persistent HPV infection), e.g., capable of stimulating a T cell response in the subject.
  • a HPV infection such as a persistent HPV infection
  • One or more antigens can be immunogenic in a subject at risk of an infection (e.g., a HPV infection, such as a persistent HPV infection), e.g., capable of stimulating a T cell response in the subject that provides immunological protection (i.e., immunity) against the infection, e.g., such as stimulating the production of memory T cells.
  • One or more antigens can include a combination of antigens capable of stimulating a T cell response (e.g., peptides including predicted T cell epitope sequences) and distinct antigens capable of stimulating a B cell response (e.g., full-length proteins, protein subunits, protein domains).
  • a T cell response e.g., peptides including predicted T cell epitope sequences
  • distinct antigens capable of stimulating a B cell response e.g., full-length proteins, protein subunits, protein domains.
  • the size of at least one antigenic peptide molecule can comprise, but is not limited to, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120 or greater amino molecule residues, and any range derivable therein.
  • the antigenic peptide molecules are equal to or less than 50 amino acids.
  • Antigenic peptides and polypeptides can be: for MHC Class 1 15 residues or less in length and usually consist of between about 8 and about 11 residues, particularly 9 or 10 residues; for MHC Class II, 6-30 residues, inclusive.
  • a longer peptide can be designed in several ways.
  • a longer peptide could consist of either: (1) individual presented peptides with an extensions of 2-5 amino acids toward the N- and C-terminus of each corresponding gene product; (2) a concatenation of some or all of the presented peptides with extended sequences for each.
  • sequencing reveals a long (>10 residues) epitope sequence present, a longer peptide would consist of: (3) the entire stretch of novel infectious disease-specific amino acids— thus bypassing the need for computational or in vitro test-based selection of the strongest HLA-presented shorter peptide.
  • Longer peptides can also include a full-length protein, a protein subunit, a protein domain, and combinations thereof of a peptide, such as those expressed in an infectious disease organism. Longer peptides (e.g. , full-length protein, protein subunit, or protein domain) and combinations thereof can be included to stimulate a B cell response.
  • Antigenic peptides and polypeptides can be presented on an HLA protein. In some aspects antigenic peptides and polypeptides are presented on an HLA protein with greater affinity than a wild-type peptide. In some aspects, an antigenic peptide or polypeptide can have an IC50 of at least less than 5000 nM, at least less than 1000 nM, at least less than 500 nM, at least less than 250 nM, at least less than 200 nM, at least less than 150 nM, at least less than 100 nM, at least less than 50 nM or less.
  • antigenic peptides and polypeptides do not stimulate an autoimmune response and/or invoke immunological tolerance when administered to a subject.
  • compositions comprising at least two or more antigenic peptides.
  • the composition contains at least two distinct peptides. At least two distinct peptides can be derived from the same polypeptide. By distinct polypeptides is meant that the peptide vary by length, amino acid sequence, or both.
  • the peptides can be derived from any polypeptide known to or suspected to be associated with an infectious disease organism, or peptides derived from any polypeptide known to or have been found to have altered expression in an infected cell in comparison to a normal cell or tissue (e.g., an infectious disease polynucleotide or polypeptide, including infectious disease polynucleotides or polypeptides with expression restricted to a host cell).
  • infectious disease polynucleotide or polypeptide including infectious disease polynucleotides or polypeptides with expression restricted to a host cell.
  • Antigenic peptides and polypeptides having a desired activity or property can be modified to provide certain desired attributes, e.g., improved pharmacological characteristics, while increasing or at least retaining substantially all of the biological activity of the unmodified peptide to bind the desired MHC molecule and activate the appropriate T cell.
  • antigenic peptide and polypeptides can be subject to various changes, such as substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use, such as improved MHC binding, stability or presentation.
  • conservative substitutions is meant replacing an amino acid residue with another which is biologically and/or chemically similar, e.g., one hydrophobic residue for another, or one polar residue for another.
  • substitutions include combinations such as Gly, Ala; Vai, He, Leu, Met; Asp, Glu; Asn, Gin; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • the effect of single amino acid substitutions may also be probed using D-amino acids.
  • Such modifications can be made using well known peptide synthesis procedures, as described in e.g., Merrifield, Science 232:341-347 (1986), Barany & Merrifield, The Peptides, Gross & Meienhofer, eds. (N.Y., Academic Press), pp. 1-284 (1979); and Stewart & Young, Solid Phase Peptide Synthesis, (Rockford, Ill., Pierce), 2d Ed. (1984).
  • Modifications of peptides and polypeptides with various amino acid mimetics or unnatural amino acids can be particularly useful in increasing the stability of the peptide and polypeptide in vivo. Stability can be assayed in a number of ways. For instance, peptidases and various biological media, such as human plasma and serum, have been used to test stability. See, e.g., Verhoef el al., Eur. J. Drug Metab Pharmacokin. 11:291-302 (1986). Half-life of the peptides can be conveniently determined using a 25% human serum (v/v) assay. The protocol is generally as follows.
  • pooled human serum (Type AB, non-heat inactivated) is delipidated by centrifugation before use. The serum is then diluted to 25% with RPMI tissue culture media and used to test peptide stability. At predetermined time intervals a small amount of reaction solution is removed and added to either 6% aqueous trichloracetic acid or ethanol. The cloudy reaction sample is cooled (4 degrees C) for 15 minutes and then spun to pellet the precipitated serum proteins. The presence of the peptides is then determined by reversed-phase HPLC using stability-specific chromatography conditions.
  • the peptides and polypeptides can be modified to provide desired attributes other than improved serum half-life. For instance, the ability of the peptides to stimulate CTL activity can be enhanced by linkage to a sequence which contains at least one epitope that is capable of inducing a T helper cell response.
  • Immunogenic peptides/T helper conjugates can be linked by a spacer molecule.
  • the spacer is typically comprised of relatively small, neutral molecules, such as amino acids or amino acid mimetics, which are substantially uncharged under physiological conditions.
  • the spacers are typically selected from, e.g., Ala, Gly, or other neutral spacers of nonpolar amino acids or neutral polar amino acids.
  • the optionally present spacer need not be comprised of the same residues and thus can be a hetero- or homooligomer.
  • the spacer will usually be at least one or two residues, more usually three to six residues.
  • the peptide can be linked to the T helper peptide without a spacer.
  • Polypeptides encoding antigens can be modified to alter processing of the polypeptides, such as protease cleavage and/or other post- translation processing. Polypeptides encoding antigens can be modified such that the antigen favors a specific conformation. Polypeptides encoding antigens can be modified such that the mutations (e.g., one or more missense mutations) disrupt a specific conformation in the antigen, such as through the introduction of prolines that disrupt secondary and tertiary structures (e.g. , alpha-helix or betasheet formation). Altering, reducing, or eliminating processing or conformation changes may, in some instances, bias the antigen to favor states favorable to neutralizing antibody production.
  • Modified polypeptide sequences can be at least 60%, 70%, 80%, or 90% identical to a native HPV polypeptide sequence. Modified polypeptide sequences can be at least 91%, 92%, 93%, or 94% identical to a native HPV polypeptide sequence. Modified polypeptide sequences can be at least 95%, 96%, 97%, 98%, or 99% identical to a native HPV polypeptide sequence.
  • Modified polypeptide sequences can be at least 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a native HPV polypeptide sequence.
  • An antigenic peptide can be linked to the T helper peptide either directly or via a spacer either at the amino or carboxy terminus of the peptide.
  • the amino terminus of either the antigenic peptide or the T helper peptide can be acylated.
  • Exemplary T helper peptides include tetanus toxoid 830-843, influenza 307-319, malaria circumsporozoite 382-398 and 378-389.
  • Proteins or peptides can be made by any technique known to those of skill in the art, including the expression of proteins, polypeptides or peptides through standard molecular biological techniques, the isolation of proteins or peptides from natural sources, or the chemical synthesis of proteins or peptides.
  • the nucleotide and protein, polypeptide and peptide sequences corresponding to various genes have been previously disclosed, and can be found at computerized databases known to those of ordinary skill in the art.
  • One such database is the National Center for Biotechnology Information's Genbank and GenPept databases located at the National Institutes of Health website.
  • the coding regions for known genes can be amplified and/or expressed using the techniques disclosed herein or as would be known to those of ordinary skill in the art.
  • various commercial preparations of proteins, polypeptides and peptides are known to those of skill in the art.
  • an antigen includes a nucleic acid (e.g. polynucleotide) that encodes an antigenic peptide or portion thereof.
  • the polynucleotide can be, e.g., DNA, cDNA, PNA, CNA, RNA (e.g., mRNA), either single- and/or double-stranded, or native or stabilized forms of polynucleotides, such as, e.g., polynucleotides with a phosphorothioate backbone, or combinations thereof and it may or may not contain introns.
  • a polynucleotide sequence encoding an antigen can be sequence-optimized to improve expression, such as through improving transcription, translation, post-transcriptional processing, and/or RNA stability.
  • polynucleotide sequence encoding an antigen can be codon-optimized.
  • Codonoptimization herein refers to replacing infrequently used codons, with respect to codon bias of a given organism, with frequently used synonymous codons.
  • Polynucleotide sequences can be optimized to improve post-transcriptional processing, for example optimized to reduce unintended splicing, such as through removal of splicing motifs (e.g.
  • Exogenous intron sequences include, but are not limited to, those derived from SV40 (e.g. , an SV40 mini-intron) and derived from immunoglobulins (e.g., human P-globin gene). Exogenous intron sequences can be incorporated between a promoter/enhancer sequence and the antigen(s) sequence.
  • Exogenous intron sequences for use in expression vectors are described in more detail in Callendret et al. (Virology. 2007 Jul 5; 363(2): 288-302), herein incorporated by reference for all purposes.
  • Polynucleotide sequences can be optimized to improve transcript stability, for example through removal of RNA instability motifs (e.g., AU- rich elements and 3’ UTR motifs) and/or repetitive nucleotide sequences.
  • Polynucleotide sequences can be optimized to improve accurate transcription, for example through removal of cryptic transcriptional initiators and/or terminators.
  • Polynucleotide sequences can be optimized to improve translation and translational accuracy, for example through removal of cryptic AUG start codons, premature polyA sequences, and/or secondary structure motifs. Polynucleotide sequences can be optimized to improve nuclear export of transcripts, such as through addition of a Constitutive Transport Element (CTE), RNA Transport Element (RTE), or Woodchuck Posttranscriptional Regulatory Element (WPRE). Nuclear export signals for use in expression vectors are described in more detail in Callendret et al. (Virology. 2007 Jul 5; 363(2): 288-302), herein incorporated by reference for all purposes.
  • CTE Constitutive Transport Element
  • RTE RNA Transport Element
  • WPRE Woodchuck Posttranscriptional Regulatory Element
  • Polynucleotide sequences can be optimized with respect to GC content, for example to reflect the average GC content of a given organism. Sequence optimization can balance one or more sequence properties, such as transcription, translation, post-transcriptional processing, and/or RNA stability. Sequence optimization can generate an optimal sequence balancing each of transcription, translation, post-transcriptional processing, and RNA stability. Sequence optimization algorithms are known to those of skill in the art, such as GeneArt (Thermo Fisher), Codon Optimization Tool (IDT), Cool Tool, SGI- DNA (La Jolla California). One or more regions of an antigen-encoding protein can be sequence-optimized separately.
  • a method disclosed herein can also include identifying one or more T cells that are antigen- specific for at least one of the antigens in the subset.
  • the identification comprises co-culturing the one or more T cells with one or more of the antigens in the subset under conditions that expand the one or more antigen-specific T cells.
  • the identification comprises contacting the one or more T cells with a tetramer comprising one or more of the antigens in the subset under conditions that allow binding between the T cell and the tetramer.
  • the method disclosed herein can also include identifying one or more T cell receptors (TCR) of the one or more identified T cells.
  • TCR T cell receptors
  • identifying the one or more T cell receptors comprises sequencing the T cell receptor sequences of the one or more identified T cells.
  • the method disclosed herein can further comprise genetically engineering a plurality of T cells to express at least one of the one or more identified T cell receptors; culturing the plurality of T cells under conditions that expand the plurality of T cells; and infusing the expanded T cells into the subject.
  • genetically engineering the plurality of T cells to express at least one of the one or more identified T cell receptors comprises cloning the T cell receptor sequences of the one or more identified T cells into an expression vector; and transfecting each of the plurality of T cells with the expression vector.
  • the method disclosed herein further comprises culturing the one or more identified T cells under conditions that expand the one or more identified T cells; and infusing the expanded T cells into the subject.
  • an isolated T cell that is antigen-specific for at least one selected antigen in the subset.
  • a still further aspect provides an expression vector capable of expressing a polypeptide or portion thereof.
  • Expression vectors for different cell types are well known in the art and can be selected without undue experimentation.
  • DNA is inserted into an expression vector, such as a plasmid, in proper orientation and correct reading frame for expression. If necessary, DNA can be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognized by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Guidance can be found e.g. in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • an immunogenic composition e.g., a vaccine composition, capable of raising a specific immune response, e.g., an infectious disease organism-specific immune response.
  • Vaccine compositions typically comprise one or a plurality of antigens, e.g., selected using a method described herein. Vaccine compositions can also be referred to as vaccines.
  • a vaccine can contain between 1 and 30 peptides, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 different peptides, 6, 7, 8, 9, 10 11, 12, 13, or 14 different peptides, or 12, 13 or 14 different peptides.
  • Peptides can include post-translational modifications.
  • a vaccine can contain between 1 and 100 or more nucleotide sequences, 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,
  • a vaccine can contain between 1 and 30 antigen sequences, 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,
  • Antigen-encoding nucleic acid sequences can refer to the antigen encoding portion of an “antigen cassette.” Features of an antigen cassette are described in greater detail herein.
  • An antigen-encoding nucleic acid sequence can contain one or more epitope-encoding nucleic acid sequences (e.g., an antigen-encoding nucleic acid sequence encoding concatenated T cell epitopes).
  • a vaccine can contain between 1 and 30 distinct epitope-encoding nucleic acid sequences, 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,
  • Epitope-encoding nucleic acid sequences can refer to sequences for individual epitope sequences, such as each of the T cell epitopes in an antigen-encoding nucleic acid sequence encoding concatenated T cell epitopes.
  • a vaccine can contain at least two repeats of an epitope-encoding nucleic acid sequence.
  • a “repeat” refers to two or more iterations of an identical nucleic acid epitope-encoding nucleic acid sequence (inclusive of the optional 5’ linker sequence and/or the optional 3 ’ linker sequences described herein) within an antigen-encoding nucleic acid sequence.
  • the antigen-encoding nucleic acid sequence portion of a cassette encodes at least two repeats of an epitope-encoding nucleic acid sequence.
  • the antigen-encoding nucleic acid sequence portion of a cassette encodes more than one distinct epitope, and at least one of the distinct epitopes is encoded by at least two repeats of the nucleic acid sequence encoding the distinct epitope (z. ⁇ ?., at least two distinct epitopeencoding nucleic acid sequences).
  • an antigen-encoding nucleic acid sequence encodes epitopes A, B, and C encoded by epitope-encoding nucleic acid sequences epitope-encoding sequence A (EA), epitope-encoding sequence B (EB), and epitopeencoding sequence C (Ec), and examplary antigen-encoding nucleic acid sequences having repeats of at least one of the distinct epitopes are illustrated by, but is not limited to, the formulas below:
  • EA-EB-EC-EA-EB-EC Repeat of multiple distinct epitopes (repeats of epitopes A, B, and C): EA-EB-EC-EA-EB-EC; or EA-EA-EB -EB-EC-EC
  • Multiple repeats of multiple distinct epitopes (repeats of epitopes A, B, and C): EA-EB-EC-EA-EB-EC-EA-EB-EC; or EA-EA-EA-EB-EB-EC-EC-EC-EC
  • the antigen-encoding nucleic acid sequences having repeats of at least one of the distinct epitopes can encode each of the distinct epitopes in any order or frequency.
  • the order and frequency can be a random arangement of the distinct epitopes, e.g., in an example with epitopes A, B, and C, by the formula EA-EB-EC-EC-EA-EB-EA-EC-EA-EC-EC-EC-E B .
  • an antigen-encoding cassette having at least one antigen-encoding nucleic acid sequence described, from 5’ to 3’, by the formula:
  • Each E or E N can independently comprise any epitope-encoding nucleic acid sequence described herein (e.g. , a nucleotide sequence encoding a polypeptide sequence as set forth in Tables C1-C9).
  • Repeats of an epitope-encoding nucleic acid sequences can be linearly linked directly to one another (e.g. , EA-EA-. .. as illustrated above). Repeats of an epitope-encoding nucleic acid sequences can be separated by one or more additional nucleotides sequences. In general, repeats of an epitope-encoding nucleic acid sequences can be separated by any size nucleotide sequence applicable for the compositions described herein.
  • repeats of an epitopeencoding nucleic acid sequences can be separated by a separate distinct epitope-encoding nucleic acid sequence (e.g., EA-EB-EC-EA..., as illustrated above).
  • each epitopeencoding nucleic acid sequences (inclusive of optional 5’ linker sequence and/or the optional 3’ linker sequences) encodes a peptide 25 amino acids in length
  • the repeats can be separated by 75 nucleotides, such as in antigen-encoding nucleic acid represented by EA-EB-EA. .
  • EA is separated by 75 nucleotides.
  • Trpl VTNTEMFVTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDTVTNTEMF VTAPDNLGYMYEVQWPGQTQPQIANCSVYDFFVWLHYYSVRDT
  • Trpl the repeats of Trpl are separated by the 25mer Trp2 and thus the repeats of the Trpl epitope-encoding nucleic acid sequences are separated the 75 nucleotide Trp2 epitope-encoding nucleic acid sequence.
  • repeats are separated by 2, 3, 4, 5, 6, 7, 8, or 9 separate distinct epitope-encoding nucleic acid sequence, and each epitope-encoding nucleic acid sequences (inclusive of optional 5 ’ linker sequence and/or the optional 3 ’ linker sequences) encodes a peptide 25 amino acids in length
  • the repeats can be separated by 150, 225, 300, 375, 450, 525, 600, or 675 nucleotides, respectively.
  • different peptides and/or polypeptides or nucleotide sequences encoding them are selected so that the peptides and/or polypeptides capable of associating with different MHC molecules, such as different MHC class I molecules and/or different MHC class II molecules.
  • one vaccine composition comprises coding sequence for peptides and/or polypeptides capable of associating with the most frequently occurring MHC class I molecules and/or different MHC class II molecules.
  • vaccine compositions can comprise different fragments capable of associating with at least 2 preferred, at least 3 preferred, or at least 4 preferred MHC class I molecules and/or different MHC class II molecules.
  • the vaccine composition can stimulate a specific cytotoxic T-cell response and a specific helper T-cell response.
  • a combination of vaccine compositions can stimulate a specific cytotoxic T-cell response and/or a specific helper T-cell response.
  • Vaccine compositions can be homologous and stimulate T-cell response and/or a specific helper T-cell response in combination.
  • Vaccine compositions can be homologous and stimulate a specific T-cell response and a specific helper T-cell response in combination.
  • Vaccine compositions can be heterologous and stimulate a specific T-cell response and/or a specific helper T-cell response in combination.
  • Vaccine compositions can be heterologous and stimulate a specific T-cell response and a specific helper T-cell response in combination.
  • Heterologous vaccines include an identical antigen cassette encoded by different vaccine platforms, e.g., a viral vaccine (e.g., a ChAdV-based platform) and a mRNA vaccine (e.g., a SAM-based platform).
  • a viral vaccine e.g., a ChAdV-based platform
  • a mRNA vaccine e.g., a SAM-based platform
  • Heterologous vaccines include different antigen cassettes (e.g., aE6/E7 fusion protein cassette and a separate T cell epitope encoding cassette, and/or epitopes/antigens derived from different HPV strains, such as E6/E7 fusion protein variants from a HPV16 strain and a HPV18 strain) encoded by the same vaccine platform, e.g., either a viral vaccine (e.g., a ChAdV-based platform) or a mRNA vaccine (e.g., a SAM-based platform).
  • a viral vaccine e.g., a ChAdV-based platform
  • mRNA vaccine e.g., a SAM-based platform
  • Heterologous vaccines include different antigen cassettes (e.g., an E6/E7 fusion protein cassette and a separate T cell epitope encoding cassette, and/or epitopes/antigens derived from different HPV strains, such as E6/E7 fusion protein variants from a HPV16 strain and a HPV18 strain) encoded by different vaccine platforms, e.g., a viral vaccine (e.g., a ChAdV- based platform) and a mRNA vaccine (e.g. , a SAM-based platform).
  • a viral vaccine e.g., a ChAdV- based platform
  • a mRNA vaccine e.g. , a SAM-based platform
  • a vaccine composition can further comprise an adjuvant and/or a carrier.
  • an adjuvant and/or a carrier examples of useful adjuvants and carriers are given herein below.
  • a composition can be associated with a carrier such as e.g. a protein or an antigen-presenting cell such as, e.g., a dendritic cell (DC) capable of presenting the peptide to a T-cell.
  • a carrier such as e.g. a protein or an antigen-presenting cell such as, e.g., a dendritic cell (DC) capable of presenting the peptide to a T-cell.
  • DC dendritic cell
  • Adjuvants are any substance whose admixture into a vaccine composition increases or otherwise modifies the immune response to an antigen.
  • Carriers can be scaffold structures, for example a polypeptide or a polysaccharide, to which an antigen, is capable of being associated.
  • adjuvants are conjugated covalently or non-covalently.
  • an adjuvant to increase an immune response to an antigen is typically manifested by a significant or substantial increase in an immune-mediated reaction, or reduction in disease symptoms.
  • an increase in humoral immunity is typically manifested by a significant increase in the titer of antibodies raised to the antigen
  • an increase in T-cell activity is typically manifested in increased cell proliferation, or cellular cytotoxicity, or cytokine secretion.
  • An adjuvant may also alter an immune response, for example, by changing a primarily humoral or Th response into a primarily cellular, or Th response.
  • Suitable adjuvants include, but are not limited to 1018 ISS, alum, aluminum salts, Amplivax, AS 15, BCG, CP-870,893, CpG7909, CyaA, dSLIM, GM-CSF, IC30, IC31, Imiquimod, ImuFact 1MP321, IS Patch, ISS, 1SCOMATR1X, Juvlmmune, LipoVac, MF59, monophosphoryl lipid A, Montanide IMS 1312, Montanide ISA 206, Montanide ISA 50V, Montanide ISA-51, OK-432, OM-174, OM-197-MP-EC, ONTAK, PepTel vector system, PLG microparticles, resiquimod, SRL172, Virosomes and other Virus-like particles, YF-17D, VEGF trap, R848, beta-glucan, Pam3Cys, Aquila's QS21 stimulon (Aquila Biotech
  • Adjuvants such as incomplete Freund's or GM-CSF are useful.
  • GM-CSF Several immunological adjuvants (e.g., MF59) specific for dendritic cells and their preparation have been described previously (Dupuis M, et al., Cell Immunol. 1998; 186(1): 18-27; Allison A C; Dev Biol Stand. 1998; 92:3-11).
  • cytokines can be used.
  • cytokines have been directly linked to influencing dendritic cell migration to lymphoid tissues (e.g., TNF-alpha), accelerating the maturation of dendritic cells into efficient antigen-presenting cells for T-lymphocytes (e.g., GM-CSF, IL-1 and IL-4) (U.S. Pat. No. 5,849,589, specifically incorporated herein by reference in its entirety) and acting as immunoadjuvants (e.g., IL-12) (Gabrilovich D I, et al., J Immunother Emphasis Tumor Immunol. 1996 (6):414-418).
  • CpG immunostimulatory oligonucleotides have also been reported to enhance the effects of adjuvants in a vaccine setting.
  • Other TLR binding molecules such as RNA binding TLR 7, TLR 8 and/or TLR 9 may also be used.
  • CpGs e.g. CpR, Idera
  • Poly(I:C)(e.g. polyi:CI2U) non-CpG bacterial DNA or RNA
  • immunoactive small molecules and antibodies such as cyclophosphamide, sunitinib, bevacizumab, celebrex, NCX-4016, sildenafil, tadalafil, vardenafil, sorafinib, XL- 999, CP- 547632, pazopanib, ZD2171, AZD2171, ipilimumab, tremelimumab, and SC58175, which may act therapeutically and/or as an adjuvant.
  • CpGs e.g. CpR, Idera
  • Poly(I:C)(e.g. polyi:CI2U) e.g. polyi:CI2U
  • non-CpG bacterial DNA or RNA as well as immunoactive small molecules and antibodies such as
  • adjuvants and additives can readily be determined by the skilled artisan without undue experimentation.
  • Additional adjuvants include colony-stimulating factors, such as Granulocyte Macrophage Colony Stimulating Factor (GM-CSF, sargramostim).
  • GM-CSF Granulocyte Macrophage Colony Stimulating Factor
  • a vaccine composition can comprise more than one different adjuvant.
  • a therapeutic composition can comprise any adjuvant substance including any of the above or combinations thereof. It is also contemplated that a vaccine and an adjuvant can be administered together or separately in any appropriate sequence.
  • a carrier can be present independently of an adjuvant.
  • the function of a carrier can for example be to increase the molecular weight of a particular mutant to increase activity or immunogenicity, to confer stability, to increase the biological activity, or to increase serum half-life.
  • a carrier can aid presenting peptides to T-cells.
  • a carrier can be any suitable carrier known to the person skilled in the art, for example a protein or an antigen presenting cell.
  • a carrier protein could be but is not limited to keyhole limpet hemocyanin, serum proteins such as transferrin, bovine serum albumin, human serum albumin, thyroglobulin or ovalbumin, immunoglobulins, or hormones, such as insulin or palmitic acid.
  • the carrier is generally a physiologically acceptable carrier acceptable to humans and safe.
  • tetanus toxoid and/or diphtheria toxoid are suitable carriers.
  • the carrier can be dextrans for example Sepharose.
  • Cytotoxic T-cells recognize an antigen in the form of a peptide bound to an MHC molecule rather than the intact foreign antigen itself.
  • the MHC molecule itself is located at the cell surface of an antigen presenting cell.
  • an activation of CTLs is possible if a trimeric complex of peptide antigen, MHC molecule, and APC is present.
  • it may enhance the immune response if not only the peptide is used for activation of CTLs, but if additionally APCs with the respective MHC molecule are added. Therefore, in some embodiments a vaccine composition additionally contains at least one antigen presenting cell.
  • Antigens can also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.
  • viral vector-based vaccine platforms such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus
  • this approach can deliver one or more nucleotide sequences that encode one or more antigen peptides.
  • the sequences may be flanked by non-mutated sequences, may be separated by linkers or may be preceded with one or more sequences targeting a subcellular compartment (See, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science.
  • antigen cassette or “cassette” is meant the combination of a selected antigen or plurality of antigens (e.g., antigen-encoding nucleic acid sequences) and the other regulatory elements necessary to transcribe the antigen(s) and express the transcribed product.
  • the selected antigen or plurality of antigens can refer to distinct epitope sequences, e.g., an antigen-encoding nucleic acid sequence in the cassette can encode an epitope-encoding nucleic acid sequence (or plurality of epitope-encoding nucleic acid sequences) such that the epitopes are transcribed and expressed.
  • An antigen or plurality of antigens can be operatively linked to regulatory components in a manner which permits transcription. Such components include conventional regulatory elements that can drive expression of the antigen(s) in a cell transfected with the viral vector.
  • the antigen cassette can also contain a selected promoter which is linked to the antigen(s) and located, with other, optional regulatory elements, within the selected viral sequences of the recombinant vector.
  • a cassette can have one or more antigen-encoding nucleic acid sequences, such as a cassette containing multiple antigen-encoding nucleic acid sequences each independently operably linked to separate promoters and/or linked together using other multicistonic systems, such as 2A ribosome skipping sequence elements (e.g., E2A, P2A, F2A, or T2A sequences) or Internal Ribosome Entry Site (IRES) sequence elements.
  • a linker can also have a cleavage site, such as a TEV or furin cleavage site. Linkers with cleavage sites can be used in combination with other elements, such as those in a multicistronic system.
  • a furin protease cleavage site can be used in conjuction with a 2A ribosome skipping sequence element such that the furin protease cleavage site is configured to facilitate removal of the 2A sequence following translation.
  • each antigen-encoding nucleic acid sequence can contain one or more epitopeencoding nucleic acid sequences (e.g., an antigen-encoding nucleic acid sequence encoding concatenated T cell epitopes).
  • cassettes encoding HPV antigens are configured as follows: (1) endogenous 26S promoter - E6/E7 fusion protein - T2A - concatenated T-cell epitopes (TCE), or (2) endogenous 26S promoter - E6/E7 fusion protein - 26S promoter - concatenated TCE..
  • Combinations of multicistronic formats can also be used, such as in illustrative example: endogenous 26S promoter - HPV 16 E6/E7 fusion protein - T2A - HPV 16 concatenated TCE - 26S promoter - HPV18 E6/E7 fusion protein - T2A - HPV 18 concatenated TCE.
  • Useful promoters can be constitutive promoters or regulated (inducible) promoters, which will enable control of the amount of antigen(s) to be expressed.
  • a desirable promoter is that of the cytomegalovirus immediate early promoter/enhancer [see, e.g., Boshart et al, Cell, 41:521-530 (1985)].
  • Another desirable promoter includes the Rous sarcoma virus LTR promoter/enhancer.
  • Still another promoter/enhancer sequence is the chicken cytoplasmic betaactin promoter [T. A. Kost et al, Nucl. Acids Res., 11(23):8287 (1983)].
  • Other suitable or desirable promoters can be selected by one of skill in the art.
  • the antigen cassette can also include nucleic acid sequences heterologous to the viral vector sequences including sequences providing signals for efficient polyadenylation of the transcript (poly(A), poly-A or pA) and introns with functional splice donor and acceptor sites.
  • a common poly-A sequence which is employed in the exemplary vectors of this invention is that derived from the papovavirus SV-40.
  • the poly-A sequence generally can be inserted in the cassette following the antigen-based sequences and before the viral vector sequences.
  • a common intron sequence can also be derived from SV-40, and is referred to as the SV-40 T intron sequence.
  • An antigen cassette can also contain such an intron, located between the promoter/enhancer sequence and the antigen(s).
  • An antigen cassette can have one or more antigens.
  • a given cassette can include 1-10, 1-20, 1-30, 10-20, 15-25, 15-20, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more antigens.
  • Antigens can be linked directly to one another.
  • Antigens can also be linked to one another with linkers.
  • Antigens can be in any orientation relative to one another including N to C or C to N.
  • the antigen cassette can be located in the site of any selected deletion in the viral vector backbone, such as the site of the El gene region deletion or E3 gene region deletion of a ChAd-based vector or the deleted structural proteins of a VEE backbone, among others which may be selected.
  • the antigen encoding sequence e.g., cassette or one or more of the nucleic acid sequences encoding an immunogenic polypeptide in the cassette
  • the antigen encoding sequence can be described using the following formula to describe the ordered sequence of each element, from 5’ to 3’:
  • the corresponding N c is a distinct HPV derived nucleic acid sequence.
  • the corresponding Uf is a distinct universal MHC class II epitope-encoding nucleic acid sequence or a distinct MHC class II HPV derived epitope-encoding nucleic acid sequence.
  • the above antigen encoding sequence formula in some instances only describes the portion of an antigen cassette encoding concatenated epitope sequences, such as concatenated T cell epitopes.
  • the above antigen encoding sequence formula describes the concatenated T cell epitopes and separately the cassette encodes one or more full-length HPV proteins that are linked optionally using a multicistonic system, such as 2A ribosome skipping sequence elements (e.g. , E2A, P2A, F2A, or T2A sequences), a Internal Ribosome Entry Site (IRES) sequence elements, and/or independently operably linked to a separate promoter.
  • 2A ribosome skipping sequence elements e.g. , E2A, P2A, F2A, or T2A sequences
  • IVS Internal Ribosome Entry Site
  • antigen encoding sequence can be described using the following formula to describe the ordered sequence of each element, from 5’ to 3’:
  • N comprises one of the HPV derived nucleic acid sequences described herein (e.g., N encodes a polypeptide sequence as set forth in Tables C1-C9)
  • L5 comprises a 5’ linker sequence
  • L3 comprises a 3’ linker sequence
  • G5 comprises a nucleic acid sequences encoding an amino acid linker
  • G3 comprises one of the at least one nucleic acid sequences encoding an amino acid linker
  • U comprises an MHC class II epitope-encoding nucleic acid sequence, where for each X the corresponding Nc is a HPV derived nucleic acid sequence, where for each Y the corresponding Uf is a (1) universal MHC class II epitope-encoding nucleic acid sequence, optionally wherein the at least one universal sequence
  • the vector backbone such as an RNA alphavirus backbone
  • 10 epitopes are present, a 5’ linker is present for each N, a 3’ linker is present for each N, 2 MHC class II epitopes are present, a linker is present linking the two MHC class II epitopes, a linker is present linking the 5’ end of the two MHC class II epitopes to the 3’ linker of the final MHC class I epitope, and a linker is present linking the 3’ end of the two MHC class II epitopes to the to the vector backbone.
  • linking the 3 ’ end of the antigen cassette to the vector backbone examples include linking directly to the 3’ UTR elements provided by the vector backbone, such as a 3’ 19-nt CSE.
  • linking the 5’ end of the antigen cassette to the vector backbone examples include linking directly to a promoter or 5’ UTR element of the vector backbone, such as a 26S promoter sequence, an alphavirus 5’ UTR, a 51 -nt CSE, or a 24-nt CSE of an alphavirus vector backbone.
  • each MHC class I epitope that is present can have a 5’ linker, a 3 ’ linker, neither, or both.
  • some MHC class I epitopes may have both a 5’ linker and a 3’ linker, while other MHC class I epitopes may have either a 5’ linker, a 3’ linker, or neither.
  • some MHC class I epitopes may have either a 5’ linker or a 3’ linker, while other MHC class I epitopes may have either a 5’ linker, a 3’ linker, or neither.
  • MHC class II epitopes may have both a 5’ linker and a 3’ linker, while other MHC class 11 epitopes may have either a 5 ’ linker, a 3 ’ linker, or neither.
  • some MHC class II epitopes may have either a 5 ’ linker or a 3 ’ linker, while other MHC class II epitopes may have either a 5’ linker, a 3’ linker, or neither.
  • each antigen that is present can have a 5’ linker, a 3’ linker, neither, or both.
  • some antigens may have both a 5’ linker and a 3’ linker, while other antigens may have either a 5’ linker, a 3’ linker, or neither.
  • some antigens may have either a 5’ linker or a 3’ linker, while other antigens may have either a 5’ linker, a 3’ linker, or neither.
  • the promoter nucleotide sequences P and/or P2 can be the same as a promoter nucleotide sequence provided by the vector backbone, such as a RNA alphavirus backbone.
  • the promoter sequence provided by the vector backbone, Pn and P2 can each comprise a 26S subgenomic promoter or a CMV promoter.
  • the promoter nucleotide sequences P and/or P2 can be different from the promoter nucleotide sequence provided by the vector backbone, as well as can be different from each other.
  • the 5’ linker L5 can be a native sequence or a non-natural sequence.
  • Non-natural sequence include, but are not limited to, AAY, RR, and DPP.
  • the 3’ linker L3 can also be a native sequence or a non-natural sequence. Additionally, L5 and L3 can both be native sequences, both be non-natural sequences, or one can be native and the other non-natural.
  • the amino acid linkers can be 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,
  • the amino acid linkers can be also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.
  • the amino acid linker G5, for each Y can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100 or more amino acids in length.
  • the amino acid linkers can be also be at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.
  • the amino acid linker G3 can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,
  • G3 can be also be at least 3, at least 4, at least
  • each N can encode a MHC class I epitope, a MHC class II epitope, an epitope capable of stimulating a B cell response, or a combination thereof.
  • N can encode a combination of a MHC class I epitope, a MHC class II epitope, and an epitope capable of stimulating a B cell response.
  • N can encode a combination of a MHC class I epitope and a MHC class II epitope.
  • N can encode a combination of a MHC class I epitope and an epitope capable of stimulating a B cell response.
  • N can encode a combination of a MHC class II epitope and an epitope capable of stimulating a B cell response.
  • each N can encode a MHC class I epitope 7-15 amino acids in length.
  • each N can also encodes a MHC class I epitope 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
  • each N can also encodes a MHC class I epitope at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 amino acids in length.
  • each N can encode a MHC class II epitope.
  • each N can encode an epitope capable of stimulating a B cell response.
  • the cassette encoding the one or more antigens can be 700 nucleotides or less.
  • the cassette encoding the one or more antigens can be 700 nucleotides or less and encode 2 distinct epitope-encoding nucleic acid sequences (e.g., encode 2 distinct HPV derived nucleic acid sequence encoding an immunogenic polypeptide).
  • the cassette encoding the one or more antigens can be 700 nucleotides or less and encode at least 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 700 nucleotides or less and encode 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 700 nucleotides or less and encode at least 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 700 nucleotides or less and include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • the cassette encoding the one or more antigens can be between 375-700 nucleotides in length.
  • the cassette encoding the one or more antigens can be between 375-700 nucleotides in length and encode 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-700 nucleotides in length and encode at least 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-700 nucleotides in length and encode 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens be between 375-700 nucleotides in length and encode at least 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-700 nucleotides in length and include 1- 10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and encode 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and encode at least 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and encode 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and encode at least 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be 600, 500, 400, 300, 200, or 100 nucleotides in length or less and include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375-400 nucleotides in length.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375-400 nucleotides in length and encode 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375-400 nucleotides in length and encode at least 2 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375- 400 nucleotides in length and encode 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375-400 nucleotides in length and encode at least 3 distinct epitope-encoding nucleic acid sequences.
  • the cassette encoding the one or more antigens can be between 375-600, between 375-500, or between 375-400 nucleotides in length and include 1-10, 1-5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more antigens.
  • an integrated multi-dimensional model can be considered that places candidate antigens in a space with at least the following axes and optimizes selection using an integrative approach.
  • HLA genes large number of HLA molecules involved in the presentation of a set of antigens may lower the probability that an infected cell will escape immune attack via downregulation or mutation of HLA molecules
  • antigens can be deprioritized (e.g., excluded) from the vaccination if they are predicted to be presented by HLA alleles lost or inactivated in either all or part of the patient’ s infected cell.
  • HLA allele loss can occur by either somatic mutation, loss of heterozygosity, or homozygous deletion of the locus.
  • Methods for detection of HLA allele somatic mutation are well known in the art, e.g. (Shukla et al., 2015). Methods for detection of somatic LOH and homozygous deletion (including for HLA locus) are likewise well described.
  • Antigens can also be deprioritized if mass-spectrometry data indicates a predicted antigen is not presented by a predicted HLA allele.
  • all self-amplifying RNA (SAM) vectors contain a self-amplifying backbone derived from a self-replicating virus.
  • self-amplifying backbone refers to minimal sequence(s) of a self-replicating virus that allows for self-replication of the viral genome.
  • minimal sequences that allow for self-replication of an alphavirus can include conserved sequences for nonstructural protein-mediated amplification (e.g., a nonstructural protein 1 (nsPl) gene, a nsP2 gene, a nsP3 gene, a nsP4 gene, and/or a polyA sequence).
  • a self- amplifying backbone can also include sequences for expression of subgenomic viral RNA (e.g. , a 26S promoter element for an alphavirus).
  • SAM vectors can be positive-sense RNA polynucleotides or negative-sense RNA polynucleotides, such as vectors with backbones derived from positive-sense or negative-sense self-replicating viruses.
  • Self-replicating viruses include, but are not limited to, alphaviruses, flaviviruses e.g., Kunjin virus), measles viruses, and rhabdoviruses (e.g., rabies virus and vesicular stomatitis virus).
  • SAM vector systems derived from self-replicating viruses are described in greater detail in Lundstrom (Molecules. 2018 Dec 13;23(12). pii: E3310. doi: 10.3390/molecules23123310), herein incorporated by reference for all purposes.
  • Alphaviruses are members of the family Togaviridae, and are positive-sense single stranded RNA viruses. Members are typically classified as either Old World, such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses, or New World, such as eastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbrial Review 1994).
  • Old World such as Sindbis, Ross River, Mayaro, Chikungunya, and Semliki Forest viruses
  • New World such as eastern equine encephalitis, Aura, Fort Morgan, or Venezuelan equine encephalitis virus and its derivative strain TC-83 (Strauss Microbrial Review 1994).
  • a natural alphavirus genome is typically around 12kb in length, the first two-thirds of which contain genes encoding nonstructural proteins (nsPs) that form RNA replication complexes for self-replication of the viral genome, and the last third of which contains a subgenomic expression cassette encoding structural proteins for virion production (Frolov RNA 2001).
  • nsPs nonstructural proteins
  • a model lifecycle of an alphavirus involves several distinct steps (Strauss Microbrial Review 1994, Jose Future Microbiol 2009). Following virus attachment to a host cell, the virion fuses with membranes within endocytic compartments resulting in the eventual release of genomic RNA into the cytosol.
  • the genomic RNA which is in a plus-strand orientation and comprises a 5’ methylguanylate cap and 3’ polyA tail, is translated to produce non-structural proteins nsPl-4 that form the replication complex. Early in infection, the plus-strand is then replicated by the complex into a minus-stand template.
  • the replication complex is further processed as infection progresses, with the resulting processed complex switching to transcription of the minus-strand into both full-length positive-strand genomic RNA, as well as the 26S subgenomic positive-strand RNA containing the structural genes.
  • CSEs conserved sequence elements of alphavirus have been identified to potentially play a role in the various RNA replication steps including; a complement of the 5’ UTR in the replication of plus-strand RNAs from a minus-strand template, a 51 -nt CSE in the replication of minus-strand synthesis from the genomic template, a 24-nt CSE in the junction region between the nsPs and the 26S RNA in the transcription of the subgenomic RNA from the minus-strand, and a 3’ 19-nt CSE in minus-strand synthesis from the plus-strand template.
  • CSEs conserved sequence elements
  • virus particles are then typically assembled in the natural lifecycle of the virus.
  • the 26S RNA is translated and the resulting proteins further processed to produce the structural proteins including capsid protein, glycoproteins El and E2, and two small polypeptides E3 and 6K (Strauss 1994). Encapsidation of viral RNA occurs, with capsid proteins normally specific for only genomic RNA being packaged, followed by virion assembly and budding at the membrane surface.
  • Alphavirus as a delivery vector
  • Alphaviruses can be used to generate alphavirus-based delivery vectors (also be referred to as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors).
  • alphavirus vectors also be referred to as alphavirus vectors, alphavirus viral vectors, alphavirus vaccine vectors, self-replicating RNA (srRNA) vectors, or self-amplifying RNA (samRNA) vectors.
  • Alphaviruses have previously been engineered for use as expression vector systems (Pushko 1997, Rheme 2004). Alphaviruses offer several advantages, particularly in a vaccine setting where heterologous antigen expression can be desired.
  • alphavirus vectors Due to its ability to self-replicate in the host cytosol, alphavirus vectors are generally able to produce high copy numbers of the expression cassette within a cell resulting in a high level of heterologous antigen production. Additionally, the vectors are generally transient, resulting in improved biosafety as well as reduced induction of immunological tolerance to the vector.
  • the public in general, also lacks pre-existing immunity to alphavirus vectors as compared to other standard viral vectors, such as human adenovirus.
  • Alphavirus based vectors also generally result in cytotoxic responses to infected cells. Cytotoxicity, to a certain degree, can be important in a vaccine setting to properly illicit an immune response to the heterologous antigen expressed.
  • an antigen expression vector described herein can utilize an alphavirus backbone that allows for a high level of antigen expression, stimulates a robust immune response to antigen, does not stimulate an immune response to the vector itself, and can be used in a safe manner.
  • the antigen expression cassette can be designed to stimulate different levels of an immune response through optimization of which alphavirus sequences the vector uses, including, but not limited to, sequences derived from VEE or its attenuated derivative TC- 83.
  • RNA is produced that expresses the heterologous protein.
  • all the elements for production of infectious virions are present and, therefore, repeated rounds of infection of the expression vector in non-infected cells can occur.
  • helper virus systems Pushko 1997.
  • the structural proteins are replaced by a heterologous gene.
  • the 26S subgenomic RNA provides for expression of the heterologous protein.
  • additional vectors that expresses the structural proteins are then supplied in trans, such as by co-transfection of a cell line, to produce infectious virus.
  • a system is described in detail in USPN 8,093,021, which is herein incorporated by reference in its entirety, for all purposes.
  • the helper vector system provides the benefit of limiting the possibility of forming infectious particles and, therefore, improves biosafety.
  • helper vector system reduces the total vector length, potentially improving the replication and expression efficiency.
  • an example of an antigen expression vector described herein can utilize an alphavirus backbone wherein the structural proteins are replaced by an antigen cassette, the resulting vector both reducing biosafety concerns, while at the same time promoting efficient expression due to the reduction in overall expression vector size.
  • RNA polymerase promoter at the 5’ end of the sequence desired to be transcribed into RNA (e.g., SAM). Promoters include, but are not limited to, bacteriophage polymerase promoters such as T3, T7, KI 1, or SP6.
  • RNA polymerase promoter can be referred to by the sequence TAATACGACTCACTATAGG, in which an IVT reaction using the DNA template TAATACGACTCACTATAGGN for the production of desired sequence N will result in the mRNA sequence GG-N.
  • T7 polymerase more efficiently transcribes RNA transcripts beginning with guanosine.
  • the RNA polymerase promoter contained in the DNA template can be a sequence the results in transcripts containing only the 5’ nucleotides of the desired sequence, e.g., a SAM having the native 5’ sequence of the self-replicating virus from which the SAM vector is derived.
  • a minimal T7 promoter can be referred to by the sequence TAATACGACTCACTATA, in which an IVT reaction using the DNA template TAATACGACTCACTATAN for the production of desired sequence N will result in the mRNA sequence N.
  • a minimal SP6 promoter referred to by the sequence ATTTAGGTGACACTATA can be used to generate transcripts without additional 5’ nucleotides.
  • the DNA template is incubated with the appropriate RNA polymerase enzyme, buffer agents, and nucleotides (NTPs).
  • RNA polynucleotide can optionally be further modified including, but limited to, addition of a 5’ cap structure such as 7-methylguanosine or a related structure, and optionally modifying the 3’ end to include a poly adenylate (poly A) tail.
  • a 5’ cap structure such as 7-methylguanosine or a related structure
  • poly A poly adenylate
  • RNA is capped with a 5’ cap structure co-transcriptionally through the addition of cap analogues during IVT.
  • Cap analogues can include dinucleotide (m 7 G-ppp-N) cap analogues or trinucleotide (m 7 G-ppp-N-N) cap analogues, where N represents a nucleotide or modified nucleotide (e.g., ribonucleosides including, but not limited to, adenosine, guanosine, cytidine, and uradine).
  • ribonucleosides including, but not limited to, adenosine, guanosine, cytidine, and uradine.
  • Exemplary cap analogues and their use in IVT reactions are also described in greater detail in U.S. Pat. No. 10,519,189, herein incorporated by reference for all purposes. As discussed, T7 polymerase more efficiently transcribes RNA transcripts beginning with guanosine.
  • a trinucleotide cap analogue (m 7 G-ppp-N-N) can be used.
  • the trinucleotide cap analogue can increase transcription efficiency 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20- fold or more relative to an IVT reaction using a dinucleotide cap analogue (m 7 G-ppp-N).
  • a 5’ cap structure can also be added following transcription, such as using a vaccinia capping system (e.g., NEB Cat. No. M2080) containing mRNA 2’-O-methyltransferase and S- Adenosyl methionine.
  • a vaccinia capping system e.g., NEB Cat. No. M2080
  • RNA polynucleotide can optionally be further modified separately from or in addition to the capping techniques described including, but limited to, modifying the 3’ end to include a poly adenylate (poly A) tail.
  • the RNA can then be purified using techniques well-known in the field, such as phenol-chloroform extraction.
  • An important aspect to consider in vaccine vector design is immunity against the vector itself (Riley 2017). This may be in the form of preexisting immunity to the vector itself, such as with certain human adenovirus systems, or in the form of developing immunity to the vector following administration of the vaccine. The latter is an important consideration if multiple administrations of the same vaccine are performed, such as separate priming and boosting doses, or if the same vaccine vector system is to be used to deliver different antigen cassettes.
  • alphavirus vectors In the case of alphavirus vectors, the standard delivery method is the previously discussed helper virus system that provides capsid, El, and E2 proteins in trans to produce infectious viral particles. However, it is important to note that the El and E2 proteins are often major targets of neutralizing antibodies (Strauss 1994). Thus, the efficacy of using alphavirus vectors to deliver antigens of interest to target cells may be reduced if infectious particles are targeted by neutralizing antibodies.
  • Nanomaterials can be made of non-immunogenic materials and generally avoid stimulating immunity to the delivery vector itself.
  • These materials can include, but are not limited to, lipids, inorganic nanomaterials, and other polymeric materials.
  • Lipids can be cationic, anionic, or neutral. The materials can be synthetic or naturally derived, and in some instances biodegradable.
  • Lipids can include fats, cholesterol, phospholipids, lipid conjugates including, but not limited to, polyethyleneglycol (PEG) conjugates (PEGylated lipids), waxes, oils, glycerides, and fat soluble vitamins.
  • PEG polyethyleneglycol
  • Lipid nanoparticles are an attractive delivery system due to the amphiphilic nature of lipids enabling formation of membranes and vesicle like structures (Riley 2017). In general, these vesicles deliver the expression vector by absorbing into the membrane of target cells and releasing nucleic acid into the cytosol. In addition, LNPs can be further modified or functionalized to facilitate targeting of specific cell types. Another consideration in LNP design is the balance between targeting efficiency and cytotoxicity. Lipid compositions generally include defined mixtures of cationic, neutral, anionic, and amphipathic lipids.
  • lipid composition can influence overall LNP size and stability.
  • the lipid composition comprises dilinoleylmethyl- 4-dimethylaminobutyrate (MC3) or MC3-like molecules.
  • MC3 and MC3-like lipid compositions can be formulated to include one or more other lipids, such as a PEG or PEG- conjugated lipid, a sterol, or neutral lipids.
  • Nucleic-acid vectors such as expression vectors, exposed directly to serum can have several undesirable consequences, including degradation of the nucleic acid by serum nucleases or off-target stimulation of the immune system by the free nucleic acids. Therefore, encapsulation of the alphavirus vector can be used to avoid degradation, while also avoiding potential off-target effects.
  • an alphavirus vector is fully encapsulated within the delivery vehicle, such as within the aqueous interior of an LNP. Encapsulation of the alphavirus vector within an LNP can be carried out by techniques well-known to those skilled in the art, such as microfluidic mixing and droplet generation carried out on a microfluidic droplet generating device.
  • Such devices include, but are not limited to, standard T-junction devices or flow-focusing devices.
  • the desired lipid formulation such as MC3 or MC3-like containing compositions
  • the droplet generating device can control the size range and size distribution of the LNPs produced.
  • the LNP can have a size ranging from 1 to 1000 nanometers in diameter, e.g., 1, 10, 50, 100, 500, or 1000 nanometers.
  • the delivery vehicles encapsulating the expression vectors can be further treated or modified to prepare them for administration.
  • V.D.l. Viral delivery with chimpanzee adenovirus
  • Vaccine compositions for delivery of one or more antigens can be created by providing adenovirus nucleotide sequences of chimpanzee origin, a variety of novel vectors, and cell lines expressing chimpanzee adenovirus genes.
  • a nucleotide sequence of a chimpanzee C68 adenovirus also referred to herein as ChAdV68
  • ChAdV68 a chimpanzee C68 adenovirus
  • Use of C68 adenovirus derived vectors is described in further detail in USPN 6,083,716, which is herein incorporated by reference in its entirety, for all purposes.
  • a recombinant adenovirus comprising the DNA sequence of a chimpanzee adenovirus such as C68 and an antigen cassette operatively linked to regulatory sequences directing its expression.
  • the recombinant virus is capable of infecting a mammalian, preferably a human, cell and capable of expressing the antigen cassette product in the cell.
  • the native chimpanzee El gene, and/or E3 gene, and/or E4 gene can be deleted.
  • An antigen cassette can be inserted into any of these sites of gene deletion.
  • the antigen cassette can include an antigen against which a primed immune response is desired.
  • a mammalian cell infected with a chimpanzee adenovirus such as C68 is provided herein.
  • a novel mammalian cell line which expresses a chimpanzee adenovirus gene (e.g., from C68) or functional fragment thereof.
  • a method for delivering an antigen cassette into a mammalian cell comprising the step of introducing into the cell an effective amount of a chimpanzee adenovirus, such as C68, that has been engineered to express the antigen cassette.
  • Still another aspect provides a method for stimulating an immune response in a mammalian host.
  • the method can comprise the step of administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, comprising an antigen cassette that encodes one or more antigens from the infection against which the immune response is targeted.
  • a recombinant chimpanzee adenovirus such as C68
  • Still another aspect provides a method for stimulating an immune response in a mammalian host to treat or prevent a disease in a subject, such as an infectious disease.
  • the method can comprise the step of administering to the host an effective amount of a recombinant chimpanzee adenovirus, such as C68, comprising an antigen cassette that encodes one or more antigens, such as from the infectious disease against which the immune response is targeted.
  • a non-simian mammalian cell that expresses a chimpanzee adenovirus gene obtained from the sequence of SEQ ID NO: 1.
  • the gene can be selected from the group consisting of the adenovirus E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4 and L5 of SEQ ID NO: 1.
  • nucleic acid molecule comprising a chimpanzee adenovirus DNA sequence comprising a gene obtained from the sequence of SEQ ID NO: 1.
  • the gene can be selected from the group consisting of said chimpanzee adenovirus E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4 and L5 genes of SEQ ID NO: 1.
  • the nucleic acid molecule comprises SEQ ID NO: 1.
  • the nucleic acid molecule comprises the sequence of SEQ ID NO: 1, lacking at least one gene selected from the group consisting of E1A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4 and L5 genes of SEQ ID NO: 1.
  • a vector comprising a chimpanzee adenovirus DNA sequence obtained from SEQ ID NO: 1 and an antigen cassette operatively linked to one or more regulatory sequences which direct expression of the cassette in a heterologous host cell, optionally wherein the chimpanzee adenovirus DNA sequence comprises at least the cis- elements necessary for replication and virion encapsidation, the czs-elements flanking the antigen cassette and regulatory sequences.
  • the chimpanzee adenovirus DNA sequence comprises a gene selected from the group consisting of El A, E1B, E2A, E2B, E3, E4, LI, L2, L3, L4 and L5 gene sequences of SEQ ID NO: 1.
  • the vector can lack the E1A and/or E1B gene.
  • an adenovirus vector comprising: a partially deleted E4 gene comprising a deleted or partially-deleted E4orf2 region and a deleted or partially-deleted E4orf3 region, and optionally a deleted or partially-deleted E4orf4 region.
  • the partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,916 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1.
  • the partially deleted E4 can comprise an E4 deletion of at least a partial deletion of nucleotides 34,916 to 34,942 of the sequence shown in SEQ ID NO: 1, at least a partial deletion of nucleotides 34,952 to 35,305 of the sequence shown in SEQ ID NO: 1, and at least a partial deletion of nucleotides 35,302 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1
  • the partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,980 to 36,516 of the sequence shown in SEQ ID NO:1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO:1.
  • the partially deleted E4 can comprise an E4 deletion of at least nucleotides 34,979 to 35,642 of the sequence shown in SEQ ID NO:1, and wherein the vector comprises at least nucleotides 2 to 36,518 of the sequence set forth in SEQ ID NO: 1.
  • the partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E40rf2, a fully deleted E40rf3, and at least a partial deletion of E40rf4.
  • the partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E40rf2, at least a partial deletion of E40rf3, and at least a partial deletion of E40rf4.
  • the partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E4Orf 1 , a fully deleted E40rf2, and at least a partial deletion of E40rf3.
  • the partially deleted E4 can comprise an E4 deletion of at least a partial deletion of E40rf2 and at least a partial deletion of E40rf3.
  • the partially deleted E4 can comprise an E4 deletion between the start site of E4Orfl to the start site of E40rf5.
  • the partially deleted E4 can be an E4 deletion adjacent to the start site of E4Orfl.
  • the partially deleted E4 can be an E4 deletion adjacent to the start site of E40rf2.
  • the partially deleted E4 can be an E4 deletion adjacent to the start site of E40rf3.
  • the partially deleted E4 can be an E4 deletion adjacent to the start site of E40rf4.
  • the E4 deletion can be at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, at least 1200, at least 1300, at least 1400, at least 1500, at least 1600, at least 1700, at least 1800, at least 1900, or at least 2000 nucleotides.
  • the E4 deletion can be at least 700 nucleotides.
  • the E4 deletion can be at least 1500 nucleotides.
  • the E4 deletion can be 50 or less, 100 or less, 200 or less, 300 or less, 400 or less, 500 or less, 600 or less, 700 or less, 800 or less, 900 or less, 1000 or less, 1100 or less, 1200 or less, 1300 or less, 1400 or less, 1500 or less, 1600 or less, 1700 or less, 1800 or less, 1900 or less, or 2000 or less nucleotides.
  • the E4 deletion can be 750 nucleotides or less.
  • the E4 deletion can be at least 1550 nucleotides or less.
  • Also disclosed herein is a host cell transfected with a vector disclosed herein such as a C68 vector engineered to expression an antigen cassette. Also disclosed herein is a human cell that expresses a selected gene introduced therein through introduction of a vector disclosed herein into the cell.
  • a vector disclosed herein such as a C68 vector engineered to expression an antigen cassette.
  • a human cell that expresses a selected gene introduced therein through introduction of a vector disclosed herein into the cell.
  • Also disclosed herein is a method for delivering an antigen cassette to a mammalian cell comprising introducing into said cell an effective amount of a vector disclosed herein such as a C68 vector engineered to expression the antigen cassette.
  • Also disclosed herein is a method for producing an antigen comprising introducing a vector disclosed herein into a mammalian cell, culturing the cell under suitable conditions and producing the antigen.
  • the function of the deleted gene region if essential to the replication and infectivity of the virus, can be supplied to the recombinant virus by a helper virus or cell line, i.e., a complementation or packaging cell line.
  • a helper virus or cell line i.e., a complementation or packaging cell line.
  • a cell line can be used which expresses the El gene products of the human or chimpanzee adenovirus; such a cell line can include HEK293 or variants thereof.
  • the protocol for the generation of the cell lines expressing the chimpanzee El gene products (Examples 3 and 4 of USPN 6,083,716) can be followed to generate a cell line which expresses any selected chimpanzee adenovirus gene.
  • An AAV augmentation assay can be used to identify a chimpanzee adenovirus El- expressing cell line. This assay is useful to identify El function in cell lines made by using the El genes of other uncharacterized adenoviruses, e.g., from other species. That assay is described in Example 4B of USPN 6,083,716.
  • a selected chimpanzee adenovirus gene can be under the transcriptional control of a promoter for expression in a selected parent cell line.
  • Inducible or constitutive promoters can be employed for this purpose.
  • inducible promoters are included the sheep metallothionine promoter, inducible by zinc, or the mouse mammary tumor virus (MMTV) promoter, inducible by a glucocorticoid, particularly, dexamethasone.
  • MMTV mouse mammary tumor virus
  • Other inducible promoters such as those identified in International patent application WO95/13392, incorporated by reference herein can also be used in the production of packaging cell lines.
  • Constitutive promoters in control of the expression of the chimpanzee adenovirus gene can be employed also.
  • a parent cell can be selected for the generation of a novel cell line expressing any desired C68 gene.
  • a parent cell line can be HeLa [ATCC Accession No. CCL 2], A549 [ATCC Accession No. CCL 185], KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75] cells.
  • Other suitable parent cell lines can be obtained from other sources.
  • Parent cell lines can include CHO, HEK293 or variants thereof, 911, HeLa, A549, LP- 293, PER.C6, or AEl-2a.
  • An El-expressing cell line can be useful in the generation of recombinant chimpanzee adenovirus El deleted vectors.
  • Cell lines constructed using essentially the same procedures that express one or more other chimpanzee adenoviral gene products are useful in the generation of recombinant chimpanzee adenovirus vectors deleted in the genes that encode those products.
  • cell lines which express other human Ad El gene products are also useful in generating chimpanzee recombinant Ads.
  • compositions disclosed herein can comprise viral vectors, that deliver at least one antigen to cells.
  • viral vectors comprise a chimpanzee adenovirus DNA sequence such as C68 and an antigen cassette operatively linked to regulatory sequences which direct expression of the cassette.
  • the C68 vector is capable of expressing the cassette in an infected mammalian cell.
  • the C68 vector can be functionally deleted in one or more viral genes.
  • An antigen cassette comprises at least one antigen under the control of one or more regulatory sequences such as a promoter.
  • Optional helper viruses and/or packaging cell lines can supply to the chimpanzee viral vector any necessary products of deleted adenoviral genes.
  • the term "functionally deleted” means that a sufficient amount of the gene region is removed or otherwise altered, e.g., by mutation or modification, so that the gene region is no longer capable of producing one or more functional products of gene expression. Mutations or modifications that can result in functional deletions include, but are not limited to, nonsense mutations such as introduction of premature stop codons and removal of canonical and non- canonical start codons, mutations that alter mRNA splicing or other transcriptional processing, or combinations thereof. If desired, the entire gene region can be removed. [00311] Modifications of the nucleic acid sequences forming the vectors disclosed herein, including sequence deletions, insertions, and other mutations may be generated using standard molecular biological techniques and are within the scope of this invention.
  • the chimpanzee adenovirus C68 vectors useful in this invention include recombinant, defective adenoviruses, that is, chimpanzee adenovirus sequences functionally deleted in the Ela or Elb genes, and optionally bearing other mutations, e.g., temperaturesensitive mutations or deletions in other genes. It is anticipated that these chimpanzee sequences are also useful in forming hybrid vectors from other adenovirus and/or adeno-associated virus sequences. Homologous adenovirus vectors prepared from human adenoviruses are described in the published literature [see, for example, Kozarsky T and TT, cited above, and references cited therein, U.S. Pat. No. 5,240,846].
  • a range of adenovirus nucleic acid sequences can be employed in the vectors.
  • a vector comprising minimal chimpanzee C68 adenovirus sequences can be used in conjunction with a helper virus to produce an infectious recombinant virus particle.
  • the helper virus provides essential gene products required for viral infectivity and propagation of the minimal chimpanzee adenoviral vector.
  • the deleted gene products can be supplied in the viral vector production process by propagating the virus in a selected packaging cell line that provides the deleted gene functions in trans.
  • a minimal chimpanzee Ad C68 virus is a viral particle containing just the adenovirus cis-elements necessary for replication and virion encapsidation. That is, the vector contains the cis-acting 5' and 3' inverted terminal repeat (ITR) sequences of the adenoviruses (which function as origins of replication) and the native 5' packaging/enhancer domains (that contain sequences necessary for packaging linear Ad genomes and enhancer elements for the El promoter).
  • ITR inverted terminal repeat
  • Recombinant, replication-deficient adenoviruses can also contain more than the minimal chimpanzee adenovirus sequences.
  • Ad vectors can be characterized by deletions of various portions of gene regions of the virus, and infectious virus particles formed by the optional use of helper viruses and/or packaging cell lines.
  • suitable vectors may be formed by deleting all or a sufficient portion of the C68 adenoviral immediate early gene Ela and delayed early gene E lb, so as to eliminate their normal biological functions.
  • Replication-defective El-deleted viruses are capable of replicating and producing infectious virus when grown on a chimpanzee adenovirus- transformed, complementation cell line containing functional adenovirus Ela and Elb genes which provide the corresponding gene products in trans.
  • the resulting recombinant chimpanzee adenovirus is capable of infecting many cell types and can express antigen(s), but cannot replicate in most cells that do not carry the chimpanzee El region DNA unless the cell is infected at a very high multiplicity of infection.
  • all or a portion of the C68 adenovirus delayed early gene E3 can be eliminated from the chimpanzee adenovirus sequence which forms a part of the recombinant virus.
  • Chimpanzee adenovirus C68 vectors can also be constructed having a deletion of the E4 gene. Still another vector can contain a deletion in the delayed early gene E2a.
  • Deletions can also be made in any of the late genes LI through L5 of the chimpanzee C68 adenovirus genome. Similarly, deletions in the intermediate genes IX and IVa2 can be useful for some purposes. Other deletions may be made in the other structural or non-structural adenovirus genes.
  • deletions can be used individually, i.e., an adenovirus sequence can contain deletions of El only. Alternatively, deletions of entire genes or portions thereof effective to destroy or reduce their biological activity can be used in any combination.
  • the adenovirus C68 sequence can have deletions of the El genes and the E4 gene, or of the El, E2a and E3 genes, or of the El and E3 genes, or of El, E2a and E4 genes, with or without deletion of E3, and so on.
  • deletions can be used in combination with other mutations, such as temperature-sensitive mutations, to achieve a desired result.
  • the cassette comprising antigen(s) be inserted optionally into any deleted region of the chimpanzee C68 Ad virus.
  • the cassette can be inserted into an existing gene region to disrupt the function of that region, if desired.
  • helper Viruses Depending upon the chimpanzee adenovirus gene content of the viral vectors employed to carry the antigen cassette, a helper adenovirus or non-replicating virus fragment can be used to provide sufficient chimpanzee adenovirus gene sequences to produce an infective recombinant viral particle containing the cassette.
  • Useful helper viruses contain selected adenovirus gene sequences not present in the adenovirus vector construct and/or not expressed by the packaging cell line in which the vector is transfected.
  • a helper virus can be replication-defective and contain a variety of adenovirus genes in addition to the sequences described above.
  • the helper virus can be used in combination with the El -expressing cell lines described herein.
  • the "helper" virus can be a fragment formed by clipping the C terminal end of the C68 genome with SspI, which removes about 1300 bp from the left end of the virus. This clipped virus is then co-transfected into an El -expressing cell line with the plasmid DNA, thereby forming the recombinant virus by homologous recombination with the C68 sequences in the plasmid.
  • Helper viruses can also be formed into poly-cation conjugates as described in Wu et al, J. Biol. Chem., 264:16985-16987 (1989); K. J. Fisher and J. M. Wilson, Biochem. J., 299:49 (Apr. 1, 1994).
  • Helper virus can optionally contain a reporter gene.
  • a number of such reporter genes are known to the art.
  • the presence of a reporter gene on the helper virus which is different from the antigen cassette on the adenovirus vector allows both the Ad vector and the helper virus to be independently monitored. This second reporter is used to enable separation between the resulting recombinant virus and the helper virus upon purification.
  • Assembly of the selected DNA sequences of the adenovirus, the antigen cassette, and other vector elements into various intermediate plasmids and shuttle vectors, and the use of the plasmids and vectors to produce a recombinant viral particle can all be achieved using conventional techniques.
  • Such techniques include conventional cloning techniques of cDNA, in vitro recombination techniques (e.g., Gibson assembly), use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence.
  • Standard transfection and co-transfection techniques are employed, e.g., CaPO4 precipitation techniques or liposome-mediated transfection methods such as lipofectamine.
  • the vector can be transfected in vitro in the presence of a helper virus into the packaging cell line. Homologous recombination occurs between the helper and the vector sequences, which permits the adenovirus-antigen sequences in the vector to be replicated and packaged into virion capsids, resulting in the recombinant viral vector particles.
  • the resulting recombinant chimpanzee C68 adenoviruses are useful in transferring an antigen cassette to a selected cell.
  • the El -deleted recombinant chimpanzee adenovirus demonstrates utility in transferring a cassette to a non-chimpanzee, preferably a human, cell.
  • the resulting recombinant chimpanzee C68 adenovirus containing the antigen cassette (produced by cooperation of the adenovirus vector and helper virus or adenoviral vector and packaging cell line, as described above) thus provides an efficient gene transfer vehicle which can deliver antigen(s) to a subject in vivo or ex vivo.
  • a chimpanzee viral vector bearing an antigen cassette can be administered to a patient, preferably suspended in a biologically compatible solution or pharmaceutically acceptable delivery vehicle.
  • a suitable vehicle includes sterile saline.
  • Other aqueous and non-aqueous isotonic sterile injection solutions and aqueous and non-aqueous sterile suspensions known to be pharmaceutically acceptable carriers and well known to those of skill in the art may be employed for this purpose.
  • the chimpanzee adenoviral vectors are administered in sufficient amounts to transduce the human cells and to provide sufficient levels of antigen transfer and expression to provide a therapeutic benefit without undue adverse or with medically acceptable physiological effects, which can be determined by those skilled in the medical arts.
  • Conventional and pharmaceutically acceptable routes of administration include, but are not limited to, direct delivery to the liver, intranasal, intravenous, intramuscular, subcutaneous, intradermal, oral and other parental routes of administration. Routes of administration may be combined, if desired.
  • Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients.
  • Recombinant, replication defective adenoviruses can be administered in a "pharmaceutically effective amount", that is, an amount of recombinant adenovirus that is effective in a route of administration to transfect the desired cells and provide sufficient levels of expression of the selected gene to provide a vaccinal benefit, i.e., some measurable level of protective immunity.
  • C68 vectors comprising an antigen cassette can be co- administered with adjuvant.
  • Adjuvant can be separate from the vector (e.g., alum) or encoded within the vector, in particular if the adjuvant is a protein. Adjuvants are well known in the art.
  • routes of administration include, but are not limited to, intranasal, intramuscular, intratracheal, subcutaneous, intradermal, rectal, oral and other parental routes of administration. Routes of administration may be combined, if desired, or adjusted depending upon the immunogen or the disease. For example, in prophylaxis of rabies, the subcutaneous, intratracheal and intranasal routes are preferred. The route of administration primarily will depend on the nature of the disease being treated.
  • the levels of immunity to antigen(s) can be monitored to determine the need, if any, for boosters. Following an assessment of antibody titers in the serum, for example, optional booster immunizations may be desired.
  • an infectious disease organism-specific e.g. a HPV specific
  • a subject has been diagnosed with an infection or is at risk of an infection (e.g. one or more of cervical cancer, anal cancer, oropharyngeal cancer, oral cancer, penile cancer, vaginal cancer, and/or vulvar cancer due to a HPV infection), such as age, geographical/travel, and/or work-related increased risk of or predisposition to an infection, or at risk to a seasonal and/or novel disease infection.
  • an infection e.g. one or more of cervical cancer, anal cancer, oropharyngeal cancer, oral cancer, penile cancer, vaginal cancer, and/or vulvar cancer due to a HPV infection
  • a subject is immunocompromised, such as diagnosed with and/or suspected of having cancer.
  • a subject can include those treated with a therapy resulting in immunosuppression.
  • a subject can include those diagnosed with a hematopoietic malignancy and treated with a hematopoietic cell targeting therapy, such as a B cell malignancy treated with an anti-CD20 therapy (e.g., rituximab).
  • an anti-CD20 therapy e.g., rituximab
  • a subject can include those diagnosed with multiple sclerosis [e.g., Relapsing-remitting multiple sclerosis (RRMS), Secondary-progressive multiple sclerosis (SPMS), or Primary-progressive multiple sclerosis (PPMS)] and treated with an anti-CD20 therapy.
  • multiple sclerosis e.g., Relapsing-remitting multiple sclerosis (RRMS), Secondary-progressive multiple sclerosis (SPMS), or Primary-progressive multiple sclerosis (PPMS)
  • RRMS Relapsing-remitting multiple sclerosis
  • SPMS Secondary-progressive multiple sclerosis
  • PPMS Primary-progressive multiple sclerosis
  • An antigen can be administered in an amount sufficient to stimulate a CTL response.
  • An antigen can be administered in an amount sufficient to stimulate a T cell response.
  • An antigen can be administered in an amount sufficient to stimulate a B cell response.
  • An antigen can be administered alone or in combination with other therapeutic agents.
  • Therapeutic agents can include those that target an infectious disease organism, such as an anti-viral or antibiotic agent.
  • an antigen or its variant can be prepared for intravenous (i.v.) injection, sub-cutaneous (s.c.) injection, intradermal (i.d.) injection, intraperitoneal (i.p.) injection, intramuscular (i.m.) injection.
  • Methods of injection include s.c., i.d., i.p., i.m., and i.v.
  • Methods of DNA or RNA injection include i.d., i.m., s.c., i.p. and i.v.
  • Other methods of administration of the vaccine composition are known to those skilled in the art.
  • a vaccine can be compiled so that the selection, number and/or amount of antigens present in the composition is/are tissue, infectious disease, and/or patient-specific. For instance, the exact selection of peptides can be guided by expression patterns of the parent proteins in a given tissue or guided by mutation or disease status of a patient. The selection can be dependent on the specific infectious disease (e.g. the specific HPV strain the subject is infected with or at risk for infection by), the status of the disease, the goal of the vaccination (e.g. , preventative or targeting an ongoing disease), earlier treatment regimens, the immune status of the patient, and, of course, the HLA-haplotype of the patient.
  • a vaccine can contain individualized components, according to personal needs of the particular patient. Examples include varying the selection of antigens according to the expression of the antigen in the particular patient or adjustments for secondary treatments following a first round or scheme of treatment.
  • a patient can be identified for administration of an antigen vaccine through the use of various diagnostic methods, e.g., patient selection methods described further below.
  • Patient selection can involve identifying mutations in, or expression patterns of, one or more genes.
  • Patient selection can involve identifying the infectious disease of an ongoing infection (e.g. the presence of a HPV infection and/or the specific HPV strain).
  • Patient selection can involve identifying risk of an infection by an infectious disease.
  • patient selection involves identifying the haplotype of the patient.
  • the various patient selection methods can be performed in parallel, e.g., a sequencing diagnostic can identify both the mutations and the haplotype of a patient.
  • the various patient selection methods can be performed sequentially, e.g.
  • one diagnostic test identifies the mutations and separate diagnostic test identifies the haplotype of a patient, and where each test can be the same (e.g., both high-throughput sequencing) or different (e.g. , one high-throughput sequencing and the other Sanger sequencing) diagnostic methods.
  • each test can be the same (e.g., both high-throughput sequencing) or different (e.g. , one high-throughput sequencing and the other Sanger sequencing) diagnostic methods.
  • the respective pharmaceutical composition for treatment of this infection can be present in high amounts and/or more than one antigen specific for this particularly antigen or pathway of this antigen can be included.
  • compositions comprising an antigen can be administered to an individual already having (e.g., diagnosed with) an infection.
  • compositions are administered to a patient in an amount sufficient to stimulate an effective CTL response to the infectious disease organism antigen and to cure or at least partially arrest symptoms and/or complications.
  • An amount adequate to accomplish this is defined as "therapeutically effective dose.” Amounts effective for this use will depend on, e.g., the composition, the manner of administration, the stage and severity of the disease being treated, the weight and general state of health of the patient, and the judgment of the prescribing physician.
  • compositions can generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations, especially when the infectious disease organism has induced organ damage and/or other immune pathology.
  • life-threatening or potentially life threatening situations especially when the infectious disease organism has induced organ damage and/or other immune pathology.
  • compositions can generally be employed in serious disease states, that is, life-threatening or potentially life threatening situations, especially when the infectious disease organism has induced organ damage and/or other immune pathology.
  • administration can begin at the detection or treatment of an infection. This can be followed by boosting doses until at least symptoms are substantially abated and for a period thereafter.
  • compositions for therapeutic treatment are intended for parenteral, topical, nasal, oral or local administration.
  • a pharmaceutical compositions can be administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly.
  • the compositions can be administered to target specific infected tissues and/or cells of a subject.
  • compositions for parenteral administration which comprise a solution of the antigen and vaccine compositions are dissolved or suspended in an acceptable carrier, e.g., an aqueous carrier.
  • aqueous carriers can be used, e.g., water, buffered water, 0.9% saline, 0.3% glycine, hyaluronic acid and the like. These compositions can be sterilized by conventional, well known sterilization techniques, or can be sterile filtered. The resulting aqueous solutions can be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration.
  • compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.
  • Antigens can also be administered via liposomes, which target them to a particular cells tissue, such as lymphoid tissue. Liposomes are also useful in increasing half-life. Liposomes include emulsions, foams, micelles, insoluble monolayers, liquid crystals, phospholipid dispersions, lamellar layers and the like. In these preparations the antigen to be delivered is incorporated as part of a liposome, alone or in conjunction with a molecule which binds to, e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • a molecule which binds to e.g., a receptor prevalent among lymphoid cells, such as monoclonal antibodies which bind to the CD45 antigen, or with other therapeutic or immunogenic compositions.
  • liposomes filled with a desired antigen can be directed to the site of lymphoid cells, where the liposomes then deliver the selected therapeutic/immunogenic compositions.
  • Liposomes can be formed from standard vesicle-forming lipids, which generally include neutral and negatively charged phospholipids and a sterol, such as cholesterol. The selection of lipids is generally guided by consideration of, e.g., liposome size, acid lability and stability of the liposomes in the blood stream. A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9; 467 (1980), U.S. Pat. Nos. 4,235,871, 4,501,728, 4,501,728, 4,837,028, and 5,019,369.
  • a ligand to be incorporated into the liposome can include, e.g., antibodies or fragments thereof specific for cell surface determinants of the desired immune system cells.
  • a liposome suspension can be administered intravenously, locally, topically, etc. in a dose which varies according to, inter alia, the manner of administration, the peptide being delivered, and the stage of the disease being treated.
  • nucleic acids encoding a peptide and optionally one or more of the peptides described herein can also be administered to the patient.
  • a number of methods are conveniently used to deliver the nucleic acids to the patient.
  • the nucleic acid can be delivered directly, as "naked DNA". This approach is described, for instance, in Wolff et al., Science 247: 1465-1468 (1990) as well as U.S. Pat. Nos. 5,580,859 and 5,589,466.
  • the nucleic acids can also be administered using ballistic delivery as described, for instance, in U.S. Pat. No. 5,204,253.
  • Particles comprised solely of DNA can be administered.
  • DNA can be adhered to particles, such as gold particles.
  • Approaches for delivering nucleic acid sequences can include viral vectors, mRNA vectors, and DNA vectors with or without electroporation.
  • the nucleic acids can also be delivered complexed to cationic compounds, such as cationic lipids.
  • cationic compounds such as cationic lipids.
  • Lipid-mediated gene delivery methods are described, for instance, in 9618372WOAWO 96/18372; 9324640WOAWO 93/24640; Mannino & Gould-Fogerite, BioTechniques 6(7): 682-691 (1988); U.S. Pat. No. 5,279,833 Rose U.S. Pat. No. 5,279,833; 9106309WOAWO 91/06309; and Feigner et al., Proc. Natl. Acad. Sci. USA 84: 7413-7414 (1987).
  • Antigens can also be included in viral vector-based vaccine platforms, such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus, including but not limited to second, third or hybrid second/third generation lentivirus and recombinant lentivirus of any generation designed to target specific cell types or receptors (See, e.g., Hu et al., Immunization Delivered by Lentiviral Vectors for Cancer and Infectious Diseases, Immunol Rev.
  • viral vector-based vaccine platforms such as vaccinia, fowlpox, self-replicating alphavirus, marabavirus, adenovirus (See, e.g., Tatsis et al., Adenoviruses, Molecular Therapy (2004) 10, 616 — 629), or lentivirus
  • this approach can deliver one or more nucleotide sequences that encode one or more antigen peptides.
  • the sequences may be flanked by non-mutated sequences, may be separated by linkers or may be preceded with one or more sequences targeting a subcellular compartment (See, e.g., Gros et al., Prospective identification of neoantigen-specific lymphocytes in the peripheral blood of melanoma patients, Nat Med. (2016) 22 (4):433-8, Stronen et al., Targeting of cancer neoantigens with donor-derived T cell receptor repertoires, Science.
  • infected cells Upon introduction into a host, infected cells express the antigens, and thereby stimulate a host immune (e.g., CTL) response against the peptide(s).
  • Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Pat. No. 4,722,848.
  • Another vector is BCG (Bacille Calmette Guerin). BCG vectors are described in Stover et al. (Nature 351:456-460 (1991)).
  • a means of administering nucleic acids uses minigene constructs encoding one or multiple epitopes.
  • the amino acid sequences of the epitopes are reverse translated.
  • a human codon usage table is used to guide the codon choice for each amino acid.
  • minigene design To optimize expression and/or immunogenicity, additional elements can be incorporated into the minigene design.
  • amino acid sequence that could be reverse translated and included in the minigene sequence include: helper T lymphocyte, epitopes, a leader (signal) sequence, and an endoplasmic reticulum retention signal.
  • MHC presentation of CTL epitopes can be improved by including synthetic (e.g. poly-alanine) or naturally-occurring flanking sequences adjacent to the CTL epitopes.
  • the minigene sequence is converted to DNA by assembling oligonucleotides that encode the plus and minus strands of the minigene.
  • Overlapping oligonucleotides (30-100 bases long) are synthesized, phosphorylated, purified and annealed under appropriate conditions using well known techniques. The ends of the oligonucleotides are joined using T4 DNA ligase. This synthetic minigene, encoding the CTL epitope polypeptide, can then cloned into a desired expression vector.
  • Purified plasmid DNA can be prepared for injection using a variety of formulations. The simplest of these is reconstitution of lyophilized DNA in sterile phosphate- buffer saline (PBS). A variety of methods have been described, and new techniques can become available. As noted above, nucleic acids are conveniently formulated with cationic lipids. In addition, glycolipids, fusogenic liposomes, peptides and compounds referred to collectively as protective, interactive, non-condensing (PINC) could also be complexed to purified plasmid DNA to influence variables such as stability, intramuscular dispersion, or trafficking to specific organs or cell types.
  • PINC protective, interactive, non-condensing
  • Also disclosed is a method of manufacturing a vaccine comprising performing the steps of a method disclosed herein; and producing a vaccine comprising a plurality of antigens or a subset of the plurality of antigens.
  • Antigens disclosed herein can be manufactured using methods known in the art.
  • a method of producing an antigen or a vector (e.g., a vector including at least one sequence encoding one or more antigens) disclosed herein can include culturing a host cell under conditions suitable for expressing the antigen or vector wherein the host cell comprises at least one polynucleotide encoding the antigen or vector, and purifying the antigen or vector.
  • Standard purification methods include chromatographic techniques, electrophoretic, immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing techniques.
  • Host cells can include a Chinese Hamster Ovary (CHO) cell, NSO cell, yeast, or a HEK293 cell.
  • Host cells can be transformed with one or more polynucleotides comprising at least one nucleic acid sequence that encodes an antigen or vector disclosed herein, optionally wherein the isolated polynucleotide further comprises a promoter sequence operably linked to the at least one nucleic acid sequence that encodes the antigen or vector.
  • the isolated polynucleotide can be cDNA.
  • a vaccination protocol can be used to dose a subject with one or more antigens and/or epitopes.
  • a priming vaccine and a boosting vaccine can be used to dose the subject.
  • the priming vaccine can be based on C68 (e.g., the sequences shown in SEQ ID NO:1 or 2) or srRNA (e.g., the sequences shown in SEQ ID NO:3 or 4) and the boosting vaccine can be based on C68 (e.g., the sequences shown in SEQ ID NO: 1 or 2) or srRNA/samRNA (e.g., the sequences shown in SEQ ID NO: 3 or 4).
  • Each vector typically includes a cassette that includes antigens and/or epitopes.
  • Cassettes can include about 20 epitopes (or antigens from which the epitopes are derived), separated by spacers such as the natural sequence that normally surrounds each epitope or other non-natural spacer sequences such as AAY. Cassettes can also include MHCII antigens/epitopes such a tetanus toxoid antigen and PADRE antigen, which can be considered universal class II antigens. Cassettes can also include a targeting sequence such as a ubiquitin targeting sequence.
  • a priming vaccine can be injected (e.g., intramuscularly) in a subject. Bilateral injections per dose can be used.
  • one or more injections of ChAdV68 (C68) can be used (e.g., total dose IxlO 12 viral particles); one or more injections of self- amplifying RNA (samRNA or SAM), such as a dose of 3 pg, lOpg, 30pg, lOOpg, or 300pg RNA can be used.
  • a SAM priming dose of 30pg or less can be used.
  • a SAM priming dose of lOpg or less can be used.
  • a SAM priming dose of 3 pg or less can be used.
  • One or more injections of samRNA at a dose of 30pg or less can be used.
  • a dose of 30pg or less can represent the total content of RNA/samRNA administered.
  • a dose of 30pg or less can represent the total content of RNA/samRNA administered and include only a single distinct samRNA construct.
  • a SAM prime of between 10-30pg, 10-100pg, 10-300pg, 30-100pg, 30-300pg, or 100-300pg RNA can be administered.
  • a SAM prime of between 10-500pg, lO-lOOOpg, 30-500pg, 30-1000pg, or 500-1000pg RNA can be administered.
  • a SAM prime of at least 400pg, at least 500pg, at least 600pg, at least 700pg, at least 800pg, at least 900pg, at least lOOOpg RNA can be administered.
  • a SAM prime of lOpg, 30pg, lOOpg, or 300pg RNA can be administered.
  • a SAM prime of 300 pg RNA can be administered.
  • a SAM prime of lOOpg RNA can be administered.
  • a SAM prime of 30pg RNA can be administered.
  • a SAM prime of 20 pg RNA can be administered.
  • a SAM prime of lOpg RNA can be administered.
  • ig RNA can be administered.
  • a SAM prime of at least 300pg RNA can be administered.
  • a SAM prime of at least lOOpg RNA can be administered.
  • a SAM prime of at least 30pg RNA can be administered.
  • a SAM prime of at least lOpg RNA can be administered.
  • a SAM prime of at least 3pg RNA can be administered.
  • a SAM prime of less than or equal to 300 pg RNA can be administered.
  • a SAM prime of less than or equal to lOOpg RNA can be administered.
  • For ChAdV68 priming IxlO 12 or less of viral particles can be administered.
  • For ChAdV68 priming 3x 10" or less of the viral particles can be administered.
  • At least IxlO 11 of the viral particles can be administered.
  • For ChAdV68 priming between IxlO 11 and IxlO 12 , between 3xl0 n and IxlO 12 , or between IxlO 11 and 3x10' 1 of the viral particles can be administered.
  • For ChAdV68 priming IxlO 11 , 3xl0 n , or IxlO 12 of the viral particles can be administered.
  • the viral particles can be at a concentration of at 5xl0 n vp/mL.
  • a vaccine boost (boosting vaccine) can be injected (e.g., intramuscularly) after prime vaccination.
  • a boosting vaccine can be administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, e.g., every 4 weeks and/or 8 weeks after the prime.
  • a boosting vaccine can be administered 4 weeks after the prime.
  • a boosting vaccine can be administered a month after the prime.
  • a boosting vaccine can be administered 28 days after the prime.
  • a boosting vaccine can be administered 28 days after the prime.
  • a boosting vaccine can be administered 113 days after the prime.
  • a boosting vaccine can be administered at least 4 weeks after the prime.
  • a boosting vaccine can be administered at least a month after the prime.
  • a boosting vaccine can be administered at least 28 days after the prime.
  • a boosting vaccine can be administered at least 28 days after the prime.
  • a boosting vaccine can be administered at least 113 days after the prime.
  • a boosting vaccine can be administered between 28 to 113 days after the prime.
  • Bilateral injections per dose can be used.
  • one or more injections of ChAdV68 (C68) can be used (e.g., total dose IxlO 12 viral particles); one or more injections of self- amplifying RNA (samRNA or SAM), such as a dose of 3 pg, lOpg, 30pg, lOOpg, or 300pg RNA can be used.
  • a SAM boosting dose of 30pg or less can be used.
  • a SAM boosting dose of lOpg or less can be used.
  • a SAM boosting dose of 3pg or less can be used.
  • One or more injections of samRNA at a dose of 30pg or less can be used.
  • a dose of 30pg or less can represent the total content of RNA/samRNA administered.
  • a dose of 30pg or less can represent the total content of RNA/samRNA administered and include only a single distinct samRNA construct.
  • a SAM boost of between 10-30pg, 10-100pg, 10-300pg, 30-100pg, 30-300pg, or 100-300pg RNA can be administered.
  • a SAM boost of between 10-500pg, 10-lOOOpg, 30-500pg, 30-1000pg, or 500- lOOOpg RNA can be administered.
  • a SAM boost of at least 400pg, at least 500pg, at least 600(ig, at least 700pg, at least 800pg, at least 900(tg, at least lOOOpg RNA can be administered.
  • a SAM boost of lOpg, 30(ig, 100(ig, or 300pg RNA can be administered.
  • a SAM boost of 300pg RNA can be administered.
  • a SAM boost of lOOjrg RNA can be administered.
  • a SAM boost of 30pg RNA can be administered.
  • a SAM boost of 20[ig RNA can be administered.
  • a SAM boost of lOpg RNA can be administered.
  • a SAM boost of 3 pg RNA can be administered.
  • a SAM boost of at least 300pg RNA can be administered.
  • a SAM boost of at least lOOpg RNA can be administered.
  • a SAM boost of at least 30pg RNA can be administered.
  • a SAM boost of at least lOpg RNA can be administered.
  • a SAM boost of at least 3(ig RNA can be administered.
  • a SAM boost of less than or equal to 300(ig RNA can be administered.
  • a SAM boost of less than or equal to lOOpg RNA can be administered.
  • a vaccine boost can include the same antigen cassette (e.g., encode the same HPV immunogenic polypeptide) as a priming dose.
  • a vaccine boost can include a different antigen cassette as a priming dose.
  • a vaccine boost can include the same composition as a priming dose.
  • Any of the samRNA-based and/or ChAdV68-based vaccine composition described herein can be used in combination with commercially available HPV vaccines, e.g., Gardasil® and/or Cervarix®.
  • a dose of can represent the total content of RNA/samRNA administered.
  • a dose of can represent the total content of RNA/samRNA administered and include only a single distinct samRNA construct.
  • Immune monitoring can be performed before, during, and/or after vaccine administration. Such monitoring can inform safety and efficacy, among other parameters.
  • PBMCs are commonly used. PBMCs can be isolated before prime vaccination, and after prime vaccination (e.g. 4 weeks and 8 weeks). PBMCs can be harvested just prior to boost vaccinations and after each boost vaccination (e.g. 4 weeks and 8 weeks).
  • Immune responses can be assessed as part of an immune monitoring protocol. For example, the ability of a vaccine composition described herein to stimulate an immune response can be monitored and/or assessed.
  • “stimulate an immune response” refers to any increase in a immune response, such as initiating an immune response (e.g., a priming vaccine stimulating the initiation of an immune response in a naive subject) or enhancement of an immune response (e.g., a boosting vaccine stimulating the enhancement of an immune response in a subject having a pre-existing immune response to an antigen, such as a pre-existing immune response initiated by a priming vaccine).
  • Enhancing an immune response can include stimulating an immune response in a convalescent subject (e.g. , a boosting vaccine stimulating the enhancement of an immune response in a subject with persistent HPV infection, e.g., non-malignant).
  • a subject can include an HIV positive subject.
  • T cell responses can be measured using one or more methods known in the art such as ELISpot, intracellular cytokine staining, cytokine secretion and cell surface capture, T cell proliferation, MHC multimer staining, or by cytotoxicity assay.
  • T cell responses to epitopes encoded in vaccines can be monitored from PBMCs by measuring induction of cytokines, such as IFN-gamma, using an ELISpot assay.
  • Specific CD4 or CD8 T cell responses to epitopes encoded in vaccines can be monitored from PBMCs by measuring induction of cytokines captured intracellularly or extracellularly, such as IFN-gamma, using flow cytometry.
  • Specific CD4 or CD8 T cell responses to epitopes encoded in the vaccines can be monitored from PBMCs by measuring T cell populations expressing T cell receptors specific for epitope/MHC class I complexes using MHC multimer staining.
  • CD4 or CD8 T cell responses to epitopes encoded in the vaccines can be monitored from PBMCs by measuring the ex vivo expansion of T cell populations following 3H-thymidine, bromodeoxyuridine and carboxyfluoresceine-diacetate- succinimidylester (CFSE) incorporation.
  • the antigen recognition capacity and lytic activity of PBMC-derived T cells that are specific for epitopes encoded in vaccines can be assessed functionally by chromium release assay or alternative colorimetric cytotoxicity assays.
  • B cell responses can be measured using one or more methods known in the art such as assays used to determine B cell differentiation (e.g., differentiation into plasma cells), B cell or plasma cell proliferation, B cell or plasma cell activation (e.g. , upregulation of costimulatory markers such as CD80 or CD86), antibody class switching, and/or antibody production (e.g., an ELISA).
  • assays used to determine B cell differentiation e.g., differentiation into plasma cells
  • B cell or plasma cell proliferation e.g. , B cell or plasma cell proliferation
  • B cell or plasma cell activation e.g. , upregulation of costimulatory markers such as CD80 or CD86
  • antibody class switching e.g., an ELISA
  • Vaccination regimens can include assessing neutralizing antibody titers against the vaccine composition of interest, such as a ChAdV68-based vaccine.
  • a ChAdV68- based vaccine can be administered as a priming dose, and re-administration of the ChAdV68- based vaccine as a boosting dose follows determining ChAdV-specific neutralizing antibody titers are below a neutralization threshold prior to re-administration.
  • the neutralizing antibody titer can be an NT50 value calculated as a minimum dilution of sera from the immunized subject that neutralizes a ChAdV virus by 50%.
  • Determining the neutralizing antibody titer can include the steps of: (1) contacting one or more dilutions of sera from the immunized subject with a ChAdV virus under conditions sufficient for neutralization of the ChAdV virus; and (2) assessing neutralization of the ChAdV virus relative to a non-neutralized virus.
  • Isolation of HLA-peptide molecules was performed using classic immunoprecipitation (IP) methods after lysis and solubilization of the tissue sample (55-58). Examples and methods are described in more detail in international patent application publication WO/2018/208856, herein incorporated by reference, in its entirety, for all purposes.
  • Presentation models can be used to identify likelihoods of peptide presentation in subjects.
  • Various presentation models are known to those skilled in the art, for example the presentation models described in more detail in US Pat No. 10,055,540, US Application Pub. No. US20200010849A1 and US20110293637, and international patent application publications WO/2018/195357, WO/2018/208856, and WO2016187508, each herein incorporated by reference, in their entirety, for all purposes.
  • An exemplary epitope presentation model, and methods for using the same, in a viral context is described in International PCT Application Publication WO2023196966A1, herein incorporated by reference for all purposes.
  • Training modules can be used to construct one or more presentation models based on training data sets that generate likelihoods of whether peptide sequences will be presented by MHC alleles associated with the peptide sequences.
  • Various training modules are known to those skilled in the art, for example the presentation models described in more detail in US Pat No. 10,055,540, US Application Pub. No. US20200010849A1, and international patent application publications WO/2018/195357 and WO/2018/208856, each herein incorporated by reference, in their entirety, for all purposes.
  • An exemplary training model for prediction, and methods for using the same, in a viral context is described in International PCT Application Publication WO2023196966A1, herein incorporated by reference for all purposes.
  • a training module can construct a presentation model to predict presentation likelihoods of peptides on a per-allele basis.
  • a training module can also construct a presentation model to predict presentation likelihoods of peptides in a multiple- allele setting where two or more MHC alleles are present.
  • a prediction module can be used to receive sequence data and select candidate antigens in the sequence data using a presentation model.
  • the sequence data may be DNA sequences, RNA sequences, and/or protein sequences extracted from infected cells patients or infectious disease organisms themselves (e.g., HPV).
  • a prediction module may identify candidate antigens that are pathogen-derived peptides (e.g., HPV derived), such as by comparing sequence data extracted from normal tissue cells of a patient with the sequence data extracted from infected cells of the patient to identify portions containing one or more infectious disease organism associated antigens.
  • a prediction module may identify candidate antigens that are expressed in an infected cell or infected tissue in comparison to a normal cell or tissue by comparing sequence data extracted from normal tissue cells of a patient with the sequence data extracted from infected tissue cells of the patient to identify expressed candidate antigens (e.g., identifying expressed polynucleotides and/or polypeptides specific to an infectious disease).
  • expressed candidate antigens e.g., identifying expressed polynucleotides and/or polypeptides specific to an infectious disease.
  • a presentation module can apply one or more presentation model to processed peptide sequences to estimate presentation likelihoods of the peptide sequences.
  • the prediction module may select one or more candidate antigen peptide sequences that are likely to be presented on infected cell HLA molecules by applying presentation models to the candidate antigens.
  • the presentation module selects candidate antigen sequences that have estimated presentation likelihoods above a predetermined threshold.
  • the presentation model selects the N candidate antigen sequences that have the highest estimated presentation likelihoods (where N is generally the maximum number of epitopes that can be delivered in a vaccine).
  • a vaccine including the selected candidate antigens for a given patient can be injected into the patient to stimulate immune responses.
  • a cassette design module can be used to generate a vaccine cassette sequence based on selected candidate peptides for injection into a patient.
  • a cassette design module can be used to generate a sequence encoding concatenated epitope sequences, such as concatenated T cell epitopes.
  • concatenated epitope sequences such as concatenated T cell epitopes.
  • cassette design modules are known to those skilled in the art, for example the cassette design modules described in more detail in US Pat No. 10,055,540, US Application Pub. No. US20200010849A1, and international patent application publications WO/2018/195357 and WO/2018/208856, each herein incorporated by reference, in their entirety, for all purposes.
  • a set of therapeutic epitopes may be generated based on the selected peptides determined by a prediction module associated with presentation likelihoods above a predetermined threshold, where the presentation likelihoods are determined by the presentation models.
  • the set of therapeutic epitopes may be generated based on any one or more of a number of methods (alone or in combination), for example, based on binding affinity or predicted binding affinity to HLA class I or class II alleles of the patient, binding stability or predicted binding stability to HLA class I or class II alleles of the patient, random sampling, and the like.
  • Therapeutic epitopes may correspond to selected peptides themselves. Therapeutic epitopes may also include C- and/or N-terminal flanking sequences in addition to the selected peptides. N- and C-terminal flanking sequences can be the native N- and C-terminal flanking sequences of the therapeutic vaccine epitope in the context of its source protein. Therapeutic epitopes can represent a fixed-length epitope Therapeutic epitopes can represent a variablelength epitope, in which the length of the epitope can be varied depending on, for example, the length of the C- or N-flanking sequence. For example, the C-terminal flanking sequence and the N-terminal flanking sequence can each have varying lengths of 2-5 residues, resulting in 16 possible choices for the epitope.
  • a cassette design module can also generate cassette sequences by taking into account presentation of junction epitopes that span the junction between a pair of therapeutic epitopes in the cassette.
  • Junction epitopes are novel non-self but irrelevant epitope sequences that arise in the cassette due to the process of concatenating therapeutic epitopes and linker sequences in the cassette.
  • the novel sequences of junction epitopes are different from the therapeutic epitopes of the cassette themselves.
  • a cassette design module can generate a cassette sequence that reduces the likelihood that junction epitopes are presented in the patient. Specifically, when the cassette is injected into the patient, junction epitopes have the potential to be presented by HLA class I or HLA class II alleles of the patient, and stimulate a CD8 or CD4 T-cell response, respectively. Such reactions are often times undesirable because T-cells reactive to the junction epitopes have no therapeutic benefit, and may diminish the immune response to the selected therapeutic epitopes in the cassette by antigenic competition. 76
  • a cassette design module can iterate through one or more candidate cassettes, and determine a cassette sequence for which a presentation score of junction epitopes associated with that cassette sequence is below a numerical threshold.
  • the junction epitope presentation score is a quantity associated with presentation likelihoods of the junction epitopes in the cassette, and a higher value of the junction epitope presentation score indicates a higher likelihood that junction epitopes of the cassette will be presented by HLA class I or HLA class II or both.
  • a cassette design module may determine a cassette sequence associated with the lowest junction epitope presentation score among the candidate cassette sequences. [00381] A cassette design module may iterate through one or more candidate cassette sequences, determine the junction epitope presentation score for the candidate cassettes, and identify an optimal cassette sequence associated with a junction epitope presentation score below the threshold.
  • a cassette design module may further check the one or more candidate cassette sequences to identify if any of the junction epitopes in the candidate cassette sequences are self-epitopes for a given patient for whom the vaccine is being designed. To accomplish this, the cassette design module checks the junction epitopes against a known database such as BLAST. In one embodiment, the cassette design module may be configured to design cassettes that avoid junction self-epitopes.
  • a cassette design module can perform a brute force approach and iterate through all or most possible candidate cassette sequences to select the sequence with the smallest junction epitope presentation score.
  • the number of such candidate cassettes can be prohibitively large as the capacity of the vaccine increases.
  • the cassette design module has to iterate through ⁇ 10 18 possible candidate cassettes to determine the cassette with the lowest junction epitope presentation score. This determination may be computationally burdensome (in terms of computational processing resources required), and sometimes intractable, for the cassette design module to complete within a reasonable amount of time to generate the vaccine for the patient.
  • accounting for the possible junction epitopes for each candidate cassette can be even more burdensome.
  • a cassette design module may select a cassette sequence based on ways of iterating through a number of candidate cassette sequences that are significantly smaller than the number of candidate cassette sequences for the brute force approach.
  • a cassette design module can generate a subset of randomly or at least pseudo- randomly generated candidate cassettes, and selects the candidate cassette associated with a junction epitope presentation score below a predetermined threshold as the cassette sequence. Additionally, the cassette design module may select the candidate cassette from the subset with the lowest junction epitope presentation score as the cassette sequence. For example, the cassette design module may generate a subset of ⁇ 1 million candidate cassettes for a set of 20 selected epitopes, and select the candidate cassette with the smallest junction epitope presentation score.
  • a cassette design module can determine an improved cassette configuration by formulating the epitope sequence for the cassette as an asymmetric traveling salesman problem (TSP).
  • TSP traveling salesman problem
  • the TSP determines a sequence of nodes associated with the shortest total distance to visit each node exactly once and return to the original node. For example, given cities A, B, and C with known distances between each other, the solution of the TSP generates a closed sequence of cities, for which the total distance traveled to visit each city exactly once is the smallest among possible routes.
  • the asymmetric version of the TSP determines the optimal sequence of nodes when the distance between a pair of nodes are asymmetric. For example, the “distance” for traveling from node A to node B may be different from the “distance” for traveling from node B to node A.
  • the cassette design module can find a cassette sequence that results in a reduced presentation score across the junctions between epitopes of the cassette.
  • the solution of the asymmetric TSP indicates a sequence of therapeutic epitopes that correspond to the order in which the epitopes should be concatenated in a cassette to minimize the junction epitope presentation score across the junctions of the cassette.
  • a cassette sequence determined through this approach can result in a sequence with significantly less presentation of junction epitopes while potentially requiring significantly less computational resources than the random sampling approach, especially when the number of generated candidate cassette sequences is large.
  • Illustrative examples of different computational approaches and comparisons for optimizing cassette design are described in more detail in US Pat No. 10,055,540, US Application Pub. No. US20200010849A1, and international patent application publications WO/2018/195357 and WO/2018/208856, each herein incorporated by reference, in their entirety, for all purposes.
  • a cassette design module can also generate cassette sequences by taking into account additional protein sequences encoded in the vaccine.
  • a cassette design module used to generate a sequence encoding concatenated T cell epitopes can take into account T cell epitopes already encoded by additional protein sequences present in the vaccine (e.g. , full-length protein sequences), such as by removing T cell epitopes already encoded by the additional protein sequences from the list of candidate sequences.
  • a cassette design module can also generate cassette sequences by taking into account the size of the sequences. Without wishing to be bound by theory, in general, increased cassette size can negatively impact vaccine aspects, such as vaccine production and/or vaccine efficacy.
  • the cassette design module can take into account overlapping sequences, such as overlapping T cell epitope sequences.
  • overlapping T cell epitope sequences In general, a single sequence containing overlapping T cell epitope sequences (also referred to as a “frame”) is more efficient than separately linking individual T cell epitope sequences as it reduces the sequence size needed to encode the multiple peptides.
  • a cassette design module used to generate a sequence encoding concatenated T cell epitopes can take into account the cost/benefit of extending a candidate T cell epitope to encode one or more additional T cell epitopes, such as determining the benefit gained in additional population coverage for an MHC presenting the additional T cell epitope versus the cost of increasing the size of the sequence.
  • a cassette design module can also generate cassette sequences by taking into account the magnitude of stimulation of an immune response generated by validated epitopes.
  • a cassette design module can also generate cassette sequences by taking into account presentation of encoded epitopes across a population, for example that at least one immunogenic epitope is presented by at least one HLA across a proportion of a population, for example by at least 85%, 90%, or 95% of a population (e.g., HLA-A, HLA-B and HLA-C genes over four major ethnic groups, namely European (EUR), African American (AFA), Asian and Pacific Islander (APA) and Hispanic (HIS)).
  • European European
  • AFA African American
  • APA Asian and Pacific Islander
  • HIS Hispanic
  • a cassette design module can also generate cassette sequences such that at least one HLA is present at least across 85%, 90%, or 95% of a population that presents at least one validated epitope or presents at least 4, 5, 6, or 7 predicted epitopes.
  • a cassette design module can also generate cassette sequences by taking into account other aspects that improve potential safety, such as limiting encoding or the potential to encode a functional protein, functional protein domain, functional protein subunit, or functional protein fragment potentially presenting a safety risk.
  • a cassette design module can limit sequence size of encoded peptides such that are less than 50%, less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 45%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, or less than 33% of the translated, corresponding full-length protein.
  • a cassette design module can limit sequence size of encoded peptides such that a single contiguous sequence is less than 50% of the translated, corresponding full-length protein, but more than one sequence may be derived from the same translated, corresponding full-length protein and together encode more than 50%.
  • a single sequence containing overlapping T cell epitope sequences (“frame”) is larger than 50% of the translated, corresponding full-length protein, the frame can be split into multiple frames (e.g. , fl, f2 etc.) such that each frame is less than 50% of the translated, corresponding full-length protein.
  • a cassette design module can also limit sequence size of encoded peptides such that a single contiguous sequence is less than 49%, less than 48%, less than 47%, less than 46%, less than 45%, less than 45%, less than 43%, less than 42%, less than 41%, less than 40%, less than 39%, less than 38%, less than 37%, less than 36%, less than 35%, less than 34%, or less than 33% of the translated, corresponding full-length protein.
  • the multiple frames can have overlapping sequences with each other, in other words each separately encode the same sequence.
  • the two or more nucleic acid sequences derived from the same gene can be ordered such that a first nucleic acid sequence cannot be immediately followed by or linked to a second nucleic acid sequence if the second nucleic acid sequence follows, immediately or not, the first nucleic acid sequence in the corresponding gene. For example, if there are 3 frames within the same gene (f 1 ,f2,f3 in increasing order of amino acid position):
  • cassette orderings are not allowed: o fl immediately followed by f2 o f2 immediately followed by f3 o fl immediately followed by f3
  • cassette orderings are allowed: o f3 immediately followed by f2 o f2 immediately followed by fl
  • a computer can be used for any of the computational methods described herein.
  • One skilled in the art will recognize a computer can have different architectures. Examples of computers are known to those skilled in the art, for example the computers described in more detail in US Pat No. 10,055,540, US Application Pub. No. US20200010849A1, and international patent application publications WO/2018/195357 and WO/2018/208856, each herein incorporated by reference, in their entirety, for all purposes.
  • the first step in the vaccine design was to obtain a set of potential epitopes to be included in the vaccine, which are short amino-acid sequences with length 8 through 12.
  • Two sets of epitopes were defined: predicted using prediction algorithms; and validated, which are experimentally identified epitopes documented in the literature or in public databases.
  • CD8+ epitopes were predicted using a machine learning EDGE platform (see US Pat No. 10,055,540, herein incorporated by reference for all purposes), which was shown to be best- in-class [Bulik-Sullivan et al. (2016).
  • the amino acid sequences of all CD8+ epitopes identified using the machine learning EDGE platform for each of HPV strains 16, 18, 31, 33, 35, 45, 52, and 58 are shown in Tables G1-G8 and Tables Q1-Q8 of the Appendix, respectively.
  • the Appendix is hereby incorporated by reference, in its entirety, for all purposes. Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification.
  • Each epitope selected is associated to one or more class I HLA allele, which were either predicted or experimentally demonstrated to present the respective epitope in the surface of antigen presentation cells.
  • the set of validated epitopes was aligned to the reference proteome, requiring an additional set of k flanking amino acids on either side of the epitope.
  • the proteome regions containing aligned epitopes were defined as the initial footprint and characterized the building foundation of the vaccine cassette (FIG. 15A).
  • the initial footprint was constrained by parameters that specify the minimum proportion of the epitope length (min_proportion ) that have a minimum required number of overlapping (min_overlap) epitopes necessary to accept an epitope as part of the initial footprint or not. These two parameters controled the size of the initial footprint.
  • the algorithm performed several ranking iterations that consider all remaining validated and predicted epitopes.
  • every epitope was ranked by the fraction c/f where c is the added population coverage provided by the epitope and f is the total increase in cassette size as consequence of the epitope addition.
  • the epitope with the lowest value was added to the cassette and the process repeated until a maximum cassette design was reached or until all epitopes were included in the cassette (FIG. 15A).
  • the coverage provided by a given epitope is calculated by the frequencies of haplotypes in a reference population that cover at least one allele associated with the epitope (FIG. 15B).
  • FIG. 16 shows the overall performance that 85 of 214 predicted epitopes were at least provisionally validated to be presented by a single HLA. For the 20 alleles tested for the HLA lines created at the time of assesment, a minimum of 2 unique TCEs was detected for each cell line tested.
  • Population coverage was estimated by counting how many epitopes-HLA pairs are required for individuals to be protected (degree of protection). Predicted epitopes received a smaller weight than validated epitopes in the population coverage estimation. By updating the validated epitopes with the mass-spectrometry data, as shown in FIG. 17, an improvement in population coverage estimates was observed, even for higher degree of protection levels.
  • HPV vaccines against HPV were designed to assess efficacy for eliciting T cell responses.
  • Initial HPV vaccines were designed targeting HPV strains HPV 16 and HPV18 that included E6 and E7 whole fusions and T cell epitope (TCE) sequences from El, E2 and E5.
  • TCE T cell epitope
  • HPV strains 16 and 18 were initially chosen for assessment (e.g., as a proof of principal) given they are common in US/Europe.
  • HPV genomes contain ORFs that are all transcribed from a single DNA strand that include the early (E) region encoding proteins E1-E7, the late (L) region encoding structural proteins L1-L2, and a generally non-coding long control region (LCR) region.
  • HPV antigens E6 and E7 were chosen for inclusion in their entirety (except for deletions and other modifications described below, e.g., in Table B) as a fusion protein given they are generally considered to be the most highly expressed proteins post-transformation, as well as due to their contribution to the oncogenic process and expression within transformed cell.
  • T-cell epitope T-cell epitope
  • MHC I epitope identification algorithms described herein (see also the machine learning EDGE platform described in US Pat No. 10,055,540 and Bulik-Sullivan et al. [Deep learning using tumor HLA peptide mass spectrometry datasets improves neoantigen identification. Nature Biotechnology 2018, 57(1)], each of which is herein incorporated by reference for all purposes).
  • the epitopes were selected based on their ability to provide broad HLA coverage for major global populations, including those in specific low middle income countries, with an initial focus on covering HLA diversity within Sub-Saharan Africa (but noteworthy later determined that cassettes were predicted to cover global diversity more broadly, e.g., see FIG. 17).
  • E6/E7 fusion proteins linked by a Furin cleavage site for each of HPV strains 16, 18, 31, 33, 35, 45, 52, and 58 are shown in Table A, including with leader sequences for expression, with both amino acid and nucleotide sequences provided, where nucleotide sequences were codon optimized.
  • the E6 and E7 proteins also featured additional modifications referred to by amino acid position relative to the identified wildtype strain sequence, as shown in Table B, which include deletions to render the respective proteins inactive, e.g., for safety considerations.
  • the amino acid sequences selected for inclusion in the TCE cassettes with their protein origin (both HPV gene and frame) for each of HPV strains 16, 18, 31, 33, 35, 45, 52, and 58 are shown in Tables C1-C8, respectively.
  • the full TCE cassette sequences for each of HPV strains 16, 18, 31, 33, 35, 45, 52, and 58 are shown in Table D, including with leader sequences and universal MHC Class II PADRE sequence (inclusive of GPGPG linkers), with both amino acid and nucleotide sequences provided.
  • Vaccines were constructed using both an alphavirus-based self- amplifying mRNA (samRNA) vector and a chimpanzee adenovirus (ChAd) vector, as described below.
  • samRNA alphavirus-based self- amplifying mRNA
  • ChAd chimpanzee adenovirus
  • the E6/E7 fusion protein and TCE cassette were expressed from separate alphavirus subgenomic promoters (SGP or “26S”) in samRNA contructs, and expressed using a T2A ribosome skipping sequence element in ChAd constructs.
  • SGP alphavirus subgenomic promoters
  • T2A ribosome skipping sequence element T2A ribosome skipping sequence element in ChAd constructs.
  • Table E The order of the E6/E7 fusion protein and the TCE cassette for the initial vaccines assessed for each of individual HPV strains 16, 18, 31, 33, 35, 45, 52, and 58 is shown in Table
  • Multi-strain vaccines encoding E6/E7 fusion proteins and TCE cassettes from both HPV16 and HPV18 were also constructed and assessed.
  • samRNA multi-strain vaccines a combination of separate alphavirus subgenomic promoters (SGP/26S) and T2A ribosome skipping sequence elements was used, with the initial construct assessed having the following format: 18.E6E7-T2A-16.TCE-18.TCE-SGP-16.E6E7.
  • a T2A ribosome skipping sequence element was used, with the initial construct assessed having the following format: 16.E6E7-18.E6E7-T2A-16.TCE-18.TCE.
  • TCE T-cell Epitope
  • TCE T-cell Epitope
  • RNA alphavirus backbone for the antigen expression system was generated from a self-replicating Venezuelan Equine Encephalitis (VEE) virus (Kinney, 1986, Virology 152: 400- 413) by deleting the structural proteins of VEE located 3’ of the 26S sub-genomic promoter (VEE sequences 7544 to 11,175 deleted; numbering based on Kinney et al 1986; SEQ ID NO:6).
  • VEE sequences 7544 to 11,175 deleted; numbering based on Kinney et al 1986; SEQ ID NO:6 To generate the self-amplifying mRNA (“SAM”) vector, the deleted sequences are replaced by antigen sequences.
  • a representative SAM vector containing 20 model antigens is “VEE-MAG25mer” (SEQ ID NO:4).
  • the vectors featuring the antigen cassettes described having the MAG25mer cassette can be replaced by the HPV cassettes and/or full-length proteins described herein.
  • SAM vectors were generated as “AU-SAM” vectors.
  • a modified T7 RNA polymerase promoter (TAATACGACTCACTATA), which lacks the canonical 3’ dinucleotide GG, was added to the 5’ end of the SAM vector to generate the in vitro transcription template DNA (7544 to 11,175 deleted without an inserted antigen cassette).
  • lx transcription buffer 40 mM Tris-HCL [pH7.9], 10 mM dithiothreitol, 2 mM spermidine, 0.002% Triton X-100, and 27 mM magnesium chloride
  • E2040S final concentrations of lx T7 RNA polymerase mix
  • 0.025 mg/mL DNA transcription template linearized by restriction digest
  • 8 mM CleanCap Reagent AU Cat. No. N-7114
  • a 7-methylguanosine or a related 5’ cap structure can be enzymatically added following transcription using a vaccinia capping system (NEB Cat. No. M2080) containing mRNA 2’-O-methyltransferase and S- Adenosyl methionine.
  • a vaccinia capping system NEB Cat. No. M2080
  • a modified ChAdV68 vector (“chAd68-Empty-E4deleted” SEQ ID NO:57) for the antigen expression system was generated based on AC_000011.1 with El (nt 577 to 3403), E3 (nt 27,125- 31,825), and E4 region (nt 34,916 to 35,642) sequences deleted and the corresponding ATCC VR-594 (Independently sequenced Full-Length VR-594 C68 SEQ ID NO: 10) nucleotides substituted at five positions.
  • a transcriptionally reglated E4- deletion ChAdV68 vector was used. Relevant ChAdV68 vectors and sequences are described in U.S. Pat. No.
  • ChAdV68.5WTnt The full-length ChAdV68 AC_000011.1 sequence with corresponding ATCC VR-594 nucleotides substituted at five positions is referred to as “ChAdV68.5WTnt” (SEQ ID NO: 1). Antigen cassettes under the control of the CMV promoter/enhancer are inserted in place of deleted El sequences.
  • ChAdV68 virus production are performed in 293F cells grown in 293 FreeStyleTM (ThermoFisher) media in an incubator at 8% CO2. On the day of infection cells are diluted to 10 6 cells per mL, with 98% viability and 400 mL are used per production run in IL Shake flasks (Corning). 4 mL of the tertiary viral stock with a target MOI of >3.3 is used per infection. The cells are incubated for 48-72h until the viability was ⁇ 70% as measured by Trypan blue.
  • the infected cells are then harvested by centrifugation, full speed bench top centrifuge and washed in 1XPBS, re-centrifuged and then re-suspended in 20 mL of lOmM Tris pH7.4.
  • the cell pellet is lysed by freeze thawing 3X and clarified by centrifugation at 4,300Xg for 5 minutes.
  • Viral DNA is purified by CsCl centrifugation. Two discontinuous gradient runs are performed. The first to purify virus from cellular components and the second to further refine separation from cellular components and separate defective from infectious particles.
  • the tube are then removed to a laminar flow cabinet and the virus band pulled using an 18 gauge needle and a 10 mL syringe. Care is taken not to remove contaminating host cell DNA and protein.
  • the band is then diluted at least 2X with 10 mM Tris pH 8.0 and layered as before on a discontinuous gradient as described above. The run is performed as described before except that this time the run is performed overnight. The next day the band is pulled with care to avoid pulling any of the defective particle band.
  • the virus is then dialyzed using a Slide-a-LyzerT M Cassette (Pierce) against ARM buffer (20 mM Tris pH 8.0, 25 mM NaCl, 2.5% Glycerol). This is performed 3X, Ih per buffer exchange. The virus is then aliquoted for storage at -80°C.
  • VP concentration is performed by using an OD 260 assay based on the extinction coefficient of l.lx 10 12 viral particles (VP) is equivalent to an Absorbance value of 1 at OD260 nm.
  • Two dilutions (1:5 and 1:10) of adenovirus are made in a viral lysis buffer (0.1% SDS, 10 mM Tris pH 7.4, ImM EDTA).
  • OD is measured in duplicate at both dilutions and the VP concentration/ mL is measured by multiplying the OD260 value X dilution factor X l.lx 10 12 VP.
  • An infectious unit (IU) titer is calculated by a limiting dilution assay of the viral stock.
  • the virus is initially diluted 100X in DMEM/5% NS/ IX PS and then subsequently diluted using 10-fold dilutions down to lx 10‘ 7 .
  • 100 pL of these dilutions are then added to 293 A cells that were seeded at least an hour before at 3e5 cells/ well of a 24 well plate. This is performed in duplicate. Plates are incubated for 48h in a CO2 (5%) incubator at 37 °C. The cells are then washed with 1XPBS and are then fixed with 100% cold methanol (-20 °C).
  • the plates are then incubated at -20 °C for a minimum of 20 minutes.
  • the wells are washed with 1XPBS then blocked in 1XPBS/O.1% BSA for 1 h at room temperature.
  • a rabbit anti- Ad antibody (Abeam, Cambridge, MA) is added at 1:8,000 dilution in blocking buffer (0.25 ml per well) and incubated for 1 h at room temperature.
  • the wells are washed 4X with 0.5 mL PBS per well.
  • a HRP conjugated Goat anti-Rabbit antibody (Bethyl Labs, Montgomery Texas) diluted 1000X is added per well and incubated for Ih prior to a final round of washing.
  • the number of infectious viruses/ mL can be determined by the number of stained cells per grid multiplied by the number of grids per field of view multiplied by a dilution factor 10. Similarly, when working with GFP expressing cells florescent can be used rather than capsid staining to determine the number of GFP expressing virions per mL.
  • mice All mouse studies were conducted at Murigenics under IACUC approved protocols.
  • Balb/c mice Envigo
  • C57B1/6 Envigo
  • FVB Teconic mice 6-8 weeks old were used depending on the study.
  • Vaccines were stored at -80°C, thawed at room temperature on the day of immunization.
  • samRNA vaccines were then diluted to 0.1 pg/mL or 0.05 pg/mL with PBS and filtered through a 0.2 micron filter.
  • Blended vaccines were combined prior to diluting each to 0. 1 pg/mL or 0.5 pg/mL. Filtered formulations were stored at 4°C and injected within 4 hours of preparation.
  • chAd68 vectors were diluted to 3el0 VP/100 pL in 20 mM Tris, 25mM NaCl, 2.5% and 100 pL was injected per animal.
  • both ChAd lots were diluted such that each lot was equivalent to 3el0 VP/100 pL or 6el0 VP/100 pL combined. Glycerol. All immunizations were bilateral intramuscular to the tibialis anterior, 2 injections of 50 pL each, 100 pL total.
  • mice spleens were extracted at various timepoints following immunization. Note that in some studies immunizations were staggered to enable spleens to be collected at the same time and compared. Spleens were collected and analyzed by IFNyELISpot and ICS. Spleens were suspended in RPMI complete (RPMI + 10% FBS) and dissociated using the gentleMACS Dissociator (Miltenyi Biotec). Dissociated cells were filtered using a 40 pm strainer and red blood cells were lysed with ACK lysing buffer (150 mM NH4CI, 10 mM KHCO , 0.1 mM EDTA). Following lysis, cells were filtered with a 30 pm strainer and resuspended in RPMI complete.
  • IFNy ELISpot assays were performed using pre-coated 96- well plates (MAbtech, Mouse IFNy ELISpot PLUS, ALP) following manufacturer’s protocol. Samples were stimulated overnight with peptide pools at a final concentration of 1 pg/mL per peptide. E6 and E7 peptides are commercially available (JPT Peptide Technologies). El and E2 peptides were derived from the sequences included in the respective TCE cassettes. A DMSO only control was plated for each sample and cell number.
  • AdjustedSpots RawSpots + 2*(RawSpots*Saturation/(100-Saturation)
  • FIG. 1A-C various samRNA vaccine format architectures were contemplated for expression of both an E6/E7 fusion protein together with a TCE cassette.
  • samRNA vaccine constructs for HPV 16 format architecture of FIG. IB
  • HPV 18 format architecture of FIG. 1A
  • Ch Ad vaccine format architectures were contemplated for expression of both an E6/E7 fusion protein together with a TCE cassette.
  • ChAd vaccine constructs for HPV 16 (format architecture of FIG. 3 A) and for HPV 18 (format architecture of FIG. 3 A) elicited immunogenicity against both E6/E7 components and El components in C57/B16 mice.
  • FIG. 5A-C various samRNA vaccine format architectures were contemplated for expression of both an E6/E7 fusion protein together with a TCE cassette from multiple HPV strains in a single vector. As shown in FIG.
  • the multi-strain samRNA vaccine construct for HPV16 and HPV 18 (format architecture of FIG. 5A) elicited immunogenicity against both E6/E7 components and El components from both HPV strains in C57/B16 mice (Multi-Strain Vaccine) comparable to mice immunized with a combination of separate singlestrain samRNA vaccine constructs for each of HPV16 and HPV18 (Blended).
  • FIG. 7A-B various ChAd vaccine format architectures were contemplated for expression of both an E6/E7 fusion protein together with a TCE cassette from multiple HPV strains in a single vector.
  • the multi-strain ChAd vaccine construct for HPV16 and HPV18 (format architecture of FIG. 7A) elicited immunogenicity against both E6/E7 components and El components from both HPV strains in C57/B16 mice (Multi-Strain Vaccine) comparable to mice immunized with a combination of separate singlestrain ChAd vaccine constructs for each of HPV16 and HPV18 (Blended).
  • a therapeutic HPV vaccine regimen comprised of a samRNA formulated in a lipid nanoparticle (LNP) and/or ChAd vaccine (GRT-C9XX) that express full-length E6 and E7 proteins as well as predicted and validated epitopes from the HPV El, E2 and E5 proteins that are assembled into TCE cassettes are assessed in a First-in-Human (FIH) study.
  • a Prime-Boost strategy is initially assessed, with a possibility to improve to a 1-dose regimen. Constructs include those described above.
  • a phase I study tests HPV16/18 vaccines in the US, with the goal of starting a study in low middle income countries (LMIC) (staggered start) with either the same HPV16/18 vaccine or with a vaccine that targets additional HPV viruses for subsequent studies in LMIC.
  • the vaccine candidates is administered via intramuscular (IM) injection in heterologous primeboost schedules with both ChAd and samRNA vaccines, a regimen known for driving robust CD8+ T cell responses.
  • cohorts may be tested with a homologous prime/boost vaccine regimen to generate data head-to-head to the heterologous prime/boost approach.
  • the homologous samRNA vaccine regimen might offer an attractive vaccine approach for LMICs (ease of manufacture and distribution) if proven sufficiently potent in driving CD8+ T cell responses.
  • the initial study is in women with documented HPV+ for 2 or more years (e.g., with lower level of spontaneous viral clearance), i.e., high-risk HPV, to assess safety, tolerability, immunogenicity, HPV/HLA subtyping, HPV DNA clearance in women 18-50yo (FIH-US trial).
  • HPV and HLA subtyping include but not limited to HPV and HLA subtyping, CD4/CD8 T cell responses (systemically and locally), cytokine profiling, etc.
  • selected clinical investigational sites in the US are recommended for the FIH study, that allows first interpretable results approximately six months after last vaccination.
  • Chronic HPV infection can be defined as persistent of the same type of HR-HPV (HPV 16 and/or 18) infection for a period of time of > 2 years documented by > 2 consecutive examinations (by detection of the same type) with HPV genotyping prior to screening Available history of high-risk (HR)-HPV positivity and HPV 16 and/or HPV18 positivity will be recorded and contraceptive (birth control) use by participants should be consistent with local regulations regarding the acceptable methods of contraception for those participating in clinical studies and agrees not to donate blood (outside of the study protocol) until 12 months after receiving the last dose of study vaccine.
  • HR-HPV positivity high-risk
  • HPV 16 and/or HPV18 positivity will be recorded and contraceptive (birth control) use by participants should be consistent with local regulations regarding the acceptable methods of contraception for those participating in clinical studies and agrees not to donate blood (outside of the study protocol) until 12 months after receiving the last dose of study vaccine.
  • Symptomatic vaginal or genital infection as confirmed by physician or investigator.
  • Study Endpoints Primary endpoints: Solicited local and systemic adverse events within 7 days of vaccination and tolerability; Secondary endpoints: Unsolicited adverse events within 30 days of vaccination.
  • CMI Cell mediated immunity
  • HPV clearance Exploratory endpoints: HPV genotyping, HPV viral load, HLA subtyping, T cell immune monitoring specific for antigens/epitopes within the vaccine cassette.
  • Cohorts Sample size: A total of 120 subjects, targeting 20 subjects/cohort
  • samRNA doses total dose is indicated.
  • individual components add up to the total dose (10 or 20pg).
  • Blood draws for safety and immunogenicity will be outlined in detail in study protocol and schedule of assessments.
  • Bodini, M. et al. The hidden genomic landscape of acute myeloid leukemia: subclonal structure revealed by undetected mutations. Blood 125, 600-605 (2015).
  • RNA CoMPASS a dual approach for pathogen and host transcriptome analysis of RNA-seq datasets. PloS One 9, e89445 (2014).
  • HLA-DR monoclonal antibodies inhibit the proliferation of normal and chronic granulocytic leukaemia myeloid progenitor cells. Br J Haematol. 1982 Nov;52(3):411-20.
  • TCR reconstitution in Jurkat reporter cells facilitates the identification of novel tumor antigens by cDNA expression cloning. Int J Cancer 99, 7-13.

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Abstract

La présente invention concerne des compositions vaccinales qui comprennent des cassettes codant pour des épitopes de CMH du VPH et/ou des protéines de VPH de pleine longueur. L'invention concerne également des nucléotides, des cellules et des méthodes associés aux compositions, y compris leur utilisation en tant que vaccins.
PCT/US2025/039540 2024-07-29 2025-07-28 Vaccins contre le papillomavirus humain (vph) Pending WO2026030253A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202463676658P 2024-07-29 2024-07-29
US63/676,658 2024-07-29

Publications (1)

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WO2026030253A1 true WO2026030253A1 (fr) 2026-02-05

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PCT/US2025/039540 Pending WO2026030253A1 (fr) 2024-07-29 2025-07-28 Vaccins contre le papillomavirus humain (vph)

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Country Link
WO (1) WO2026030253A1 (fr)

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