WO2023282771A1 - Coronavirus vaccine composition - Google Patents

Coronavirus vaccine composition Download PDF

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Publication number
WO2023282771A1
WO2023282771A1 PCT/NZ2022/050092 NZ2022050092W WO2023282771A1 WO 2023282771 A1 WO2023282771 A1 WO 2023282771A1 NZ 2022050092 W NZ2022050092 W NZ 2022050092W WO 2023282771 A1 WO2023282771 A1 WO 2023282771A1
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WIPO (PCT)
Prior art keywords
seq
composition according
vaccine composition
multivalent vaccine
sequence
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PCT/NZ2022/050092
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French (fr)
Inventor
Robert Graham Feldman
Andrew Brian Herbert
Andrew James HOLLINGS
Giovanni MAZZOCCO
Alexander MYRONOV
Iga NIEMIEC
Piotr STĘPNIAK
Oliver KOLBACHER
Benjamin SCHUBERT
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Covid-19 Vaccine Corporation Limited
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Priority claimed from GBGB2109947.8A external-priority patent/GB202109947D0/en
Priority claimed from GBGB2109945.2A external-priority patent/GB202109945D0/en
Priority claimed from GBGB2109944.5A external-priority patent/GB202109944D0/en
Priority claimed from GBGB2112763.4A external-priority patent/GB202112763D0/en
Priority claimed from GBGB2205960.4A external-priority patent/GB202205960D0/en
Application filed by Covid-19 Vaccine Corporation Limited filed Critical Covid-19 Vaccine Corporation Limited
Publication of WO2023282771A1 publication Critical patent/WO2023282771A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • A61K2039/55505—Inorganic adjuvants
    • 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
    • A61K2039/55561—CpG containing adjuvants; Oligonucleotide containing adjuvants
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • CORONAVIRUS VACCINE COMPOSITION The present disclosure relates to a vaccine composition comprising T cell epitopes designed to be presented on a diverse range of HLA molecules (such as MHC class I), oligopeptides, polypeptides and/or polynucleotide constructs encoding one or more of the same employed in the said compositions; use of any one of the same in the prophylaxis of coronavirus and in particular COVID-19, and manufacture of said composition.
  • HLA molecules such as MHC class I
  • use of any one of the same in the prophylaxis of coronavirus and in particular COVID-19 and manufacture of said composition.
  • Some of the first vaccines were RNA or viral vector based and targeted an immune response to the spike protein from the surface of coronavirus SARS-CoV-2 because use of this protein induces neutralising antibodies from B cells.
  • Work by David Montefiori and Bette Korber demonstrates the spike protein is mutating and the mutation D614G is appearing. Mutation in the spike protein may assist the coronavirus evading immune responses including immune responses in vaccinated patients.
  • Vaccines designed to generate neutralising antibodies to pathogen proteins act extracellularly and may have limited to ability to counteract viruses in the phase when they have hijacked and infected a cell.
  • cytotoxic T cells CD8+ T cells
  • activated CTCs cytotoxic T cells
  • CD8+ T cells cytotoxic T cells
  • kill multiple cells serial killing
  • the current vaccines are not suitable for use in every individual because they can illicit rare but serious adverse events, such as myocarditis, embolisms, strokes and the like.
  • vaccines for example “employing peptides”, such as to multiple proteins, may be desirable; in particular, those optimised to generate T cell responses, more specifically those optimised to generate cytotoxic T cell responses.
  • those optimised to generate T cell responses may be desirable; in particular, those optimised to generate T cell responses, more specifically those optimised to generate cytotoxic T cell responses.
  • targeting multiple parts of the pathogen’s genome including several proteins simultaneously will minimise the impact of mutations in viral proteins.
  • MHC 1 Major Histocompatibility Complex class 1 (MHC 1), is involved in generating cytotoxic T cells.
  • MHC 1 has three subgroups: MHC-A, MHC-B and MHC-C.
  • HLA genes (the human version of MHC) are extremely polymorphic resulting in a vast diversity of peptide-binding HLA specificities and a low population coverage for any given peptide-HLA specificity. Thus, for a given peptide only a percentage of the population will have HLA that can process it and thus present it to a T cell.
  • HLA-A*02:01 which is present in 39.08% of the global population with every other allele occurring more rarely and some very rarely in less than 1% of the global population. It would be useful to have alternative formats of coronavirus vaccines available, for example which are suitable for generating immune responses: • in a diverse population of HLA haplotypes and/or • covering multiple parts of the viral genome from a number of different viral proteins.
  • the present inventors have designed a combination of T cell epitopes, in particular to a variety of viral proteins across the viral genome, that are able to bind HLA molecules in a high percentage of the patient population, for example at least 95% of the population, such as 99% of the population or more, in particular 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7 or 99.8%, in particular 99.7% of the population.
  • This is achieved by having a pool of T cell epitopes, preferably to a number of antigens, with a high level of redundancy. Surprisingly, this coverage seems to be maintained in the presence of new variants.
  • This vaccine may be employed alone or in combination with one or more other vaccines (in particular other coronavirus vaccines, such as a SARS-CoV2 vaccine(s)).
  • other vaccines in particular other coronavirus vaccines, such as a SARS-CoV2 vaccine(s)
  • SUMMARY OF THE DISCLOSURE 1.
  • a multivalent vaccine composition characterised in that it elicits broad spectrum protection against at least one strain of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617.
  • said vaccine comprising: a pool of T cell epitopes derived from at least one viral protein wherein the vaccine has a calculated world population HLA coverage of at least 95%, for example 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 9 9.7%, or 99.8%, in particular 99.7%. 1A.
  • a multivalent vaccine composition comprising a pool of T cell epitopes derived from at least two viral proteins, such as 3, 4, 5 etc.
  • composition elicits broad spectrum protection against multiple strains of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants] B.1.1.7 and/or B.1.1.529 [omicron]).
  • coronavirus such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants] B.1.1.7 and/or B.1.1.529 [omicron]
  • a multivalent vaccine composition comprising at least one construct of formula (I): A -X-B-X-J-X-(U)q-X-W-X-Z (I) w herein:
  • A is sequence independently selected from SEQ ID NO: 1 to 37;
  • X each occurrence is independently a linker, in particular a cleaveable linker, such a s an amino acid linker in particular as disclosed herein;
  • B comprises a sequence independently selected from SEQ ID NO: 1 to 37;
  • J comprises a sequence independently selected from SEQ ID NO: 1 to 37;
  • U comprises a sequence independently selected from SEQ ID NO: 1 to 37;
  • W comprises a sequence independently selected from SEQ ID NO: 1 to 37;
  • Z comprises a sequence independently selected from SEQ ID NO: 1 to 37; and
  • q is 0 or an integer 1 to 40, such as 1 to 31, for example 2, 3, 4, 5,6 ,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,
  • a multivalent vaccine composition according to paragraphs 1, 1A or 1B comprising 2-100 T cell epitopes, for example 2 -50, (such as 5 to 40 or 7 to 36 or 37) in particular 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 or 37, more specifically 5, 6 or 7, especially 6 or 7. 3.
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 3, wherein the T cell epitope peptides are encoded in one or more viral vectors or as a transcribable polynucleotide, such as RNA or DNA, in particular RNA. 5.
  • HLA such as MHC 1
  • cells such as antigen presenting cells, in particular cells autologous to the patient.
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 3 wherein the T cells epitopes are provided as isolated (individual i.e. unlinked sequences) peptides. 7.
  • a multivalent vaccine composition according to paragraphs 7 or 8, where the epitopes (i.e two or more such as all) are linked via a linker or linkers, for example a peptide linker or linkers (including independently selected linkers) 1 to 30 amino acids in length, in particular a linker independently selected from a cleavable linker (such as a proteolytic cleavage site, for example comprising a proteasome dependent site, more specifically AAY, or a furin dependent site such as REKR [SEQ ID NO: 41]) and a linker of formula (II): B 1-W1-Y1-Z1 formula (II) wherein B1 is independently selected from A, R, S and P; W 1 is independently selected from D, L, I and T; Y1 is independently selected from L, G and A Z1 is independently selected from V, K and A.
  • a linker or linkers for example a peptide linker or linkers (including independently selected linkers) 1 to 30 amino acids in length,
  • a multivalent vaccine composition paragraphs to any one of claims 7 to 9, where the e pitopes are linked via a linker or linkers, and at least one has a formula (IIIA): B 2-W2-C0-1-K0-1-(Y2)0-1-Z2 (IIIA) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R. Z2 is independently selected from W and Y 0 means the entity is absent, and 1 means the entity is present. 11.
  • the T cell epitope comprises the sequence shown in SEQ ID NO: 25.
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 67, wherein the linked epitopes have a sequence comprising SEQ ID NO: 47 or a derivative thereof wherein the leader sequence (in italics) is absent: MGVTGILQLPRDRREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRREK RSTFNVPMEKREKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRYLFDESGEFKLREKRM RPNFTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNA PRITFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTY VREKRETKAIVSTIQRRE
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 67, wherein the linked epitopes have a sequence comprising SEQ ID NO: 48 or a derivative thereof wherein the leader sequence (in italics) is absent: MGVTGILQLPRDRREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRREK RSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRMRPNFTIKGSFREKRY DPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNAPRITFREKRVYMPAS WVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTYVREKRETKAIVSTIQ RREKRTMADLVYALREKRHFPREG
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 68, wherein the linked epitopes have a sequence comprising any one of SEQ ID NO: 49, 50 and 71 to 76, wherein each X is a linker, such as a cleavable linker, for example independently selected from a linker disclosed herein, such as AAY,REKR, formula (II), formula (III) and formula (IIIA), such as AAY and REKR .
  • linker such as a cleavable linker
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to11 and 15 to 71, wherein the linked epitopes have a sequence comprising SEQ ID NO: 71, wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, such as AAY, such as REKR. 73.
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 74, wherein the linked epitopes have a sequence comprising SEQ ID NO: 74 or a derivative thereof wherein the leader sequence (in italics) is removed: MDAMK RGLCCVLLLCGAVFVDSVTGXLVQAGNVQLRXLDGISQYSLRXGVYYPDKVFRXFGADPIHSLRXSTFNVPM EKXFYDFAVSKGFXSAPHGVVFLXYLFDESGEFKLXMRPNFTIKGSFXYDPLQPELXVFVSNGTHWFXQFAY ANRNRFXKVDGVDVELXNQRNAPRITFXVYMPASWVMRIXKPLEFGATSAXSDNIALLVXASAFFGMSRIXV VFLHVTYVXETKAIVSTIQRXTMADLVYALXHFPREGVFVSXKMFDAYVNTFXRTIAFGGCVFXYAFEHIVY
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 75, wherein the linked epitopes have a sequence comprising SEQ ID NO: 75 or a derivative thereof wherein the leader sequence (in italics) is removed: MGVT GILQLPRDRXLVQAGNVQLRXLDGISQYSLRXGVYYPDKVFRXFGADPIHSLRXSTFNVPMEKXFYDFAVSK GFXSAPHGVVFLXYLFDESGEFKLXYLFDESGEFKLXMRPNFTIKGSFXYDPLQPELXVFVSNGTHWFXQFA YANRNRFXKVDGVDVELXNQRNAPRITFXVYMPASWVMRIXKPLEFGATSAREKRSDNIALLVXASAFFGMS RIXVVFLHVTYVXETKAIVSTIQRXTMADLVYALXHFPREGVFVSXKMFDAYVNTFXRTIAFGGCVFXYAFE HIV
  • each X is independently a cleavable linker, for example an amino acid linker, such as independently selected from AAY and REKR, in particular AAY, in particular REKR.
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 77, wherein the linked epitopes have a sequence comprising SEQ ID NO: 65 or a derivative thereof wherein the leader sequence (in italics) is absent: MDAMKRGLCC VLLLCGAVFVDSVTGREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRR EKRSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRYLFDESGEFKLREK RMRPNFTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQR NAPRITFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHV TYVRE
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 1 5 to 78, wherein the linked epitopes have a sequence comprising SEQ ID NO: 67 or derivative thereof wherein the leader sequence (in italics) is absent: MDAMKRGLCCVLLLCGAVFVDSVTGREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRF GADPIHSLRREKRSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRMRPN FTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNAPRI TFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTYVRE KRETKAIVSTIQRREKRTM
  • a multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 87, w herein the composition comprises an addition sequence independently selected from: a. an intracellular translocation sequence, b. a sequence that increases proteolytic processing and/or proteasomal cleavage efficiency, c. a sequence that enhances immunological responses and/or immunogenicity, d. a sequence that enhances epitope presentation, such as a PADRE sequence, e. a sequence that induces, increases or sustains activity of one or more immune cells (such as CD4+, CD8+ and antigen presenting cells); and/or f. combinations of 2 or more of the same. 89.
  • a multivalent vaccine composition according to paragraph 87 wherein the composition comprises an intracellular translocation domain, for example comprises sequence MDAMKRGLCCVLLLCGAVFVDSVTG [SEQ ID NO: 38] and/or MGVTGILQLPRDR [SEQ ID NO: 39].
  • 90. A multivalent vaccine composition according to paragraph 88 or 89, wherein the composition comprises a sequence associated with enhanced immunological response and/or immunogenicity.
  • 91. A multivalent vaccine composition according to any one of paragraphs 86 to 88, wherein the composition comprises a sequence which enhances epitope presentation such as a PADRE sequence, such as AKFVAAWTLKAAA [SEQ ID NO: 40]. 92.
  • the composition comprises a sequence that increases proteolytic processing efficiency, for example a cleavable linker, such as disclosed herein.
  • the composition comprises an adjuvant.
  • a multivalent vaccine composition according to paragraph 96 wherein at least one dose of said further vaccine (for example all the doses) is administered prior to the multivalent vaccine composition, for example 1-6 days, 1 to 3 weeks, a month or more prior.
  • a multivalent vaccine composition according to paragraph 95 or 96 wherein at least one dose of said further vaccine (for example all the doses) is administered concomitantly with the multivalent vaccine composition i.e. in same dosing regimen.
  • 102. A multivalent vaccine composition according to paragraph 100, wherein the two or more vaccines are provided as a kit, i.e. together, in particular including instructions.
  • 103. A multivalent vaccine composition according to any one of paragraphs 96 to 100, wherein the vaccines are co-formulated.
  • 104. A multivalent vaccine composition according to any one of paragraphs 96 to 102, for use in a population of patients already vaccinated against coronavirus. 105.
  • a second dose is administered, for example intramuscularly or subcutaneously.
  • a multivalent vaccine composition for the manufacture of a medicament for the treatment of coronavirus, in particular COVID-19. 110.
  • a method of treatment comprising administered to a human multivalent vaccine composition as defined in any one of paragraphs 1 to 108.
  • the vaccine composition according to the present disclosure comprises a sequence or combination of sequences disclosed in the sequence listing herein.
  • the present disclosure provides a coronavirus vaccine, in particular a pan SARS-CoV-2
  • the multivalent vaccine comprises 5, 10 or more SARS-CoV-2 T cell epitopes, 15 or more SARS-CoV-2 T cell epitopes, 20 or more SARS-CoV-2 T cell epitopes, 25 or more SARS-CoV-2 T cell epitopes, 30 or more SARS-CoV-2 T cell epitopes, 35 or more SARS-CoV- 2 T cell epitopes, 40 or more SARS-CoV-2 T cell epitopes, 45 or more SARS-CoV-2 T cell epitopes, 50 or more SARS-CoV-2 T cell epitopes, or 55 or more SARS-CoV-2 T cell epitopes.
  • q in formula (I) is an integer 1 to 40, such as 1 to 31, for example 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 or 30.
  • q is 38, 39 or 40 then one or more of the epitopes 1 to 37 will be repeated within the construct.
  • A, B, J, each U, Wand Z will be selected to be different peptides.
  • A, B, J, U, W and Z all have different amino acid sequences.
  • the invention also extends to polynucleotides encoding said construct of the present disclosure including of formula (I).
  • the vaccines of the present disclosure generate T cell responses, for example CD8+ T cell responses.
  • the peptides components in the constructs of the disclosure are all MHC I restricted.
  • one or more elements of the vaccine composition of the present disclosure is able to be presented on one or more of the HLA alleles selected from the group consisting of: A01:01, A02:01, A02:02, A02:06, A02:07, A11:01, A23:01, A24:02, A24:41, A24:51, A26:01, A30:02, A31:01, A31:29, A32:01, A34:01, A68:01, B07:02, B08:01, B15:01, B15:93, B18:01, B27:05, B35:01, B38:01, B39:01, B39:54, B40:01, B40:10, B48:01, B51:01, B54:18, B55:02, B56:02, B56:43, B58:01, C01:02, C01:57, C02
  • T he present disclosure also includes combinations of peptides (for example as mixtures or linked) as disclosed herein and a polynucleotide or polynucleotides encoding said one or more peptides and/or combinations thereof (in particular a sequence disclosed herein), vectors and/or cells comprising said polynucleotide or polynucleotides.
  • the present disclosure also includes a polypeptide disclosed herein, a polynucleotide encoding same, a vector and/or cell comprising said polynucleotide.
  • the vaccine composition according to the present disclosure does not comprise a polynucleotide. In one embodiment the vaccine composition according to the present disclosure does not comprise a vector, such as a viral vector. In one embodiment the vaccine composition according to the present disclosure does not comprise a cell, such as an immune cell.
  • the sequence is selected from any one of sequences 71 to 76: C1_p generic linker SEQ ID NO: 71 wherein X is a linker, for example an amino acid linker (including a linker of a formula or sequence disclosed herein), such as a cleavable linker (including a proteolytic cleavage), in particular selected from AAY and REKR, more specifically AAY; C1_p.3 generic linker SEQ ID NO 72 wherein X is a linker, for example an amino acid linker, such as a cleavable linker (including a proteolytic cleavage), in particular selected from AAY and REKR, more specifically AAY; C2_f generic linker SEQ ID NO: 73 or a derivative thereof minus the leader sequence wherein X is a linker, for example an amino acid linker, such as a cleavable linker (include proteolytic cleavage), in particular selected from AAY and REKR, more specifically REK
  • X is Xaa, wherein Xaa is an amino acid linker, for example as disclosed herein, including a formula (II) and/or (III).
  • X in SEQ ID NO: 49 is Xaa.
  • X in SEQ ID NO: 50 is Xaa.
  • X in SEQ ID NO: 71 is Xaa.
  • X in SEQ ID NO: 72 is Xaa.
  • X in SEQ ID NO: 73 is Xaa.
  • X in SEQ ID NO: 74 is Xaa.
  • X in SEQ ID NO: 75 is Xaa.
  • X in SEQ ID NO: 76 is Xaa.
  • the invention provides a sequence or construct of a formula disclosed herein.
  • the present disclosure includes a polynucleotide encoding a construct herein or a codon optimised version thereof, for a mammalian cell, for example a human cell or CHO.
  • the polynucleotide is a sequence disclosed herein.
  • the polynucleotide is for use in manufacturing a peptide, oligopeptide or polypeptide according to the present disclosure.
  • the polynucleotide is optimised for a cell selected from an E. coli cell and an insect cell, in particular E. coli.
  • the polynucleotide is a vaccine construct for use in a vaccine composition according to the present disclosure.
  • the polynucleotide is RNA.
  • the polynucleotide is DNA.
  • the disclosure extends to any sequence of construct according to the present invention (in particular disclosed herein), including where they are provided as a pharmaceutical formulation (in particular a vaccine composition).
  • the pharmaceutical formulation is filled into a vial, for example containing 1 to 8 doses.
  • the disclosure also includes use of all aspects in treatment (which included prophylaxis and vaccination), in particular treatment of coronavirus, such as SARS-CoV-2.
  • compositions of the present disclosure include the ability to generate a T cell response in a high percentage of the population and thus the vaccine has worldwide utility.
  • Compositions of the present disclosure have high levels of redundancy built in. This means the composition includes multiple epitopes for a given viral protein and/or target multiple virus proteins. Thus, vaccination with the composition is likely to provide immunity even if the virus mutates.
  • the level of redundancy is the numerical value calculated for any one of the constructs disclosed herein. In one embodiment this calculated value from an “example” is combined with an independent functional claim defining the invention. Thus, these numerical values may be calculated and combined with one or more claims and are NOT limited to being associated with only one individual sequence.
  • the order of the epitopes can be important, for example it may have an impact on expression.
  • the order of the epitopes has been optimised in constructs according to the present disclosure, in particular optimised for expression.
  • linkers have been optimised, for example to remove neo-epitopes and/or optimise recovery of the component epitopes, i.e. to optimise cleavage and/or processing by cells in vivo.
  • the percentage disorder of a construct according to the present disclosure is in the range 10 to 75 (or 20 to 60%), for example 15, 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 or 75%.
  • the constructs according to the present disclosure do not comprise a bead (such as a biobead, in particular, as disclosed in the Examples herein).
  • constructs are optimised for expression in vitro and/or in vivo, for example are provided as multiple fragments (oligopeptides as defined herein).
  • the constructs of the present disclosure are optimised for expression in a cell, for example a host cell used in recombinant expression, in particular E. coli (for example strain BL21(DE3).
  • E. coli for example strain BL21(DE3).
  • T he linkers can be the same or different. Multiple constructs of this format may be provided in a single vaccine composition.
  • Figure 3 Shows pentamer immune profiling analysis of antigen-specific CD8+ T c ells
  • Figure 4 Shows pentamer immune profiling analysis of antigen-specific CD8+ T c ells
  • Figure 5 Shows ELISPOT analysis
  • Figure 6 Shows percentage recovery and disorder for certain constructs according t o the present disclosure.
  • Figure 7 Shows spot forming units for certain constructs according to the present d isclosure
  • Figure 8 Shows spot forming units for certain constructs Figure 9 Polyacrylamide gels of E.
  • Figure 10 Shows gating strategy for ICFC analysis of ex vivo restimulated s plenocytes from Group 1-11 mice
  • Figure 11 Shows frequencies of IFN ⁇ + CD8+ T cells in the splenocytes from G11 c ontrol mice (immunized with HPV peptide) after the ex vivo stimulation with 5 ⁇ M HPV peptide
  • Figure 12A Shows frequencies of peptide-specific IFN ⁇ + CD8+ T cells in the s plenocytes after the ex vivo stimulation with HLA-A*02:01 peptide pool (5 ⁇ M per peptide)
  • Figure 12B Summarises the number of mice with positive peptide-specific IFN ⁇ + C D8+ T cell responses within each group
  • Figure 13 Shows gating strategy for ICFC analysis of ex vivo restimulated s plenocytes.
  • Figure 14A Shows restimulation with HLA-A* 02:01 peptide pool
  • Figure 14B Shows restimulation with HLA-A* 11:01 peptide 9
  • Figure 14C Shows restimulation with HLA-A* 11:01 peptide 10
  • Figure 14D Shows restimulation with HLA-B* 07:02 peptide 11
  • Figure 15 Shows % of peptide specific IFN ⁇ + cells in CD3 + & CD8 + T cells
  • Null sequence SW is a linker SEQ ID NO: 1-36 T cell epitopes shown in Table 1 SEQ ID NO: 37 LLFNKVTLA peptide for inducing a specific CD8+ T cell response in i ndividuals possessing the MHC class I A*02:01 allele
  • SEQ ID NO: 38 MDAMKRGLCCVLLLCGAVFVDSVTG intracellular translocation domain
  • SEQ ID NO: 39 MGVTGILQLPRDR intracellular translocation domain
  • SEQ ID NO: 77 Polynucleotide encoding C1_p3 (also known as T1.2) SEQ ID NO: 78 Polypeptide with bespoke linkers, designated design 0 SEQ ID NO: 79 Polypeptide with bespoke linkers, designated design 1 SEQ ID NO: 80 Polypeptide with bespoke linkers, designated design 2 SEQ ID NO: 81 Polypeptide with bespoke linkers, designated design 3 SEQ ID NO: 82 Polypeptide with bespoke linkers, designated design 4 SEQ ID NO: 83 Polypeptide with bespoke linkers, designated design 5 SEQ ID NO: 84 Polypeptide with bespoke linkers, designated design 6 SEQ ID NO: 85 Polypeptide with bespoke linkers, designated design 7 SEQ ID NO: 86 Polypeptide with bespoke linkers, designated design 8 SEQ ID NO: 87 Polypeptide with bespoke linkers, designated design 9 SEQ ID NO: 88 Oligopeptide with bespoke linkers, designated fragment 1: MRPNFTIKGSF-PWRW-HFPREGVF
  • Broad spectrum protection refers to protection against at least two variants of a coronavirus, but preferably more i.e. so-called pan protection, and/or protection in a high percentage of the patient population.
  • Pan SARS-CoV-2 vaccine as employed herein refers to the ability to provide some level of protection against essentially all SARS-CoV2 viruses.
  • Immunity refers to the ability to protect patients. Immunity as employed herein refers to the ability of a patient to resist infection or minimise adverse events associated with infection, for example to minimise viral load and/or symptoms, avoid respiratory distress, avoid cytokine storm, avoid organ failure and the like. In one embodiment immunity is the ability to prevent infection.
  • Coronavirus refers to a virus, which is in the family of Coronaviridae (order Nidovirales and realm Ribovira), for example with a lineage identified by Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN). Coronaviruses constitute the subfamily Coronaviridae. Under this subfamily is the genera Beta-CoV. This contains the lineages SARS-CoV, SARS-CoV-2, MHV and MERS-CoV.
  • SARS-CoV-2 viruses as employed herein refers to viruses with a lineage of SARS-CoV-2, for example where the lineage is designated using PANGOLIN.
  • Lineage includes clades and strains. Lineages using the WHO label currently include alpha, beta, gamma, epsilon, eta, lota, kappa, zeta, mu.
  • PANGO lineage include A.1, A.2, A.3, A.4, A.5, A.6, B.1, B.2, B.3, B.4, B.5, B.6, B.7, B.9, B.10, B.13, B.14, B.15, B.16, such as B.1.1, B.1.22, B.1.26, B.1.37 B.1.3-B1.66, B.1.5- B.1.72, B.1.1.177 and B.1.13.
  • Notable sequences include WIV04/2019, B.1.617 (such as B.1.617.3), B.1.427/B.1.429, B.1.525, B.1.351 (20H/501Y.V2 or 501.V2), B.1.1.207 C.37), P.1, P.2, B.1.1.7 (VOC-202012/01), VOC-20DEC-01 AND20I/501Y.V1.
  • Omicron is Pango lineage B.1.1.29 (GISAID clade GR/484A), Nextstrain clade 21K, 21L, 21M, 22A, 22B, 22C, additional amino acid change +S:R346K, +SL452X, +S: F486V.
  • Strain as employed herein is genetic variant within a virus lineage, for example Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2, AY.1, AY.2 & AY.3), Zeta ⁇ (P.2), Eta (B.1.525 & B.1.1.318), Theta ⁇ (P.3), Kappa (B.1.617.1, B.1.617.3, AV.1 & C.36.3), Lambda (C37 & B.1.621), Epsilon ⁇ (B.1.427/B.1.429, B.1.1.7 with S494P & A.27), Iota (B.1.526, B.1.1.7 with Q667H, B.1.620, B.1.214.2, R.1, B.1 with 214insQAS, AT.1, A.30, B.1.630, P.1+N501T and E484Q, B.1.619, B.1.629, C.1.2, B
  • the patient is a human, for example aged 18 years or over. In one embodiment the patient is an infant or child, for example age 3 months to 18 years.
  • the construct may further comprise (including encoding) a leader sequence, to assist polypeptide (including expressed polypeptide) processing within various cellular compartments.
  • the construct when the T cell epitopes are linked by one or more furin cleavage sites (e.g. where linkers are furin cleavage sites) said construct will generally comprise a leader sequence or a translocation domain.
  • the construct will exclude a leader sequence or translocation domain.
  • constructs of the present disclosure comprise a PADRE sequence, for example at the C-terminal.
  • the constructs of the present disclosure comprise a label, such as a HisTag.
  • the constructs of the disclosure do not comprise a label, such as HisTag.
  • the disclosure extends to sequences comprising a HisTag disclosed herein (such as T2.1) where said Tag is absent.
  • X in sequences according to the present disclosure is a linker, for example a cleavable linker (including a proteolytic cleavage site), such as independently selected from a linker explicitly disclosed herein, more specifically AAY and REKR.
  • constructs of the disclosure comprises multiple X sequences
  • the sequence of each X is selected independently, for example such that some or all of the linkers are different.
  • all the X’s have the same amino acid sequence (including where the same amino acid sequence is encoded by different polynucleotide sequences).
  • the selection/optimisation of the linker can increase immune responses.
  • Independently (such as independently selected) as employed herein refers to the fact the multiples of the entity X can be the same or different.
  • sequences of the present disclosure are isolated, for example isolated from the virus or body (or a cell), i.e. removed form a native environment. Isolation may include being in a storage container or the like in a suitable media or formulation.
  • isolated refers to where the construct is essential free of contaminants and other extraneous materials, in particular the construct is not contained within a body or naturally occurring. Recombinant synthesis is not considered to be “naturally occurring” within the meaning of the current invention.
  • isolated constructs include constructs formulated with excipients, diluents, carriers and the like.
  • the sequences of the present disclosure are recombinant.
  • Recombinant as employed herein refers to where the sequence is made using recombinant techniques.
  • the sequences of the present disclosure are purified. Purified as employed herein refers to free from toxins and contaminations, for example 95, 96, 97, 98 or 99% pure as assessed by a suitable assay.
  • Sequence as employed herein can refer to polynucleotide sequences, peptide, oligopeptide, or polypeptide sequences (including protein sequences) and peptide sequences. In one embodiment the disclosure extends to a sequence (such as an amino acid sequence disclosed herein) at least 95% similar or identical to a sequence disclosed herein, for example 96%, 97%, 98% or 99%, for example over the whole length of the sequence in question.
  • Polynucleotide sequence as employed herein refers to RNA and/or DNA, including incorporated into a vector, (such as a viral vector) or within a cell.
  • polynucleotide sequences are codon optimised, for example for a mammalian cell, such as a human cell or a CHO cell.
  • Polypeptide as employed herein refers to a sequence of 50 amino acids or greater.
  • Oligopeptide also referred to an oligo-epitopes as employed herein overlaps with the definition of polypeptide and peptide, and is intended to refer to construct with at least two epitopes linked, for example with a linker as disclosed here, and no more than 10 linked epitopes.
  • one or more oligopeptides are employed in combination, for example in totally comprising the same number of epitopes discussed elsewhere herein, such as 37.
  • oligopeptides are employed in the same combination, for example in the same formulation, in particular 1 to 6 oligiopetides selected from SEQ ID NO: 88 to 93.
  • Peptide as employed herein refers to a sequence of 2 to 49 amino acids.
  • Multipeptide vaccine refers to a vaccine comprising two or more peptides or oligopeptides, in particular where each peptide or oligopeptide contains two or more epitopes.
  • the vaccine composition according to the present disclosure employs one or more sequences set forth in any one of SEQ ID NO: 42 to 50, 65 to 68 or 71 to 76 or 88 to 98 including a sequence 95%, or more similar or identical thereto, such as 96%, 97%, 98% or 99% similar or identical thereto or a polynucleotide, encoding the same.
  • the vaccine composition according to the present disclosure employs sequence set forth in any one of SEQ ID NO: 51 to 64 or 77 or 99 to 104, sequence identical or similar thereto encoding the same amino acid, and/or a codon optimised version thereof, for example optimised for a mammalian cell, such as human cell or CHO cell.
  • Linker(s) as employed herein refer to something other than a simple bond linking the epitopes.
  • one or more linkers will independently be an amino acid sequence, for example 2 to 20 amino acids long (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, in particular 2,3, 4, 5 or 6).
  • the linker is cleavable.
  • constructs of the present disclosure comprise from 1 to 40 linkers, for example 2 to 36, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, in particular 5, 35 or 36.
  • Linkers suitable for use in the present constructs are disclosed herein.
  • Additional linkers for use in the constructs of the present disclosure include: • Proteasome dependent, for example LRA, RLRA [SEQ ID NO: 70] and a linker of formula ( II): B 1-W1-Y1-Z1 (II) wherein B1 is independently selected from A, R, S and P, particularly P; W1 is independently selected from D, L, I and T; Y1 is independently selected from L, G and A Z1 is independently selected from V, K and A (See Bazhan et al 2019); and an optimised site such as that described in Schubert & Kohlbacher, 2016 Genome Medicine (2016) 8:9 specifically incorporated herein by reference.
  • B1 is independently selected from A, R, S and P, particularly P
  • W1 is independently selected from D, L, I and T
  • Y1 is independently selected from L
  • G and A Z1 is independently selected from V, K and A (See Bazhan et al 2019); and an optimised site such as that described in Schubert & Kohlbacher, 2016
  • cleavage site algorithm (1) on page 3 col 2 the immunogenicity model algorithm (2) also on page 3 col 2, algorithm (3) and (4) on page 4, are specifically incorporated and may be used as basis for amendments.
  • a linker of formula (III) is employed in constructs of the present disclosure (including the construct of formula (I) and sequences disclosed herein comprising X): B 2-W2-C0-1-K0-1-(Y2)0-1-(Z2)0-1 (III) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P.
  • W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R.
  • Z2 is independently selected from M, W and Y 0 means the entity is absent, and 1 means the entity is present.
  • the linker of formula (III) is independently selected from: PWMW (SEQ ID NO: 107), PWNW (SEQ ID NO: 108), PWQK (SEQ ID NO: 109), PWQW (SEQ ID NO: 110), PWQY (SEQ ID NO: 111), PWRW (SEQ ID NO: 112), PWRY (SEQ ID NO: 113), PWSW (SEQ ID NO: 114), PWTW (SEQ ID NO: 115), MWCW (SEQ ID NO: 116), MWKW (SEQ ID NO: 117), WWCW (SEQ ID NO: 118), YWCYM (SEQ ID NO: 119), GWKW (SEQ ID NO: 120), SWTW (SEQ ID NO: 121), PNCKQY (SEQ ID NO: 122), PNCKRY (SEQ ID NO: 123), YWCW (SEQ ID NO: 124), GWCW (SEQ ID NO: 125), WWCY (SEQ ID
  • linkers are independently selected and at least one (for example 2 or more or all) is/are SW.
  • a linker of formula (IIIA) is employed in constructs of the present disclosure (including the construct of formula (I) and sequences disclosed herein comprising X): B 2-W2-C0-1-K0-1-(Y2)0-1-Z2 (IIIA) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R.
  • Z2 is independently selected from W and Y 2 means the entity is absent, and 3 means the entity is present.
  • the linker of formula (IIIA) is independently selected from: PWMW (SEQ ID NO: 107), PWNW (SEQ ID NO: 108), PWQK (SEQ ID NO: 109), PWQW (SEQ ID NO: 110), PWQY (SEQ ID NO: 111), PWRW (SEQ ID NO: 112), PWRY (SEQ ID NO: 113), PWSW (SEQ ID NO: 114), PWTW (SEQ ID NO: 115), MWCW (SEQ ID NO: 116), MWKW (SEQ ID NO: 117), WWCW (SEQ ID NO: 118), GWKW (SEQ ID NO: 120), SWTW (SEQ ID NO: 121), PNCKQY (SEQ ID NO: 122), PNCKRY (SEQ ID NO: 123), YWCW (SEQ ID NO: 124), GWCW (
  • the linker or linkers has/have 4 amino acids in length, such as PWMW, PWNW, PWQK, PWQW, PWQY, PWRW, PWRY, PWSW, PWTW, MWCW, MWKW, WWCW, YWCYM GWKW, SWTW.
  • the linker or linkers has/have 6 amino acids in length, such as PNCKQY and PNCKRY.
  • all the linkers in the fragment/construct are the same length, such as 4 amino acids in length or 6 amino acids in length, including where linkers are the same sequence or different sequences.
  • the linkers in the fragment/construct are at least two different lengths, for example 2 different lengths of linkers are employed.
  • the linkers are independently selected from amino acid sequences 2 and 4 amino acids in length, for example those explicitly disclosed herein.
  • the same amino acid sequence is employed in the linkers of the constructs according to the disclosure.
  • different amino acid sequences are employed in constructs of the disclosure, for example 2, 3, 4, or 5 different amino acid sequences are employed within the same construct, such as 2 (in particular SW & GWCW, PWRW & PWQW, or PWQY & PWRY, or MWCW & WWCW, or PNCKRY & PNCKQY), 3 (in particular SW, PWSW & PWTW) 4 or 5 different amino acid sequences (in particular YWCY, PWSW, PWTW, PWNW & WWCY or PWQY, MWKW, GWKW, SWTW & PWMW)
  • the first one or two linkers at the N-terminal end of the protein are one type and the remaining linkers are a different type.
  • the linker or linkers at the N terminal and C terminals are the same and the intervening linker or linkers is/are different.
  • the linkers in a construct appears in blocks of the same type i.e. at least two consecutive linkers are of the same type, such as 2 to 4 consecutive linkers are the same type. In one embodiment no two consecutive linkers in a construct are the same. In one embodiment all the linkers are the same amino acid sequence.
  • each X is independently selected from a linker disclosed herein, for example each X is independently selected from formula (II) (III) and (IIIA), in particular independently selected from formula (IIIA).
  • the linker between each epitope is a different sequence, for example a different nucleotide sequence and/or different amino acid sequence.
  • High redundancy as employed herein refers to levels of 0.900 or above, for example in the range 0.900 to 0.999, such as 0.905, 0.91, 0.915, 0.92, 0.925, 0.93, 0.935, 0.94, 0.945, 0.95, 0.955, 0.96, 0.965, 0.97, 0.975, 0.98, 0.985, 0.99, 0.995, in particular 0.966.
  • redundancy is calculated as set out herein, for example as shown in Example 12.
  • the compositions of the present disclosure are generally provided as a parenteral formulation, for example for intramuscular or subcutaneous delivery.
  • compositions including vaccines
  • sweetman S. C. (Ed.). Martindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483 pp.; Aulton, M. E. (Ed.) Pharmaceutics. The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and, Ansel, H. C, Allen, L. V. and Popovich, N. G. Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999, 676 pp.
  • Excipients employed in the manufacture of drug delivery systems are described in various publications known to those skilled in the art including, for example, Kibbe, E. H.
  • HLA alleles with allele frequency (in decimals) > 0.05 from two M ⁇ ori populations identified in the HLA allele frequencies database (http://www.allelefrequencies.net), and HLA alleles prevalent in the global population, was developed for use in epitope selection.
  • SARS-Cov-2 proteins were then analysed to identify suitable protein-derived epitopes including those having low mutation frequency.
  • Epitopes were then selected for vaccine construct preparation using a combinatorial approach to provide target global population coverage. The selected epitopes and their corresponding HLA alleles is shown below in Table 1.
  • T he epitopes depicted in Table 1 cover 52 unique HLA alleles present in the global population, providing a predicted 99.7% coverage of the global population. Additionally, the applicants predict, that a multivalent construct consisting of the epitopes LVQAGNVQLR [SEQ ID NO: 1], LDGISQYSLR [SEQ ID NO: 2], GVYYPDKVFR [SEQ ID NO: 3], FGADPIHSLR [SEQ ID NO: 4], STFNVPMEK [SEQ ID NO: 5], FYDFAVSKGF [SEQ ID NO: 6], and SAPHGVVFL [SEQ ID NO: 7] would provide coverage of ⁇ 94.6% of the global population.
  • Example 2 Construct structure This example describes the preparation of a multivalent vaccine composition comprising selected SARS-CoV-2 epitopes as described above in Example 1, together with additional functional components.
  • a general schema of the construction of the multivalent construct is shown in Figure 1 and 2, and comprises from the N-terminus of the polypeptide to the C- terminus the following functionalities: • an optional leader sequence, typically comprising an intracellular translocation domain, for example to elicit translocation of the polypeptide into the endoplasmic reticulum (ER) for proteasome-independent protein (e.g. furin-dependent) processing.
  • ER endoplasmic reticulum
  • R epresentative leader sequences associated with furin-dependent processing include the TPA activator signal sequence [SEQ ID NO: 38] and an alternative leader sequence [SEQ ID NO: 39]; • selected epitope sequences, for example the amino acid sequences set out in Table 1 above [SEQ ID NOs: 1 – 36], or the HLA A*02:01-presented SARS-CoV-2 epitope of SEQ ID NO: 37, optionally separated by spacer sequences comprising proteolytic cleavage s ites.
  • linker sequences such as the AAY spacer for proteasome-dependent processing, and the REKR [SEQ ID NO: 42] spacer for proteasome- independent/furin-dependent peptide processing, are specifically contemplated herein; • an amino acid sequence associated with enhanced immunological response or immunogenicity, such as the promiscuous Pan HLA DR-binding Epitope (PADRE) s equence [SEQ ID NO: 40].
  • PADRE promiscuous Pan HLA DR-binding Epitope
  • Representative full-length multivalent vaccine constructs include: • C1_p [SEQ ID NO 42], C1_p.2 [SEQ ID NO: 43], and C1_p.3 [SEQ ID NO:44] polypeptide c onstructs, which are optimized for proteasome-dependent processing, have no leader sequence, and comprise the AAY spacer; • C2_f [SEQ ID NO: 45] and C2_f.2 [SEQ ID NO: 46] polypeptide constructs, which are o ptimized for furin-dependent processing, include the TPA leader peptide, and comprise the REKR spacer; • C3_f [SEQ ID NO: 47] and C3_f.2 [SEQ ID NO: 48] polypeptide constructs, which are o ptimized for furin-dependent processing with an alternative leader peptide, and comprise the REKR spacer; and • C4_pr [SEQ ID NO: 49] and C4_pr.2 [SEQ ID NO: 50] polypeptide constructs,
  • Representative cDNA coding sequences include: C1_n [SEQ ID NO: 51], C1_n.2 [SEQ ID NO: 52], C2_n [SEQ ID NO: 53], C2_n.2 [SEQ ID NO: 54], C3_n [SEQ ID NO: 55], C3_n.2 [SEQ ID NO: 56] and C1_p.3 [SEQ ID NO: 77].
  • the C1_p, the C1_p.2, and the C1_p.3 polypeptide constructs each target proteasome-dependent processing, but differ in the number or order of epitopes they comprise.
  • Example 3 Multivalent construct production This example describes the preparation of a prokaryotic host capable of expressing a multivalent polypeptide construct as described herein.
  • the polypeptide construct referred to in these examples as T2.1, is equivalent to the C2_f polypeptide construct discussed herein and presented as SEQ ID NO: 45.
  • the genetic construct expressing T2.1 is referred to in these examples sometimes as CVC4.
  • a plasmid (CVC4_pET9-T2-phaC) containing the C2_f polypeptide construct was used as a template.
  • Primers were designed to amplify the coding region and introduce a C-terminal His6 tag for cloning into expression vector pET22b.
  • Primers T2F (5’-TAAGAAGGAGATATACATATGGATGCTATGAAAAGGGGACTATGC [SEQ ID NO. 57]
  • T2R 5’-CTCGAGTGCGGCCGCAAGCTTATCAGTGATGGTGATGG TGGTGAGAGCCTCCACCGCCAGAGC [SEQ ID NO.
  • Cloning into pET22b was performed with an In-Fusion® HD Cloning kit (Takara 638910) using 1 ⁇ L of linearised pET22b (NdeI/HindIII) and 1 ⁇ L of purified PCR product to produce plasmid pET-22b-CVC4-T2-His6 before transformation into E. coli Stellar cells, according to manufacturer’s instructions. Selection was on LB agar+100 ⁇ g/mL carbenicillin (LB/carb).
  • the selected CVC4-encoded construct (T2-His6) have the following characteristics: Length: 579 aa; Molecular weight: 69.482 kDa; Isoelectric point: 11.30; Charge at pH 7: 77.12; Extinction Coefficient: 59,610
  • construct CVC4 plasmid DNA (1 ⁇ L) was transformed into E. coli BL21-AI (ThermoFisher C607003) according to manufacturer’s instructions and grown on LB agar/carb overnight.
  • Example 4 Production of vaccine containing T2.1 protein conjugate
  • This example describes the production of a multivalent polypeptide construct conjugate, comprising the T2.1 multivalent construct conjugated to a phaC polymer particle-forming protein, in ClearColiTM BL21 (DE3) using a kanamycin resistant T2.1 phaC plasmid and tetracycline resistant phaA / phaB plasmid, and the resulting production of T2.1 biobeads – a representative example of the polypeptide conjugates contemplated herein.
  • a tetracycline resistant phaA and phaB plasmid was created containing the phaA and phaB genes from Cupriavidus necator downstream of a lac promoter.
  • a chloramphenicol resistant (CmR) plasmid containing phaA and phaB genes is called pMCS69.
  • An IDT g-block was designed to contain the tetracycline resistance promoter along with the tetR gene from pBR322 as a fragment.
  • the pMCS69 backbone fragment and the promoter-TetR gBlock were assembled with NEBuilder.
  • the tetR fragment was inserted into the 6,116 bp pMCS69 fragment using the Gibson/Infusion/homology cloning protocol and transformants plated on Tet agar plates. This resulted in many Tetracycline resistant colonies.
  • Plasmid from several clones were isolated and the tetR, phaA and phaB (and promoter) were sequences and confirmed to be correct.
  • the resulting plasmid has been named “p69Tet”.
  • a kanamycin resistant plasmid containing the phaC gene from Cupriavidus necator was created (WT phaC plasmid).
  • a gene for phaC protein was codon optimized, synthesized by Genscript and inserted in to the NdeI and BamHI cloning sites of plasmid pET-9a.
  • a T7 pET- based kanamycin resistant plasmid expressing T2.1 as a N-terminal translational fusion to phaC from Cupriavidus necator was created.
  • the T2.1 antigen was cloned such that it was translationally fused to the phaC via a SGGGGSGGGGS linker [SEQ ID NO: 69].
  • the T2.1 sequence was codon optimized and translationally fused to phaC.
  • the kanamycin resistant WT phaC plasmid was used as a backbone for constructing the T2.1-linker-phaC plasmid.
  • the amino acid sequence of the T2.1-phaC polypeptide is presented herein as SEQ ID NO: 65.
  • Plasmid p69Tet was electroporated into electrocompetent ClearColi® BL21 (DE3) strains obtained from Lucigen, according to the manufacturer’s instructions and selected on MTB agar with 10 ⁇ g/ml tetracycline. These cells were used to make a batch of electrocompetent cells (CCPT1E) used for the electroporation of T2.1-linker-phaC plasmid.
  • CPT1E electrocompetent cells
  • a T2.1-phaC biobead producing organism was created by electroporation of the kanamycin resistant T2.1-linker- phaC plasmid into the ClearColi® cells already containing p69Tet. Transformed cells were selected by growth on media containing kanamycin and Tetracycline.
  • transformed cells were inoculated into Modified Terrific Broth with 1% NaCl and 1% glucose, tetracycline and kanamycin and allowed to grow at 37°C with shaking until an OD600nm of 1.0 was achieved, after which 0.5 mM IPTG was added, and the temperature dropped to 25°C.
  • the cultures were grown for a further 48 h after which they were harvested.
  • Cells were harvested by centrifugation, resuspended in 50 mM Tris, 10 mM EDTA, 0.08% SDS, pH 11 and passed through a microfluidizer twice.
  • Insoluble material containing biobeads was sedimented at 15,000 g for 20 min and washed once with 50 mM Tris, 150 mM NaCl, 1 mM EDTA.
  • the isolated bead material was subjected to SDS-PAGE on NuPage bis-tris 4-18 % gels with MOPS buffer and stained with Coomassie Blue. A band of the correct molecular weight for the T2.1-linker-phaC construct was observed. Tryptic digest and mass spectroscopy analysis of the peptides identified the fusion protein as T2.1-phaC.
  • T1.1 [SEQ ID NO: 66] multivalent construct conjugated to a phaC polymer particle-forming protein in ClearColiTM BL21 (DE3) using a kanamycin resistant T1.1 phaC plasmid and tetracycline resistant phaA / phaB plasmid, and the resulting production of T1.1 biobeads – another representative example of the polypeptide conjugates contemplated herein.
  • T2.1-phaC the T1.1 antigen was cloned such that it was translationally fused to the phaC via a SGGGGSGGGGS linker [SEQ ID NO: 69].
  • the T1.1 sequence was codon optimized and translationally fused to phaC.
  • Media component and concentrations were as follows: pea hydrolysates, 12g/L; yeast extract, 24 g/L; NaCl, 10 g/L; K2HPO4, 8 g/L; glycerol, 30 g/L.
  • Additions added when media had cooled were as follows: pluronic antifoam, 0.02 ml/L; tetracycline, 0.01 g/L; kanamycin, 0.05 g/L.
  • the fermenter was inoculated from pre-culture flasks grown at 37oC. Fermenter temperature was controlled at 25oC. Dissolved oxygen was controlled at 30% air saturation. The fermenter was maintained at pH 6.8.
  • Biobeads were sedimented by centrifugation and washed several times with the same buffer by resuspension and sedimentation. Biobeads were further purified by resuspension in 0.1N NaOH containing 0.01% Brij58 and subsequent sedimentation and resuspension in buffer containing 25 mM Tris, 10mM EDTA, pH 10, and concentrated by tangential flow filtration using a 0.1 micron porosity membrane (Cytiva, CFP-1-E). Purified biobeads were placed into a monodispersion by passing the suspension through a microfluidizer (Microfluidics, M110P) at 25,000 psi.
  • a microfluidizer Microfluidics, M110P
  • Vaccine containing multivalent vaccine conjugates as biobeads was formulated to contain different doses of antigen. Sterile adjuvant was added to biobead suspension Vaccine was aseptically filled into pharmaceutical vials (Type 1 borosilicate glass), with butyl rubber stoppers and aluminium crimp seals. Vials of vaccine containing multivalent vaccine conjugates as biobeads were stored at 2 – 8 °C.
  • Example 5 Production of vaccine containing multivalent polypeptide T his example describes the production of a vaccine comprising soluble T2.1 HisTag multivalent polypeptide construct as described herein. E .coli BL21-AI-CVC4 was grown in 10L or 100L fermenters. Media was prepared and autoclaved in the fermenter.
  • Media component and concentrations were as follows: pea hydrolysates, 12g/L; yeast extract, 24 g/L; NaCl, 5 g/L; K2HPO4, 6 g/L; glycerol, 10 g/L; MgSO4.7H2O, 2.5 g/L.
  • Additions added when media had cooled were as follows: pluronic antifoam, 0.2ml / 10L; Carbenicillin, 1g / 10L.
  • the fermenter was inoculated from pre-culture flasks grown at 37oC, for 3 hours. Temperature was controlled at 37oC for initial growth phase, and then cooled down to 21oC for induction. Dissolved oxygen was controlled to 85% air saturation and then reduced to 30% for induction.
  • the fermenter was maintained at pH 6.8.
  • a media bolus was prepared and autoclaved for 15 minutes at 121oC.
  • Bolus composition was glycerol, 25% (w/v); yeast extract, 12% (w/v).
  • the biomass was harvested by centrifugation approximately 20 hours post induction. Biomass was frozen at -20oC until further processing. Biomass was thawed overnight and resuspended in lysis buffer (50mM Tris pH 7.5), using a Miccra D-9 rotor-stator.
  • Lysis was performed using a Microfluidics, M110P microfluidiser M110P. Process pressure was 25,000 PSI, and 3 complete passes were performed. Insoluble material was collected by centrifugation at 12,000g for 30 minutes. The pellet (i.e., “insoluble” fraction) was collected for further processing. The lysate pellet (i.e., ‘insoluble’ fraction) was subjected to solubilisation in 1% SDS with sonication on ice. The solubilised fraction containing T2.1 was centrifuged to remove debris. The solubilised fraction was diluted into IMAC loading conditions (50mM Tris pH 8.4, 500mM NaCl, 0.2% SDS).
  • the diluted material was loaded onto a 20mL His-Prep column, with a 5mL His-Trap column attached (i.e., 25mL total IMAC column volume).
  • the IMAC columns were washed with loading buffer (50mM Tris pH 8.4, 500mM NaCl, 0.2% SDS), and T2.1 recovered with elution buffer (50mM Tris pH 8.4, 500mM NaCl, 400mM Imidazole, 0.2% SDS).
  • Elution fractions were analysed by SDS-PAGE for presence of T2.1, and fractions found to contain T2.1 protein were pooled. Pooled elution fractions were concentrated and buffer exchanged to remove imidazole in a 5,000 molecular weight cut off (MWCO) centrifugal concentrator.
  • MWCO molecular weight cut off
  • T2.1 fraction was subjected to endotoxin removal using Sartobind-Q membrane filter capsules.
  • the T2.1 fraction was passed through a 0.45 ⁇ m membrane disk filter followed by the Sartobind-Q capsule and then a 0.2 ⁇ m membrane disk filter.
  • Vaccine containing T2.1 was formulated to contain approximately 0.11% SDS. Adjuvant was added.
  • Vaccine was aseptically filled into pharmaceutical vials (Type 1 borosilicate glass), with butyl rubber stoppers and aluminium crimp seals. Vials of T2.1 vaccine were stored at 2 – 8 °C.
  • Example 6 In vivo immunogenesis This example describes an evaluation of the immunogenic effect of the T2.1 multivalent construct in humanised mice, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Cytokine-supplemented humanised mice were injected with T2.1 multivalent construct vaccine at 10 ⁇ g and 50 ⁇ g dose, with adjuvant at Day 0 and Day 21 before being terminated for downstream pentamer staining analysis to evaluate COVID-19 specific CD8+ T cell response at Day 28.
  • mice All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study.
  • Humanized mice were generated by injecting sub-lethally irradiated immune-deficient pups with CD34+ cordblood cells via intrahepatic route. Mice were submandibularly bled at 12 weeks post-engraftment to determine the levels of human immune reconstitution via flow cytometry. Mice with more than 10% human immune cell reconstitution (calculated based on the proportion of human CD45 relative to the sum of human and mouse CD45) in the peripheral blood were used in this study.
  • inoculant preparation and Dosing regimen Except for LPS, inoculants for Groups 1 to 3 were prepared as above. Mice from Group 1-3 were given the first inoculation on Day 0, followed by a booster on Day 21 post injection. At Day 21, mice from Group 1 and 2 were injected with T2.1 10 ⁇ g, adjuvant. Details of dosing regimen for each inoculant used in the study was listed in Table 3. Mice from Group 1-3 were injected with 150 ⁇ l per inoculant via intraperitoneal route. Table 3: Doses used for each inoculant during study. Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint.
  • splenocytes were first stained with live/dead and human Fc receptor block (BD; 564220) and incubated at room temperature for 10 mins. After which, splenocytes from each mouse were stained with three individual pentamers-containing panels (P1, P2 or P3, 10 ⁇ l per peptide-loaded pentamer) and incubated for 15 mins at room temperature. Cells were washed with FACS buffer containing PBS, 0.2% bovine serum albumin (GE Healthcare) and 0.05% sodium azide (Merck) and stained with antibodies in the core panel for 30 mins at 4 °C.
  • FACS buffer containing PBS, 0.2% bovine serum albumin (GE Healthcare) and 0.05% sodium azide (Merck)
  • Streptavidin BV421 (2 ⁇ l) was added to cells stained with panel containing biotinylated pentamer (P1). After washing, cells were fixed in 4% paraformaldehyde (Mouse FoxP3 buffer set; BD 5604094) for 15 min at 4 °C. Cells were then washed and data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analyzed using FlowJo software (version 10; Tree Star Inc). All graphs were plotted using GraphPad Prism 8.0 software (GraphPad Software Inc).
  • Example 7 in vivo immunogenesis using biobead delivery This example describes an evaluation of the immunogenic effect of the T1.1 and T2.1 multivalent construct on biobeads in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response.
  • Mice were injected with multivalent biobead vaccines at 4 ⁇ g and 12 ⁇ g doses, without adjuvant at Day 0, 1, 21 and 22 before being terminated for downstream pentamer staining analysis to evaluate COVID-19 specific CD8+ T cell response at Day 29.
  • the effective initial and booster doses were 8 ⁇ g and 24 ⁇ g Methods
  • the materials and reagents used in this example were as follows: Table 5: Materials and reagents T he animal experiment was conducted according to the Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485]. Up to five mice of the same gender were housed in a cage.
  • mice All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study.
  • Inoculant preparation and Dosing regimen Groups 1 to 6 were prepared as above. Details of dosing regimen for each inoculant used in the study was listed in Table 3. Mice from Group 1-6 were injected with 200 ⁇ l per inoculant via intraperitoneal route as in table 6. Table 6: Doses used for each inoculant during study. Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint.
  • Table 7 FACS panel for immune profiling at endpoint. B riefly, splenocytes were first stained with live/dead (Life Technologies) and mouse Fc receptor block (BD Bioscience) and incubated at room temperature for 10 mins. After which, splenocytes from each mouse were stained with two individual pentamers-containing panels (Panel 1 or Panel 2, 5 ⁇ l per peptide-loaded pentamer). Pentamer panels are listed in Table 7, specifically No. 11-15 inclusive and incubated for 15 mins at room temperature.
  • splenocytes from Group 1-6 were stained with core antibodies and five HLA-A*02:01 peptide loaded pentamers stated in Table 6 in two individual panels. Briefly, the gating strategy was as follows. After removal of doublets and dead cells, the levels of mouse CD45+ leucocytes were determined using the mouse CD45 gating.
  • B cells CD45+/CD19+
  • CD4+ T cells CD45+/CD3+/CD4+
  • CD8+ T cells CD45+/CD3+/CD8+
  • na ⁇ ve T cells CD45+/CD3+/CD4+ or CD8+/CD62L+/CD44-
  • central memory CM
  • EM effector memory
  • CD69 a T cell activation marker
  • CD8+ T cells levels of CD19/CD3 double negative immune subset (“others”), consisting of natural killer cells (NK), dendritic cells (DCs), myeloid (neutrophils and monocytes), was also included in the analysis.
  • NK natural killer cells
  • DCs dendritic cells
  • myeloid myeloid (neutrophils and monocytes)
  • Pentamer immune profiling analysis of splenocytes at endpoint To detect for the presence of antigen-specific CD8+T cells, splenocytes from Group 1-6 were stained with core antibodies and pentamers stated in Table 6 in three individual panels.
  • splenocytes were re- stimulated with the peptide pool ex vivo and their recall responses detected by direct and indirect mouse IFN- ⁇ ELISPOT analysis.
  • re-stimulations of pooled cells with concanavalin A (T cell activator) or EBV peptide (an irrelevant peptide) were also included as positive and negative controls, respectively.
  • concanavalin A T cell activator
  • EBV peptide an irrelevant peptide
  • T2.1 biobeads also induced positive CD8+ T cell responses in G2 and G4 mice in which approximately 60 IFN ⁇ SFUs per million cells were also detected. O verall, significantly higher numbers of IFN ⁇ SFUs were detected in the HLA-peptide pool re-stimulated splenocytes/CD8+ T cells from G1-G4, when compared to that in the re- stimulated cells from the control groups, G5 and 6 ( Figure 5). This thus confirms the induction of HLA-A1 peptide-specific CD8+ T cell responses in the mice that had been immunised with T1.1 or T2.1 biobeads. Comparatively, T1.1 biobeads induced greater peptide-specific response than T2.1 biobeads.
  • Example 8 Highly redundant multivalent constructs This example describes the selection of SARS-CoV-2 epitopes for use in multivalent vaccine constructs as contemplated herein to provide redundancy in HLA coverage and to mitigate against vaccine escape. Global population coverage and HLA redundancy was calculated (using the IEDB population coverage online service (http://tools.iedb.org/population/) for the following multivalent constructs comprising various subsets of the epitopes selected in Example 1 above.
  • Table 9 Various exemplary multivalent constructs are presented below in Tables 9 to 11.
  • Table 9 Global coverage achieved with short multivalent constructs A s can be seen in Table 10, a substantial increase in global coverage is achieved via the inclusion of selected additional SARS-CoV2 epitopes in accordance with this disclosure. Notably, a predicted global coverage of almost 95% is achieved using the seven epitopes identified in Table 9 above.
  • the addition of the 29 further epitopes set out in Table 1 has a meaningful impact on global coverage, raising predicted coverage to ⁇ 99.75%.
  • the epitopes and constructs contemplated herein provide substantial redundancy in HLA coverage. As shown in Table 6 below, the exclusion of one or a subset of epitopes from the construct has a limited impact on both HLA coverage and predicted global coverage. Table 10. Redundancy in HLA coverage
  • the epitopes and constructs contemplated herein provide substantial r edundancy in global coverage.
  • Table 11 the exclusion of an epitope capable of binding to or predicted to bind to a given HLA class 1 variant is predicted to have a limited impact on global coverage.
  • Table 11 Redundancy in global coverage N otably, as can be recognised for example from the data presented in Table 11, the vaccine constructs contemplated herein are highly redundant. The removal of a single epitope from the construct has minimal impact on predicted global population coverage achieved by the resulting variant construct.
  • Example 9 Design of Optimised Peptides with Bespoke Linkers Ten peptides (SEQ ID NO: 78 to 87 were derived using algorithms whereby the individual peptides are re-ordered and bespoke linkers are computed. Scores are compared above with the starting polypeptide, T1.2 (SEQ ID NO: 44).
  • Example 10 in vivo immunogenesis using biobead delivery This example describes an evaluation of the immunogenic effect of the T1.1 and T1.2 multivalent construct on biobeads in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response.
  • mice were injected with multivalent biobead vaccines at 0.5 ⁇ g, 2 ⁇ g and 8 ⁇ g doses, with adjuvant at Day 0 and 21 before being terminated for downstream analysis to evaluate COVID- 19 specific CD8+ T cell response at Day 28.
  • Methods The materials and reagents used in this example were as follows: Table 12 Materials and reagents
  • mice All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study.
  • Inoculant preparation and Dosing regimen G roups 1 to 8 were prepared as above. Details of dosing regimen for each inoculant used in the study are listed in Table 13. Table 13: Doses used for each inoculant during study. Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for 45 mins before processed to serum and stored at -80oC.
  • ELISPOTs Mouse IFN- ⁇ ELISPOT was performed using the kits from Mabtech.2 x 105 of splenocytes from each mouse were plated in the pre-coated ELISPOT plate and re-stimulated with the peptide pool [5 ⁇ M per peptide, specifically no. 1 – 8 inclusive (Table 14)] for approximately 32 hours. Re-stimulations of pooled splenocytes with either 5 ⁇ g/ml of Concanavalin A (T cell activator; Sigma), 5 ⁇ M of EBV peptide (GLCTLVAML), or 5 ⁇ M of HPV peptide (VQSTHVDIRTLEDLLMGTLGIVCPI) were also included as positive and negative controls, respectively.
  • Concanavalin A T cell activator; Sigma
  • GLCTLVAML 5 ⁇ M of EBV peptide
  • HPV peptide VQSTHVDIRTLEDLLMGTLGIVCPI
  • the HPV peptide- immunized group did not respond to the re-stimulation with the peptide pool but had detectable levels of IFN- ⁇ SFUs upon re-stimulation with the HPV peptide.
  • the results indicate the presence of positive HLA-A2:01 peptide-specific CD8+ T cell responses in mice that were immunized with the biobeads.
  • Splenocytes from G1 mice i.e.
  • Example 11 Production of vaccine constructs using bespoke cleavable linkers
  • the design of optimal linkers or spacers for a multi-epitope T cell vaccine has been described by Schubert and Kohlbacher, Genome Medicine (2016) 8:9. Using their described algorithm, it is possible to design linkers that will cleave optimally.
  • Optimal cleavage has the advantage of maximising the amount of peptide availability for MHC class I display, minimising partial cleavage and preventing neo-epitope formation.
  • Cleavage Score The average cleavage score at the C-terminal ends of epitopes and spacer. Range [-23, 3.04]; more positive is better.
  • Spacer Cleavage Score The average predicted cleavage score of C-terminal cleavage sites within a spacer sequence.
  • Fragments 1 to 6 SEQ ID NO 89- 94
  • Fragment 4 includes the PADRE sequence.
  • Bespoke linkers were designed as above for each of the six fragments as above with the following characteristics: G enes coding for Fragments 1 - 6 (SEQ ID NO: 88 to 93) were cloned into the expression vector pD451, containing T7 inducible promoter.
  • Purified plasmid containing the genes to be tested was transformed into chemically competent BL21(DE3) cells via heat shock and plated on non-inducing agar with 100 mg/L kanamycin. Plates were incubated overnight at 37°C. One colony from each transformation was picked and grown into 0.82 mL of a non-inducing medium containing 100 mg/L kanamycin, then incubated overnight at 37°C. Overnight culture was inoculated into 0.6 mL of auto-induction media containing 100 mg/L kanamycin. Three sets of auto-induction media were inoculated from the same starter culture and incubated at 30°C overnight (24 hours). Cells were harvested by centrifugation and frozen.
  • Example 12 Production of mRNA vaccine constructs DNA template preparation Fully synthesized constructs (5’UTR-ORF-3’UTR-polyA) were cloned into the pIVTScripTM template backbone plasmid for T1.2, T3, T4, T5, T6 and T7 (SEQ ID NO: 100-105). Sequence were verified by Sanger sequencing. The DNA template then linearized.
  • NTPs Nucleotide triphosphates
  • 10 ⁇ IVT buffer 10 ⁇ IVT buffer
  • linearized plasmid transcription t emplate were brought to room temperature. NTPs and 10 ⁇ IVT buffer were vortexed until homogenous. Repeat warming and mixing was performed until no precipitate was observed.
  • I n vitro transcription reaction The components in the table below were combined in RNase-free tubes. For a 1-ml reaction, the q uantities listed in the table were used. E ach reaction was mixed using gentle pipetting until homogenous.
  • Tubes were then sealed with parafilm and incubated at 37°C in a water bath or heat block for 2-3 hr.
  • T he reaction was incubated at 37°C for a minimum of 15 minutes.
  • P urification L iCl was added to the IVT reactions to achieve a final concentration of 2.5 M.
  • the preparations were then mixed by gentle inversion or swirling before chilling at ⁇ 20 °C for at least 30 min. Preparations were then centrifuged for 30 min at 18,500 ⁇ g at 4°C. Pellets from the centrifugation were washed in ⁇ 20 °C ethanol.
  • LNP encapsulation LNPs were prepared by mixing appropriate volumes of lipid stock solutions in ethanol with an aqueous solution of mRNA employing a microfluidic micromixer. Preparation mRNA was dissolved in 25 mM sodium acetate buffer, pH 4.0. The following molar ratios of lipids were dissolved in ethanol: LNP encapsulation of mRNA 1 volume of the lipid preparation was mixed with 3 volumes of mRNA solution in the mixer using a syringe pump.
  • a flow rates at 0.5 mL/min for the lipid/ethanol stream and 1.5 mL/min for the mRNA/aqueous stream was used to drive a combined flow rate of 2 mL/min through a microfluidics micromixer.
  • the output was dialyzed for 4 h against 1000 volumes of 50 mM MES/50 mM sodium citrate buffer (pH 6.7). Subsequently the preparation was dialyzed overnight against 1000 volumes of 1 ⁇ phosphate buffered saline, pH 7.4. Quality was confirmed by measuring particle size analysis by dynamic light scattering, polydispersity index by dynamic light scattering, mRNA concentration using RiboGreen and presence of prokaryotic contamination.
  • Example 13 Redundancy Calculation This example describes a metric to estimate the degree of redundancy, in terms of HLA coverage, that a multipeptide vaccine could have when subjected to multiple point mutations.
  • This HLA redundancy score is then calculated for five SARS-CoV-2 published vaccine compositions is addition to design described in this specification. I t is assumed that these simulated mutations render the resulting peptides useless. Therefore, the mutated peptides are removed from their corresponding list.
  • the global HLA coverage of such a modified vaccine is subsequently determined using the IEDB coverage prediction tool (http://tools.iedb.org/population/). T his simulation is repeated 10 times for each vaccine composition and the resulting global coverage is reported (with statistics such as median, mean and standard deviation).
  • the vaccine design described in the specification is designated DA in this example.
  • Simulation of peptide mutation. Method and parameters The objective of the implemented method is to generate peptides with one or more mutations using a procedure that is consistent with some basic biological aspects of mutagenesis. Since mutations occur at the nucleotide level and the number of codons encoding each amino acid are not equal.
  • the analysis was started by encoding each peptide into its nucleotide sequence.
  • the host (human) codon usage bias data for SARS-CoV-2 was extracted from the codon usage bias database (http://www.kazusa.or.jp/codon/) and the most frequent codon for each amino acid was used to encode its corresponding nucleotide.
  • the probability of mutation for each nucleotide estimated per cycle was selected according to the study by Amicone et al., 2021 and was set to be equal to 1.25 * 10-6 nt-1 cycle-1. Each nucleotide was then randomly mutated according to the biased mutation frequencies reported by Pathan et al., 2020.
  • Transition matrix defining the probability of nucleotide mutation.
  • Table 15 Transition matrix defining the mutation probabilities computed according to the parameters defined in Borges et al. 2021 and Pathan et al.2020 M utation events were computed independently for each nucleotide for a given number of cycles (e.g. 25,000 cycles) defined by the user. At each cycle, individual mutation calls were made separately for each nucleotide and were then combined together in a full nucleotide sequence. At the end of each simulation every peptide has a list of associated nucleotide sequences whose size is equal to the selected number of cycles. Those sequences are then encoded back to their amino acid representation using the appropriate codon map.
  • the large majority of the resulting peptides are identical to the initial peptide, in some cases they’re different. If at least one mutant peptide is present in a given peptide pool, the original peptide is removed. It needs to be noticed that a higher number of cycles increases the chances of generating mutated peptides and thus the chances for any original peptide to be removed from its list is also increased.
  • the procedure described above is performed for each peptide in a list (e.g. D1) and results in a pool of “surviving peptides” which are referred to as a sample. The number of s amples generated for each list is also a parameter determined by the user.
  • f ollows where is the coefficien t of variation, a statistical measure of the relative dispersion of data points in a data series around the mean as defined by the following equation: T he weight values and are parameters allowing to specify the relative importance of the t wo components o f the scoring function: the average and the coefficient of variation for HLA c overage.
  • the weights are defined between 0 and 1 and they sum up to 1: T he mean of the HLA coverage ⁇ is a percentage value and thus it is possible to express it as a number between 0 and 1.
  • Example 14 in vivo immunogenesis using biobead and mRNA delivery
  • This example describes an evaluation of the immunogenic effect of the T1.2 multivalent construct on biobeads and various mRNA multivalent constructs in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response.
  • M ice were immunized with 10 ⁇ g of each mRNA vaccine constructs at day 0 and day 21 via the intramuscular route.
  • Three groups of mice received varying doses of T1.2 biobeads (8 ⁇ g, 2 ⁇ g or 0.5 ⁇ g) adjuvanted with alum and ODN1826 at day 0 and day 21 via the intraperitoneal route.
  • mice were immunized with 50 ⁇ g of human papillomavirus (HPV) peptide, adjuvanted with alum and ODN1826, at day 0 and day 21. Following the same dosing regimen, a group of control mice received adjuvanted naked biobeads. At Day 28 or 7 days after the last immunization, the mice were sacrificed for downstream mouse IFN- ⁇ intracellular flow cytometry (ICFC) and immune profiling analysis to evaluate peptide-specific CD8+ T cell responses.
  • ICFC intracellular flow cytometry
  • mice T he animal experiment was conducted according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guideline and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485] on 23 November 2021. Up to five mice were housed in a cage. Each mouse was identified using ear punching identification system and grouped as listed in Table 02. All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12- hour light-dark cycle with ad libitum access to normal diet and drinking water.
  • ABSL2 Animal Biosafety Level 2
  • A*STAR Agency for Science, Technology and Research, Singapore
  • Table 18 Study groups IM – intramuscular injection (50 ⁇ L per hindlimb, 100 ⁇ L per mouse), IP – intraperitoneal injection (200 ⁇ L per mouse) Table19 Immunization regimen. Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for at least 45 minutes before being processed to serum and stored in -20°C. Isolation of splenocytes At endpoint, spleen was collected and meshed through 70 ⁇ m strainer with 5ml of RPMI media. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco).
  • splenocytes were counted using trypan blue staining protocol (Gibco). Immune profiling analysis using FACS Immune profiling was performed on approximately 3 ⁇ 106 mouse splenocytes using core antibodies listed in Table 20. Briefly, splenocytes were first stained with live/dead dye (Life Technologies) for 10 mins at room temperature. After which, cells were incubated with mouse Fc receptor block (BD Bioscience) at 1:100 dilution in 50 ⁇ l staining volume and incubated at room temperature for 10 minutes before staining with core antibodies (final dilution of 1:200) for 30 mins at 4°C.
  • live/dead dye Life Technologies
  • mouse Fc receptor block BD Bioscience
  • mouse Fc receptor block (BD Bioscience) was added to the cells at 1:100 dilution in 50 ⁇ l staining volume and the mixture incubated at room temperature for 10 minutes.
  • Core surface antibodies namely, mCD45, CD3, CD19, CD4 and CD8 (as listed in Table 20) were added, and the suspension incubated at 4°C for approximately 1 hour.
  • FACS buffer After washing with FACS buffer, the cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD). Subsequently, intracellular staining for IFN- ⁇ were performed with the BV421 anti-mouse IFN- ⁇ antibody (Biolegend) for 30 minutes at room temperature.
  • Positive responders for each type of restimulation are defined as those samples with values that were at least 3 SDs higher than the mean frequencies of peptide-specific IFN ⁇ + CD8+ T cells for splenocytes from G11 (negative control).
  • immunization with the T1.2 biobeads resulted in a more consistent peptide-specific IFN ⁇ + CD8+ T cell responses when compared to the immunization with mRNA constructs.
  • T1.2, T3, T4 and T5 all gave a significant response in 20% or more of the mice tested.
  • the T cell vaccine can be delivered via mRNA.
  • each of the T1.2, T3, T4 and T5 constructs all have different orders of epitopes and possess different linkers. Responses to the designs in this specification are therefore not restricted to any particular order or any one linker.
  • Example 15 in vivo immunogenesis using biobead with three human MHC Class I alleles
  • This example describes an evaluation of the immunogenic effect of the T1.2 multivalent biobead construct in transgenic mice possessing the human MHC class I A*02:01, A*11:01 and B*07:01 alleles, and particularly to investigate the induction of a strong human CD8+ T cell- mediated cytotoxic immune response in these different alleles.
  • M ice received 2 ⁇ g T1.2 biobeads adjuvanted with alum and ODN1826 at day 0 and day 21 via the intraperitoneal route. Following the same dosing regimen, a group of control mice received adjuvanted naked biobeads.
  • mice were sacrificed for downstream mouse IFN- ⁇ intracellular flow cytometry (ICFC) to evaluate peptide-specific CD8+ T cell responses.
  • ICFC intracellular flow cytometry
  • mice T he animal experiment was conducted according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guideline and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485] on 23 November 2021. Up to five mice were housed in a cage. Each mouse was identified using ear punching identification system and grouped as listed in Table 02. All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12- hour light-dark cycle with ad libitum access to normal diet and drinking water.
  • ABSL2 Animal Biosafety Level 2
  • A*STAR Agency for Science, Technology and Research, Singapore
  • Table 23 Study groups IP – intraperitoneal injection (200 ⁇ L per mouse) Isolation of splenocytes A t endpoint, spleen was collected and meshed through 70 ⁇ m strainer with 5ml of RPMI media. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco). The number of splenocytes was counted using trypan blue staining protocol (Gibco). Immune profiling analysis using FACS Immune profiling was performed on approximately 3 ⁇ 106 mouse splenocytes using core antibodies listed in Table 24 Briefly, splenocytes were first stained with live/dead dye (Life Technologies) for 10 mins at room temperature.
  • ICFC Intracellular Flow Cytometry
  • ICFC Intracellular Flow Cytometry
  • ICFC Intracellular Flow Cytometry
  • IFN- ⁇ intracellular staining for IFN- ⁇ were performed with the BV421 anti-mouse IFN- ⁇ antibody (Biolegend) for 30 minutes at room temperature. Subsequently, cells were washed with FACS buffer before acquiring the data using a LSR II flow cytometer (BD) with FACSDiva software. The FACS data were analyzed with FlowJo software (version 10; Tree Star Inc). The frequencies of peptide-specific IFN ⁇ + CD8+ T cells were determined by subtracting the basal frequencies of IFN ⁇ + CD8+ T cells in the unstimulated samples from that of the peptide-restimulated samples.
  • positive responses for each type of restimulation within each group are defined as those samples with values that were at least 3 SDs (standard deviations) higher than the mean frequency of peptide-specific IFN ⁇ + CD8+ T cells for splenocytes from G11 (negative control).
  • Table 25 Peptides for ICFC Analysis All graphs were plotted using the GraphPad Prism 9.3.1 software (GraphPad Software Inc). Statistical significance was assigned when the p values are below 0.05.
  • RESULTS IFN ⁇ intracellular cytokine flow cytometry (ICFC) To evaluate the levels of peptide-specific CD8+ T cells response, splenocytes from the immunized mice were restimulated with various peptide combinations as listed in Table 25.
  • HLA-A*02:01 Transgenic Mice To account for any background cytokine secretion, the levels of peptide-specific responses in the immunized mice were determined by subtracting the basal frequencies of IFN ⁇ + CD8+ T cells in the unstimulated samples from that of the peptide-restimulated samples. Significantly higher frequencies of peptide-specific IFN ⁇ + CD8+ T cells were detected in the HLA-A*02:01 peptide pool restimulated splenocytes from mice when compared to that in the restimulated cells from G2 negative control group (median value of 0%, as shown in Figure 14A).
  • HLA-A*11:01 Transgenic Mice Recall CD8+ T cell responses were determined by restimulating the splenocytes from the HLA-A*11:01 transgenic mice (G3-G4) with peptide 9 (ASAFFGMSRI) or peptide 10 (SSTFNVPMEKL). Immunization with T1.2 biobeads generated positive peptide-specific CD8+ T-cell responses greater than the negative control group for both peptides ( Figures 14B and 14C). HLA-B*07:02 Transgenic Mice For the HLA-B*07:02 mice (G5-G6), immunization with T1.2 biobeads generated positive peptide 11-specific CD8+ T cell responses higher than the control group ( Figure 14D).
  • Example 16 mRNA vaccine containing two multiepitope mRNA sequences - multiepitope mRNA sequence preparation This example describes the preparation of two multiepitope antigen mRNA sequences as described herein.
  • F3-8His and F6-8His are equivalent to the polypeptide constructs discussed herein and presented as SEQ ID NO: 91 and SEQ ID No: 94, respectively, both HIS tagged on the C-terminus.
  • F3-8His is a polypeptide antigen containing seven MHC class I epitopes with linkers between them and bearing eight histidine residues at the polypeptide C-terminus.
  • F6-8His is a polypeptide antigen containing six MHC class I epitopes with linkers between them and bearing eight histidine residues at the polypeptide C-terminus.
  • RNA sequences for use in a vaccine were prepared according to the following design.
  • the five prime (5’) end of the sequences included a 31-nucleotide length 5’ untranslated region (UTR) of human haemoglobin subunit alpha 1 and the Kozak consensus sequence (CCCGCCACC).
  • the open reading frame (ORF) contained the sequences for either F3-8His or F6-8His.
  • the three prime (3’) UTR included a 295-nucleotide 3’ UTR of amino- terminal enhancer of split (AES) together with non-coding RNA (ncRNA) from mitochondrially encoded 12S ribosomal RNA (rRNA).
  • a poly-adenosine tail up to 113- nucleotides in length was included at the three-prime end of the construct.
  • Template DNA for use in in vitro transcription was prepared as follows. The DNA sequences for the mRNA sequence for vaccine component containing multiepitope F3-8His and F6- 8His are presented as SEQ ID NO: 125 and SEQ ID No: 126, respectively. Copy DNA (cDNA) for the desired RNA sequences was prepared and inserted into an E. coli expression vector plasmid pJ214 (ATUM). The cDNA included a restriction endonuclease SapI binding site for linearization during subsequent processing. To amplify the DNA, plasmids were transformed into E.
  • coli cell NEB® Stable and single colonies were picked from the transformation plates for inoculation of a 24-hour starter cultures.
  • the starter cultures were used to inoculate 2.5 L of LB media containing the appropriate antibiotics in 5 L Optimum GrowthTM shake flasks (Thomson).
  • the overnight cultures in LB media were incubated at 30 °C at 200 rpm shaker speed.
  • Amplification cultures were harvested by centrifugation at 6,000 ⁇ g for 30 min at 4 ⁇ C. Cell pellets were stored at -80 ⁇ C until purification.
  • Cell pellets from 2.5 L culture was resuspended with 125 mL of 25 mM Tris, 10 mM EDTA, 55 mM Dextrose, pH 8.0 and lysed by alkaline lysis with 125 mL of each of 0.24 M Sodium Hydroxide and 1% SDS for 5 minutes while gently mixing.
  • the lysates were centrifuged at 12,000 g for 20 minutes to isolate the soluble fraction containing the plasmid.
  • the soluble fractions were diluted with Milli-Q water to adjust the solutions conductivities to 35 mS/cm.
  • the diluted samples were passed through a 0.45 ⁇ m filter and purified using CIMmultus DEAE (Sartorius) anion exchange (AEX) chromatography columns.
  • the AEX eluates were collected and loaded onto HiPrep 26/10 Desalting (Cytiva) gel filtration desalting columns. The highest purity fractions corresponding to the target plasmid were pooled.
  • Purified supercoiled plasmids were linearised using BspQI at 2:1 units/ ⁇ g overnight at 37°C, 300 rpm. Linearised plasmids were mixed with 4 M ammonium sulphate (3:1 v/v) and purified using hydrophobic interaction chromatography (HIC) on CIMmultus C4 HLD columns (Sartorius). The highest purity fractions corresponding to the linearised plasmid DNA were pooled.
  • HIC hydrophobic interaction chromatography
  • NTP nucleoside triphosphates
  • ATP adenosine triphosphate
  • CTP cytosine triphosphate
  • GTP guanine triphosphate
  • ⁇ TP N1-methyl-pseudouridine triphosphate
  • RNA constructs coding for multiepitope antigens F3-8His and F6-8His were prepared as a vaccine for use in vivo as follows. Messenger RNA solutions for both F3-8His and F6-8His were diluted with buffer and mixed with the transfection agent in vivo-jetRNA® (Polyplus S.A.) at a ratio of 1 ⁇ g mRNA to 1 ⁇ L transfection agent solution, according to the manufacturer’s instructions.
  • Example 17 in vivo immunogenesis using fragments administer as mRNA This example describes an evaluation of the immunogenic effect of an mRNA construct of two fragments (F3, SEQ ID NO: 90 and F6, SEQ ID NO: 93) in transgenic mice possessing the human MHC class I A*02:01allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response.
  • mice received 2 ⁇ g or 0.5 ⁇ g of mRNA delivered using in vivo-jetRNA® at day 0 and day 21 via the intramuscular route. Following the same dosing regimen, a group of control mice received 2 ⁇ g of a green fluorescent protein mRNA construct also delivered using in vivo- jetRNA®. At Day 28 or 7 days after the last immunization, the mice were sacrificed for downstream mouse IFN- ⁇ intracellular flow cytometry (ICFC) to evaluate peptide-specific CD8+ T cell responses. Protocols followed were essentially the same as that in example 15 and IFN ⁇ intracellular cytokine flow cytometry was performed as described previously with harvested splenocytes stimulated with the peptide YLFDESGEFKL (SEQ ID NO: 8).
  • ICFC mouse IFN- ⁇ intracellular flow cytometry
  • splenocytes from each mouse were plated into each well of 48 well plates and restimulated with the respective combinations of peptides (refer to next slide for more details) After approximately one hour of incubation at 37°C, ER-Golgi stop, Brefeldin A, was added to the cells and the cells were further incubated overnight Internal negative and positive controls were included by restimulating the cells with media only or PMA/ionomycin.
  • the cells were first transferred to 96 well v bottomed plates before staining with live/dead dye 1:400 dilution in PBS) at room temperature for 10 minutes After which, mouse Fc receptor blocking reagent (diluted in FACS buffer) were added and the mixture incubated at room temperature for 10 minutes to block non-specific binding Core surface antibodies, namely mCD45, CD3, CD19, CD4 and CD8 were then added, and the suspension incubated at 44°C for approximately one hour.
  • mouse Fc receptor blocking reagent diluted in FACS buffer
  • Study group were as follows: G1: F3&F6 mRNA, in vivo JetRNA, 2 ⁇ g G2: F4&F8 mRNA, in vivo JetRNA, 0.5 ⁇ g G3: GFP mRNA, in vivo JetRNA, 2 ⁇ g Results Results are shown in Figure 15 which demonstrate a dose dependent specific CD8+ response to the peptide YLFDESGEFKL but not the control green fluorescent protein construct. This shows that fragment vaccine constructs are capable of inducing SARS-CoV- 2 specific cytotoxic T cell responses.

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Abstract

The present disclosure relates to a multivalent vaccine composition characterised in that it elicits broad spectrum protection against at least one strain of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants], B.1.1.7 and/or omicron), said vaccine comprising: a pool of T cell epitopes derived from at least one viral protein wherein the vaccine has a calculated world population HLA coverage of at least 95%, for example 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%. The disclosure also relates to constructs disclosed herein, methods or preparing same and use in treatment including prophylaxis.

Description

CORONAVIRUS VACCINE COMPOSITION The present disclosure relates to a vaccine composition comprising T cell epitopes designed to be presented on a diverse range of HLA molecules (such as MHC class I), oligopeptides, polypeptides and/or polynucleotide constructs encoding one or more of the same employed in the said compositions; use of any one of the same in the prophylaxis of coronavirus and in particular COVID-19, and manufacture of said composition. BACKGROUND In 2020 viruses, such as coronavirus in particular those causing COVID-19, became one of the top challenges to the way of life we have all become accustomed to. Many companies started working on vaccines to prevent the spread of coronavirus. Some of the first vaccines were RNA or viral vector based and targeted an immune response to the spike protein from the surface of coronavirus SARS-CoV-2 because use of this protein induces neutralising antibodies from B cells. Work by David Montefiori and Bette Korber demonstrates the spike protein is mutating and the mutation D614G is appearing. Mutation in the spike protein may assist the coronavirus evading immune responses including immune responses in vaccinated patients. Vaccines designed to generate neutralising antibodies to pathogen proteins act extracellularly and may have limited to ability to counteract viruses in the phase when they have hijacked and infected a cell. However, cytotoxic T cells (CD8+ T cells) are very important in the fight against viruses because activated CTCs (cytotoxic T cells) are able to: • attack cells infected by viruses, shut down viral synthesis by the cell and initiate apoptosis and disposal of said cell; • induce caspase killing of infected cells mediated by FasL; and • kill multiple cells (serial killing) whilst minimising the impact on healthy cells. It may also be that antibodies generated by exiting vaccines to coronavirus are waning and that sustained protection requires cellular responses. In addition, the current vaccines are not suitable for use in every individual because they can illicit rare but serious adverse events, such as myocarditis, embolisms, strokes and the like. Thus, alternative vaccines for example “employing peptides”, such as to multiple proteins, may be desirable; in particular, those optimised to generate T cell responses, more specifically those optimised to generate cytotoxic T cell responses. What is more, targeting multiple parts of the pathogen’s genome including several proteins simultaneously will minimise the impact of mutations in viral proteins. However, there is a barrier to the development of vaccines employing peptides because it is difficult to generate cytotoxic T cell responses to provide maximum population coverage. Major Histocompatibility Complex class 1 (MHC 1), is involved in generating cytotoxic T cells. MHC 1 has three subgroups: MHC-A, MHC-B and MHC-C. These proteins are present on nucleated cells and sample the environment and present peptide antigens (usually intracellular in the case of MHC class 1) to T cells. It is this system in humans that requires blood and organ donors to be matched to prevent rejection of implanted tissue. HLA genes (the human version of MHC) are extremely polymorphic resulting in a vast diversity of peptide-binding HLA specificities and a low population coverage for any given peptide-HLA specificity. Thus, for a given peptide only a percentage of the population will have HLA that can process it and thus present it to a T cell. The most common HLA allele is HLA-A*02:01 which is present in 39.08% of the global population with every other allele occurring more rarely and some very rarely in less than 1% of the global population. It would be useful to have alternative formats of coronavirus vaccines available, for example which are suitable for generating immune responses: • in a diverse population of HLA haplotypes and/or • covering multiple parts of the viral genome from a number of different viral proteins. The present inventors have designed a combination of T cell epitopes, in particular to a variety of viral proteins across the viral genome, that are able to bind HLA molecules in a high percentage of the patient population, for example at least 95% of the population, such as 99% of the population or more, in particular 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7 or 99.8%, in particular 99.7% of the population. This is achieved by having a pool of T cell epitopes, preferably to a number of antigens, with a high level of redundancy. Surprisingly, this coverage seems to be maintained in the presence of new variants. This vaccine may be employed alone or in combination with one or more other vaccines (in particular other coronavirus vaccines, such as a SARS-CoV2 vaccine(s)). SUMMARY OF THE DISCLOSURE 1. A multivalent vaccine composition characterised in that it elicits broad spectrum protection against at least one strain of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants] B.1.1.7 and/or B.1.1.529 [omicron]), said vaccine comprising: a pool of T cell epitopes derived from at least one viral protein wherein the vaccine has a calculated world population HLA coverage of at least 95%, for example 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, or 99.8%, in particular 99.7%. 1A. A multivalent vaccine composition comprising a pool of T cell epitopes derived from at least two viral proteins, such as 3, 4, 5 etc. wherein the composition elicits broad spectrum protection against multiple strains of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants] B.1.1.7 and/or B.1.1.529 [omicron]). 1B A multivalent vaccine composition comprising at least one construct of formula (I): A-X-B-X-J-X-(U)q-X-W-X-Z (I) wherein: A is sequence independently selected from SEQ ID NO: 1 to 37; X each occurrence is independently a linker, in particular a cleaveable linker, such as an amino acid linker in particular as disclosed herein; B comprises a sequence independently selected from SEQ ID NO: 1 to 37; J comprises a sequence independently selected from SEQ ID NO: 1 to 37; U comprises a sequence independently selected from SEQ ID NO: 1 to 37; W comprises a sequence independently selected from SEQ ID NO: 1 to 37; Z comprises a sequence independently selected from SEQ ID NO: 1 to 37; and q is 0 or an integer 1 to 40, such as 1 to 31, for example 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 or 30, in particular q is selected from 0, 1 or 2, especially 0, 1, 2 or 32. 2. A multivalent vaccine composition according to paragraphs 1, 1A or 1B, comprising 2-100 T cell epitopes, for example 2 -50, (such as 5 to 40 or 7 to 36 or 37) in particular 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 or 37, more specifically 5, 6 or 7, especially 6 or 7. 3. A multivalent vaccine composition according to paragraphs 1, 1A, 1B or 2, wherein each T cell epitope is in the range 7 to 15 amino acids in length (e.g. 8 or 9 to 11 amino acids or 8, 9, 10 ,11, 13 in particular a mixture of the latter), such as independently 7, 8, 9, 10, 11, 12, 13, 14, 15, In one embodiment the epitopes are different lengths within the range. In one embodiment the epitopes are the same length. 4. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 3, wherein the T cell epitope peptides are encoded in one or more viral vectors or as a transcribable polynucleotide, such as RNA or DNA, in particular RNA. 5. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 4, wherein the T cell epitopes are presented on HLA (such as MHC 1), for example on cells, such as antigen presenting cells, in particular cells autologous to the patient. 6. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 3, wherein the T cells epitopes are provided as isolated (individual i.e. unlinked sequences) peptides. 7. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 4, wherein one or more T cell, epitopes are linked, for example 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the epitopes are linked, in particular 5, 6 or 7 epitopes are linked. 8. A multivalent vaccine composition according to claim 7, wherein two or more of said T cell epitopes (such as all said T cell epitopes) are linked via a bond, such as an amide bond. 9. A multivalent vaccine composition according to paragraphs 7 or 8, where the epitopes (i.e two or more such as all) are linked via a linker or linkers, for example a peptide linker or linkers (including independently selected linkers) 1 to 30 amino acids in length, in particular a linker independently selected from a cleavable linker (such as a proteolytic cleavage site, for example comprising a proteasome dependent site, more specifically AAY, or a furin dependent site such as REKR [SEQ ID NO: 41]) and a linker of formula (II): B1-W1-Y1-Z1 formula (II) wherein B1 is independently selected from A, R, S and P; W1 is independently selected from D, L, I and T; Y1 is independently selected from L, G and A Z1 is independently selected from V, K and A. 10. A multivalent vaccine composition paragraphs to any one of claims 7 to 9, where the epitopes are linked via a linker or linkers, and at least one has a formula (IIIA): B2-W2-C0-1-K0-1-(Y2)0-1-Z2 (IIIA) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R. Z2 is independently selected from W and Y 0 means the entity is absent, and 1 means the entity is present. 11. A multivalent vaccine composition according to any one of paragraphs 7 to11, wherein the linked T cell epitopes are provided as an isolated polypeptide. 12. A multivalent vaccine composition according to any one of claims 7 to 11, wherein the linked epitopes are provided as at least one oligopeptide, for example provided as multiple oligopeptides. 13. A multivalent vaccine composition according to paragraph 11, wherein the composition comprises 2-10 oligopetides, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular 5, 6 or 7. 14. A multivalent vaccine composition according to paragraphs 12 or 13, wherein the oligopeptide(s) is/are selected from SEQ ID NO: 88, 89, 90, 91, 92 and 93 and combinations thereof, for example 2, 3, 4, 5 or 6, in particular comprises all said sequences. 15. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 14, wherein the one or more T cell epitopes have a low propensity to mutate in the wild-type viral protein (for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the epitopes have a low propensity to mutate). 16. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 15, wherein the composition comprises T cell epitopes to multiple viral proteins. 17. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 16, wherein the viral protein is independently selected from ORF1a polyprotein, ORF1ab polyprotein, ORF8 protein, membrane glycoprotein, nucleocapsid phosphoprotein, and spike surface glycoprotein. 18. A multivalent vaccine composition according to paragraph 16 or 17, wherein the viral protein is ORF1a polyprotein. 19. A multivalent vaccine composition according to paragraph 16 to 18, wherein the viral protein is ORF1ab polyprotein. 20. A multivalent vaccine composition according to any one of paragraphs 16 to 19, wherein the viral protein is ORF8 protein. 21. A multivalent vaccine composition according to any one of paragraphs 16 to 20, wherein the viral protein is membrane glycoprotein. 22. A multivalent vaccine composition according to any one of paragraphs 16 to 21, wherein the viral protein is nucleocapsid phosphoprotein. 23. A multivalent vaccine composition according to any one of paragraphs 16 to 22, wherein the viral protein is spike surface glycoprotein. 24. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 23, wherein the T cell epitope is disclosed herein and combinations of 2 or more thereof, such as comprising one or more sequences shown in SEQ ID NO: 1 to 37. 25. A multivalent vaccine composition according to paragraph 24, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 1. 26. A multivalent vaccine composition according to paragraph 24 or 25, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 2. 27. A multivalent vaccine composition according to any one of paragraphs 24 to 26, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 3. 28. A multivalent vaccine composition according to any one of paragraphs 24 to 27, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 4. 29. A multivalent vaccine composition according to any one of paragraphs 24 to 28, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 5. 30. A multivalent vaccine composition according to any one of paragraphs 24 to 29, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 6. 31. A multivalent vaccine composition according to any one of paragraphs 24 to 30, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 7. 32. A multivalent vaccine composition according to any one of paragraphs 24 to 31, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 8. 33. A multivalent vaccine composition according to any one of paragraphs 24 to 32, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 9. 34. A multivalent vaccine composition according to any one of paragraphs 24 to 33, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 10. 35. A multivalent vaccine composition according to any one of paragraphs 24 to 34, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 11. 36. A multivalent vaccine composition according to any one of paragraphs 24 to 35, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 12. 37. A multivalent vaccine composition according to any one of paragraphs 24 to 36, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 13. 38. A multivalent vaccine composition according to any one of paragraphs 24 to 37, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 14. 39. A multivalent vaccine composition according to any one of paragraphs 24 to 38, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 15. 40. A multivalent vaccine composition according to any one of paragraphs 24 to 39, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 16. 41. A multivalent vaccine composition according to any one of paragraphs 24 to 40, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 17. 42. A multivalent vaccine composition according to any one of paragraphs 24 to 41, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 18. 43. A multivalent vaccine composition according to any one of paragraphs 24 to 42, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 19. 44. A multivalent vaccine composition according to any one of paragraphs 24 to 43, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 20. 45. A multivalent vaccine composition according to any one of paragraphs 24 to 44, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 21. 46. A multivalent vaccine composition according to any one of paragraphs 24 to 45, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 22. 47. A multivalent vaccine composition according to any one of paragraphs 24 to 46, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 23. 48. A multivalent vaccine composition according to any one of paragraphs 24 to 47, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 24. 49. A multivalent vaccine composition according to any one of paragraphs 24 to 47, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 25. 50. A multivalent vaccine composition according to any one of paragraphs 24 to 48, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 26. 51. A multivalent vaccine composition according to any one of paragraphs 24 to 50, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 27. 52. A multivalent vaccine composition according to any one of paragraphs 24 to 51, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 28. 53. A multivalent vaccine composition according to any one of paragraphs 24 to 52, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 29. 54. A multivalent vaccine composition according to any one of paragraphs 24 to 53, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 30. 55. A multivalent vaccine composition according to any one of paragraphs 24 to 54, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 31. 56. A multivalent vaccine composition according to any one of paragraphs 24 to 55, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 32. 57. A multivalent vaccine composition according to any one of paragraphs 24 to 56, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 33. 58. A multivalent vaccine composition according to any one of paragraphs 24 to 57, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 34. 59. A multivalent vaccine composition according to any one of paragraphs 24 to 58, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 35. 60. A multivalent vaccine composition according to any one of paragraphs 24 to 59, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 36. 61. A multivalent vaccine composition according to any one of paragraphs 24 to 60, wherein the T cell epitope comprises the sequence shown in SEQ ID NO: 37. 62. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 61, wherein the T cell epitopes are linked and have a sequence shown in SEQ ID NO: 42. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 62, wherein the T cell epitopes are linked and have a sequence shown in SEQ ID NO: 43. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 63, wherein the T cell epitopes are linked and have a sequence shown in SEQ ID NO: 44. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 64 wherein the T cell epitopes are linked and have a sequence comprising SEQ ID NO: 45. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 65, wherein the linked epitopes have a sequence comprising SEQ ID NO: 46. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 67, wherein the linked epitopes have a sequence comprising SEQ ID NO: 47 or a derivative thereof wherein the leader sequence (in italics) is absent: MGVTGILQLPRDRREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRREK RSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRYLFDESGEFKLREKRM RPNFTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNA PRITFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTY VREKRETKAIVSTIQRREKRTMADLVYALREKRHFPREGVFVSREKRKMFDAYVNTFREKRRTIAFGGCVFR EKRYAFEHIVYREKRRHFDEGNCDTLREKRAPHGHVMVELREKRRPQGLPNNTAREKRFLLPSLATVREKRL PFFSNVTWREKRLEPLVDLPIGIREKRALWEIQQVREKRSSTFNVPMEKLREKRFLGIITTVREKRSMWALI ISVREKRALWEIQQVVREKRLLFNKVTLAREKRAKFVAAWTLKAAA. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 67, wherein the linked epitopes have a sequence comprising SEQ ID NO: 48 or a derivative thereof wherein the leader sequence (in italics) is absent: MGVTGILQLPRDRREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRREK RSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRMRPNFTIKGSFREKRY DPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNAPRITFREKRVYMPAS WVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTYVREKRETKAIVSTIQ RREKRTMADLVYALREKRHFPREGVFVSREKRKMFDAYVNTFREKRRTIAFGGCVFREKRYAFEHIVYREKR RHFDEGNCDTLREKRAPHGHVMVELREKRRPQGLPNNTAREKRFLLPSLATVREKRLPFFSNVTWREKRLEP LVDLPIGIREKRALWEIQQVREKRSSTFNVPMEKLREKRFLGIITTVREKRSMWALIISVREKRALWEIQQV VREKRLLFNKVTLAREKRAKFVAAWTLKAAA. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 68, wherein the linked epitopes have a sequence comprising any one of SEQ ID NO: 49, 50 and 71 to 76, wherein each X is a linker, such as a cleavable linker, for example independently selected from a linker disclosed herein, such as AAY,REKR, formula (II), formula (III) and formula (IIIA), such as AAY and REKR . A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 11 and 15 to 69, wherein the linked epitopes have a sequence comprising SEQ ID NO :49, wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, such as AAY, such as REKR. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 70, wherein the linked epitopes have a sequence comprising SEQ ID NO: 50, wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, such as AAY, such as REKR. 72. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to11 and 15 to 71, wherein the linked epitopes have a sequence comprising SEQ ID NO: 71, wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, such as AAY, such as REKR. 73. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 72, wherein the linked epitopes have a sequence comprising SEQ ID NO: 72 wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, such as AAY, such as REKR. 74. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 73, wherein the linked epitopes have a sequence comprising SEQ ID NO: 73 wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, in particular AAY, in particular REKR. 75. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 74, wherein the linked epitopes have a sequence comprising SEQ ID NO: 74 or a derivative thereof wherein the leader sequence (in italics) is removed: MDAMK RGLCCVLLLCGAVFVDSVTGXLVQAGNVQLRXLDGISQYSLRXGVYYPDKVFRXFGADPIHSLRXSTFNVPM EKXFYDFAVSKGFXSAPHGVVFLXYLFDESGEFKLXMRPNFTIKGSFXYDPLQPELXVFVSNGTHWFXQFAY ANRNRFXKVDGVDVELXNQRNAPRITFXVYMPASWVMRIXKPLEFGATSAXSDNIALLVXASAFFGMSRIXV VFLHVTYVXETKAIVSTIQRXTMADLVYALXHFPREGVFVSXKMFDAYVNTFXRTIAFGGCVFXYAFEHIVY XRHFDEGNCDTLXAPHGHVMVELXRPQGLPNNTAXFLLPSLATVXLPFFSNVTWXLEPLVDLPIGIXALWEI QQVXSSTFNVPMEKLXFLGIITTVXSMWALIISVXALWEIQQVVXLLFNKVTLAXAKFVAAWTLKAAA wherein each X is independently a cleavable linker, for example an amino acid sequence, such as independently selected from AAY and REKR, in particular AAY, in particular REKR. 76. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 75, wherein the linked epitopes have a sequence comprising SEQ ID NO: 75 or a derivative thereof wherein the leader sequence (in italics) is removed: MGVT GILQLPRDRXLVQAGNVQLRXLDGISQYSLRXGVYYPDKVFRXFGADPIHSLRXSTFNVPMEKXFYDFAVSK GFXSAPHGVVFLXYLFDESGEFKLXYLFDESGEFKLXMRPNFTIKGSFXYDPLQPELXVFVSNGTHWFXQFA YANRNRFXKVDGVDVELXNQRNAPRITFXVYMPASWVMRIXKPLEFGATSAREKRSDNIALLVXASAFFGMS RIXVVFLHVTYVXETKAIVSTIQRXTMADLVYALXHFPREGVFVSXKMFDAYVNTFXRTIAFGGCVFXYAFE HIVYXRHFDEGNCDTLXAPHGHVMVELXRPQGLPNNTAXFLLPSLATVXLPFFSNVTWXLEPLVDLPIGIXA LWEIQQVXSSTFNVPMEKLXFLGIITTVXSMWALIISVXALWEIQQVVXLLFNKVTLAXAKFVAAWTLKAA 77. wherein each X is independently a cleavable linker, for example an amino acid linker, such as independently selected from AAY and REKR, in particular AAY, in particular REKR. 78. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 76, wherein the linked epitopes have a sequence comprising SEQ ID NO: 76 or a derivative thereof wherein the leader sequence (in italics) is removed: 79. MGVTGILQLPRDRXLVQAGNVQLRXLDGISQYSLRXGVYYPDKVFRXFGADPIHSLRXSTFNVPMEKXFYDF AVSKGFXSAPHGVVFLXYLFDESGEFKLXMRPNFTIKGSFXYDPLQPELXVFVSNGTHWFXQFAYANRNRFX KVDGVDVELXNQRNAPRITFXVYMPASWVMRIXKPLEFGATSAXSDNIALLVXASAFFGMSRIXVVFLHVTY VXETKAIVSTIQRXTMADLVYALXHFPREGVFVSXKMFDAYVNTFXRTIAFGGCVFXYAFEHIVYXRHFDEG NCDTLXAPHGHVMVELXRPQGLPNNTAXFLLPSLATVXLPFFSNVTWXLEPLVDLPIGIXALWEIQQVXSST FNVPMEKLXFLGIITTVXSMWALIISVXALWEIQQVVXLLFNKVTLAXAKFVAAWTLKAAA wherein each X is independently a cleavable linker, for example an amino acid linker such as independently selected from AAY and REKR, in particular AAY, in particular REKR. 80. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 77, wherein the linked epitopes have a sequence comprising SEQ ID NO: 65 or a derivative thereof wherein the leader sequence (in italics) is absent: MDAMKRGLCC VLLLCGAVFVDSVTGREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRFGADPIHSLRR EKRSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRYLFDESGEFKLREK RMRPNFTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQR NAPRITFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHV TYVREKRETKAIVSTIQRREKRTMADLVYALREKRHFPREGVFVSREKRKMFDAYVNTFREKRRTIAFGGCV FREKRYAFEHIVYREKRRHFDEGNCDTLREKRAPHGHVMVELREKRRPQGLPNNTAREKRFLLPSLATVREK RLPFFSNVTWREKRLEPLVDLPIGIREKRALWEIQQVREKRSSTFNVPMEKLREKRFLGIITTVREKRSMWA LIISVREKRALWEIQQVVREKRLLFNKVTLAREKRAKFVAAWTLKAAASGGGGSGGGGSATGKGAAASTQEG KSQPFKVTPGPFDPATWLEWSRQWQGTEGNGHAAASGIPGLDALAGVKIAPAQLGDIQQRYMKDFSALWQAM AEGKAEATGPLHDRRFAGDAWRTNLPYRFAAAFYLLNARALTELADAVEADAKTRQRIRFAISQWVDAMSPA NFLATNPEAQRLLIESGGESLRAGVRNMMEDLTRGKISQTDESAFEVGRNVAVTEGAVVFENEYFQLLQYKP LTDKVHARPLLMVPPCINKYYILDLQPESSLVRHVVEQGHTVFLVSWRNPDASMAGSTWDDYIEHAAIRAIE VARDISGQDKINVLGFCVGGTIVSTALAVLAARGEHPAASVTLLTTLLDFADTGILDVFVDEGHVQLREATL GGGAGAPCALLRGLELANTFSFLRPNDLVWNYVVDNYLKGNTPVPFDLLFWNGDATNLPGPWYCWYLRHTYL QNELKVPGKLTVCGVPVDLASIDVPTYIYGSREDHIVPWTAAYASTALLANKLRFVLGASGHIAGVINPPAK NKRSHWTNDALPESPQQWLAGAIEHHGSWWPDWTAWLAGQAGAKRAAPANYGNARYRAIEPAPGRYVKAKA. 81. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 78, wherein the linked epitopes have a sequence comprising SEQ ID NO: 67 or derivative thereof wherein the leader sequence (in italics) is absent: MDAMKRGLCCVLLLCGAVFVDSVTGREKRLVQAGNVQLRREKRLDGISQYSLRREKRGVYYPDKVFRREKRF GADPIHSLRREKRSTFNVPMEKREKRFYDFAVSKGFREKRSAPHGVVFLREKRYLFDESGEFKLREKRMRPN FTIKGSFREKRYDPLQPELREKRVFVSNGTHWFREKRQFAYANRNRFREKRKVDGVDVELREKRNQRNAPRI TFREKRVYMPASWVMRIREKRKPLEFGATSAREKRSDNIALLVREKRASAFFGMSRIREKRVVFLHVTYVRE KRETKAIVSTIQRREKRTMADLVYALREKRHFPREGVFVSREKRKMFDAYVNTFREKRRTIAFGGCVFREKR YAFEHIVYREKRRHFDEGNCDTLREKRAPHGHVMVELREKRRPQGLPNNTAREKRFLLPSLATVREKRLPFF SNVTWREKRLEPLVDLPIGIREKRALWEIQQVREKRSSTFNVPMEKLREKRFLGIITTVREKRSMWALIISV REKRALWEIQQVVREKRLLFNKVTLAREKRAKFVAAWTLKAAASGGGGSGGGGSATGKGAAASTQEGKSQPF KVTPGPFDPATWLEWSRQWQGTEGNGHAAASGIPGLDALAGVKIAPAQLGDIQQRYMKDFSALWQAMAEGKA EATGPLHDRRFAGDAWRTNLPYRFAAAFYLLNARALTELADAVEADAKTRQRIRFAISQWVDAMSPANFLAT NPEAQRLLIESGGESLRAGVRNMMEDLTRGKISQTDESAFEVGRNVAVTEGAVVFENEYFQLLQYKPLTDKV HARPLLMVPPCINKYYILDLQPESSLVRHVVEQGHTVFLVSWRNPDASMAGSTWDDYIEHAAIRAIEVARDI SGQDKINVLGFCVGGTIVSTALAVLAARGEHPAASVTLLTTLLDFADTGILDVFVDEGHVQLREATLGGGAG APCALLRGLELANTFSFLRPNDLVWNYVVDNYLKGNTPVPFDLLFWNGDATNLPGPWYCWYLRHTYLQNELK VPGKLTVCGVPVDLASIDVPTYIYGSREDHIVPWTAAYASTALLANKLRFVLGASGHIAGVINPPAKNKRSH WTNDALPESPQQWLAGAIEHHGSWWPDWTAWLAGQAGAKRAAPANYGNARYRAIEPAPGRYVKAKA 82. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 81, wherein the linked epitopes comprise a sequence shown in the attached sequence listing, and combinations of two or more of the same. 83. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11 and 15 to 82 wherein the sequence is independently selected from SEQ ID NO: 78, 79, 80, 81, 82, 83, 84, 85, 86 and 87. 84. A multivalent vaccine composition according to to any one of paragraphs 1, 1A, 1B to 11 and 15 to 83, wherein the sequence comprise SEQ ID NO: 79 (optimised sequence design no.1). 85. A multivalent vaccine composition according to to any one of paragraphs 1, 1A, 1B to 11and 15 to 84, wherein the sequence comprise SEQ ID NO: 81 (optimised sequence design no.3). 86. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 11and 15 to 83, wherein the sequence comprises SEQ ID NO: 86 (optimised sequence design no. 8). 87. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 86, wherein one or more (e.g. all) of the T cell epitopes are capable of presentation on at least one HLA molecule, in particular HLA type 1 including HLA type 1 variant. 88. A multivalent vaccine composition according to any one of paragraphs 1, 1A, 1B to 87, wherein the composition comprises an addition sequence independently selected from: a. an intracellular translocation sequence, b. a sequence that increases proteolytic processing and/or proteasomal cleavage efficiency, c. a sequence that enhances immunological responses and/or immunogenicity, d. a sequence that enhances epitope presentation, such as a PADRE sequence, e. a sequence that induces, increases or sustains activity of one or more immune cells (such as CD4+, CD8+ and antigen presenting cells); and/or f. combinations of 2 or more of the same. 89. A multivalent vaccine composition according to paragraph 87, wherein the composition comprises an intracellular translocation domain, for example comprises sequence MDAMKRGLCCVLLLCGAVFVDSVTG [SEQ ID NO: 38] and/or MGVTGILQLPRDR [SEQ ID NO: 39]. 90. A multivalent vaccine composition according to paragraph 88 or 89, wherein the composition comprises a sequence associated with enhanced immunological response and/or immunogenicity. 91. A multivalent vaccine composition according to any one of paragraphs 86 to 88, wherein the composition comprises a sequence which enhances epitope presentation such as a PADRE sequence, such as AKFVAAWTLKAAA [SEQ ID NO: 40]. 92. A multivalent vaccine composition according to any one of paragraphs 88 to 91, wherein the composition comprises a sequence of part e) induces or increases activity of CD8+ cells, for example the sequence comprises LLFNKVTLA [SEQ ID NO: 37]. 93. A multivalent vaccine composition according to any one of paragraphs 88 to 92, wherein the composition comprises a sequence that increases proteolytic processing efficiency, for example a cleavable linker, such as disclosed herein. 94. A multivalent vaccine peptide or polypeptide composition according to any one of paragraphs 1 to 93, wherein the composition comprises an adjuvant. 95. A multivalent vaccine peptide or polypeptide composition according to paragraph 94, wherein the adjuvant is selected from: • metal salts such as aluminium hydroxide or aluminium phosphate, • oil in water emulsions, • toll like receptors agonist, (such as toll like receptor 2 agonist, toll like receptor 3 agonist, toll like receptor 4 agonist, toll like receptor 7 agonist, toll like receptor 8 agonist and toll like receptor 9 agonist), • saponins, for example Quil A and its derivatives such as QS7 and/or QS21, • CpG containing oligonucleotides, • 3D –MPL, • (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o- phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D- glucopyranosyldihydrogenphosphate), • DP (3S, 9R) –3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)- 3-hydroxytetradecanoylamino]decan-1,10-diol,1,10- bis(dihydrogenophosphate), • MP-Ac DP (3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9- [(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1 -dihydrogenophosphate 10-(6-aminohexanoate), and • combinations thereof. 96. A multivalent vaccine composition according to any one of paragraphs 1 to 94, for use in treatment, such as prophylaxis, in particular prophylaxis to generate immunity to a coronavirus (more specifically SARS-CoV2). 97. A multivalent vaccine composition according to paragraph 95, wherein the vaccine is employed in combination with further coronavirus vaccine, such as a further COVID-19 vaccine, in particular where the further vaccine is based on the spike protein (further coronavirus vaccine may include Pfizer/BioNTech Comirnity® (Tozinameran) vaccine, Oxford/AstraZeneca Vaxzevria® COVID-19 Vaccine (ChAdOx1-S [recombinant]) Moderna Spikevax® (COVID-19 Vaccine, mRNA), Janssen COVID-19 VACCINE JANSSEN® Ad26.COV2.S and combinations thereof). 98. A multivalent vaccine composition according to paragraph 96, wherein at least one dose of said further vaccine (for example all the doses) is administered prior to the multivalent vaccine composition, for example 1-6 days, 1 to 3 weeks, a month or more prior. 99. A multivalent vaccine composition according to paragraph 95 or 96, wherein at least one dose of said further vaccine (for example all the doses) is administered concomitantly with the multivalent vaccine composition i.e. in same dosing regimen. 100. A multivalent vaccine composition according to any one of paragraphs 96 to 98, wherein at least one dose of said further vaccine (for example all the doses) is administered after the multivalent vaccine composition, for example 1-6 days, 1 to 3 weeks, a month or more after. 101. A multivalent vaccine composition according to any one of paragraphs 96 to 99, wherein said two or more vaccines are separate formulations. 102. A multivalent vaccine composition according to paragraph 100, wherein the two or more vaccines are provided as a kit, i.e. together, in particular including instructions. 103. A multivalent vaccine composition according to any one of paragraphs 96 to 100, wherein the vaccines are co-formulated. 104. A multivalent vaccine composition according to any one of paragraphs 96 to 102, for use in a population of patients already vaccinated against coronavirus. 105. A multivalent vaccine composition according to any one of paragraphs 96 to 103, wherein the dose administered is in the range 5 to 100µg, such as 10, 20, 30, 40, 50, 60, 70, 80 or 90µgs, in particular 50µg. 106. A multivalent vaccine composition according to any one of paragraphs 96 to 104, wherein a first dose of the multivalent vaccine composition is administered, for example intramuscularly or subcutaneously. 107. A multivalent vaccine composition according to paragraph 105, wherein a second dose is administered, for example intramuscularly or subcutaneously. 108. A multivalent vaccine composition according to paragraph 106, wherein the second dose is administered between 2 and 12 weeks after the first dose, such as 3 weeks. 109. Use of a multivalent vaccine composition according to any one of paragraphs 196 to 106, for the manufacture of a medicament for the treatment of coronavirus, in particular COVID-19. 110. A method of treatment comprising administered to a human multivalent vaccine composition as defined in any one of paragraphs 1 to 108. In one embodiment the vaccine composition according to the present disclosure comprises a sequence or combination of sequences disclosed in the sequence listing herein. In one embodiment the present disclosure provides a coronavirus vaccine, in particular a pan SARS-CoV-2 In one embodiment, the multivalent vaccine comprises 5, 10 or more SARS-CoV-2 T cell epitopes, 15 or more SARS-CoV-2 T cell epitopes, 20 or more SARS-CoV-2 T cell epitopes, 25 or more SARS-CoV-2 T cell epitopes, 30 or more SARS-CoV-2 T cell epitopes, 35 or more SARS-CoV- 2 T cell epitopes, 40 or more SARS-CoV-2 T cell epitopes, 45 or more SARS-CoV-2 T cell epitopes, 50 or more SARS-CoV-2 T cell epitopes, or 55 or more SARS-CoV-2 T cell epitopes. In one embodiment q in formula (I) is an integer 1 to 40, such as 1 to 31, for example 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 or 30. Thus, when q is 38, 39 or 40 then one or more of the epitopes 1 to 37 will be repeated within the construct. Generally, A, B, J, each U, Wand Z will be selected to be different peptides. In one embodiment A, B, J, U, W and Z all have different amino acid sequences. The invention also extends to polynucleotides encoding said construct of the present disclosure including of formula (I). Advantageously the vaccines of the present disclosure generate T cell responses, for example CD8+ T cell responses. In one embodiment the peptides components in the constructs of the disclosure are all MHC I restricted. In one embodiment one or more elements of the vaccine composition of the present disclosure is able to be presented on one or more of the HLA alleles selected from the group consisting of: A01:01, A02:01, A02:02, A02:06, A02:07, A11:01, A23:01, A24:02, A24:41, A24:51, A26:01, A30:02, A31:01, A31:29, A32:01, A34:01, A68:01, B07:02, B08:01, B15:01, B15:93, B18:01, B27:05, B35:01, B38:01, B39:01, B39:54, B40:01, B40:10, B48:01, B51:01, B54:18, B55:02, B56:02, B56:43, B58:01, C01:02, C01:57, C02:02, C03:04, C03:13, C04:01, C04:03, C04:43, C05:01, C06:02, C07:01, C07:02, C08:01, C12:03, C14:02, and C15:02, for example an element in the vaccine is suitable for presentation on 90% or more of said alleles. The present inventors have show that the vaccines of the present invention can be delivered in different formats, all of which provide the desired results. The present disclosure also includes combinations of peptides (for example as mixtures or linked) as disclosed herein and a polynucleotide or polynucleotides encoding said one or more peptides and/or combinations thereof (in particular a sequence disclosed herein), vectors and/or cells comprising said polynucleotide or polynucleotides. In an independent aspect the present disclosure also includes a polypeptide disclosed herein, a polynucleotide encoding same, a vector and/or cell comprising said polynucleotide. In one embodiment the vaccine composition according to the present disclosure does not comprise a polynucleotide. In one embodiment the vaccine composition according to the present disclosure does not comprise a vector, such as a viral vector. In one embodiment the vaccine composition according to the present disclosure does not comprise a cell, such as an immune cell. In one embodiment the sequence is selected from any one of sequences 71 to 76: C1_p generic linker SEQ ID NO: 71 wherein X is a linker, for example an amino acid linker (including a linker of a formula or sequence disclosed herein), such as a cleavable linker (including a proteolytic cleavage), in particular selected from AAY and REKR, more specifically AAY; C1_p.3 generic linker SEQ ID NO 72 wherein X is a linker, for example an amino acid linker, such as a cleavable linker (including a proteolytic cleavage), in particular selected from AAY and REKR, more specifically AAY; C2_f generic linker SEQ ID NO: 73 or a derivative thereof minus the leader sequence wherein X is a linker, for example an amino acid linker, such as a cleavable linker (include proteolytic cleavage), in particular selected from AAY and REKR, more specifically REKR; C2_f.2 generic linker SEQ ID NO: 74 or a derivative thereof minus the leader sequence wherein X is a linker, for example an amino acid sequence, such as a cleavable linker (include proteolytic cleavage), in particular selected from AAY and REKR, more specifically REKR; C3_f generic linker SEQ ID NO: 75 wherein X is a linker, for example an amino acid linker, such as a cleavable linker (include proteolytic cleavage), in particular selected from AAY and REKR, more specifically REKR; C3_f.2 generic linker SEQ ID NO: 76 wherein X is a linker, for example an amino acid linker, such as a cleavable linker (include proteolytic cleavage), in particular selected from AAY and REKR, more specifically REKR. In one embodiment X is Xaa, wherein Xaa is an amino acid linker, for example as disclosed herein, including a formula (II) and/or (III). In one embodiment X in SEQ ID NO: 49 is Xaa. In one embodiment X in SEQ ID NO: 50 is Xaa. In one embodiment X in SEQ ID NO: 71 is Xaa. In one embodiment X in SEQ ID NO: 72 is Xaa. In one embodiment X in SEQ ID NO: 73 is Xaa. In one embodiment X in SEQ ID NO: 74 is Xaa. In one embodiment X in SEQ ID NO: 75 is Xaa. In one embodiment X in SEQ ID NO: 76 is Xaa. In an independent aspect the invention provides a sequence or construct of a formula disclosed herein. In an independent aspect the present disclosure includes a polynucleotide encoding a construct herein or a codon optimised version thereof, for a mammalian cell, for example a human cell or CHO. In one embodiment the polynucleotide is a sequence disclosed herein. In one embodiment the polynucleotide is for use in manufacturing a peptide, oligopeptide or polypeptide according to the present disclosure. In one embodiment the polynucleotide is optimised for a cell selected from an E. coli cell and an insect cell, in particular E. coli. In one embodiment the polynucleotide is a vaccine construct for use in a vaccine composition according to the present disclosure. In one embodiment the polynucleotide is RNA. In one embodiment the polynucleotide is DNA. Thus, the disclosure extends to any sequence of construct according to the present invention (in particular disclosed herein), including where they are provided as a pharmaceutical formulation (in particular a vaccine composition). In one embodiment the pharmaceutical formulation is filled into a vial, for example containing 1 to 8 doses. The disclosure also includes use of all aspects in treatment (which included prophylaxis and vaccination), in particular treatment of coronavirus, such as SARS-CoV-2. Advantages of the composition and constructs of the present disclosure include the ability to generate a T cell response in a high percentage of the population and thus the vaccine has worldwide utility. Compositions of the present disclosure have high levels of redundancy built in. This means the composition includes multiple epitopes for a given viral protein and/or target multiple virus proteins. Thus, vaccination with the composition is likely to provide immunity even if the virus mutates. In one embodiment the level of redundancy is the numerical value calculated for any one of the constructs disclosed herein. In one embodiment this calculated value from an “example” is combined with an independent functional claim defining the invention. Thus, these numerical values may be calculated and combined with one or more claims and are NOT limited to being associated with only one individual sequence. When the epitopes are linked the order of the epitopes can be important, for example it may have an impact on expression. The order of the epitopes has been optimised in constructs according to the present disclosure, in particular optimised for expression. In some embodiments linkers have been optimised, for example to remove neo-epitopes and/or optimise recovery of the component epitopes, i.e. to optimise cleavage and/or processing by cells in vivo. In one embodiment the percentage disorder of a construct according to the present disclosure is in the range 10 to 75 (or 20 to 60%), for example 15, 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 or 75%. In some embodiments the constructs according to the present disclosure do not comprise a bead (such as a biobead, in particular, as disclosed in the Examples herein). In one embodiment constructs are optimised for expression in vitro and/or in vivo, for example are provided as multiple fragments (oligopeptides as defined herein). In one embodiment the constructs of the present disclosure are optimised for expression in a cell, for example a host cell used in recombinant expression, in particular E. coli (for example strain BL21(DE3). BRIEF DESCRIPTION OF THE FIGURES Figure 1 & 2 Show constructs according to and employed in the present disclosure. The linkers can be the same or different. Multiple constructs of this format may be provided in a single vaccine composition. Figure 3 Shows pentamer immune profiling analysis of antigen-specific CD8+ T cells Figure 4 Shows pentamer immune profiling analysis of antigen-specific CD8+ T cells Figure 5 Shows ELISPOT analysis Figure 6 Shows percentage recovery and disorder for certain constructs according to the present disclosure. Figure 7 Shows spot forming units for certain constructs according to the present disclosure Figure 8 Shows spot forming units for certain constructs Figure 9 Polyacrylamide gels of E. coli cell lysates showing high expression levels of six fragments (SEQ ID No 88 to 93) Figure 10 Shows gating strategy for ICFC analysis of ex vivo restimulated splenocytes from Group 1-11 mice Figure 11 Shows frequencies of IFNγ+ CD8+ T cells in the splenocytes from G11 control mice (immunized with HPV peptide) after the ex vivo stimulation with 5µM HPV peptide Figure 12A Shows frequencies of peptide-specific IFNγ+ CD8+ T cells in the splenocytes after the ex vivo stimulation with HLA-A*02:01 peptide pool (5µM per peptide) Figure 12B Summarises the number of mice with positive peptide-specific IFNγ+ CD8+ T cell responses within each group Figure 13 Shows gating strategy for ICFC analysis of ex vivo restimulated splenocytes. Figure 14A Shows restimulation with HLA-A* 02:01 peptide pool Figure 14B Shows restimulation with HLA-A* 11:01 peptide 9 Figure 14C Shows restimulation with HLA-A* 11:01 peptide 10 Figure 14D Shows restimulation with HLA-B* 07:02 peptide 11 Figure 15 Shows % of peptide specific IFN ^^^^+ cells in CD3+ & CD8+ T cells SEQUENCE LISTING Null sequence AAY proteasome-dependent processing site Null sequence SW is a linker SEQ ID NO: 1-36 T cell epitopes shown in Table 1 SEQ ID NO: 37 LLFNKVTLA peptide for inducing a specific CD8+ T cell response in individuals possessing the MHC class I A*02:01 allele SEQ ID NO: 38 MDAMKRGLCCVLLLCGAVFVDSVTG intracellular translocation domain SEQ ID NO: 39 MGVTGILQLPRDR intracellular translocation domain SEQ ID NO: 40 AKFVAAWTLKAAA a carrier epitope for enhancing T cell responses (PADRE sequence) SEQ ID NO: 41 A furin cleavage site SEQ ID NO: 42 C1_p (also referred to as T1.1) polypeptide constructs according to and employed in the present disclosure SEQ ID NO: 43 C1_p.2 polypeptide construct according to and employed in the present disclosure SEQ ID NO: 44 C1_p.3 (also referred to as T1.2) polypeptide constructs according to and employed in the present disclosure SEQ ID NO: 45 C2_f (also referred to as T2.1) polypeptide construct according to and employed in the present disclosure SEQ ID NO: 46 C2_f.2 (also referred to as T2.2) polypeptide construct according to and employed in the present disclosure SEQ ID NO: 47 C3_f polypeptide construct according to and employed in the present disclosure SEQ ID NO: 48 C3_f.2 polypeptide construct according to and employed in the present disclosure SEQ ID NO: 49 C4_pr polypeptide construct according to and employed in the present disclosure SEQ ID NO: 50 C4_pr.2 polypeptide construct according to and employed in the present disclosure SEQ ID NO: 51 C1_n polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 52 C1_n.2 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 53 C2_n polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 54 C2_n.2 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 55 C3_n polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 56 C3_n.2 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 57 T2F polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 58 T2R polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 59 MS008 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 60 MS009 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 61 T2F1 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 62 T2F2 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 63 T2R1 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 64 T2R2 polynucleotide construct according to and employed in the present disclosure SEQ ID NO: 65- T2.1-phaC fusion polypeptide construct according to and employed in the present disclosure SEQ ID NO 66 T1.1-phaC fusion polypeptide construct according and employed in the present disclosure SEQ ID NO 67 T2.2-phaC fusion polypeptide construct according and employed in the present disclosure SEQ ID NO 68 T1.2-phaC fusion polypeptide construct according and employed in the present disclosure SEQ ID NO: 69 Linker sequence SEQ ID NO: 70 Linker sequence SEQ ID NO 71 to 76 are generic structures of the constructs according to the present disclosure. SEQ ID NO: 77 Polynucleotide encoding C1_p3 (also known as T1.2) SEQ ID NO: 78 Polypeptide with bespoke linkers, designated design 0 SEQ ID NO: 79 Polypeptide with bespoke linkers, designated design 1 SEQ ID NO: 80 Polypeptide with bespoke linkers, designated design 2 SEQ ID NO: 81 Polypeptide with bespoke linkers, designated design 3 SEQ ID NO: 82 Polypeptide with bespoke linkers, designated design 4 SEQ ID NO: 83 Polypeptide with bespoke linkers, designated design 5 SEQ ID NO: 84 Polypeptide with bespoke linkers, designated design 6 SEQ ID NO: 85 Polypeptide with bespoke linkers, designated design 7 SEQ ID NO: 86 Polypeptide with bespoke linkers, designated design 8 SEQ ID NO: 87 Polypeptide with bespoke linkers, designated design 9 SEQ ID NO: 88 Oligopeptide with bespoke linkers, designated fragment 1: MRPNFTIKGSF-PWRW-HFPREGVFVS-PWRW-LDGISQYSLR-PWQW- VYMPASWVMRI-PWQW-RPQGLPNNTA-PWQW-FYDFAVSKGF SEQ ID NO: 89 Oligopeptide with bespoke linkers, designated fragment 2 SAPHGVVFL-PWQY-KPLEFGATSA-PWQY-LPFFSNVTW-PWRY- ETKAIVSTIQR-PWQY-LEPLVDLPIGI-PWQY-FLLPSLATV-PWQY- FGADPIHSLR SEQ ID NO: 90 Oligopeptide with bespoke linkers, designated fragment 3 AKFVAAWTLKAAA-PWRW-YLFDESGEFKL-PWQW-KVDGVDVEL-PWRW- ALWEIQQV-PWRW-RHFDEGNCDTL-PWQW-GVYYPDKVFR-PWRW- SDNIALLV SEQ ID NO: 91 Oligopeptide with bespoke linkers, designated fragment 4 STFNVPMEK-PWNW-LVQAGNVQLR-PWNW-QFAYANRNRF-PWRW- KMFDAYVNTF-PWTW-VVFLHVTYV-PWNW-APHGHVMVEL SEQ ID NO: 92 Oligopeptide with bespoke linkers, designated fragment 5 ALWEIQQVV-PNCKRY-TMADLVYAL-PNCKRY-YAFEHIVY-PNCKRY- SSTFNVPMEKL-PNCKRY-ASAFFGMSRI-PNCKRY-VFVSNGTHWF SEQ ID NO: 93 Oligopeptide with bespoke linkers, designated fragment 6 SMWALIISV-PNCKRY-YDPLQPEL-PNCKQY-NQRNAPRITF-PNCKQY- FLGIITTV-PNCKQY-LLFNKVTLA-PNCKQY-RTIAFGGCVF SEQ ID NO: 94 Polypeptide designated T3 SEQ ID NO: 95 Polypeptide designated T4 SEQ ID NO: 96 Polypeptide designated T5 SEQ ID NO: 97 Polypeptide designated T6 SEQ ID NO: 98 Polypeptide designated T7 SEQ ID NO: 99 Polynucleotide designated T1.2 SEQ ID NO: 100 Polynucleotide designated T3 SEQ ID NO: 101 Polynucleotide designated T4 SEQ ID NO: 102 Polynucleotide designated T5 SEQ ID NO: 103 Polynucleotide designated T6 SEQ ID NO: 104 Polynucleotide designated T7 SEQ ID NO: 105 Cytotoxic T cell epitope from Epstein Barr virus used as a positive control SEQ ID NO: 106 Cytotoxic T cell epitope from human papilloma virus used as a positive control SEQ ID NOs: 107-127 Linkers SEQ ID NO: 128 C1_p generic linker SEQ ID NO: 129 C1_p.3 generic linker SEQ ID NO: 130 C3_f generic linker SEQ ID NO: 131 Template sequence of IVTScrip™ mRNA-T1.2 SEQ ID NO: 132 DNA nucleotide sequence for mRNA vaccine construct containing F3-8His SEQ ID NO: 133 DNA nucleotide sequence for mRNA vaccine construct containing F6-8His DETAILED DISCLOSURE Population coverage may be calculated, for example using http://tools.iedb.org/population. Broad spectrum protection as employed herein refers to protection against at least two variants of a coronavirus, but preferably more i.e. so-called pan protection, and/or protection in a high percentage of the patient population. Pan SARS-CoV-2 vaccine as employed herein refers to the ability to provide some level of protection against essentially all SARS-CoV2 viruses. Immunity refers to the ability to protect patients. Immunity as employed herein refers to the ability of a patient to resist infection or minimise adverse events associated with infection, for example to minimise viral load and/or symptoms, avoid respiratory distress, avoid cytokine storm, avoid organ failure and the like. In one embodiment immunity is the ability to prevent infection. Protection as employed herein refers to the ability to protect patients from severe reactions/symptoms to infection with coronavirus, such as cytokine storm, particularly in the vulnerable and those with underlying health issues. Protection does not necessarily prevent infection with coronavirus. In one embodiment there is provided protection from infection. Coronavirus as employed herein refers to a virus, which is in the family of Coronaviridae (order Nidovirales and realm Ribovira), for example with a lineage identified by Phylogenetic Assignment of Named Global Outbreak Lineages (PANGOLIN). Coronaviruses constitute the subfamily Coronaviridae. Under this subfamily is the genera Beta-CoV. This contains the lineages SARS-CoV, SARS-CoV-2, MHV and MERS-CoV. Essentially all SARS-CoV-2 viruses as employed herein refers to viruses with a lineage of SARS-CoV-2, for example where the lineage is designated using PANGOLIN. Lineage includes clades and strains. Lineages using the WHO label currently include alpha, beta, gamma, epsilon, eta, lota, kappa, zeta, mu. PANGO lineage include A.1, A.2, A.3, A.4, A.5, A.6, B.1, B.2, B.3, B.4, B.5, B.6, B.7, B.9, B.10, B.13, B.14, B.15, B.16, such as B.1.1, B.1.22, B.1.26, B.1.37 B.1.3-B1.66, B.1.5- B.1.72, B.1.1.177 and B.1.13. Notable sequences include WIV04/2019, B.1.617 (such as B.1.617.3), B.1.427/B.1.429, B.1.525, B.1.351 (20H/501Y.V2 or 501.V2), B.1.1.207 C.37), P.1, P.2, B.1.1.7 (VOC-202012/01), VOC-20DEC-01 AND20I/501Y.V1. Omicron is Pango lineage B.1.1.29 (GISAID clade GR/484A), Nextstrain clade 21K, 21L, 21M, 22A, 22B, 22C, additional amino acid change +S:R346K, +SL452X, +S: F486V. Strain as employed herein is genetic variant within a virus lineage, for example Alpha (B.1.1.7), Beta (B.1.351), Gamma (P.1), Delta (B.1.617.2, AY.1, AY.2 & AY.3), Zeta^ (P.2), Eta (B.1.525 & B.1.1.318), Theta^ (P.3), Kappa (B.1.617.1, B.1.617.3, AV.1 & C.36.3), Lambda (C37 & B.1.621), Epsilon^ (B.1.427/B.1.429, B.1.1.7 with S494P & A.27), Iota (B.1.526, B.1.1.7 with Q667H, B.1.620, B.1.214.2, R.1, B.1 with 214insQAS, AT.1, A.30, B.1.630, P.1+N501T and E484Q, B.1.619, B.1.629, C.1.2, B.1.630 & B.1.631/B.1.628), Zeta (P.2), Mu (B.1.621 and B.1.621.1) and combinations of two or more of the same)are strains or variants of SARS-CoV2. In one embodiment the patient is a human, for example aged 18 years or over. In one embodiment the patient is an infant or child, for example age 3 months to 18 years. In one embodiment when the T cell epitopes are linked, the construct may further comprise (including encoding) a leader sequence, to assist polypeptide (including expressed polypeptide) processing within various cellular compartments. In one embodiment when the T cell epitopes are linked by one or more furin cleavage sites (e.g. where linkers are furin cleavage sites) said construct will generally comprise a leader sequence or a translocation domain. In one embodiment when the T cell epitope(s) are linked by only a protease cleavage site then generally the construct will exclude a leader sequence or translocation domain. In one embodiment constructs of the present disclosure comprise a PADRE sequence, for example at the C-terminal. In one embodiment the constructs of the present disclosure comprise a label, such as a HisTag. In one embodiment the constructs of the disclosure do not comprise a label, such as HisTag. The disclosure extends to sequences comprising a HisTag disclosed herein (such as T2.1) where said Tag is absent. X in sequences according to the present disclosure is a linker, for example a cleavable linker (including a proteolytic cleavage site), such as independently selected from a linker explicitly disclosed herein, more specifically AAY and REKR. In one embodiment, wherein constructs of the disclosure comprises multiple X sequences, the sequence of each X is selected independently, for example such that some or all of the linkers are different. In one embodiment all the X’s have the same amino acid sequence (including where the same amino acid sequence is encoded by different polynucleotide sequences). Surprisingly, the selection/optimisation of the linker can increase immune responses. Independently (such as independently selected) as employed herein, refers to the fact the multiples of the entity X can be the same or different. In one embodiment sequences of the present disclosure are isolated, for example isolated from the virus or body (or a cell), i.e. removed form a native environment. Isolation may include being in a storage container or the like in a suitable media or formulation. Thus “isolated” as employed herein refers to where the construct is essential free of contaminants and other extraneous materials, in particular the construct is not contained within a body or naturally occurring. Recombinant synthesis is not considered to be “naturally occurring” within the meaning of the current invention. However isolated constructs include constructs formulated with excipients, diluents, carriers and the like. In one embodiment the sequences of the present disclosure are recombinant. Recombinant as employed herein refers to where the sequence is made using recombinant techniques. In one embodiment the sequences of the present disclosure are purified. Purified as employed herein refers to free from toxins and contaminations, for example 95, 96, 97, 98 or 99% pure as assessed by a suitable assay. Purified sequences will generally be stored in a suitable media or formulation. Sequence as employed herein can refer to polynucleotide sequences, peptide, oligopeptide, or polypeptide sequences (including protein sequences) and peptide sequences. In one embodiment the disclosure extends to a sequence (such as an amino acid sequence disclosed herein) at least 95% similar or identical to a sequence disclosed herein, for example 96%, 97%, 98% or 99%, for example over the whole length of the sequence in question. Polynucleotide sequence as employed herein refers to RNA and/or DNA, including incorporated into a vector, (such as a viral vector) or within a cell. In one embodiment polynucleotide sequences are codon optimised, for example for a mammalian cell, such as a human cell or a CHO cell. Polypeptide as employed herein refers to a sequence of 50 amino acids or greater. Oligopeptide (also referred to an oligo-epitopes) as employed herein overlaps with the definition of polypeptide and peptide, and is intended to refer to construct with at least two epitopes linked, for example with a linker as disclosed here, and no more than 10 linked epitopes. In one embodiment one or more oligopeptides are employed in combination, for example in totally comprising the same number of epitopes discussed elsewhere herein, such as 37. In one embodiment 2, 3, 4, 5, 6, 7, 8, 9 or 10 oligopeptides (such as 4, 5 or 6) are employed in the same combination, for example in the same formulation, in particular 1 to 6 oligiopetides selected from SEQ ID NO: 88 to 93. Peptide as employed herein refers to a sequence of 2 to 49 amino acids. Multipeptide vaccine refers to a vaccine comprising two or more peptides or oligopeptides, in particular where each peptide or oligopeptide contains two or more epitopes. In one embodiment the vaccine composition according to the present disclosure employs one or more sequences set forth in any one of SEQ ID NO: 42 to 50, 65 to 68 or 71 to 76 or 88 to 98 including a sequence 95%, or more similar or identical thereto, such as 96%, 97%, 98% or 99% similar or identical thereto or a polynucleotide, encoding the same. In one embodiment the vaccine composition according to the present disclosure employs sequence set forth in any one of SEQ ID NO: 51 to 64 or 77 or 99 to 104, sequence identical or similar thereto encoding the same amino acid, and/or a codon optimised version thereof, for example optimised for a mammalian cell, such as human cell or CHO cell. Employs as used herein refers to the fact the entity may be provided as an amino acid sequence or encoded as a polynucleotide (including a polynucleotide in a vector). Linker(s) as employed herein refer to something other than a simple bond linking the epitopes. Generally, one or more linkers will independently be an amino acid sequence, for example 2 to 20 amino acids long (such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, in particular 2,3, 4, 5 or 6). In one embodiment the linker is cleavable. In one embodiment constructs of the present disclosure comprise from 1 to 40 linkers, for example 2 to 36, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36, in particular 5, 35 or 36. Linkers suitable for use in the present constructs are disclosed herein. Additional linkers for use in the constructs of the present disclosure, such as in a construct of formula (I) and specific sequences disclosed herein comprising X include: • Proteasome dependent, for example LRA, RLRA [SEQ ID NO: 70] and a linker of formula (II): B1-W1-Y1-Z1 (II) wherein B1 is independently selected from A, R, S and P, particularly P; W1 is independently selected from D, L, I and T; Y1 is independently selected from L, G and A Z1 is independently selected from V, K and A (See Bazhan et al 2019); and an optimised site such as that described in Schubert & Kohlbacher, 2016 Genome Medicine (2016) 8:9 specifically incorporated herein by reference. In particular the cleavage site algorithm (1) on page 3 col 2, the immunogenicity model algorithm (2) also on page 3 col 2, algorithm (3) and (4) on page 4, are specifically incorporated and may be used as basis for amendments. In one embodiment a linker of formula (III) is employed in constructs of the present disclosure (including the construct of formula (I) and sequences disclosed herein comprising X): B2-W2-C0-1-K0-1-(Y2)0-1-(Z2)0-1 (III) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R. Z2 is independently selected from M, W and Y 0 means the entity is absent, and 1 means the entity is present. n one embodiment the linker of formula (III) is independently selected from: PWMW (SEQ ID NO: 107), PWNW (SEQ ID NO: 108), PWQK (SEQ ID NO: 109), PWQW (SEQ ID NO: 110), PWQY (SEQ ID NO: 111), PWRW (SEQ ID NO: 112), PWRY (SEQ ID NO: 113), PWSW (SEQ ID NO: 114), PWTW (SEQ ID NO: 115), MWCW (SEQ ID NO: 116), MWKW (SEQ ID NO: 117), WWCW (SEQ ID NO: 118), YWCYM (SEQ ID NO: 119), GWKW (SEQ ID NO: 120), SWTW (SEQ ID NO: 121), PNCKQY (SEQ ID NO: 122), PNCKRY (SEQ ID NO: 123), YWCW (SEQ ID NO: 124), GWCW (SEQ ID NO: 125), WWCY (SEQ ID NO: 126), YWCY (SEQ ID NO: 127) and SW. In one embodiment linkers are independently selected and at least one (for example 2 or more or all) is/are SW. In one embodiment a linker of formula (IIIA) is employed in constructs of the present disclosure (including the construct of formula (I) and sequences disclosed herein comprising X): B2-W2-C0-1-K0-1-(Y2)0-1-Z2 (IIIA) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R. Z2 is independently selected from W and Y 2 means the entity is absent, and 3 means the entity is present. In one embodiment the linker of formula (IIIA) is independently selected from: PWMW (SEQ ID NO: 107), PWNW (SEQ ID NO: 108), PWQK (SEQ ID NO: 109), PWQW (SEQ ID NO: 110), PWQY (SEQ ID NO: 111), PWRW (SEQ ID NO: 112), PWRY (SEQ ID NO: 113), PWSW (SEQ ID NO: 114), PWTW (SEQ ID NO: 115), MWCW (SEQ ID NO: 116), MWKW (SEQ ID NO: 117), WWCW (SEQ ID NO: 118), GWKW (SEQ ID NO: 120), SWTW (SEQ ID NO: 121), PNCKQY (SEQ ID NO: 122), PNCKRY (SEQ ID NO: 123), YWCW (SEQ ID NO: 124), GWCW (SEQ ID NO: 125), WWCY (SEQ ID NO: 126), YWCY (SEQ ID NO: 127) In one embodiment the linker or linkers has/have 2 amino acids in length, such as SW. In one embodiment the linker or linkers has/have 4 amino acids in length, such as PWMW, PWNW, PWQK, PWQW, PWQY, PWRW, PWRY, PWSW, PWTW, MWCW, MWKW, WWCW, YWCYM GWKW, SWTW. In one embodiment the linker or linkers has/have 6 amino acids in length, such as PNCKQY and PNCKRY. In one embodiment all the linkers in the fragment/construct are the same length, such as 4 amino acids in length or 6 amino acids in length, including where linkers are the same sequence or different sequences. In one embodiment the linkers in the fragment/construct are at least two different lengths, for example 2 different lengths of linkers are employed. In one embodiment the linkers are independently selected from amino acid sequences 2 and 4 amino acids in length, for example those explicitly disclosed herein. In one embodiment the same amino acid sequence is employed in the linkers of the constructs according to the disclosure. In one embodiment different amino acid sequences are employed in constructs of the disclosure, for example 2, 3, 4, or 5 different amino acid sequences are employed within the same construct, such as 2 (in particular SW & GWCW, PWRW & PWQW, or PWQY & PWRY, or MWCW & WWCW, or PNCKRY & PNCKQY), 3 (in particular SW, PWSW & PWTW) 4 or 5 different amino acid sequences (in particular YWCY, PWSW, PWTW, PWNW & WWCY or PWQY, MWKW, GWKW, SWTW & PWMW) In one embodiment the first one or two linkers at the N-terminal end of the protein are one type and the remaining linkers are a different type. In one embodiment the linker or linkers at the N terminal and C terminals are the same and the intervening linker or linkers is/are different. In one embodiment the linkers in a construct appears in blocks of the same type i.e. at least two consecutive linkers are of the same type, such as 2 to 4 consecutive linkers are the same type. In one embodiment no two consecutive linkers in a construct are the same. In one embodiment all the linkers are the same amino acid sequence. In one embodiment each X is independently selected from a linker disclosed herein, for example each X is independently selected from formula (II) (III) and (IIIA), in particular independently selected from formula (IIIA). In one embodiment the linker between each epitope is a different sequence, for example a different nucleotide sequence and/or different amino acid sequence. High redundancy as employed herein refers to levels of 0.900 or above, for example in the range 0.900 to 0.999, such as 0.905, 0.91, 0.915, 0.92, 0.925, 0.93, 0.935, 0.94, 0.945, 0.95, 0.955, 0.96, 0.965, 0.97, 0.975, 0.98, 0.985, 0.99, 0.995, in particular 0.966. In one embodiment redundancy is calculated as set out herein, for example as shown in Example 12. The compositions of the present disclosure are generally provided as a parenteral formulation, for example for intramuscular or subcutaneous delivery. Examples of formulations of compositions, including vaccines, may be found in Sweetman, S. C. (Ed.). Martindale. The Complete Drug Reference, 33rd Edition, Pharmaceutical Press, Chicago, 2002, 2483 pp.; Aulton, M. E. (Ed.) Pharmaceutics. The Science of Dosage Form Design. Churchill Livingstone, Edinburgh, 2000, 734 pp.; and, Ansel, H. C, Allen, L. V. and Popovich, N. G. Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th Ed., Lippincott 1999, 676 pp. Excipients employed in the manufacture of drug delivery systems are described in various publications known to those skilled in the art including, for example, Kibbe, E. H. Handbook of Pharmaceutical Excipients, 3rd Ed., American Pharmaceutical Association, Washington, 2000. In the context of this specification "comprising" is to be interpreted as "including". Embodiments of the invention comprising certain features/elements are also intended to extend to alternative embodiments "consisting" or "consisting essentially" of the relevant elements/features. Where technically appropriate, embodiments of the invention may be combined. Technical references such as patents and applications are incorporated herein by reference. Any embodiments specifically and explicitly recited herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments. Plural includes singular and vice versa unless the context indicates otherwise. Amendments may be based on the background section of this specification. The present application claims priority from GB2109947.8, GB2109945.2, GB2109944.5, GB2112763.4 and GB2205960.4 each incorporated by reference. Said documents may be used as basis to make corrections. The present invention is further described by way of illustration only in the following examples, which refer to the accompanying Figures. EXAMPLES This example describes the identification and selection of SARS-Cov-2 epitopes for use in multivalent vaccine compositions contemplated herein. A set of class I HLA alleles, including HLA alleles with allele frequency (in decimals) > 0.05 from two Māori populations identified in the HLA allele frequencies database (http://www.allelefrequencies.net), and HLA alleles prevalent in the global population, was developed for use in epitope selection. SARS-Cov-2 proteins were then analysed to identify suitable protein-derived epitopes including those having low mutation frequency. Epitopes were then selected for vaccine construct preparation using a combinatorial approach to provide target global population coverage. The selected epitopes and their corresponding HLA alleles is shown below in Table 1.
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
The epitopes depicted in Table 1 cover 52 unique HLA alleles present in the global population, providing a predicted 99.7% coverage of the global population. Additionally, the applicants predict, that a multivalent construct consisting of the epitopes LVQAGNVQLR [SEQ ID NO: 1], LDGISQYSLR [SEQ ID NO: 2], GVYYPDKVFR [SEQ ID NO: 3], FGADPIHSLR [SEQ ID NO: 4], STFNVPMEK [SEQ ID NO: 5], FYDFAVSKGF [SEQ ID NO: 6], and SAPHGVVFL [SEQ ID NO: 7] would provide coverage of ~94.6% of the global population. Example 2: Construct structure This example describes the preparation of a multivalent vaccine composition comprising selected SARS-CoV-2 epitopes as described above in Example 1, together with additional functional components. A general schema of the construction of the multivalent construct is shown in Figure 1 and 2, and comprises from the N-terminus of the polypeptide to the C- terminus the following functionalities: • an optional leader sequence, typically comprising an intracellular translocation domain, for example to elicit translocation of the polypeptide into the endoplasmic reticulum (ER) for proteasome-independent protein (e.g. furin-dependent) processing. Representative leader sequences associated with furin-dependent processing include the TPA activator signal sequence [SEQ ID NO: 38] and an alternative leader sequence [SEQ ID NO: 39]; • selected epitope sequences, for example the amino acid sequences set out in Table 1 above [SEQ ID NOs: 1 – 36], or the HLA A*02:01-presented SARS-CoV-2 epitope of SEQ ID NO: 37, optionally separated by spacer sequences comprising proteolytic cleavage sites. Representative linker sequences, such as the AAY spacer for proteasome- dependent processing, and the REKR [SEQ ID NO: 42] spacer for proteasome- independent/furin-dependent peptide processing, are specifically contemplated herein; • an amino acid sequence associated with enhanced immunological response or immunogenicity, such as the promiscuous Pan HLA DR-binding Epitope (PADRE) sequence [SEQ ID NO: 40]. Representative full-length multivalent vaccine constructs include: • C1_p [SEQ ID NO 42], C1_p.2 [SEQ ID NO: 43], and C1_p.3 [SEQ ID NO:44] polypeptide constructs, which are optimized for proteasome-dependent processing, have no leader sequence, and comprise the AAY spacer; • C2_f [SEQ ID NO: 45] and C2_f.2 [SEQ ID NO: 46] polypeptide constructs, which are optimized for furin-dependent processing, include the TPA leader peptide, and comprise the REKR spacer; • C3_f [SEQ ID NO: 47] and C3_f.2 [SEQ ID NO: 48] polypeptide constructs, which are optimized for furin-dependent processing with an alternative leader peptide, and comprise the REKR spacer; and • C4_pr [SEQ ID NO: 49] and C4_pr.2 [SEQ ID NO: 50] polypeptide constructs, which can be optimized for desired proteolytic processing by appropriate selection of the spacer/proteolytic cleavage site and inclusion or exclusion of the optional leader peptide. Representative cDNA coding sequences include: C1_n [SEQ ID NO: 51], C1_n.2 [SEQ ID NO: 52], C2_n [SEQ ID NO: 53], C2_n.2 [SEQ ID NO: 54], C3_n [SEQ ID NO: 55], C3_n.2 [SEQ ID NO: 56] and C1_p.3 [SEQ ID NO: 77]. Notably, the C1_p, the C1_p.2, and the C1_p.3 polypeptide constructs each target proteasome-dependent processing, but differ in the number or order of epitopes they comprise. Similarly, the furin-dependent multivalent constructs C2_f and C2_f.2 differ in the number of epitopes they comprise, as do the C3_f and the C3_f.2 constructs. Without wishing to be bound by any theory, the applicant believes that the construction of the multivalent vaccine constructs herein enables the particular epitopes included in the construct to be varied without compromising efficacy and/or population coverage. Example 3: Multivalent construct production This example describes the preparation of a prokaryotic host capable of expressing a multivalent polypeptide construct as described herein. The polypeptide construct, referred to in these examples as T2.1, is equivalent to the C2_f polypeptide construct discussed herein and presented as SEQ ID NO: 45. The genetic construct expressing T2.1 is referred to in these examples sometimes as CVC4. A plasmid (CVC4_pET9-T2-phaC) containing the C2_f polypeptide construct was used as a template. Primers were designed to amplify the coding region and introduce a C-terminal His6 tag for cloning into expression vector pET22b. Primers T2F (5’-TAAGAAGGAGATATACATATGGATGCTATGAAAAGGGGACTATGC [SEQ ID NO. 57]) and T2R (5’-CTCGAGTGCGGCCGCAAGCTTATCAGTGATGGTGATGG TGGTGAGAGCCTCCACCGCCAGAGC [SEQ ID NO. 58]) were used to amplify the coding region using CloneAmp MasterMix (Takara, #639298) according to the manufacturer’s instructions with a 1/500 dilution of plasmid diluted in water as a template. 10 µL of PCR product was run on a 1% agarose gel (1× TAE) until separated. Bands of an appropriate size (1780bp) were visualised and excised on a DarkReader blue light table before purification using a NucleoSpin Gel and PCR Purification kit (Macherey-Nagel #740609) according to manufacturer’s instructions. Cloning into pET22b was performed with an In-Fusion® HD Cloning kit (Takara 638910) using 1 µL of linearised pET22b (NdeI/HindIII) and 1 µL of purified PCR product to produce plasmid pET-22b-CVC4-T2-His6 before transformation into E. coli Stellar cells, according to manufacturer’s instructions. Selection was on LB agar+100 µg/mL carbenicillin (LB/carb). The selected CVC4-encoded construct (T2-His6) have the following characteristics: Length: 579 aa; Molecular weight: 69.482 kDa; Isoelectric point: 11.30; Charge at pH 7: 77.12; Extinction Coefficient: 59,610 For creation of an organism capable of expressing T2.1, construct CVC4 plasmid DNA (1 µL) was transformed into E. coli BL21-AI (ThermoFisher C607003) according to manufacturer’s instructions and grown on LB agar/carb overnight. Example 4: Production of vaccine containing T2.1 protein conjugate This example describes the production of a multivalent polypeptide construct conjugate, comprising the T2.1 multivalent construct conjugated to a phaC polymer particle-forming protein, in ClearColiTM BL21 (DE3) using a kanamycin resistant T2.1 phaC plasmid and tetracycline resistant phaA / phaB plasmid, and the resulting production of T2.1 biobeads – a representative example of the polypeptide conjugates contemplated herein. A tetracycline resistant phaA and phaB plasmid was created containing the phaA and phaB genes from Cupriavidus necator downstream of a lac promoter. A chloramphenicol resistant (CmR) plasmid containing phaA and phaB genes is called pMCS69. An IDT g-block was designed to contain the tetracycline resistance promoter along with the tetR gene from pBR322 as a fragment. The pMCS69 backbone fragment and the promoter-TetR gBlock were assembled with NEBuilder. The tetR fragment was inserted into the 6,116 bp pMCS69 fragment using the Gibson/Infusion/homology cloning protocol and transformants plated on Tet agar plates. This resulted in many Tetracycline resistant colonies. Plasmid from several clones were isolated and the tetR, phaA and phaB (and promoter) were sequences and confirmed to be correct. The resulting plasmid has been named “p69Tet”. A kanamycin resistant plasmid containing the phaC gene from Cupriavidus necator was created (WT phaC plasmid). A gene for phaC protein was codon optimized, synthesized by Genscript and inserted in to the NdeI and BamHI cloning sites of plasmid pET-9a. A T7 pET- based kanamycin resistant plasmid expressing T2.1 as a N-terminal translational fusion to phaC from Cupriavidus necator was created. The T2.1 antigen was cloned such that it was translationally fused to the phaC via a SGGGGSGGGGS linker [SEQ ID NO: 69]. The T2.1 sequence was codon optimized and translationally fused to phaC. The kanamycin resistant WT phaC plasmid was used as a backbone for constructing the T2.1-linker-phaC plasmid. The amino acid sequence of the T2.1-phaC polypeptide is presented herein as SEQ ID NO: 65. Plasmid p69Tet was electroporated into electrocompetent ClearColi® BL21 (DE3) strains obtained from Lucigen, according to the manufacturer’s instructions and selected on MTB agar with 10 µg/ml tetracycline. These cells were used to make a batch of electrocompetent cells (CCPT1E) used for the electroporation of T2.1-linker-phaC plasmid. A T2.1-phaC biobead producing organism was created by electroporation of the kanamycin resistant T2.1-linker- phaC plasmid into the ClearColi® cells already containing p69Tet. Transformed cells were selected by growth on media containing kanamycin and Tetracycline. To produce T2.1-phaC biobeads, transformed cells were inoculated into Modified Terrific Broth with 1% NaCl and 1% glucose, tetracycline and kanamycin and allowed to grow at 37°C with shaking until an OD600nm of 1.0 was achieved, after which 0.5 mM IPTG was added, and the temperature dropped to 25°C. The cultures were grown for a further 48 h after which they were harvested. Cells were harvested by centrifugation, resuspended in 50 mM Tris, 10 mM EDTA, 0.08% SDS, pH 11 and passed through a microfluidizer twice. Insoluble material containing biobeads was sedimented at 15,000 g for 20 min and washed once with 50 mM Tris, 150 mM NaCl, 1 mM EDTA. The isolated bead material was subjected to SDS-PAGE on NuPage bis-tris 4-18 % gels with MOPS buffer and stained with Coomassie Blue. A band of the correct molecular weight for the T2.1-linker-phaC construct was observed. Tryptic digest and mass spectroscopy analysis of the peptides identified the fusion protein as T2.1-phaC. A similar preparative strategy was employed to prepare a T1.1 [SEQ ID NO: 66] multivalent construct conjugated to a phaC polymer particle-forming protein, in ClearColiTM BL21 (DE3) using a kanamycin resistant T1.1 phaC plasmid and tetracycline resistant phaA / phaB plasmid, and the resulting production of T1.1 biobeads – another representative example of the polypeptide conjugates contemplated herein. As for T2.1-phaC above, the T1.1 antigen was cloned such that it was translationally fused to the phaC via a SGGGGSGGGGS linker [SEQ ID NO: 69]. The T1.1 sequence was codon optimized and translationally fused to phaC. Further representative multivalent vaccine conjugates were prepared using the amino acid sequences of the T2.2 [SEQ ID NO: 67] and T1.2 [SEQ ID NO: 68] multivalent constructs conjugated as described above to a phaC polymer particle-forming protein. The codon optimized T2.2 and T1.2 sequences were thus translationally fused to phaC, then expressed and purified as described above. The amino acid sequence of the T2.2-phaC polypeptide is SEQ ID NO: 67, and the amino acid sequence of the T1.2-phaC polypeptide is SEQ ID NO: 69. To produce vaccine containing multivalent vaccine conjugates as biobeads, the following steps were followed. Transformed cells were grown in 100L fermenters. Media was prepared and autoclaved in the fermenter. Media component and concentrations were as follows: pea hydrolysates, 12g/L; yeast extract, 24 g/L; NaCl, 10 g/L; K2HPO4, 8 g/L; glycerol, 30 g/L. Additions added when media had cooled were as follows: pluronic antifoam, 0.02 ml/L; tetracycline, 0.01 g/L; kanamycin, 0.05 g/L. The fermenter was inoculated from pre-culture flasks grown at 37oC. Fermenter temperature was controlled at 25oC. Dissolved oxygen was controlled at 30% air saturation. The fermenter was maintained at pH 6.8. Expression of multivalent vaccine conjugates as biobeads was induced using 1mM IPTG. After 20 hours of induction, the temperature was adjusted to 15 °C. Cells were harvested into a 50mM Tris, 10mM EDTA, pH 7.5 buffer by tangential flow filtration using a 500KDa MWCO membrane (Cytiva, UFP- 500-E). Cells were diluted in buffer containing 50mM Tris, 10mM EDTA, 0.5% Brij58, pH 7.5 and lysed by multiple passes through a microfluidizer (Microfluidics, M110P) at 25,000 psi. Biobeads were sedimented by centrifugation and washed several times with the same buffer by resuspension and sedimentation. Biobeads were further purified by resuspension in 0.1N NaOH containing 0.01% Brij58 and subsequent sedimentation and resuspension in buffer containing 25 mM Tris, 10mM EDTA, pH 10, and concentrated by tangential flow filtration using a 0.1 micron porosity membrane (Cytiva, CFP-1-E). Purified biobeads were placed into a monodispersion by passing the suspension through a microfluidizer (Microfluidics, M110P) at 25,000 psi. Vaccine containing multivalent vaccine conjugates as biobeads was formulated to contain different doses of antigen. Sterile adjuvant was added to biobead suspension Vaccine was aseptically filled into pharmaceutical vials (Type 1 borosilicate glass), with butyl rubber stoppers and aluminium crimp seals. Vials of vaccine containing multivalent vaccine conjugates as biobeads were stored at 2 – 8 °C. Example 5: Production of vaccine containing multivalent polypeptide This example describes the production of a vaccine comprising soluble T2.1 HisTag multivalent polypeptide construct as described herein. E.coli BL21-AI-CVC4 was grown in 10L or 100L fermenters. Media was prepared and autoclaved in the fermenter. Media component and concentrations were as follows: pea hydrolysates, 12g/L; yeast extract, 24 g/L; NaCl, 5 g/L; K2HPO4, 6 g/L; glycerol, 10 g/L; MgSO4.7H2O, 2.5 g/L. Additions added when media had cooled were as follows: pluronic antifoam, 0.2ml / 10L; Carbenicillin, 1g / 10L. The fermenter was inoculated from pre-culture flasks grown at 37oC, for 3 hours. Temperature was controlled at 37oC for initial growth phase, and then cooled down to 21oC for induction. Dissolved oxygen was controlled to 85% air saturation and then reduced to 30% for induction. The fermenter was maintained at pH 6.8. A media bolus was prepared and autoclaved for 15 minutes at 121oC. Bolus composition was glycerol, 25% (w/v); yeast extract, 12% (w/v). Bolus was added into the bioreactor at T = 4.5 hours. Expression of T2.1 antigen was induced at OD600 = 2.2 using 2g/L arabinose and 0.5mM IPTG. The biomass was harvested by centrifugation approximately 20 hours post induction. Biomass was frozen at -20oC until further processing. Biomass was thawed overnight and resuspended in lysis buffer (50mM Tris pH 7.5), using a Miccra D-9 rotor-stator. Lysis was performed using a Microfluidics, M110P microfluidiser M110P. Process pressure was 25,000 PSI, and 3 complete passes were performed. Insoluble material was collected by centrifugation at 12,000g for 30 minutes. The pellet (i.e., “insoluble” fraction) was collected for further processing. The lysate pellet (i.e., ‘insoluble’ fraction) was subjected to solubilisation in 1% SDS with sonication on ice. The solubilised fraction containing T2.1 was centrifuged to remove debris. The solubilised fraction was diluted into IMAC loading conditions (50mM Tris pH 8.4, 500mM NaCl, 0.2% SDS). The diluted material was loaded onto a 20mL His-Prep column, with a 5mL His-Trap column attached (i.e., 25mL total IMAC column volume). The IMAC columns were washed with loading buffer (50mM Tris pH 8.4, 500mM NaCl, 0.2% SDS), and T2.1 recovered with elution buffer (50mM Tris pH 8.4, 500mM NaCl, 400mM Imidazole, 0.2% SDS). Elution fractions were analysed by SDS-PAGE for presence of T2.1, and fractions found to contain T2.1 protein were pooled. Pooled elution fractions were concentrated and buffer exchanged to remove imidazole in a 5,000 molecular weight cut off (MWCO) centrifugal concentrator. Concentrated T2.1 fraction was subjected to endotoxin removal using Sartobind-Q membrane filter capsules. The T2.1 fraction was passed through a 0.45µm membrane disk filter followed by the Sartobind-Q capsule and then a 0.2 µm membrane disk filter. Vaccine containing T2.1 was formulated to contain approximately 0.11% SDS. Adjuvant was added. Vaccine was aseptically filled into pharmaceutical vials (Type 1 borosilicate glass), with butyl rubber stoppers and aluminium crimp seals. Vials of T2.1 vaccine were stored at 2 – 8 °C. Example 6: In vivo immunogenesis This example describes an evaluation of the immunogenic effect of the T2.1 multivalent construct in humanised mice, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Cytokine-supplemented humanised mice were injected with T2.1 multivalent construct vaccine at 10μg and 50μg dose, with adjuvant at Day 0 and Day 21 before being terminated for downstream pentamer staining analysis to evaluate COVID-19 specific CD8+ T cell response at Day 28. Methods The materials and reagents used in this example were as follows:
Figure imgf000034_0001
The animal experiment was conducted according to the Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485]. Up to five mice of the same gender were housed in a cage. Each mouse was identified using ear punching identification system and grouped as follows: Group 1: T2.1 10μg, adjuvant (n = 7); Group 2: T2.150μg+adjuvant (n = 7); Group 3: adjuvant (n = 7). All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study. Humanized mice were generated by injecting sub-lethally irradiated immune-deficient pups with CD34+ cordblood cells via intrahepatic route. Mice were submandibularly bled at 12 weeks post-engraftment to determine the levels of human immune reconstitution via flow cytometry. Mice with more than 10% human immune cell reconstitution (calculated based on the proportion of human CD45 relative to the sum of human and mouse CD45) in the peripheral blood were used in this study. Inoculant preparation and Dosing regimen Except for LPS, inoculants for Groups 1 to 3 were prepared as above. Mice from Group 1-3 were given the first inoculation on Day 0, followed by a booster on Day 21 post injection. At Day 21, mice from Group 1 and 2 were injected with T2.1 10μg, adjuvant. Details of dosing regimen for each inoculant used in the study was listed in Table 3. Mice from Group 1-3 were injected with 150μl per inoculant via intraperitoneal route. Table 3: Doses used for each inoculant during study.
Figure imgf000035_0001
Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for 45 mins before processed to serum and stored in -80oC. Isolation of splenocytes for Pentamer staining analysis At endpoint, spleen was collected, meshed through 70μm strainer with 5ml of completed RPMI media containing 10% FBS. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco). The number of splenocytes was counted using trypan blue staining before Pentamer staining. Pentamer staining analysis using FACS Pentamer staining was performed on 6×105 mouse splenocytes from Group 1 to 3 mice using a 3-layer approach with the panels of antibodies listed in Table 4 below. Table 4: FACS panel for immune profiling at endpoint.
Figure imgf000035_0002
Figure imgf000036_0001
Briefly, splenocytes were first stained with live/dead and human Fc receptor block (BD; 564220) and incubated at room temperature for 10 mins. After which, splenocytes from each mouse were stained with three individual pentamers-containing panels (P1, P2 or P3, 10μl per peptide-loaded pentamer) and incubated for 15 mins at room temperature. Cells were washed with FACS buffer containing PBS, 0.2% bovine serum albumin (GE Healthcare) and 0.05% sodium azide (Merck) and stained with antibodies in the core panel for 30 mins at 4 °C. Streptavidin BV421 (2μl) was added to cells stained with panel containing biotinylated pentamer (P1). After washing, cells were fixed in 4% paraformaldehyde (Mouse FoxP3 buffer set; BD 5604094) for 15 min at 4 °C. Cells were then washed and data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analyzed using FlowJo software (version 10; Tree Star Inc). All graphs were plotted using GraphPad Prism 8.0 software (GraphPad Software Inc). Results Pentamer immune profiling analysis of splenocytes at endpoint To detect for the presence of antigen-specific CD8+T cells, splenocytes from Group 1-3 were stained with core antibodies and pentamers stated in Table 4 in three individual panels. Positive pentamer staining was detected in A*02:01-ALWEIQQVV and B*35:01-LPFFSNVTW (Figure 3), for Group 2 mice at were notably higher than the basal signal observed in control (adjuvant only) Group 3 mice. The results indicate that T2.1 multivalent construct did modulate the immune profiles in antigen-specific CD8+ T cell response in Group 2 (the high dose test group). The up- modulation demonstrated that the antigen induces a specific human cytotoxic immune response likely to lead to immunity to COVID-19 infection. Example 7: in vivo immunogenesis using biobead delivery This example describes an evaluation of the immunogenic effect of the T1.1 and T2.1 multivalent construct on biobeads in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Mice were injected with multivalent biobead vaccines at 4μg and 12μg doses, without adjuvant at Day 0, 1, 21 and 22 before being terminated for downstream pentamer staining analysis to evaluate COVID-19 specific CD8+ T cell response at Day 29. The effective initial and booster doses were 8μg and 24μg Methods The materials and reagents used in this example were as follows: Table 5: Materials and reagents
Figure imgf000037_0001
Figure imgf000038_0001
The animal experiment was conducted according to the Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485]. Up to five mice of the same gender were housed in a cage. Each mouse was identified using ear punching identification system and grouped as follows: Group 1: T1.1 biobead, 8μg antigen(n = 5); Group 2: T2.1 biobead, 8μg antigen (n = 5); Group 3: T1.1 biobead, 24μg antigen (n = 5); Group 4: T2.1 biobead, 24μg antigen (n = 5); Group 5: naked biobead (no antigen) (n = 5); Group 6: HPV positive control peptide, 50μg (n=5, given only on days 1 and 21). All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study. Inoculant preparation and Dosing regimen Groups 1 to 6 were prepared as above. Details of dosing regimen for each inoculant used in the study was listed in Table 3. Mice from Group 1-6 were injected with 200μl per inoculant via intraperitoneal route as in table 6. Table 6: Doses used for each inoculant during study.
Figure imgf000038_0002
Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for 45 mins before processed to serum and stored in -80oC. Isolation of splenocytes for Pentamer staining analysis At endpoint, spleen was collected, meshed through 70μm strainer with 5ml of completed RPMI media containing 10% FBS. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco). The number of splenocytes was counted using trypan blue staining before Pentamer staining. Pentamer staining analysis using FACS Pentamer staining was performed on 1 × 106 mouse splenocytes using a 2-layer approach with the panels of antibodies listed in Table 7. Table 7: FACS panel for immune profiling at endpoint.
Figure imgf000039_0001
Briefly, splenocytes were first stained with live/dead (Life Technologies) and mouse Fc receptor block (BD Bioscience) and incubated at room temperature for 10 mins. After which, splenocytes from each mouse were stained with two individual pentamers-containing panels (Panel 1 or Panel 2, 5μl per peptide-loaded pentamer). Pentamer panels are listed in Table 7, specifically No. 11-15 inclusive and incubated for 15 mins at room temperature. Cells were washed with FACS buffer containing PBS, 0.2% bovine serum albumin (GE Healthcare) and 0.05% sodium azide (Sigma) and stained with antibodies in the core panel at 1:200 dilution in 50μl staining volume for 30 mins at 4°C. Core antibody panels are listed in Table 6, specifically No. 1-9 inclusive. Streptavidin BV421 (2μl) was added to cells stained with panel containing biotinylated pentamer (Panel 1). After washing, cells were fixed in 4% paraformaldehyde (Mouse FoxP3 buffer set; BD) for 15 mins at 4°C. Cells were then washed with FACS buffer and data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software, and analyzed using FlowJo software (version 10; Tree Star Inc). To detect for the presence of antigen-specific CD8+ T cells, splenocytes from Group 1-6 were stained with core antibodies and five HLA-A*02:01 peptide loaded pentamers stated in Table 6 in two individual panels. Briefly, the gating strategy was as follows. After removal of doublets and dead cells, the levels of mouse CD45+ leucocytes were determined using the mouse CD45 gating. From the mouse CD45+ leucocytes, the following leucocyte subsets were gated: B cells (CD45+/CD19+), CD4+ T cells (CD45+/CD3+/CD4+), CD8+ T cells (CD45+/CD3+/CD8+), naïve T cells (CD45+/CD3+/CD4+ or CD8+/CD62L+/CD44-), central memory (CM) T cells (CD45+/CD3+/CD4+ or CD8+/CD62L+/CD44+), effector memory (EM) T cells (CD45+/CD3+/CD4+ or CD8+/CD62L-/CD44+), and effector T cells (CD45+/CD3+/CD4+ or CD8+/CD62L-/CD44-). The expression levels of CD69, a T cell activation marker, was evaluated on both CD4+ and CD8+ T cells, while levels of individual HLA-A*02:01 peptide-loaded pentamer was evaluated on CD8+ T cells. A CD19/CD3 double negative immune subset (“others”), consisting of natural killer cells (NK), dendritic cells (DCs), myeloid (neutrophils and monocytes), was also included in the analysis. Results Pentamer immune profiling analysis of splenocytes at endpoint To detect for the presence of antigen-specific CD8+T cells, splenocytes from Group 1-6 were stained with core antibodies and pentamers stated in Table 6 in three individual panels. Despite being at the limit of detection, positive pentamer staining was detected for Group 1 with A*02:01-ALWEIQQVV and A*02:01 - YLFDESGEFKL (Fig. 4) significantly above background. It is noteworthy that positive pentamer staining for these two peptides was also seen in EG.6. The results suggest that T1.1 multivalent construct did modulate the immune profiles in antigen-specific CD8+ T cell response in Group 1. The up-modulation supports that the antigen induced a specific human cytotoxic immune response likely to lead to immunity to COVID-19 infection. ELISPOTs Mouse IFN-γ ELISPOT was performed using the kits from Mabtech. 2 x 105 of splenocytes from each mouse were plated in the pre-coated ELISPOT plate and re-stimulated with the peptide pool [5μM per peptide, specifically no.1 – 8 inclusive (Table 7)] for approximately 32 hours. Re- stimulations of pooled splenocytes with either 5μg/ml of Concanavalin A (T cell activator; Sigma), 5μM of EBV peptide (GLCTLVAML), or 5μM of HPV peptide (VQSTHVDIRTLEDLLMGTLGIVCPI) were also included as positive and negative controls, respectively. The plates were then developed according to manufacturer’s instructions. The wells were imaged and number of Spot Forming Units (SFUs) counted on the CTL Immunospot® Analyzer (BioSpot® Software version 7.0). Data were expressed as SFUs per million cells after subtracting the background counts (determined from the negative controls). Table 8. Peptide pool/controls for ELISPOT analysis.
Figure imgf000041_0001
All graphs were plotted using GraphPad Prism 8.0 software (GraphPad Software Inc). Pairwise comparison was performed using Mann-Whitney U test (2-tail) and p values below 0.05 are considered statistically significant. Specifically, the groups receiving T1.1 and T2.1 antigen (i.e. G1, G2, G3, G4) were compared to G5 (“Naked” biobead). Results To evaluate the levels of antigen-specific CD8+ T cells response, splenocytes were re- stimulated with the peptide pool ex vivo and their recall responses detected by direct and indirect mouse IFN-γ ELISPOT analysis. In parallel, re-stimulations of pooled cells with concanavalin A (T cell activator) or EBV peptide (an irrelevant peptide) were also included as positive and negative controls, respectively. As seen from Figure 4, there were detectable levels of IFNγ SFUs (averaging at about 120 SFUs per million cells) from the peptide pool-restimulated splenocytes and enriched CD8+ T cells from G1 and G3. Similarly, T2.1 biobeads also induced positive CD8+ T cell responses in G2 and G4 mice in which approximately 60 IFNγ SFUs per million cells were also detected. Overall, significantly higher numbers of IFNγ SFUs were detected in the HLA-peptide pool re-stimulated splenocytes/CD8+ T cells from G1-G4, when compared to that in the re- stimulated cells from the control groups, G5 and 6 (Figure 5). This thus confirms the induction of HLA-A1 peptide-specific CD8+ T cell responses in the mice that had been immunised with T1.1 or T2.1 biobeads. Comparatively, T1.1 biobeads induced greater peptide-specific response than T2.1 biobeads. Between the low and high dose groups, there is no strong dose-dependent effect as majority of the mice in the high dose groups (G3 and G4) had similar levels of IFNγ SFUs as their corresponding low dose groups (Figure 4). This suggests a maximal immune response is achieved at the lower dose tested. Example 8: Highly redundant multivalent constructs This example describes the selection of SARS-CoV-2 epitopes for use in multivalent vaccine constructs as contemplated herein to provide redundancy in HLA coverage and to mitigate against vaccine escape. Global population coverage and HLA redundancy was calculated (using the IEDB population coverage online service (http://tools.iedb.org/population/) for the following multivalent constructs comprising various subsets of the epitopes selected in Example 1 above. Various exemplary multivalent constructs are presented below in Tables 9 to 11. Table 9. Global coverage achieved with short multivalent constructs
Figure imgf000042_0001
As can be seen in Table 10, a substantial increase in global coverage is achieved via the inclusion of selected additional SARS-CoV2 epitopes in accordance with this disclosure. Notably, a predicted global coverage of almost 95% is achieved using the seven epitopes identified in Table 9 above. Advantageously, the addition of the 29 further epitopes set out in Table 1 has a meaningful impact on global coverage, raising predicted coverage to ~99.75%. Importantly, the epitopes and constructs contemplated herein provide substantial redundancy in HLA coverage. As shown in Table 6 below, the exclusion of one or a subset of epitopes from the construct has a limited impact on both HLA coverage and predicted global coverage. Table 10. Redundancy in HLA coverage
Figure imgf000042_0002
Figure imgf000043_0001
Without wishing to be bound by any theory, the applicants believe such epitopic redundancy is expected to mitigate against vaccine escape. Further, the epitopes and constructs contemplated herein provide substantial redundancy in global coverage. As shown in Table 11 below, the exclusion of an epitope capable of binding to or predicted to bind to a given HLA class 1 variant is predicted to have a limited impact on global coverage. Table 11. Redundancy in global coverage
Figure imgf000043_0002
Notably, as can be recognised for example from the data presented in Table 11, the vaccine constructs contemplated herein are highly redundant. The removal of a single epitope from the construct has minimal impact on predicted global population coverage achieved by the resulting variant construct. For example, the removal of epitope VYMPASWVMRI, which has the largest impact on predicted global coverage, reduces predicted global coverage by only ~0.25%. Removal of any one of the other epitopes presented in Table 11 has a less detrimental impact on predicted global coverage. These data support the utility of the highly redundant vaccine constructs contemplated herein. Example 9 Design of Optimised Peptides with Bespoke Linkers Ten peptides (SEQ ID NO: 78 to 87 were derived using algorithms whereby the individual peptides are re-ordered and bespoke linkers are computed. Scores are compared above with the starting polypeptide, T1.2 (SEQ ID NO: 44). Three peptides with high predicted recoveries were chosen for testing: • one of the least disordered, and • one of the most disordered, and • one with a disorder between least and most The results are shown in Figure 6. Example 10: in vivo immunogenesis using biobead delivery This example describes an evaluation of the immunogenic effect of the T1.1 and T1.2 multivalent construct on biobeads in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Mice were injected with multivalent biobead vaccines at 0.5μg, 2μg and 8μg doses, with adjuvant at Day 0 and 21 before being terminated for downstream analysis to evaluate COVID- 19 specific CD8+ T cell response at Day 28. Methods The materials and reagents used in this example were as follows: Table 12 Materials and reagents
Figure imgf000044_0001
Figure imgf000045_0001
The animal experiment was conducted according to the Singapore National Advisory Committee for Laboratory Animal Research (NACLAR) guidelines and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485]. Up to five mice of the same gender were housed in a cage. Each mouse was identified using ear punching identification system and grouped as follows: Group 1: T1.1 biobead, 8μg antigen(n = 6); Group 2: T1.2 biobead, 8μg antigen (n = 6); Group 3: T1.1 biobead, 2μg antigen(n = 7); Group 4: T1.2 biobead, 2μg antigen (n = 7); Group 5: T1.1 biobead, 0.5μg antigen(n = 6); Group 6: T1.2 biobead, 0.5μg antigen (n = 6); Group 7: naked biobead (no antigen) (n = 6); Group 8: HPV positive control peptide, 50μg (n=6). All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12 hour light-dark cycle with ad libitum access to normal diet and drinking water during the course of study. Inoculant preparation and Dosing regimen Groups 1 to 8 were prepared as above. Details of dosing regimen for each inoculant used in the study are listed in Table 13. Table 13: Doses used for each inoculant during study.
Figure imgf000045_0002
Figure imgf000046_0001
Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for 45 mins before processed to serum and stored at -80oC. ELISPOTs Mouse IFN-γ ELISPOT was performed using the kits from Mabtech.2 x 105 of splenocytes from each mouse were plated in the pre-coated ELISPOT plate and re-stimulated with the peptide pool [5μM per peptide, specifically no. 1 – 8 inclusive (Table 14)] for approximately 32 hours. Re-stimulations of pooled splenocytes with either 5μg/ml of Concanavalin A (T cell activator; Sigma), 5μM of EBV peptide (GLCTLVAML), or 5μM of HPV peptide (VQSTHVDIRTLEDLLMGTLGIVCPI) were also included as positive and negative controls, respectively. The plates were then developed according to manufacturer’s instructions. The wells were imaged and number of Spot Forming Units (SFUs) counted on the CTL Immunospot® Analyzer (BioSpot® Software version 7.0). Data were expressed as SFUs per million cells after subtracting the background counts (determined from the negative controls). Table 14. Peptide pool/controls for ELISPOT analysis. No. Peptide pool/controls Sequence/Antibody
Figure imgf000046_0002
All graphs were plotted using GraphPad Prism 9.1.2 software (GraphPad Software Inc). For simplicity, the graphs for FACS results only show the values of G7 obtained with FACS panel 1. Pairwise comparison was performed using Mann-Whitney U test (2-tail) and p values below 0.05 are considered statistically significant. Specifically, all the groups were compared to the control group, G7 (“Naked” biobead, Alum+ODN1826). In addition, for FACS results, the statistical significance of G8 was obtained by comparing it with the values of G7 obtained with same FACS panel (i.e. panel 2). Results To evaluate the levels of antigen-specific CD8+ T cells response, splenocytes were re- stimulated with the peptide pool containing all eight HLA-A2:01 peptides ex vivo and subjected to mouse IFN-γ ELISPOT analysis. In parallel, re-stimulations of pooled cells with concanavalin A (T cell activator) or EBV peptide (an irrelevant peptide) were also included as positive and negative controls, respectively. As seen from Figure 7, there were significantly higher levels of IFN-γ SFUs from the peptide pool-restimulated splenocytes from all the mice receiving either T1.1 or T1.2 biobeads when compared to that of the G7 negative controls. The median IFN-γ SFUs for the sample groups range from about 26 for G6 to about 75 for G1 while that for the control G7 and G8 groups are 6 and 0 respectively. On the other hand, the HPV peptide- immunized group (G8) did not respond to the re-stimulation with the peptide pool but had detectable levels of IFN-γ SFUs upon re-stimulation with the HPV peptide. Taken together, the results indicate the presence of positive HLA-A2:01 peptide-specific CD8+ T cell responses in mice that were immunized with the biobeads. When comparing the magnitude of responses induced by the different doses, there seemed to be a possible dose-dependent effect for the immunization with T1.1 biobeads. Splenocytes from G1 mice (i.e. immunized with the highest dose of 8μg) generally responded with higher levels of IFN-γ SFUs when compared to the lower dose groups (i.e. G3 and G5). Example 11: Production of vaccine constructs using bespoke cleavable linkers The design of optimal linkers or spacers for a multi-epitope T cell vaccine has been described by Schubert and Kohlbacher, Genome Medicine (2016) 8:9. Using their described algorithm, it is possible to design linkers that will cleave optimally. Optimal cleavage has the advantage of maximising the amount of peptide availability for MHC class I display, minimising partial cleavage and preventing neo-epitope formation. Individually designed linkers were designed for Seq IDs 1 to 37 with the order of epitopes rearranged compared to SEQ ID 44. Five alternative sets of linkers were designed and designated T3, T 4, T5, T6 and T7 (SEQ ID NO: 100-105). In summary, the iterative approached used is as follows: • Analysis with Epytope (github.com/Kohlbacherlab/epytope) • Spacer Design with OptiVac (github.com/SchubertLab/OptiVac) • Reshuffling derivative of model published in Dorigatti, E., & Schubert, B. (2020). PLoS Comp. Bio, 16(10), e1008237. • Optimal arrangement with Epytope • Proteasomal Cleavage Prediction with: PCM • Add secondary structure prediction: PSIPred • Generation of the top 10 top-scoring designs per configuration • Added cleavage prediction for N-terminal fMet cleavage based on the published human/E. coli MetAP1/2 cleavage probabilities The characteristics of the chosen constructs is as follows: Cleavage Score (C terminal): The average cleavage score at the C-terminal ends of epitopes and spacer. Range [-23, 3.04]; more positive is better. Spacer Cleavage Score (C spacer): The average predicted cleavage score of C-terminal cleavage sites within a spacer sequence. Range [-23.59, 3.44]; more negative is better
Figure imgf000048_0001
Furthermore, in order to improve levels of expression, the epitopes (SEQ ID NOs 1 to 37) were distributed into six separate oligopeptides designated Fragments 1 to 6 (SEQ ID NO 89- 94). Fragment 4 includes the PADRE sequence. Bespoke linkers were designed as above for each of the six fragments as above with the following characteristics:
Figure imgf000048_0002
Genes coding for Fragments 1 - 6 (SEQ ID NO: 88 to 93) were cloned into the expression vector pD451, containing T7 inducible promoter. Purified plasmid containing the genes to be tested was transformed into chemically competent BL21(DE3) cells via heat shock and plated on non-inducing agar with 100 mg/L kanamycin. Plates were incubated overnight at 37°C. One colony from each transformation was picked and grown into 0.82 mL of a non-inducing medium containing 100 mg/L kanamycin, then incubated overnight at 37°C. Overnight culture was inoculated into 0.6 mL of auto-induction media containing 100 mg/L kanamycin. Three sets of auto-induction media were inoculated from the same starter culture and incubated at 30°C overnight (24 hours). Cells were harvested by centrifugation and frozen. Cell pellets were lysed and then centrifuged to separate total and soluble fractions. Total and soluble protein fractions were denatured and run on polyacrylamide gels under reduced conditions and Coomassie stained. All six Fragments expressed at high levels detectable in total cell fractions (Figure 9). Example 12: Production of mRNA vaccine constructs DNA template preparation Fully synthesized constructs (5’UTR-ORF-3’UTR-polyA) were cloned into the pIVTScrip™ template backbone plasmid for T1.2, T3, T4, T5, T6 and T7 (SEQ ID NO: 100-105). Sequence were verified by Sanger sequencing. The DNA template then linearized. Template plasmids were purified by phenol/chloroform extraction followed by ethanol precipitation to remove the enzymes and reaction components. Preparation Nucleotide triphosphates (NTPs), 10× IVT buffer, and linearized plasmid transcription template were brought to room temperature. NTPs and 10× IVT buffer were vortexed until homogenous. Repeat warming and mixing was performed until no precipitate was observed. In vitro transcription reaction The components in the table below were combined in RNase-free tubes. For a 1-ml reaction, the quantities listed in the table were used.
Figure imgf000049_0001
Each reaction was mixed using gentle pipetting until homogenous. Tubes were then sealed with parafilm and incubated at 37°C in a water bath or heat block for 2-3 hr. Thaw 10× DNase I reaction buffer and vortexed followed by DNase I and mixed gently until homogenous using the following amounts or equivalent ratios:
Figure imgf000049_0002
The reaction was incubated at 37°C for a minimum of 15 minutes. Purification LiCl was added to the IVT reactions to achieve a final concentration of 2.5 M. The preparations were then mixed by gentle inversion or swirling before chilling at −20 °C for at least 30 min. Preparations were then centrifuged for 30 min at 18,500 × g at 4°C. Pellets from the centrifugation were washed in −20 °C ethanol. The preparation was centrifuged, once again, for 5 minutes at 12,000 × g at 4 °C. The ethanol was replaced, and centrifugation was then repeated. Pellets were then dried for 5 to 10 minutes. The presence of RNA was confirmed by gel analysis. LNP encapsulation LNPs were prepared by mixing appropriate volumes of lipid stock solutions in ethanol with an aqueous solution of mRNA employing a microfluidic micromixer. Preparation mRNA was dissolved in 25 mM sodium acetate buffer, pH 4.0. The following molar ratios of lipids were dissolved in ethanol:
Figure imgf000050_0001
LNP encapsulation of mRNA 1 volume of the lipid preparation was mixed with 3 volumes of mRNA solution in the mixer using a syringe pump. A flow rates at 0.5 mL/min for the lipid/ethanol stream and 1.5 mL/min for the mRNA/aqueous stream was used to drive a combined flow rate of 2 mL/min through a microfluidics micromixer. The output was dialyzed for 4 h against 1000 volumes of 50 mM MES/50 mM sodium citrate buffer (pH 6.7). Subsequently the preparation was dialyzed overnight against 1000 volumes of 1× phosphate buffered saline, pH 7.4. Quality was confirmed by measuring particle size analysis by dynamic light scattering, polydispersity index by dynamic light scattering, mRNA concentration using RiboGreen and presence of prokaryotic contamination. Example 13: Redundancy Calculation This example describes a metric to estimate the degree of redundancy, in terms of HLA coverage, that a multipeptide vaccine could have when subjected to multiple point mutations. This HLA redundancy score is then calculated for five SARS-CoV-2 published vaccine compositions is addition to design described in this specification. It is assumed that these simulated mutations render the resulting peptides useless. Therefore, the mutated peptides are removed from their corresponding list. The global HLA coverage of such a modified vaccine is subsequently determined using the IEDB coverage prediction tool (http://tools.iedb.org/population/). This simulation is repeated 10 times for each vaccine composition and the resulting global coverage is reported (with statistics such as median, mean and standard deviation). This procedure allows the evaluation of the HLA coverage robustness in vaccine compositions when subjected to multiple single point mutations in the SARS-CoV-2 genome. The five published vaccines designs analysed are Adam K. M., 2021 (D1), Almofti, Y. A., Abd-Elrahman, K. A., & Eltilib, E., 2021 (D2), Kar, T., Narsaria, U., Basak, S., Deb, D., Castiglione, F., Mueller, D. M., & Srivastava, A. P., 2020 (D3). Cun, Y., Li, C., Shi, L., Sun, M., Dai, S., Sun, L., Shi, L., & Yao, Y., 2021 (D4) and Sarkar, B., Ullah, M. A., Araf, Y., Islam, N. N., & Zohora, U. S., 2021 (D5). The vaccine design described in the specification is designated DA in this example. Simulation of peptide mutation. Method and parameters The objective of the implemented method is to generate peptides with one or more mutations using a procedure that is consistent with some basic biological aspects of mutagenesis. Since mutations occur at the nucleotide level and the number of codons encoding each amino acid are not equal. The analysis was started by encoding each peptide into its nucleotide sequence. The host (human) codon usage bias data for SARS-CoV-2 was extracted from the codon usage bias database (http://www.kazusa.or.jp/codon/) and the most frequent codon for each amino acid was used to encode its corresponding nucleotide. The probability of mutation for each nucleotide estimated per cycle, was selected according to the study by Amicone et al., 2021 and was set to be equal to 1.25 * 10-6 nt-1 cycle-1. Each nucleotide was then randomly mutated according to the biased mutation frequencies reported by Pathan et al., 2020. Combining these two sources of information we defined a transition matrix defining the probability of nucleotide mutation. Table 15: Transition matrix defining the mutation probabilities computed according to the parameters defined in Borges et al. 2021 and Pathan et al.2020
Figure imgf000051_0001
Mutation events were computed independently for each nucleotide for a given number of cycles (e.g. 25,000 cycles) defined by the user. At each cycle, individual mutation calls were made separately for each nucleotide and were then combined together in a full nucleotide sequence. At the end of each simulation every peptide has a list of associated nucleotide sequences whose size is equal to the selected number of cycles. Those sequences are then encoded back to their amino acid representation using the appropriate codon map. Although the large majority of the resulting peptides are identical to the initial peptide, in some cases they’re different. If at least one mutant peptide is present in a given peptide pool, the original peptide is removed. It needs to be noticed that a higher number of cycles increases the chances of generating mutated peptides and thus the chances for any original peptide to be removed from its list is also increased. The procedure described above is performed for each peptide in a list (e.g. D1) and results in a pool of “surviving peptides” which are referred to as a sample. The number of samples generated for each list is also a parameter determined by the user. For each peptide list, the Worldwide population HLA coverage was computed for each sample, using the IEDB coverage tool (http://tools.iedb.org/population/). Scoring function Summary statistics such as the mean µd and standard deviation σd were computed for the HLA coverage values obtained from each list ^^^^ (e.g. D1). In order to compare the robustness of each peptide list when subjected to mutations, a custom scoring function accounting for both the average HLA coverage and its coefficient of variation across multiple samples was defined as follows: where is the coefficien
Figure imgf000052_0001
t of variation, a statistical measure of the relative dispersion of data points in a data series around the mean as defined by the following equation:
Figure imgf000052_0002
The weight values and
Figure imgf000052_0005
are parameters allowing to specify the relative importance of the two components o
Figure imgf000052_0004
f the scoring function: the average and the coefficient of variation for HLA coverage. The weights are defined between 0 and 1 and they sum up to 1:
Figure imgf000052_0003
The mean of the HLA coverage
Figure imgf000052_0006
µ is a percentage value and thus it is possible to express it as a number between 0 and 1. Moreover in these calculations, the
Figure imgf000052_0007
is always smaller than
Figure imgf000052_0008
hence is also bound to the 0 to 1 interval. Therefore, is also defined between 0 and 1. In
Figure imgf000052_0009
particular is close to 1 when both the average HLA coverage is close to 1 and the coefficient of variation is close to 0. On the other hand,
Figure imgf000052_0010
is closer to 0 when the HLA coverage is low and the coefficient of variation across the samples is high (close to 1). Results The results of the simulation are given in Table 16.
Figure imgf000052_0011
Where “Invention” are the epitopes disclosed in this specification. Doc 1 Adam Tropical Diseases, Travel Medicine and Vaccines (2021)7:22 Doc 2 Almofti et al BMC Immunology (2021) 22:22 Doc 3 Kar et al Scientific Reports (2020) 10:10895 Doc 4 Cun et al Human Vaccines & Immunotherapeutics 2021, Vol 17, No.41097-1108 Doc 5 Sarkar et al Expert Review of Vaccines doi.org/10.1080/14760584.2021.1874925 Table 16: Table containing the results of the simulation performed for 10,000 cycles and 10 samples per peptide sequence. The weights w1 and w2 were equalized giving to the average HLA coverage and the coefficient of variation equal importance in their contribution to determine the value of the stability score Rd. Conclusions A procedure for assessing the stability of worldwide coverage of HLA when subject to mutagenesis has been developed. The simulation was designed to be consistent with some basic biological aspects of mutagenesis such as SARS-Cov-2 nucleotide-specific mutation and codon usage biases. After simulating the mutation events, the global HLA coverage for each peptide list was calculated and its degree of redundancy was evaluated by a specially designed scoring function. In the simulation performed, the highest score for HLA coverage redundancy is attributed to the vaccine construct described in this specification (“Invention” in Table 16) with an advantage of around 11% over the second scoring design (Doc 2) in terms of ^^^^d score. Example 14: in vivo immunogenesis using biobead and mRNA delivery This example describes an evaluation of the immunogenic effect of the T1.2 multivalent construct on biobeads and various mRNA multivalent constructs in transgenic mice possessing the human MHC class I A*02:01 allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Mice were immunized with 10μg of each mRNA vaccine constructs at day 0 and day 21 via the intramuscular route. Three groups of mice received varying doses of T1.2 biobeads (8μg, 2μg or 0.5μg) adjuvanted with alum and ODN1826 at day 0 and day 21 via the intraperitoneal route. In parallel, positive control mice were immunized with 50μg of human papillomavirus (HPV) peptide, adjuvanted with alum and ODN1826, at day 0 and day 21. Following the same dosing regimen, a group of control mice received adjuvanted naked biobeads. At Day 28 or 7 days after the last immunization, the mice were sacrificed for downstream mouse IFN-γ intracellular flow cytometry (ICFC) and immune profiling analysis to evaluate peptide-specific CD8+ T cell responses. Methods The materials and reagents used in this example were as follows: Table 17 Materials and reagents
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
The animal experiment was conducted according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guideline and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485] on 23 November 2021. Up to five mice were housed in a cage. Each mouse was identified using ear punching identification system and grouped as listed in Table 02. All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12- hour light-dark cycle with ad libitum access to normal diet and drinking water. Table 18: Study groups
Figure imgf000055_0002
Figure imgf000056_0001
IM – intramuscular injection (50µL per hindlimb, 100µL per mouse), IP – intraperitoneal injection (200µL per mouse) Table19 Immunization regimen.
Figure imgf000056_0002
Blood sample collection and processing Peripheral blood was collected in 1.7ml Eppendorf tube via facial vein bleed at endpoint. Whole blood was allowed to clot for at least 45 minutes before being processed to serum and stored in -20°C. Isolation of splenocytes At endpoint, spleen was collected and meshed through 70μm strainer with 5ml of RPMI media. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco). The number of splenocytes was counted using trypan blue staining protocol (Gibco). Immune profiling analysis using FACS Immune profiling was performed on approximately 3 × 106 mouse splenocytes using core antibodies listed in Table 20. Briefly, splenocytes were first stained with live/dead dye (Life Technologies) for 10 mins at room temperature. After which, cells were incubated with mouse Fc receptor block (BD Bioscience) at 1:100 dilution in 50µl staining volume and incubated at room temperature for 10 minutes before staining with core antibodies (final dilution of 1:200) for 30 mins at 4°C. Cells were then washed with FACS buffer and data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software and analysed using FlowJo software (version 10; Tree Star Inc). Table 20 Core antibodies and Intracellular Flow Cytometry (ICFC) panel
Figure imgf000057_0001
IFNγ intracellular cytokine flow cytometry (ICFC) Approximately 2 million splenocytes from each mouse were plated into each well of the 48-well plates and restimulated with either HLA-A*02:01 peptide pool (5μM per peptide, Table 21) or 5μM of individual peptides (Peptide no. 1-8 for cells from G1-G6 and G11; Peptide no. 3, 5 and 7 for cells from G7-G9, as listed in Table 21). For the splenocytes from the HPV control group (G10), the splenocytes were restimulated with 5μM of HPV peptide (Peptide no. 9 in Table 21). After approximately one hour of incubation, ER-Golgi stop, Brefeldin A (Biolegend), were added to the cells and the cells were further incubated overnight. Internal negative and positive controls were included by restimulating the cells with media only or PMA/ionomycin (Biolegend). After the overnight incubation, the cells were harvested and washed with FACS buffer before staining with live/dead dye (Life Technologies) for 10 mins at room temperature. After which, mouse Fc receptor block (BD Bioscience) was added to the cells at 1:100 dilution in 50µl staining volume and the mixture incubated at room temperature for 10 minutes. Core surface antibodies, namely, mCD45, CD3, CD19, CD4 and CD8 (as listed in Table 20) were added, and the suspension incubated at 4°C for approximately 1 hour. After washing with FACS buffer, the cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD). Subsequently, intracellular staining for IFN-γ were performed with the BV421 anti-mouse IFN-γ antibody (Biolegend) for 30 minutes at room temperature. Subsequently, cells were washed with FACS buffer before acquiring the data using a LSR II flow cytometer (BD) with FACSDiva software. The FACS data were analyzed with FlowJo software (version 10; Tree Star Inc). The frequencies of peptide-specific IFNγ+ CD8+ T cells were determined by subtracting the basal frequencies of IFNγ+ CD8+ T cells in the unstimulated samples from that of the peptide-restimulated samples. In addition, positive responses for each type of restimulation within each group are defined as those samples with values that were at least 3 SDs (standard deviations) higher than the mean frequency of peptide-specific IFNγ+ CD8+ T cells for splenocytes from G11 (negative control).
Figure imgf000058_0001
Analysis All graphs were plotted using the GraphPad Prism 9.3.1 software (GraphPad Software Inc). Pairwise comparisons were performed using the Mann-Whitney U test (2-tail) and with respect to the negative control group (G11). Statistical significance was assigned when the p values are below 0.05. RESULTS IFNγ intracellular cytokine flow cytometry (ICFC) To evaluate the levels of peptide-specific CD8+ T cells response, splenocytes from the immunized mice were first restimulated with either the peptide pool containing all eight HLA- A*02:01 peptides ex vivo and subjected to IFN-γ ICFC analysis. In parallel, restimulations of pooled cells with PMA/ionomycin or unstimulated cells were also included as positive and negative controls, respectively. The gating strategy for quantifying the levels of IFNγ+ CD8+ T cells in these ex vivo restimulated splenocytes is shown in Figure 10. Briefly, CD45+ mouse leucocytes were gated out after removal of doublets and dead cells. From these mouse CD45+ leucocytes, the CD8+ T cells (CD45+/CD3+/CD8+) were subsequently identified from the T cell population (CD3+/CD19-). The expression levels of IFNγ were then determined on these CD8+ T cells. With the data from the G10 HPV control mice (as shown in Figure11), we confirm that the ICFC setup and gating strategy can detect IFNγ+ CD8+ T cells. The HPV peptide-restimulated splenocytes from G10 control mice had significantly higher frequencies of IFNγ+ CD8+ T cells when compared to the unstimulated cells (media only). To account for any background cytokine secretion, the levels of peptide-specific responses in the immunized mice were determined by subtracting the basal frequencies of IFNγ+ CD8+ T cells in the unstimulated samples from that of the peptide- restimulated samples. As seen from Figure 12A, significantly higher frequencies of peptide-specific IFNγ+ CD8+ T cells were detected in the HLA-peptide pool restimulated splenocytes from the mice immunized with T1.2 biobeads (median value of 0.044%, 0.058% and 0.036% for G7, G8 and G9 respectively) when compared to that in the restimulated cells from the negative control group (G11, naked biobeads). On the other hand, the recall CD8+ T cells responses were only present in a minority of the splenocytes from the mice that were immunized with mRNA constructs. Given that the level of responses can vary within the same group (especially so for the mRNA groups), the number of positive responders for the different peptide restimulation was determined. Positive responders for each type of restimulation are defined as those samples with values that were at least 3 SDs higher than the mean frequencies of peptide-specific IFNγ+ CD8+ T cells for splenocytes from G11 (negative control). For the restimulation with HLA- A*02:01 peptide pool, immunization with the T1.2 biobeads resulted in a more consistent peptide-specific IFNγ+ CD8+ T cell responses when compared to the immunization with mRNA constructs. This is evident from the fact that almost all the mice in G7-G9 were positive responders (Figure12B). On the other hand, only one to two mice in each of the mRNA group had positive IFNγ+ CD8+ T cell responses upon restimulation with the HLA peptide pool. Data in Figure 12B are presented as mean frequencies ± SEM. Pairwise comparison using two- tailed Mann-Whitney U test with respect to G11. *p < 0.05, **p<0.01. 1 Frequencies of peptide- specific IFNγ+ CD8+ T cells were determined by subtracting the basal values in the unstimulated samples from that of the peptide pool-restimulated samples.2 Dotted line at 0.014 denotes the cut- off value above which the samples are considered to have a positive peptide-specific response. CONCLUSIONS As with other examples, CVC’s T1.2 construct induced a robust specific CD8+ T cell response. Expression in both prokaryotic and eukaryotic systems was not optimized for mRNA constructs tests. Nevertheless, even with low levels of expression, in around 20% of the mice tested, we saw a significant specific cytotoxic T cell response. Notably, mRNA constructs of T1.2, T3, T4 and T5 all gave a significant response in 20% or more of the mice tested. This demonstrates that in principle, the T cell vaccine can be delivered via mRNA. Furthermore, each of the T1.2, T3, T4 and T5 constructs all have different orders of epitopes and possess different linkers. Responses to the designs in this specification are therefore not restricted to any particular order or any one linker. Example 15: in vivo immunogenesis using biobead with three human MHC Class I alleles This example describes an evaluation of the immunogenic effect of the T1.2 multivalent biobead construct in transgenic mice possessing the human MHC class I A*02:01, A*11:01 and B*07:01 alleles, and particularly to investigate the induction of a strong human CD8+ T cell- mediated cytotoxic immune response in these different alleles. Mice received 2μg T1.2 biobeads adjuvanted with alum and ODN1826 at day 0 and day 21 via the intraperitoneal route. Following the same dosing regimen, a group of control mice received adjuvanted naked biobeads. At Day 28 or 7 days after the last immunization, the mice were sacrificed for downstream mouse IFN-γ intracellular flow cytometry (ICFC) to evaluate peptide-specific CD8+ T cell responses. Methods The materials and reagents used in this example were as follows: Table 22 Materials and reagents
Figure imgf000060_0001
Figure imgf000061_0001
The animal experiment was conducted according to the National Advisory Committee for Laboratory Animal Research (NACLAR) guideline and approved by Institutional Animal Care and Use Committee (IACUC) [IACUC approval number: 191485] on 23 November 2021. Up to five mice were housed in a cage. Each mouse was identified using ear punching identification system and grouped as listed in Table 02. All mice were housed under Animal Biosafety Level 2 (ABSL2) in Biological Resource Centre, Agency for Science, Technology and Research, Singapore (A*STAR) on controlled 12- hour light-dark cycle with ad libitum access to normal diet and drinking water. Table 23: Study groups
Figure imgf000061_0002
IP – intraperitoneal injection (200µL per mouse) Isolation of splenocytes At endpoint, spleen was collected and meshed through 70μm strainer with 5ml of RPMI media. Red blood cells in the enriched immune cells suspension were lysed with ACK lysis buffer (Gibco). The number of splenocytes was counted using trypan blue staining protocol (Gibco). Immune profiling analysis using FACS Immune profiling was performed on approximately 3 × 106 mouse splenocytes using core antibodies listed in Table 24 Briefly, splenocytes were first stained with live/dead dye (Life Technologies) for 10 mins at room temperature. After which, cells were incubated with mouse Fc receptor block (BD Bioscience) at 1:100 dilution in 50µl staining volume and incubated at room temperature for 10 minutes before staining with core antibodies (final dilution of 1:200) for 30 mins at 4°C. Cells were then washed with FACS buffer and data was acquired using a LSR II flow cytometer (BD Biosciences) with FACSDiva software and analysed using FlowJo software (version 10; Tree Star Inc). Table 24 Core antibodies and Intracellular Flow Cytometry (ICFC) panel
Figure imgf000062_0001
IFNγ intracellular cytokine flow cytometry (ICFC) Approximately 2 million splenocytes from each mouse were plated into each well of the 48-well plates and restimulated with either HLA-A*02:01 peptide pool (5μM per peptide, Table 25) or 5μM of individual peptides (Peptide no.9 and 10 for cells from G3 and G4; Peptide no.11 for cells from G5 and G6, as listed in Table 23). After approximately one hour of incubation, ER- Golgi stop, Brefeldin A (Biolegend), were added to the cells and the cells were further incubated overnight. Internal negative and positive controls were included by restimulating the cells with media only or PMA/ionomycin (Biolegend). After the overnight incubation, the cells were harvested and washed with FACS buffer before staining with live/dead dye (Life Technologies) for 10 mins at room temperature. After which, mouse Fc receptor block (BD Bioscience) was added to the cells at 1:100 dilution in 50µl staining volume and the mixture incubated at room temperature for 10 minutes. Core surface antibodies, namely, mCD45, CD3, CD19, CD4 and CD8 (as listed in Table 22 were added, and the suspension incubated at 4°C for approximately 1 hour. After washing with FACS buffer, the cells were fixed and permeabilized with the Mouse FoxP3 buffer set (BD). Subsequently, intracellular staining for IFN-γ were performed with the BV421 anti-mouse IFN-γ antibody (Biolegend) for 30 minutes at room temperature. Subsequently, cells were washed with FACS buffer before acquiring the data using a LSR II flow cytometer (BD) with FACSDiva software. The FACS data were analyzed with FlowJo software (version 10; Tree Star Inc). The frequencies of peptide-specific IFNγ+ CD8+ T cells were determined by subtracting the basal frequencies of IFNγ+ CD8+ T cells in the unstimulated samples from that of the peptide-restimulated samples. In addition, positive responses for each type of restimulation within each group are defined as those samples with values that were at least 3 SDs (standard deviations) higher than the mean frequency of peptide-specific IFNγ+ CD8+ T cells for splenocytes from G11 (negative control). Table 25. Peptides for ICFC
Figure imgf000063_0001
Analysis All graphs were plotted using the GraphPad Prism 9.3.1 software (GraphPad Software Inc). Statistical significance was assigned when the p values are below 0.05. RESULTS IFNγ intracellular cytokine flow cytometry (ICFC) To evaluate the levels of peptide-specific CD8+ T cells response, splenocytes from the immunized mice were restimulated with various peptide combinations as listed in Table 25. In parallel, restimulations of pooled cells with PMA/ionomycin or unstimulated cells were also included as positive and negative controls, respectively. The gating strategy for quantifying the levels of IFNγ+ CD8+ T cells in these ex vivo restimulated splenocytes is shown in Figure 13. Briefly, CD45+ mouse leucocytes were gated out after removal of doublets and dead cells. From these mouse CD45+ leucocytes, the CD8+ T cells (CD45+/CD3+/CD8+) were subsequently identified from the T cell population (CD3+/CD19-). The expression levels of IFNγ were then determined on these CD8+ T cells. HLA-A*02:01 Transgenic Mice To account for any background cytokine secretion, the levels of peptide-specific responses in the immunized mice were determined by subtracting the basal frequencies of IFNγ+ CD8+ T cells in the unstimulated samples from that of the peptide-restimulated samples. Significantly higher frequencies of peptide-specific IFNγ+ CD8+ T cells were detected in the HLA-A*02:01 peptide pool restimulated splenocytes from mice when compared to that in the restimulated cells from G2 negative control group (median value of 0%, as shown in Figure 14A). HLA-A*11:01 Transgenic Mice Recall CD8+ T cell responses were determined by restimulating the splenocytes from the HLA-A*11:01 transgenic mice (G3-G4) with peptide 9 (ASAFFGMSRI) or peptide 10 (SSTFNVPMEKL). Immunization with T1.2 biobeads generated positive peptide-specific CD8+ T-cell responses greater than the negative control group for both peptides (Figures 14B and 14C). HLA-B*07:02 Transgenic Mice For the HLA-B*07:02 mice (G5-G6), immunization with T1.2 biobeads generated positive peptide 11-specific CD8+ T cell responses higher than the control group (Figure 14D). The results indicate that T1.2 multivalent construct induced a specific cytotoxic T cell response in all the MHC class I alleles tested. These data suggest the T cell construct will lead to immunity to COVID-19 infection in a broad population group. Example 16: mRNA vaccine containing two multiepitope mRNA sequences - multiepitope mRNA sequence preparation This example describes the preparation of two multiepitope antigen mRNA sequences as described herein. The multiepitope antigen sequences coded by the mRNA used as vaccine referred to in these examples as F3-8His and F6-8His, are equivalent to the polypeptide constructs discussed herein and presented as SEQ ID NO: 91 and SEQ ID No: 94, respectively, both HIS tagged on the C-terminus. F3-8His is a polypeptide antigen containing seven MHC class I epitopes with linkers between them and bearing eight histidine residues at the polypeptide C-terminus. F6-8His is a polypeptide antigen containing six MHC class I epitopes with linkers between them and bearing eight histidine residues at the polypeptide C-terminus. Messenger RNA sequences for use in a vaccine were prepared according to the following design. The five prime (5’) end of the sequences included a 31-nucleotide length 5’ untranslated region (UTR) of human haemoglobin subunit alpha 1 and the Kozak consensus sequence (CCCGCCACC). The open reading frame (ORF) contained the sequences for either F3-8His or F6-8His. The three prime (3’) UTR included a 295-nucleotide 3’ UTR of amino- terminal enhancer of split (AES) together with non-coding RNA (ncRNA) from mitochondrially encoded 12S ribosomal RNA (rRNA). A poly-adenosine tail up to 113- nucleotides in length was included at the three-prime end of the construct. Template DNA for use in in vitro transcription was prepared as follows. The DNA sequences for the mRNA sequence for vaccine component containing multiepitope F3-8His and F6- 8His are presented as SEQ ID NO: 125 and SEQ ID No: 126, respectively. Copy DNA (cDNA) for the desired RNA sequences was prepared and inserted into an E. coli expression vector plasmid pJ214 (ATUM). The cDNA included a restriction endonuclease SapI binding site for linearization during subsequent processing. To amplify the DNA, plasmids were transformed into E. coli cell NEB® Stable and single colonies were picked from the transformation plates for inoculation of a 24-hour starter cultures. The starter cultures were used to inoculate 2.5 L of LB media containing the appropriate antibiotics in 5 L Optimum Growth™ shake flasks (Thomson). The overnight cultures in LB media were incubated at 30 °C at 200 rpm shaker speed. Amplification cultures were harvested by centrifugation at 6,000 × g for 30 min at 4˚C. Cell pellets were stored at -80˚C until purification. Cell pellets from 2.5 L culture was resuspended with 125 mL of 25 mM Tris, 10 mM EDTA, 55 mM Dextrose, pH 8.0 and lysed by alkaline lysis with 125 mL of each of 0.24 M Sodium Hydroxide and 1% SDS for 5 minutes while gently mixing. The lysates were centrifuged at 12,000 g for 20 minutes to isolate the soluble fraction containing the plasmid. The soluble fractions were diluted with Milli-Q water to adjust the solutions conductivities to 35 mS/cm. The diluted samples were passed through a 0.45 μm filter and purified using CIMmultus DEAE (Sartorius) anion exchange (AEX) chromatography columns. The AEX eluates were collected and loaded onto HiPrep 26/10 Desalting (Cytiva) gel filtration desalting columns. The highest purity fractions corresponding to the target plasmid were pooled. Purified supercoiled plasmids were linearised using BspQI at 2:1 units/μg overnight at 37°C, 300 rpm. Linearised plasmids were mixed with 4 M ammonium sulphate (3:1 v/v) and purified using hydrophobic interaction chromatography (HIC) on CIMmultus C4 HLD columns (Sartorius). The highest purity fractions corresponding to the linearised plasmid DNA were pooled. The pooled samples from HIC purified fractions were loaded onto HiPrep 26/10 (Cytiva) desalting gel filtration chromatography columns. The highest purity fractions corresponding to the target linearised plasmid were pooled and concentrated using 3 M sodium acetate and 100% ethanol precipitation (1.2.5 v/v) for one hour at -20 °C. Purified linear plasmids were resuspended in Tris buffer containing EDTA (TE) buffer. Messenger RNA production by in vitro transcription (IVT) was undertaken as follows. Linearised plasmid DNA (pDNA) was used as template for mRNA IVT using T7 RNA polymerase (ΝΕΒ) and Cap1 analogue CleanCap® AG (TriLink). Template pDNA, enzymes and IVT reagents were incubated at 37°C for 3hr. The nucleoside triphosphates (NTP) used during IVT were adenosine triphosphate, (ATP), cytosine triphosphate (CTP) guanine triphosphate (GTP), and N1-methyl-pseudouridine triphosphate (ΨTP). The mRNA IVT reactions were stopped by mixing the sample with DNaseI and incubation at 37°C for 15 min to digest the pDNA template. The mRNA products of IVT were purified using Monarch® RNA Cleanup Kits (New England Biolabs). Procedure was performed according to the manufacturer’s protocol and the purified mRNA was eluted in 175 μL of nuclease-free water. Purified mRNA was filter-sterilised through a 0.22 μm filter, aliquoted and stored at -80˚C. Messenger RNA constructs coding for multiepitope antigens F3-8His and F6-8His were prepared as a vaccine for use in vivo as follows. Messenger RNA solutions for both F3-8His and F6-8His were diluted with buffer and mixed with the transfection agent in vivo-jetRNA® (Polyplus S.A.) at a ratio of 1 µg mRNA to 1 µL transfection agent solution, according to the manufacturer’s instructions. The mixture was incubated at room temperature for 15 minutes and injected into test animals within one hour of preparation. DNA nucleotide sequence for mRNA vaccine construct containing F3-8His: SEQ ID NO: 132 DNA nucleotide sequence for mRNA vaccine construct containing F6-8His: SEQ ID NO: 133. Example 17: in vivo immunogenesis using fragments administer as mRNA This example describes an evaluation of the immunogenic effect of an mRNA construct of two fragments (F3, SEQ ID NO: 90 and F6, SEQ ID NO: 93) in transgenic mice possessing the human MHC class I A*02:01allele, and particularly to investigate the induction of a strong human CD8+ T cell-mediated cytotoxic immune response. Mice received 2μg or 0.5μg of mRNA delivered using in vivo-jetRNA® at day 0 and day 21 via the intramuscular route. Following the same dosing regimen, a group of control mice received 2μg of a green fluorescent protein mRNA construct also delivered using in vivo- jetRNA®. At Day 28 or 7 days after the last immunization, the mice were sacrificed for downstream mouse IFN-γ intracellular flow cytometry (ICFC) to evaluate peptide-specific CD8+ T cell responses. Protocols followed were essentially the same as that in example 15 and IFNγ intracellular cytokine flow cytometry was performed as described previously with harvested splenocytes stimulated with the peptide YLFDESGEFKL (SEQ ID NO: 8). Briefly, Approximately 2 million splenocytes from each mouse were plated into each well of 48 well plates and restimulated with the respective combinations of peptides (refer to next slide for more details) After approximately one hour of incubation at 37°C, ER-Golgi stop, Brefeldin A, was added to the cells and the cells were further incubated overnight Internal negative and positive controls were included by restimulating the cells with media only or PMA/ionomycin. After the overnight incubation, the cells were first transferred to 96 well v bottomed plates before staining with live/dead dye 1:400 dilution in PBS) at room temperature for 10 minutes After which, mouse Fc receptor blocking reagent (diluted in FACS buffer) were added and the mixture incubated at room temperature for 10 minutes to block non-specific binding Core surface antibodies, namely mCD45, CD3, CD19, CD4 and CD8 were then added, and the suspension incubated at 44°C for approximately one hour. After washing with FACS buffer, the cells were fixed and permeabilized with the Mouse FoxP 3 buffer set (Subsequently, intracellular staining for IFN γ was performed with the BV 421 anti-mouse IFN γ antibody for 30 minutes at room temperature. Subsequently, cells were washed with FACS buffer before acquiring the data using a LSR II flow cytometer (BD Biosciences) with FACSDiva software The FACS data were analyzed with FlowJo software (version 10 Tree Star Inc). Study group were as follows: G1: F3&F6 mRNA, in vivo JetRNA, 2 µg G2: F4&F8 mRNA, in vivo JetRNA, 0.5 µg G3: GFP mRNA, in vivo JetRNA, 2µg Results Results are shown in Figure 15 which demonstrate a dose dependent specific CD8+ response to the peptide YLFDESGEFKL but not the control green fluorescent protein construct. This shows that fragment vaccine constructs are capable of inducing SARS-CoV- 2 specific cytotoxic T cell responses.

Claims

Claims 1. A multivalent vaccine composition characterised in that it elicits broad spectrum protection against at least one strain of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants], B.1.1.7 and/or omicron), said vaccine comprising: a pool of T cell epitopes derived from at least one viral protein wherein the vaccine has a calculated world population HLA coverage of at least 95%, for example 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%. 1A. A multivalent vaccine composition comprising a pool of T cell epitopes derived from at least two viral proteins, such as 3, 4, 5 etc. wherein the composition elicits broad spectrum protection against multiple strains of coronavirus (such as a viral lineage, in particular SARS-Cov-2, more specifically selected from B.1.617. 2 [delta and/or kappa variants] and/or B.1.1.7). 1B A multivalent vaccine composition comprising at least one construct of formula (I): A-X-B-X-J-X-(U)q-X-W-X-Z (I) wherein: A is sequence independently selected from SEQ ID NO: 1 to 37; X each occurrence is independently a linker, in particular a cleaveable linker, such as an amino acid linker in particular as disclosed herein; B comprises a sequence independently selected from SEQ ID NO: 1 to 37; J comprises a sequence independently selected from SEQ ID NO: 1 to 37; U comprises a sequence independently selected from SEQ ID NO: 1 to 37; W comprises a sequence independently selected from SEQ ID NO: 1 to 37; Z comprises a sequence independently selected from SEQ ID NO: 1 to 37; and q is 0 or an integer 1 to 40, such as 1 to 31, for example 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 or 30. 2. A multivalent vaccine composition according to claim 1, 1A or 1B, comprising 2-100 T cell epitopes, for example 2 -50, (such as 5 to 40 or 7 to 36 or 37) in particular 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 or 37, more specifically 5, 6 or 7. 3. A multivalent vaccine composition according to claim 1, 1A, 1B or 2, wherein each T cell epitope is in the range 7 to 15 amino acids in length (e.g. 8 or 9 to 11 amino acids), such as 7, 8, 9, 10, 11, 12, 13, 14, 15. 4. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 3, wherein the T cell epitope peptides are encoded in one or more viral vectors or as a transcribable polynucleotide, such as RNA. 5. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 4, wherein the T cell epitopes are presented on HLA, for example on cells, such as antigen presenting cells, in particular cells autologous to the patient.
6. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 3, wherein the T cells epitopes are provided as isolated (individual i.e. unlinked sequences) peptides. 7. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 4, wherein one or more T cell, epitopes are linked, for example 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100% of the epitopes are linked. 8. A multivalent vaccine composition according to claim 7, wherein two or more of said T cell epitopes (such as all said T cell epitopes) are linked via a bond, such as an amide bond. 9. A multivalent vaccine composition according to claim 7 or 8, where the epitopes are linked via a linker or linkers, for example a peptide linker or linkers (including independently selected linkers) 1 to 30 amino acids in length, in particular a linker independently selected from a cleavable linker (such as a proteolytic cleavage site, for example comprising a proteasome dependent site, more specifically AAY, or a furin dependent site such as REKR [SEQ ID NO: 41]) and a linker of formula (II): B1-W1-Y1-Z1 formula (II) wherein B1 is independently selected from A, R, S and P; W1 is independently selected from D, L, I and T; Y1 is independently selected from L, G and A Z1 is independently selected from V, K and A. 10. A multivalent vaccine composition according to any one of claims 7 to 9, where the epitopes are linked via a linker or linkers, and at least one has a formula (IIIA): B2-W2-C0-1-K0-1-(Y2)0-1-Z2 (IIIA) wherein: B2 is independently selected from G, M, P, S, W and Y, in particular P. W2 is independently selected from N and W, in particular W; C is cysteine K is lysine Y2 is independently selected from M, N, Q, R, S and T, such as N, Q, R and T in particular Q or R. Z2 is independently selected from W and Y 0 means the entity is absent, and 1 means the entity is present. 11. A multivalent vaccine composition according to any one of claims 1 to 4 and 7 to11, wherein linked T cell epitopes are provided as an isolated polypeptide. 12. A multivalent vaccine composition according to any one of claims 1 to 4 and 7 to 11, wherein linked epitopes are provided as at least one oligopeptide, for example provided as multiple oligopeptides. 13. A multivalent vaccine composition according to claim 11, wherein the composition comprises 2-10 oligopetides, such as 2, 3, 4, 5, 6, 7, 8, 9 or 10, in particular 5, 6 or 7. 14. A multivalent vaccine composition according to claim 12 or 13, wherein the oligopeptide(s) is/are selected from SEQ ID NO: 88, 89, 90, 91, 92 and 93, for example comprises all said sequences.
15. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 14, wherein the one or more T cell epitopes have a low propensity to mutate in the wild-type viral protein (for example 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the epitopes have a low propensity to mutate). 16. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 15, wherein the composition comprises T cell epitopes to multiple viral proteins. 17. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 16, wherein the viral protein is independently selected from ORF1a polyprotein, ORF1ab polyprotein, ORF8 protein, membrane glycoprotein, nucleocapsid phosphoprotein, and spike surface glycoprotein. 18. A multivalent vaccine composition according to claim 17, wherein the viral protein is ORF1a polyprotein. 19. A multivalent vaccine composition according to claim 17 or 18, wherein the viral protein is ORF1ab polyprotein. 20. A multivalent vaccine composition according to any one of claims 16 to 19, wherein the viral protein is ORF8 protein. 21. A multivalent vaccine composition according to any one of claims 16 to 20, wherein the viral protein is membrane glycoprotein. 22. A multivalent vaccine composition according to any one of claims 16 to 21, wherein the viral protein is nucleocapsid phosphoprotein. 23. A multivalent vaccine composition according to any one of claims 16 to 22, wherein the viral protein is spike surface glycoprotein. 24. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 23, wherein the T cell epitope is disclosed herein and combinations of 2 or more thereof, such as comprising one or more sequences shown in SEQ ID NO: 1 to 37. 25. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 81, wherein one or more (e.g. all) of the T cell epitopes are capable of presentation on at least one HLA molecule, in particular HLA type 1 including HLA type 1 variant. 26. A multivalent vaccine composition according to any one of claims 1, 1A, 1B to 85, wherein the composition comprises an addition sequence independently selected from: a. an intracellular translocation sequence, b. a sequence that increases proteolytic processing efficiency, c. a sequence that enhances immunological responses and/or immunogenicity, d. a sequence that enhances epitope presentation, such as a PADRE sequence, e. a sequence that induces, increases or sustains activity of one or more immune cells (such as CD4+, CD8+ and antigen presenting cells); and/or f. combinations of 2 or more of the same. 27. A multivalent vaccine composition according to claim 82, wherein the intracellular translocation domain comprises sequence MDAMKRGLCCVLLLCGAVFVDSVTG [SEQ ID NO: 38] and/or MGVTGILQLPRDR [SEQ ID NO: 39].
28. A multivalent vaccine composition according to claim 86 or 87, wherein the sequence associated with enhanced immunological response and/or immunogenicity comprises a PADRE sequence, such as AKFVAAWTLKAAA [SEQ ID NO: 40]. 29. A multivalent vaccine composition according to any one of claims 86 to 88, wherein the sequence of part e) induces or increases activity of CD8+ cells, for example the sequence comprises LLFNKVTLA [SEQ ID NO: 37]. 30. A multivalent vaccine peptide or polypeptide composition according to claims 1 or 89, wherein the composition comprises an adjuvant. 31. A multivalent vaccine peptide or polypeptide composition according to claim 90, wherein the adjuvant is selected from: • metal salts such as aluminium hydroxide or aluminium phosphate, • oil in water emulsions, • toll like receptors agonist, (such as toll like receptor 2 agonist, toll like receptor 3 agonist, toll like receptor 4 agonist, toll like receptor 7 agonist, toll like receptor 8 agonist and toll like receptor 9 agonist), • saponins, for example Quil A and its derivatives such as QS7 and/or QS21, • CpG containing oligonucleotides, • 3D –MPL, • (2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetra-decanoylamino]-4-o- phosphono-β-D-glucopyranosyl]-2-[(R)-3-hydroxytetradecanoylamino]-α-D- glucopyranosyldihydrogenphosphate), • DP (3S, 9R) –3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)- 3-hydroxytetradecanoylamino]decan-1,10-diol,1,10- bis(dihydrogenophosphate), • MP-Ac DP ( 3S-, 9R) -3-[(R) -dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9- [(R)-3-hydroxytetradecanoylamino]decan-1,10-diol,1 -dihydrogenophosphate 10-(6-aminohexanoate), and • combinations thereof. 32. A multivalent vaccine composition according to any one of claims 1 to 31, for use in treatment, such as prophylaxis, in particular prophylaxis to generate immunity to a coronavirus (more specifically SARS-CoV2). 33. A multivalent vaccine composition according to claim 92, wherein the vaccine is employed in combination with further coronavirus vaccine, such as a further COVID-19 vaccine, in particular where the further vaccine is based on the spike protein (further coronavirus vaccine may include Pfizer/BioNTech Comirnity® (Tozinameran) vaccine, Oxford/AstraZeneca Vaxzevria® COVID-19 Vaccine (ChAdOx1-S [recombinant]) Moderna Spikevax® (COVID-19 Vaccine, mRNA), Janssen COVID-19 VACCINE JANSSEN® Ad26.COV2.S and combinations thereof).
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Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
JAIN NEHA; SHANKAR UMA; MAJEE PRATIVA; KUMAR AMIT: "Scrutinizing the SARS-CoV-2 protein information for designing an effective vaccine encompassing both the T-cell and B-cell epitopes", INFECTION , GENETICS AND EVOLUTION, ELSEVIER, AMSTERDAM, NL, vol. 87, 29 November 2020 (2020-11-29), NL , XP086438310, ISSN: 1567-1348, DOI: 10.1016/j.meegid.2020.104648 *
MEDHA;PARUL BHATT;PRIYANKA;MONIKA SHARMA;SADHNA SHARMA: "Prediction and identification of T cell epitopes of COVID-19 with balanced cytokine response for the development of peptide based vaccines", IN SILICO PHARMACOLOGY, BIOMED CENTRAL LTD, LONDON, UK, vol. 9, no. 1, 28 June 2021 (2021-06-28), London, UK , pages 1 - 17, XP021293086, DOI: 10.1007/s40203-021-00098-7 *
SARKAR BISHAJIT; ULLAH MD. ASAD; JOHORA FATEMA TUZ; TANIYA MASUMA AFRIN; ARAF YUSHA: "Immunoinformatics-guided designing of epitope-based subunit vaccines against the SARS Coronavirus-2 (SARS-CoV-2)", IMMUNOBIOLOGY, URBAN UND FISCHER VERLAG, DE, vol. 225, no. 3, 1 May 2020 (2020-05-01), DE , XP086169024, ISSN: 0171-2985, DOI: 10.1016/j.imbio.2020.151955 *
TAHIR UL QAMAR MUHAMMAD, REHMAN ABDUR, TUSLEEM KISHVER, ASHFAQ USMAN, QASIM MUHAMMAD, ZHU XITONG, FATIMA ISRAR, SHAHID FARAH, CHEN: "Designing of a next generation multiepitope based vaccine (MEV) against SARS-COV-2: Immunoinformatics and in silico approaches", PLOS ONE, PUBLIC LIBRARY OF SCIENCE, vol. 15, no. 12, 22 December 2020 (2020-12-22), pages e0244176, XP055827088, DOI: 10.1371/journal.pone.0244176 *

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