WO2023179514A1 - Composition de vaccin contre le coronavirus, méthode et utilisation associées - Google Patents

Composition de vaccin contre le coronavirus, méthode et utilisation associées Download PDF

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WO2023179514A1
WO2023179514A1 PCT/CN2023/082378 CN2023082378W WO2023179514A1 WO 2023179514 A1 WO2023179514 A1 WO 2023179514A1 CN 2023082378 W CN2023082378 W CN 2023082378W WO 2023179514 A1 WO2023179514 A1 WO 2023179514A1
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dose
coronavirus
vaccines
seq
sars
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梁朋
梁果
宿丹梅
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四川三叶草生物制药有限公司
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
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    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
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    • A61P31/14—Antivirals for RNA viruses
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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    • 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
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    • A61K2039/55511—Organic adjuvants
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    • 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
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
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    • 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
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    • 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

  • the surface antigens may be the same or different in the recombinant polypeptides of the protein.
  • vaccines comprising an immunogenic composition provided herein and an optional adjuvant, wherein the vaccine is optionally a subunit vaccine.
  • the vaccine is a prophylactic and/or therapeutic vaccine.
  • Optional adjuvants may be used in priming and/or boosting doses.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g., in water Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG
  • a protein comprising a coronavirus antigen or immunogen is capable of generating an immune response, e.g., to SARS-CoV or SARS-CoV-2 S protein Immune responses to peptides.
  • the immune response inhibits or reduces replication of the coronavirus in a subject (eg, a patient).
  • the immune response includes the production of one or more neutralizing antibodies, such as polyclonal and/or monoclonal antibodies.
  • neutralizing antibodies inhibit or reduce replication of coronavirus in a subject (eg, a patient).
  • linking the coronavirus S protein peptide to the C-terminal propeptide of collagen results in a self-trimerizing recombinant polypeptide.
  • the proteins provided herein include multiple self-trimerized propeptides of coronavirus S protein peptides and collagen recombinant polypeptides.
  • the trimeric nature of the recombinant protein contributes to protein stability.
  • the trimeric nature of the recombinant protein contributes to the protein's ability to generate an immune response.
  • the trimeric nature of the recombinant protein and/or the macrostructure of the plurality of self-trimerized recombinant proteins contributes to the protein's ability to generate an immune response.
  • the coronavirus spike (S) protein is a class I fusion glycoprotein initially synthesized as a precursor protein.
  • the single precursor S polypeptide forms a homotrimer and is glycosylated and processed in the Golgi to remove the signal peptide and cleaved by cellular proteases to produce separate S1 and S2 polypeptide chains, which remain in the homotrimer Binds as an S1/S2 protomer and is therefore a trimer of heterodimers.
  • the S1 subunit is located at the distal end of the viral membrane and contains a receptor-binding domain (RBD) that mediates attachment of the virus to its host receptor.
  • RBD receptor-binding domain
  • the viral antigen or immunogen includes the spike glycoprotein sequence of Hu-1 coronavirus (eg, NC_045512). In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of Cluster 5 ( ⁇ FVI-Spike) virus. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.7 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.1.207 lineage.
  • Hu-1 coronavirus eg, NC_045512
  • the viral antigen or immunogen includes the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of Cluster 5 ( ⁇ FVI-Spike) virus. In some embodiments, the viral antigen or immunogen includes the spike glycoprotein sequence of a
  • the viral antigen or immunogen includes any one, two, or three selected from the group consisting of N440K, L452R, S477G, S477N, E484K, E484Q, N501Y, D614G, H655Y, P681H, P681R, and A701V , four, five or more mutations.
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 27, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:30. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence included in an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO:30
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 32, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:33. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence included in an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of 701, 716, 888, 982, 1027, 1118 and 1176 (
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 34, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 36, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 39. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence included in an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 39.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 40. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence included in an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, Contains a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO: 40.
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:41. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81% , 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 % or 99% sequence identity of the amino acid sequence included in the amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400 , 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (about SEQ ID Sequences containing substitutions, deletions and/or insertions at one or more amino acid positions
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 42, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes the sequence set forth in SEQ ID NO:43. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of the amino acid sequence included in an amino acid sequence selected from the group consisting of 13, 18, 20, 26, 69, 70, 80, 138, 142, 144, 152, 190, 215, 242, 243, 244, 246, 400, 401, 402, 417, 440, 452, 477, 484, 501, 570, 614, 655, Comprises a substitution at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to the amino acid position of SEQ ID NO:43
  • the viral antigen or immunogen includes a variant of SEQ ID NO: 48, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144( ⁇ Y), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G , H655Y, P681H , any one, two, three, four, five or more mutations in the group consisting of , P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118
  • the viral antigen or immunogen includes a signal peptide. In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 53. In some embodiments, the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity. In some embodiments, the viral antigen or immunogen includes the sequence set forth in SEQ ID NO: 54.
  • the viral antigen or immunogen includes at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89 Amino acid sequences with %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.
  • one or more peptide linkers can be used to link the recombinant viral antigen or immunogen to the multimerization domain.
  • the trimer may include any of the stabilizing mutations provided herein (or combinations thereof).
  • C-propeptide complexes are stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain registration is unclear.
  • the triple helical repeat of glycine then spreads from the associated C-terminus to the N-terminus in a zipper-like manner.
  • This knowledge creates a non-natural type of collagen matrix by using recombinant DNA technology to exchange the C-propeptides of different collagen chains.
  • Non-collagenous proteins such as cytokines and growth factors
  • C-propeptide needs to be cleaved before reorganized collagen fibers can be assembled into an insoluble cellular matrix.
  • the C-terminal propeptide is human collagen. In some embodiments, the C-terminal propeptide includes pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2(I), pro ⁇ 2(V), pro ⁇ 2(XI ) or the C-terminal polypeptide of pro ⁇ 3(XI) or a fragment thereof. In some embodiments, the C-terminal propeptide is or includes the C-terminal polypeptide of proal(I).
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 67. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO: 67. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 68. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO: 68. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO: 69.
  • the C-terminal propeptide exhibits an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:74. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:75. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:75. In some embodiments, the C-terminal propeptide is or includes SEQ ID The amino acid sequence described in NO:76. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:76.
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 77. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:77. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO: 78. In some embodiments, the C-terminal propeptide is an amino acid sequence that has at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:78. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO:79.
  • the C-terminal propeptide can include a sequence comprising a glycine-X-Y repeat, wherein X and Y are independently any amino acid, or are at least 85%, 90%, 92%, 95%, or 97% identical thereto
  • the amino acid sequence can form disulfide bonds between polypeptides and trimerize the recombinant polypeptide.
  • X and Y are independently proline or hydroxyproline.
  • a trimerized recombinant polypeptide includes a single recombinant polypeptide comprising the same viral antigen or immunogen. In some embodiments, a trimerized recombinant polypeptide includes a single recombinant polypeptide, each recombinant polypeptide comprising a different viral antigen or immunogen than the other recombinant polypeptides. In some embodiments, a trimerized recombinant polypeptide includes a single recombinant polypeptide, wherein one of the single recombinant polypeptides includes a different viral antigen or immunogen than the other recombinant polypeptides.
  • a trimerized recombinant polypeptide includes a single recombinant polypeptide, wherein two of the single recombinant polypeptides include the same viral antigen or immunogen, and the viral antigen or immunogen is different from the virus contained in the remaining recombinant polypeptide. Antigen or immunogen.
  • a stabilized recombinant coronavirus e.g., SARS-CoV or SARS-CoV-2
  • S ectodomain trimer in a prefusion conformation includes a single-chain S ectodomain protomer comprising S1 /Mutation of S2 and/or S2′ protease cleavage sites to prevent protease cleavage at these sites.
  • the SARS-CoV-2 S protein peptide includes a mutation from 685R to 685A. Exemplary protease cleavage sites for various viruses are shown below:
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • the above-mentioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO: 54 (MFVFLVLLPLLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 87 (1507aa):
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:88 (1507aa):
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 9, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H6 55Y, P681H, Any one, two, three, four, five or more mutations in the group consisting of P681R, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H
  • the recombinant polypeptide is or includes a variant of SEQ ID NO: 15, and the variant includes a variant selected from the group consisting of S13I, L18F, T20N, P26S, ⁇ 69-70 ( ⁇ HV), D80A, D138Y, G142D, ⁇ 144 ( ⁇ Y ), W152C, R190S, D215G, ⁇ 242-244( ⁇ LAL), R246I, ⁇ 400-402( ⁇ FVI), K417T, K417N, N440K, L452R, S477N, S477G, E484K, E484Q, N501Y, A570D, D614G, H655Y, P681H, P681R, R68 2G, R683S, R685G, Any one, two, three, four, five or more mutations of the group consisting of A701V, T716I, F888L, S982A, T1027I, D1118H
  • a protein comprising a plurality of recombinant polypeptides described herein is an immunogen.
  • a protein comprising a plurality of recombinant polypeptides described herein is comprised in a nanoparticle.
  • the protein is directly attached to the nanoparticle, such as a protein nanoparticle.
  • the protein is indirectly linked to the nanoparticle.
  • a protein comprising a plurality of recombinant polypeptides described herein is contained in a virus-like particle (VLP).
  • VLP virus-like particle
  • complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or fragments, variants or mutants thereof, in any suitable combination.
  • complexes comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide Trimers are formed through trimerization of disulfide bonds between polypeptides.
  • the first coronavirus and the second coronavirus are independently selected from the group consisting of B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P. 1.
  • the mutation that inactivates the S1/S2 cleavage site can comprise replacing RRAR (682-685 in SEQ ID NO:55) with GSAG (SEQ ID NO:60), and turning Mutations in the region may include the double mutations K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G.
  • the truncation of HR2 entails deleting one or more residues set forth in SEQ ID NO: 65 at the C-terminus of the wild-type soluble S sequence.
  • polynucleotides encoding the coronavirus antigens or immunogens and recombinant polypeptides provided herein, as well as vectors for genetically engineering cells to express such coronavirus antigens or immunogens and recombinant polypeptides.
  • polynucleotides encoding recombinant polypeptides provided herein are provided.
  • a polynucleotide comprises a single nucleic acid sequence, such as a nucleic acid sequence encoding a recombinant polypeptide.
  • multi-core The nucleotides comprise a first nucleic acid sequence encoding a recombinant polypeptide, in particular a coronavirus antigen or immunogen, and a second nucleic acid sequence encoding a recombinant polypeptide comprising a different coronavirus antigen or immunogen.
  • a polynucleotide encoding a recombinant polypeptide includes at least one promoter operably linked to control expression of the recombinant polypeptide. In some embodiments, the polynucleotide includes two, three, or more promoters operably linked to control expression of the recombinant polypeptide.
  • a polynucleotide encoding a recombinant polypeptide is introduced into a composition containing a cultured cell (eg, a host cell), such as by retroviral transduction, transfection, or transformation. In some embodiments, this may allow expression (eg, production) of recombinant polypeptides. In some embodiments, the expressed recombinant polypeptide is purified.
  • the polynucleotides (nucleic acid molecules) provided herein encode a coronavirus antigen or immunogen as described herein. In some embodiments, the polynucleotides (nucleic acid molecules) provided herein encode a recombinant polypeptide comprising a coronavirus antigen or immunogen (eg, coronavirus S protein peptide) as described herein.
  • a coronavirus antigen or immunogen eg, coronavirus S protein peptide
  • a vector or construct includes one or more promoters operably linked to a nucleic acid molecule encoding a recombinant polypeptide to drive expression thereof.
  • the promoter is operably linked to one or more nucleic acid molecules, such as nucleic acid molecules encoding recombinant polypeptides containing different coronavirus antigens or immunogens.
  • a single promoter directs the expression of an RNA containing two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF) that Genes are separated from each other by sequences encoding self-cleaving peptides (eg, 2A sequences) or protease recognition sites (eg, furin).
  • ORF open reading frame
  • the ORF encodes a single polypeptide, which is processed into a single protein either during translation (in the case of 2A) or after translation.
  • peptides such as T2A can cause ribosomes to skip (ribosome hopping) the synthesis of the C-terminal peptide bond of the 2A element, resulting in dissociation between the end of the 2A sequence and the next peptide downstream (see, e.g., de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004), which are incorporated by reference in their entirety for all purposes).
  • Many 2A components are known in the art.
  • adjuvants may include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g., in water Adjuvant containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG
  • a vaccine comprising an immunogenic composition described herein is a multivalent vaccine.
  • the antigenic material for incorporation into the multivalent vaccine composition is derived from a coronavirus strain or type, for example as described herein (see, for example, Section 1).
  • Antigens for incorporation into multivalent vaccine compositions can be derived from one strain or multiple strains (eg, between two and five strains) of coronavirus to provide a broader spectrum of protection.
  • the antigens used for incorporation into the multivalent vaccine composition are derived from multiple strains of coronavirus.
  • antigens include live, attenuated and inactivated viruses, such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), influenza A Attenuated strains of hepatitis virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J.
  • viruses such as inactivated poliovirus (Jiang et al., J. Biol. Stand., (1986) 14:103-9), influenza A Attenuated strains of hepatitis virus (Bradley et al., J. Med. Virol., (1984) 14:373-86), attenuated measles virus (James et al., N. Engl. J.
  • the vaccines presented here are universal vaccines.
  • a universal vaccine is one that protects against multiple strains of the same virus (eg, multiple strains of coronavirus). Developing an effective universal coronavirus vaccine would reduce costs and labor, such as using seasonal vaccine formulations, and allow for stronger pandemic prevention.
  • a universal vaccine is a vaccine composed of multiple epitopes derived from different strains of the virus.
  • a universal vaccine consists of a single epitope that is conserved across different strains of the virus.
  • a universal vaccine could be based on relatively conserved domains of the S protein.
  • Immunogenic combinations comprising a disclosed immunogen (e.g., a disclosed recombinant coronavirus S antigen or a nucleic acid molecule encoding a protomer of a disclosed recombinant coronavirus S antigen) and a pharmaceutically acceptable carrier are also provided. things.
  • immunogenic compositions include a trimerized recombinant polypeptide provided herein and optionally a pharmaceutically acceptable carrier.
  • an immunogenic composition includes a trimerized recombinant polypeptide provided herein and disodium hydrogen phosphate, e.g., disodium hydrogen phosphate dihydrate, sodium phosphate dibasic, e.g., disodium hydrogen phosphate monohydrate, sodium chloride , and Twain 80.
  • 1.0 mL of an aqueous immunogenic composition solution includes 720 ⁇ g of a trimerized recombinant polypeptide provided herein and 0.62 mg disodium hydrogen phosphate dihydrate, 0.62 mg disodium hydrogen phosphate monohydrate, 9.0 mg sodium chloride, and 0.2mg Tween 80.
  • immunogenic compositions include a protein comprising a plurality of trimerized recombinant polypeptides provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include protein nanoparticles provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include VLPs provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include an isolated nucleic acid provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include a carrier provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include a virus provided herein and optionally a pharmaceutically acceptable carrier.
  • immunogenic compositions include pseudoviruses provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, immunogenic compositions include cells provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition as described herein is a vaccine. In some embodiments, the vaccine is a prophylactic vaccine. In some embodiments, the vaccine is a therapeutic vaccine. In some embodiments, the vaccines are prophylactic and therapeutic vaccines. Such pharmaceutical compositions may be administered to a subject via a variety of modes of administration known to those of ordinary skill in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, nasal.
  • compositions including one or more disclosed immunogens are immunogenic compositions.
  • Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described, for example, in Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995. are described in more detail.
  • Proteins, peptides or hydrolysates eg, albumin, gelatin
  • sugars eg, sucrose, lactose, sorbitol
  • amino acids eg, sodium glutamate
  • the resulting aqueous solution can be packaged and used as is or lyophilized. Lyophilized formulations are mixed with sterile solutions prior to single or multiple dose administration.
  • Formulated compositions may contain bacteriostatic agents to prevent or minimize degradation during storage, including but not limited to benzyl alcohol, phenol, m-cresol, Chlorobutanol, methylparaben and/or propylparaben.
  • Bacteriostatic agents may be contraindicated in some patients; therefore, lyophilized preparations may be reconstituted in solutions with or without such ingredients.
  • the immunogenic composition of the present invention may contain pharmaceutically acceptable carrier substances required to approximate physiological conditions, such as pH adjusters and buffers, tension adjusters, wetting agents, etc., such as sodium acetate, sodium lactate, sodium chloride , potassium chloride, calcium chloride, sorbitan monolaurate and triethanolamine oleate.
  • Immunogenic compositions may optionally include adjuvants to enhance the host's immune response.
  • Suitable adjuvants are, for example, toll-like receptor (TLR) agonists, alum, AlPO 4 , alhydrogel, lipid-A and its derivatives or variants, oil emulsions, saponins, neutral liposomes , liposomes containing vaccines and cytokines, non-ionic block copolymers and chemokines.
  • TLR toll-like receptor
  • Nonionic block polymers containing polyoxyethylene (POE) and polyoxypropylene (POP) may be used as adjuvants, among many other suitable adjuvants well known in the art, such as POE-POP-POE blocks.
  • immunogenic compositions of the invention may include or be administered with more than one adjuvant. In some embodiments, immunogenic compositions of the invention may include or be administered with two adjuvants.
  • the immunogenic compositions of the present invention may include or be administered with various adjuvants.
  • a vaccine such as one comprising an immunogenic composition provided herein may include or be administered in conjunction with a variety of adjuvants.
  • suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-1, one or a combination of any of them may be combined with a compound selected from the group consisting of SEQ ID NO: 85 - Trimers of recombinant polypeptides or fragments, variants or mutants of the group consisting of -92 are used together.
  • the vaccine compositions or nanoparticle immunogens disclosed herein eg, SARS-COV-2 vaccine compositions
  • Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.
  • the immunogenic composition of the present invention includes a recombinant polypeptide selected from the group consisting of SEQ ID NO: 85-92, or a trimer of fragments, variants or mutants thereof, wherein the recombinant polypeptide is by polypeptide Trimerization of metadisulfide bonds to form trimers may include an adjuvant formulation containing a metabolizable oil (e.g., squalene) and alpha-tocopherol (e.g., DL-alpha- Tocopherol), and polyoxyethylene sorbitan monooleate (Tween-80).
  • a metabolizable oil e.g., squalene
  • alpha-tocopherol e.g., DL-alpha- Tocopherol
  • Tween-80 polyoxyethylene sorbitan monooleate
  • the adjuvant formulation may include about 2% to about 10% squalene, about 2% to about 10% alpha-tocopherol (eg, D-alpha-tocopherol), and about 0.3% to about 3% Polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation can include about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate.
  • immunogenic compositions of the invention may comprise QS21, 3D-MPL and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: a metabolizable oil, such as keratin Squalene, alpha-tocopherol, and Tween-80 and/or Span 85.
  • the immunogenic compositions of the present invention may include an adjuvant in the form of a liposome composition.
  • TLR9 (CD289) recognizes the unmethylated cytidine-phosphate-guanosine (CpG) motif found in microbial DNA, which can be mimicked using synthetic CpG-containing oligodeoxynucleotides (CpG-ODN).
  • CpG-ODN is known to enhance antibody production and stimulate T helper 1 (Th1) cell responses (Coffman et al., Immunity, 33:492-503, 2010, incorporated by reference in its entirety for all purposes).
  • the oligonucleotide includes a combination of phosphate linkages in the phosphate backbone, such as a combination of phosphodiester linkages and phosphorothioate linkages.
  • Oligonucleotides with a phosphorothioate backbone can be more immunogenic than oligonucleotides with a phosphodiester backbone and appear to be more resistant to degradation after injection into the host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995, are incorporated by reference in their entirety for all purposes).
  • CpG oligonucleotides of the invention include at least one, two or three internucleotide phosphorothioate linkages.
  • two stereoisomers of the phosphorothioate bond are present in the multiple CpG oligonucleotide molecules. in the nucleotide molecule.
  • all of the internucleotide linkages of the CpG oligonucleotide are phosphorothioate linkages, or in other words, the CpG oligonucleotide has a phosphorothioate backbone.
  • any suitable CpG oligodeoxynucleotide (ODN) or combination thereof may be used as an adjuvant.
  • K-type ODN also known as B-type
  • Type K ODN can be based on the following sequence TCCATGGA CG TTCCTGAG CG TT.
  • the use of phosphorothioate nucleotides increases resistance to nuclease digestion compared to natural phosphodiester nucleotides, resulting in a significantly longer half-life in vivo.
  • K-type ODN triggers pDC differentiation and production of TNF- ⁇ , and triggers B cell proliferation and secretion of IgM.
  • Type C ODN may be based on the following sequence T CG T CG TT CG AA CG A CG TTGAT. This type of ODN stimulates B cells to secrete IL-6 and pDC to produce IFN- ⁇ .
  • P-type ODN contains two palindromic sequences, allowing them to form a higher ordered structure.
  • P-type ODN may be based on the following sequence T CG T CG A CG AT CG G CGCGCG C CG .
  • P-type ODN activates B cells and PDC, and induce greater IFN- ⁇ production.
  • boldface letters in ODN sequences indicate self-complementary palindromes, and CpG motifs are underlined.
  • One or more adjuvants may be used in combination, including but not limited to alum (aluminum salt), oil-in-water emulsions, water-in-oil emulsions, liposomes, and microparticles, such as poly(lactide-co-glycolide) ) particles (Shah et al., Methods Mol Biol, 1494:1-14, 2017, incorporated by reference in its entirety for all purposes).
  • the immunogenic composition further includes an aluminum salt adjuvant that adsorbs SARS-CoV-2 antigen.
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 1000 ⁇ g of one or more adjuvants, preferably from about 25 ⁇ g to about 500 ⁇ g of one or more adjuvants, preferably from about 50 ⁇ g to about 300 ⁇ g of one or more adjuvants, preferably about 100 ⁇ g to about 250 ⁇ g of one or more adjuvants, preferably about 150 ⁇ g to about 225 ⁇ g of one or more adjuvants.
  • a unit dose of the immunogenic composition may include from about 10 ⁇ g to about 500 ⁇ g CpG adjuvant, preferably from about 25 ⁇ g to about 300 ⁇ g CpG adjuvant, preferably from about 50 ⁇ g to about 250 ⁇ g CpG adjuvant, preferably from about 75 ⁇ g to about 200 ⁇ g CpG adjuvant, preferably about 100 ⁇ g to about 175 ⁇ g CpG adjuvant, preferably about 150 ⁇ g CpG adjuvant adjuvant, such as CpG 1018A.
  • a unit dose of the immunogenic composition may include about 50 ⁇ g to about 100 ⁇ g of aluminum-containing adjuvant and about 75 ⁇ g to about 200 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition may include about 50 ⁇ g to about 100 ⁇ g of aluminum-containing adjuvant and about 100 ⁇ g to about 175 ⁇ g of CpG adjuvant. In some embodiments, a unit dose of the immunogenic composition can include about 75 ⁇ g to about 100 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 150 ⁇ g of a CpG adjuvant, such as CpG 1018A.
  • the dose contains 30 ⁇ g of SARS-CoV-2 S fusion protein or S-trimer.
  • a unit dose of the immunogenic composition may include about 30 ⁇ g of SARS-CoV-2 S-fusion protein or S-trimer, about 75 ⁇ g to about 100 ⁇ g of an aluminum-containing adjuvant, such as Alum, and about 150 ⁇ g of CpG adjuvant. agents, such as CpG 1018A.
  • the immunogenic composition may include a tonicity adjusting agent.
  • Suitable tonicity adjusting agents include, for example, glucose, glycerol, sodium chloride, glycerin and mannitol.
  • Immunogenic compositions may include preservatives. Suitable preservatives include, for example, antioxidants and antibacterial agents. However, in preferred embodiments, the immunogenic composition is prepared under sterile conditions and in disposable containers and therefore does not need to contain a preservative.
  • kits for generating an immune response to a coronavirus surface antigen in a subject wherein the surface antigen includes S protein or an antigenic fragment thereof, and the method includes administering to the subject an effective amount of A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 85-92, optionally as a primary series, an additional dose, and /or the use of a homologous or heterologous booster dose, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose, additional doses, or
  • the heterologous booster is used in conjunction with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • adjuvants in any priming agent, additional agent, and/or booster agent may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants.
  • adjuvants such as adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • CpG-ODN CpG oligodeoxynucleotides
  • TLR9 agonists such as an adjuvant containing metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Twe
  • the adjuvants in any priming agent and/or booster agent may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants, such as those containing Adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; adjuvants containing metabolizable oil, alpha-tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween- 80), such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as alum and/or aluminum hydroxide-containing adjuvants
  • oligonucleotide-containing adjuvants such as those
  • Subjects may be selected for treatment who have or are at risk of contracting coronavirus, for example because of exposure or potential exposure to coronavirus. Following administration of the disclosed immunogens, the subject can be monitored for infection or coronavirus-related symptoms, or both.
  • the disclosed immunogens may be used for prophylactic or therapeutic purposes.
  • the disclosed therapeutic agents are provided prior to any symptoms, such as prior to infection.
  • Prophylactic administration of the disclosed therapeutic agents serves to prevent or ameliorate any subsequent infection.
  • the disclosed therapeutic agents are provided at or after the onset of symptoms of disease or infection, for example, after the development of symptoms of coronavirus infection corresponding to the coronavirus S antigen, or after diagnosis of coronavirus infection. Accordingly, therapeutic agents may be provided prior to anticipated exposure to the coronavirus in order to attenuate the expected severity, duration, or extent of infection and/or associated disease symptoms following exposure or suspected exposure to the virus, or after actual onset of infection.
  • plasmid DNA vaccines are used to express the disclosed immunogens in a subject.
  • nucleic acid molecules encoding the disclosed immunogens can be administered to a subject to induce an immune response to the coronavirus S antigen.
  • the nucleic acid molecule can be included on a plasmid vector for DNA immunization, such as the pVRC8400 vector (as described in Barouch et al., J. Virol, 79, 8828-8834, 2005, for all purposes The full text is incorporated by reference).
  • Embodiment 25 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 28 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide includes SEQ ID NO: 74 or an amino acid sequence at least 90% identical thereto, which is capable of forming inter-polypeptide disulfide bonds and enabling recombination. Peptide trimerization.
  • Embodiment 32 The protein according to any one of embodiments 1-31, wherein the surface antigen in each recombinant polypeptide includes any one of SEQ ID NOs: 27-66 and 81-84 or an amino acid that is at least 80% identical thereto sequence.
  • Embodiment 42 A vector comprising an isolated nucleic acid according to any one of embodiments 37-41.
  • Embodiment 46 A vaccine comprising an immunogenic composition according to embodiment 45 and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine, and/or optionally wherein the vaccine is prophylactic and/or or a therapeutic vaccine, optionally administered as a priming dose and/or as a booster dose, such as a second and/or third booster dose.
  • Embodiment 50 A method for generating an immune response to a coronavirus surface antigen in a subject, comprising administering to the subject an effective amount of the protein, immunogen of any one of embodiments 1-47 and 49 , protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines to generate an immune response.
  • Embodiment 52 The method according to embodiment 50 or 51, wherein generating an immune response inhibits or reduces coronavirus replication in the subject.
  • Embodiment 57 The method according to any one of embodiments 50-56, further comprising a priming step and/or a boosting step.
  • Embodiment 60 The method according to any one of embodiments 50-59, wherein the effective amount is administered without an adjuvant.
  • Embodiment 61 The method according to any one of embodiments 50-59, wherein the effective amount is administered with the adjuvant or adjuvants.
  • Embodiment 62 A method comprising administering to a subject an effective amount of a protein according to any one of embodiments 1-33 to produce neutralizing antibodies or neutralizing antisera against coronavirus in the subject.
  • Embodiment 65 The method according to embodiment 64, further comprising administering to a human subject an effective amount of an isolated neutralizing antibody or neutralizing antisera by passive immunization to prevent or treat coronavirus infection.
  • Embodiment 66 The method according to any one of embodiments 62-65, wherein the neutralizing antibody or neutralizing antisera comprises a polyclonal antibody directed against a coronavirus surface antigen, optionally wherein the neutralizing antibody or neutralizing antisera does not Contains or essentially contains no antibodies directed against the C-terminal propeptide of collagen.
  • Embodiment 67 The method according to any one of embodiments 62-65, wherein the neutralizing antibody comprises a monoclonal antibody directed against a coronavirus surface antigen, optionally wherein the neutralizing antibody does not contain or substantially contains no C-specific antibodies against collagen. Antibodies to terminal propeptides.
  • Embodiment 69 Use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine according to any one of embodiments 1-47 and 49, For inducing an immune response to coronavirus in a subject, and/or for treating or preventing coronavirus infection.
  • Embodiment 71 A method for analyzing a sample, comprising contacting the sample with the protein of any one of embodiments 1-33, and detecting the interaction between the protein and an analyte capable of specifically binding to a coronavirus surface antigen. combine.
  • Embodiment 73 The method according to embodiment 71 or 72, wherein the binding indicates the presence of the analyte in the sample and/or the presence of a coronavirus infection in the subject from which the sample is derived.
  • Embodiment 74 A kit comprising the protein of any one of embodiments 1-33 and a substrate, plate or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or lateral flow detection kit ( lateral flow assay kit).
  • Embodiment 2 The method of embodiment 1, wherein the coronavirus infection is a severe acute respiratory syndrome (SARS)-coronavirus 2 (SARS-CoV-2) infection.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-2 severe acute respiratory syndrome-2
  • Embodiment 4 The method according to any one of embodiments 1-3, wherein the coronavirus surface antigen comprises SARS-CoV-2 spike (S) extracellular domain peptide or a fragment or epitope thereof, optionally the S extracellular domain peptide or its fragments or epitopes include the SARS-CoV-2 delta (delta, B.1.617.2) variant S ectodomain peptide or its fragments, variants or mutants, such as those containing the delta variant affected by Chimeric sequences of body binding domain (RBD) and Hu-1 or other variant S protein peptide sequences.
  • S SARS-CoV-2 spike
  • RBD body binding domain
  • Hu-1 Hu-1
  • Embodiment 7 The method according to any one of embodiments 1-6, wherein the coronavirus surface antigen comprises the SARS-CoV-2 spike (S) S1 peptide or a fragment or epitope thereof, optionally the S1 The peptide is the S1 peptide of the SARS-CoV-2 delta (delta, B.1.617.2) variant or its fragment, variant or mutant.
  • S SARS-CoV-2 spike
  • the peptide is the S1 peptide of the SARS-CoV-2 delta (delta, B.1.617.2) variant or its fragment, variant or mutant.
  • Embodiment 12 The method of any one of embodiments 9-11, wherein the mutation comprises consecutive site amino acid substitutions, such as 986K ⁇ 986P and 987V ⁇ 987P.
  • Embodiment 13 The method according to any one of embodiments 1-12, wherein the recombinant subunit vaccine includes the sequence described in any one of SEQ ID NO:81-92 or is identical to SEQ ID NO:81-92 Any one of the sequences has an amino acid sequence identity of at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99%.
  • Embodiment 14 The method of any one of embodiments 1-13, wherein the recombinant subunit vaccine includes or is at least or about 80%, 85%, 90% SEQ ID NO: 85 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 16 The method of any one of embodiments 1-15, wherein the recombinant subunit vaccine includes or is at least or about 80%, 85%, 90% SEQ ID NO:87 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 18 The method according to any one of embodiments 1-17, wherein the recombinant subunit vaccine comprises the sequence of any one of SEQ ID NO:89-92 or is identical to SEQ ID NO:89-92 Any one of the sequences has an amino acid sequence identity of at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99%.
  • Embodiment 19 The method according to any one of embodiments 1-18, wherein the recombinant subunit vaccine includes SEQ ID NO: 91 or is at least or about 80%, 85%, 90% with SEQ ID NO: 91 , 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Embodiment 24 The method of any one of embodiments 1-23, wherein the recombinant subunit vaccine is administered without an adjuvant, optionally as a primary series, Additional doses, and/or homologous or heterologous booster doses are used, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally
  • the initial dose, additional dose, or heterologous booster dose is used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • Adjuvants containing glycosides such as CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan Adjuvants to monooleate (Tween-80), such as adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an oil-in-water emulsion; or any adjuvant agent combination.
  • CpG-ODN CpG oligodeoxynucleotides
  • TLR9 agonists metabolizable oils, alpha-tocopherol, and/or polyoxyethylene sorbitan Adjuvants to monooleate (Tween-80), such as adjuvants containing squalene, alpha-tocopherol, and Tween-80 and/or Span 85 in the form of an
  • Embodiment 28 The method of embodiment 27, wherein the soluble coronavirus surface antigen is S protein or peptide.
  • Embodiment 34 The method of claim 29, wherein the neutralizing antibody is of SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myelin, ometron, and/or other strains Monoclonal antibody to S protein.
  • the neutralizing antibody is of SARS-CoV-2 Hu-1, alpha, beta, gamma, delta, myelin, ometron, and/or other strains Monoclonal antibody to S protein.
  • Embodiment 35 The method of embodiment 29, wherein the neutralizing antibody is a monoclonal antibody to the S protein of SARS-CoV-2 delta (B.1.617.2).
  • Embodiment 37 A complex comprising a trimer of a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, wherein the recombinant polypeptide trimers through inter-polypeptide disulfide bonds to form a trimer.
  • Embodiment 38 An immunogenic composition comprising a trimer of a recombinant polypeptide or a combination of any two or more trimers, the recombinant polypeptide comprising a trimer selected from the group consisting of SEQ ID NO: 85 - A sequence of groups of 92.
  • Embodiment 39 The immunogenic composition of embodiment 38, comprising a trimer of a recombinant polypeptide having the sequence set forth in SEQ ID NO: 91.
  • Embodiment 40 A method for generating an immune response to a coronavirus surface antigen in a subject, the method comprising administering to the subject an effective amount of a complex comprising a compound selected from SEQ ID NO: 85 - Recombinant polypeptides of the group consisting of 92, optionally the complex is used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, such as the first dose , second dose, third dose, fourth dose, and/or more doses, optionally the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA Any one or more of vaccines, adenovirus vector vaccines, and inactivated virus vaccines can be used in combination.
  • a complex comprising a compound selected from SEQ ID NO: 85 - Recombinant polypeptides of the group consisting of 92, optionally the complex is used as a primary series, an additional dose, and
  • the method includes administering to the subject an effective amount of a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 85-92, optionally as a primary series, additional agents (additional dose), and/or homologous or heterologous booster dose (booster dose) use, such as a first dose, a second dose, a third dose, a fourth dose, and/or more doses, optionally the initial dose
  • a dose, an additional dose, or a heterologous booster dose may be used in combination with any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • Embodiment 43 A method for generating an immune response to a coronavirus surface antigen in a subject
  • the surface antigen includes the S protein of coronavirus or an antigenic fragment thereof, and optionally, the surface antigen includes a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-84 or an antigenic fragment thereof, and
  • the surface antigen includes S protein or an antigenic fragment thereof
  • Embodiment 46 A fusion protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising from the amino to the carboxyl terminus:
  • Embodiment 48 The fusion protein of embodiment 47, wherein the third region includes the S1 domain of a third coronavirus, wherein the third coronavirus is the same as or different from the first coronavirus or the second coronavirus.
  • Embodiment 2 The fusion protein of any one of embodiments 46-51, wherein the second region includes one or more amino acid residues that are different from the corresponding amino acid residues in the first coronavirus.
  • Embodiment 53 The fusion protein of any one of embodiments 46-52, wherein the first and second coronaviruses are different variants or strains of the same coronavirus.
  • Embodiment 54 The fusion protein of embodiment 53, wherein the first region includes the NTD of the first coronavirus, the second region includes the RBD of the second coronavirus, and the first and second coronaviruses are SARS- Different variants of CoV-2.
  • Embodiment 56 A trimeric fusion protein comprising three recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus:
  • NTD N-terminal domain
  • Embodiment 57 A method for preventing coronavirus infection in a mammal, the method comprising immunizing the mammal with an effective amount of the fusion protein according to any one of claims 46-56.
  • Embodiment 59 The method of embodiment 58, wherein the first and second coronaviruses are different variants of SARS-CoV-2, and the neutralizing antibodies produced in the mammal neutralize B.1.1.529, Two or more SARS of the B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P.1, B.1.1.7, B.1.617 and A.23.1 lineages -CoV-2 virus.
  • Embodiment 60 The method of embodiment 59, wherein the neutralizing antibody produced in the mammal neutralizes B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2 , B.1.351, P.1, B.1.1.7, B.1.617 and A.23.1 lineages of three or more SARS-CoV-2 viruses.
  • Embodiment 61 The method of any one of embodiments 57-60, comprising immunizing the mammal with two or more doses of the fusion protein, at least one of the two or more doses of the fusion protein
  • the protein includes the SARS-CoV-2 delta (B.1.617.2) spike protein amino acid sequence
  • the at least one dose of the fusion protein includes the sequence described in any one of SEQ ID NOs: 81-92 or with SEQ
  • the sequence described in any one of ID NOs: 81-92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity.
  • Embodiment 62 The method of any one of embodiments 57-61, wherein the fusion protein is administered as a booster after one or more doses of an immunogen comprising a protein from the same or different SARS- Spike protein peptides of NTD and RBD of CoV-2 variants, optionally the one or more doses of the immunogen comprise the sequence described in any one of SEQ ID NOs: 27-66 and 81-84 or are consistent with SEQ ID NOs: 27-66 and 81-84.
  • sequence described in any one of ID NOs: 27-66 and 81-92 has an amino acid sequence with at least or about 80%, 85%, 90%, 92%, 95%, 97%, 99% sequence identity
  • the enhancer fusion protein comprises the sequence described in any one of SEQ ID NO:81-92 or has at least or about 80% or 85% of the sequence described in any one of SEQ ID NO:81-92. , 90%, 92%, 95%, 97%, 99% sequence identity of the amino acid sequences.
  • Example 1 Generation of recombinant disulfide-linked SARS-CoV-2 S-trimer fusion protein
  • a secreted recombinant disulfide-linked polypeptide containing a SARS-CoV-2 protein peptide fused to a trimerization domain was generated as a candidate protein subunit vaccine.
  • the ectodomain of the spike protein from SARS-CoV2, including its signal peptide (SP), S1 and S2 domains is C-terminally fused in-frame to a lactating protein encoding the human C-propeptide of ⁇ 1 collagen.
  • Animal expression vectors to express secreted trimeric S-trimer fusion antigens for example, as shown in Figures 1A-1B.
  • an affinity purification protocol was developed taking advantage of the high binding affinity between the Protein TrimerizerTM tag and Endo180, which is capable of binding to type 1 pro- Collagen receptors in the C-terminal region of collagen and mature it.
  • the Endo180-Fc fusion protein is loaded onto a Protein A column and captured by the resin through high-affinity binding between Protein A and the human IgG1 Fc domain of Endo180-Fc.
  • serum-free cell culture medium containing CHO cell-secreted S-trimers was loaded into a pre-captured Endo180-Fc on protein A column.
  • the bound S-trimer is purified using a mild salt elution step without causing separation of Endo180-Fc from the Protein A column. to close to uniformity.
  • the S-trimer is further purified by low pH for prophylactic virus inactivation (VI), anion exchange chromatography to remove host cell DNA and any residual endotoxin, and nanofiltration as a prophylactic virus removal (VR) step , and finally UF/DF to the concentration required to concentrate the S-trimer into the formulation buffer, thereby obtaining the active drug (DS) of the S-trimer subunit vaccine candidate.
  • Stability analysis of the purified S-trimer showed that the S-trimer is stable in liquid solution formulations at 2-8°C.
  • Protein TrimerizationTM technology (Liang et al., Nat. Comms., 12:1346, 2021) was used to generate covalent trimers of spike antigens based on Hu-1 strains and VOC strains.
  • High-level expression of the S-trimer fusion protein is shown in Figure 2.
  • An 8% SDS-PAGE analysis of S-trimer expression in fed-batch serum-free CHO cell cultures was performed in a 10L bioreactor. Analyze 10 ⁇ L of cell-free conditioned medium from days 9 to 13 under reducing conditions, followed by Coomassie Brilliant Blue staining.
  • Figure 3A shows the delta S-trimer purification process sample and reference substance reducing SDS-PAGE Coomassie brilliant blue staining analysis.
  • Figure 3B shows the SEC-HPLC purity analysis. The purity of delta S-trimer is 97.22%.
  • CMI ELISpot S-trimer antigen-specific cell-mediated immunity
  • Figure 5 shows BALB/c mouse SARS-CoV-2 Hu-1, alpha (alpha, ⁇ , B.1.1.7), beta (beta, ⁇ , B.1.351), gamma (gamma, ⁇ , P.1), delta (delta, ⁇ , B.1.617.2), mu (mu, ⁇ , B.1.621) and Omicro (Omicro, o, B.1.1.529) strain pseudoviruses and antibody EC50 data.
  • Th1 cytokines IFN- ⁇ , IL-2
  • Th2 cytokines IL-4, IL-5
  • ELISpot kits Mabtech
  • Pseudoviral reservoirs were titrated by infecting 293T-ACE2 cells by adding the Bright-Glo Luciferase Assay System (Promega, E2650) after an incubation period of 44 to 48 h at 37°C and 5% CO using microbiome. Luciferase activity was measured with a plate reader (TECAN, Spark). Then according to the Reed-Muench method ( Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay.Nie J.et al. DOI:10.21203/rs.3.pex-941/v11, for all purposes its full text The form quoted is added) to calculate the TCID 50 of the fake virus.
  • the Reed-Muench method Quantification of SARS-CoV-2 neutralizing antibody by a pseudotyped virus based assay.Nie J.et al. DOI:10.21203/rs.3.pex-941/v11, for all purposes its full text The form quoted
  • the cDNA encoding the extracellular domain of the SARS-CoV-2 spike (S) protein from Delta was gene synthesized by GenScript using Cricetulus griseus (Chinese hamster) preferred codons. This cDNA was subcloned into the pTRIMER expression vector (GenHunter Corporation) at the Hind III and Bgl II sites to allow in-frame fusion of the soluble S protein to the Protein TrimerizationTM tag (from human type I (alpha) collagen). Amino acid residues 1156-1406) as described above.
  • the expression vector was transiently transfected into the HEK-293F cell line (Clover Biopharma) using PEI (Polyscience) and grown in OPM-293 CD05 medium (OPM) and OPM-293 proFeed supplement (OPM). S-trimeric proteins were purified from conditioned media to homogeneity using a Protein Trimerizer TM tag-specific affinity column (Clover Biopharma).
  • mice were divided into 3 groups (10 mice in each group) as a control group without boosting (Group 1), or boosted with 3 ⁇ g of different vaccine candidates, Including Hu-1 S-trimer (Group 2), Delta S-Trimer vaccine (Group 3), all of which are adjuvanted with CpG (150 ⁇ g) plus Alum (75 ⁇ g).
  • Sera were collected before the third booster dose (D56, D-1 PD3) and 14 days after the third dose (D71, D14 PD3) and tested for neutralizing antibodies against multiple mutant pseudoviruses.
  • the Delta vaccine candidate (Group 3) significantly boosted neutralizing antibodies to Hu-1, Beta, Gamma, Delta, and Omicron VOCs, including Omicron (12.1-fold boost) (Figure 7A-7G ). Enhancement of neutralizing antibody breadth was also observed in the Hu-1 S-trimer group with Alum/CpG as adjuvant (Group 2). The potentiation of certain VOC (Hu-1, Alpha, Delta, and Omicron) neutralizing antibodies by the fully adjuvanted Hu-1 S-trimer (Group 2) was not significant. This may be due to the short interval between the last two doses of the vaccine (36 days), which did not allow enough time for more memory responses to develop.
  • multivalent vaccine combinations such as bivalent, trivalent and quadrivalent.
  • Use Hu-1 S-trimer, Omicron S-trimer, Omicron strain (BA.4/5) S-trimer, Belta S-trimer, Delta-S- Trimers are combined to obtain multivalent vaccines.
  • the multivalent vaccine combinations and trial design are shown in Table 1 and Figure 9. Part of the test results are shown in Figure 10.

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Abstract

L'invention concerne une composition immunogène comprenant un peptide recombinant et une protéine, le peptide recombinant et la protéine comprenant un antigène et un immunogène de coronavirus, tel que le peptide de protéine S de souche du variant de coronavirus SARS-CoV-2 delta (delta, B.1.617.2) ou un fragment, un variant ou un mutant de celui-ci, tel qu'un antigène chimérique et un immunogène contenant un domaine de liaison au récepteur de souche du variant delta et une séquence peptidique de protéine S de Hu-1 ou d'autre variant. Selon certains aspects, la composition immunogène comprend une protéine de fusion sécrétoire. La protéine de fusion sécrétoire comprend un antigène de coronavirus soluble. La protéine d'antigène de coronavirus soluble est fusionnée par fusion intra-trame à une partie à extrémité C-terminale capable d'auto-trimérisation, de façon à former une protéine de fusion trimère liée à une liaison disulfure. Selon certains aspects, la composition immunogène peut être utilisée pour produire une réponse immunitaire, par exemple en tant que vaccin pour la prévention d'une infection à coronavirus, telle qu'une infection par le SARS-CoV-2 Hu-1, alpha, bêta, gamma, delta, mu, omicron et/ou d'autres souches. Selon certains aspects, la composition immunogène peut être utilisée dans une composition de vaccin, par exemple, en tant que partie d'un vaccin prophylactique et/ou thérapeutique. L'invention concerne en outre un procédé de production du peptide recombinant et de la protéine, une méthode prophylactique, thérapeutique et/ou de diagnostic et un kit associé.
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