WO2023142786A1 - 冠状病毒疫苗组合物、方法及其使用 - Google Patents

冠状病毒疫苗组合物、方法及其使用 Download PDF

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WO2023142786A1
WO2023142786A1 PCT/CN2022/140514 CN2022140514W WO2023142786A1 WO 2023142786 A1 WO2023142786 A1 WO 2023142786A1 CN 2022140514 W CN2022140514 W CN 2022140514W WO 2023142786 A1 WO2023142786 A1 WO 2023142786A1
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dose
coronavirus
seq
vaccines
sars
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PCT/CN2022/140514
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French (fr)
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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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Priority to CN202280082721.4A priority Critical patent/CN118369353A/zh
Publication of WO2023142786A1 publication Critical patent/WO2023142786A1/zh

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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
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    • 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
    • A61K39/385—Haptens or antigens, bound to carriers
    • 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
    • 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
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    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • 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
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    • C07—ORGANIC CHEMISTRY
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    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • 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
    • C12N2770/20022—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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present disclosure relates in some aspects to immunogenic compositions for treating and/or preventing coronavirus infection, said immunogenic compositions comprising recombinant peptides and proteins comprising coronavirus antigens and immunogens , such as coronavirus S protein peptides, including subunit vaccines based on the S protein peptides of the SARS-CoV-2 beta (B.1.351) strain and formed by disulfide bonds between trimerized TM tag polypeptides.
  • Coronaviruses infect a wide range of birds and mammals, including humans. Coronaviruses may circulate through the body annually and usually cause mild respiratory illness, although more severe in infants, young children, the elderly, and people who are immunocompromised. However, certain coronaviruses, including Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) , is highly pathogenic. The high pathogenicity, airborne transmission, high case fatality rate, and vaguely defined epidemiology of coronaviruses create an urgent need for effective vaccines and related therapeutic agents. In particular, vaccines that can rapidly induce effective immune responses against SARS-CoV-2 are urgently needed.
  • the present invention provides methods, uses and articles of manufacture that meet the above and other needs.
  • proteins comprising a plurality of recombinant polypeptides, each comprising a coronavirus surface antigen linked to a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptides forms an interpolypeptide dichotomy. sulfur bond.
  • recombinant subunit vaccines comprising an ectodomain (e.g., without transmembrane and cytoplasmic structures) from the S protein of a coronavirus (e.g., SARS-CoV-2 beta (B.1.351) or a fragment thereof domain) fused in frame to the C-propeptide of collagen capable of forming disulfide-linked homotrimers.
  • a coronavirus e.g., SARS-CoV-2 beta (B.1.351) or a fragment thereof domain
  • the resulting recombinant subunit vaccines e.g., S-trimers
  • S-trimers can be derived from transfected Expressed and purified in cells, and is expected to be in the native conformation in trimeric form.
  • the coronavirus is severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV-1), SARS coronavirus 2 (SARS-CoV-2), SARS-like coronavirus, Middle East respiratory syndrome (MERS ) coronavirus (MERS-CoV), MERS-like coronavirus, NL63-CoV, 229E-CoV, OC43-CoV, HKU1-CoV, WIV1-CoV, MHV, HKU9-CoV, PEDV-CoV or SDCV.
  • SARS severe acute respiratory syndrome
  • SARS-CoV-1 SARS coronavirus
  • SARS-CoV-2 SARS coronavirus 2
  • MERS-like coronavirus Middle East respiratory syndrome coronavirus
  • NL63-CoV 229E-CoV
  • OC43-CoV HKU1-CoV
  • WIV1-CoV WIV1-CoV
  • MHV HKU9-CoV
  • PEDV-CoV or SDCV.
  • the surface antigen may comprise a coronavirus spike (S) protein or a fragment thereof or an epitope, wherein the epitope is optionally a linear epitope or a conformational epitope, and wherein the protein comprises three recombinant polypeptides .
  • S coronavirus spike
  • the surface antigen may comprise a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.
  • the surface antigen may comprise a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 (HR1) or Heptapeptide repeat 2 (HR2) or any combination thereof.
  • RBD receptor binding domain
  • RBM receptor binding motif
  • FP fusion peptide
  • HR1 heptad repeat 1
  • HR2 Heptapeptide repeat 2
  • the surface antigen may comprise the receptor binding domain (RBD) of the S protein.
  • the surface antigen may include the S1 subunit and the S2 subunit of the S protein.
  • the surface antigen may be free of transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.
  • the surface antigen may include a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or cathepsin L.
  • the surface antigen may contain no protease cleavage sites, where the protease is optionally furin, trypsin, Factor Xa, thrombin, or cathepsin L, or may contain Cleavage Mutated protease cleavage site.
  • the surface antigen may be soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.
  • the surface antigens may be the same or different among the recombinant polypeptides of the protein.
  • the surface antigen may be fused directly to the C-terminal propeptide, or may be passed through a linker (such as a linker comprising a glycine-X-Y repeat sequence, where X and Y are independently any amino acid and optionally a proline). amino acid or hydroxyproline) to the C-terminal propeptide.
  • a linker such as a linker comprising a glycine-X-Y repeat sequence, where X and Y are independently any amino acid and optionally a proline. amino acid or hydroxyproline
  • the protein may be soluble or not directly bound to a lipid bilayer, such as a membrane or viral envelope.
  • the protein may bind to a cell surface receptor in a subject, optionally wherein the subject is a mammal, such as a primate, such as a human.
  • the cell surface receptor can be angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grab Non-integrin (DC-SIGN) or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grab Non-integrin
  • L-SIGN liver/lymph node-SIGN
  • the C-terminal propeptide may be human collagen.
  • the C-terminal propeptide may include pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2(I), pro ⁇ 2(V), C-terminal propeptide of pro ⁇ 2(XI) or pro ⁇ 3(XI) or a fragment thereof.
  • the C-terminal propeptides may be the same or different in the recombinant polypeptides.
  • the C-terminal propeptide may comprise any of SEQ ID NO:67-80, or have at least 90%, 91%, 92%, 93% of any of SEQ ID NO:67-80.
  • Amino acid sequences with %, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence homology are capable of forming disulfide bonds between polypeptides and trimerizing recombinant polypeptides.
  • the C-terminal propeptide may comprise SEQ ID NO: 67 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 68 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 69 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 70 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 71 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 72 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 73 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 74 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 75 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 76 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 77 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 78 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 79 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise SEQ ID NO: 80 or an amino acid sequence at least 95% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the C-terminal propeptide may comprise a sequence comprising a glycine-X-Y repeat sequence linked to the N-terminus of any one of SEQ ID NOs: 67-80, wherein X and Y are independently any amino acid and Optionally proline or hydroxyproline, or an amino acid sequence at least 90% identical thereto, is capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide.
  • the surface antigen in each recombinant polypeptide may be in a prefusion conformation.
  • the surface antigen in each recombinant polypeptide may be in a postfusion conformation.
  • the surface antigen in each recombinant polypeptide may comprise any one of SEQ ID NOs: 27-66 and 81-85 or an amino acid sequence at least 80% identical thereto.
  • the recombinant polypeptide may comprise any one of SEQ ID NOs: 1-26 and 86-95, or an amino acid sequence at least 80% identical thereto.
  • immunogens comprising the proteins provided herein.
  • protein nanoparticles comprising a protein provided herein attached directly or indirectly to a nanoparticle.
  • VLPs virus-like particles
  • isolated nucleic acids encoding one, two, three or more recombinant polypeptides of the proteins provided herein.
  • the polypeptide encoding the S protein peptide is fused in-frame to the polypeptide encoding the C-terminal propeptide of collagen.
  • the isolated nucleic acids provided herein are operably linked to a promoter.
  • an isolated nucleic acid provided herein is a DNA molecule. In some embodiments, an isolated nucleic acid provided herein is an RNA molecule, optionally an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplifying mRNA, or trans-amplified mRNA.
  • vectors comprising the isolated nucleic acids provided herein.
  • the vector is a viral vector.
  • viruses, pseudoviruses or cells comprising a vector provided herein, optionally, wherein the virus or cell has a recombinant genome.
  • immunogenic compositions comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell provided herein and a pharmaceutically acceptable carrier.
  • a vaccine comprising an immunogenic composition provided herein and optionally an adjuvant, wherein the vaccine is optionally a subunit vaccine.
  • the vaccine is a prophylactic and/or therapeutic vaccine.
  • Optional adjuvants may be used in the prime and/or boost.
  • adjuvants for the initial dose and/or any booster dose or doses may include: adjuvants containing aluminum, such as adjuvants containing alum and/or aluminum hydroxide; adjuvants containing oligonucleotides, such as Adjuvants containing CpG oligodeoxynucleotides (CpG-ODN); adjuvants containing TLR9 agonists; containing metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween -80), such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 in the form of an oil-in-water emulsion; or any combination of adjuvants.
  • adjuvants containing aluminum such as adjuvants containing alum and/or aluminum hydroxide
  • adjuvants containing oligonucleotides such as Adjuvants containing CpG oligode
  • provided herein are methods of producing a protein comprising: expressing an isolated nucleic acid or vector provided herein in a host cell to produce a protein provided herein; and purifying the protein.
  • proteins produced by the methods provided herein are proteins produced by the methods provided herein.
  • a method for generating an immune response to a coronavirus S protein peptide or fragment or epitope thereof in a subject comprising administering to the subject an effective amount of the protein, immunogen, protein nano Particles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines to generate an immune response.
  • the methods provided herein are used to treat or prevent coronavirus infection.
  • generating an immune response suppresses or reduces replication of the coronavirus in the subject.
  • the immune response includes a cell-mediated and/or humoral response, optionally including the production of one or more neutralizing antibodies, such as polyclonal or monoclonal antibodies.
  • the immune response is directed against the S protein peptide of coronavirus or a fragment or epitope thereof, but not against the C-terminal propeptide.
  • administration to the subject does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to one or more coronaviruses.
  • the administration does not result in antibody-dependent enhancement (ADE) in the subject upon subsequent exposure to the one or more coronaviruses.
  • the method further comprises a priming step and/or a boosting step.
  • an effective amount is administered in a single dose or in a series of doses with one or more intervals. In some embodiments, the effective amount is administered without the use of an adjuvant. In some embodiments, an effective amount is administered with an adjuvant.
  • kits for treating coronavirus infections comprising administering to a subject an effective amount of a protein provided herein to generate neutralizing antibodies or neutralizing antiserum to a coronavirus in the subject.
  • the subject is a mammal, optionally a human or non-human primate.
  • the method further comprises isolating neutralizing antibodies or neutralizing antisera from the subject.
  • the method further comprises administering to the human subject an effective amount of the isolated neutralizing antibody or neutralizing antiserum to prevent or treat coronavirus infection by passive immunization.
  • the neutralizing antibody or neutralizing antiserum against coronavirus comprises a polyclonal antibody against coronavirus S protein peptide or a fragment thereof or an epitope, optionally, wherein the neutralizing antibody or neutralizing antiserum is not Contains or is substantially free of antibodies directed against the C-terminal propeptide of collagen.
  • the neutralizing antibody comprises a monoclonal antibody against the coronavirus S protein peptide or a fragment thereof or an epitope, optionally, wherein the neutralizing antibody does not contain or substantially does not contain the C-terminal propeptide against the collagen Antibody.
  • the proteins, immunogens, protein nanoparticles, VLPs, isolated nucleic acids, vectors, viruses, pseudoviruses, cells, immunogenic compositions or vaccines provided herein are used to induce an immune response to a coronavirus in a subject , and/or for the treatment or prevention of coronavirus infection.
  • provided herein is a use of a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition, or vaccine provided herein for inducing An immune response to a coronavirus, and/or for the treatment or prevention of a coronavirus infection.
  • a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus, cell, immunogenic composition or vaccine provided herein for the manufacture of a Drugs or prophylactics for the immune response to coronaviruses in subjects, and/or for the treatment or prevention of coronavirus infections.
  • Also provided herein is a method for analyzing a sample, comprising: contacting the sample with the protein provided herein, and detecting the binding between the protein and the analyte capable of specifically binding to the S protein peptide of coronavirus or a fragment or epitope thereof.
  • the analyte is an antibody, receptor or cell that recognizes a S protein peptide or a fragment or epitope thereof.
  • binding is indicative of the presence of the analyte in the sample, and/or the presence of a coronavirus infection in a subject from which the sample was derived.
  • kits comprising a protein provided herein and a matrix, pad or vial containing or immobilizing the protein, optionally wherein the kit is an ELISA or a lateral flow assay kit.
  • Figures 1A and 1B show isolated and purified chimeric proto-S-trimers containing beta mutant RBD.
  • Figure 2 shows the receptor binding kinetics of the chimeric proto-S-trimer containing RBD of the beta variant strain to ACE2-Fc. Compared with the original strain, the chimeric original S-trimer receptor with beta (beta) variant strain RBD has higher affinity.
  • Figure 3 shows the immune protection of adjuvanted S-trimers against non-human primate (rhesus monkey) SARS-CoV-2 virus. Animals were inoculated twice intramuscularly with adjuvanted S-trimer or vehicle control. Animals were challenged with SARS-CoV-2 virus on day 35 and then immune protection was assessed.
  • the first group is the saline vehicle control
  • the second group is the original S-trimer (adjuvant 1 contains CpG 1018 plus Alum)
  • the third group is the chimeric original S-trimer containing beta (beta) mutant RBD polymer (adjuvant 1)
  • the fourth group is the chimeric original S-trimer containing beta (beta) mutant RBD (adjuvant 2 contains squalene, ⁇ -tocopherol, and Tween-80)
  • Lung tissue was collected at autopsy at 7 dpi and tested for SARS-CoV-2 viral load based on genomic RNA (gRNA). Significantly lower viral loads in lung tissue than vehicle controls were observed in the three groups containing adjuvanted S-trimer. Comparing the third and fourth groups, the adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 was similar to CpG 1018 plus Alum as an adjuvant.
  • Figure 4 shows primate neutralizing antibody responses to adjuvanted S-trimers.
  • Use original S-trimer (adjuvant 1 containing CpG 1018 plus Alum), chimeric original S-trimer containing beta (beta) mutant RBD (adjuvant 1), or containing beta (beta) variant
  • a chimeric proto-S-trimer of strain RBD (adjuvant 2 containing squalene, ⁇ -tocopherol, and Tween-80) was inoculated intramuscularly twice in animals. Serum was collected at D35 for pseudovirus neutralizing antibody test and live SARS-CoV-2 virus neutralizing antibody test, A.
  • FIG. 1 shows SARS-CoV-2Hu-1, B.1.1.7, B.1.617.2, B.1.351, P.1, and B.1.621 strain pseudovirus neutralizing antibody titer (IC 50 ) data.
  • B. Live SARS-CoV-2 virus neutralizing antibody titer data are shown. Points represent data from individual animals; horizontal lines represent geometric mean titers (GMT) ⁇ SEM for each group. Animals receiving chimeric proto-S-trimers (adjuvant 2) containing the beta variant RBD (adjuvant 2) produced 6 to 100-fold higher of neutralizing antibodies.
  • the chimeric original S-trimer (adjuvant 2) containing beta (beta) variant strain RBD has a more extensive neutralization effect, and has produced in animals the effect on SARS-CoV-2Hu-1 and various VOC or Neutralizing antibodies to VOI. Comparing the chimeric original S-trimer (adjuvant 1) containing beta (beta) mutant strain RBD and the chimeric original S-trimer (adjuvant 2) containing beta (beta) mutant strain RBD, containing The chimeric original S-trimer (adjuvant 2) of beta mutant strain RBD has similar or better neutralization effect on pseudovirus and live virus.
  • Figure 5 shows the protective effect of chimeric prototype S-trimer (adjuvant 2) containing beta (beta) variant strain RBD on Hu-1 and on Omicron strain infection in primates .
  • Animals were intramuscularly inoculated twice with adjuvanted S-trimer or vehicle control, and serum was collected on D35 for pseudovirus neutralizing antibody testing.
  • the first group is the original S-trimer (adjuvant 1 containing CpG 1018 plus Alum)
  • the second group is the chimeric original S-trimer (adjuvant 1) containing RBD of the beta variant strain
  • the third group is the chimeric proto-S-trimer containing beta (beta) mutant RBD (adjuvant 2 contains squalene, ⁇ -tocopherol, and Tween-80)
  • the fourth group is the proto-S - Trimer and bivalent vaccine (Adjuvant 1 ) containing a chimeric original S-trimer of the beta variant RBD.
  • Animals receiving chimeric proto-S-trimers (adjuvant 2) containing the beta variant RBD (adjuvant 2) produced approximately 18-fold higher Neutralizing antibodies against Omicron strains. Comparing the chimeric original S-trimer (adjuvant 1) containing beta (beta) mutant strain RBD and the chimeric original S-trimer (adjuvant 2) containing beta (beta) mutant strain RBD, containing The chimeric original S-trimer (adjuvant 2) of the RBD of the beta mutant strain had a better neutralizing effect on the Omicron strain.
  • Fig. 6 shows the humoral immune response of mice to different doses of adjuvanted S-trimer and chimeric proto-S-trimer containing beta (beta) mutant RBD on day 35.
  • Figure 7A shows the immune response of non-human primates to the third dose of S-trimer containing different doses of adjuvant 1 and the chimeric original S-trimer containing beta (beta) mutant strain RBD research experiment design.
  • Figure 7B shows the immune response of non-human primates to the third dose of S-trimer containing different doses of adjuvant 1 and the chimeric original S-trimer containing beta (beta) mutant strain RBD .
  • Fig. 8 shows the immune protection of SARS-CoV-2 virus containing adjuvant S-trimer and chimeric original S-trimer containing beta (beta) variant strain RBD -weight.
  • Fig. 9 shows the immune protection of SARS-CoV-2 virus containing adjuvant S-trimer and chimeric original S-trimer containing beta (beta) mutant strain RBD- Nasal, throat, and anal viral load assay results.
  • Control saline, 1#: original S-trimer + adjuvant 1, 2#: beta chimera + adjuvant 1, 3#: beta chimera + adjuvant 2.
  • Adjuvant 1 contains CpG 1018 plus Alum.
  • Adjuvant 2 contained squalene, ⁇ -tocopherol, and Tween-80.
  • Figure 10 shows the neutralizing antibody titers of the S-trimer containing adjuvant 1 and the chimeric original S-trimer containing adjuvant 2 containing beta (beta) variant strain RBD against true virus.
  • Adjuvant 1 contains CpG 1018 plus Alum.
  • Adjuvant 2 contained squalene, ⁇ -tocopherol, and Tween-80.
  • Figure 11 shows the effect of the first agent S-trimer containing different doses of adjuvant 2 and different doses and the chimeric original S-trimer containing beta (beta) mutant strain RBD on the effect of true virus Omicron Neutralizing antibody titers for variants BA.4 and BA.5.
  • Adjuvant 1 contains CpG 1018 plus Alum.
  • Adjuvant 2 contained squalene, ⁇ -tocopherol, and Tween-80.
  • Fig. 12 shows that the first dose of S-trimer containing different doses of adjuvant 2 and different doses and the chimeric original S-trimer containing Beta (beta) variant strain RBD have the effect on true virus Hu-1 and beta neutralizing antibody titers.
  • Adjuvant 1 contained CpG1018 plus Alum.
  • Adjuvant 2 contained squalene, ⁇ -tocopherol, and Tween-80.
  • compositions, methods and uses of fusion peptides and proteins comprising coronavirus antigens or immunogens for the treatment (eg, prophylactic, therapeutic) of coronavirus infection.
  • compositions and methods of use of recombinant soluble surface antigens from RNA viruses covalently linked in trimeric form are disclosed.
  • the resulting fusion proteins are secreted as disulfide-linked homotrimers, which are more structurally stable while retaining a conformation similar to native trimeric viral antigens, and thus can be used as a more efficient strategy against these dangerous pathogens. effective vaccine.
  • a viral antigen trimer as a vaccine or as part of a multivalent vaccine to prevent viral infection, with no or with an adjuvant, or with more than one adjuvant, optionally by Intramuscular injection or intranasal administration.
  • Viral antigen trimers can be used in prime doses, additional doses, and/or boosters. Independently, the initial dose, the additional dose, and/or any one or more booster doses may be without or with an adjuvant.
  • optional adjuvants may include: aluminum-containing adjuvants, such as those containing alum and/or aluminum hydroxide; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants; adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g. An adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as those containing alum and/or aluminum hydroxide
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants
  • disclosed herein are methods of using viral antigen trimers as antigens for diagnosing viral infection by detecting antibodies (eg, IgM or IgG) that recognize viral antigens (eg, neutralizing antibodies).
  • antibodies eg, IgM or IgG
  • viral antigens eg, neutralizing antibodies
  • disclosed herein are methods of using viral antigen trimers as antigens to generate polyclonal or monoclonal antibodies useful for passive immunization (eg, neutralizing mAbs for the treatment of coronavirus infection).
  • viral antigen trimers as vaccines or as part of a multivalent vaccine, wherein the vaccine includes multiple trimer subunit vaccines that include the same protein of the virus or viral antigens comprising two or more different proteins of one or more viruses or viral antigens of one or more strains of the same virus.
  • disclosed herein are monovalent vaccines comprising a trimer of a viral antigen disclosed herein. In some embodiments, disclosed herein are bivalent vaccines comprising a trimer of a viral antigen disclosed herein. In some embodiments, disclosed herein are trivalent vaccines comprising a trimer of a viral antigen disclosed herein. In some embodiments, disclosed herein are tetravalent vaccines comprising a trimer of a viral antigen disclosed herein.
  • disclosed herein are monovalent vaccines comprising an S-trimer disclosed herein.
  • bivalent vaccines comprising an S-trimer disclosed herein.
  • bivalent vaccines comprising at least one S-trimer comprising a first S protein antigen and at least one S-trimer comprising a second S protein antigen.
  • the first and second S protein antigens are from the same S protein of one or more viral species or strains/subtypes, or from one or more viral species or from one of the same viral species. Two or more different S proteins of one or more strains/subtypes.
  • disclosed herein are trivalent vaccines comprising an S-trimer disclosed herein.
  • an S-trimer comprising at least one S-trimer comprising a first S protein antigen, at least one S-trimer comprising a second S protein antigen, and at least one S-trimer comprising a third S protein antigen.
  • - Trivalent vaccine of the trimer the first, second and third S protein antigens are from the same S protein of one or more viral species or strain/subtype, or from one or more viral species or the same virus Two, three or more different S proteins of one or more strains/subtypes of a species.
  • disclosed herein are tetravalent vaccines comprising an S-trimer disclosed herein.
  • an S-trimer comprising at least one S-trimer comprising a first S protein antigen, at least one S-trimer comprising a second S protein antigen, at least one S-trimer comprising a third S protein antigen, - A trimer and at least one quadrivalent vaccine comprising an S-trimer of a fourth S protein antigen.
  • the first, second, third and fourth spike protein antigens are from the same spike protein of one or more viral species or strain/subtype, or from one or more viral species Or two, three, four or more different S proteins of one or more strains/subtypes of the same virus species.
  • the proteins comprising coronavirus antigens and immunogens provided herein can be used to effectively and safely treat (eg, therapeutic, prophylactic) coronavirus infections.
  • proteins provided herein comprising coronavirus antigens and immunogens treat coronavirus infection without mediated vaccine-induced enhancement of disease (VED) and/or antibody-dependent enhancement (ADE).
  • VED vaccine-induced enhancement of disease
  • AD antibody-dependent enhancement
  • the proteins provided herein comprising coronavirus antigens and immunogens are easy to produce and exhibit stability under high stress conditions such as high temperature, extreme pH, hyperosmolarity, and hypoosmolarity.
  • the proteins and immunogenic compositions provided herein circumvent and satisfy the production, stability, safety and efficacy issues that hinder the development of coronavirus vaccines.
  • the coronavirus antigens and immunogens provided herein include coronavirus spike (S) proteins or peptides, especially SARS-CoV or SARS-CoV-2 S proteins.
  • S coronavirus spike
  • SARS-CoV and SARS-CoV-2 S proteins consist of S protein trimers, which belong to the group of viral fusion glycoproteins of type I, which also includes HIV glycoprotein 160 (Env), influenza hemagglutination (HA), Paramyxovirus F and Ebola virus glycoproteins.
  • the SARS-CoV and SARS-CoV-2 S proteins each encode surface glycoprotein precursors, and the amino terminus and most of the protein are predicted to be located on the cell surface or on the outside of the virus particle.
  • the S protein includes a signal peptide at the N-terminus, an extracellular domain, a transmembrane domain and an intracellular domain. Similar to other coronaviruses, the S protein of SARS-CoV and SARS-CoV-2 can be cleaved into S1 and S2 subunits by proteases. In particular, SARS-CoV-2 contains a furin-like cleavage site that other SARS-like CoVs lack.
  • the recombinant S ectodomain trimer comprises a recombinant S ectodomain protomer from an alphacoronavirus (eg, NL63-CoV or 229E-CoV).
  • the recombinant S ectodomain trimer comprises components derived from betacoronaviruses (e.g., OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis Virus (MHV) or HKU9-CoV) S ectodomain protomer.
  • betacoronaviruses e.g., OC43-CoV, SARS-CoV, SARS-CoV-2, MERS-CoV, HKU1-CoV, WIV1-CoV, mouse hepatitis Virus (MHV) or HKU9-CoV
  • coronaviruses including SARS-CoV-2 have trimeric surface antigens on their viral envelopes to allow for receptors during infection through specific cell surfaces. body into different host cells.
  • SARS-CoV-2 uses its trimeric virus surface antigen S protein to enter the host cells of the mammalian respiratory system after binding to its specific cell surface receptor ACE2.
  • a prerequisite for the generation of effective recombinant subunit vaccines is the ability to produce similar to the native viral S antigen, in particular maintaining its trimeric conformation to elicit a sufficient number of antibodies that can bind to the receptor-binding domain (RBD) of the viral S protein , thereby preventing the virus from binding to the ACE2 receptor, thereby eliminating the virus infection.
  • RBD receptor-binding domain
  • proteins comprising coronavirus antigens or immunogens are capable of generating an immune response, e.g., to SARS-CoV or SARS-CoV-2 S protein Peptide immune response.
  • the immune response suppresses or reduces replication of the coronavirus in the subject (eg, patient).
  • the immune response includes production of one or more neutralizing antibodies, such as polyclonal and/or monoclonal antibodies.
  • the neutralizing antibody inhibits or reduces replication of the coronavirus in a subject (eg, a patient).
  • administration of the protein to the subject does not result in antibody-dependent enhancement (ADE) in the subject due to prior exposure to the coronavirus.
  • proteins comprising coronavirus antigens and immunogens are used as vaccines.
  • coronavirus antigens and immunogens are linked to proteins or peptides to form fusion proteins or recombinant polypeptides.
  • the protein or peptide linked to the coronavirus antigen or immunogen is capable of binding, eg, covalently or non-covalently, to the protein or peptide (eg, the protein or peptide of a fusion protein or recombinant polypeptide).
  • the protein or peptide linked to the coronavirus antigen or immunogen is a multimerization domain.
  • coronavirus antigens and immunogens are linked to collagen propeptides (eg, at the C-terminus of collagen propeptides) to form fusion peptides or recombinant polypeptides.
  • the proteins provided herein include recombinant polypeptides comprising coronavirus antigens and immunogens (eg, coronavirus S protein peptides or fragments or epitopes thereof), linked to the C-terminal propeptide of collagen.
  • the propeptide of the collagen is derived from the human C-propeptide of alpha 1 collagen and is capable of self-trimerization after expression.
  • linking coronavirus antigens and immunogens eg, coronavirus S protein peptide
  • immunogens eg, coronavirus S protein peptide
  • the propeptide of collagen eg, at the C-terminus of the propeptide of collagen
  • the production of recombinant proteins can preserve the tertiary and quaternary structure of the coronavirus S protein peptide, which may be important for the stability of the native conformation of the coronavirus S protein peptide, while the availability of antigenic sites on the protein surface can in turn enable Elicit an immune response, such as neutralizing antibodies.
  • linking the coronavirus S protein peptide to a protein or peptide capable of self-trimerization allows aggregation of the recombinant protein, thereby mimicking the natural homotrimeric structure of the coronavirus S protein peptide on the viral envelope.
  • linking the coronavirus S protein peptide to the C-terminal propeptide of collagen results in a self-trimeric recombinant polypeptide.
  • the proteins provided herein include a plurality of self-trimerized coronavirus S protein peptides and propeptides of collagen recombinant polypeptides.
  • the trimeric nature of recombinant proteins contributes to protein stability.
  • the trimeric nature of the recombinant protein contributes to the ability of the protein to generate an immune response.
  • the trimeric nature of the recombinant protein and/or the macrostructure of the plurality of self-trimeric recombinant proteins contributes to the ability of the protein to generate an immune response.
  • immunogenic compositions comprising the proteins provided herein, methods of producing the proteins provided herein, methods of treating subjects with the proteins and compositions provided herein, and kits.
  • the proteins provided herein include coronavirus antigens and immunogens.
  • the coronavirus antigens and immunogens contemplated herein are capable of promoting or stimulating cell-mediated and/or humoral responses.
  • the response eg, a cell-mediated or humoral response
  • the coronavirus antigen or immunogen is a coronavirus spike protein peptide.
  • Coronaviruses are a family of positive-sense single-stranded RNA viruses known to cause severe respiratory disease. They possess the largest genome (26-32kb) among known RNA viruses and are phylogenetically divided into four genera ( ⁇ , ⁇ , ⁇ , ⁇ ), with ⁇ -coronaviruses further subdivided into four lineages (A, B, C, D). It is currently known that the viruses of the coronavirus family that infect humans come from the genus ⁇ -coronavirus and ⁇ -coronavirus. In addition, it is believed that the gamma-coronavirus and delta-coronavirus genera may infect humans in the future.
  • betacoronaviruses include Middle East respiratory syndrome coronavirus (MERS-CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43( OC43-CoV), murine hepatitis virus (MHV-CoV), bat SARS-like coronavirus WIV1 (WIV1-CoV), and human coronavirus HKU9 (HKU9-CoV).
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • HKU1-CoV human coronavirus HKU1
  • MHV-CoV human coronavirus OC43( OC43-CoV)
  • MHV-CoV murine hepatitis virus
  • WIV1-CoV bat SARS-like coronavirus WIV1
  • HKU9-CoV human coronavirus HKU9
  • Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV).
  • a non-limiting example of a deltacoronavirus is porcine deltacoronavirus (SDCV).
  • This article discloses a list of SARS-associated coronaviruses:
  • Taiwan TC1 SARS coronavirus Taiwan TC1
  • Taiwan TC2 SARS coronavirus Taiwan TC2
  • Taiwan TC3 SARS coronavirus Taiwan TC3
  • the coronavirus genome is capped, polyadenylated and covered with nucleocapsid proteins.
  • the coronavirus particle includes a viral envelope comprising a type I fusion glycoprotein called the spike (S) protein.
  • S spike
  • Most coronaviruses have a common genome structure in which the replicase gene is contained in the 5' part of the genome and the structural gene is contained in the 3' part of the genome.
  • the coronavirus spike (S) protein is a class I fusion glycoprotein originally synthesized as a precursor protein.
  • a single precursor S polypeptide forms a homotrimer and is glycosylated and processed within the Golgi to remove the signal peptide and is cleaved by cellular proteases to generate separate S1 and S2 polypeptide chains, still in the homotrimer Associates as S1/S2 protomers and is therefore a trimer of heterodimers.
  • the S1 subunit is located at the distal end of the viral membrane and contains the receptor binding domain (RBD) that mediates the attachment of the virus to its host receptor.
  • RBD receptor binding domain
  • the S2 subunit contains the fusion protein machinery, such as the fusion peptide, two heptad repeats (HR1 and HR2) and central helix, a transmembrane domain and a cytosolic tail domain unique to fusion glycoproteins .
  • fusion protein machinery such as the fusion peptide, two heptad repeats (HR1 and HR2) and central helix, a transmembrane domain and a cytosolic tail domain unique to fusion glycoproteins .
  • the coronavirus antigen or immunogen is a coronavirus S protein peptide in a prefusion conformation, which is the extracellular domain of the coronavirus S protein after it is processed into the mature coronavirus S protein in the secretion system and triggered to cause the coronavirus S is the structural conformation adopted before the fusion event for the transition to the post-fusion conformation.
  • Kirchdoerfer et al. "Pre-fusion structure of a human coronavirus spike protein," Nature, 531:118-121, 2016 provides a three-dimensional structure of an exemplary coronavirus spike protein (HKU1-CoV) in a pre-fusion conformation.
  • the coronavirus antigen or immunogen includes one or more amino acid substitutions, deletions, or insertions compared to the native coronavirus S It provides increased retention of the pre-fusion conformation compared to polymers.
  • "Stabilization" of the prefusion conformation by one or more amino acid substitutions, deletions, or insertions can be, for example, energetic stabilization (e.g., reducing the energy of the prefusion conformation relative to the postfusion open conformation) and/or kinetic stabilization ( For example, reducing the conversion rate from the pre-fusion conformation to the post-fusion conformation).
  • stabilization of the coronavirus S ectodomain trimer in the prefusion conformation may include increased resistance to denaturation compared to the corresponding native coronavirus S sequence.
  • methods to determine whether the coronavirus S ectodomain trimer is in a prefusion conformation including (but not limited to) negative stain electron microscopy and antibody binding assays using antibodies specific for the prefusion conformation.
  • the coronavirus antigen or immunogen is a fragment of a S protein peptide.
  • the antigen or immunogen is an epitope of a S protein peptide.
  • An epitope includes an antigenic determinant chemical group or peptide sequence on a molecule that is antigenic so as to elicit a specific immune response, eg, an epitope is a region of an antigen to which B cells and/or T cells respond.
  • Antibodies can bind to specific antigenic epitopes, such as epitopes on the extracellular domain of coronavirus S. Epitopes can be formed by either contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • the coronavirus epitope is a linear epitope. In some embodiments, the coronavirus epitope is a conformational epitope. In some embodiments, the coronavirus epitope is a neutralizing epitope site. In some embodiments, all neutralizing epitopes of the coronavirus S protein peptide or fragment thereof are present as antigens or immunogens.
  • the viral antigen or immunogen when the viral antigen or immunogen is a fragment of the S protein peptide, only a single subunit of the S protein peptide is present, and the single subunit of the S protein peptide is trimerized.
  • the viral antigen or immunogen comprises a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.
  • viral antigens or immunogens include signal peptides, receptor binding domain (RBD) peptides, receptor binding motif (RBM) peptides, fusion peptides (FP), heptad repeat 1 (HR1) peptides or a heptad repeat 2 (HR2) peptide or any combination thereof.
  • the viral antigen or immunogen includes the receptor binding domain (RBD) of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 subunit and the S2 subunit of the S protein. In some embodiments, the viral antigen or immunogen includes the S1 subunit of the S protein, but does not include the S2 subunit. In some embodiments, the viral antigen or immunogen includes the S2 subunit of the S protein, but does not include the S1 subunit. In some embodiments, the viral antigen or immunogen is free of transmembrane (TM) domain peptides and/or cytoplasmic (CP) domain peptides.
  • TM transmembrane
  • CP cytoplasmic
  • the viral antigen or immunogen includes a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, or cathepsin L.
  • the viral antigen or immunogen does not contain a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, or cathepsin L, or contains a mutation that is not cleavable by a protease.
  • Protease cleavage site wherein the protease is optionally furin, trypsin, factor Xa, or cathepsin L, or contains a mutation that is not cleavable by a protease.
  • the viral antigen or immunogen is a SARS-CoV-2 antigen comprising at least one SARS-CoV-2 protein or fragment thereof.
  • the SARS-CoV-2 antigen is recognized by SARS-CoV-2 reactive antibodies and/or T cells.
  • the SARS-CoV-2 antigen is an inactivated whole virus.
  • the SARS-CoV-2 antigens include subunits of the virus.
  • the SARS-CoV-2 antigen comprises a structural protein of SARS-CoV-2 or a fragment thereof.
  • the structural protein of SARS-CoV-2 comprises one of the group consisting of spike (S) protein, membrane (M) protein, nucleocapsid (N) protein and envelope (E) protein or more.
  • the SARS-CoV-2 antigen comprises or further comprises a nonstructural protein of SARS-CoV-2 or a fragment thereof.
  • the nucleotide sequence of a representative SARS-CoV-2 isolate (Wuhan-Hu-1) is recorded as GenBank No. MN908947.3 (Wu et al., Nature, 579:265-269, 2020).
  • the viral antigen or immunogen comprises the sequence set forth in any one of SEQ ID NOs: 81-85. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 85%, 90%, 92%, 95%, 97%, or 99% sequence identity to SEQ ID NO: 85 as shown below sexual amino acid sequence. In some embodiments, the viral antigen or immunogen comprises an RBD trimer, e.g., the SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • RBD trimer e.g., the SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:55. In some embodiments, the viral antigen or immunogen comprises a receptor binding motif ( RBM)) Amino acid sequences having at least or about 80%, 85%, 90%, 92%, 95% or 97% sequence identity. In some embodiments, the viral antigen or immunogen comprises an RBD trimer, e.g., the SARS-CoV-2 RBD sequence linked to any one of SEQ ID Nos: 67-80.
  • RBM receptor binding motif
  • the viral antigen or immunogen comprises the spike glycoprotein sequence of the original Wuhan-Hu-1 coronavirus (eg, NC_045512). In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen comprises the Spike glycoprotein sequence of the Cluster 5 ( ⁇ FVI-spike) virus. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.1.7 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.1.207 lineage.
  • the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.1.317 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.1.318 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the P.1 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.351 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.429/CAL.20C lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.525 lineage.
  • the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.526 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.617 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.617.2 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.618 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.620 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the P.2 lineage.
  • the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the P.3 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.1.143 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the A.23.1 lineage. In some embodiments, the viral antigen or immunogen comprises the spike glycoprotein sequence of a virus in the B.1.617 lineage. In some embodiments, viral antigens or immunogens include viruses derived from Wuhan-Hu-1, viruses in the B.1.526 lineage, viruses in the B.1.1.7 lineage, viruses in the P.1 lineage, B.
  • viruses of the 1.351 lineage viruses of the P.2 lineage, viruses of the B.1.1.143 lineage, viruses of the A.23.1 lineage and viruses of the B.1.617 lineage
  • viruses of the B.1.1.143 lineage viruses of the A.23.1 lineage
  • viruses of the B.1.617 lineage The sequence of the spike glycoprotein of the virus (in any suitable combination).
  • the viral antigen or immunogen comprises T95I, G142D, ⁇ 143-145, and/or T478K, for example, as described in B.1.617.2 delta and/or B.1.1.529 Omicron var. ( For example in BA.4 and BA.5).
  • the viral antigen or immunogen comprises E484K and/or S477N, eg, as in the B.1.526 variant.
  • the viral antigen or immunogen comprises ⁇ 400-402 ( ⁇ FVI), eg, as in the Cluster 5 ( ⁇ FVI-spike) variant.
  • the viral antigen or immunogen comprises ⁇ 69-70 ( ⁇ HV), ⁇ 144 ( ⁇ Y), N501Y, A570D, D614G, P681H, T716I, S982A and/or D1118H, e.g., as in variant B.1.1.7 middle.
  • the viral antigen or immunogen comprises P681H, eg, as in the B.1.1.207 variant.
  • the viral antigen or immunogen comprises L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I and/or V1176F, e.g., as in the P.1 variant.
  • the viral antigen or immunogen comprises E484K, eg, as in the P.2 variant.
  • the viral antigen or immunogen comprises E484K and/or N501Y, eg, as in the P.3 variant.
  • the viral antigen or immunogen comprises L18F, D80A, D215G, ⁇ 242-244 ( ⁇ LAL), R246I, K417N, E484K, N501Y, D614G and/or A701V, eg, as in the B.1.351 variant.
  • the viral antigen or immunogen comprises S13I, W152C and/or L452R, eg, as in the B.1.429/CAL.20C variant.
  • viral antigens or immunogens include ⁇ 69-70 ( ⁇ HV), E484K and/or F888L, eg, as in the B.1.525 variant.
  • the viral antigen or immunogen comprises G142D, L452R, E484Q and/or P681R, eg, as in the B.1.617 variant. In some embodiments, the viral antigen or immunogen comprises G142D, L452R and/or P681R, eg, as in the B.1.617.2 variant. In some embodiments, the viral antigen or immunogen comprises E484K, eg, as in the B.1.618 variant. In some embodiments, a viral antigen or immunogen may comprise a fusion polypeptide (protomer) comprising any one or more of the foregoing mutations in any suitable combination.
  • the viral antigen or immunogen may comprise a trimer of three fusion polypeptides, and any of the three protomer fusion polypeptides may comprise any one or more of the foregoing in any suitable combination mutation.
  • two or all three of the three protomer fusion polypeptides that form the trimer may include different mutations and/or different combinations of mutations in each protomer.
  • a viral antigen or immunogen may comprise a mixture of trimers, and each trimer may comprise a different mutation and/or a different combination of mutations.
  • the viral antigen or immunogen comprises amino acid positions 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, Any one, two, three, four, five or more mutations of the group consisting of mutations (eg, substitutions, deletions and/or insertions) at 1118 and 1176.
  • mutations eg, substitutions, deletions and/or insertions
  • the viral antigen or immunogen comprises a group consisting of mutations (e.g., substitutions, deletions, and/or insertions) at amino acid positions 440, 452, 477, 484, 501, 614, 655, 681, and 701. Any one, two, three, four, five, six, seven, eight or all mutations in the group.
  • the viral antigen or immunogen comprises a chimeric polypeptide comprising sequences from a different virus, for example one or more mutations from a first variant of a coronavirus and from a second variant of a coronavirus different from the first variant of one or more mutations.
  • such chimeric viral antigens or immunogens can be used to elicit a broad immune response against the first and second variants of the coronavirus.
  • the viral antigen or immunogen comprises a virus antigen or immunogen selected from the group consisting of T95I, G142D, ⁇ 143-145, T478K, 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, H655 Y, P681H, P681R, Any one, two, three, four, five or more mutations in the group consisting of R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027
  • the viral antigen or immunogen comprises any one, two, 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 SARS-CoV-2 antigen includes a truncated S protein lacking a signal peptide, the transmembrane and cytoplasmic domains of the full-length S protein.
  • the SARS-CoV-2 antigens are recombinant proteins, while in other embodiments, the SARS-CoV-2 antigens are purified from virions. In some preferred embodiments, the SARS-CoV-2 antigen is an isolated antigen.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:27. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 27 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 27, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:28. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 28 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 28, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:29. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 29 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 29, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:30. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 30 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 30, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 31. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 31 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 31, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:32. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 32 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 32, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO: 33. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 33 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 33, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:34. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 34 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 34, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:35. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 35 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 35, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:36. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 36 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 36, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:37. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 37 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 37, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:38. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 38 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 38, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:39. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 39 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 39, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:40. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 40 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 40, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:41. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 41 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 41, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:42. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 42 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 42, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:43. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 43 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 43, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:44. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 44 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 44, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:45. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 45 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 45, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:46. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 46 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 46, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:47. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 47 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 47, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:48. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 48 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 48, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:49. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 49 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 49, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:50. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:50 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 50, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:51. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:51 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 51, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:52. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:52 Amino acid sequences having %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the viral antigen or immunogen includes a signal peptide.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:53.
  • the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:53 Amino acid sequences having %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:54.
  • the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:54 Amino acid sequences having %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:55. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:55 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Sequence comprising substitutions, deletions and/or insertions at one or more amino acid positions in the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 55, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:56. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:56 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including those 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, Comprising a substitution at one or more amino acid positions of the group consisting of 681, 682, 683, 684, 685, 701, 716, 888, 982, 1027, 1118 and 1176 (with respect to amino acid positions
  • the viral antigen or immunogen comprises a variant of SEQ ID NO: 56, and the variant comprises 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, R682G, R683S, R685G, A701V, T716I, F888L, S982A, T1027I, D1118H and V1176F any one, two, three, four, five or more mutations.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:57. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:57 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:57 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:58. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:58 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:58 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:59. In some embodiments, the viral antigen or immunogen comprises a sequence comprising substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO:59. In some embodiments, the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:60. In some embodiments, the viral antigen or immunogen comprises a sequence comprising substitutions, deletions and/or insertions at one or more amino acid positions of SEQ ID NO:60.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:61. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 61 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:61 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:62. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 62 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:62 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:63. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 63 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:63 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:64. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 64 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:64 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:65. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 65 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:65 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:81. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 81 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO: 81 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:82. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:82 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:82 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:83. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:83 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:83 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:84. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:84 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:84 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen comprises the sequence set forth in SEQ ID NO:85. In some embodiments, the viral antigen or immunogen comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:85 Sequences containing substitutions, deletions and/or insertions at amino acid positions.
  • the viral antigen or immunogen does not include a transmembrane domain such as SEQ ID NO: 66 or a portion thereof.
  • the coronavirus antigen or immunogen comprises a soluble S protein peptide.
  • the soluble S protein peptide lacks a TM domain peptide and a CP domain peptide.
  • the soluble S protein peptide does not bind to lipid bilayers, such as membranes or viral envelopes.
  • the S protein peptide is generated from a codon-optimized nucleic acid sequence. In some embodiments, the S protein peptide is generated from a non-codon-optimized nucleic acid sequence.
  • the viral antigens or immunogens referred to herein may include recombinant polypeptides or fusion peptides comprising said viral antigens or immunogens.
  • the term viral antigen or immunogen may be used to refer to a protein comprising a coronavirus antigen or immunogen.
  • the coronavirus antigen or immunogen is a coronavirus protein peptide provided herein.
  • the viral antigens or immunogens mentioned herein can be used in prime doses, additional doses, and/or booster doses. Independently, the initial dose, the additional dose, and/or any one or more booster doses may be without or with an adjuvant.
  • optional adjuvants may include: aluminum-containing adjuvants, such as those containing alum and/or aluminum hydroxide; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants; adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g. An adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as those containing alum and/or aluminum hydroxide
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants
  • coronavirus antigens and immunogens provided herein can be combined, e.g., linked, with other proteins or peptides to form recombinant polypeptides, including fusion peptides.
  • individual recombinant polypeptides (eg, monomers) provided herein are combined to form multimers, eg, trimers, of recombinant polypeptides.
  • association of individual recombinant polypeptide monomers occurs through covalent interactions.
  • association of individual recombinant polypeptide monomers occurs through non-covalent interactions.
  • interactions are effected by proteins or peptides linked to coronavirus antigens or immunogens (eg, S protein peptides).
  • coronavirus antigen or immunogen eg, S protein peptides
  • the protein or peptide to which it is linked can be selected so as to preserve the natural homotrimeric structure of the glycoprotein. This can be advantageous for eliciting a strong and potent immunogenic response to the S protein peptide.
  • retention and/or maintenance of the native conformation of a coronavirus antigen or immunogen eg, S protein peptide
  • a recombinant polypeptide comprising a S protein peptide described herein is alternatively referred to herein as a recombinant S antigen, a recombinant S immunogen, or a recombinant S protein.
  • recombinant polypeptides or multimeric recombinant polypeptides thereof aggregate or can aggregate to form proteins or complexes comprising multiple coronavirus antigens and/or immunogenic recombinant polypeptides. Formation of such proteins may facilitate the generation of strong and potent immunogenic responses to coronavirus antigens and/or immunogens.
  • the formation of proteins comprising multiple recombinant polypeptides, thereby forming multiple coronavirus antigens, such as coronavirus S protein peptides can retain the tertiary and/or quaternary structure of the viral antigens, allowing an immune response to be mounted against the native structure .
  • aggregation may confer structural stability on coronavirus antigens or immunogens, which in turn may provide access to potential antigenic sites capable of promoting immune responses.
  • a coronavirus antigen or immunogen may be linked at its C-terminus (C-terminus linkage) to a trimerization domain to facilitate trimerization of monomers.
  • trimerization stabilizes the membrane-proximal aspect of a coronavirus antigen or immunogen (eg, a coronavirus S protein peptide) in a trimer configuration.
  • Non-limiting examples of exogenous multimerization domains that promote stable trimerization of soluble recombinant proteins include: GCN4 leucine zipper (Harbury et al. 1993 Science 262:1401-1407), trimerization of pulmonary surfactant proteins Motif (Hoppe et al.1994 FEBS Lett 344:191-195), collagen (McAlinden et al.2003 J Biol Chem 278:42200-42207) and phage T4 fibritin Foldon (Miroshnikov et al.1998 Protein Eng 11:329 - 414), any of which can be linked to the coronavirus antigens or immunogens described herein (for example, by being linked to the C-terminus of the S peptide) to promote trimerization of recombinant virus antigens or immunogens.
  • one or more peptide linkers can be used to link recombinant viral antigens or immunogens to the multimerization domain.
  • the trimer can include any of the stabilizing mutations provided herein (or combinations thereof).
  • the recombinant polypeptide or fusion protein comprises the first sequence described in any one of SEQ ID NO:27-66 and 81-85, and the first sequence is connected to any one of SEQ ID NO:67-80 The second sequence, wherein the C-terminal of the first sequence is directly connected to the N-terminal of the second sequence.
  • the recombinant polypeptide or fusion protein comprises the first sequence described in any one of SEQ ID NO:27-66 and 81-85, and the first sequence is connected to any one of SEQ ID NO:67-80
  • the second sequence wherein the C-terminus of the first sequence is indirectly linked to the N-terminus of the second sequence, such as through a linker.
  • the linker includes a sequence comprising a glycine-X-Y repeat sequence.
  • the trimerized protein moiety required for biopharmaceutical design should meet the following criteria. Ideally, it should be part of a naturally secreted protein, such as immunoglobulin Fc, which is also abundant in circulation (non-toxic), derived from humans (lack of immunogenicity), relatively stable (long half-life), and capable of efficient autoimmunity. Trimerization, the self-trimerization is enhanced by interchain covalent disulfide bonds, so the trimerized coronavirus antigen or immunogen is structurally stable.
  • a naturally secreted protein such as immunoglobulin Fc, which is also abundant in circulation (non-toxic), derived from humans (lack of immunogenicity), relatively stable (long half-life), and capable of efficient autoimmunity.
  • Trimerization the self-trimerization is enhanced by interchain covalent disulfide bonds, so the trimerized coronavirus antigen or immunogen is structurally stable.
  • Collagen is a member of the fibrin family, which is a major component of the extracellular matrix. It is the most abundant protein in mammals, accounting for nearly 25% of the total protein in the body. Collagen plays an important structural role in the formation of bones, tendons, skin, cornea, cartilage, blood vessels and teeth.
  • the fibrillar types of collagens I, II, III, IV, V, and XI are all synthesized as larger trimeric precursors called procollagens, in which hundreds of "G-X-Y" repeats (or glycine repeats)
  • the central uninterrupted triple helical domain is flanked by a noncollagenous domain (NC), N-propeptide and C-propeptide.
  • NC noncollagenous domain
  • BMP-1 is a protease that recognizes a specific peptide sequence of procollagen near the junction between the glycine repeat and the collagen C-prodomain and is responsible for the removal of the propeptide.
  • the exfoliated trimeric C-propeptide of type I collagen is found in the human serum of normal adults at concentrations ranging from 50-300 ng/mL, with children having higher levels, suggesting active bone formation.
  • C-propeptide of type I collagen In people with familial high serum concentrations of C-propeptide of type I collagen, levels can be as high as 1–6 ⁇ g/mL without apparent abnormalities, suggesting that C-propeptide is nontoxic. Structural studies of the trimeric C-propeptide of collagen have shown that it is a three-lobed structure, with all three subunits brought together in a linking region near its N-terminus, where it is linked to the rest of the procollagen molecule. The geometry of this protruding protein is fused in one direction similar to Fc dimers.
  • Types I, IV, V, and XI collagens are mainly assembled into a chain consisting of two ⁇ -1 chains and one ⁇ -2 chain (for types I, IV, V) or three different chains (for type XI) Heterotrimeric forms, which are highly homologous in sequence.
  • Both type II and type III collagens are homotrimers of ⁇ -1 chains.
  • type I collagen the most abundant form of collagen
  • a stable alpha(I) homotrimer is also formed and is present at variable levels in different tissues.
  • Most of these collagen C-propeptide chains can self-assemble into homotrimers when overexpressed alone in cells.
  • the N-propeptide domain was first synthesized, the molecular assembly of trimeric collagen begins with the cognate binding of the C-propeptide.
  • the C-propeptide complex is believed to be stabilized by the formation of interchain disulfide bonds, but the necessity of disulfide bond formation for proper chain registration is unknown.
  • the triple helical repeat of glycine then propagates in a zipper-like manner from the associated C-terminus to the N-terminus.
  • This knowledge creates a non-natural type of collagen matrix by exchanging the C-propeptides of different collagen chains using recombinant DNA technology.
  • Non-collagenous proteins such as cytokines and growth factors have also been fused to the N-terminus of procollagen or mature collagen to form a new collagen matrix with the aim of slow release of non-collagenous proteins from the cellular matrix.
  • the C-propeptide needs to be cleaved before reorganizing collagen fibers to assemble into an insoluble cell matrix.
  • collagen in the recombinant polypeptides described herein has many advantages, including: (1) collagen is the most abundant protein secreted in mammals, accounting for nearly 25% of the total protein in the body; (2) collagen is the major form of natural It appears in the form of a trimeric helix, and its globular C-propeptide is responsible for the initiation of trimerization; (3) The trimeric C-propeptide of collagen released from the hydrolysis of mature collagen protein is expressed in mammalian blood at submicrograms/ mL levels occur naturally and are not known to be toxic to the body; (4) the linear triple helical region of collagen can be included as a linker, with the predicted spacing of each residue being Or excluded as part of the fusion protein so that the distance between the protein to be trimerized and the C-propeptide of the collagen can be precisely adjusted for optimal bioactivity; (5) Cleavage of the C-propeptide from the procollagen The recognition site of the down BMP1 can be mutated or deleted to prevent the destruction of the trimeric
  • a coronavirus antigen or immunogen is linked to the C-terminal propeptide of collagen to form a recombinant polypeptide.
  • the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.
  • the recombinant protein forms trimers.
  • the coronavirus antigen or immunogen is a S protein peptide as described in Section 1.
  • fusion polypeptides including the fusion polypeptide in SEQ ID NO: 1 can be produced and trimerized by interpolypeptide disulfide bonds (Cys residues that can form interpolypeptide disulfide bonds are shown in bold)
  • the signal peptide MFVFLVLLPLVSS (SEQ ID NO:54) on the N-terminus can be produced and trimerized by interpolypeptide disulfide bonds (Cys residues that can form interpolypeptide disulfide bonds are shown in bold)
  • the signal peptide MFVFLVLLPLVSS SEQ ID NO:54
  • interpolypeptide disulfide bonds may include Cys15-136, Cys131-166, Cys291-301, Cys379-432, Cys336-361, Cys391-525, Cys480-488, Cys538-590, Cys617-649, Cys662 - one or more or all of Cys743-749, Cys738-760, Cys840-851, Cys1032-1043 and Cys1082-1126, in any suitable combination.
  • the fusion polypeptide in the trimer can include one or more glycosylation sites (e.g., Asn-linked), e.g., at 17, 61, 122, 149, 165, 234, 282, At one or more or all of the Asn residues at 331, 343, 603, 616, 657, 709, 717, 801, 1074, 1098 and 1134, in any suitable combination.
  • one or more glycosylation sites e.g., Asn-linked
  • 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 a 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 pro ⁇ 1(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 having 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 having 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 having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:69. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:70. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:70. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:71. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:71.
  • the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:72. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:72. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:73. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:73. In some embodiments, the C-terminal propeptide is or is the amino acid sequence set forth in SEQ ID NO:74.
  • the C-terminal propeptide exhibits an amino acid sequence having 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 having 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 the amino acid sequence set forth in SEQ ID NO:76. In some embodiments, the C-terminal propeptide is an amino acid sequence having 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 having 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 having 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 exhibits an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:79. In some embodiments, the C-terminal propeptide is or includes the amino acid sequence set forth in SEQ ID NO:80. In some embodiments, the C-terminal propeptide is an amino acid sequence having at least or about 85%, 90%, 92%, 95%, or 97% sequence identity to SEQ ID NO:80.
  • the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human ⁇ 1(I) collagen), with an aspartic acid in the BMP-1 site (D) Substitution to asparagine (N), eg, as shown in SEQ ID NO: 68, wherein RAD is mutated to RAN .
  • a collagen trimerization domain e.g., the C-propeptide of human ⁇ 1(I) collagen
  • D aspartic acid in the BMP-1 site
  • N Substitution to asparagine
  • the C-terminal propeptide is or includes the amino acid sequence of a collagen trimerization domain (e.g., the C-propeptide of human ⁇ 1(I) collagen), with an alanine in the BMP-1 site Substitution of (A) to asparagine (N), eg, as shown in SEQ ID NO: 69, wherein R A D is mutated to R ND .
  • the C-terminal propeptide herein may include a mutated BMP-1 site, eg, RSAN instead of DDAN.
  • a C-terminal propeptide herein may include a BMP-1 site, e.g., a sequence comprising a RAD (e.g., RADDAN) sequence rather than a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) sequence
  • a BMP-1 site e.g., a sequence comprising a RAD (e.g., RADDAN) sequence rather than a RAN (e.g., RANDAN) or RND (e.g., RNDDAN) sequence
  • SEQ ID NO: 68 or 69 can be used in the fusion polypeptides disclosed herein.
  • SEQ ID NO: 27 (underlined) or fragments, variants or mutants thereof may be linked directly or indirectly to SEQ ID NO: 67 (italics) or fragments, variants or mutants thereof, e.g., to form the following fusion protein:
  • the C-terminal propeptide is or includes an amino acid sequence that is a fragment of any one of SEQ ID NOs: 67-80.
  • the C-terminal propeptide may comprise a sequence comprising a glycine-X-Y repeat sequence, where X and Y are independently any amino acid, or are at least 85%, 90%, 92%, 95%, or 97% identical thereto An amino acid sequence capable of forming inter-polypeptide disulfide bonds and trimerizing recombinant polypeptides.
  • X and Y are independently proline or hydroxyproline.
  • the recombinant polypeptide forms trimers, thereby forming a homotrimer of the S protein peptide.
  • the S protein peptide of the trimerized recombinant polypeptide is in a prefusion conformation.
  • the S protein peptide of the trimerized recombinant polypeptide is in a postfusion conformation.
  • the validation state allows access to different antigenic sites on the S protein peptide.
  • the antigenic site is an epitope, such as a linear epitope or a conformational epitope.
  • a trimerized recombinant polypeptide comprises a single recombinant polypeptide comprising the same viral antigen or immunogen.
  • the trimerized recombinant polypeptides comprise individual recombinant polypeptides, each recombinant polypeptide comprising a different viral antigen or immunogen than the other recombinant polypeptides.
  • the trimerized recombinant polypeptides comprise individual recombinant polypeptides, wherein one of the individual recombinant polypeptides comprises a different viral antigen or immunogen than the other recombinant polypeptides.
  • the trimerized recombinant polypeptides comprise a single recombinant polypeptide, wherein two of the single recombinant polypeptides comprise the same viral antigen or immunogen, and the viral antigen or immunogen is different from the virus contained in the remaining recombinant polypeptides. antigen or immunogen.
  • the recombinant polypeptide includes any of the coronavirus antigens or immunogens described in Section I. In some embodiments, the recombinant polypeptide comprises any of the coronavirus antigens or immunogens described in Section 1 linked as described herein to the C-terminal propeptide of collagen as described herein.
  • the immunogen comprises a recombinant SARS-CoV or SARS-CoV-2 S ectodomain trimer, such as a SARS-CoV-2 beta (B.1.351) coronavirus S ectodomain trimer, which includes the original A polymer comprising one or more (e.g. two, e.g. two consecutive) proline substitutions at or near the boundary between the HR1 domain and the central helical domain, the proline Acid substitution stabilizes the S ectodomain trimer in the prefusion conformation.
  • the one or more e.g. two, e.g.
  • one or more proline substitutions stabilize a coronavirus (e.g., SARS-CoV or SARS-CoV-2) S cell in a prefusion conformation.
  • An ectodomain trimer such as the SARS-CoV-2 beta (B.1.351) coronavirus S ectodomain trimer.
  • the SARS-CoV-2 spike protein peptide includes a 986K/987V to 986P/987P mutation.
  • the 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 the /S2 and/or S2' protease cleavage sites to prevent protease cleavage at these sites.
  • the SARS-CoV-2 spike protein peptide includes a 685R to 685A mutation. Exemplary protease cleavage sites for various viruses are shown below:
  • the recombinant coronavirus e.g., SARS-CoV or SARS-CoV-2
  • S ectodomain 3 in a prefusion conformation is stabilized by one or more proline substitutions (e.g., 986P/987P substitutions).
  • Protomers of the polymers include additional modifications for stabilization in the prefusion conformation, such as mutations at the protease cleavage site to prevent protease cleavage.
  • the extracellular domain includes the signal peptide (SP), which is removed during cellular processing; the N-terminal domain (NTD); the receptor binding domain (RBD); one or more S1/S2 cleavage sites; fusion peptide (FP); internal fusion peptide (IFP); heptad repeat 1/2 (HR1/2) and transmembrane domain (TM).
  • SP signal peptide
  • NTD N-terminal domain
  • RBD receptor binding domain
  • FP fusion peptide
  • IFP internal fusion peptide
  • HR1/2 heptad repeat 1/2
  • TM transmembrane domain
  • Exemplary sources of sequences can be found at ncbi.nlm.nih.gov/nuccore/MN908947.3, ncbi.nlm.nih.gov/nuccore/MN908947, ncbi.nlm.nih.gov/nuccore/MN908947.2. Additional sequences are available at ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/, including the complete genome of pneumovirus isolate Wuhan-Hu-1.
  • a protomer of a SARS-CoV-2 S ectodomain trimer that is stable prior to fusion can have NTD, RBD, S1 (at S1/S2 site 1 or S1/S2 site 2), FP, IFP, HR1, HR2 or the C-terminal residue of the extracellular domain (which can be attached to, for example, a trimerization domain or a transmembrane domain).
  • the position numbering of the S protein may vary between SARS-CoV strains, but the sequences can be aligned to identify associated domains and cleavage sites. It is understood that some residues (eg, up to 10) at the N-terminus and C-terminus of any ectodomain fragment may be removed or modified in the disclosed immunogens without reducing S ectodomain trimerization body as an immunogen.
  • the recombinant polypeptide is or includes an NTD peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an RBD peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes the NTD and RBD peptides of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an S1 domain peptide of the SARS-CoV or SARS-CoV-2 S protein. In some embodiments, the recombinant polypeptide is or includes an S2 domain peptide of the SARS-CoV or SARS-CoV-2 S protein.
  • SARS-CoV-1 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:26(1491aa):
  • the aforementioned SARS-CoV-1 S recombinant polypeptide may include the N-terminal signal provided in SEQ ID NO:53.
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO: 1 (1509aa):
  • the aforementioned SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal provided in SEQ ID NO:54.
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:86:
  • the above SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54 (MFVFLVLLPLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:87:
  • the above SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54 (MFVFLVLLPLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:88:
  • the above SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54 (MFVFLVLLPLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:89:
  • the above SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54 (MFVFLVLLPLVSS).
  • SARS-CoV-2 S recombinant polypeptide without a signal peptide is provided in SEQ ID NO:90:
  • the above SARS-CoV-2 S recombinant polypeptide may include the N-terminal signal peptide provided in SEQ ID NO:54 (MFVFLVLLPLVSS).
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:1. In some embodiments, the recombinant polypeptide is or comprises 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 amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 1, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:2. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 2 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 2, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:3. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:3 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 3, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:4. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:4 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 4, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:5. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:5 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 5, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:6. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:6 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 6, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:7. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:7 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO:7, and the variant comprises 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, H 655Y, 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 recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:8. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:8 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 8, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:9. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:9 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 9, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 10. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 10 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 10, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 11. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 11 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 11, and the variant comprises 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 recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 12. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 12 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 12, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 13. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 13 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 13, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 14. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 14 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 14, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 15. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 15 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 15, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 16. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 16 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 16, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 17. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 17 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 17, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO: 18. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 18 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 18, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 19. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 19 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 19, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:20. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 20 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 20, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO: 21. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 21 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 21, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:22. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 22 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 22, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:23. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 23 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 23, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:24. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 24 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 24, and the variant comprises 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, H 655Y, 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 the sequence set forth in SEQ ID NO:25. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 25 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences, including at one or more amino acid positions (e.g.
  • the recombinant polypeptide is or comprises a variant of SEQ ID NO: 25, and the variant comprises a variant selected from 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, H 655Y, 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 and V1176F
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:26. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 26 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:26 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:85. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO: 26 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO: 85 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:86. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:86 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO: 86 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:87. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:87 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO: 87 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:88. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:88 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence comprising one or more of SEQ ID NO:88 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:89. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:89 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO: 89 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:90. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:90 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:90 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:91. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:91 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:91 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:92. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:92 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:92 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:93. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:93 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:93 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:94. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:94 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity amino acid sequence comprising one or more of SEQ ID NO:94 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptide is or includes the sequence set forth in SEQ ID NO:95. In some embodiments, the recombinant polypeptide is or comprises at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% of SEQ ID NO:95 , 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity of amino acid sequences comprising one or more of SEQ ID NO:95 Sequences comprising substitutions, deletions and/or insertions at amino acid positions.
  • the recombinant polypeptides provided herein not only associate to form trimers, but can also aggregate or be aggregated to produce a protein comprising multiple recombinant polypeptides.
  • the formed protein has a macroscopic structure.
  • the macrostructure can confer structural stability to coronavirus antigens or immunogenic recombinant polypeptides, which in turn can provide access to potential antigenic sites capable of promoting an immune response.
  • the trimerized recombinant polypeptides aggregate to form a protein comprising a plurality of trimerized recombinant polypeptides. In some embodiments, multiple trimerized recombinant polypeptides form proteins with macroscopic structures.
  • a protein comprising a plurality of recombinant polypeptides described herein is an immunogen.
  • a protein comprising a plurality of recombinant polypeptides described herein is contained within a nanoparticle.
  • proteins are directly attached to nanoparticles, such as protein nanoparticles.
  • the protein is indirectly linked to the nanoparticle.
  • a protein comprising a plurality of recombinant polypeptides described herein is contained within a virus-like particle (VLP).
  • VLP virus-like particle
  • a complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NOs: 1-26 and 86-96, or a fragment, variant or mutant thereof, in any suitable combination.
  • a complex comprising a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 1-26 and 86-95, wherein the recombinant polypeptide Trimerization occurs through interpolypeptide disulfide bonds to form trimers.
  • fusion proteins comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus: a) a first region comprising a non-chimeric coronavirus spike located on a first coronavirus A part of the coronavirus spike protein ectodomain before the coronavirus spike protein receptor binding domain (RBD) in the protein; b) a second region comprising a second coronavirus different from the first coronavirus The coronavirus spike protein receptor binding domain (RBD); and c) the C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.
  • the fusion protein further comprises a third region between the second region and the C-terminal propeptide of the collagen.
  • 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.
  • the third region includes the S2 domain of a fourth coronavirus, wherein the fourth coronavirus is the same as or different from the first, second, or fourth coronavirus.
  • the first region includes the N-terminal domain (NTD) of the first coronavirus.
  • the first region includes one or more amino acid residues that differ from corresponding amino acid residues in the second coronavirus.
  • the second region includes one or more amino acid residues that differ from corresponding amino acid residues in the first coronavirus.
  • the first and second coronaviruses are different variants or strains of the same coronavirus.
  • the first region includes the NTD of a first coronavirus
  • the second region includes the RBD of a second coronavirus
  • the first and second coronaviruses are different variants of SARS-CoV-2.
  • the first coronavirus and the second coronavirus are independently selected from B.1.1.529, B.1.617.2, B.1.526, B.1.1.143, P.2, B.1.351, P. 1.
  • trimeric fusion proteins comprising three recombinant polypeptides, each comprising from amino to carboxyl terminus: a) a first region comprising SARS-CoV-2 of the B.1.526 lineage The coronavirus spike protein N-terminal domain (NTD); b) the second region, which includes the coronavirus spike protein receptor binding domain (RBD) of SARS-CoV-2 of the B.1.351 lineage; and c ) C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.
  • NTD coronavirus spike protein N-terminal domain
  • RBD coronavirus spike protein receptor binding domain
  • trimeric fusion proteins comprising three recombinant polypeptides, each recombinant polypeptide comprising from amino to carboxyl terminus: a) a first region comprising the SARS-CoV-2 beta coronavirus spike Spike protein N-terminal domain (NTD); b) second region, which includes the SARS-CoV-2 beta or non-SARS-CoV-2 beta coronavirus spike protein receptor binding domain of SARS-CoV-2 (RBD); and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.
  • NTD SARS-CoV-2 beta coronavirus spike Spike protein N-terminal domain
  • second region which includes the SARS-CoV-2 beta or non-SARS-CoV-2 beta coronavirus spike protein receptor binding domain of SARS-CoV-2 (RBD)
  • C-terminal propeptide of collagen wherein the C-terminal pro
  • trimeric fusion proteins comprising three recombinant polypeptides, each comprising from amino to carboxyl terminus: a) a first region comprising either a SARS-CoV-2 beta or a non-SARS- The N-terminal domain (NTD) of the SARS-CoV-2 coronavirus spike protein of CoV-2 beta; b) the second region, which includes the SARS-CoV-2 coronavirus spike of SARS-CoV-2 beta a protein receptor binding domain (RBD); and c) a C-terminal propeptide of collagen, wherein the C-terminal propeptide of the recombinant polypeptide forms an interpolypeptide disulfide bond.
  • NTD N-terminal domain
  • RBD protein receptor binding domain
  • provided herein are methods for preventing coronavirus infection in a mammal comprising immunizing the mammal with an effective amount of a fusion protein disclosed herein.
  • neutralizing antibodies against the first and second coronaviruses are produced in the mammal.
  • 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, 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 two or more SARS-CoV-2 viruses.
  • 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.
  • the method comprises immunizing the mammal with two or more doses of the fusion protein.
  • the fusion protein is administered as a reboost following one or more doses of the immunogen, including spike protein peptides comprising NTD and RBD from the same SARS-CoV-2 variant.
  • fusion polypeptides derived or modified from the Spike (S) glycoprotein of coronaviruses including SARS-CoV-1 and SARS-CoV-2.
  • the fusion polypeptides disclosed herein can be stabilized in a prefusion conformation compared to the wild-type S protein sequence of a coronavirus.
  • fusion to a trimerization domain prevents the S protein peptide in the fusion protein from forming a straight helix (eg, similar to what occurs during membrane fusion).
  • cryo-EM structure of the S-trimer subunit vaccine candidate revealed that it predominantly adopts a tightly closed prefusion state, unlike the full-length wild-type Spike protein, which forms prefusion and fusion in the presence of detergents. after state.
  • fusion proteins may include an altered soluble S sequence with modifications to inactivate the S1/S2 cleavage site; a turn region between the heptapeptide repeat 1 (HR1) region and the central helix (CH) region mutations in , which prevent HR1 and CH from forming a straight helix; and/or, in addition to stabilizing mutations, truncation of the heptapeptide repeat 2 region (HR2).
  • HR1 heptapeptide repeat 1
  • CH central helix
  • the fusion proteins herein may, but need not, include one or more mutations, such as K986G/V987G, K986P/V987P, K986G/V987P, or K986P/V987G, which are believed to stabilize the spine in its prefusion state. spike protein.
  • mutations such as K986G/V987G, K986P/V987P, K986G/V987P, or K986P/V987G are not necessary to stabilize fusion polypeptides comprising protein trimerization TM trimerization domains disclosed herein.
  • the mutation that renders the S1/S2 cleavage site inactive may comprise replacing RRAR (682-685 in SEQ ID NO:55) with GSAG (SEQ ID NO:60), and turning Mutations in the region may comprise double mutations K986G/V987G, K986P/V987P, K986G/V987P or K986P/V987G.
  • truncation of HR2 entails deletion of one or more residues shown in SEQ ID NO: 65 at the C-terminus of the wild-type soluble S sequence.
  • the immunogenic polypeptide may further comprise (a) one or more proline or glycine substitutions, and/or (b) one or more amino acid residues in the region of HR1 that interacts with HR2 base insertion.
  • the immunogenic polypeptide may have one or more substitutions selected from A942P, S943P, A944P, A942G, S943G, and A944G.
  • the insertion may be a G or GS between any residues in A942-A944.
  • Recombinant polypeptides or fragments, variants or mutants thereof mentioned herein, in any suitable combination include recombinant polypeptides or fragments, variants or mutants thereof selected from the group consisting of SEQ ID NO: 1-26 and 86-95 Trimers of mutants, wherein recombinant polypeptides are trimerized to form trimers through inter-polypeptide disulfide bonds, can be used in the starter and/or booster. Independently, the initial dose and/or any one or more booster doses may be without or with an adjuvant.
  • optional adjuvants may include: aluminum-containing adjuvants, such as those containing alum and/or aluminum hydroxide; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants; adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g. An adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as those containing alum and/or aluminum hydroxide
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants
  • 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, eg, a nucleic acid sequence encoding a recombinant polypeptide.
  • the polynucleotide comprises a first nucleic acid sequence encoding a recombinant polypeptide, particularly 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 comprises at least one promoter operably linked to control expression of the recombinant polypeptide. In some embodiments, the polynucleotide comprises two, three or more promoters operably linked to control expression of the recombinant polypeptide.
  • the polynucleotide comprises two or more nucleic acid coding sequences, such as sequences encoding recombinant polypeptides comprising different coronavirus antigens or immunogens
  • at least one promoter is operably linked to control the two sequences. Expression of one or more nucleic acid sequences.
  • the polynucleotide comprises two, three or more promoters operably linked to control expression of the recombinant polypeptide.
  • expression of the recombinant polypeptide is inducible or conditional.
  • a polynucleotide encoding a recombinant polypeptide comprises a conditional promoter, enhancer, or transactivator.
  • the conditional promoter, enhancer or transactivator is an inducible promoter, enhancer or transactivator or is a repressible promoter, enhancer or transactivator.
  • inducible or conditional promoters can be used to restrict the expression of recombinant polypeptides to specific microenvironments.
  • expression driven by an inducible or conditional promoter is regulated by exposure to exogenous agents such as heat, radiation, or drugs.
  • the polynucleotide may further comprise a nucleic acid sequence encoding a peptide between the one or more nucleic acid sequences.
  • the nucleic acid located between the nucleic acid sequences encodes a peptide that separates the translation products of the nucleic acid sequences during or after translation.
  • the peptide comprises an internal ribosome entry site (IRES), a self-cleaving peptide, or a peptide that causes ribosome skipping, such as a T2A peptide.
  • a polynucleotide encoding a recombinant polypeptide is introduced into a composition comprising cultured cells (eg, host cells), eg, by retroviral transduction, transfection, or transformation. In some embodiments, this may allow expression (eg, production) of the recombinant polypeptide. 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 recombinant polypeptides comprising coronavirus antigens or immunogens (eg, coronavirus S protein peptides) as described herein.
  • vectors or constructs comprising a nucleic acid molecule as described herein.
  • the vector or construct comprises one or more promoters operably linked to a nucleic acid molecule encoding a recombinant polypeptide to drive its expression.
  • a 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.
  • the vector is a viral vector.
  • the viral vector is a retroviral vector.
  • the retroviral vector is a lentiviral vector.
  • the retroviral vector is a gamma-retroviral vector.
  • a vector or construct includes a single promoter that drives expression of one or more nucleic acid molecules of the polynucleotide.
  • promoters may be polycistronic (dicistronic or tricistronic, see eg, US Patent No. 6,060,273).
  • the transcription unit can be engineered as a bicistronic unit containing an IRES (internal ribosomal entry site), which allows co-expression of gene products (e.g., encoding different recombinant peptides).
  • the vectors provided herein are bicistronic, allowing the vector to contain and express two nucleic acid sequences.
  • the vectors provided herein are tricistronic, allowing the vector to contain and express three nucleic acid sequences.
  • a single promoter directs the expression of RNA comprising two or three genes (e.g., encoding a chimeric signaling receptor and encoding a recombinant receptor) in a single open reading frame (ORF), these 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 that is processed into a single protein during translation (for 2A) or after translation.
  • peptides can cause ribosomes to skip (ribosome skipping) 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 downstream peptide (see, for example, de Felipe. Genetic Vaccines and Ther. 2:13 (2004) and deFelipe et al. Traffic 5:616-626 (2004)).
  • Many 2A elements are known in the art.
  • Examples of 2A sequences that can be used in the methods and nucleic acids disclosed herein include, but are not limited to, those from foot-and-mouth disease virus (F2A), equine rhinitis A virus (E2A), Thosea asigna virus (T2A) and 2A sequence of porcine teschovirus-1 (P2A).
  • F2A foot-and-mouth disease virus
  • E2A equine rhinitis A virus
  • T2A Thosea asigna virus
  • P2A sequence of porcine teschovirus-1 P2A
  • the vector is contained within a virus.
  • the virus is a pseudovirus.
  • the virus is a virus-like particle.
  • the vector is contained within a cell.
  • the virus or cell comprising the vector comprises a recombinant genome.
  • immunogenic compositions comprising trimers of recombinant polypeptides comprising sequences selected from the group consisting of SEQ ID NOs: 1-26 and 86-95, or any A combination of two or more trimers. In some embodiments, provided herein are immunogenic compositions comprising trimers of recombinant polypeptides having the sequence set forth in SEQ ID NO: 1.
  • An immunogenic composition as mentioned herein comprising a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide passes through an inter-polypeptide disulfide bond Trimerization forms trimers, which can be used in initiators and/or intensifiers. Independently, the initial dose and/or any one or more booster doses may be without or with an adjuvant.
  • optional adjuvants may include: aluminum-containing adjuvants, such as those containing alum and/or aluminum hydroxide; oligonucleotide-containing adjuvants, such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants; adjuvants containing TLR9 agonists; adjuvants containing metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80), e.g. An adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 in the form of an oil emulsion; or any combination of adjuvants.
  • aluminum-containing adjuvants such as those containing alum and/or aluminum hydroxide
  • oligonucleotide-containing adjuvants such as CpG-containing oligodeoxynucleotides (CpG- ODN) adjuvants
  • a unit dose of the immunogenic composition may comprise from about 10 ⁇ g to about 100 ⁇ g of SARS-CoV-2 antigen, preferably from about 25 ⁇ g to about 75 ⁇ g of SARS-CoV-2 antigen, preferably from about 40 ⁇ g to about 60 ⁇ g of SARS-CoV -2 antigen or about 50 ⁇ g SARS-CoV-2 antigen.
  • the dose comprises 3 ⁇ g of SARS-CoV-2 antigen.
  • the dose comprises 9 ⁇ g of SARS-CoV-2 antigen.
  • the dose comprises 30 ⁇ g of SARS-CoV-2 antigen.
  • a composition comprising a recombinant coronavirus S antigen (e.g., a trimer or protein) as described herein can be compared to the age-specific target recommended by the Advisory Committee on Immunization Practices (ACIP; cdc.gov/vaccines/acip/index.html).
  • Other vaccines such as influenza vaccine or varicella zoster vaccine
  • infants about one to six months old are administered simultaneously (usually separately) or sequentially.
  • the disclosed immunogens comprising the recombinant coronavirus S antigen described herein can be compared with, for example, hepatitis B (HepB), diphtheria, tetanus and pertussis (DTaP), pneumococcus (PCV), Haemophilus influenzae type b (Hib), polio, influenza and rotavirus vaccines administered simultaneously or sequentially.
  • HepB hepatitis B
  • DTaP diphtheria, tetanus and pertussis
  • PCV pneumococcus
  • Hib Haemophilus influenzae type b
  • polio influenza and rotavirus vaccines administered simultaneously or sequentially.
  • Multivalent or combination vaccines provide protection against multiple pathogens.
  • multivalent vaccines protect against multiple strains of the same pathogen.
  • multivalent vaccines protect against multiple pathogens, such as the combination vaccine Tdap, which protects against strains of tetanus, pertussis and diphtheria.
  • Multivalent vaccines are essential to minimize the number of immunizations required to confer protection against multiple pathogens or viral strains, reduce administrative costs, and increase coverage. This can be especially useful when vaccinating infants or children, for example.
  • 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, eg, as described herein (see, eg, Section I).
  • 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 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), A Attenuated strain of hepatitis virus (Bradley et al., J.Med.Virol., (1984) 14:373-86), attenuated measles virus (James et al., N.Engl.J.Med., ( 1995) 332:1262-6) and epitopes of pertussis virus (eg, ACEL-IMUNE cell-free DTP, Wyeth-Lederle vaccine and Pediatrics).
  • inactivated poliovirus Mara virus
  • a Attenuated strain of hepatitis virus Bradley et al., J.Med.Virol., (1984) 14:373-86
  • attenuated measles virus James et al., N.Engl.J.Med.
  • the vaccines provided herein are universal vaccines.
  • a universal vaccine is a vaccine 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, for example using seasonal vaccine formulations, and allow stronger pandemic prevention.
  • the immunogens described herein can be used as a primary series, an additional dose, and/or a homologous or heterologous booster dose, e.g., the first dose, the second dose Second dose, third dose, fourth dose, and/or more doses.
  • the immunogens described herein are useful as seasonal vaccines.
  • the immunogens described herein can be given as the first dose, the second dose, the third dose, the Four doses, and/or more doses are used.
  • a universal vaccine is a vaccine consisting of multiple epitopes derived from different strains of the virus.
  • a universal vaccine consists of a single epitope that is conserved among different strains of virus.
  • a universal vaccine could be based on a relatively conserved domain of the S protein.
  • an immunogenic composition includes a trimerized recombinant polypeptide provided herein and optionally a pharmaceutically acceptable carrier.
  • the immunogenic composition comprises a trimerized recombinant polypeptide provided herein and disodium phosphate, such as disodium phosphate dihydrate, sodium dihydrogen phosphate, such as disodium phosphate monohydrate, sodium chloride , and Tween 80.
  • an aqueous immunogenic composition comprises 720 ⁇ g of a trimerized recombinant polypeptide provided herein and 0.62 mg disodium phosphate dihydrate, 0.62 mg disodium phosphate monohydrate, 9.0 mg sodium chloride, and 0.2 mg Tween 80.
  • an immunogenic composition includes 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.
  • an immunogenic composition includes a VLP provided herein and optionally a pharmaceutically acceptable carrier.
  • an immunogenic composition includes an isolated nucleic acid provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition includes a carrier provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition includes a virus provided herein and optionally a pharmaceutically acceptable carrier. In some embodiments, an immunogenic composition includes a pseudovirus 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.
  • the vaccines are prophylactic and therapeutic vaccines.
  • Such pharmaceutical compositions can be administered to a subject by various modes of administration known to those of ordinary skill in the art, for example, intramuscular, intradermal, subcutaneous, intravenous, intraarterial, intraarticular, intraperitoneal, nasal Internal, sublingual, tonsil, oropharyngeal or other parenteral and mucosal routes.
  • a pharmaceutical composition comprising one or more of the disclosed immunogens is an immunogenic composition.
  • Actual methods for preparing administrable compositions are known or apparent to those skilled in the art and are described, for example, in such publications as Remingtons Pharmaceutical Sciences, 19th Ed., Mack Publishing Company, Easton, Pa., 1995. are described in more detail in .
  • the immunogens described herein e.g., recombinant coronavirus S antigens, e.g., trimers, proteins
  • Potential carriers include, but are not limited to, physiologically balanced media, phosphate-buffered saline, water, emulsions (e.g., oil/water or water/oil emulsions), various types of wetting agents, antifreeze additives, or stabilizers such as Proteins, peptides or hydrolysates (eg, albumin, gelatin), sugars (eg, sucrose, lactose, sorbitol), amino acids (eg, monosodium glutamate), or other protective agents.
  • the resulting aqueous solution can be packaged for use as it is or lyophilized.
  • the lyophilized preparations are mixed with sterile solutions prior to single or multiple dose administration.
  • Formulation 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.
  • Bacteriostats may be contraindicated in some patients; therefore, lyophilized formulations 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 regulators and buffers, tonicity regulators, 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, AlPO4 , 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 (polyxylpropylene, POP) can be used as adjuvants among many other suitable adjuvants well known in the art, such as POE-POP-POE block Segment copolymer, MPL TM (3-O-deacylated monophosphate A; Corixa, Hamilton, Ind.) and IL-12 (Genetics Institute, Cambridge, Mass.) (Newman et al., 1998, Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142).
  • MPL TM 3-O-deacylated monophosphate A
  • Corixa Corixa, Hamilton, Ind.
  • IL-12 Geneetics Institute, Cambridge, Mass.
  • an immunogenic composition of the invention may include or be administered with more than one adjuvant. In some embodiments, an immunogenic composition of the invention may include or be administered with two adjuvants. In some embodiments, the immunogenic compositions of the invention may include or be administered with various adjuvants. For example, in some cases, for example, vaccines comprising the immunogenic compositions provided herein may include or be administered in combination with various adjuvants.
  • Suitable adjuvants include, for example, aluminum hydroxide, lecithin, Freund's adjuvant, MPL TM and IL-1, one or any combination of several of which can be combined with a compound selected from the group consisting of SEQ ID NO:86 Trimers of recombinant polypeptides or fragments, variants or mutants of the group consisting of -95 are used in combination.
  • a vaccine composition or nanoparticle immunogen disclosed herein eg, a SARS-COV-2 vaccine composition
  • Various pharmaceutical compositions can be prepared according to standard procedures well known in the art.
  • the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide is passed through the polypeptide
  • Trimerization of inter-disulfide bonds to form trimers may comprise adjuvant formulations comprising metabolizable oils (e.g. squalene) and ⁇ -tocopherol (e.g. DL- ⁇ - tocopherol), and polyoxyethylene sorbitan monooleate (Tween-80).
  • 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 may include about 5% squalene, about 5% tocopherol, and about 0.4% polyoxyethylene sorbitan monooleate. In some embodiments, the adjuvant formulation, Adjuvant 2, is dosed at about 10.68 mg/dose of squalene, about 11.15 mg/dose of tocopherol, and about 4.86 mg/dose of polyoxyethylene sorbitan monooleate.
  • the immunogenic compositions of the invention may comprise a full dose of adjuvant 2. In some embodiments, the immunogenic compositions of the invention may comprise a half dose of adjuvant 2. In some embodiments Among them, the immunogenic composition of the present invention may contain 1/4 dose of adjuvant 2. In some embodiments, the immunogenic composition of the present invention may contain 1/3, 1/5, 1/6, 1 /7, 1/8, 1/9, 1/10 dose of adjuvant 2.
  • the immunogenic composition of the present invention may comprise 3 deO-acylated monophospholipid A (3D- MPL) and an adjuvant in the form of an oil-in-water emulsion comprising a metabolizable oil, ⁇ -tocopherol and polyoxyethylene sorbitan monooleate.
  • the immunogenic composition of the present invention may comprise QS21 (Quillaja saponaria Molina extract: component 21), 3D-MPL and an oil-in-water emulsion, wherein the oil-in-water emulsion includes metabolizable oil, ⁇ - Tocopherol and polyoxyethylene sorbitan monooleate.
  • an immunogenic composition of the invention may comprise QS21, 3D-MPL, and an oil-in-water emulsion, wherein the oil-in-water emulsion has the following composition: Metabolizable oils such as squalene, alpha-tocopherol and Tween-80.
  • the immunogenic compositions of the invention may comprise an adjuvant in the form of a liposomal composition.
  • the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide is passed through the polypeptide
  • Trimerization of meta-disulfide bonds to form trimers may include adjuvant formulations comprising metabolizable oils (e.g., squalene), polyoxyethylene sorbitan monooleate (Tween- 80) and Span 85.
  • the adjuvant formulation may include about 5% (w/v) squalene, about 0.5% (w/v) polyoxyethylene sorbitan monooleate, and about 0.5% (w/v) Span 85.
  • the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide is passed through the polypeptide Trimerization of inter-disulfide bonds to form trimers may comprise an adjuvant formulation comprising Quillaja saponin, cholesterol and phospholipids, for example, in the form of a nanoparticle composition.
  • the immunogenic compositions of the invention may comprise a mixture of separately purified fractions of Quillaja saponaria Molina, which are subsequently formulated with cholesterol and phospholipids.
  • the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide is passed through the polypeptide
  • the inter-disulfide bonds trimerize to form a trimer, which may comprise an adjuvant selected from the group consisting of MF59 TM , Matrix-A TM , Matrix-C TM , Matrix-M TM , AS01, AS02, AS03 and AS04.
  • the immunogenic composition of the present invention comprises a trimer of a recombinant polypeptide or a fragment, variant or mutant thereof selected from the group consisting of SEQ ID NO: 86-95, wherein the recombinant polypeptide is passed through the polypeptide Trimerization of inter-disulfide bonds to form trimers that may contain toll-like receptor 9 (TLR9) agonists, where TLR9 agonists are oligonucleotides 8 to 35 nucleotides in length containing unmethylated cytidine-phosphate-guanosine (also known as CpG or cytosine-phosphate-guanosine) motif, and SARS-CoV-2 antigens and oligonucleotides to effectively stimulate mammalian subjects (such as the need for SARS-CoV-2 The amount of the immune response against the SARS-CoV-2 antigen in the human subject of CoV-2 antigen and oligonucleotide) is present in the immunogenic 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).
  • Optimal oligonucleotide TLR9 agonists typically contain palindromic sequences of the general formula: 5'-purine-purine-CG-pyrimidine-pyrimidine-3' or 5'-purine-purine-CG-pyrimidine-pyrimidine-pyrimidine-CG- 3'.
  • the CpG oligonucleotides are linear. In other embodiments, the CpG oligonucleotide is circular or includes a hairpin loop. CpG oligonucleotides can be single-stranded or double-stranded. In some embodiments, CpG oligonucleotides may comprise modifications. Modifications include, but are not limited to, modifications of 3'OH or 5'OH groups, modifications of nucleotide bases, modifications of sugar components, and modifications of phosphate groups.
  • Modified bases can be included in the loop of a CpG oligonucleotide as long as the modified base retains the same specificity for its natural complement via Watson-Crick base pairing (e.g., the palindromic portion remains self-complementary). in the text sequence.
  • CpG oligonucleotides include non-classical bases.
  • CpG oligonucleotides include modified nucleosides.
  • the modified nucleoside is selected from 2'-deoxy-7-deazaguanosine (2'-deoxy-7-deazaguanosine), 2'-deoxy-6-thioguanosine, arabinosine arabinoguanosine, 2'-deoxy-2'substituted-arabinoguanosine and 2'-O-substituted-arabinoguanosine.
  • CpG oligonucleotides may contain modifications of phosphate groups.
  • phosphate modifications include, but are not limited to, methyl phosphonate, phosphorothioate, phosphoramide (bridged or unbridged), phosphotriester, and phosphorodithioate, and can be used in any combination use. Other non-phosphate linkages may also be used.
  • oligonucleotides include only phosphorothioate backbones. In some embodiments, the oligonucleotide includes only a phosphodiester backbone. In some embodiments, oligonucleotides include a combination of phosphate linkages in the phosphate backbone, eg, a combination of phosphodiester and phosphorothioate linkages.
  • Oligonucleotides with phosphorothioate backbones can be more immunogenic than oligonucleotides with phosphodiester backbones and appear to be more resistant to degradation after injection into a host (Braun et al., J Immunol, 141:2084-2089, 1988; and Latimer et al., Mol Immunol, 32:1057-1064, 1995).
  • the CpG oligonucleotides of the invention comprise at least one, two or three internucleotide phosphorothioate linkages.
  • both stereoisomers of phosphorothioate linkages are present in the plurality of CpG oligonucleotides in the nucleotide molecule.
  • all 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 can be used as an adjuvant.
  • the K-type ODN also known as the B-type
  • the K-type ODN may 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 increased half-life in vivo.
  • K-type ODN triggers pDCs to differentiate and produce TNF- ⁇ , and triggers B cells to proliferate and secrete IgM.
  • Type D ODN also known as Type A
  • Type A consists of a mixed phosphodiester/phosphorothioate backbone containing a single CpG motif flanked by palindromic sequences and polyG at the 3' and 5' ends Tails (structural motifs that promote concatemer formation).
  • a type D ODN may be based on the following sequence GGTGCAT CG ATGCAGGGGGG.
  • D-type ODN triggers pDC maturation and secretion of IFN- ⁇ , but has no effect on B cells.
  • C-type ODNs like K-types, are composed entirely of phosphorothioate nucleotides, but, like D-types, contain palindromic CpG motifs.
  • a type C ODN may be based on the following sequence T CG T CG TT CG AA CG A CG TTGAT. Such ODNs stimulate B cells to secrete IL-6 and pDCs to produce IFN- ⁇ .
  • P-type ODNs contain two palindromic sequences, enabling them to form higher ordered structures.
  • the 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 PDCs, and induces a greater amount of IFN- ⁇ production.
  • words in bold in ODN sequences indicate self-complementary palindromes and CpG motifs are underlined.
  • CpG ODNs such as CpG 7909 (5'-TCGTCGTTTTGTCGTTTTGTCGTT-3') and CpG 1018 (5'-TGACTGTGAACGTTCGAGATGA-3'), are known and disclosed in U.S. Pat. ,785,610, 8,003,115, 8,133,874, 8,114,418, 8,222,398, 8,333,980, 8,597,665, 8,669,237, 9,028,845, and 10,052,378; and Bode et al., "CpG DNA as a vaccine adjuvant" , Expert Rev Vaccines(2011), 10(4):499-511, All of these are incorporated herein by reference in their entirety for all purposes.
  • 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 ) microparticles (Shah et al., Methods Mol Biol, 1494:1-14, 2017).
  • the immunogenic composition further comprises an aluminum salt adjuvant that adsorbs the SARS-CoV-2 antigen.
  • the aluminum salt adjuvant comprises one or more of the group consisting of amorphous aluminum hydroxyphosphate sulfate, aluminum hydroxide, aluminum phosphate, and aluminum potassium sulfate.
  • the aluminum salt adjuvant includes one or both of aluminum hydroxide and aluminum phosphate.
  • the aluminum salt adjuvant comprises aluminum hydroxide.
  • a unit dose of the immunogenic composition includes about 0.25 to about 0.50 mg Al 3+ , or about 0.35 mg Al 3+ .
  • the immunogenic composition further includes an additional adjuvant.
  • Suitable adjuvants include, but are not limited to, squalene-in-water emulsions (e.g., MF59 or AS03), TLR3 agonists (e.g., polyIC or polyICLC), TLR4 agonists (e.g., bacterial lipopolysaccharide derivatives such as mono Phospholipid A (MPL), and/or saponins such as Quil A or QS-21, such as AS01 or AS02), TLR5 agonists (bacterial flagellin), and TLR7, TLR8, and/or TLR9 agonists (imidazoquinoline derivatives , such as imiquimod and resiquimod) (Coffman et al., Immunity, 33:492-503, 2010).
  • additional adjuvants include MPL and alum (eg, AS04).
  • the mitogenic components of Freund's adjuvant intact and incomplete
  • the mitogenic components of Freund's adjuvant intact and incomplete
  • the immunogenic composition includes pharmaceutically acceptable excipients including, for example, solvents, fillers, buffers, tonicity modifiers, and preservatives (Pramanick et al., Pharma Times, 45:65- 77, 2013).
  • an immunogenic composition may include an excipient that acts as one or more of a solvent, bulking agent, buffer, and tonicity modifier (e.g., sodium chloride in saline may be used simultaneously as an aqueous carrier and tonicity regulator).
  • the immunogenic composition includes an aqueous carrier as a solvent.
  • Suitable carriers include, for example, sterile water, saline, phosphate buffered saline and Ringer's solution.
  • the composition is isotonic.
  • Immunogenic compositions may include buffering agents. Buffers control pH to inhibit degradation of the active agent during handling, storage and optionally reconstitution.
  • Suitable buffers include, for example, salts comprising acetate, citrate, phosphate or sulfate.
  • Other suitable buffers include, for example, amino acids such as arginine, glycine, histidine and lysine. Buffering agents may further include hydrochloric acid or sodium hydroxide.
  • the buffer maintains the pH of the composition in the range of 6-9. In some embodiments, the pH is greater than (lower limit) 6, 7 or 8. In some embodiments, the pH is less than (upper limit) 9, 8 or 7. That is, the pH is in the range of about 6 to 9, with the lower limit being less than the upper limit.
  • Immunogenic compositions may include tonicity modifiers.
  • Suitable tonicity modifiers include, for example, dextrose, glycerol, sodium chloride, glycerin and mannitol.
  • Immunogenic compositions may include fillers.
  • Bulking agents are particularly useful when the pharmaceutical composition is to be lyophilized prior to administration.
  • fillers are protective agents that help stabilize and prevent degradation of the active agent during freezing or spray drying and/or during storage.
  • Suitable bulking agents are sugars (mono-, di- and polysaccharides) such as sucrose, lactose, trehalose, mannitol, sorbitol, dextrose and raffinose.
  • Immunogenic compositions may include preservatives. Suitable preservatives include, for example, antioxidants and antimicrobials. However, in preferred embodiments, the immunogenic compositions are prepared under sterile conditions and are in single-use containers and therefore need not contain preservatives.
  • compositions can be provided as sterile compositions.
  • Pharmaceutical compositions generally contain an effective amount of a disclosed immunogen and can be prepared by conventional techniques. Generally, the amount of immunogen per dose of immunogenic composition is selected as that amount that induces an immune response without significant adverse side effects.
  • compositions may be provided in unit dosage form for inducing an immune response in a subject.
  • a unit dosage form comprising a suitable single preselected dose for administration to a subject, or a suitably labeled or measured multiple of two or more preselected unit doses, and/or a metering mechanism for administering a unit dose or multiples thereof .
  • the composition further includes an adjuvant.
  • a method for generating an immune response to a coronavirus surface antigen in a subject comprising administering to the subject an effective amount of a compound comprising a compound selected from the group consisting of SEQ ID NO : Recombinant polypeptides of the group consisting of 1-26 and 86-95, optionally the complex is used as a primary series, an additional dose, and/or a homologous or heterologous booster dose ) using, for example, 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 with other recombinant subunit vaccines, nano Any one or several of particle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines are used in combination.
  • a method for generating an immune response to a coronavirus surface antigen in a subject comprising administering to the subject an effective amount of A complex comprising a recombinant polypeptide selected from the group consisting of SEQ ID NO: 1-26 and 86-95, optionally as a primary series, an additional dose, and and/or administration of a homologous or heterologous booster dose, such as a first dose, a second dose, a third dose, a fourth dose, and/or further doses, optionally the initial dose, an additional dose, or
  • the heterologous booster is used in combination with any one or several of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines.
  • kits for generating an immune response to a coronavirus surface antigen in a subject wherein the surface antigen comprises a sequence selected from the group consisting of SEQ ID NOs: 27-66 and 81-85 , 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 NO: 1-26 and 86-95, optionally the complex is used as an initial agent ( primary series), additional doses, and/or homologous or heterologous booster doses, such as the first, second, third, fourth, and/or further doses , optionally the initial dose, additional dose, or heterologous booster and any one or more of other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, and inactivated virus vaccines A combination of use.
  • a coronavirus surface antigen comprises the S protein of a coronavirus or an antigenic fragment thereof, and optionally, the surface antigen comprises A sequence selected from the group consisting of SEQ ID NO: 27-66 and 81-85 or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising The sequence described in any one of SEQ ID NO:86-95, optionally this compound is used as initial agent (primary series), additional agent (additional dose), and/or homologous or heterologous enhancer (booster dose) ) using, for example, 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 with other recombinant subunit vaccines, nano Any one or several of particle vaccines, mRNA vaccines,
  • adjuvants in any of the priming doses, additional doses, and/or boosters may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants; Adjuvants such as CpG oligodeoxynucleotide (CpG-ODN)-containing adjuvants; TLR9 agonist-containing adjuvants; metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 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 Adjuvants
  • a method for generating an immune response to a coronavirus surface antigen in a subject wherein the surface antigen comprises S protein or an antigenic fragment thereof, and the method comprises administering to the subject an effective amount of A complex or a combination of any two or more of them, said complex comprising a recombinant polypeptide comprising a sequence selected from the group consisting of SEQ ID NO: 1-26 and 86-95, optionally
  • the complex or combination of complexes can be used as primary series, additional dose, and/or homologous or heterologous booster dose, such as the first dose, the second dose, the third dose dose, fourth dose, and/or further doses, optionally the initial dose, additional dose, or heterologous booster dose with other recombinant subunit vaccines, nanoparticle vaccines, mRNA vaccines, DNA vaccines, adenovirus vector vaccines, It is used in conjunction with any one or several of the inactivated virus vaccines.
  • the method comprises administering to the subject an effective amount of a complex comprising a recombinant polypeptide comprising SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87 and /or the sequence described in SEQ ID NO:88.
  • adjuvants in any of the priming doses, additional doses, and/or boosters may independently include: aluminum-containing adjuvants, such as alum and/or aluminum hydroxide-containing adjuvants; oligonucleotide-containing adjuvants; Adjuvants such as CpG oligodeoxynucleotide (CpG-ODN)-containing adjuvants; TLR9 agonist-containing adjuvants; metabolizable oils, ⁇ -tocopherol, and/or polyoxyethylene sorbitan monooleate (Tween-80) adjuvant, such as an adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 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 Adjuvants
  • a disclosed immunogen e.g., a recombinant coronavirus S antigen, e.g., an S-trimer or S protein as described herein, a nucleic acid encoding a protomer of a disclosed recombinant coronavirus S antigen
  • a recombinant coronavirus S antigen e.g., an S-trimer or S protein as described herein, a nucleic acid encoding a protomer of a disclosed recombinant coronavirus S antigen
  • Molecules such as RNA molecules
  • vectors, or protein nanoparticles or virus-like particles comprising the disclosed recombinant coronavirus S antigen to induce an immune response to the corresponding coronavirus S antigen in a subject.
  • the subjects are humans.
  • the immune response may be a protective immune response, eg a response that suppresses subsequent infection with the corresponding coronavirus. Stimulation of an immune response can also be used
  • a subject may be selected for treatment as having or being at risk of contracting a coronavirus, for example because of being or likely to be exposed to a coronavirus.
  • the subject can be monitored for infection or symptoms associated with the coronavirus, or both.
  • Typical subjects to be treated with the therapies and methods of the present invention include humans as well as non-human primates and other animals.
  • acceptable screening methods are employed to determine risk factors associated with a target or suspected disease or condition, or to determine the status of an existing disease or condition in a subject .
  • These screening methods include, for example, routine testing to identify environmental, familial, occupational, and other such risk factors that may be associated with a target or suspected disease or condition, as well as diagnostic methods, such as for the detection and/or characterization of coronavirus infection Various ELISA and other immunoassay methods.
  • routine methods allow clinicians to select patients in need of treatment using the methods and pharmaceutical compositions of the invention.
  • compositions may be administered according to the teachings herein or other conventional methods, as a stand-alone prophylactic or therapeutic regimen, or as a follow-up, adjunct, or coordinated regimen to other treatments.
  • the disclosed immunogens eg, coronavirus S antigen, eg, trimers, proteins
  • the disclosed therapeutic agents are provided prior to any symptoms, eg, prior to infection.
  • Prophylactic administration of the disclosed therapeutics is used to prevent or ameliorate any subsequent infection.
  • the disclosed therapeutic agents are provided at or after the onset of symptoms of a disease or infection, for example, after the development of symptoms of a coronavirus infection corresponding to a coronavirus S antigen, or after a diagnosis of a 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 initiation of infection.
  • the immunogens and immunogenic compositions described herein are provided to the subject in an amount effective to induce or enhance the subject's (preferably human) immune response against the coronavirus S antigen.
  • Actual doses of the disclosed immunogens will depend, for example, on the subject's disease symptoms and particular state (e.g., subject's age, size, health, degree of symptoms, predisposing factors, etc.), time and route of administration, simultaneous Factors such as other drugs or treatments administered and the particular pharmacology of the composition to elicit a desired activity or biological response in a subject vary. Dosage regimens may be adjusted to provide the optimum prophylactic or therapeutic response.
  • Immunogenic compositions comprising one or more of the disclosed immunogens may be used in coordinated (or prime-boost) vaccination regimens or combined formulations.
  • novel combination immunogenic compositions and coordinated immunization regimens use separate immunogens or formulations, each of which is designed to elicit an antiviral immune response, such as immunity to the coronavirus S antigen answer.
  • the separate immunogenic compositions that elicit an antiviral immune response can be combined in a multivalent immunogenic composition administered to the subject in a single immunization step, or they can be combined in a coordinated (or prime-boost) immunization regimen Administration alone (in a monovalent immunogenic composition).
  • boosters There can be several boosters, and each booster can be a different disclosed immunogen.
  • a booster can be the same immunogen as another booster or prime.
  • the priming and boosting doses can be administered as a single dose or in multiple doses, for example, two, three, four, five, six or more doses can be administered to a subject over several days, weeks or months . Multiple boosters, such as one to five (e.g., 1, 2, 3, 4, or 5 boosters) or more may also be performed.
  • Different doses can be used in a series of serial immunizations. For example, using a relatively large dose in a primary immunization, followed by a relatively small dose for a boost.
  • the booster may be administered about two weeks, about three to eight weeks, or about four weeks after the priming, or about several months after the priming. In some embodiments, the booster can be administered about 5, about 6, about 7, about 8, about 10, about 12, about 18, about 24 months after the priming dose, or more or less time after the priming dose . Additional boosters may also be administered periodically at appropriate time points to enhance the "immunological memory" of the subject. Suitability of selected vaccine parameters, such as formulation, dosage, regimen, etc., can be determined by taking aliquots of sera from subjects and measuring antibody titers during the immunization schedule.
  • the clinical condition of the subject can be monitored for a desired effect, such as preventing infection or ameliorating a disease state (eg, reducing viral load). If such monitoring indicates that vaccination is suboptimal, the subject can be boosted with additional doses of the immunogenic composition and the vaccination parameters can be modified in a manner expected to enhance the immune response.
  • a desired effect such as preventing infection or ameliorating a disease state (eg, reducing viral load).
  • the prime-boost approach can include a DNA-primer and protein-boost vaccination regimen for the subject.
  • the method may involve two or more administrations of the nucleic acid molecule or protein.
  • each human dose will comprise 1-1000 ⁇ g of protein, such as from about 1 ⁇ g to about 100 ⁇ g, such as from about 1 ⁇ g to about 50 ⁇ g, such as about 1 ⁇ g, about 2 ⁇ g, about 5 ⁇ g, about 10 ⁇ g, about 15 ⁇ g, About 20 ⁇ g, about 25 ⁇ g, about 30 ⁇ g, about 40 ⁇ g, or about 50 ⁇ g.
  • the amount used in the immunogenic composition is selected based on the subject population (eg, infants or the elderly). The optimum amount for a particular ingredient can be determined by standard studies involving observation of antibody titers and other responses in subjects. It will be appreciated that a therapeutically effective amount of a disclosed immunogen (e.g., a disclosed recombinant coronavirus S antigen, such as a trimer, protein, viral vector, or nucleic acid molecule in an immunogenic composition) may be included in a single dose administered An amount that is ineffective in inducing an immune response but effective in multiple doses, such as in a prime-boost regimen.
  • a disclosed immunogen e.g., a disclosed recombinant coronavirus S antigen, such as a trimer, protein, viral vector, or nucleic acid molecule in an immunogenic composition
  • the subject's immune system typically responds to the immunogenic composition by producing antibodies specific to the coronavirus S protein peptide contained in the immunogen. Such a response means that an immunologically effective dose was delivered to the subject.
  • the subject's antibody response will be determined with an assessment of the effective dose/immunization regimen. In most cases, it is sufficient to assess antibody titers in serum or plasma obtained from subjects. Decisions as to whether to administer a re-boost and/or alter the amount of therapeutic agent administered to an individual can be based at least in part on antibody titer levels. Antibody titer levels can be based, for example, on an immunobinding assay that measures the concentration of antibody in serum that binds to an antigen, including, for example, a recombinant coronavirus S antigen, such as an S-trimer.
  • Methods do not need to completely eliminate, reduce or prevent coronavirus infection to be effective.
  • eliciting an immune response to a coronavirus with one or more of the disclosed immunogens can reduce or inhibit a desired amount of coronavirus infection, e.g., at least 10, compared to coronavirus infection in the absence of the immunogen. %, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, even at least 100% (elimination or prevention of detectable infected cells) .
  • coronavirus replication can be reduced or inhibited by the disclosed methods. The method does not need to completely eliminate coronavirus replication to be effective.
  • eliciting an immune response with one or more of the disclosed immunogens can reduce the replication of the corresponding coronavirus by a desired amount, e.g., at least 10%, at least 20%, compared to the replication of the coronavirus in the absence of an immune response. %, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminate or prevent detectable coronavirus replication).
  • the disclosed immunogen is administered to the subject at the same time as the adjuvant is administered. In other embodiments, a disclosed immunogen is administered to a subject after administration of the adjuvant and within a sufficient time to induce an immune response.
  • nucleic acid administration is direct immunization with plasmid DNA, such as a mammalian expression plasmid.
  • Immunization by nucleic acid constructs is well known in the art and is disclosed, for example, in U.S. Pat. No. 5,643,578 (which describes methods for immunizing vertebrates by introducing DNA encoding a desired antigen to elicit a cell-mediated or humoral response) and U.S. Pat. Patent Nos. 5,593,972 and 5,817,637 (which describe the operably linking of a nucleic acid sequence encoding an antigen to regulatory sequences capable of expression). US Patent No.
  • 5,880,103 describes several methods of delivering nucleic acids encoding immunogenic peptides or other antigens to organisms. Methods include liposomal delivery of nucleic acids (or their own synthetic peptides) and immunostimulatory constructs or ISCOMS TM , negatively charged 30-40nm sized cells that form spontaneously after mixing cholesterol and Quil A TM (saponin) cage structure.
  • ISCOMS TM immunostimulatory constructs
  • the use of ISCOMS TM as an antigen delivery vehicle has produced protective immunity in experimental models of various infections, including toxoplasmosis and Epstein-Barr virus-induced tumors (Mowat and Donachie, Immunol. Today 12:383, 1991). Antigen doses as low as 1 ⁇ g encapsulated in ISCOMS TM have been found to produce class I-mediated CTL responses (Takahashi et al., Nature 344:873, 1990).
  • plasmid DNA vaccines are used to express the disclosed immunogens in a subject.
  • a nucleic acid molecule encoding a disclosed immunogen 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, incorporated herein by reference ).
  • the disclosed recombinant coronavirus S antigens can be expressed by attenuated viral hosts or vectors or bacterial vectors.
  • Recombinant vaccinia virus, adeno-associated virus (AAV), herpes virus, retrovirus, cytogmeglo virus, or other viral vectors can be used to express peptides or proteins to elicit a CTL response.
  • AAV adeno-associated virus
  • US Patent No. 4,722,848 describes vaccinia vectors and methods useful in immunization programs.
  • Bacillus Calmette Guerin provides another vector for expressing peptides (see Stover, Nature 351:456-460, 1991).
  • nucleic acid encoding the disclosed recombinant coronavirus S antigen is introduced directly into cells.
  • nucleic acids can be loaded onto gold microspheres by standard methods and introduced into the skin by devices such as Bio-Rad's HELIOS TM Gene Gun.
  • a nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter.
  • the DNA is injected into the muscle, but it can also be injected directly into other sites.
  • Injectable doses are generally about 0.5 ⁇ g/kg to about 50 mg/kg, and typically about 0.005 mg/kg to about 5 mg/kg (see, eg, US Patent No. 5,589,466).
  • nucleic acids can be loaded onto gold microspheres by standard methods and introduced into the skin by devices such as Bio-Rad's HELIOS TM Gene Gun.
  • a nucleic acid can be "naked," consisting of a plasmid under the control of a strong promoter.
  • the DNA is injected into the muscle, but it can also be injected directly into other sites.
  • Injectable doses are generally about 0.5 ⁇ g/kg to about 50 mg/kg, and typically about 0.005 mg/kg to about 5 mg/kg (see, eg, US Patent No. 5,589,466).
  • mRNA-based immunization protocols can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigens directly into cells.
  • mRNA-based nucleic acid vaccines may provide an effective alternative to the aforementioned methods. mRNA vaccines eliminate the safety issues of DNA integration into the host genome and can be directly translated in the host cell cytoplasm. Furthermore, simple cell-free in vitro synthesis of RNA avoids manufacturing complications associated with viral vectors.
  • RNA-based vaccines Two exemplary formats of RNA-based vaccines that can be used to deliver nucleic acids encoding the disclosed recombinant coronavirus S antigen include conventional non-amplified mRNA immunization (see, e.g., Petsch et al., "Protective efficacy of in vitro synthesized, specific mRNA vaccines against influenza A virus infection," Nature biotechnology, 30(12): 1210-6, 2012) and self-amplifying mRNA immunization (see, e.g., Geall et al., "Nonviral delivery of self-amplifying RNA vaccines, ” PNAS, 109(36): 14604-14609, 2012; Magini et al., “Self-Amplifying mRNA Vaccines Expressing Multiple conserveed Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,” PLoS One, 11(8):e0161193, 2016
  • administering to a subject a therapeutically effective amount of one or more of the disclosed immunogens induces a neutralizing immune response in the subject.
  • serum can be collected from a subject at an appropriate time point after immunization of the subject, frozen and stored for neutralization assays.
  • Assays for neutralizing activity are known to those of ordinary skill in the art and are described further herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, single Period infection assay.
  • PRNT plaque reduction neutralization
  • microneutralization assays microneutralization assays
  • flow cytometry-based assays single Period infection assay.
  • a panel of coronavirus pseudoviruses can be used to determine serum neutralization activity.
  • administering to a subject a therapeutically effective amount of one or more of the disclosed immunogens induces a neutralizing immune response in the subject.
  • serum can be collected from a subject at an appropriate time point after immunization of the subject, frozen and stored for neutralization assays.
  • Assays for neutralizing activity are known to those of ordinary skill in the art and are described further herein, including, but not limited to, plaque reduction neutralization (PRNT) assays, microneutralization assays, flow cytometry-based assays, single Period infection assay.
  • PRNT plaque reduction neutralization
  • microneutralization assays microneutralization assays
  • flow cytometry-based assays single Period infection assay.
  • a panel of coronavirus pseudoviruses can be used to determine serum neutralization activity.
  • a neutralizing immune response induced by an immunogen disclosed herein generates neutralizing antibodies against a coronavirus (eg, SARS-CoV-2).
  • the neutralizing antibodies herein bind to a cellular receptor or co-receptor of a coronavirus (eg, SARS-CoV-2) or a component thereof.
  • the viral receptor or co-receptor is a coronavirus receptor or co-receptor, preferably a pneumovirus receptor or co-receptor, more preferably a human coronavirus receptor, such as SARS-CoV-2 receptor body or co-receptor.
  • the neutralizing antibodies herein modulate, reduce, antagonize, alleviate, block, inhibit, eliminate and/or interfere with at least one coronavirus (e.g., SARS-CoV) in vitro, in situ and/or in vivo -2) activity or binding, or coronavirus (e.g. SARS-CoV-2) receptor activity or binding, e.g. SARS-CoV-2 release, SARS-CoV-2 receptor signaling, membrane SARS-CoV-2 cleavage, SARS-CoV-2 activity, SARS-CoV-2 production and/or synthesis.
  • coronavirus e.g., SARS-CoV
  • coronavirus e.g. SARS-CoV-2 receptor activity or binding
  • the immunogens disclosed herein induce neutralizing antibodies against SARS-CoV-2 that modulate, reduce, antagonize, alleviate, block, inhibit, eliminate and/or interfere with the interaction of SARS-CoV-2 with SARS-CoV-2.
  • Binding of CoV-2 receptors or co-receptors such as angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell-specific intercellular adhesion molecule-3-grabbing non- Integrin (DC-SIGN) and/or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin-converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell-specific intercellular adhesion molecule-3-grabbing non- Integrin
  • L-SIGN liver/lymph node-SIGN
  • An article of manufacture may include a container and a label or package insert on or associated with the container.
  • Suitable containers include, for example, bottles, vials, syringes, test tubes, IV solution bags, and the like.
  • the container can be formed from various materials such as glass or plastic.
  • the container has a sterile access port.
  • Exemplary containers include bags of intravenous solutions, vials, including containers with stoppers piercable by injecting needles.
  • the article of manufacture or kit may further include a package insert indicating that the composition is useful for treating a particular condition, such as a condition described herein (eg, a coronavirus infection).
  • the article of manufacture or kit may further comprise another or the same container comprising a pharmaceutically acceptable buffer. It may further comprise other materials such as other buffers, diluents, filters, needles and/or syringes.
  • the label or package insert can indicate that the composition is used to treat a coronavirus infection in an individual.
  • a label or package insert on or associated with the container may indicate reconstitution and/or directions for use of the formulation.
  • the label or package insert may further indicate that the formulation is used or intended for subcutaneous, intravenous or other administration to treat or prevent coronavirus infection in an individual.
  • the container contains a composition, alone or in combination with another composition effective for the treatment, prevention and/or diagnosis of a disorder.
  • the article of manufacture or kit may comprise (a) a first container having a composition contained therein (i.e., a first medicament), wherein the composition comprises an immunogenic composition or a protein or recombinant polypeptide thereof; and (b) a second A container having a composition (i.e., a second agent) contained therein, wherein the composition includes another agent, such as an adjuvant or other therapeutic agent, and the article or kit further includes a label or package insert with the second agent Instructions for treating a subject with an effective amount of the medicament.
  • polypeptide and “protein” are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length.
  • Polypeptides can include amino acid residues, including natural and/or unnatural amino acid residues.
  • the term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation and phosphorylation.
  • polypeptides may contain modifications to native or natural sequences so long as the protein retains the desired activity. These modifications can be deliberate, such as through site-directed mutagenesis, or accidental, such as through mutations in the protein-producing host or errors due to PCR amplification.
  • a "subject" is a mammal, such as a human or other animal, and typically a human.
  • the subject (eg, patient) to which one or more agents, cells, cell populations or compositions are administered is a mammal, typically a primate, eg, a human.
  • the primate is a monkey or an ape.
  • a subject can be male or female, and can be of any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • the subject is a non-primate mammal, such as a rodent.
  • treatment means to ameliorate or reduce, in whole or in part, a disease, condition or disorder, or to associated symptoms, adverse reactions or outcomes, or phenotypes. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of disease, relieving symptoms, alleviating any direct or indirect pathological consequences of disease, preventing metastasis, reducing the rate of disease progression, ameliorating or remission of disease state, and remission or improvement of prognosis. The term does not imply a complete cure of the disease or complete elimination of any symptoms or effects on all symptoms or outcomes.
  • delaying the development of a disease refers to delaying, hindering, slowing, slowing, stabilizing, inhibiting and/or delaying the development of a disease (eg, cancer).
  • the length of delay may vary depending on the history of the disease and/or the individual being treated. In some embodiments, substantial or substantial delay may actually encompass prevention, since the individual does not develop the disease. For example, the development of advanced cancers, such as metastases, may be delayed.
  • prevention includes providing prevention of disease occurrence or recurrence in subjects who may be predisposed to the disease but have not yet been diagnosed with the disease.
  • provided cells and compositions are used to delay the development of a disease or slow the progression of a disease.
  • inhibiting a function or activity means reducing the function or activity when compared to otherwise identical conditions other than the condition or parameter of interest or compared to another condition. For example, cells that inhibit tumor growth reduce the rate at which a tumor grows compared to the rate at which it would grow without the cells.
  • an "effective amount" of an agent refers to an effective amount at a dose/amount and for a period of time required to achieve a desired effect (eg, a therapeutic or prophylactic effect).
  • a “therapeutically effective amount” of an agent refers to the dose and dose required to achieve the desired therapeutic effect (e.g., for the treatment of a disease, condition or disorder) and/or the pharmacokinetic or pharmacodynamic effect of the treatment.
  • a therapeutically effective amount can vary depending on factors such as the subject's disease state, age, sex, and weight, and the cell population administered.
  • provided methods involve administering cells and/or compositions in an effective amount (eg, a therapeutically effective amount).
  • prophylactically effective amount refers to an effective amount at the dose and time period required to achieve the desired prophylactic effect. Usually, but not necessarily, a prophylactically effective amount will be less than a therapeutically effective amount because the prophylactic dose is for a subject in a pre- or early-stage disease. In cases where the tumor burden is low, the prophylactically effective amount for some aspects will be higher than the therapeutically effective amount.
  • An effective amount of a vaccine or other agent is sufficient to produce a desired response, such as reduction or elimination of signs or symptoms of a disorder or disease, such as pneumonia. For example, this may be the amount necessary to inhibit viral replication or to measurably alter the external symptoms of viral infection.
  • an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of any condition or disease, eg, for the treatment of a coronavirus infection.
  • the effective amount is a therapeutically effective amount.
  • an effective amount is an amount that prevents the development of one or more signs or symptoms of a particular disease or disorder, such as one or more signs or symptoms associated with a coronavirus infection.
  • the terms "antigen” or “immunogen” are used interchangeably to refer to a substance, usually a protein, capable of inducing an immune response in a subject.
  • the term also refers to an immunologically active protein, that is, capable of eliciting a humoral and/or cell-type immune response against the protein once administered to the subject (either directly or by administering to the subject a nucleotide sequence encoding the protein or a vector) .
  • the term “vaccine immunogen” is used interchangeably with “protein antigen” or "immunogenic polypeptide”.
  • conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
  • conservatively modified variants refers to variants having conservative amino acid substitutions, in which amino acid residues are replaced by other amino acid residues having side chains of similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art.
  • These families include those with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., , glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine acid, proline, phenylalanine, methionine, tryptophan), beta branched chains (e.g. threonine, valine, isoleucine) and aromatic side chains (e.g. tyramine acid, phenylalanine, tryptophan, histidine).
  • basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule so they elicit a specific immune response, for example, an epitope is an area of an antigen to which B cells and/or T cells respond.
  • An epitope can be formed by either contiguous amino acids or non-contiguous amino acids juxtaposed by the tertiary folding of the protein.
  • fusion proteins are recombinant proteins that contain the amino acid sequences of at least two unrelated proteins linked together by peptide bonds to form a single protein. Therefore, it does not contain the naturally occurring coronavirus surface antigen, the fusion (F) protein described herein. Unrelated amino acid sequences can be linked directly to each other, or they can be linked using a linker sequence. As used herein, proteins are unrelated if their amino acid sequences are not normally linked together by peptide bonds in their natural environment (eg, within a cell). For example, the amino acid sequence of a viral antigen and the amino acid sequence of a collagen or procollagen are not usually linked together by peptide bonds.
  • An immunogen is a protein, or portion thereof, capable of inducing an immune response in a mammal, such as a mammal infected or at risk of infection by a pathogen. Administration of the immunogen can result in protective and/or active immunity against the pathogen of interest.
  • immunogenic composition a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or that induces a measurable B cell response against the immunogenic polypeptide Response (e.g. production of antibodies).
  • Sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity; the higher the percent, the more identical the sequences. Two sequences share a specified percentage of amino acid residues in common when compared and aligned for maximum correspondence through the comparison window or specified regions measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection or nucleotides, the two sequences are "substantially identical” (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity).
  • the identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably 100 to 500 or 1000 or more nucleotides (or 20 amino acids) in length. , 50, 200 or more amino acids).
  • Vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject.
  • the immune response is a protective immune response.
  • vaccines induce antigen-specific immune responses against antigens of pathogens (eg, viral pathogens) or cellular components associated with pathological conditions.
  • a vaccine may comprise a polynucleotide (eg, a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (eg, a disclosed antigen), a virus, a cell, or one or more cellular components.
  • the vaccine or vaccine immunogen or vaccine composition is expressed from the fusion construct and self-assembles into nanoparticles displaying the immunogenic polypeptide or protein on the surface.
  • VLPs refer to non-replicative viral shells derived from any of several viruses.
  • VLPs typically consist of one or more viral proteins, such as, but not limited to, proteins known as capsid, coat, coat, surface and/or envelope proteins, or particle-forming polypeptides derived from these proteins. After recombinantly expressing the protein in an appropriate expression system, VLPs can form spontaneously. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using routine techniques known in the art, eg, by electron microscopy, biophysical characterization, and the like. See, eg, Baker et al. (1991) Biophys. J.
  • VLPs can be isolated by density gradient centrifugation and/or identified by characteristic density bands.
  • cryo-EM can be performed on vitrified aqueous samples prepared by the VLP in question and images recorded under appropriate exposure conditions.
  • composition refers to any mixture of two or more products, substances or compounds, including cells. It can be a solution, suspension, liquid, powder, paste, aqueous, non-aqueous, or any combination thereof.
  • vector refers to a nucleic acid molecule capable of propagating another nucleic acid to which it has been linked.
  • the term includes vectors that are self-replicating nucleic acid structures as well as vectors that incorporate into the genome of a host cell into which the vector has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Embodiment 1 A protein comprising a plurality of recombinant polypeptides, each recombinant polypeptide comprising a coronavirus SARS-CoV-2 beta (B.1.351) surface antigen linked to the C-terminal propeptide of collagen, wherein the C of the recombinant polypeptide - The terminal propeptide forms an interpolypeptide disulfide bond.
  • Embodiment 2 The protein according to embodiment 1, wherein the coronavirus infection is a SARS-coronavirus 2 (SARS-CoV-2) infection.
  • SARS-CoV-2 SARS-coronavirus 2
  • Embodiment 3 The protein according to embodiment 1 or 2, wherein the surface antigen comprises a coronavirus spike (S) protein or fragment or epitope thereof, wherein the epitope is optionally a linear epitope or a conformational epitope, and wherein the protein Contains three recombinant polypeptides.
  • S coronavirus spike
  • the epitope is optionally a linear epitope or a conformational epitope
  • the protein Contains three recombinant polypeptides.
  • Embodiment 4 The protein according to embodiment 3, wherein the surface antigen comprises a signal peptide, an S1 subunit peptide, an S2 subunit peptide, or any combination thereof.
  • Embodiment 5 The protein according to embodiment 3, wherein the surface antigen comprises a signal peptide, a receptor binding domain (RBD) peptide, a receptor binding motif (RBM) peptide, a fusion peptide (FP), a heptad repeat 1 ( HR1) peptide or heptad repeat 2 (HR2) peptide or any combination thereof.
  • RBD receptor binding domain
  • RBM receptor binding motif
  • FP fusion peptide
  • HR1 heptad repeat 1
  • HR2 heptad repeat 2
  • Embodiment 6 The protein according to any one of embodiments 3-5, wherein the surface antigen comprises the receptor binding domain (RBD) of the S protein.
  • RBD receptor binding domain
  • Embodiment 7 The protein according to any one of embodiments 3-6, wherein the surface antigen comprises the S1 subunit and the S2 subunit of the S protein.
  • Embodiment 8 The protein according to any one of embodiments 3-7, wherein the surface antigen does not comprise a transmembrane (TM) domain peptide and/or a cytoplasmic (CP) domain peptide.
  • TM transmembrane
  • CP cytoplasmic
  • Embodiment 9 The protein according to any one of embodiments 3-8, wherein the surface antigen comprises a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or cathepsin L.
  • Embodiment 10 The protein according to any one of embodiments 3-8, wherein the surface antigen does not include a protease cleavage site, wherein the protease is optionally furin, trypsin, factor Xa, thrombin or tissue Protease L.
  • Embodiment 11 The protein according to any one of embodiments 1-10, wherein the surface antigen is soluble or not directly bound to a lipid bilayer, eg a membrane or a viral envelope.
  • a lipid bilayer eg a membrane or a viral envelope.
  • Embodiment 12 The protein according to any one of embodiments 1-11, wherein the surface antigens are the same or different in the recombinant polypeptides of the protein.
  • Embodiment 13 The protein according to any one of embodiments 1-12, wherein the surface antigen is fused directly to the C-terminal propeptide, or linked to the C-terminal propeptide by a linker, for example a linker comprising a glycine-X-Y repeat sequence , wherein X and Y are independently any amino acid, and are optionally proline or hydroxyproline.
  • a linker for example a linker comprising a glycine-X-Y repeat sequence , wherein X and Y are independently any amino acid, and are optionally proline or hydroxyproline.
  • Embodiment 14 The protein according to any one of embodiments 1-13, which is soluble or not directly bound to a lipid bilayer, eg a membrane or a viral envelope.
  • a lipid bilayer eg a membrane or a viral envelope.
  • Embodiment 15 The protein according to any one of embodiments 1-14, wherein the protein is capable of binding to a cell surface receptor in a subject, optionally wherein the subject is a mammal, such as a primate, such as a human .
  • Embodiment 16 The protein according to embodiment 15, wherein the cell surface receptor is angiotensin converting enzyme 2 (ACE2), dipeptidyl peptidase 4 (DPP4), dendritic cell specific intercellular adhesion molecule-3 - grab non-integrin (DC-SIGN) or liver/lymph node-SIGN (L-SIGN).
  • ACE2 angiotensin converting enzyme 2
  • DPP4 dipeptidyl peptidase 4
  • DC-SIGN dendritic cell specific intercellular adhesion molecule-3 - grab non-integrin
  • L-SIGN liver/lymph node-SIGN
  • Embodiment 17 The protein according to any one of embodiments 1-16, wherein the C-terminal propeptide is human collagen.
  • Embodiment 18 The protein according to any one of embodiments 1-17, wherein the C-terminal propeptide comprises pro ⁇ 1(I), pro ⁇ 1(II), pro ⁇ 1(III), pro ⁇ 1(V), pro ⁇ 1(XI), pro ⁇ 2 (I), a C-terminal polypeptide of pro ⁇ 2(V), pro ⁇ 2(XI) or pro ⁇ 3(XI), or a fragment thereof.
  • Embodiment 19 The protein according to any one of embodiments 1-18, wherein the C-terminal propeptides are the same or different in the recombinant polypeptides.
  • Embodiment 20 The protein according to any one of embodiments 1-19, wherein the C-terminal propeptide comprises any one of SEQ ID NOs: 67-80 or an amino acid sequence at least 90% identical thereto, which is capable of forming an interpolypeptide disulfide bonds and trimerizes recombinant polypeptides.
  • Embodiment 21 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 67 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 22 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 68 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 23 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 69 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 24 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 70 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 25 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 71 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 26 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 72 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 27 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 73 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 28 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 74 or an amino acid sequence at least 90% identical thereto, which is capable of forming interpolypeptide disulfide bonds and enabling recombination Polypeptide trimerization.
  • Embodiment 29 The protein according to any one of embodiments 1-20, wherein the C-terminal propeptide comprises SEQ ID NO: 75 or SEQ ID NO: 76 or an amino acid sequence at least 90% identical thereto, which is capable of forming an interpolypeptide disulfide bonds and trimerizes recombinant polypeptides.
  • Embodiment 30 The protein according to any one of embodiments 1-29, wherein the C-terminal propeptide comprises a sequence comprising a glycine-X-Y repeat sequence linked to the N-terminus of any one of SEQ ID NOs: 67-80 , wherein X and Y are independently any amino acid, and optionally proline or hydroxyproline, or an amino acid sequence at least 90% identical thereto, capable of forming interpolypeptide disulfide bonds and trimerizing the recombinant polypeptide .
  • Embodiment 31 The protein according to any one of embodiments 1-30, wherein the surface antigen in each recombinant polypeptide is in a prefusion conformation or a postfusion conformation.
  • Embodiment 32 The protein according to any one of embodiments 1-31, wherein the surface antigen in each recombinant polypeptide comprises any one of SEQ ID NOs: 27-66 and 81-85 or amino acids at least 80% identical thereto sequence.
  • Embodiment 33 The protein according to any one of embodiments 1-32, wherein the recombinant polypeptide comprises any one of SEQ ID NOs: 1-26 and 86-95 or an amino acid sequence at least 80% identical thereto.
  • Embodiment 34 An immunogen comprising a protein according to any one of embodiments 1-33, optionally for use as a vaccine priming dose and/or as a booster dose, such as a second dose and/or a third dose Strengthen injections.
  • Embodiment 35 A protein nanoparticle comprising the protein of any one of embodiments 1-33, directly or indirectly linked to the nanoparticle, optionally as a vaccine prime and/or as a booster Use, for example, a second and/or third booster dose.
  • Embodiment 36 A virus-like particle (VLP) comprising the protein of any one of embodiments 1-33, optionally for use as a vaccine priming dose and/or as a booster, such as a second dose and / or a third booster dose.
  • VLP virus-like particle
  • Embodiment 37 An isolated nucleic acid encoding one, two, three or more of the recombinant polypeptides of the protein according to any one of embodiments 1-33.
  • Embodiment 38 The isolated nucleic acid according to embodiment 37, wherein the polypeptide encoding the surface antigen is fused in frame to the polypeptide encoding the C-terminal propeptide of collagen.
  • Embodiment 39 The isolated nucleic acid according to embodiment 37 or 38, which is operably linked to a promoter.
  • Embodiment 40 The isolated nucleic acid according to any one of embodiments 37-39, which is a DNA molecule.
  • Embodiment 41 The isolated nucleic acid according to any one of embodiments 37-39, which is an RNA molecule, optionally an mRNA molecule, such as nucleoside-modified mRNA, non-amplified mRNA, self-amplified mRNA or trans-amplified mRNA. increase mRNA.
  • Embodiment 42 A vector comprising the isolated nucleic acid according to any one of embodiments 37-41.
  • Embodiment 43 The vector according to embodiment 42, which is a viral vector.
  • Embodiment 44 A virus, pseudovirus or cell comprising a vector according to embodiment 42 or 43, optionally wherein the virus or cell has a recombinant genome.
  • Embodiment 45 An immunogenic composition comprising a protein, immunogen, protein nanoparticle, VLP, isolated nucleic acid, vector, virus, pseudovirus or cell according to any one of embodiments 1-44 and pharmaceutically acceptable carrier.
  • 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 the vaccine is used as an initial dose and/or as a booster dose, such as a second dose and/or a third booster dose.
  • Embodiment 47 The vaccine according to embodiment 46, wherein the vaccine comprises a plurality of different adjuvants.
  • Embodiment 48 A method of producing a protein, comprising: expressing the isolated nucleic acid or vector according to any one of embodiments 37-43 in a host cell to produce the protein according to any one of embodiments 1-33; and Purify protein.
  • Embodiment 49 A protein produced by the method of embodiment 48.
  • 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 51 A method according to embodiment 50 for treating or preventing a coronavirus infection.
  • Embodiment 52 The method according to embodiment 50 or 51, wherein generating an immune response suppresses or reduces coronavirus replication in the subject.
  • Embodiment 53 The method according to any one of embodiments 50-52, wherein the immune response comprises a cell-mediated and/or humoral response, optionally comprising production of one or more neutralizing antibodies, e.g. polyclonal antibodies or monoclonal antibodies.
  • the immune response comprises a cell-mediated and/or humoral response, optionally comprising production of one or more neutralizing antibodies, e.g. polyclonal antibodies or monoclonal antibodies.
  • Embodiment 54 The method according to any one of embodiments 50-53, wherein the immune response is directed against a surface antigen of the coronavirus, but not against the C-terminal propeptide.
  • Embodiment 55 The method according to any one of embodiments 50-54, wherein the administering does not result in antibody-dependent enhancement (ADE) in the subject due to previous exposure to one or more coronaviruses.
  • ADE antibody-dependent enhancement
  • Embodiment 56 The method according to any one of embodiments 50-55, wherein the administration does not result in antibody-dependent enhancement (ADE) in the subject when subsequently exposed to one or more coronaviruses.
  • ADE antibody-dependent enhancement
  • Embodiment 57 The method according to any one of embodiments 50-56, further comprising a priming step and/or a boosting step.
  • Embodiment 58 The method according to any one of embodiments 50-57, wherein by topical, transdermal, subcutaneous, intradermal, oral, intranasal (eg, intranasal spray), intratracheal, sublingual, buccal, Rectal, vaginal, inhalation, intravenous (eg, IV), intraarterial, intramuscular (eg, intramuscular), intracardiac, intraosseous, intraperitoneal, transmucosal, intravitreal, subretinal, intraarticular, joint
  • the administration step is carried out by peripheral, topical or epicutaneous administration.
  • Embodiment 59 The method according to any one of embodiments 50-58, wherein the effective amount is administered in a single dose or in a series of doses with one or more intervals.
  • 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 an adjuvant or adjuvants.
  • Embodiment 62 A method comprising administering to a subject an effective amount of the protein according to any one of embodiments 1-33 to generate neutralizing antibodies or neutralizing antiserum to a coronavirus in the subject.
  • Embodiment 63 The method according to embodiment 62, wherein the subject is a mammal, optionally a human or a non-human primate.
  • Embodiment 64 The method according to embodiment 62 or 63, further comprising isolating the neutralizing antibody or neutralizing antiserum from the subject.
  • Embodiment 65 The method according to embodiment 64, further comprising administering an effective amount of the isolated neutralizing antibody or neutralizing antiserum to the human subject 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 antiserum comprises polyclonal antibodies directed against coronavirus surface antigens, optionally wherein the neutralizing antibody or neutralizing antiserum is not Contains or is substantially free of 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 is free or substantially free of C- Antibody to terminal propeptide.
  • Embodiment 68 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 use in Inducing an immune response to a coronavirus in a subject, and/or treating or preventing a coronavirus infection.
  • 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 70 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 the manufacture of a medicament or prophylactic for inducing an immune response to a coronavirus in a subject, and/or for treating or preventing a 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 combined.
  • Embodiment 72 The method according to embodiment 71, wherein the analyte is an antibody, a receptor or a cell that recognizes a surface antigen.
  • Embodiment 73 The method according to embodiment 71 or 72, wherein 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 was 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 a lateral flow assay kit ( lateral flow assay kit).
  • Example 1 Generation of recombinant disulfide bonded SARS-CoV-2 S-trimeric fusion proteins
  • Production of secreted recombinant disulfide-linked polypeptides comprising SARS-CoV-2 protein peptides fused to trimerization domains as protein subunit vaccine candidates.
  • the complete ectodomain of the native Spike protein from SARS-CoV2, such as the Hu-1 strain and the VOC beta strain, including its signal peptide (SP), S1 and S2 domains, is fused in frame at the C-terminus to the encoding ⁇ 1 Mammalian expression vector of human C-propeptide of collagen to enable expression of secreted trimer S-trimer fusion antigen PCT/CN2020/095269, PCT/CN2021/087066, PCT/CN2021/093895 and PCT/CN2021 /099285 is incorporated by reference in its entirety for all purposes.
  • an affinity purification protocol was developed to take advantage of the high binding affinity between the protein trimerized TM tag and Endo180, which is capable of binding to type 1 pre C-terminal region of collagen and collagen receptor for collagen maturation.
  • the Endol80-Fc fusion protein was 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 Endol80-Fc.
  • serum-free cell culture medium containing S-trimer secreted by CHO cells was loaded onto a protein A column with pre-captured Endol80-Fc.
  • bound S-trimers are purified in one step using a mild salt elution under conditions that do not separate Endol80-Fc from the Protein A column to near uniformity. Further purification of S-trimer by low pH for prophylactic viral inactivation (VI), anion exchange chromatography to remove host cell DNA and any residual endotoxin, nanofiltration as a prophylactic viral removal (VR) step , and finally UF/DF to concentrate the S-trimer into the formulation buffer to the concentration required to obtain the active drug (DS) of the S-trimer subunit vaccine candidate. Stability analysis of the purified S-trimer indicated that the S-trimer was stable in liquid solution formulations at 2-8°C.
  • SDS-PAGE analysis under non-reducing and reducing conditions confirmed that the purified S-trimer was a disulfide-linked trimer and was partially cleaved at the S1/S2 boundary by furin produced by CHO cells.
  • S-trimers occur in various high molecular weight forms, likely as a result of partial cleavage of the antigen, releasing non-covalently linked and cleaved S1 during sample processing.
  • Figures 1A and 1B show the expression and purification of S-trimer antigens.
  • Figure 1A shows depth-filtered S-trimeric antigenic protein;
  • Figure 1B shows purified S-trimeric antigenic protein.
  • the protein trimerization TM technology (Liang et al., Nat. Comms., 12:1346, 2021) was used to generate covalent trimers of spike antigens based on Hu-1 strain and VOC beta strain.
  • S-trimer expression in fed-batch serum-free CHO cell cultures was analyzed by 8% SDS-PAGE in a 10 L bioreactor. Cell-free conditioned medium was analyzed under reducing conditions followed by Coomassie brilliant blue staining.
  • the binding affinity of the S-trimer to ACE2 was assessed by Bio-Layer interferometry on a ForteBio Octet QKe (Pall).
  • ACE2-Fc (10 ⁇ g/mL) was immobilized on a protein A (ProA) biosensor (Pall).
  • Real-time receptor binding curves were obtained by applying the sensor to two-fold serial dilutions of S-trimer (22.5-36 ⁇ g/mL in PBS).
  • Kinetic parameters (K on and K off ) and affinity (K D ) were analyzed using Octet software version 12.0. Assuming a 1:1 binding model of the S-trimer (protoform or B.1.351) to ACE2-Fc, the dissociation constant ( KD ) was determined using steady state analysis.
  • a 96-well plate (Corning) was coated with 1 ⁇ g/mL ACE2-Fc (100 ⁇ L/well) overnight at 4°C and blocked with 2% skim milk for 2 hours at 37°C. After washing 3 times with PBST, incubate with 100 ng/mL S-trimer (original type, B.1.351, B.1.1.7 or P.1) mixed with serially diluted antiserum at 37°C for 1 Hour.
  • the plate was incubated with rabbit anti-protein trimerized TM tag antibody (Clover Biopharmaceuticals) at a dilution of 1:5000 for 1 hour at 37 °C, then washed 3 times with PBST, and then washed with 1 : 20000 dilutions of goat anti-rabbit IgG-HRP (Southern Biotech) incubation. After washing 3 times with PBST, TMB (Thermo Scientific) was added for signal development.
  • rabbit anti-protein trimerized TM tag antibody (Clover Biopharmaceuticals) at a dilution of 1:5000 for 1 hour at 37 °C
  • the IC50 for a given serum sample was defined as the reciprocal of the dilution at which the sample showed 50% competition.
  • the original (Wuhan-Hu-1) strain and variants B.1.1.7, B.1.351 and P.1 strains and other strains were tested for neutralization of SARS-CoV-2 pseudoviruses.
  • the samples were first heat-inactivated for 30 min and serially diluted (3-fold), incubated with an equal amount of 650 TCID 50 pseudovirus at 37°C for 1 h, Both virus alone (positive control) and cells alone (negative control) were used. Then, fresh trypsinized ACE2 overexpression-293 cells were added to each well at 20,000 cells/well.
  • luciferase activity was determined by luciferase assay system (Beyotime).
  • the EC50 neutralizing antibody titer for a given serum sample was defined as the reciprocal of the dilution in which the sample exhibited a 50% reduction in relative light units (RLU) compared to virus alone control wells.
  • Th1 cytokines IFN- ⁇ , IL-2
  • Th2 cytokines IL-5
  • ELISpot kit Mabtech
  • splenocytes of the immunized mice or the PBMCs of the immunized mice were collected. Stimulate 5 ⁇ 10 5 splenocytes (96-well plate ). Phorbol 12-myristate 13-acetate (PMA) and ionomycin were added as non-specific stimuli to positive control wells, while negative control wells received no stimuli.
  • PMA Phorbol 12-myristate 13-acetate
  • ionomycin were added as non-specific stimuli to positive control wells, while negative control wells received no stimuli.
  • S-trimer Detection of SARS-CoV-2 specific binding and neutralizing antibodies in convalescent serum by S-trimer.
  • the S-trimer was used as an antigen to detect the presence of SARS-CoV-2 spike protein-binding antibodies and ACE2-competitive antibodies in human convalescent serum samples collected from recovered COVID-19 patients. High levels of S-trimer-binding antibodies and ACE2 competitive titers were detected in convalescent sera, as well as high neutralizing antibody titers using a pseudovirus neutralization assay.
  • Antibody titers were observed to correlate with disease severity, with lower antibody titers observed in patients with mild COVID-19 disease and higher titers in severe cases. Antibody titers also appeared to correlate moderately with patient age, but no gender differences were observed.
  • mice received two intramuscular injections of S-trimer (without adjuvant or with various adjuvants).
  • the effect of adjuvants on humoral immunogenicity was evident, as the S-trimer-binding antibody titers, ACE2 competitive titers, and neutralizing antibody titers were significantly higher in the adjuvanted group at the corresponding antigen dose levels in unadjuvanted vaccines.
  • S-trimers with different adjuvants elicited ACE2-competitive and pseudovirus-neutralizing antibody titers similar to or higher than those observed in human convalescent serum samples. Similar results were observed in S-trimer immunized rats.
  • the immunogenicity of adjuvanted S-trimers was further studied in non-human primates (rhesus monkeys). Animals were inoculated twice intramuscularly with adjuvanted S-trimer or PBS vehicle control. The animals were then challenged with the SARS-CoV-2 virus on day 35, after which immune protection was assessed by various parameters.
  • FIG 3 shows the immune protection of adjuvanted S-trimers against human primate (rhesus macaque) SARS-CoV-2 virus. Animals were inoculated twice intramuscularly with adjuvanted S-trimer or vehicle control. Animals were challenged with SARS-CoV-2 virus on day 35 and then immune protection was assessed.
  • the first group is the saline vehicle control
  • the second group is the original S-trimer (adjuvant 1 contains CpG 1018 plus Alum)
  • the third group is the chimeric original S-trimer containing beta (beta) mutant RBD polymer (adjuvant 1)
  • the fourth group is the chimeric original S-trimer containing beta (beta) mutant RBD (adjuvant 2 contains squalene, ⁇ -tocopherol, and Tween-80)
  • Lung tissue was collected at autopsy at 7 dpi and tested for SARS-CoV-2 viral load based on genomic RNA (gRNA). Significantly lower viral loads in lung tissue than vehicle controls were observed in the three groups containing adjuvanted S-trimer. Comparing the third and fourth groups, the adjuvant containing squalene, ⁇ -tocopherol, and Tween-80 was similar to CpG 1018 plus Alum as an adjuvant.
  • Figure 4 shows primate neutralizing antibody responses to adjuvanted S-trimers.
  • Use original S-trimer (adjuvant 1 containing CpG 1018 plus Alum), chimeric original S-trimer containing beta (beta) mutant RBD (adjuvant 1), or containing beta (beta) variant
  • the chimeric proto-S-trimer of strain RBD (Adjuvant 2 containing squalene, ⁇ -tocopherol, and Tween-80) was inoculated intramuscularly twice in animals. Both antigens were administered at a dose of 30 ⁇ g per dose. Serum was collected at D35 for pseudovirus neutralizing antibody test and live SARS-CoV-2 virus neutralizing antibody test, A.
  • FIG. 1 shows SARS-CoV-2Hu-1, B.1.1.7, B.1.617.2, B.1.351, P.1, and B.1.621 strain pseudovirus neutralizing antibody titer (IC50) data.
  • B. Live SARS-CoV-2 virus neutralizing antibody titer data are shown. Points represent data from individual animals; horizontal lines represent geometric mean titers (GMT) ⁇ SEM for each group. Animals receiving chimeric proto-S-trimers (adjuvant 2) containing the beta variant RBD (adjuvant 2) produced 6 to 100-fold higher of neutralizing antibodies.
  • the chimeric original S-trimer (adjuvant 2) containing beta (beta) variant strain RBD has a more extensive neutralization effect, and has produced in animals the effect on SARS-CoV-2Hu-1 and various VOC or Neutralizing antibodies to VOI. Comparing the chimeric original S-trimer (adjuvant 1) containing beta (beta) mutant strain RBD and the chimeric original S-trimer (adjuvant 2) containing beta (beta) mutant strain RBD, containing The chimeric original S-trimer (adjuvant 2) of beta mutant strain RBD has similar or better neutralization effect on pseudovirus and live virus.
  • Figure 5 shows the protective effect of chimeric prototype S-trimer (adjuvant 2) containing beta (beta) variant strain RBD on Hu-1 and on Omicron strain infection in primates .
  • Animals were intramuscularly inoculated twice with adjuvanted S-trimer or vehicle control, and serum was collected on D35 for pseudovirus neutralizing antibody testing.
  • the first group is the original S-trimer (adjuvant 1 containing CpG 1018 plus Alum)
  • the second group is the chimeric original S-trimer (adjuvant 1) containing RBD of the beta variant strain
  • the third group is the chimeric proto-S-trimer containing beta (beta) mutant RBD (adjuvant 2 contains squalene, ⁇ -tocopherol, and Tween-80)
  • the fourth group is the proto-S - Trimer and bivalent vaccine (Adjuvant 1 ) containing a chimeric original S-trimer of the beta variant RBD.
  • Animals receiving chimeric proto-S-trimers (adjuvant 2) containing the beta variant RBD (adjuvant 2) produced approximately 18-fold higher Neutralizing antibodies against Omicron strains. Comparing the chimeric original S-trimer (adjuvant 1) containing beta (beta) mutant strain RBD and the chimeric original S-trimer (adjuvant 2) containing beta (beta) mutant strain RBD, containing The chimeric original S-trimer (adjuvant 2) of the RBD of the beta mutant strain had a better neutralizing effect on the Omicron strain.
  • Embodiment 3 the immunogenicity research of the SARS-CoV-2 vaccine containing different adjuvant doses
  • Chimeric proto-S-trimers containing beta variant RBD also included other groups in which animals in group 2 received a booster (dose 2), administered with aluminum alone or without any adjuvant dose (antigen only).
  • dose 2 administered with aluminum alone or without any adjuvant dose (antigen only).
  • both the original S-trimer and the chimeric original S-trimer vaccine group containing beta (beta) variant strain RBD received the full dose of CAS-1 as an adjuvant Primer and booster doses.
  • Chimeric proto-S-trimers containing BETA variant RBD also included other groups in which animals received priming and boosting with half or quarter doses of CAS-1 or no adjuvant (only antigen).
  • the neutralizing antibody titer based on the pseudovirus neutralization test was evaluated, and the virus strains included the original strain (Wuhan-Hu-1), the Beta (Beta) variant (B. 1.351), the Alpha variant (B.1.1.7), and the Gamma variant (P.1).
  • Phase 1 clinical study is a double-blind, randomized, defined-dose study evaluating chimeric proto-S-trimer containing beta variant RBD in combination with CpG1018/aluminum adjuvant and CAS-1 Safety and immunogenicity when used with adjuvants. Including original S-trimer (CpG 1018/Alum) and chimeric original S-trimer and CpG 1018/Alum containing RBD of Beta mutant strain. All vaccine formulations were administered as a two-dose regimen, 21 days apart. These interim study results are based on 70 adults (ages 18 to 75) in South Africa with high levels of existing immunity to previous infections and vaccinations.
  • Fig. 10 shows that the chimeric original S-trimer vaccine containing 9 ⁇ g of beta mutant strain RBD with the full dose of CAS-1 has a good immune response and broad spectrum of antibodies after 1 dose.
  • the neutralizing antibody of the vaccine was significantly higher than that of the neutralizing antibody containing 30 ⁇ g of the original S-trimer and CpG 1018/alum adjuvant, about 1.5 to 2.3 times.
  • the vaccine formulation consisting of 9 ⁇ g of the chimeric original S-trimer antigen of the beta variant strain RBD and the full dose of CAS-1 elicited a higher response to Omicron than 30 ⁇ g of the original S-trimer combined with CpG 1018/Alum Stronger immune responses for BA.4 and BA.5.
  • Participants who synthesized the prototype S-trimer had a 2.9-fold increase in the immune response to BA.5 and a 3.9-fold increase in the immune response to Omicron BA.4. This data suggests that the use of an Adjuvant 2 system may help to conserve antigenic agent.
  • the data in Figure 11 shows that relative to the level before the boost, 30 ⁇ g of the original S-trimer (CpG1018/Alum) increased the antibody titer against Omicron BA.5 by 4.5 times, and against Omicron BA. The neutralizing antibody of 4 also increased by 4.5 times.
  • other formulations containing less (3 ug) chimeric proto-S-trimer containing beta variant RBD or less (half) adjuvant 2 also elicited resistance to the tested SARS-CoV-2 variants. Massive immune response.

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Abstract

提供了包括重组肽和蛋白质的免疫原性组合物，所述重组肽和蛋白质包含冠状病毒抗原和免疫原，例如SARS-CoV-2冠状病毒贝塔（beta，B.1.351）变异株S蛋白肽或其片段、变体或突变体，例如含贝塔变异株受体结合域及原始型或其它变异株S蛋白肽序列的嵌合抗原和免疫原。所述免疫原性组合物包括分泌性融合蛋白，所述分泌性融合蛋白包括可溶性冠状病毒抗原，所述可溶性冠状病毒抗原蛋白通过框内融合连接到能够自三聚化的胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。提供的免疫原性组合物可用于产生免疫应答，例如作为疫苗用于预防冠状病毒感染，例如SARS-CoV-2原始型（Hu-1）、阿尔法、贝塔、伽马、德尔塔、奥密克戎和/或其它毒株的感染。提供的免疫原性组合物可用于疫苗组合物中，例如，作为预防性和/或治疗性疫苗的一部分。还提供了用于产生重组肽和蛋白质的方法、预防、治疗和/或诊断方法以及相关试剂盒。

Description

冠状病毒疫苗组合物、方法及其使用 技术领域
本公开内容在一些方面涉及用于治疗和/或预防冠状病毒感染的免疫原性组合物，所述免疫原性组合物包括重组肽和蛋白质，所述重组肽和蛋白质包括冠状病毒抗原和免疫原，例如冠状病毒S蛋白肽，包括基于SARS-CoV-2贝塔(B.1.351)毒株的S蛋白肽由三聚体化 TM标签多肽间二硫键连接形成的亚单位疫苗。
背景技术
冠状病毒感染广泛范围的鸟类和哺乳动物，包括人类。冠状病毒可能每年在人体内传播，通常会引起轻微的呼吸系统疾病，尽管在婴幼儿、老年人和免疫功能低下的人群中严重程度更高。但是，某些冠状病毒，包括中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征冠状病毒(SARS-CoV-1)和严重急性呼吸综合征冠状病毒2(SARS-CoV-2)，具有高致病性。冠状病毒的高致病性、空气传播性、高病死率和模糊确定的流行病学使得迫切需要有效的疫苗和相关的治疗药剂。特别是，迫切需要能够迅速诱导针对SARS-CoV-2的有效免疫应答的疫苗。本发明提供了满足上述和其它需要的方法、用途和制品(article of manufacture)。
发明内容
在一些实施方案中，本文公开了包含多种重组多肽的蛋白质，每种重组多肽包括连接到胶原的C-末端前肽的冠状病毒表面抗原，其中重组多肽的C-末端前肽形成多肽间二硫键。
在一些实施方案中，本文公开了重组亚单位疫苗，其包括来自冠状病毒(例如SARS-CoV-2贝塔(B.1.351)的S蛋白或其片段的胞外域(例如，没有跨膜和细胞质结构域)，其在框内与能够形成二硫键连接的同源三聚体的胶原的C-前肽融合。由此产生的重组亚单位疫苗(例如S-三聚体)可以从转染的细胞中表达和纯化，并且预期为三聚体形式的天然构象。这解决了当病毒抗原以可溶性形式表达为重组肽或蛋白质而没有跨膜和/或细胞质结构域时经常遇到的病毒抗原错误折叠问题。这种错误折叠的病毒抗原不能忠实地保持天然的病毒抗原构象，并且常常不能产生中和抗体。
在一些实施方案中，冠状病毒是严重急性呼吸综合征(SARS)冠状病毒(SARS-CoV-1)、SARS冠状病毒2(SARS-CoV-2)、SARS样冠状病毒、中东呼吸综合征(MERS)冠状病毒(MERS-CoV)、MERS样冠状病毒、NL63-CoV、229E-CoV、OC43-CoV、HKU1-CoV、WIV1-CoV、MHV、HKU9-CoV、PEDV-CoV或SDCV。
在前述任一实施方案中，表面抗原可以包括冠状病毒刺突(S)蛋白或其片段或表位，其中表位可选地是线性表位或构象表位，并且其中蛋白质包括三种重组多肽。
在前述任一实施方案中，表面抗原可以包括信号肽、S1亚单位肽、S2亚单位肽或其任意组合。
在前述任一实施方案中，表面抗原可以包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)或七肽重复序列2(HR2)或其任意组合。
在前述任一实施方案中，表面抗原可以包括S蛋白的受体结合结构域(RBD)。
在前述任一实施方案中，表面抗原可以包括S蛋白的S1亚单位和S2亚单位。
在前述任一实施方案中，表面抗原可以不含跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
在前述任一实施方案中，表面抗原可以包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
在前述任一实施方案中，表面抗原可以不含蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L，或者可以包含不可被蛋白酶裂解的突变的蛋白酶裂解位点。
在前述任一实施方案中，表面抗原可以是可溶解的或不直接结合到脂质双层，例如膜或病毒包膜。
在前述任一实施方案中，在蛋白质的重组多肽中表面抗原可以相同或不同。
在前述任一实施方案中，表面抗原可以直接融合到C-末端前肽，或可以通过接头(例如包含甘氨酸-X-Y重复序列的接头，其中X和Y独立地是任何氨基酸并且可选地是脯氨酸或羟脯氨酸)连接到C-末端前肽。
在前述任一实施方案中，蛋白质可以是可溶解的或不直接结合到脂质双层，例如膜或病毒包膜。
在前述任一实施方案中，蛋白质可以结合到受试者的细胞表面受体，可选地其中受试者是哺乳动物，例如灵长类动物，例如人类。
在前述任一实施方案中，细胞表面受体可以是血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)或肝脏/淋巴结-SIGN(L-SIGN)。
在前述任一实施方案中，C-末端前肽可以是人胶原。
在前述任一实施方案中，C-末端前肽可以包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、proα2(XI)或proα3(XI)的C-末端前肽或其片段。
在前述任一实施方案中，在重组多肽中C-末端前肽可以相同或不同。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:67-80中任一个，或与SEQ ID NO:67-80中任一个具有至少90％、91％、92％、93％、94％、95％、96％、 97％、98％、99％或99.5％序列同源性的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:67或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:68或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:69或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:70或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:71或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:72或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:73或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:74或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:75或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:76或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:77或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:78或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:79或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括SEQ ID NO:80或与其至少95％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，C-末端前肽可以包括包含甘氨酸-X-Y重复序列连接到SEQ ID NO:67-80中任一个的N-末端的序列，其中X和Y独立地是任何氨基酸并且可选地是脯氨酸或羟脯氨酸，或与其至少90％相同的氨基酸序列，能够形成多肽间二硫键并使重组多肽三聚化。
在前述任一实施方案中，每种重组多肽中的表面抗原可以为融合前构象。
在前述任一实施方案中，每种重组多肽中的表面抗原可以为融合后构象。
在前述任一实施方案中，每种重组多肽中的表面抗原可以包括SEQ ID NO:27-66和81-85中任一个或与其至少80％相同的氨基酸序列。
在前述任一实施方案中，重组多肽可以包括SEQ ID NO:1-26和86-95中任一个或与其至少80％相同的氨基酸序列。
本文还提供了包含本文提供的蛋白质的免疫原。本文提供了包含直接或间接连接到纳米颗粒的本文提供的蛋白质的蛋白质纳米颗粒。本文提供了包含本文提供的蛋白质的类病毒颗粒(VLP)。
本文还提供了编码本文提供的蛋白质的一种、两种、三种或更多种重组多肽的分离核酸。在一些实施方案中，编码S蛋白肽的多肽在框内与编码胶原的C-末端前肽的多肽融合。在一些实施方案中，本文提供的分离核酸可操作地连接到启动子。
在一些实施方案中，本文提供的分离核酸是DNA分子。在一些实施方案中，本文提供的分离核酸是RNA分子，可选地是mRNA分子，例如核苷修饰的mRNA、非扩增mRNA、自扩增mRNA或反式扩增mRNA。
本文还提供了包含本文提供的分离核酸的载体。在一些实施方案中，载体是病毒载体。
在一些方面，本文提供了包含本文提供的载体的病毒、假病毒或细胞，可选地，其中病毒或细胞具有重组基因组。在一些方面，本文提供了免疫原性组合物，其包含本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒或细胞以及药学上可接受的载体。
本文还提供了包含本文提供的免疫原性组合物和可选佐剂的疫苗，其中疫苗可选地是亚单位疫苗。在一些实施方案中，疫苗是预防性和/或治疗性疫苗。可选佐剂可在初始剂和/或加强剂中使用。独立地，初始剂和/或任意一剂或多剂加强剂的佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
在一些方面，本文提供了产生蛋白质的方法，包括：在宿主细胞中表达本文提供的分离核酸或载体，以产生本文提供的蛋白质；并纯化蛋白质。本文提供了通过本文提供的方法产生的蛋白质。
本文提供了用于在受试者中产生对冠状病毒的S蛋白肽或其片段或表位的免疫应答的方法，包括向受试者施用有效量的如本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，以产生免疫应答。在一些实施方案中，本文提供的方法用于治疗或预防冠状病毒感染。在一些实施方案中，产生免疫应答抑制或减少了受试者中冠状病毒的复制。在一些实施方案中，免疫应答包括细胞介导的应答和/或体液应答，可选地包括产生一种或更多种中和抗体，例如多克隆抗体或单克隆抗体。在一些实施方案中，免疫应答针对冠状病毒的S蛋白肽 或其片段或表位，但不针对C-末端前肽。在一些实施方案中，由于先前暴露于一种或更多种冠状病毒，对受试者的施用不会导致受试者中的抗体依赖性增强(ADE)。在一些实施方案中，当随后暴露于一种或更多种冠状病毒时，施用不会导致受试者中的抗体依赖性增强(ADE)。在一些实施方案中，方法进一步包括初免步骤(priming step)和/或加强步骤(boosting step)。在一些实施方案中，通过局部、经皮、皮下、皮内、口服、鼻内(例如，鼻内喷雾剂)、气管内、舌下、口腔、直肠、阴道、吸入、静脉内(例如，静脉注射)、动脉内、肌肉内(例如，肌肉注射)、心内、骨内、腹腔内、跨粘膜、玻璃体内、视网膜下、关节内、关节周围、局部或应用皮肤(epicutaneous)给药来进行给药步骤。在一些实施方案中，以单剂量或具有一个间隔或更多个间隔的一系列剂量施用有效量。在一些实施方案中，在不使用佐剂的情况下施用有效量。在一些实施方案中，有效量与佐剂一起施。
本文提供了方法，该方法包括向受试者施用有效量的本文提供的蛋白质，以在受试者中产生针对冠状病毒的中和抗体或中和抗血清。在一些实施方案中，受试者是哺乳动物，可选地是人类或非人类灵长类动物。在一些实施方案中，方法进一步包括从受试者分离中和抗体或中和抗血清。在一些实施方案中，方法进一步包括通过被动免疫向人类受试者施用有效量的分离中和抗体或中和抗血清，以预防或治疗冠状病毒感染。在一些实施方案中，针对冠状病毒的中和抗体或中和抗血清包括针对冠状病毒S蛋白肽或其片段或表位的多克隆抗体，可选地，其中中和抗体或中和抗血清不含或基本不含针对胶原的C-末端前肽的抗体。在一些实施方案中，中和抗体包括针对冠状病毒S蛋白肽或其片段或表位的单克隆抗体，可选地，其中中和抗体不含或基本不含针对胶原的C-末端前肽的抗体。
在一些方面，本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。
在一些方面，本文提供了本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。在一些方面，本文提供了本文提供的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于制造用于诱导受试者中对冠状病毒的免疫应答的药物或预防剂，和/或用于治疗或预防冠状病毒感染。
本文还提供了用于分析样品的方法，包括：将样品与本文提供的蛋白质接触，并检测蛋白质与能够特异性结合冠状病毒的S蛋白肽或其片段或表位的分析物之间的结合。在一些实施方案中，分析物是识别S蛋白肽或其片段或表位的抗体、受体或细胞。在一些实施方案中，结合指示样品中存在分析物，和/或样品来源于的受试者中存在冠状病毒感染。
本文提供了试剂盒，其包括本文提供的蛋白质和含有或固定蛋白质的基质、垫或小瓶，可选地其中试剂盒是ELISA或侧向流动检测试剂盒(lateral flow assay kit)。
附图说明
图1A和图1B示出了分离和纯化的含贝塔(beta)变异株RBD的嵌合原始型S-三聚体。
图2示出了含贝塔(beta)变异株RBD的嵌合原始型S-三聚体与ACE2-Fc的受体结合动力学。与原始株相比，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体受体亲和力更高。
图3示出了含佐剂S-三聚体对非人类灵长类动物(恒河猴)SARS-CoV-2病毒的免疫保护。用含佐剂S-三聚体或载体对照对动物肌肉内接种两次。在第35天用SARS-CoV-2病毒攻击动物，然后评估免疫保护。第一组为盐水载体对照，第二组为原始型S-三聚体(佐剂1含CpG 1018加Alum)，第三组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，第四组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、和吐温-80)。在7dpi尸检时采集肺组织，并基于基因组RNA(gRNA)检测SARS-CoV-2病毒载量。在含佐剂S-三聚体的三组中观察到肺组织中的病毒载量明显低于载体对照。比较第三组和第四组，含角鲨烯、α-生育酚、和吐温-80的佐剂与CpG 1018加Alum作为佐剂效果类似。
图4示出了灵长类动物对含佐剂S-三聚体中和抗体应答。用原始型S-三聚体(佐剂1含CpG 1018加Alum)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，或含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、和吐温-80)对动物肌肉内接种两次。在D35收集血清，进行假病毒中和抗体测试和活SARS-CoV-2病毒中和抗体测试，A.示出了SARS-CoV-2Hu-1、B.1.1.7、B.1.617.2、B.1.351、P.1、和B.1.621毒株假病毒中和抗体滴度(IC 50)数据。B.示出了活SARS-CoV-2病毒中和抗体滴度数据。点代表个体动物的数据；横线表示每组的几何平均滴度(GMT)±SEM。对比接受原始型S-三聚体(佐剂1)的动物，接受含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)的动物产生了6到100倍高的中和抗体。并且含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)具有更广泛的中和作用，在动物中产生了对SARS-CoV-2Hu-1及多种VOC或VOI的中和抗体。比较含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)对假病毒和活病毒中和效果类似或更好。
图5示出了含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)在灵长类动物中对Hu-1和对奥密克戎毒株感染的保护作用。用含佐剂S-三聚体或载体对照对动物肌肉内接种两次，然后在D35收集血清，进行假病毒中和抗体测试。第一组为原始型S-三聚体(佐剂1含CpG 1018加Alum)，第二组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，第三组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、和吐温-80)，第四组为原始型S-三聚体和含贝塔(beta)变 异株RBD的嵌合原始型S-三聚体的双价疫苗(佐剂1)。对比接受原始型S-三聚体(佐剂1)的动物，接受含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)的动物产生了约18倍高的对奥密克戎毒株的中和抗体。比较含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)对奥密克戎毒株中和效果更好。
图6示出了鼠对含不同剂量的佐剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体在第35天的体液免疫应答。
图7A示出了非人类灵长类动物对含不同剂量的佐剂1的第三剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体的免疫应答的研究试验设计。图7B示出了非人类灵长类动物对含不同剂量的佐剂1的第三剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体的免疫应答。与含贝塔(Beta)变异株RBD的嵌合原始型S-三聚体CpG/Alum)全剂量初免/增强/增强相比，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体只加铝剂(-CpG增强)，在第3次增强后的中和抗体方面没有显着差异。即使使用1/3的CpG剂量，我们也观察到针对所有VOC，尤其是Omicron和Delta的类似增强的中和抗体。
图8示出了含佐剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体对非人类灵长类动物的的SARS-CoV-2病毒的免疫保护-体重。
图9示出了含佐剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体对非人类灵长类动物的SARS-CoV-2病毒的免疫保护-鼻，咽喉，和肛门病毒载量测定结果。对照：盐水，1#：原始型S-三聚体+佐剂1，2#：塔嵌合型+佐剂1，3#：贝塔嵌合型+佐剂2。佐剂1含CpG 1018加Alum。佐剂2含角鲨烯、α-生育酚、和吐温-80。
图10示出了含佐剂1的S-三聚体和含佐剂2的含贝塔(beta)变异株RBD的嵌合原始型S-三聚体对真病毒中和抗体滴度。佐剂1含CpG 1018加Alum。佐剂2含角鲨烯、α-生育酚、和吐温-80。
图11示出了含不同剂量的佐剂2和不同剂量第一剂剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体对真病毒奥密克戎变种BA.4和BA.5的中和抗体滴度。佐剂1含CpG 1018加Alum。佐剂2含角鲨烯、α-生育酚、和吐温-80。
图12示出了含不同剂量的佐剂2和不同剂量第一剂S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体对真病毒Hu-1和贝塔的中和抗体滴度。佐剂1含CpG1018加Alum。佐剂2含角鲨烯、α-生育酚、和吐温-80。
具体实施方式
本文提供了包含冠状病毒抗原或免疫原的融合肽和蛋白质的免疫原性组合物、方法和用途，用于冠状病毒感染的治疗(例如预防性、治疗性)。在一些实施方案中，公开了来自共价连接三聚体形式的RNA病毒的重组可溶性表面抗原的组合物和使用方法。 在一些实施方案中，产生的融合蛋白作为二硫键连接的同源三聚体分泌，其结构更稳定，同时保留类天然三聚体病毒抗原的构象，因此可以用作对抗这些危险病原体的更有效疫苗。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为疫苗或作为多价疫苗的一部分来预防病毒感染的方法，不使用或使用佐剂，或使用一种以上佐剂，可选地通过肌肉内注射或鼻内施用。病毒抗原三聚体可在初始剂，额外剂，和/或加强剂中使用。独立地，初始剂，额外剂，和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为抗原用于通过检测识别病毒抗原(例如中和抗体)的抗体(例如IgM或IgG)来诊断病毒感染的方法。
在一些实施方案中，本文公开了使用病毒抗原三聚体作为抗原来产生可用于被动免疫的多克隆或单克隆抗体(例如，用于治疗冠状病毒感染的中和mAb)的方法。
在一些实施方案中，本文公开了作为疫苗或作为多价疫苗的一部分的病毒抗原三聚体，其中疫苗包括多种三聚体亚单位疫苗，所述三聚体亚单位疫苗包括病毒的相同蛋白质的病毒抗原，或包括一种或更多种病毒的两种或更多种不同蛋白质的病毒抗原或相同病毒的一种或更多种毒株的病毒抗原。
在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的单价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的二价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的三价疫苗。在一些实施方案中，本文公开了包含本文公开的病毒抗原三聚体的四价疫苗。
在一些实施方案中，本文公开了包含本文公开的S-三聚体的单价疫苗。在一些实施方案中，本文公开了包含本文公开的S-三聚体的二价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体和至少一个包含第二S蛋白抗原的S-三聚体的二价疫苗。在一些实施方案中，第一和第二S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种或更多种不同S蛋白。在一些实施方案中，本文公开了包含本文公开的S-三聚体的三价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体、至少一个包含第二S蛋白抗原的S-三聚体和至少一个包含第三S蛋白抗原的S-三聚体的三价疫苗。在一些实施方案中，第一、第二和第三S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种、三种或更多种不同S蛋白。在一些实施方案中，本文公开了包含本文公开的S-三聚体的四价疫苗。在一些实施方案中，本文公开了包含至少一个包含第一S蛋白抗原的S-三聚体、至少一个包含第二S蛋白抗原 的S-三聚体、至少一个包含第三S蛋白抗原的S-三聚体和至少一个包含第四S蛋白抗原的S-三聚体的四价疫苗。在一些实施方案中，第一、第二、第三和第四S蛋白抗原来自一种或更多种病毒物种或毒株/亚型的相同S蛋白，或来自一种或更多种病毒物种或相同病毒物种的一种或更多种毒株/亚型的两种、三种、四种或更多种不同S蛋白。
本文提供的包含冠状病毒抗原和免疫原的蛋白质，包括重组多肽和融合蛋白，可用于有效且安全地治疗(例如，治疗性、预防性)冠状病毒感染。例如，本文提供的包含冠状病毒抗原和免疫原的蛋白质治疗冠状病毒感染，而没有介导的(mediated)疫苗诱导的疾病增强(VED)和/或抗体依赖性增强(ADE)。此外，本文提供的包含冠状病毒抗原和免疫原的蛋白质容易产生，并且在例如高温、极端pH、高渗透压和低渗透压等高应激条件下表现出稳定性。因此，本文提供的蛋白质和免疫原性组合物规避并满足了阻碍冠状病毒疫苗开发的生产、稳定性、安全性和有效性问题。
在一些方面，本文提供的冠状病毒抗原和免疫原包括冠状病毒刺突(S)蛋白或肽，尤其是SARS-CoV或SARS-CoV-2 S蛋白。SARS-CoV和SARS-CoV-2的刺突由S蛋白三聚体组成，S蛋白三聚体属于I类病毒融合糖蛋白的组，该组还包括HIV糖蛋白160(Env)、流感血凝素(HA)、副粘病毒F和埃博拉病毒糖蛋白。SARS-CoV和SARS-CoV-2S蛋白各自编码表面糖蛋白前体，并且预测氨基端和大部分蛋白质位于细胞表面或病毒颗粒的外侧。S蛋白包括位于N末端的信号肽、细胞外结构域、跨膜结构域和细胞内结构域。与其它冠状病毒相似，SARS-CoV和SARS-CoV-2的S蛋白可以被蛋白酶裂解成S1和S2亚基。特别是，SARS-CoV-2包含弗林蛋白酶(furin)样裂解位点，这是其它SARS样CoV所缺乏的。
在一些实施方案中，本文提供了重组S胞外域三聚体。在一些实施方案中，重组S胞外域三聚体包括来自α-冠状病毒(例如，NL63-CoV或229E-CoV)的重组S胞外域原聚体。在一些实施方案中，重组S胞外域三聚体包括来自β-冠状病毒(例如OC43-CoV、SARS-CoV、SARS-CoV-2、MERS-CoV、HKU1-CoV、WIV1-CoV、小鼠肝炎病毒(MHV)或HKU9-CoV)的S胞外域原聚体。
与其它包膜RNA病毒(例如HIV、RSV和流感)类似，包括SARS-CoV-2在内的冠状病毒在其病毒包膜上都有三聚体表面抗原，以便在感染期间通过特定的细胞表面受体进入不同的宿主细胞。与SARS-CoV-1一样，SARS-CoV-2利用其三聚体病毒表面抗原S蛋白与其特异性细胞表面受体ACE2结合后进入哺乳动物呼吸系统的宿主细胞。产生有效的重组亚单位疫苗的先决条件是能够产生类似天然的病毒S抗原，特别是保持其三聚体构象，以引起足够数量的抗体可以结合到病毒S蛋白的受体结合结构域(RBD)，从而阻止病毒与ACE2受体结合，从而消除病毒感染。
在一些实施方案中，包含冠状病毒抗原或免疫原(例如，SARS-CoV或SARS-CoV-2 S蛋白肽)的蛋白质能够产生免疫应答，例如，对SARS-CoV或SARS-CoV-2 S蛋白肽的免疫应答。在一些实施方案中，免疫应答抑制或减少受试者(例如患者)中冠状病毒的复制。在一些实施方案中，免疫应答包括产生一种或更多种中和抗体，例如多克隆 和/或单克隆抗体。在一些实施方案中，中和抗体抑制或减少受试者(例如患者)中冠状病毒的复制。在一些实施方案中，例如作为免疫原性组合物向受试者施用蛋白质不会由于先前暴露于冠状病毒而导致受试者中的抗体依赖性增强(ADE)。在一些方面，包含冠状病毒抗原和免疫原的蛋白质用作疫苗。
在一些实施方案中，冠状病毒抗原和免疫原(例如，SARS-CoV或SARS-CoV-2 S蛋白肽)与蛋白质或肽连接以形成融合蛋白或重组多肽。在一些实施方案中，与冠状病毒抗原或免疫原连接的蛋白质或肽能够与蛋白质或肽(例如，融合蛋白或重组多肽的蛋白质或肽)结合，例如共价或非共价连接。因此，在一些情况下，与冠状病毒抗原或免疫原连接的蛋白质或肽是多聚结构域。
在一些实施方案中，冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽)连接到胶原的前肽(例如在胶原的前肽的C-末端)，以形成融合肽或重组多肽。因此，在一些实施方案中，本文提供的蛋白质包括含有冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽或其片段或表位)的重组多肽，连接到胶原的C-末端前肽。在一些实施方案中，胶原的前肽来源于α1胶原的人类C-前肽，表达后能够自三聚体化。
在一些实施方案中，将冠状病毒抗原和免疫原(例如冠状病毒S蛋白肽)连接到胶原的前肽(例如在胶原的前肽的C-末端)有助于蛋白质产生免疫应答的能力。例如，重组蛋白的产生可保留冠状病毒S蛋白肽的三级和四级结构，这可能对冠状病毒S蛋白肽的天然构象的稳定性很重要，而蛋白质表面上抗原位点的可用性反过来能够引发免疫应答，例如中和抗体。此外，将冠状病毒S蛋白肽连接到能够自三聚体化的蛋白质或肽使得重组蛋白聚集，从而模仿病毒包膜上冠状病毒S蛋白肽的天然同源三聚体结构。
在一些实施方案中，将冠状病毒S蛋白肽连接到胶原的C-末端前肽导致自三聚体化的重组多肽。在一些实施方案中，本文提供的蛋白质包括多种自三聚体化的冠状病毒S蛋白肽和胶原重组多肽的前肽。在一些实施方案中，重组蛋白的三聚体性质有助于蛋白质的稳定性。在一些实施方案中，重组蛋白的三聚体性质有助于蛋白质产生免疫应答的能力。在一些实施方案中，重组蛋白的三聚体性质和/或多种自三聚体化的重组蛋白的宏观结构有助于蛋白质产生免疫应答的能力。
本文还提供了包含本文提供的蛋白质的免疫原性组合物、生产本文提供的蛋白质的方法、用本文提供的蛋白质和组合物治疗受试者的方法以及试剂盒。
本申请中提及的所有出版物，包括专利文件、科学文章和数据库，均出于所有目的以其全文形式被援引加入，其程度与各单独出版物被单独援引加入的程度相同。如果本文所述定义与被援引加入本文的专利、申请、公开申请和其它出版物中所述定义相反或不一致，则本文所述定义优先于被援引加入本文的定义。本文使用的章节标题仅出于组织的目的，不得解释为限制所描述的主题。
I.病毒抗原和免疫原
本文提供的蛋白质包括冠状病毒抗原和免疫原。本文所预期的冠状病毒抗原和免疫原能够促进或刺激细胞介导的应答和/或体液应答。在一些实施方案中，应答(例如，细胞介导的或体液应答)包括抗体(例如，中和抗体)的产生。在一些实施方案中，冠状病毒抗原或免疫原是冠状病毒刺突蛋白肽。
冠状病毒是正义单链RNA病毒家族，正义单链RNA病毒已知可引起严重呼吸道疾病。它们拥有已知RNA病毒中最大的基因组(26-32kb)，在系统发生上分为四个属(α、β、γ、δ)，其中β-冠状病毒进一步细分为四个谱系(A、B、C、D)。目前已知冠状病毒家族感染人类的病毒来自α-冠状病毒和β-冠状病毒属。此外，人们认为，γ-冠状病毒和δ-冠状病毒属将来可能会感染人类。β-冠状病毒的非限制性示例包括中东呼吸综合征冠状病毒(MERS-CoV)、严重急性呼吸综合征冠状病毒(SARS-CoV)、人类冠状病毒HKU1(HKU1-CoV)、人类冠状病毒OC43(OC43-CoV)、鼠肝炎病毒(MHV-CoV)、蝙蝠SARS样冠状病毒WIV1(WIV1-CoV)和人类冠状病毒HKU9(HKU9-CoV)。α-冠状病毒的非限制性示例包括人类冠状病毒229E(229E-CoV)、人类冠状病毒NL63(NL63-CoV)、猪流行性腹泻病毒(PEDV)和传染性胃肠炎冠状病毒(TGEV)。δ-冠状病毒的非限制性示例是猪δ-冠状病毒(SDCV)。
本文公开了严重急性呼吸综合征相关冠状病毒的列表：
■ 蝙蝠冠状病毒Cp/Yunnan2011
■ 蝙蝠冠状病毒RaTG13
■ 蝙蝠冠状病毒Rp/Shaanxi2011
○ 蝙蝠SARS冠状病毒HKU3
■ 蝙蝠SARS冠状病毒HKU3-1
■ 蝙蝠SARS冠状病毒HKU3-10
■ 蝙蝠SARS冠状病毒HKU3-11
■ 蝙蝠SARS冠状病毒HKU3-12
■ 蝙蝠SARS冠状病毒HKU3-13
■ 蝙蝠SARS冠状病毒HKU3-2
■ 蝙蝠SARS冠状病毒HKU3-3
■ 蝙蝠SARS冠状病毒HKU3-4
■ 蝙蝠SARS冠状病毒HKU3-5
■ 蝙蝠SARS冠状病毒HKU3-6
■ 蝙蝠SARS冠状病毒HKU3-7
■ 蝙蝠SARS冠状病毒HKU3-8
■ 蝙蝠SARS冠状病毒HKU3-9
■ 蝙蝠SARS冠状病毒Rp1
■ 蝙蝠SARS冠状病毒Rp2
○ 蝙蝠SARS CoV Rf1/2004
■ 蝙蝠CoV 273/2005
○ 蝙蝠SARS CoV Rm1/2004
■ 蝙蝠CoV 279/2005
■ 蝙蝠SARS CoV Rp3/2004
■ 蝙蝠SARS样冠状病毒
■ 蝙蝠SARS样冠状病毒Rs3367
■ 蝙蝠SARS样冠状病毒RsSHC014
■ 蝙蝠SARS样冠状病毒WIV1
■ 蝙蝠SARS样冠状病毒YNLF_31C
■ 蝙蝠SARS样冠状病毒YNLF_34C
■ BtRf-BetaCoV/HeB2013
■ BtRf-BetaCoV/JL2012
■ BtRf-BetaCoV/SX2013
■ BtRs-BetaCoV/GX2013
■ BtRs-BetaCoV/HuB2013
■ BtRs-BetaCoV/YN2013
■ Civet SARS CoV 007/2004
■ Civet SARS CoV SZ16/2003
■ Civet SARS CoV SZ3/2003
○ 重组SARSr-CoV
■ SARS冠状病毒ExoN1
■ SARS冠状病毒MA15
■ SARS冠状病毒MA15 ExoN1
■ SARS冠状病毒wtic-MB
■ 中菊头蝠冠状病毒
■ SARS蝙蝠冠状病毒
■ SARS冠状病毒A001
■ SARS冠状病毒A013
■ SARS冠状病毒A021
■ SARS冠状病毒A022
■ SARS冠状病毒A030
■ SARS冠状病毒A031
■ SARS冠状病毒AS
■ SARS冠状病毒B012
■ SARS冠状病毒B024
■ SARS冠状病毒B029
■ SARS冠状病毒B033
■ SARS冠状病毒B039
■ SARS冠状病毒B040
■ SARS冠状病毒BJ01
■ SARS冠状病毒BJ02
■ SARS冠状病毒BJ03
■ SARS冠状病毒BJ04
■ SARS冠状病毒BJ162
■ SARS冠状病毒BJ182-12
■ SARS冠状病毒BJ182-4
■ SARS冠状病毒BJ182-8
■ SARS冠状病毒BJ182a
■ SARS冠状病毒BJ182b
■ SARS冠状病毒BJ202
■ SARS冠状病毒BJ2232
■ SARS冠状病毒BJ302
■ SARS冠状病毒C013
■ SARS冠状病毒C014
■ SARS冠状病毒C017
■ SARS冠状病毒C018
■ SARS冠状病毒C019
■ SARS冠状病毒C025
■ SARS冠状病毒C028
■ SARS冠状病毒C029
■ SARS冠状病毒CDC#200301157
■ SARS冠状病毒civet010
■ SARS冠状病毒civet014
■ SARS冠状病毒civet019
■ SARS冠状病毒civet020
■ SARS冠状病毒CS21
■ SARS冠状病毒CS24
■ SARS冠状病毒CUHK-AG01
■ SARS冠状病毒CUHK-AG02
■ SARS冠状病毒CUHK-AG03
■ SARS冠状病毒CUHK-L2
■ SARS冠状病毒CUHK-Su10
■ SARS冠状病毒CUHK-W1
■ SARS冠状病毒cw037
■ SARS冠状病毒cw049
■ SARS冠状病毒ES191
■ SARS冠状病毒ES260
■ SARS冠状病毒FRA
○  SARS冠状病毒Frankfurt 1
■ SARS冠状病毒Frankfurt1-v01
■ SARS冠状病毒GD01
■ SARS冠状病毒GD03T0013
■ SARS冠状病毒GD322
■ SARS冠状病毒GD69
■ SARS冠状病毒GDH-BJH01
■ SARS冠状病毒GZ-A
■ SARS冠状病毒GZ-B
■ SARS冠状病毒GZ-C
■ SARS冠状病毒GZ-D
■ SARS冠状病毒GZ02
■ SARS冠状病毒GZ0401
■ SARS冠状病毒GZ0402
■ SARS冠状病毒GZ0403
■ SARS冠状病毒GZ43
■ SARS冠状病毒GZ50
■ SARS冠状病毒GZ60
■ SARS冠状病毒HB
■ SARS冠状病毒HC/SZ/61/03
■ SARS冠状病毒HGZ8L1-A
■ SARS冠状病毒HGZ8L1-B
■ SARS冠状病毒HGZ8L2
■ SARS冠状病毒HHS-2004
■ SARS冠状病毒HKU-36871
■ SARS冠状病毒HKU-39849
■ SARS冠状病毒HKU-65806
■ SARS冠状病毒HKU-66078
■ SARS冠状病毒Hong Kong/03/2003
■ SARS冠状病毒HPZ-2003
■ SARS冠状病毒HSR 1
■ SARS冠状病毒HSZ-A
■ SARS冠状病毒HSZ-Bb
■ SARS冠状病毒HSZ-Bc
■ SARS冠状病毒HSZ-Cb
■ SARS冠状病毒HSZ-Cc
■ SARS冠状病毒HSZ2-A
■ SARS冠状病毒HZS2-Bb
■ SARS冠状病毒HZS2-C
■ SARS冠状病毒HZS2-D
■ SARS冠状病毒HZS2-E
■ SARS冠状病毒HZS2-Fb
■ SARS冠状病毒HZS2-Fc
■ SARS冠状病毒JMD
■ SARS冠状病毒LC1
■ SARS冠状病毒LC2
■ SARS冠状病毒LC3
■ SARS冠状病毒LC4
■ SARS冠状病毒LC5
■ SARS冠状病毒LLJ-2004
■ SARS冠状病毒NS-1
■ SARS冠状病毒P2
■ SARS冠状病毒PC4-115
■ SARS冠状病毒PC4-127
■ SARS冠状病毒PC4-13
■ SARS冠状病毒PC4-136
■ SARS冠状病毒PC4-137
■ SARS冠状病毒PC4-145
■ SARS冠状病毒PC4-199
■ SARS冠状病毒PC4-205
■ SARS冠状病毒PC4-227
■ SARS冠状病毒PC4-241
■ SARS冠状病毒PUMC01
■ SARS冠状病毒PUMC02
■ SARS冠状病毒PUMC03
■ SARS冠状病毒Rs_672/2006
■ SARS冠状病毒sf098
■ SARS冠状病毒sf099
■ SARS冠状病毒ShanghaiQXC1
■ SARS冠状病毒ShanghaiQXC2
■ SARS冠状病毒Shanhgai LY
■ SARS冠状病毒Sin0409
■ SARS冠状病毒Sin2500
■ SARS冠状病毒Sin2677
■ SARS冠状病毒Sin2679
■ SARS冠状病毒Sin2748
■ SARS冠状病毒Sin2774
■ SARS冠状病毒Sin3408
■ SARS冠状病毒Sin3408L
■ SARS冠状病毒Sin3725V
■ SARS冠状病毒Sin3765V
■ SARS冠状病毒Sin842
■ SARS冠状病毒Sin845
■ SARS冠状病毒Sin846
■ SARS冠状病毒Sin847
■ SARS冠状病毒Sin848
■ SARS冠状病毒Sin849
■ SARS冠状病毒Sin850
■ SARS冠状病毒Sin852
■ SARS冠状病毒Sin_WNV
■ SARS冠状病毒Sino1-11
■ SARS冠状病毒Sino3-11
■ SARS冠状病毒SinP1
■ SARS冠状病毒SinP2
■ SARS冠状病毒SinP3
■ SARS冠状病毒SinP4
■ SARS冠状病毒SinP5
■ SARS冠状病毒SoD
■ SARS冠状病毒SZ1
■ SARS冠状病毒SZ13
■ SARS冠状病毒Taiwan
■ SARS冠状病毒Taiwan JC-2003
■ SARS冠状病毒Taiwan TC1
■ SARS冠状病毒Taiwan TC2
■ SARS冠状病毒Taiwan TC3
■ SARS冠状病毒TJ01
■ SARS冠状病毒TJF
■ SARS冠状病毒Tor2
○  SARS冠状病毒TW
■ SARS冠状病毒TW-GD1
■ SARS冠状病毒TW-GD2
■ SARS冠状病毒TW-GD3
■ SARS冠状病毒TW-GD4
■ SARS冠状病毒TW-GD5
■ SARS冠状病毒TW-HP1
■ SARS冠状病毒TW-HP2
■ SARS冠状病毒TW-HP3
■ SARS冠状病毒TW-HP4
■ SARS冠状病毒TW-JC2
■ SARS冠状病毒TW-KC1
■ SARS冠状病毒TW-KC3
■ SARS冠状病毒TW-PH1
■ SARS冠状病毒TW-PH2
■ SARS冠状病毒TW-YM1
■ SARS冠状病毒TW-YM2
■ SARS冠状病毒TW-YM3
■ SARS冠状病毒TW-YM4
■ SARS冠状病毒TW1
■ SARS冠状病毒TW10
■ SARS冠状病毒TW11
■ SARS冠状病毒TW2
■ SARS冠状病毒TW3
■ SARS冠状病毒TW4
■ SARS冠状病毒TW5
■ SARS冠状病毒TW6
■ SARS冠状病毒TW7
■ SARS冠状病毒TW8
■ SARS冠状病毒TW9
■ SARS冠状病毒TWC
■ SARS冠状病毒TWC2
■ SARS冠状病毒TWC3
■ SARS冠状病毒TWH
■ SARS冠状病毒TWJ
■ SARS冠状病毒TWK
■ SARS冠状病毒TWS
■ SARS冠状病毒TWY
■ SARS冠状病毒Urbani
■ SARS冠状病毒Vietnam
■ SARS冠状病毒WF188
■ SARS冠状病毒WH20
■ SARS冠状病毒WHU
■ SARS冠状病毒xw002
■ SARS冠状病毒ZJ01
■ SARS冠状病毒ZJ02
■ SARS冠状病毒ZJ0301
■ SARS冠状病毒ZMY 1
■ SARS冠状病毒ZS-A
■ SARS冠状病毒ZS-B
■ SARS冠状病毒ZS-C
■ SARS相关蝙蝠冠状病毒RsSHC014
■ SARS相关β-冠状病毒Rp3/2004
■ 严重急性呼吸综合征冠状病毒2
下表示出了示例性SARS CoV-2毒株。
Figure PCTCN2022140514-appb-000001
冠状病毒基因组被加帽(capped)、多聚腺苷酸化并被核衣壳蛋白覆盖。冠状病毒粒子包括包含称为刺突(S)蛋白的I型融合糖蛋白的病毒包膜。大多数冠状病毒具有共同的基因组结构，其中复制酶基因包含在基因组的5′部分，而结构基因包含在基因组的3′部分。
冠状病毒刺突(S)蛋白是最初作为前体蛋白合成的I类融合糖蛋白。单个前体S多肽形成同源三聚体，并在高尔基体内进行糖基化并加工以去除信号肽，并被细胞蛋白酶裂解以产生单独的S1和S2多肽链，在同源三聚体中仍然作为S1/S2原聚体结合，因此是异二聚体的三聚体。S1亚单位位于病毒膜的远端，包含受体结合结构域(RBD)，该受体结合结构域介导病毒与其宿主受体的附着(attachment)。S2亚单位包含融合蛋白机制(machinery)，例如融合肽、融合糖蛋白特有的两个七肽重复序列(HR1和HR2)和中心螺旋、跨膜结构域和细胞溶质尾部结构域(cytosolic tail domain)。
在一些情况下，冠状病毒抗原或免疫原是处于融合前构象的冠状病毒S蛋白肽，它是冠状病毒S蛋白的胞外域在分泌系统中加工成成熟冠状病毒S蛋白之后并且在触发导致冠状病毒S向融合后构象转变的融合事件之前采用的结构构象。Kirchdoerfer et  al.,“Pre-fusion structure of a human coronavirus spike protein,”Nature,531:118-121,2016中提供了处于融合前构象的示例性冠状病毒S蛋白(HKU1-CoV)的三维结构。
在一些情况下，冠状病毒抗原或免疫原包括一个或更多个与天然冠状病毒S序列相比的氨基酸取代、缺失或插入，与由相应的天然冠状病毒S序列形成的冠状病毒S胞外域三聚体相比，其提供了增加的融合前构象保留。通过一个或更多个氨基酸取代、缺失或插入的融合前构象的“稳定”可以是，例如，能量稳定(例如，降低融合前构象相对于融合后开放构象的能量)和/或动力学稳定(例如，降低从融合前构象到融合后构象的转化率)。此外，与相应的天然冠状病毒S序列相比，处于融合前构象的冠状病毒S胞外域三聚体的稳定可以包括增加对变性的抗性。本文提供了确定冠状病毒S胞外域三聚体是否处于融合前构象的方法，包括(但不限于)使用融合前构象特异性抗体的负染色电子显微镜和抗体结合分析。
在一些情况下，冠状病毒抗原或免疫原是S蛋白肽的片段。在一些实施方案中，抗原或免疫原是S蛋白肽的表位。表位包括分子上的抗原决定簇化学基团或肽序列，它们是抗原性的，从而引发特定的免疫应答，例如，表位是B细胞和/或T细胞作出应答的抗原区域。抗体可以结合到特定的抗原表位，例如冠状病毒S胞外域上的表位。表位既可以由连续氨基酸形成，也可以由通过蛋白质三级折叠而并列的非连续氨基酸形成。在一些实施方案中，冠状病毒表位是线性表位。在一些实施方案中，冠状病毒表位是构象表位。在一些实施方案中，冠状病毒表位是中和表位位点。在一些实施方案中，冠状病毒S蛋白肽或其片段的所有中和表位作为抗原或免疫原存在。
在一些情况下，例如，当病毒抗原或免疫原是S蛋白肽的片段时，仅存在S蛋白肽的单个亚单位，并且S蛋白肽的单个亚单位是三聚化的。在一些实施方案中，病毒抗原或免疫原包括信号肽、S1亚单位肽、S2亚单位肽或其任意组合。在一些实施方案中，病毒抗原或免疫原包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)肽或七肽重复序列2(HR2)肽或其任意组合。在一些实施方案中，病毒抗原或免疫原包括S蛋白的受体结合结构域(RBD)。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S1亚单位和S2亚单位。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S1亚单位，但不包括S2亚单位。在一些实施方案中，病毒抗原或免疫原包括S蛋白的S2亚单位，但不包括S1亚单位。在一些实施方案中，病毒抗原或免疫原不含跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
在一些实施方案中，病毒抗原或免疫原包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa或组织蛋白酶L。
在一些实施方案中，病毒抗原或免疫原不含蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa或组织蛋白酶L，或者包含不可被蛋白酶裂解的突变的蛋白酶裂解位点。
在一些实施方案中，病毒抗原或免疫原是包含至少一种SARS-CoV-2蛋白质或其片段的SARS-CoV-2抗原。在一些实施方案中，SARS-CoV-2抗原被SARS-CoV-2反 应性抗体和/或T细胞识别。在一些实施方案中，SARS-CoV-2抗原是灭活的全病毒。在一些实施方案中，SARS-CoV-2抗原包括病毒的亚单位。在一些实施方案中，SARS-CoV-2抗原包括SARS-CoV-2的结构蛋白或其片段。在一些实施方案中，SARS-CoV-2的结构蛋白包括由刺突(S)蛋白、膜(M)蛋白、核衣壳(N)蛋白和包膜(E)蛋白组成的组中的一种或更多种。在一些实施方案中，SARS-CoV-2抗原包括或进一步包括SARS-CoV-2的非结构蛋白或其片段。代表性SARS-CoV-2分离菌(isolate)(Wuhan-Hu-1)的核苷酸序列记载为GenBank号MN908947.3(Wu et al.,Nature,579:265-269,2020)。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:81-85中任意一个所述的序列。在一些实施方案中，病毒抗原或免疫原包括与如下所示的SEQ ID NO:85具有至少或约80％、85％、90％、92％、95％、97％、或99％的序列同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2RBD序列。
Figure PCTCN2022140514-appb-000002
Figure PCTCN2022140514-appb-000003
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与如下所示的SEQ ID NO:55(下划线序列表示Thr333-Gly526(粗体)的受体结合结构域(RBD)内的受体结合基序(RBM))具有至少或约80％、85％、90％、92％、95％或97％的序列同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括RBD三聚体，例如，与SEQ ID No:67-80中的任何一个连接的SARS-CoV-2RBD序列。
Figure PCTCN2022140514-appb-000004
在一些实施方案中，病毒抗原或免疫原包括原始Wuhan-Hu-1冠状病毒的刺突糖蛋白序列(例如NC_045512)。在一些实施方案中，病毒抗原或免疫原包括B.1.526谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括Cluster 5(ΔFVI-刺突)病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.7谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.207 谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.317谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.318谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.1谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.351谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.429/CAL.20C谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.525谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.526谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617.2谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.618谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.620谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.2谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括P.3谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.1.143谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括A.23.1谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括B.1.617谱系中的病毒的刺突糖蛋白序列。在一些实施方案中，病毒抗原或免疫原包括来源于选自由Wuhan-Hu-1、B.1.526谱系中的病毒、B.1.1.7谱系中的病毒、P.1谱系中的病毒、B.1.351谱系中的病毒、P.2谱系中的病毒、B.1.1.143谱系中的病毒、A.23.1谱系中的病毒和B.1.617谱系中的病毒组成的组的任何两种或更多种病毒(以任何合适的组合)的刺突糖蛋白的序列。
在一些实施方案中，病毒抗原或免疫原包括T95I，G142D，Δ143-145，和/或T478K，例如，如在B.1.617.2德尔塔和/或B.1.1.529奥密克戎变种(例如BA.4和BA.5)中。
在一些实施方案中，病毒抗原或免疫原包括E484K和/或S477N，例如，如在B.1.526变种中。在一些实施方案中，病毒抗原或免疫原包括Δ400-402(ΔFVI)，例如，如在Cluster 5(ΔFVI-刺突)变体中。在一些实施方案中，病毒抗原或免疫原包括Δ69-70(ΔHV)、Δ144(ΔY)、N501Y、A570D、D614G、P681H、T716I、S982A和/或D1118H，例如，如在B.1.1.7变种中。在一些实施方案中，病毒抗原或免疫原包括P681H，例如，如在B.1.1.207变种中。在一些实施方案中，病毒抗原或免疫原包括L18F、T20N、P26S、D138Y、R190S、K417T、E484K、N501Y、D614G、H655Y、T1027I和/或V1176F，例如，如在P.1变种中。在一些实施方案中，病毒抗原或免疫原包括E484K，例如，如在P.2变种中。在一些实施方案中，病毒抗原或免疫原包括E484K和/或N501Y，例如，如在P.3变种中。在一些实施方案中，病毒抗原或免疫原包括L18F、D80A、D215G，Δ242-244(ΔLAL)、R246I、K417N、E484K、N501Y、D614G和/或A701V，例如，如在B.1.351变种中。在一些实施方案中，病毒抗原或免疫原包括S13I、W152C和/或L452R， 例如，如在B.1.429/CAL.20C变种中。在一些实施方案中，病毒抗原或免疫原包括Δ69-70(ΔHV)、E484K和/或F888L，例如，如在B.1.525变种中。在一些实施方案中，病毒抗原或免疫原包括G142D、L452R、E484Q和/或P681R，例如，如在B.1.617变种中。在一些实施方案中，病毒抗原或免疫原包括G142D、L452R和/或P681R，例如，如在B.1.617.2变种中。在一些实施方案中，病毒抗原或免疫原包括E484K，例如，如在B.1.618变种中。在一些实施方案中，病毒抗原或免疫原可以包括融合多肽(原聚体)，所述融合多肽(原聚体)包括以任何适当组合的任何一种或更多种前述突变。在一些实施方案中，病毒抗原或免疫原可以包括三种融合多肽的三聚体，并且三种原聚体融合多肽中的任何一种可以包括以任何适当组合的任何一种或更多种前述突变。在一些实施方案中，形成三聚体的三种原聚体融合多肽中的两种或全部三种可以在每个原聚体中包括不同的突变和/或不同的突变组合。在一些实施方案中，病毒抗原或免疫原可以包括三聚体的混合物，并且每个三聚体可以包括不同的突变和/或不同的突变组合。
在一些实施方案中，病毒抗原或免疫原包括选自由SEQ ID NO:55的氨基酸位置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和1176处的突变(例如，取代、缺失和/或插入)组成的组中的任何一个、两个、三个、四个、五个或更多个突变。在一些实施方案中，病毒抗原或免疫原包括选自由在氨基酸位置440、452、477、484、501、614、655、681和701处的突变(例如，取代、缺失和/或插入)组成的组中的任何一个、两个、三个、四个、五个、六个、七个、八个或所有突变。在一些实施方案中，病毒抗原或免疫原包括包含来自不同病毒的序列的嵌合多肽，例如来自冠状病毒第一变种的一个或更多个突变以及来自不同于第一变种的冠状病毒第二变种的一个或更多个突变。在一些实施方案中，此类嵌合病毒抗原或免疫原(或嵌合病毒抗原或免疫原的组合)可用于引发针对冠状病毒的第一和第二变种的广泛免疫应答。
在一些实施方案中，病毒抗原或免疫原包括选自由T95I，G142D，Δ143-145，T478K，S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。在一些实施方案中，病毒抗原或免疫原包括选自由N440K、L452R、S477G、S477N、E484K、E484Q、N501Y、D614G、H655Y、P681H、P681R和A701V组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，SARS-CoV-2抗原包括缺乏信号肽的截短的S蛋白、全长S蛋白的跨膜和细胞质结构域。在一些实施方案中，SARS-CoV-2抗原是重组蛋白，而在 其它实施方案中，SARS-CoV-2抗原从病毒粒子中纯化。在一些优选的实施方案中，SARS-CoV-2抗原是分离抗原。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:27中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:27具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:27的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:28中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:28具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:28的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:29中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:29具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:29的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:30中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:30具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:30的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:31中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:31具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:31的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:32中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:32具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:32的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:33中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:33具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:33的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:34中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:34具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中 ，病毒抗原或免疫原包括SEQ ID NO:34的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:35中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:35具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:35的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:36中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:36具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:36的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:37中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:37具有至少或约80％、81％ 、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:37的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:38中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:38具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:38的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:39中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:39具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:39的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、 D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:40中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:40具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:40的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:41中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:41具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:41的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:42中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:42具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:42的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:43中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:43具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:43的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:44中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:44具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:44的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H 、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:45中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:45具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:45的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:46中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:46具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:46的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:47中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:47具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:47的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:48中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:48具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:48的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:49中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:49具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:49的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:50中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:50具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:50的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:51中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:51具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:51的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:52中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:52具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，病毒抗原或免疫原包括信号肽。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:53中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:53具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列 同一性的氨基酸序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:54中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:54具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:55具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:55的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:56中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:56具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在选自由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和1176(关于SEQ ID NO:55的氨基酸位置)组成的组中的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:56的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:57中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:57具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、 95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:57的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:58中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:58具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:58的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:59中所述的序列。在一些实施方案中，病毒抗原或免疫原包括在SEQ ID NO:59的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:60中所述的序列。在一些实施方案中，病毒抗原或免疫原包括在SEQ ID NO:60的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:61中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:61具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:61的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:62中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:62具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:62的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:63中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:63具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:63的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:64中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:64具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:64的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:65中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:65具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、 95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:65的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:81中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:81具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:81的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:82中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:82具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:82的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:83中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:83具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:83的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:84中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:84具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:84的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原包括SEQ ID NO:85中所述的序列。在一些实施方案中，病毒抗原或免疫原包括与SEQ ID NO:85具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:85的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，病毒抗原或免疫原不包括例如SEQ ID NO:66或其一部分的跨膜结构域。在一些实施方案中，冠状病毒抗原或免疫原包括可溶性S蛋白肽。在一些实施方案中，可溶性S蛋白肽缺少TM结构域肽和CP结构域肽。在一些实施方案中，可溶性S蛋白肽不结合到脂质双层，例如膜或病毒包膜。
在一些实施方案中，S蛋白肽由经密码子优化的核酸序列产生。在一些实施方案中，S蛋白肽由未经密码子优化的核酸序列产生。
在一些实施方案中，本文提及的病毒抗原或免疫原可包括包含所述病毒抗原或免疫原的重组多肽或融合肽。术语病毒抗原或免疫原可用于指包含冠状病毒抗原或免疫原的蛋白质。在某些情况下，冠状病毒抗原或免疫原是本文提供的冠状病毒蛋白肽。 本文提及的病毒抗原或免疫原可在初始剂，额外剂，和/或加强剂中使用。独立地，初始剂，额外剂，和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
II.重组肽和蛋白质
可以预期，本文提供的冠状病毒抗原和免疫原，例如S蛋白肽(参见第I节)，可以与其它蛋白质或肽结合，例如连接，以形成重组多肽，包括融合肽。在一些实施方案中，本文提供的单个重组多肽(例如单体)结合形成重组多肽的多聚体，例如三聚体。在一些实施方案中，单个重组多肽单体的结合通过共价相互作用发生。在一些实施方案中，单个重组多肽单体的结合通过非共价相互作用发生。在一些实施方案中，相互作用(例如共价或非共价)受连接冠状病毒抗原或免疫原(例如S蛋白肽)的蛋白质或肽影响。在一些实施方案中，例如，当冠状病毒抗原或免疫原是如本文所述的S蛋白肽时，可选择将其连接到的蛋白质或肽，从而保留糖蛋白的天然同源三聚体结构。这可以有利于激发对S蛋白肽的强而有效的免疫原性应答。例如，冠状病毒抗原或免疫原(例如S蛋白肽)的天然构象的保留和/或维持可改善或允许进入能够产生免疫应答的抗原位点。在一些情况下，包含本文所述S蛋白肽的重组多肽(例如，参见第I节)在本文中可选地被称为重组S抗原、重组S免疫原或重组S蛋白。
进一步预期，在一些情况下，重组多肽或其多聚重组多肽聚集或可聚集以形成包含多个冠状病毒抗原和/或免疫原重组多肽的蛋白质或复合物。这种蛋白质的形成可有利于产生对冠状病毒抗原和/或免疫原的强而有效的免疫原性应答。例如，形成包含多个重组多肽的蛋白质，从而形成多个冠状病毒抗原，例如冠状病毒S蛋白肽，可保留病毒抗原的三级和/或四级结构，允许针对天然结构引发(mounted)免疫应答。在一些情况下，聚集可赋予冠状病毒抗原或免疫原的结构稳定性，这反过来又可提供对能够促进免疫应答的潜在抗原位点的进入。
1.融合肽和重组多肽
在一些实施方案中，冠状病毒抗原或免疫原可在其C-末端(C-末端连接)连接至三聚化结构域，以促进单体的三聚化。在一些实施方案中，三聚化以三聚体构型稳定冠状病毒抗原或免疫原(例如冠状病毒S蛋白肽)的膜近端方面。
促进可溶性重组蛋白稳定三聚体的外源性多聚结构域的非限制性示例包括：GCN4亮氨酸拉链(Harbury et al.1993 Science 262:1401-1407)、肺表面活性物质蛋白的三聚基序(Hoppe et al.1994 FEBS Lett 344:191-195)、胶原(McAlinden et al.2003 J Biol Chem 278:42200-42207)和噬菌体T4 fibritin Foldon(Miroshnikov et al.1998 Protein Eng 11:329-414)，其中任何一种都可连接到本文所述的冠状病毒抗原或免疫原(例如，通过连 接到S肽的C末端)以促进重组病毒抗原或免疫原的三聚化。另请参见美国专利号7,268,116、7,666,837、7,691,815、10,618,949、10,906,944和10,960,070，以及US2020/0009244，这些专利出于所有目的以其全文形式被援引加入本文。
在一些实施方案中，一个或更多个肽接头(例如，甘氨酸-丝氨酸接头，例如，10个氨基酸的甘氨酸-丝氨酸肽接头)可用于将重组病毒抗原或免疫原连接到多聚化结构域。只要重组病毒抗原或免疫原三聚体保持所需性质(例如，融合前构象)，三聚体可包括本文提供的任何稳定突变(或其组合)。在一些实施方案中，重组多肽或融合蛋白包括SEQ ID NO:27-66和81-85中任一个所述的第一序列，所述第一序列连接到SEQ ID NO:67-80中任一个所述的第二序列，其中第一序列的C末端直接连接到第二序列的N末端。在一些实施方案中，重组多肽或融合蛋白包括SEQ ID NO:27-66和81-85中任一个所述的第一序列，所述第一序列连接到SEQ ID NO:67-80中任一个所述的第二序列，其中第一序列的C末端间接连接到第二序列的N末端，例如通过接头。在一些实施方案中，接头包括包含甘氨酸-X-Y重复序列的序列。
为了在治疗上可行，生物药物设计所需的三聚化蛋白部分应满足以下标准。理想情况下，它应该是天然分泌蛋白的一部分，如免疫球蛋白Fc，其还是在循环中丰富(无毒)、来源于人类(缺乏免疫原性)、相对稳定(半衰期长)并且能够有效自三聚化，所述自三聚化通过链间共价二硫键加强，因此三聚化的冠状病毒抗原或免疫原在结构上是稳定的。
胶原是纤维蛋白家族，纤维蛋白家族是细胞外基质的主要成分。它是哺乳动物中含量最丰富的蛋白质，占身体总蛋白质的近25％。胶原在骨骼、肌腱、皮肤、角膜、软骨、血管和牙齿的形成中起着重要的结构作用。胶原I、II、III、IV、V和XI的纤维状类型都合成为较大的三聚体前体，称为前胶原，其中由数百个“G-X-Y”重复序列(或甘氨酸重复序列)组成的中心不间断的三螺旋结构域两侧是非胶原结构域(NC)、N-前肽和C-前肽。C-末端和N-末端延伸都在前胶原分泌后进行蛋白质水解处理，这一事件触发成熟蛋白质组装成胶原原纤维，形成不溶性细胞基质。BMP-1是识别甘氨酸重复序列和胶原C-前结构域之间的连接处附近的前胶原的特定肽序列并负责去除前肽的蛋白酶。在正常成人的人类血清中发现I型胶原的脱落三聚体C-前肽的浓度在50-300ng/mL范围内，儿童具有更高的水平，这表明活跃的骨形成。在I型胶原的C-前肽的家族性高血清浓度人群中，其水平可高达1-6μg/mL而无明显异常，表明C-前肽是无毒的。对胶原的三聚体C-前肽的结构研究表明，它是三叶结构，所有三个亚单位在靠近其N-末端的连接区域聚集在一起，连接到前胶原分子的其余部分。这种突出蛋白质的几何形状在一个方向上的融合与Fc二聚体相似。
I型、Ⅳ型、V型和XI型胶原主要组装成由两个α-1链和一个α-2链(对于Ⅰ、Ⅳ、Ⅴ型)或三个不同的链(对于XI型)组成的异源三聚体形式，它们在序列上高度同源。Ⅱ型和Ⅲ型胶原均为α-1链的同源三聚体。对于I型胶原(最丰富的胶原形式)，也形成稳定的α(I)同源三聚体，并且在不同组织中以可变水平存在。当单独在细胞中过表达时， 这些胶原C-前肽链中的大多数可以自组装成同源三聚体。虽然N-前肽结构域是首先合成的，但三聚体胶原的分子组装始于C-前肽的配准结合。据信，C-前肽复合物通过链间二硫键的形成而稳定，但二硫键形成对于适当的链配准的必要性尚不清楚。甘氨酸的三螺旋重复，然后以类似拉链的方式从相关的C-末端传播到N-末端。通过使用重组DNA技术交换不同胶原链的C-前肽，这一知识创造了非天然类型的胶原基质。非胶原蛋白质(例如细胞因子和生长因子)也已融合到前胶原或成熟胶原的N-末端，以形成新的胶原基质，其目的是使非胶原蛋白质从细胞基质中缓慢释放。然而，在这两种情况下，在重组胶原纤维组装成不溶性细胞基质之前，C-前肽都需要被裂解。
尽管其它蛋白质三聚化结构域(例如来自细菌噬菌体T4的酵母原纤蛋白(fibritin)的GCN4和大肠杆菌的天冬氨酸转氨基甲酰酶的那些)先前已经描述过允许异源蛋白质的三聚化，但这些三聚体蛋白均不是人类性质的，它们也不是天然分泌的蛋白质。因此，任何三聚体融合蛋白都必须在细胞内合成，这不仅可能导致天然分泌蛋白(例如可溶性受体)的错误折叠，而且也使得从数千种其它细胞内蛋白中纯化此类融合蛋白变得困难。此外，在三聚体生物药物设计中使用此类非人类蛋白质三聚化结构域(例如来自酵母、细菌噬菌体和细菌)的致命缺点是其在人体内的假定免疫原性，使得此类融合蛋白在注射到人体后不久就失效。
因此，在本文所述的重组多肽中使用胶原具有许多优点，包括：(1)胶原是哺乳动物体内分泌的最丰富的蛋白质，占体内总蛋白质的近25％；(2)胶原的主要形式天然地以三聚体螺旋形式出现，其球状C-前肽负责三聚化的启动；(3)从成熟胶原蛋白质水解释放的胶原的三聚体C-前肽在哺乳动物血液中以亚微克/毫升的水平天然存在，并且不知道对身体有毒；(4)胶原的线性三螺旋区域可以作为接头包含在内，预测每个残基的间距为
Figure PCTCN2022140514-appb-000005
或排除为融合蛋白的一部分，以便可精确调整待三聚化的蛋白质和胶原的C-前肽之间的距离，以实现最佳生物活性；(5)将C-前肽从原胶原上裂解下来的BMP1的识别位点可以被突变或删除，以防止三聚体融合蛋白的破坏；(6)C-前肽结构域通过二硫键自三聚化，并且它提供通用的亲和标签，可用于纯化产生的任何分泌性融合蛋白。在一些实施方案中，与冠状病毒抗原和免疫原(例如S蛋白肽)结合的胶原的C-前肽能够重组产生可溶性的、共价连接的同源三聚体融合蛋白。
在一些实施方案中，冠状病毒抗原或免疫原连接到胶原的C-末端前肽以形成重组多肽。在一些实施方案中，重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，重组蛋白形成三聚体。在一些实施方案中，冠状病毒抗原或免疫原是如第I节中所述的S蛋白肽。
例如，可通过多肽间二硫键(可形成多肽间二硫键的Cys残基用粗体显示)产生融合多肽并使融合多肽三聚化，所述融合多肽包括SEQ ID NO:1中融合多肽的N-末端上的信号肽MFVFLVLLPLVSS(SEQ ID NO:54)。
Figure PCTCN2022140514-appb-000006
Figure PCTCN2022140514-appb-000007
在一些实施方案中，多肽间二硫键可包括Cys15-136、Cys131-166、Cys291-301、Cys379-432、Cys336-361、Cys391-525、Cys480-488、Cys538-590、Cys617-649、Cys662-671、Cys743-749、Cys738-760、Cys840-851、Cys1032-1043和Cys1082-1126中的一个或多个或全部，以任何合适的组合。在一些实施方案中，三聚体中的融合多肽可包括一个或多个糖基化位点(例如，Asn-连接的)，例如，在17、61、122、149、165、234、282、331、343、603、616、657、709、717、801、1074、1098和1134处的一个或多个或全部Asn残基处，以任何合适的组合。
在一些实施方案中，C-末端前肽为人胶原。在一些实施方案中，C-末端前肽包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、 proα2(XI)或proα3(XI)的C-末端多肽或其片段。在一些实施方案中，C-末端前肽是或包括proα1(I)的C-末端多肽。
在一些实施方案中，C-端前肽是或包括SEQ ID NO:67所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:67具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:68所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:68具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:69所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:69具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:70所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:70具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:71所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:71具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括SEQ ID NO:72所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:72具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:73所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:73具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:74所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:74具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:75所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:75具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:76所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:76具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括SEQ ID NO:77所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:77具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:78所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:78具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或是SEQ ID NO:79所述的氨基酸序列。在一些实施方案中，C-末端前肽显示与SEQ ID NO:79具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。在一些实施方案中，C-末端前肽是或包括SEQ ID NO:80所述的氨基酸序列。在一些实施方案中，C-末端前肽是与SEQ ID NO:80具有至少或约85％、90％、92％、95％或97％序列同一性的氨基酸序列。
在一些实施方案中，C-末端前肽是或包括胶原三聚结构域(例如，人α1(I)胶原的C-前肽)的氨基酸序列，在BMP-1位点中具有天冬氨酸(D)到天冬酰胺(N)的取代，例如，如SEQ ID NO:68中所示，其中RA D突变为RA N。在一些实施方案中，C-末端前肽是或包括胶原三聚化结构域(例如，人α1(I)胶原的C-前肽)的氨基酸序列，在BMP-1位点中具有丙氨酸(A)到天冬酰胺(N)的取代，例如，如SEQ ID NO:69中所示，其中R AD突变为R ND。在一些实施方案中，本文中的C-末端前肽可包括突变的BMP-1位点，例如，RSAN而不是DDAN。在一些实施方案中，本文中的C-末端前肽可包括BMP-1位点，例如，包含RAD(例如，RADDAN)序列而非RAN(例如，RANDAN)或RND(例如，RNDDAN)的序列(例如，SEQ ID NO:68或69)可用于本文中公开的融合多肽中。例如，SEQ ID NO:27(带下划线)或其片段、变体或突变体可直接或间接连接到SEQ ID NO:67(斜体)或其片段、变体或突变体，例如，以形成以下融合蛋白：
Figure PCTCN2022140514-appb-000008
在一些实施方案中，C-末端前肽是或包括氨基酸序列，所述氨基酸序列是SEQ ID NO:67-80中任何一个的片段。
在一些实施方案中，C-末端前肽可包括包含甘氨酸-X-Y重复序列的序列，其中X和Y独立地为任何氨基酸，或与其至少85％、90％、92％、95％或97％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。在一些实施方案中，X和Y独立地为脯氨酸或羟脯氨酸。
在S蛋白肽连接到C-末端前肽以形成重组多肽的一些情况下，重组多肽形成三聚体，从而形成S蛋白肽的同源三聚体。在一些实施方案中，三聚化重组多肽的S蛋白肽处于融合前构象。在一些实施方案中，三聚化重组多肽的S蛋白肽处于融合后构象。在一些实施方案中，确认状态允许进入S蛋白肽上的不同抗原位点。在一些实施方案中， 抗原位点是表位，例如线性表位或构象表位。具有所述三聚化重组多肽的优点是可以针对多种潜在和多样的抗原位点进行免疫应答。
在一些实施方案中，三聚化重组多肽包括包含相同的病毒抗原或免疫原的单个重组多肽。在一些实施方案中，三聚化重组多肽包括单个重组多肽，每个重组多肽包含不同于其它重组多肽的病毒抗原或免疫原。在一些实施方案中，三聚化重组多肽包括单个重组多肽，其中单个重组多肽中的一个包括不同于其它重组多肽的病毒抗原或免疫原。在一些实施方案中，三聚化重组多肽包括单个重组多肽，其中单个重组多肽中的两个包括相同的病毒抗原或免疫原，并且该病毒抗原或免疫原不同于包含在剩余重组多肽中的病毒抗原或免疫原。
在一些实施方案中，重组多肽包括第I节中所述的任何冠状病毒抗原或免疫原。在一些实施方案中，重组多肽包括如本文所述连接到如本文所述的胶原的C-末端前肽的第I节中所述的任何冠状病毒抗原或免疫原。
在一些实施方案中，免疫原包括重组SARS-CoV或SARS-CoV-2 S胞外域三聚体，例如SARS-CoV-2贝塔(B.1.351)冠状病毒S胞外域三聚体，其包括原聚体，所述原聚体在HR1结构域和中心螺旋结构域之间的边界处或附近包含一个或更多个(例如两个，例如两个连续的)脯氨酸取代，所述脯氨酸取代稳定了处于融合前构象的S胞外域三聚体。在一些这样的实施方案中，稳定了处于融合前构象的S胞外域的一个或更多个(例如两个，例如两个连续的)脯氨酸取代位于HR1的C-末端残基的15位氨基酸N-末端和中心螺旋的N-末端残基的5位氨基酸C-末端之间。
在一些实施方案中，一个或更多个(例如两个，例如两个连续的)脯氨酸取代稳定了处于融合前构象的冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体，例如SARS-CoV-2贝塔(B.1.351)冠状病毒S胞外域三聚体。在一些实施方案中，SARS-CoV-2 S蛋白肽包括986K/987V向986P/987P的突变。
在一些实施方案中，处于融合前构象的被稳定了的重组冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体包括单链S胞外域原聚体，其包含S1/S2和/或S2′蛋白酶裂解位点的突变，以防止这些位点处的蛋白酶裂解。在一些实施方案中，SARS-CoV-2 S蛋白肽包括685R向685A的突变。各种病毒的示例性蛋白酶裂解位点如下所示：
冠状病毒 S1/S2,位点1 S1/S2,位点2 S2’
2019-nCov SPRRAR↓SVAS IAY↓TMS SKPSKR↓SF
CoV-ZX21 TASILR↓STGQ IAY↓TMS SKPSKR↓SF
Bat-AC45 TASILR↓STGQ IAY↓TMS SKPSKR↓SF
SARS-Cov TVSLLR↓STGQ IAY↓TMS LKPTKR↓SF
BM48-31 SSTLVR↓SGGH LAY↓TMS LKPTKR↓SF
HKU9-1 ADSLPR↓LQLV VNY↓DPL GATTYR↓SA
MERS-CoV TPRSCR↓SVPG   GSRSAR↓SA
HKU1 SRRKRR↓SISA   CGSSSR↓SF
HCoV-OC43 KNRRSR↓GAITT   SKASSR↓SA
HCoV-229E IAVQPR↓NVSYD   SRVAGR↓SA
HCoV-NL63 IPVRPR↓NSSDN   SRIAGR↓SA
在一些实施方案中，通过一个或更多个脯氨酸取代(例如986P/987P取代)稳定了处于融合前构象的重组冠状病毒(例如，SARS-CoV或SARS-CoV-2)S胞外域三聚体的原聚体包括另外的修饰，用于处于融合前构象的稳定，例如在蛋白酶裂解位点处发生突变以阻止蛋白酶裂解。
参考作为SEQ ID NO:55提供的SARS-CoV-2 S蛋白质序列，胞外域包括信号肽(SP)，其在细胞加工过程中被去除；N-末端结构域(NTD)；受体结合结构域(RBD)；一个或更多个S1/S2裂解位点；融合肽(FP)；内部融合肽(IFP)；七肽重复序列1/2(HR1/2)和跨膜结构域(TM)。序列的示例性来源可在ncbi.nlm.nih.gov/nuccore/MN908947.3、ncbi.nlm.nih.gov/nuccore/MN908947、ncbi.nlm.nih.gov/nuccore/MN908947.2上找到。另外的序列可在ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/上找到，包括肺炎病毒分离株Wuhan-Hu-1的完整基因组。
在一些实施方案中，融合前稳定的SARS-CoV-2 S胞外域三聚体的原聚体，例如SARS-CoV-2贝塔(B.1.351)冠状病毒S胞外域三聚体的原聚体，可具有NTD、RBD、S1(在S1/S2位点1或S1/S2位点2处)、FP、IFP、HR1、HR2或胞外域的C-末端残基的C-末端残基(其可连接到例如三聚化结构域或跨膜结构域)。S蛋白的位置编号可能在SARS-CoV毒株之间变化，但序列可以对齐以确定相关的结构域和裂解位点。应了解，任何胞外域片段的N-末端和C-末端上的一些残基(例如多达10个)可在所公开的免疫原中被移除或修饰，而不会降低S胞外域三聚体作为免疫原的效用。
在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的NTD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的RBD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的NTD肽和RBD肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的S1结构域肽。在一些实施方案中，重组多肽是或包括SARS-CoV或SARS-CoV-2 S蛋白的S2结构域肽。
SEQ ID NO:26(1491aa)中提供了不含信号肽的示例性SARS-CoV-1 S重组多肽：
Figure PCTCN2022140514-appb-000009
上述SARS-CoV-1 S重组多肽可包括SEQ ID NO:53中提供的N-末端信号。
SEQ ID NO:1(1509aa)中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000010
Figure PCTCN2022140514-appb-000011
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54中提供的N-末端信号。
SEQ ID NO:86中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000012
Figure PCTCN2022140514-appb-000013
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:87中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000014
Figure PCTCN2022140514-appb-000015
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:88中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000016
Figure PCTCN2022140514-appb-000017
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:89中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000018
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
SEQ ID NO:90中提供了不含信号肽的示例性SARS-CoV-2 S重组多肽：
Figure PCTCN2022140514-appb-000019
Figure PCTCN2022140514-appb-000020
上述SARS-CoV-2 S重组多肽可包括SEQ ID NO:54(MFVFLVLLPLVSS)中提供的N-末端信号肽。
在一些实施方案中，重组多肽是或包括SEQ ID NO:1中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:1具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸 位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:1的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:2中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:2具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:2的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:3中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:3具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:3的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:4中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:4具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:4的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:5中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:5具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:5的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:6中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:6具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽 是或包括SEQ ID NO:6的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:7中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:7具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:7的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:8中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:8具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:8的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:9中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:9具有至少或约80％、81％、82％、83％ 、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:9的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:10中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:10具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:10的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:11中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:11具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:11的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、 Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:12中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:12具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:12的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:13中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:13具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:13的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:14中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:14具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例 如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:14的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:15中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:15具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:15的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:16中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:16具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:16的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、 R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:17中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:17具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:17的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:18中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:18具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:18的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:19中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:19具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:19的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:20中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:20具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:20的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:21中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:21具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:21的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:22中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:22具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:22的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:23中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:23具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:23的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:24中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:24具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组 多肽是或包括SEQ ID NO:24的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:25中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:25具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在一个或更多个氨基酸位置(例如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和/或1176(关于SEQ ID NO:55的氨基酸位置)或其任何组合)处包含取代、缺失和/或插入的序列。在一些实施方案中，重组多肽是或包括SEQ ID NO:25的变体，并且变体包括选自由S13I、L18F、T20N、P26S、Δ69-70(ΔHV)、D80A、D138Y、G142D、Δ144(ΔY)、W152C、R190S、D215G、Δ242-244(ΔLAL)、R246I、Δ400-402(ΔFVI)、K417T、K417N、N440K、L452R、S477N、S477G、E484K、E484Q、N501Y、A570D、D614G、H655Y、P681H、P681R、R682G、R683S、R685G、A701V、T716I、F888L、S982A、T1027I、D1118H和V1176F或其任何组合组成的组中的任何一个、两个、三个、四个、五个或更多个突变。
在一些实施方案中，重组多肽是或包括SEQ ID NO:26中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:26具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:26的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:85中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:26具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:85的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:86中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:86具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:86的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:87中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:87具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:87的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:88中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:88具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:88的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:89中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:89具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:89的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:90中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:90具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:90的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:91中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:91具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:91的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:92中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:92具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:92的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:93中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:93具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:93的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:94中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:94具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:94的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
在一些实施方案中，重组多肽是或包括SEQ ID NO:95中所述的序列。在一些实施方案中，重组多肽是或包括与SEQ ID NO:95具有至少或约80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％序列同一性的氨基酸序列，包括在SEQ ID NO:95的一个或更多个氨基酸位置处包含取代、缺失和/或插入的序列。
如上所述，在一些实施方案中，本文提供的重组多肽不仅结合形成三聚体，而且还可以聚集或被聚集以产生包含多种重组多肽的蛋白质。在一些实施方案中，形成的蛋白质具有宏观结构。在一些情况下，宏观结构可赋予冠状病毒抗原或免疫原重组多肽的结构稳定性，其反过来又可提供进入能够促进免疫应答的潜在抗原位点。
在一些实施方案中，三聚化重组多肽聚集形成包含多种三聚化重组多肽的蛋白质。在一些实施方案中，多种三聚化重组多肽形成具有宏观结构的蛋白质。
在一些实施方案中，本文所述的包含多种重组多肽的蛋白质是免疫原。在一些实施方案中，本文所述的包含多种重组多肽的蛋白质包含在纳米颗粒中。例如，在一些实施方案中，蛋白质直接连接到纳米颗粒，例如蛋白质纳米颗粒。在一些实施方案中，蛋白质间接连接到纳米颗粒。在一些实施方案中，本文所述的包含多种重组多肽的蛋白质包含在病毒样颗粒(VLP)中。
在一些实施方案中，本文提供了复合物，其包括选自由SEQ ID NO:1-26和86-96组成的组的重组多肽或其片段、变体或突变体，以任何合适的组合。在一些实施方案中，本文提供了复合物，其包括选自由SEQ ID NO:1-26和86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体。
在一些实施方案中，本文提供了包含多个重组多肽的融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括位于第一冠状病毒的非嵌合冠状病毒刺突蛋白中的冠状病毒刺突蛋白受体结合结构域(RBD)之前的冠状病毒刺突蛋白胞外域的一部分；b)第二区域，其包括不同于所述第一冠状病毒的第二冠状病毒的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，融合蛋白进一步包括第二区域和胶原的C-末端前肽之间的第三区域。在一些实施方案中，第三区域包括第三冠状病毒的S1结构域，其中第三冠状病毒与第一冠状病毒或第二冠状病毒相同或不同。在一些实施方案中，第三区域包括第四冠状病毒的S2结构域，其中第四冠状病毒与第一、第二或第四冠状病毒相同或不同。在一些实施方案中，第一区域包括第一冠状病毒的N-末端结构域(NTD)。在 一些实施方案中，第一区域包括与第二冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。在一些实施方案中，第二区域包括与第一冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。在一些实施方案中，第一和第二冠状病毒是相同冠状病毒的不同变种或毒株。在一些实施方案中，第一区域包括第一冠状病毒的NTD，第二区域包括第二冠状病毒的RBD，并且第一和第二冠状病毒是SARS-CoV-2的不同变种。在一些实施方案中，第一冠状病毒和第二冠状病毒独立选自由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和A.23.1谱系的SARS-CoV-2病毒组成的组。
在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括B.1.526谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括B.1.351谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括SARS-CoV-2贝塔的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括SARS-CoV-2贝塔或非SARS-CoV-2贝塔的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。在一些实施方案中，本文提供了包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：a)第一区域，其包括SARS-CoV-2贝塔或非SARS-CoV-2贝塔的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；b)第二区域，其包括SARS-CoV-2贝塔的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和c)胶原的C-末端前肽，其中重组多肽的C-末端前肽形成多肽间二硫键。
在一些实施方案中，本文提供了用于预防哺乳动物中冠状病毒感染的方法，包括用本文公开的有效量的融合蛋白来免疫哺乳动物。在一些实施方案中，在哺乳动物中产生针对第一和第二冠状病毒的中和抗体。在一些实施方案中，第一和第二冠状病毒是SARS-CoV-2的不同变种，并且在哺乳动物中产生的中和抗体中和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和A.23.1谱系的两种或更多种SARS-CoV-2病毒。在一些实施方案中，在哺乳动物中产生的中和抗体中和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和A.23.1谱系的三种或更多种SARS-CoV-2病毒。在一些实施方案中，方法包括用两剂或更多剂的融合蛋白免疫哺乳动物。在一些实施方案中，在一剂或更多剂的免疫原(包括包含来自相同SARS-CoV-2变种的NTD和RBD的刺突蛋白肽)之后，以再次加强剂施用融合蛋白。
在一些实施方案中，本文提供了从包括SARS-CoV-1和SARS-CoV-2的冠状病毒的刺突(S)糖蛋白衍生或修饰的工程化融合多肽。在一些实施方案中，与冠状病毒的野生型S蛋白序列相比，本文公开的融合多肽可稳定在融合前构象中。在一些实施方案 中，与三聚化结构域的融合可防止融合蛋白中的S蛋白肽形成直螺旋(例如，类似于膜融合过程中发生的情况)。例如，S-三聚体亚单位候选疫苗的冷冻EM结构表明，它主要采用紧密闭合的融合前状态，与全长野生型刺突蛋白不同，后者在去污剂存在下形成融合前和融合后状态。Ma et al.,J Virol(2021)doi:10.1128/JVI.00194-21。在一些实施方案中，融合蛋白可以包括改变的可溶性S序列，具有使S1/S2裂解位点失活的修饰；七肽重复序列1(HR1)区域和中心螺旋(CH)区域之间的转角区域中的突变，阻止HR1和CH形成直螺旋；和/或除稳定突变外，七肽重复序列2区域(HR2)的截断。在一些实施方案中，本文中的融合蛋白可以但不必须包括一个或更多个突变，例如K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G，它们被认为稳定处于融合前状态的刺突蛋白。在一些实施方案中，突变例如K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G对于稳定本文公开的包含蛋白质三聚体化 TM三聚体化结构域的融合多肽不是必需的。
在这些实施方案中的一些实施方案中，使得S1/S2裂解位点失活的突变可以包含用GSAG(SEQ ID NO:60)取代RRAR(SEQ ID NO:55中的682-685)，并且转角区域中的突变可以包含双突变K986G/V987G、K986P/V987P、K986G/V987P或K986P/V987G。在一些实施方案中，HR2的截断必须在野生型可溶性S序列的C-末端删除SEQ ID NO:65中所示的一个或更多个残基。在一些实施方案中，免疫原多肽可以进一步包括在与HR2相互作用的HR1区域中的(a)一个或更多个脯氨酸或甘氨酸取代，和/或(b)一个或更多个氨基酸残基的插入。在这些实施方案中的一些实施方案中，免疫原多肽可以具有选自A942P、S943P、A944P、A942G、S943G和A944G的一个或更多个取代。在这些实施方案中的一些实施方案中，插入可以是在A942-A944中的任何残基之间插入G或GS。
本文提及的重组多肽或其片段、变体或突变体，以任何合适的组合，其包括选自由SEQ ID NO:1-26和86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可在初始剂和/或加强剂中使用。独立地，初始剂和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
2.多核苷酸和载体
还提供了编码本文提供的冠状病毒抗原或免疫原和重组多肽的多核苷酸(核酸分子)，以及用于基因工程化细胞以表达这种冠状病毒抗原或免疫原和重组多肽的载体。
在一些实施方案中，提供了编码本文提供的重组多肽的多核苷酸。在一些方面，多核苷酸包含单个核酸序列，例如编码重组多肽的核酸序列。在其它实例中，多核 苷酸包含编码重组多肽特别是冠状病毒抗原或免疫原的第一核酸序列和编码包含不同冠状病毒抗原或免疫原的重组多肽的第二核酸序列。
在一些实施方案中，编码重组多肽的多核苷酸包含至少一个启动子，所述启动子可操作地连接以控制重组多肽的表达。在一些实施方案中，多核苷酸包含两个、三个或更多个启动子，所述启动子可操作地连接以控制重组多肽的表达。
在一些实施方案中，例如当多核苷酸包含两个或更多个核酸编码序列，例如编码包含不同冠状病毒抗原或免疫原的重组多肽的序列时，至少一个启动子可操作地连接以控制两个或更多个核酸序列的表达。在一些实施方案中，多核苷酸包含两个、三个或更多个启动子，所述启动子可操作地连接以控制重组多肽的表达。
在一些实施方案中，重组多肽的表达是可诱导的或有条件的。因此，在一些方面，编码重组多肽的多核苷酸包含条件启动子、增强子或反式激活子。在一些这样的方面中，条件启动子、增强子或反式激活子是诱导型启动子、增强子或反式激活子或者是可抑制启动子、增强子或反式激活子。例如，在一些实施方案中，诱导型或条件启动子可用于将重组多肽的表达限制在特定微环境中。在一些实施方案中，由诱导型或条件启动子驱动的表达通过暴露于外源剂(例如热、辐射或药物)来调节。
在多核苷酸包含一个以上编码重组多肽的核酸序列的情况下，多核苷酸可以进一步包括编码一个或更多个核酸序列之间的肽的核酸序列。在一些情况下，位于核酸序列之间的核酸编码肽，所述肽在翻译期间或之后分离核酸序列的翻译产物。在一些实施方案中，肽包含内部核糖体进入位点(IRES)、自裂解肽或引起核糖体跳跃(skipping)的肽，例如T2A肽。
在一些实施方案中，编码重组多肽的多核苷酸被引入含有培养细胞(例如宿主细胞)的组合物中，例如通过逆转录病毒转导、转染或转化。在一些实施方案中，这可允许重组多肽的表达(例如产生)。在一些实施方案中，对表达的重组多肽进行纯化。
在一些实施方案中，本文提供的多核苷酸(核酸分子)编码如本文所述的冠状病毒抗原或免疫原。在一些实施方案中，本文提供的多核苷酸(核酸分子)编码如本文所述的包含冠状病毒抗原或免疫原(例如冠状病毒S蛋白肽)的重组多肽。
还提供了含有如本文所述的核酸分子的载体或构建体。在一些实施方案中，载体或构建体包含一个或更多个启动子，所述启动子可操作地连接到编码重组多肽的核酸分子以驱动其表达。在一些实施方案中，启动子可操作地连接到一个或一个以上核酸分子，例如编码含有不同冠状病毒抗原或免疫原的重组多肽的核酸分子。
在一些实施方案中，载体是病毒载体。在一些实施方案中，病毒载体是逆转录病毒载体。在一些实施方案中，逆转录病毒载体是慢病毒载体。在一些实施方案中，逆转录病毒载体是γ-逆转录病毒载体。
在一些实施方案中，载体或构建体包括单个启动子，其驱动多核苷酸的一个或更多个核酸分子的表达。在一些实施方案中，此类启动子可以是多顺反子(双顺反子或三顺反子，参见例如美国专利号6,060,273)。例如，在一些实施方案中，转录单元可 以被工程化为包含IRES(内部核糖体进入位点)的双顺反子单元，其允许通过来自单个启动子的消息来共表达基因产物(例如，编码不同的重组多肽)。在一些实施方案中，本文提供的载体为双顺反子，允许载体包含并表达两个核酸序列。在一些实施方案中，本文提供的载体为三顺反子，允许载体包含并表达三个核酸序列。
在一些实施方案中，单个启动子指导RNA的表达，所述RNA在单个开放阅读框(ORF)中包含两个或三个基因(例如编码嵌合信号传导受体和编码重组受体)，这些基因通过编码自裂解肽的序列(例如2A序列)或蛋白酶识别位点(例如弗林蛋白酶)彼此分离。因此，ORF编码单个多肽，在翻译过程中(对于2A而言)或翻译后，该多肽被加工成单个蛋白质。在一些情况下，肽(例如T2A)可导致核糖体跳过(核糖体跳跃)2A元件C-末端肽键的合成，导致2A序列末端与下游下一个肽之间的分离(参见，例如，de Felipe.Genetic Vaccines and Ther.2:13(2004)和deFelipe et al.Traffic 5:616-626(2004))。许多2A元件是本领域已知的。可用于本文公开的方法和核酸中的2A序列的实例包括但不限于如美国专利公开号20070116690中所述的来自口蹄疫病毒(F2A)、马鼻炎A病毒(E2A)、Thosea asigna病毒(T2A)和猪捷申病毒(porcine teschovirus)-1(P2A)的2A序列。
在一些实施方案中，载体包含在病毒中。在一些实施方案中，病毒为假病毒。在一些实施方案中，病毒为病毒样颗粒。在一些实施方案中，载体包含在细胞中。在一些实施方案中，包含载体的病毒或细胞包含重组基因组。
III.免疫原性组合物和制剂
在一些实施方案中，本文提供了免疫原性组合物，其包括重组多肽的三聚体，所述重组多肽包括选自由SEQ ID NO:1-26和86-95组成的组的序列，或任何两种或更多种三聚体的组合。在一些实施方案中，本文提供了包含具有SEQ ID NO:1中所述的序列的重组多肽的三聚体的免疫原性组合物。本文提及的免疫原性组合物，其包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可在初始剂和/或加强剂中使用。独立地，初始剂和/或任意一剂或多剂加强剂可不使用或使用佐剂。如果使用佐剂，可选佐剂可包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
在一些实施方案中，单位剂量的免疫原性组合物可包括约10μg至约100μg SARS-CoV-2抗原、优选约25μg至约75μg SARS-CoV-2抗原、优选约40μg至约60μg SARS-CoV-2抗原或约50μg SARS-CoV-2抗原。在一些实施方案中，剂量包含3μg SARS-CoV-2抗原。在其它实施方案中，剂量包含9μg SARS-CoV-2抗原。在进一步的实施方案中，剂量包含30μg SARS-CoV-2抗原。
在一些情况下，可能需要将所公开的免疫原与诱导对其它试剂的保护性应答的其它制药产品(例如疫苗)联合。例如，包括如本文所述的重组冠状病毒S抗原(例如三聚体或蛋白质)的组合物可以与免疫实践咨询委员会(ACIP；cdc.gov/vaccines/acip/index.html)推荐的针对目标年龄组(例如，大约一到六个月大的婴儿)的其它疫苗(例如流感疫苗或水痘带状疱疹疫苗)同时(通常单独)或顺序施用。因此，包括本文所述的重组冠状病毒S抗原的公开的免疫原可以与针对例如乙型肝炎(HepB)、白喉、破伤风和百日咳(DTaP)、肺炎球菌(PCV)、b型流感嗜血杆菌(Hib)、脊髓灰质炎、流感和轮状病毒的疫苗同时或顺序施用。
多价或联合疫苗提供对多种病原体的抵御。在一些方面，多价疫苗可以抵御同一病原体的多种毒株。在一些方面，多价疫苗抵御多种病原体，例如联合疫苗Tdap，它抵御破伤风、百日咳和白喉的毒株。多价疫苗对于使对多种病原体或致病毒株赋予抵御所需的免疫接种的数量最小化是非常必要的，以降低管理成本，并提高覆盖率。例如，在为婴儿或儿童接种疫苗时，这可能特别有用。
在一些实施方案中，例如包含本文所述的免疫原性组合物的疫苗是多价疫苗。在一些实施方案中，用于并入多价疫苗组合物的抗原材料来源于冠状病毒毒株或类型，例如如本文所述(参见，例如，第一节)。用于并入多价疫苗组合物的抗原可以来源于冠状病毒的一个毒株或多个毒株(例如，两个和五个毒株之间)，以提供更广泛的保护范围。在一个实施方案中，用于并入多价疫苗组合物的抗原来源于冠状病毒的多个毒株。其它有用的抗原包括活的、减毒的和灭活的病毒，例如灭活的脊髓灰质炎病毒(Jiang et al.,J.Biol.Stand.,(1986)14:103-9)、甲型肝炎病毒的减毒株(Bradley et al.,J.Med.Virol.,(1984)14:373-86)、减毒的麻疹病毒(James et al.,N.Engl.J.Med.,(1995)332:1262-6)以及百日咳病毒的表位(例如，ACEL-IMUNE无细胞DTP、Wyeth-Lederle疫苗和Pediatrics)。
在一些方面，本文提供的疫苗是通用疫苗。在一些实施方案中，通用疫苗是针对同一病毒的多种毒株(例如冠状病毒的多种毒株)进行保护的疫苗。开发有效的通用冠状病毒疫苗将降低成本和劳动力，例如使用季节性疫苗配方，并允许更强有力的大流行预防。
在一些实施方案中，本文所述的免疫原可作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂。在一些实施方案中，本文所述的免疫原可作为季节性疫苗使用。在一些实施方案中，本文所述的免疫原可作为五年内，四年内，三年内，两年内，一年内，和/或六个月内的第一剂，第二剂，第三剂，第四剂，和/或更多剂使用。
在一些方面，通用疫苗是由来源于不同病毒株的多个表位组成的疫苗。在一些方面，通用疫苗由在不同病毒株中保守的单个表位组成。例如，通用疫苗可以基于S蛋白的相对保守的结构域。
还提供了包含所公开的免疫原(例如，所公开的重组冠状病毒S抗原或编码所公开的重组冠状病毒S抗原的原聚体的核酸分子)和药学上可接受的载体的免疫原性组合物。在一些实施方案中，免疫原性组合物包括本文提供的三聚化重组多肽和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的三聚化重组多肽和磷酸氢二钠，例如二水合磷酸氢二钠，磷酸二氢钠，例如一水合磷酸氢二钠，氯化钠，和吐温80。在一些实施方案中，1.0mL免疫原性组合物水溶液包括720μg本文提供的三聚化重组多肽和0.62mg二水合磷酸氢二钠，0.62mg一水合磷酸氢二钠，9.0mg氯化钠，和0.2mg吐温80。在一些实施方案中，免疫原性组合物包括包含本文提供的多个三聚化重组多肽的蛋白质以及可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的蛋白质纳米颗粒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的VLP和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的分离核酸和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的载体和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的病毒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的假病毒和可选的药学上可接受的载体。在一些实施方案中，免疫原性组合物包括本文提供的细胞和可选的药学上可接受的载体。在一些实施方案中，如本文所述的免疫原性组合物是疫苗。在一些实施方案中，疫苗是预防性疫苗。在一些实施方案中，疫苗是治疗性疫苗。在一些实施方案中，疫苗是预防性疫苗和治疗性疫苗。此类药物组合物可通过本领域普通技术人员已知的多种给药模式向受试者给药，例如，肌肉内、皮内、皮下、静脉内、动脉内、关节内、腹腔内、鼻内、舌下、扁桃体、口咽或其它肠胃外和粘膜途径。在几个实施方案中，包括一个或更多个所公开的免疫原的药物组合物是免疫原性组合物。用于制备可给药组合物的实际方法对于本领域技术人员来说是已知的或显而易见的，并且在例如Remingtons Pharmaceutical Sciences,19th Ed.,Mack Publishing Company,Easton,Pa.,1995这种出版物中进行了更详细的描述。
因此，本文所述的免疫原，例如重组冠状病毒S抗原，例如三聚体、蛋白质，可与药学上可接受的载体调配，以帮助保持生物活性，同时也促进在可接受温度范围内储存期间的稳定性增加。潜在的载体包括但不限于生理平衡培养基、磷酸盐缓冲盐溶液、水、乳液(例如，油/水或水/油乳液)、各种类型的润湿剂、防冷冻添加剂或稳定剂，例如蛋白质、肽或水解产物(例如，白蛋白、明胶)、糖(例如，蔗糖、乳糖、山梨醇)、氨基酸(例如，谷氨酸钠)或其它保护剂。所得的水溶液可按原样包装使用或冻干。冻干制剂在单剂量或多剂量给药前与无菌溶液混合。
配方组合物，尤其是液体制剂，可含有抑菌剂，以防止或最小化储存期间的降解，包括但不限于有效浓度(通常为1％w/v)的苯甲醇、苯酚、间甲酚、氯丁醇、对羟基苯甲酸甲酯和/或对羟基苯甲酸丙酯。一些患者可能禁用抑菌剂；因此，冻干制剂可在含有或不含有此类成分的溶液中重新组合。
本发明的免疫原性组合物可包含近似生理条件所需的药学上可接受的载体物质，例如pH调节剂和缓冲剂、张力调节剂、润湿剂等，例如醋酸钠、乳酸钠、氯化钠、氯化钾，氯化钙、山梨醇酐单月桂酸酯和油酸三乙醇胺。免疫原性组合物可以可选地包括佐剂以增强宿主的免疫应答。合适的佐剂例如为toll样受体(TLR)激动剂、明矾、AlPO 4、水凝胶(alhydrogel)、脂质-A及其衍生物或变体、油乳剂、皂甙、中性脂质体、含有疫苗和细胞因子的脂质体、非离子嵌段共聚物和趋化因子。本领域众所周知的许多其它合适的佐剂中的含有聚氧乙烯(polyoxyethylene,POE)和聚氧丙烯(polyxylpropylene,POP)的非离子嵌段聚合物可作为佐剂使用，例如POE-POP-POE嵌段共聚物、MPL TM(3-O-脱酰基单磷酸脂A；Corixa,Hamilton,Ind.)和IL-12(Genetics Institute,Cambridge,Mass.)(Newman et al.,1998,Critical Reviews in Therapeutic Drug Carrier Systems 15:89-142)。这些佐剂的优点在于，它们有助于以非特异性的方式刺激免疫系统，从而增强对药物产品的免疫应答。在一些实施方案中，本发明的免疫原性组合物可包括或与一种以上佐剂一起施用。在一些实施方案中，本发明的免疫原性组合物可包括或与两种佐剂一起施用。在一些实施方案中，本发明的免疫原性组合物可包括或与多种佐剂一起施用。例如，在一些情况下，例如包含本文提供的免疫原性组合物的疫苗可包括或与多种佐剂联合施用。
对于疫苗组合物，合适佐剂的实例包括，例如氢氧化铝、卵磷脂、Freund’s佐剂、MPL TM和IL-1，其一种或任意几种的组合可与包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体配合使用。在一些实施方案中，本文公开的疫苗组合物或纳米颗粒免疫原(例如，SARS-COV-2疫苗组合物)可配制为控释或缓释制剂。这可以通过含有缓释聚合物的组合物或通过微胶囊递送系统或生物粘附凝胶来实现。各种药物组合物可根据本领域众所周知的标准程序制备。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含水包油乳液形式的可代谢油(例如角鲨烯)和α-生育酚(例如DL-α-生育酚)，以及聚氧乙烯山梨醇酐单油酸酯(吐温-80)。在一些实施方案中，佐剂制剂可包括约2％至约10％角鲨烯、约2％至约10％α-生育酚(例如，D-α-生育酚)和约0.3％至约3％聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，佐剂制剂可包括约5％角鲨烯、约5％生育酚和约0.4％聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，佐剂制剂,即佐剂2，的剂量约10.68mg/剂角鲨烯、约11.15mg/剂生育酚和约4.8 6mg/剂聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含整剂量的佐剂2.在一些实施方案中，本发明的免疫原性组合物可包含半剂量的佐剂2.在一些实施方案中，本发明的免疫原性组合物可包含1/4剂量的佐剂2.在一些实施方案中，本发明的免疫原性组合物可包含1/3、1/5、1/6、1/7、1/8、1/9、1/10剂量的佐剂2.在一些实施方案中，本发明的免疫原性组合物可包含3个脱O-酰化单磷酸脂A(3D-MPL)和水包油乳液形式的佐剂，该佐剂包含可代谢油、 α-生育酚和聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含QS21(Quillaja saponaria Molina提取物：组分21)、3D-MPL和水包油乳液，其中水包油乳液包括可代谢油、α-生育酚和聚氧乙烯山梨醇酐单油酸酯。在一些实施方案中，本发明的免疫原性组合物可包含QS21、3D-MPL和水包油乳液，其中水包油乳液具有以下组合物：可代谢油，例如角鲨烯、α-生育酚和吐温-80。在一些实施方案中，本发明的免疫原性组合物可包含脂质体组合物形式的佐剂。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含可代谢的油(例如，角鲨烯)、聚氧乙烯山梨醇酐单油酸酯(吐温-80)和Span 85。在一些实施方案中，佐剂制剂可包括约5％(w/v)角鲨烯、约0.5％(w/v)聚氧乙烯山梨醇酐单油酸酯和约0.5％(w/v)Span 85。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含佐剂制剂，所述佐剂制剂包含皂树(Quillaja)皂苷、胆固醇和磷脂，例如，以纳米颗粒组合物的形式。在一些实施方案中，本发明的免疫原性组合物可包含单独纯化的Quillaja saponaria Molina部分的混合物，其中随后与胆固醇和磷脂调配。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含选自由MF59 TM、Matrix-A TM、Matrix-C TM、Matrix-M TM、AS01、AS02、AS03和AS04组成的组的佐剂。
在一些实施方案中，本发明的免疫原性组合物，包括选自由SEQ ID NO:86-95组成的组的重组多肽或其片段、变体或突变体的三聚体，其中重组多肽通过多肽间二硫键三聚化形成三聚体，可包含toll样受体9(TLR9)激动剂，其中TLR9激动剂为长度为8至35个核苷酸的寡核苷酸，包含未甲基化的胞苷-磷酸-鸟苷(也称为CpG或胞嘧啶-磷酸-鸟苷)基序，并且SARS-CoV-2抗原和寡核苷酸以有效刺激哺乳动物受试者(例如需要SARS-CoV-2抗原和寡核苷酸的人类受试者)中针对SARS-CoV-2抗原的免疫应答的量存在于免疫原性组合物中。TLR9(CD289)识别微生物DNA中发现的未甲基化的胞苷-磷酸-鸟苷(CpG)基序，可使用合成的含CpG的寡脱氧核苷酸(CpG-ODN)对其进行模仿。已知CpG-ODN可增强抗体产生并刺激T辅助细胞1(Th1)细胞应答(Coffman et al.,Immunity,33:492-503,2010)。最佳寡核苷酸TLR9激动剂通常包含以下通式的回文序列：5’-嘌呤-嘌呤-CG-嘧啶-嘧啶-3’或5’-嘌呤-嘌呤-CG-嘧啶-嘧啶-CG-3’。美国专利号6,589,940，其以其全文形式被援引加入本文。在一些实施方案中，CpG寡核苷酸是线性的。在其它实施方案中，CpG寡核苷酸是环状的或包括发夹环。CpG寡核苷酸可以是单链的或双链的。在一些实施方案中，CpG寡核苷酸可包含修饰。修饰包括但不限于3’OH 或5’OH基团的修饰、核苷酸碱基的修饰、糖组分的修饰和磷酸基团的修饰。只要经修饰的碱基通过Watson-Crick碱基配对对其天然补体保持相同的特异性(例如，回文部分仍然是自我互补的)，经修饰的碱基可以包括在CpG寡核苷酸的回文序列中。在一些实施方案中，CpG寡核苷酸包括非经典碱基。在一些实施方案中，CpG寡核苷酸包括经修饰的核苷。在一些实施方案中，经修饰的核苷选自由2’-脱氧-7-脱氮鸟苷(2’-deoxy-7-deazaguanosine)、2’-脱氧-6-硫代鸟苷、阿糖鸟苷(arabinoguanosine)、2’-脱氧-2’取代-阿糖鸟苷和2’-O-取代-阿糖鸟苷。CpG寡核苷酸可能包含磷酸基团的修饰。例如，除了磷酸二酯键之外，磷酸修饰包括但不限于膦酸甲酯、硫代磷酸酯、磷酰胺(桥接或非桥接)、磷酸三酯和二硫代磷酸酯，并且可以以任何组合使用。也可使用其它非磷酸键。在一些实施方案中，寡核苷酸仅包括硫代磷酸酯主链。在一些实施方案中，寡核苷酸仅包括磷酸二酯主链。在一些实施方案中，寡核苷酸包括磷酸主链中的磷酸键的组合，例如磷酸二酯键和硫代磷酸酯键的组合。具有硫代磷酸酯主链的寡核苷酸可以比具有磷酸二酯主链的寡核苷酸具有更高的免疫原性，并且在注射到宿主中后似乎更耐降解(Braun et al.,J Immunol,141:2084-2089,1988；和Latimer et al.,Mol Immunol,32:1057-1064,1995)。本发明的CpG寡核苷酸包括至少一个、两个或三个核苷酸间硫代磷酸酯键。在一些实施方案中，当多个CpG寡核苷酸分子存在于包含至少一种赋形剂的药物组合物中时，硫代磷酸酯键的两个立体异构体存在于多个CpG寡核苷酸分子中。在一些实施方案中，CpG寡核苷酸的所有核苷酸间键均为硫代磷酸酯键，或者换句话说，CpG寡核苷酸具有硫代磷酸酯主链。
在本发明中，任何合适的CpG寡脱氧核苷酸(ODN)或其组合可用作佐剂。例如，K型ODN(也称为B型)编码硫代磷酸酯主链上的多个CpG基序。K型ODN可基于以下序列TCCATGGA CGTTCCTGAG CGTT。与天然磷酸二酯核苷酸相比，硫代磷酸酯核苷酸的使用增强了对核酸酶消化的抵抗力，从而导致体内半衰期显著延长。K型ODN引发pDC分化并产生TNF-α，并引发B细胞增殖并分泌IgM。D型ODN(也称为A型)由混合的磷酸二酯/硫代磷酸酯主链构成，包含单个CpG基序，两侧为回文序列，并在3’和5’端具有多聚G尾巴(促进串联体形成的结构基序)。D型ODN可基于以下序列GGTGCAT CGATGCAGGGGGG。D型ODN引发pDC成熟并分泌IFN-α，但对B细胞无影响。C型ODN与K型相似，完全由硫代磷酸酯核苷酸组成，但与D型ODN相似，包含回文CpG基序。C型ODN可基于以下序列T CGT CGTT CGAA CGA CGTTGAT。这类ODN刺激B细胞分泌IL-6和pDC以产生IFN-α。P型ODN包含两个回文序列，使得它们能够形成更高的有序结构。P型ODN可基于以下序列T CGT CGA CGAT CGG CGCGCGC CG。与C型ODN相比，P型ODN激活B细胞和PDC，并诱导更大量IFN-α产生。在本段中，ODN序列中的粗体字表示自我互补回文，CpG基序加下划线。
示例性CpG ODN，例如CpG 7909(5′-TCGTCGTTTTGTCGTTTTGTCGTT-3’)和CpG 1018(5’-TGACTGTGAACGTTCGAGATGA-3’)，是已知的并且公开于美国专利号7,255,868、7,491,706、7,479,285、7,745,598、7,785,610、8,003,115、8,133,874 、8,114,418、8,222,398、8,333,980、8,597,665、8,669,237、9,028,845和10,052,378；和Bode et al.,“CpG DNA as a vaccine adjuvant”,Expert Rev Vaccines(2011),10(4):499-511中，所有这些均出于所有目的以其全文形式被援引加入本文。
一种或更多种佐剂可组合使用，包括但不限于明矾(铝盐)、水包油乳液、油包水乳液、脂质体和微粒，例如聚(丙交酯-共-乙交酯)微粒(Shah et al.,Methods Mol Biol,1494:1-14,2017)。在一些实施方案中，免疫原性组合物进一步包括吸附SARS-CoV-2抗原的铝盐佐剂。在一些实施方案中，铝盐佐剂包括由无定形羟基磷酸铝硫酸盐、氢氧化铝、磷酸铝和硫酸铝钾组成的组中的一种或更多种。在一些实施方案中，铝盐佐剂包括氢氧化铝和磷酸铝中的一种或两者。在一些实施方案中，铝盐佐剂包括氢氧化铝。在一些实施方案中，单位剂量的免疫原性组合物包括约0.25至约0.50mg Al 3+，或约0.35mg Al 3+。在一些实施方案中，免疫原性组合物进一步包括另外的佐剂。其它合适的佐剂包括但不限于水包角鲨烯乳液(例如，MF59或AS03)、TLR3激动剂(例如，聚IC或聚ICLC)、TLR4激动剂(例如，细菌脂多糖衍生物，如单磷酸脂A(MPL)，和/或皂甙，如Quil A或QS-21，如AS01或AS02)、TLR5激动剂(细菌鞭毛蛋白)和TLR7、TLR8和/或TLR9激动剂(咪唑喹啉衍生物，例如咪喹莫特和瑞喹莫特)(Coffman et al.,Immunity,33:492-503,2010)。在一些实施方案中，另外的佐剂包括MPL和明矾(例如AS04)。对于兽医用途和对于非人类动物抗体的生产，可以使用Freund’s佐剂的促有丝分裂成分(完整的和不完整的)。
在一些实施方案中，免疫原性组合物包括药学上可接受的赋形剂，包括例如溶剂、填充剂、缓冲剂、张力调节剂和防腐剂(Pramanick et al.,Pharma Times,45:65-77,2013)。在一些实施方案中，免疫原性组合物可包括赋形剂，其充当溶剂、填充剂、缓冲剂和张力调节剂中的一种或更多种(例如，盐水中的氯化钠可同时用作水性载体和张力调节剂)。
在一些实施方案中，免疫原性组合物包括作为溶剂的水性载体。合适的载体包括例如无菌水、盐水、磷酸盐缓冲盐水和Ringer’s溶液。在一些实施方案中，组合物是等渗的。
免疫原性组合物可包括缓冲剂。缓冲剂控制pH，以抑制在处理、储存和可选地重构期间活性剂的降解。合适的缓冲剂包括例如包含醋酸盐、柠檬酸盐、磷酸盐或硫酸盐的盐。其它合适的缓冲剂包括例如氨基酸，例如精氨酸、甘氨酸、组氨酸和赖氨酸。缓冲剂可以进一步包括盐酸或氢氧化钠。在一些实施方案中，缓冲剂将组合物的pH保持在6到9的范围内。在一些实施方案中，pH大于(下限)6、7或8。在一些实施方案中，pH小于(上限)9、8或7。也就是说，pH在约6到9的范围内，其中下限小于上限。
免疫原性组合物可包括张力调节剂。合适的张力调节剂包括例如葡萄糖、甘油(glycerol)、氯化钠、甘油(glycerin)和甘露醇。
免疫原性组合物可包括填充剂。当在给药前将药物组合物冻干时，填充剂特别有用。在一些实施方案中，填充剂是保护剂，有助于在冷冻或喷雾干燥期间和/或在储存期间稳定和防止活性剂降解。合适的填充剂为糖(单、双和多糖)，例如蔗糖、乳糖、海藻糖、甘露醇、山梨醇、葡萄糖和棉子糖。
免疫原性组合物可包括防腐剂。合适的防腐剂包括例如抗氧化剂和抗菌剂。然而，在优选实施方案中，免疫原性组合物在无菌条件下制备并在一次性容器中，因此不需要包含防腐剂。
在一些实施方案中，组合物可作为无菌组合物提供。药物组合物通常包含有效量的所公开的免疫原，并且可以通过常规技术制备。通常，选择每剂量免疫原性组合物中免疫原的量作为诱导免疫应答而无显著不良副作用的量。在一些实施方案中，组合物可以单位剂型提供，用于诱导受试者的免疫应答。单位剂型包含用于给受试者施用的合适的单个预选剂量，或两个或更多个预选单位剂量的合适标记的或测量的倍数，和/或用于施用单位剂量或其倍数的计量机构。在其它实施方案中，组合物进一步包括佐剂。
IV.诱导免疫应答的方法
在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，包括向受试者施用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和86-95组成的组的重组多肽，可选地该复合物作为作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括S蛋白或其抗原片段，并且方法包括向受试者施用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和86-95组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括选自由SEQ ID NO:27-66和81-85组成的组的序列，并且方法包括向受试者施用有效量的复合物，所述复合物包括选自由SEQ ID NO:1-26和86-95组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫 苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括冠状病毒的S蛋白或其抗原片段，并且可选地，表面抗原包括选自由SEQ ID NO:27-66和81-85组成的组的序列或其抗原片段，并且方法包括向受试者施用有效量的复合物，所述复合物包括重组多肽，所述重组多肽包括SEQ ID NO:86-95中任一项所述的序列，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。可选地，任意初始剂，额外剂，和/或加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
在一些实施方案中，本文提供了用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，其中表面抗原包括S蛋白或其抗原片段，并且方法包括向受试者施用有效量的复合物或复合物中任何两种或更多种的组合，所述复合物包括重组多肽，所述重组多肽包括选自由SEQ ID NO:1-26和86-95组成的组的序列，可选地该复合物或复合物组合作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。在一些实施方案中，方法包括向受试者施用有效量的复合物，所述复合物包括重组多肽，所述重组多肽包括SEQ ID NO:85、SEQ ID NO:86、SEQ ID NO:87和/或SEQ ID NO:88中所述的序列。可选地，任意初始剂，额外剂，和/或加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
可以向受试者施用所公开的免疫原(例如，重组冠状病毒S抗原，例如，本文所述的S-三聚体或S蛋白，编码所公开的重组冠状病毒S抗原的原聚体的核酸分子(例如RNA分子)或载体，或包含所公开的重组冠状病毒S抗原的蛋白质纳米颗粒或病毒样颗粒)，以诱导对受试者中相应冠状病毒S抗原的免疫应答。在特定的示例中，受试者是人。免疫应答可以是保护性免疫应答，例如抑制随后相应冠状病毒感染的应答。免疫应答的激发也可用于治疗或抑制与相应冠状病毒相关的感染和疾病。
可以选择受试者进行具有或处于感染冠状病毒的风险中的治疗，例如因为暴露于或可能暴露于冠状病毒。在施用所公开的免疫原后，可以监测受试者的感染或与冠状病毒相关的症状，或两者。
拟用本发明的疗法和方法进行治疗的典型受试者包括人类以及非人类灵长类和其它动物。为了鉴定根据本发明的方法进行预防或治疗的受试者，采用可接受的筛查方法来确定与目标或疑似疾病或病症相关的风险因素，或确定受试者中现有疾病或病症的状态。这些筛查方法包括，例如常规检查，以确定可能与目标或疑似疾病或病症相关的环境、家族、职业和其它此类风险因素，以及诊断方法，例如用于检测和/或表征冠状病毒感染的各种ELISA和其它免疫分析方法。这些和其它常规方法允许临床医生使用本发明的方法和药物组合物选择需要治疗的患者。根据这些方法和原则，可以根据本文的教导或其它常规方法施用组合物，作为独立的预防或治疗方案，或作为其它治疗的后续、辅助或协调治疗方案。
施用所公开的免疫原(例如，冠状病毒S抗原，例如，三聚体、蛋白质)可用于预防性或治疗性目的。当预防性地提供时，所公开的治疗剂在任何症状之前提供，例如在感染之前。所公开的治疗剂的预防性施用用于预防或改善任何后续感染。当治疗性地提供时，所公开的治疗剂在疾病或感染症状开始时或之后提供，例如，在与冠状病毒S抗原对应的冠状病毒感染症状发展之后，或在诊断冠状病毒感染之后。因此，可以在预期暴露于冠状病毒之前提供治疗剂，以便在暴露于或怀疑暴露于病毒之后，或在实际开始感染之后，减弱感染和/或相关疾病症状的预期严重程度、持续时间或程度。
本文所述的免疫原及其免疫原性组合物以有效诱导或增强受试者(优选为人类)针对冠状病毒S抗原的免疫应答的量提供给受试者。所公开的免疫原的实际剂量将根据例如受试者的疾病迹象和特定状态(例如，受试者的年龄、大小、健康状况、症状程度、易感因素等)、给药时间和途径、同时给药的其它药物或治疗等因素以及组合物在受试者中激发所需活性或生物反应的特定药理学而变化。剂量方案可以调整，以提供最佳的预防或治疗反应。
包括一种或更多种所公开的免疫原的免疫原性组合物可用于协调(或初免-加强)疫苗接种方案或组合制剂中。在某些实施方案中，新的组合免疫原性组合物和协调免疫方案使用单独的免疫原或制剂，每种免疫原或制剂均旨在引发抗病毒免疫应答，例如对冠状病毒S抗原的免疫应答。引发抗病毒免疫应答的单独免疫原性组合物可以在单个免疫步骤中组合在向受试者施用的多价免疫原性组合物中，或者它们可以在协调(或初免-加强)免疫方案中单独施用(在单价免疫原性组合物中)。
可以有几个加强剂，每个加强剂可以是不同的所公开的免疫原。在一些示例中，加强剂可以是与另一加强剂或初免剂相同的免疫原。初免剂和加强剂可作为单剂量或多剂量施用，例如，可在几天、几周或几个月内向受试者施用两剂、三剂、四剂、五剂、六剂或更多。也可以进行多次加强剂，例如一到五次(例如，1、2、3、4或5次加 强剂)或更多次。不同剂量可用于一系列连续免疫。例如，在初级免疫中使用相对较大的剂量，然后使用相对较小的剂量进行增强。
在一些实施方案中，可在初免剂后约两周、约三到八周或约四周或者初免后约几个月施用加强剂。在一些实施方案中，可在初免剂后约5、约6、约7、约8、约10、约12、约18、约24个月或者初免剂后或多或少时间施用加强剂。也可以在适当的时间点定期使用另外的加强剂，以增强受试者的“免疫记忆”。选择的疫苗参数的适合性，例如制剂、剂量、方案等，可通过从受试者中取出等份血清并在免疫程序期间测定抗体滴度来确定。此外，可监测受试者的临床状况以获得预期效果，例如预防感染或改善疾病状态(例如降低病毒载量)。如果此类监测表明疫苗接种是次优的，则可以使用额外剂量的免疫原性组合物使受试者得到加强，并且可以以预期增强免疫应答的方式改进疫苗接种参数。
在一些实施方案中，初免-加强方法可以包括针对受试者的DNA-引物和蛋白质-加强疫苗接种方案。该方法可以包括两次或更多次施用核酸分子或蛋白质。
对于蛋白质疗法，通常，每个人体剂量将包括1-1000μg的蛋白质，例如从约1μg到约100μg，例如从约1μg到约50μg，例如约1μg、约2μg、约5μg、约10μg、约15μg、约20μg、约25μg、约30μg、约40μg或约50μg。
免疫原性组合物中使用的量基于受试人群(例如，婴儿或老年人)进行选择。通过涉及观察受试者中的抗体滴度和其它反应的标准研究，可以确定特定成分的最佳用量。应理解，所公开的免疫原(例如所公开的重组冠状病毒S抗原，例如免疫原性组合物中的三聚体、蛋白质、病毒载体或核酸分子)的治疗有效量可以包括在通过单剂量给药诱导免疫应答时无效但在多剂量给药时有效的量，例如在初免-加强给药方案中。
在施用本发明所公开的免疫原后，受试者的免疫系统通常通过产生对免疫原中包含的冠状病毒S蛋白肽具有特异性的抗体来对免疫原性组合物作出反应。这种反应意味着向受试者递送了免疫有效剂量。
在一些实施方案中，将在评估有效剂量/免疫方案的情况下确定受试者的抗体反应。在大多数情况下，评估从受试者获得的血清或血浆中的抗体滴度就足够了。关于是否给予再次加强接种和/或改变给予个体的治疗剂的量的决定可以至少部分地基于抗体滴度水平。抗体滴度水平可以基于例如免疫结合分析，该免疫结合分析测量血清中结合抗原(包括例如重组冠状病毒S抗原，例如S-三聚体)的抗体的浓度。
无需完全消除、减少或防止冠状病毒感染，方法即可有效。例如，与在没有免疫原的情况下的冠状病毒感染相比，用一种或更多种所公开的免疫原激发对冠状病毒的免疫应答可以减少或抑制希望量的冠状病毒感染，例如至少10％、至少20％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少98％、甚至至少100％(消除或防止可检测到的感染的细胞)。在另外的示例中，可以通过所公开的方法减少或抑制冠状病毒复制。不需要完全消除冠状病毒复制，方法即可有效。例如，与在没有免疫应答的情况下的冠状病毒复制相比，用一种或更多种所公开的免疫原激发免疫应答可以减 少希望量的相应冠状病毒复制，例如，至少10％、至少20％、至少50％、至少60％、至少70％、至少80％、至少90％、至少95％、至少98％、甚至至少100％(消除或防止可检测到的冠状病毒复制)。
在一些实施方案中，在施用佐剂的同时向受试者施用所公开的免疫原。在其它实施方案中，在施用佐剂之后并在足够的时间内向受试者施用所公开的免疫原以诱导免疫应答。
核酸给药的一种方法是用质粒DNA直接免疫，例如用哺乳动物表达质粒。通过核酸构建体进行免疫是本领域众所周知的，并且披露于例如美国专利号5,643,578(其描述了通过引入编码所需抗原的DNA以引发细胞介导的或体液应答来免疫脊椎动物的方法)以及美国专利号5,593,972和5,817,637(其描述了将编码抗原的核酸序列可操作地连接到能够表达的调控序列)。美国专利号5,880,103描述了几种将编码免疫原性肽或其它抗原的核酸递送至生物体的方法。方法包括核酸(或它们自己的合成肽)和免疫刺激构建体或ISCOMS TM的脂质体递送，ISCOMS TM是胆固醇和Quil A TM(皂甙)混合后自发形成的30-40nm大小的带负电荷的笼状结构。使用ISCOMS TM作为抗原的递送载体已经在各种感染(包括弓形虫病和EB病毒诱导的肿瘤)的实验模型中产生了保护性免疫(Mowat and Donachie,Immunol.Today 12:383,1991)。已发现低至1μg的封装在ISCOMS TM中的抗原剂量产生I类介导的CTL反应(Takahashi et al.,Nature 344:873,1990)。
在一些实施方案中，质粒DNA疫苗用于在受试者中表达所公开的免疫原。例如，可将编码所公开的免疫原的核酸分子施用给受试者以诱导对冠状病毒S抗原的免疫应答。在一些实施方案中，核酸分子可以包括在用于DNA免疫的质粒载体上，例如pVRC8400载体(如Barouch et al.,J.Virol,79,8828-8834,2005中所述，其被援引加入本文)。
在使用核酸进行免疫的另一种方法中，所公开的重组冠状病毒S抗原(例如三聚体、蛋白质)可以通过减毒的病毒宿主或载体或细菌载体来表达。重组牛痘病毒、腺病毒相关病毒(AAV)、疱疹病毒、逆转录病毒、巨细胞病毒(cytogmeglo virus)或其它病毒载体可用于表达肽或蛋白质，从而引发CTL反应。例如，美国专利号4,722,848中描述了在免疫规划中有用的牛痘载体和方法。卡介苗(Bacillus Calmette Guerin)提供了另一种用于表达肽的载体(参见Stover,Nature 351:456-460,1991)。
在一个实施方案中，将编码所公开的重组冠状病毒S抗原的核酸直接引入细胞中。例如，可以通过标准方法将核酸装载到金微球上，并通过诸如Bio-Rad的HELIOS TM基因枪等设备将其引入皮肤中。核酸可以是“裸露的”，由强启动子控制下的质粒组成。通常，DNA被注射到肌肉中，但也可以直接注射到其它部位。注射剂量通常约为0.5μg/kg至约50mg/kg，并且典型地约为0.005mg/kg至约5mg/kg(参见，例如，美国专利号5,589,466)。
例如，可以通过标准方法将核酸装载到金微球上，并通过诸如Bio-Rad的HELIOS TM基因枪等设备将其引入皮肤中。核酸可以是“裸露的”，由强启动子控制下的质 粒组成。通常，DNA被注射到肌肉中，但也可以直接注射到其它部位。注射剂量通常约为0.5μg/kg至约50mg/kg，并且典型地约为0.005mg/kg至约5mg/kg(参见，例如，美国专利号5,589,466)。
在另一实施方案中，基于mRNA的免疫方案可用于将编码所公开的重组冠状病毒S抗原的核酸直接递送到细胞中。在一些实施方案中，基于mRNA的核酸疫苗可提供对前述方法的有效替代。mRNA疫苗排除了DNA整合到宿主基因组的安全性问题，并且可以直接在宿主细胞细胞质中翻译。此外，RNA的简单无细胞的体外合成避免了与病毒载体相关的制造并发症。可用于递送编码所公开的重组冠状病毒S抗原的核酸的两种基于RNA的疫苗的示例性形式包括常规非扩增mRNA免疫(参见，例如，Petsch et al.,“Protective efficacy of in vitro synthesized,specific mRNA vaccines against influenza A virus infection,”Nature biotechnology,30(12):1210-6,2012)和自扩增mRNA免疫(参见，例如，Geall et al.,“Nonviral delivery of self-amplifying RNA vaccines,”PNAS,109(36):14604-14609,2012；Magini et al.,“Self-Amplifying mRNA Vaccines Expressing Multiple Conserved Influenza Antigens Confer Protection against Homologous and Heterosubtypic Viral Challenge,”PLoS One,11(8):e0161193,2016；和Brito et al.,“Self-amplifying mRNA vaccines,”Adv Genet.,89:179-233,2015)。
在一些实施方案中，向受试者施用治疗有效量的一种或更多种所公开的免疫原可诱导受试者中的中和免疫应答。为评估中和活性，在受试者免疫后，可在适当的时间点从受试者收集血清，冷冻并储存以进行中和试验。中和活性的测定方法是本领域普通技术人员已知的，并在此进一步描述，包括但不限于蚀斑减少中和(PRNT)测定、微量中和测定、基于流式细胞术的测定、单周期感染测定。在一些实施方案中，可使用一组冠状病毒假病毒来测定血清中和活性。
在一些实施方案中，向受试者施用治疗有效量的一种或更多种所公开的免疫原可诱导受试者中的中和免疫应答。为评估中和活性，在受试者免疫后，可在适当的时间点从受试者收集血清，冷冻并储存以进行中和试验。中和活性的测定方法是本领域普通技术人员已知的，并在此进一步描述，包括但不限于蚀斑减少中和(PRNT)测定、微量中和测定、基于流式细胞术的测定、单周期感染测定。在一些实施方案中，可使用一组冠状病毒假病毒来测定血清中和活性。
在一些实施方案中，由本文公开的免疫原诱导的中和免疫应答产生针对冠状病毒(例如SARS-CoV-2)的中和抗体。在一些实施方案中，本文的中和抗体结合到冠状病毒(例如SARS-CoV-2)或其组分的细胞受体或共同受体。在一些实施方案中，病毒受体或共同受体是冠状病毒受体或共同受体，优选是肺炎病毒受体或共同受体，更优选是人冠状病毒受体，例如SARS-CoV-2受体或共同受体。在一些实施方案中，本文的中和抗体在体外、在原位和/或在体内调节、减少、拮抗、减轻、阻断、抑制、消除和/或干扰至少一种冠状病毒(例如SARS-CoV-2)活性或结合，或冠状病毒(例如SARS-CoV-2)受体活 性或结合，例如SARS-CoV-2释放、SARS-CoV-2受体信号传导、膜SARS-CoV-2裂解、SARS-CoV-2活性、SARS-CoV-2产生和/或合成。在一些实施方案中，本文公开的免疫原诱导针对SARS-CoV-2的中和抗体，其调节、减少、拮抗、减轻、阻断、抑制、消除和/或干扰SARS-CoV-2与SARS-CoV-2受体或共同受体的结合，例如血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)和/或肝脏/淋巴结-SIGN(L-SIGN)。
V.制品或试剂盒
还提供了含有所提供的重组多肽、蛋白质和免疫原性组合物的制品或试剂盒。制品可以包括容器以及容器上或与容器相关的标签或包装说明书。合适的容器包括，例如瓶子、小瓶、注射器、试管、IV溶液袋等。容器可以由各种材料(例如玻璃或塑料)形成。在一些实施方案中，容器具有无菌进入口。示例性容器包括静脉注射溶液袋、小瓶，包括带有可被注射用针刺穿的塞子的容器。制品或试剂盒可以进一步包括包装说明书，其指示组合物可用于治疗特定病症，例如本文所述的病症(例如冠状病毒感染)。或者，或者另外地，制品或试剂盒可以进一步包括包含药学上可接受的缓冲液的另一或相同容器。它可以进一步包括其它材料，例如其它缓冲液、稀释剂、过滤器、针头和/或注射器。
标签或包装说明书可以表明组合物用于治疗个体的冠状病毒感染。容器上或与容器相关的标签或包装说明书可以表明制剂的重组和/或使用说明。标签或包装说明书可以进一步表明制剂用于或目的在于皮下、静脉或其它给药方式，以治疗或预防个体中的冠状病毒感染。
在一些实施方案中，容器容纳组合物，该组合物是单独地或与另一种有效治疗、预防和/或诊断病症的组合物组合。制品或试剂盒可以包括(a)第一容器，具有包含在其中的组合物(即，第一药剂)，其中组合物包括免疫原性组合物或其蛋白质或重组多肽；和(b)第二容器，具有包含在其中的组合物(即，第二药剂)，其中组合物包括另一种药剂，例如佐剂或其它治疗剂，并且该物品或试剂盒进一步包括标签或包装说明书上用第二药剂以有效量治疗受试者的说明。
术语
除非另有定义，否则本文中使用的所有专用术语、符号和其它技术和科学术语(term)或术语(terminology)的含义旨在与所要求保护的主题所属领域的普通技术人员通常理解的含义相同。在一些情况下，为了清楚和/或便于参考，本文定义了具有通常理解的含义的术语，并且本文中包含的此类定义不一定表示与本领域中通常理解的内容的实质性差异。
术语“多肽”和“蛋白质”可互换地用于指代氨基酸残基的聚合物，且不限于最小长度。多肽(包括所提供的受体和其它多肽，例如接头或肽)可包括氨基酸残基， 包括天然和/或非天然氨基酸残基。术语还包括多肽的表达后修饰，例如糖基化、唾液酸化、乙酰化和磷酸化。在一些方面，只要蛋白质保持所需的活性，多肽可包含关于天然(native)或天然(natural)序列的修饰。这些修饰可以是故意的，如通过定点突变，也可以是偶然的，如通过产生蛋白质的宿主的突变或由于PCR扩增产生的错误。
如本文所使用的，“受试者”是哺乳动物，例如人类或其它动物，并且通常是人类。在一些实施方案中，向其施用一种或多种药剂、细胞、细胞群或组合物的受试者(例如患者)是哺乳动物，通常是灵长类动物，例如人类。在一些实施方案中，灵长类动物是猴子或猿。受试者可以是男性或女性，并且可以是任何合适的年龄段，包括婴儿、少年、青少年、成人和老年受试者。在一些实施方案中，受试者是非灵长类哺乳动物，例如啮齿动物。
如本文所使用的，“治疗(treatment)”(及其语法变体，如“治疗(treat)”或“治疗(treating)”)是指完全或部分改善或减少疾病、病症或紊乱，或与之相关的症状、不良反应或结果、或表型。治疗的理想效果包括但不限于预防疾病的发生或复发、缓解症状、减轻疾病的任何直接或间接病理后果、预防转移、降低疾病进展速度、改善或缓解疾病状态以及缓解或改善预后。该术语并不意味着完全治愈疾病或完全消除任何症状或对所有症状或结果的影响。
如本文所使用的，“延缓疾病的发展”是指延缓、阻碍、减缓、减慢、稳定、抑制和/或延迟疾病(例如癌症)的发展。取决于疾病史和/或接受治疗的个体，延缓的时间长度可能不同。在一些实施方案中，充分或显著的延缓实际上可以包含预防，因为个体不会发生疾病。例如，晚期癌症，如转移的发展，可能会延缓。
如本文所使用的，“预防”包括针对可能易患疾病但尚未被诊断患有疾病的受试者的疾病发生或复发提供预防。在一些实施方案中，所提供的细胞和组合物用于延缓疾病的发展或减缓疾病的进展。
如本文所使用的，“抑制”功能或活性是指当与除感兴趣的条件或参数之外的其它相同条件相比或者与另一条件相比时，降低功能或活性。例如，与没有细胞时的肿瘤生长速度相比，抑制肿瘤生长的细胞会降低肿瘤的生长速度。
在给药的情况下，药剂(例如药物制剂、细胞或组合物)的“有效量”是指达到预期效果(例如治疗或预防效果)所需的剂量/量和时间段的有效量。
药剂(例如药物制剂或细胞)的“治疗有效量”是指达到预期治疗效果(例如用于治疗疾病、病症或紊乱)和/或治疗的药代动力学或药效学效果所需的剂量和时间段的有效量。治疗有效量可根据例如受试者的疾病状态、年龄、性别和体重以及施用的细胞群等因素而变化。在一些实施方案中，所提供的方法涉及以有效量(例如，治疗有效量)施用细胞和/或组合物。
“预防有效量”是指达到预期预防效果所需的剂量和时间段的有效量。通常但不一定的是，因为预防剂量用于疾病之前或早期阶段的受试者，因此预防有效量将小于治疗有效量。在肿瘤负担较低的情况下，一些方面的预防有效量将高于治疗有效量。 疫苗或其它药剂的有效量，足以产生期望的反应，如减少或消除病症或疾病的病征或症状，如肺炎。例如，这可能是抑制病毒复制或可测定地改变病毒感染的外部症状所必需的量。一般来说，这一数量将足以可测定地抑制病毒(例如，SARS-CoV-2)复制或传染性。当向受试者给药时，通常使用的剂量将达到目标组织浓度，该浓度已被证明能够在体外实现病毒复制的抑制。在一些实施方案中，“有效量”是治疗(包括预防)任何病症或疾病的一个或更多个症状和/或潜在原因的量，例如用于治疗冠状病毒感染。在一些实施方案中，有效量是治疗有效量。在一些实施方案中，有效量是防止特定疾病或病症的一个或更多个病征或症状发展的量，例如与冠状病毒感染相关的一个或更多个病征或症状。
如本文所使用的，术语“抗原”或“免疫原”可互换地用于指代能够在受试者中诱导免疫应答的物质，通常为蛋白质。该术语还指免疫活性蛋白质，即一旦施用给受试者(直接或通过向受试者施用编码蛋白质的核苷酸序列或载体)，能够引发针对该蛋白质的体液和/或细胞类型的免疫应答。除非另有说明，否则术语“疫苗免疫原”可与“蛋白质抗原”或“免疫原多肽”互换使用。
术语“保守修饰的变体”适用于氨基酸和核酸序列。关于特定核酸序列，保守修饰的变体是指编码相同或基本相同的氨基酸序列的那些核酸，或在核酸不编码氨基酸序列的情况下，指基本相同的序列。由于遗传密码的简并性，大量功能相同的核酸编码任何给定的蛋白质。对于多肽序列，“保守修饰的变体”是指具有保守氨基酸取代的变体，氨基酸残基被具有有着类似电荷的侧链的其它氨基酸残基替换。本领域已经定义了具有有着类似电荷的侧链的氨基酸残基家族。这些家族包括具有碱性侧链(例如，赖氨酸、精氨酸、组氨酸)、酸性侧链(例如，天冬氨酸、谷氨酸)、不带电荷的极性侧链(例如，甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如，丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β支链(例如，苏氨酸、缬氨酸、异亮氨酸)和芳香族侧链(例如，酪氨酸、苯丙氨酸、色氨酸、组氨酸)。
表位是指抗原决定簇。这些是抗原分子上的特定化学基团或肽序列，因此它们引发特异性免疫应答，例如，表位是B细胞和/或T细胞应答的抗原区域。表位既可以由连续氨基酸形成，也可以由通过蛋白质的三级折叠而并列的非连续氨基酸形成。
除非另有说明，否则融合蛋白是重组蛋白，含有至少两种不相关蛋白质的氨基酸序列，这些蛋白质通过肽键连接在一起形成单个蛋白质。因此，它不包含天然存在的冠状病毒表面抗原，即本文所述的融合(F)蛋白。不相关的氨基酸序列可以直接相互连接，或者它们可以使用接头序列连接。如本文所使用的，如果蛋白质的氨基酸序列在其自然环境中(例如，在细胞内)通常不通过肽键连接在一起，则蛋白质是不相关的。例如，病毒抗原的氨基酸序列和胶原或前胶原的氨基酸序列通常不会通过肽键连接在一起。
免疫原是蛋白质或其一部分，能够在哺乳动物中诱导免疫应答，例如被病原体感染或有病原体感染风险的哺乳动物。免疫原的施用可导致针对目标病原体的保护性免疫和/或主动免疫。
免疫原性组合物是指包含免疫原性多肽的组合物，该免疫原性多肽诱导针对表达免疫原性多肽的病毒的可测量的CTL反应，或诱导针对免疫原性多肽的可测量的B细胞应答(例如产生抗体)。
两个或更多个核酸序列或两个或更多个氨基酸序列之间的序列同一性或相似性按照序列之间的同一性或相似性表示。序列一致性可以按照百分比一致性来衡量；百分比越高，序列越相同。当通过比较窗口或使用以下序列比较算法之一或通过手动比对和目视检查所测量的指定区域进行比较和比对以获得最大对应时，如果两个序列具有指定百分比的相同的氨基酸残基或核苷酸，则两个序列是“基本相同的”(即，在指定区域上，或者在未指定的情况下在整个序列上，60％同一性，优选65％、70％、75％、80％、85％、90％、95％或99％同一性)。可选地，同一性存在于长度至少约为50个核苷酸(或10个氨基酸)的区域上，或更优选地存在于长度为100至500或1000或更多个核苷酸(或20、50、200或更多个氨基酸)的区域上。
疫苗是指在受试者中引发预防性或治疗性免疫应答的药物组合物。在一些情况下，免疫应答是保护性免疫应答。通常，疫苗针对病原体的抗原(例如病毒病原体)或与病理状况相关的细胞成分诱发抗原特异性的免疫应答。疫苗可包括多核苷酸(例如编码所公开的抗原的核酸)、肽或多肽(例如所公开的抗原)、病毒、细胞或一种或更多种细胞成分。在一些实施方案中，疫苗或疫苗免疫原或疫苗组合物从融合构建体表达并自组装成在表面上显示免疫原多肽或蛋白质的纳米颗粒。
类病毒颗粒(VLP)是指不可复制的病毒外壳，来源于几种病毒中的任何一种。VLP通常由一种或更多种病毒蛋白质组成，例如但不限于称为衣壳、包被、外壳、表面和/或包膜蛋白的蛋白质，或来源于这些蛋白质的颗粒形成多肽。在适当的表达系统中重组表达蛋白质后，VLP可以自发形成。用于生产特定VLP的方法是本领域已知的。在病毒蛋白质的重组表达之后的VLP的存在可以使用本领域已知的常规技术进行检测，例如通过电子显微镜、生物物理表征等。参见例如Baker et al.(1991)Biophys.J.60:1445-1456；和Hagensee et al.(1994)J.Virol.68:4503-4505。例如，VLP可以通过密度梯度离心进行分离和/或通过特征密度带进行鉴定。或者，低温电子显微镜可以在讨论中的VLP制备的玻璃化含水样品上进行，并在适当的曝光条件下记录图像。
本文使用的术语“大约/约”是指本技术领域的技术人员容易知道的各个值的通常误差范围。本文中对“大约/约”值或参数的提及包括(并描述)针对该值或参数本身的实施方案。
如本文所使用的，单数形式“一个/一种(a)”、“一个/一种(an)”和“所述/该(the)”包括复数指代，除非上下文另有明确规定。例如，“一个/一种(a)”或“一个/一种(an)”表示“至少一个/一种”或“一个或更多个/一种或更多种”。
在本文中，所要求保护的主题的各个方面以范围格式呈现。应当理解，范围格式的描述仅仅是为了方便和简洁，不应当被解释为对所要求保护的主题的范围的不灵活的限制。因此，应当认为范围的描述已经具体地公开了所有可能的子范围以及该范围 内的各个数值。例如，在提供值的范围的情况下，应当理解，在该范围的上限和下限之间与该范围内的任何其它陈述或介入值的每个介入值都包含在所要求保护的主题内。这些较小范围的上限和下限可以独立地包括在较小范围内，并且也包括在所要求保护的主题内，受制于所述范围内的任何明确排除的限制。如果所述范围包括一个或两个限制，则不包括其中一个或两个那些包括在内的限制的范围也包括在所要求保护的主题内。这适用于任何范围的宽度。
如本文所使用的，组合物指两种或更多种产品、物质或化合物(包括细胞)的任何混合物。它可以是溶液、悬浮液、液体、粉末、糊状物、水性的、非水性的或其任何组合。
本文使用的术语“载体”是指能够传播与其连接的另一种核酸的核酸分子。该术语包括作为自我复制核酸结构的载体以及并入已引入载体的宿主细胞基因组中的载体。某些载体能够引导与它们可操作地连接的核酸的表达。此类载体在本文中称为“表达载体”。
示例性实施方案
实施方案1.一种包含多个重组多肽的蛋白质，每个重组多肽包含连接到胶原的C-末端前肽的冠状病毒SARS-CoV-2贝塔(B.1.351)表面抗原，其中重组多肽的C-末端前肽形成多肽间二硫键。
实施方案2.根据实施方案1的蛋白质，其中冠状病毒感染为SARS-冠状病毒2(SARS-CoV-2)感染。
实施方案3.根据实施方案1或2的蛋白质，其中表面抗原包括冠状病毒刺突(S)蛋白或其片段或表位，其中表位可选地为线性表位或构象表位，并且其中蛋白质包括三个重组多肽。
实施方案4.根据实施方案3的蛋白质，其中表面抗原包括信号肽、S1亚单位肽、S2亚单位肽或其任何组合。
实施方案5.根据实施方案3的蛋白质，其中表面抗原包括信号肽、受体结合结构域(RBD)肽、受体结合基序(RBM)肽、融合肽(FP)、七肽重复序列1(HR1)肽或七肽重复序列2(HR2)肽或其任何组合。
实施方案6.根据实施方案3-5中任一项的蛋白质，其中表面抗原包括S蛋白的受体结合结构域(RBD)。
实施方案7.根据实施方案3-6中任一项的蛋白质，其中表面抗原包括S蛋白的S1亚单位和S2亚单位。
实施方案8.根据实施方案3-7中任一项的蛋白质，其中表面抗原不包括跨膜(TM)结构域肽和/或细胞质(CP)结构域肽。
实施方案9.根据实施方案3-8中任一项的蛋白质，其中表面抗原包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
实施方案10.根据实施方案3-8中任一项的蛋白质，其中表面抗原不包括蛋白酶裂解位点，其中蛋白酶可选地为弗林蛋白酶(furin)、胰蛋白酶、因子Xa、凝血酶或组织蛋白酶L。
实施方案11.根据实施方案1-10中任一项的蛋白质，其中表面抗原是可溶的或不直接结合到脂质双层，例如膜或病毒包膜。
实施方案12.根据实施方案1-11中任一项的蛋白质，其中在蛋白质的重组多肽中表面抗原相同或不同。
实施方案13.根据实施方案1-12中任一项的蛋白质，其中表面抗原直接融合到C-末端前肽，或通过接头(例如包含甘氨酸-X-Y重复序列的接头)连接到C-末端前肽，其中X和Y独立地是任何氨基酸，并且可选地是脯氨酸或羟脯氨酸。
实施方案14.根据实施方案1-13中任一项的蛋白质，其是可溶的或不直接结合到脂质双层，例如膜或病毒包膜。
实施方案15.根据实施方案1-14中任一项的蛋白质，其中蛋白质能够结合到受试者的细胞表面受体，可选地其中受试者是哺乳动物，例如灵长类动物，例如人类。
实施方案16.根据实施方案15的蛋白质，其中细胞表面受体为血管紧张素转换酶2(ACE2)、二肽基肽酶4(DPP4)、树突状细胞特异性细胞间粘附分子-3-抓取非整合素(DC-SIGN)或肝脏/淋巴结-SIGN(L-SIGN)。
实施方案17.根据实施方案1-16中任一项的蛋白质，其中C-末端前肽是人胶原。
实施方案18.根据实施方案1-17中任一项的蛋白质，其中C-末端前肽包括proα1(I)、proα1(II)、proα1(III)、proα1(V)、proα1(XI)、proα2(I)、proα2(V)、proα2(XI)或proα3(XI)的C-末端多肽或其片段。
实施方案19.根据实施方案1-18中任一项的蛋白质，其中在重组多肽中C-末端前肽相同或不同。
实施方案20.根据实施方案1-19中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:67-80中的任一个或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案21.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:67或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案22.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:68或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案23.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:69或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案24.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:70或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案25.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:71或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案26.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:72或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案27.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:73或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案28.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:74或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案29.根据实施方案1-20中任一项的蛋白质，其中C-末端前肽包括SEQ ID NO:75或SEQ ID NO:76或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案30.根据实施方案1-29中任一项的蛋白质，其中C-端前肽包括包含连接到SEQ ID NO:67-80中任一种的N-末端的甘氨酸-X-Y重复序列的序列，其中X和Y独立地是任何氨基酸，并且可选地是脯氨酸或羟脯氨酸，或与其至少90％相同的氨基酸序列，其能够形成多肽间二硫键并使重组多肽三聚化。
实施方案31.根据实施方案1-30中任一项的蛋白质，其中每个重组多肽中的表面抗原为融合前构象或融合后构象。
实施方案32.根据实施方案1-31中任一项的蛋白质，其中每个重组多肽中的表面抗原包括SEQ ID NO:27-66和81-85中的任一个或与其至少80％相同的氨基酸序列。
实施方案33.根据实施方案1-32中任一项的蛋白质，其中重组多肽包括SEQ ID NO:1-26和86-95中的任一个或与其至少80％相同的氨基酸序列。
实施方案34.一种包含实施方案1-33中任一项的蛋白质的免疫原，可选地该免疫原作为疫苗初始剂和/或作为加强剂使用，例如第二剂和/或第三剂加强针剂。
实施方案35.一种包含实施方案1-33中任一项的蛋白质的蛋白质纳米颗粒，所述蛋白质直接或间接连接到纳米颗粒，可选地该纳米颗粒作为疫苗初始剂和/或作为加强剂使用，例如第二剂和/或第三剂加强针剂。
实施方案36.一种包含实施方案1-33中任一项的蛋白质的病毒样颗粒(VLP)，可选地该病毒样颗粒作为疫苗初始剂和/或作为加强剂使用，例如第二剂和/或第三剂加强针剂。
实施方案37.一种分离核酸，其编码根据实施方案1-33中任一项的蛋白质的重组多肽中的一个、两个、三个或更多个。
实施方案38.根据实施方案37的分离核酸，其中编码表面抗原的多肽在框架内融合到编码胶原的C-末端前肽的多肽。
实施方案39.根据实施方案37或38的分离核酸，其可操作地连接到启动子。
实施方案40.根据实施方案37-39中任一项的分离核酸，其是DNA分子。
实施方案41.根据实施方案37-39中任一项的分离核酸，其为RNA分子，可选地为mRNA分子，例如核苷修饰的mRNA、非扩增mRNA、自扩增mRNA或反式扩增mRNA。
实施方案42.一种包含根据实施方案37-41中任一项的分离核酸的载体。
实施方案43.根据实施方案42的载体，其为病毒载体。
实施方案44.一种包含根据实施方案42或43的载体的病毒、假病毒或细胞，可选地其中病毒或细胞具有重组基因组。
实施方案45.一种免疫原性组合物，其包含根据实施方案1-44中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒或细胞以及药学上可接受的载体。
实施方案46.一种疫苗，其包含根据实施方案45的免疫原性组合物和可选的佐剂，其中疫苗可选地为亚单位疫苗，和/或可选地其中疫苗为预防性和/或治疗性疫苗，可选地该疫苗作为初始剂和/或作为加强剂使用，例如第二剂和/或第三剂加强针剂。
实施方案47.根据实施方案46的疫苗，其中疫苗包括多种不同佐剂。
实施方案48.一种产生蛋白质的方法，包括：在宿主细胞中表达根据实施方案37-43中任一项的分离核酸或载体，以产生根据实施方案1-33中任一项的蛋白质；和纯化蛋白质。
实施方案49.通过实施方案48的方法产生的蛋白质。
实施方案50.一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，包括向受试者施用有效量的实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，以产生免疫应答。
实施方案51.根据实施方案50的方法，用于治疗或预防冠状病毒感染。
实施方案52.根据实施方案50或51的方法，其中产生免疫应答抑制或减少受试者中的冠状病毒复制。
实施方案53.根据实施方案50-52中任一项的方法，其中免疫应答包括细胞介导的应答和/或体液应答，可选地包括产生一种或更多种中和抗体，例如多克隆抗体或单克隆抗体。
实施方案54.根据实施方案50-53中任一项的方法，其中免疫应答针对冠状病毒的表面抗原，但不针对C-末端前肽。
实施方案55.根据实施方案50-54中任一项的方法，其中施用不会由于先前暴露于一种或更多种冠状病毒而导致受试者中的抗体依赖性增强(ADE)。
实施方案56.根据实施方案50-55中任一项的方法，其中当随后暴露于一种或更多种冠状病毒时，施用不会导致受试者中的抗体依赖性增强(ADE)。
实施方案57.根据实施方案50-56中任一项的方法，进一步包括初免步骤和/或加强步骤。
实施方案58.根据实施方案50-57中任一项的方法，其中通过局部、经皮、皮下、皮内、口服、鼻内(例如，鼻内喷雾剂)、气管内、舌下、口腔、直肠、阴道、吸入、静脉内(例如，静脉注射)、动脉内、肌肉内(例如，肌内注射)、心内、骨内、腹腔内、跨粘膜、玻璃体内、视网膜下、关节内、关节周围、局部或应用皮肤(epicutaneous)给药来进行给药步骤。
实施方案59.根据实施方案50-58中任一项的方法，其中以单剂量或具有一个间隔或更多个间隔的一系列剂量施用有效量。
实施方案60.根据实施方案50-59中任一项的方法，其中在没有佐剂的情况下施用有效量。
实施方案61.根据实施方案50-59中任一项的方法，其中有效量与佐剂或多种佐剂一起施用。
实施方案62.一种方法，包括向受试者施用有效量的根据实施方案1-33中任一项的蛋白质，以在受试者中产生针对冠状病毒的中和抗体或中和抗血清。
实施方案63.根据实施方案62的方法，其中受试者为哺乳动物，可选地为人类或非人类灵长类。
实施方案64.根据实施方案62或63的方法，进一步包括从受试者分离中和抗体或中和抗血清。
实施方案65.根据实施方案64的方法，进一步包括通过被动免疫向人类受试者施用有效量的分离的中和抗体或中和抗血清，以预防或治疗冠状病毒感染。
实施方案66.根据实施方案62-65中任一项的方法，其中中和抗体或中和抗血清包括针对冠状病毒表面抗原的多克隆抗体，可选地其中中和抗体或中和抗血清不含或基本不含针对胶原的C-末端前肽的抗体。
实施方案67.根据实施方案62-65中任一项的方法，其中中和抗体包括针对冠状病毒表面抗原的单克隆抗体，可选地其中中和抗体不含或基本不含针对胶原的C-末端前肽的抗体。
实施方案68.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗，用于诱导受试者中对冠状病毒的免疫应答，和/或治疗或预防冠状病毒感染。
实施方案69.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于诱导受试者中对冠状病毒的免疫应答，和/或用于治疗或预防冠状病毒感染。
实施方案70.根据实施方案1-47和49中任一项的蛋白质、免疫原、蛋白质纳米颗粒、VLP、分离核酸、载体、病毒、假病毒、细胞、免疫原性组合物或疫苗的用途，用于制造用于诱导受试者中对冠状病毒的免疫应答的药物或预防剂，和/或用于治疗或预防冠状病毒感染。
实施方案71.一种用于分析样品的方法，包括：将样品与实施方案1-33中任一项的蛋白质接触，并检测蛋白质与能够特异性结合到冠状病毒表面抗原的分析物之间的结合。
实施方案72.根据实施方案71的方法，其中分析物为识别表面抗原的抗体、受体或细胞。
实施方案73.根据实施方案71或72的方法，其中结合表明样品中存在分析物，和/或样品来源于的受试者中存在冠状病毒感染。
实施方案74.一种试剂盒，包括实施方案1-33中任一项的蛋白质和含有或固定蛋白质的底物、板或小瓶，可选地其中试剂盒是ELISA或侧向流动检测试剂盒(lateral flow assay kit)。
实施例
包括以下实施例仅用于说明目的，并不旨在限制本发明的范围。
方法
蛋白表达和纯化
实施例1：产生重组二硫键连接的SARS-CoV-2 S-三聚体融合蛋白
产生分泌型重组二硫键连接的多肽，其包含融合到三聚化结构域SARS-CoV-2蛋白肽，作为候选蛋白亚单位疫苗。来自SARS-CoV2,例如Hu-1毒株和VOC贝塔毒株，的包括其信号肽(SP)、S1和S2结构域的天然刺突蛋白的完整胞外域在框内在C-末端融合到编码α1胶原的人类C-前肽的哺乳动物表达载体，以使分泌的三聚体S-三聚体融合抗原得以表达PCT/CN2020/095269,PCT/CN2021/087066,PCT/CN2021/093895和PCT/CN2021/099285出于所有目的以其全文形式被援引加入。
为了快速表达S-三聚体抗原，采用了蛋白质三聚体化TM技术(Liu et al.,Scientific Reports,7(1):8953,2017，出于所有目的以其全文形式被援引加入)。编码SARS-CoV-2刺突(S)蛋白的胞外域的cDNA被亚克隆到pTRIMER哺乳动物表达载体中， 以允许在框内融合到蛋白质三聚体化TM标签，蛋白质三聚体化TM标签能够通过二硫键自我三聚化。在稳定转染到CHO细胞中、随后筛选高滴度生产克隆和广泛的工艺优化后，开发了生物反应器中的补料分批无血清细胞培养工艺，导致S-三聚体作为分泌蛋白的高水平表达。Liang et al.,Nat.Comms.,12:1346,2021；Richmond et al.,Lancet,397:682-694,2021，出于所有目的以其全文形式被援引加入。
为了获得用于疫苗研究的高纯度形式的S-三聚体，利用蛋白质三聚体化TM标签和Endo180之间的高结合亲和力的优势开发了亲和纯化方案，Endo180是能够结合到1型前胶原的C-末端区域并使胶原成熟的胶原受体。将Endo180-Fc融合蛋白装载到蛋白A柱上，并通过蛋白A与Endo180-Fc的人IgG1Fc结构域之间的高亲和力结合被树脂捕获。然后，将含有CHO细胞分泌的S-三聚体的无血清细胞培养基装载到具有预捕获的Endo180-Fc的蛋白A柱上。在冲洗掉任何未结合的污染宿主细胞蛋白质(HCP)和其它杂质后，在不使Endo180-Fc从蛋白A柱分离的条件下，使用温和的盐洗脱一步将结合的S-三聚体纯化至接近均一性。进一步纯化S-三聚体，通过低pH值用于预防性病毒灭活(VI)，阴离子交换层析以去除宿主细胞DNA和任何残留的内毒素，纳滤作为预防性病毒去除(VR)步骤，以及最终UF/DF以将S-三聚体浓缩到制剂缓冲液中所需的浓度，从而获得S-三聚体亚单位候选疫苗的活性药物(DS)。纯化的S-三聚体的稳定性分析表明，S-三聚体在2-8℃的液体溶液制剂中是稳定的。
非还原和还原条件下的SDS-PAGE分析证实，纯化的S-三聚体是二硫键连接的三聚体，并在S1/S2边界被CHO细胞产生的弗林蛋白酶部分裂解。在非还原条件下，S-三聚体以多种高分子量形式出现，可能是抗原的部分裂解的结果，在样品处理期间释放非共价连接的和裂解的S1。
图1A和1B示出了S-三聚体抗原表达纯化。图1A示出了经深层过滤的S-三聚体抗原蛋白；图1B示出了纯化的S-三聚体抗原蛋白。
基于Hu-1毒株和VOC毒株的刺突抗原的生产和表征S-三聚体与人类ACE2的受体结合研究
利用蛋白质三聚体化TM技术(Liang et al.,Nat.Comms.,12:1346,2021)产生基于Hu-1毒株和VOC贝塔毒株的刺突抗原共价三聚体。在10L生物反应器中对补料分批无血清CHO细胞培养物中的S-三聚体表达进行了8％SDS-PAGE分析。在还原条件下分析无细胞条件培养基，然后进行考马斯亮蓝染色。
通过Bio-Layer干涉法测量在ForteBio Octet QKe(Pall)上评估S-三聚体与ACE2的结合亲和力。ACE2-Fc(10μg/mL)固定在蛋白质A(ProA)生物传感器(Pall)上。通过将传感器应用于S-三聚体(PBS中为22.5-36μg/mL)的两倍连续稀释液中来获得实时受体结合曲线。使用Octet软件12.0版本分析动力学参数(K on和K off)和亲和力(K D)。假设S-三聚体(原始型或B.1.351)与ACE2-Fc的结合模型为1:1，使用稳态分析确定解离常数(K D)。在图2中，使用蛋白质A传感器通过Fortebio BioLayer干涉测量法评估含贝塔 (beta)变异株RBD的嵌合原始型S-三聚体与ACE2-Fc的受体结合动力学。与原始株相比，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体受体亲和力更高。
ACE2竞争性ELISA试验
96孔板(康宁)在4℃下涂有1μg/mL ACE2-Fc(100μL/孔)过夜，用2％脱脂乳在37℃下封闭2小时。在用PBST洗涤3次后，用与连续稀释的抗血清混合的100ng/mL S-三聚体(原始型、B.1.351、B.1.1.7或P.1)在37℃下温育1小时。用PBST洗涤3次后，将板与1:5000稀释度的兔抗蛋白质三聚体化 TM标签抗体(三叶草生物制药)在37℃下温育1小时，然后用PBST洗涤3次，然后用1:20000稀释度的山羊抗兔IgG-HRP(Southern Biotech)温育。用PBST洗涤3次后，加入TMB(Thermo Scientific)进行信号显影。抑制百分比计算如下：％抑制＝[(A-空白)-(P-空白)]/(A-空白)×100，其中A是不存在血清时S-三聚体与ACE2-Fc结合的最大OD信号，P是在给定稀释度下存在血清时S-三聚体与ACE2-Fc结合的OD信号。给定血清样品的IC 50被定义为稀释度的倒数，其中样品显示50％竞争。
假病毒中和试验
对原始(Wuhan-Hu-1)毒株和变种B.1.1.7、B.1.351和P.1毒株和其它毒株进行SARS-CoV-2假病毒中和试验。为了评估抗血清的SARS-CoV-2假病毒中和活性，首先将样品热灭活30分钟，并连续稀释(3倍)，与等量的650TCID 50假病毒在37℃下温育1小时，同时使用单独病毒(阳性对照)和单独细胞(阴性对照)。然后，将新鲜的胰蛋白酶化的ACE2过表达-293细胞以20000个细胞/孔添加到每个孔中。在5％CO 2培养箱中在37℃下温育24小时后，根据生产商的方案，裂解细胞，并通过荧光素酶分析系统(Beyotime)测定荧光素酶活性。给定血清样品的EC 50中和抗体滴度被定义为稀释度的倒数，其中样品显示与单独病毒对照孔相比，相对光单位(RLU)减少50％。
脾细胞刺激和ELISpot试验
为了检测抗原特异性的T-细胞应答，按照生产商的说明使用ELISpot试剂盒(Mabtech)测量Th1细胞因子(IFN-γ、IL-2)和Th2细胞因子(IL-5)。在第三次免疫后2周，收集免疫小鼠的脾细胞或免疫小鼠的PBMC。用2μg/mL原始SARS-CoV-2 S1肽库(pool)、SARS-CoV S1肽库、B.1.351 RBD肽库或P.1 RBD肽库在体外刺激5×10 5脾细胞(96孔板)。将佛波醇12-肉豆蔻酸酯13-醋酸酯(Phorbol 12-myristate 13-acetate，PMA)和离子霉素作为非特异性刺激物添加到阳性对照孔中，而阴性对照孔未接受刺激物。温育24-48小时后，加入ELISpot试剂盒和SA-ALP/SA-HRP中的生物素化的检测抗体。通过添加BCIP/NBT或AEC底物溶液来形成斑点，在黑暗中温育5-30分钟后产生色斑。使用自动ELISpot读出器(CTL)对IFN-γ、IL-2和IL-5斑点形成细胞(SFC)进行计数。
实施例2：含佐剂SARS-CoV-2疫苗的免疫原性
用S-三聚体检测康复期血清中SARS-CoV-2特异性结合和中和抗体。S-三聚体被用作抗原，以检测从康复的COVID-19患者采集的人类恢复期血清样品中SARS-CoV-2刺突蛋白结合抗体和ACE2竞争性抗体的存在。在恢复期血清中检测到高水平的S-三聚体结合抗体和ACE2竞争性滴度，以及使用假病毒中和试验检测到高中和抗体滴度。在早期新染
Figure PCTCN2022140514-appb-000021
人类志愿者的血清中未检测到S-三聚体结合抗体，而在COVID-19康复期血清和新染
Figure PCTCN2022140514-appb-000022
血清中均检测到与流感血凝素(HA)-三聚体结合的抗体，暗示所有测试的受试者均有流感感染史，但在COVID-19康复期受试者中仅发现SARS-CoV-2感染史。在最近的新的临床1期实验中，在新染
Figure PCTCN2022140514-appb-000023
人类志愿者的血清中检测到了S-三聚体结合抗体，表明所有测试的受试者均有新冠感染史(图10-12)。这些结果支持了试验的特异性，并证明了S-三聚体用于检测康复期血清中SARS-CoV-2刺突蛋白特异性的抗体的能力，进一步证实了S-三聚体中刺突抗原的天然类似构象。
观察到抗体滴度与疾病严重程度相关，在患有轻度COVID-19疾病的患者中观察到抗体滴度较低，在严重病例中滴度较高。抗体滴度似乎也与患者年龄中度相关，但未观察到性别差异。
评估BALB/c小鼠中S-三聚体的免疫原性。在两剂初免-加强方案(第0天和第21天)中，小鼠被两次肌肉注射S-三聚体(无佐剂或具有各种佐剂)。佐剂对体液免疫原性的影响是明显的，因为在相应的抗原剂量水平下，含佐剂组的S-三聚体结合抗体滴度、ACE2竞争性滴度和中和抗体滴度显著高于无佐剂疫苗。具有不同佐剂的S-三聚体引发与在人类康复期血清样品中观察到的水平相似或更高的ACE2竞争性抗体滴度和假病毒中和抗体滴度。在S-三聚体免疫的大鼠中观察到类似的结果。
在非人类灵长类动物(恒河猴)中进一步研究了含佐剂S-三聚体的免疫原性。用含佐剂S-三聚体或PBS载体对照对动物肌肉内接种两次。然后在第35天用SARS-CoV-2病毒攻击动物，然后通过各种参数评估免疫保护。
图3示出了含佐剂S-三聚体对人类灵长类动物(恒河猴)SARS-CoV-2病毒的免疫保护。用含佐剂S-三聚体或载体对照对动物肌肉内接种两次。在第35天用SARS-CoV-2病毒攻击动物，然后评估免疫保护。第一组为盐水载体对照，第二组为原始型S-三聚体(佐剂1含CpG 1018加Alum)，第三组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，第四组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、和吐温-80)。在7dpi尸检时采集肺组织，并基于基因组RNA(gRNA)检测SARS-CoV-2病毒载量。在含佐剂S-三聚体的三组中观察到肺组织中的病毒载量明显低于载体对照。比较第三组和第四组，含角鲨烯、α-生育酚、和吐温-80的佐剂与CpG 1018加Alum作为佐剂效果类似。
图4示出了灵长类动物对含佐剂S-三聚体中和抗体应答。用原始型S-三聚体(佐剂1含CpG 1018加Alum)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，或含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、 和吐温-80)对动物肌肉内接种两次。两种抗原的给药剂量均为每剂30μg。在D35收集血清，进行假病毒中和抗体测试和活SARS-CoV-2病毒中和抗体测试，A.示出了SARS-CoV-2Hu-1、B.1.1.7、B.1.617.2、B.1.351、P.1、和B.1.621毒株假病毒中和抗体滴度(IC50)数据。B.示出了活SARS-CoV-2病毒中和抗体滴度数据。点代表个体动物的数据；横线表示每组的几何平均滴度(GMT)±SEM。对比接受原始型S-三聚体(佐剂1)的动物，接受含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)的动物产生了6到100倍高的中和抗体。并且含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)具有更广泛的中和作用，在动物中产生了对SARS-CoV-2Hu-1及多种VOC或VOI的中和抗体。比较含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)对假病毒和活病毒中和效果类似或更好。
通过收集被处死的免疫的小鼠的脾细胞，然后用S-三聚体抗原刺激并通过ELISpot检测Th1(IL-2和IFNγ)和Th2(IL-4和IL-5)细胞因子，来研究S-三聚体抗原特异性的细胞介导的免疫(CMI)。实验结果显示三个动物实验组的CMI应答没有显著的不同。
图5示出了含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)在灵长类动物中对Hu-1和对奥密克戎毒株感染的保护作用。用含佐剂S-三聚体或载体对照对动物肌肉内接种两次，然后在D35收集血清，进行假病毒中和抗体测试。第一组为原始型S-三聚体(佐剂1含CpG 1018加Alum)，第二组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)，第三组为含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2含角鲨烯、α-生育酚、和吐温-80)，第四组为原始型S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体的双价疫苗(佐剂1)。对比接受原始型S-三聚体(佐剂1)的动物，接受含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)的动物产生了约18倍高的对奥密克戎毒株的中和抗体。比较含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂1)和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)，含贝塔(beta)变异株RBD的嵌合原始型S-三聚体(佐剂2)对奥密克戎毒株中和效果更好.
在用原始型SARS-CoV-2病毒进行病毒攻击后，对照组的动物体重呈持续下降趋势。虽然所有接种疫苗的动物都经历了体重增加，然后逐渐恢复(图8)。与对照组相比，所有接种组的鼻腔、咽喉和肛门拭子中的病毒载量均显着降低(图9)。
实施例3:含不同佐剂剂量的SARS-CoV-2疫苗的免疫原性研究
探索性地使用不同的佐剂剂量进行了的鼠的免疫原性研究。以3μg/剂的抗原与指定的佐剂一起给药，每只动物接受两次i.m.注射，在第0天注射一次，在第21天注射第二次。所有组的动物接受以CpG 1018/铝剂为佐剂的初始剂量。在原始型S-三聚体和含贝塔(BETA)变异株RBD的嵌合原始型S-三聚体组中，第二针加强剂和CpG1018/铝剂一起使用。(数据没有显示)。
含贝塔(beta)变异株RBD的嵌合原始型S-三聚体还包括其它组，其中第2组的动物接受的加强剂(剂2)，只与铝剂一起给药或没有和任何佐剂(仅抗原)给药。在图6的第1组中，原始型S-三聚体和含贝塔(beta)变异株RBD的嵌合原始型S-三聚体疫苗组均接受了以全剂量CAS-1为佐剂的初免和加强剂量。含贝塔(BETA)变异株RBD的嵌合原始型S-三聚体还包括其它组，其中动物接受初免和加强，辅以一半或四分之一剂量的CAS-1或无佐剂(仅抗原)。在第35天(第2剂后2周)评估基于假病毒中和试验的中和抗体滴度，病毒毒株包括原始毒株(Wuhan-Hu-1)、贝塔(Beta)变体(B.1.351)、阿尔法(Alpha)变体(B.1.1.7)和伽马(Gamma)变体(P.1).
虽然原始型S-三聚体诱导了针对武汉-Hu-1和阿尔法Alpha(B.1.1.7)菌株的高水平中和抗体(图6)，但观察到针对贝塔Beta(B.1.351)和伽马(Gamma)(P.1)变体的中和抗体较低且变化较大，与武汉-Hu-1相比，针对Beta(B.1.351)的滴度低约20倍。相比之下，含贝塔(Beta变异株RBD的嵌合原始型S-三聚体显著地诱导出对所有变体的高反应。其中接受含贝塔(BETA)变异株RBD的嵌合原始型S-三聚体的初始剂量辅以CpG 1018/铝剂，给予含贝塔(BETA)变异株RBD的嵌合原始型S-三聚体的加强剂辅以单独使用铝剂或无佐剂也诱导高水平的中和抗体，表明第二剂CpG 1018的作用可能较低。
实施例4：1期临床试验及结果
1期临床研究是一项双盲、随机、确定剂量的研究，其评估了含贝塔(beta))变异株RBD的嵌合原始型S-三聚体分别与CpG1018/铝佐剂和CAS-1佐剂一起使用时的安全性和免疫原性。包括原始型S-三聚体(CpG 1018/Alum)以及含贝塔(Beta)变异株RBD的嵌合原始型S-三聚体与CpG 1018/Alum。所有疫苗制剂均作为两剂方案给药，间隔21天。这些中期研究结果基于南非的70名成年人(18至75岁)，奥密克戎他们对以前的感染和疫苗接种具有高水平的现有免疫力。
图10的数据表明，带有全剂量CAS-1的含9μg贝塔变异株RBD的嵌合原始型S-三聚体疫苗接种1剂后具有良好的免疫反应和抗体的广谱性。该疫苗的中和抗体明显高于施用含30μg原始型S-三聚体和CpG 1018/明矾佐剂的中和抗体，约1.5～2.3倍。
图10-图11的数据显示，含有CAS-1佐剂的含贝塔(beta))变异株RBD的嵌合原始型S-三聚体引发了对奥密克戎BA.4和BA.5的强烈免疫反应。具体来说，相对于加强前水平，带有全剂量CAS-1的含9μg贝塔变异株RBD的嵌合原始型S-三聚体导致针对奥密克戎BA.5的抗体滴度增加了5.0倍，针对奥密克戎BA.4的抗体滴度增加了6.6倍。该由9μg贝塔变异株RBD的嵌合原始型S-三聚体抗原与全剂量CAS-1组成疫苗制剂，比30μg原始型S-三聚体与CpG 1018/Alum组合引发了对奥密克戎BA.4和BA.5的更强烈免疫反应。与施用含30微克贝塔变异株RBD的嵌合原始型S-三聚体和CpG 1018/明矾佐剂的参与者相比，施用了带有全剂量佐剂2的含9ug贝塔变异株RBD的 嵌合原始型S-三聚体的参与者对BA.5的免疫反应增加了2.9倍，对奥密克戎BA.4的免疫反应增加了3.9倍。该数据表明，使用佐剂2系统可能有助于节约抗原剂。
图11的数据表明，相对于加强前水平，30μg原始型S-三聚体(CpG1018/Alum)使针对奥密克戎BA.5的抗体滴度增加了4.5倍，针对奥密克戎BA.4的中和抗体也增加了4.5倍。此外，其它含有更少(3ug)含贝塔变异株RBD的嵌合原始型S-三聚体或更少(一半)佐剂2的制剂也引发了对所测试的SARS-CoV-2变体的大量免疫反应。
图12的数据表明，相对于加强前水平，带有CAS-1的含贝塔变异株RBD的嵌合原始型S-三聚体导致针对原始Hu-1和贝塔变体产生了强烈的免疫反应。
总之，这些结果表明含贝塔变异株RBD的嵌合原始型S-三聚体与佐剂2作为原始型S-三聚体的补充选项(CpG 1018/Alum)的潜力。
本发明并不旨在将范围限于特定的公开实施方案，这些实施方案被提供例如用于阐明本发明的各个方面。通过本文的描述和教导，对所述组合物和方法的各种改动将变得显而易见。可以在不偏离本发明的真实范围和精神的情况下实施这些变化，并且这些变化旨在落入本发明的范围。
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Figure PCTCN2022140514-appb-000024
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Figure PCTCN2022140514-appb-000027
Figure PCTCN2022140514-appb-000028
Figure PCTCN2022140514-appb-000029
Figure PCTCN2022140514-appb-000030
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Figure PCTCN2022140514-appb-000032
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Figure PCTCN2022140514-appb-000037
Figure PCTCN2022140514-appb-000038
Figure PCTCN2022140514-appb-000039
Figure PCTCN2022140514-appb-000040
Figure PCTCN2022140514-appb-000041
Figure PCTCN2022140514-appb-000042
Figure PCTCN2022140514-appb-000043
Figure PCTCN2022140514-appb-000044
Figure PCTCN2022140514-appb-000045
Figure PCTCN2022140514-appb-000046
Figure PCTCN2022140514-appb-000047
Figure PCTCN2022140514-appb-000048
Figure PCTCN2022140514-appb-000049

Claims (77)

  1. 一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的重组亚单位疫苗免疫哺乳动物，该重组亚单位疫苗包括可溶性冠状病毒SARS-CoV-2贝塔(B.1.351)表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
  2. 根据权利要求1所述的方法，其中冠状病毒感染是严重急性呼吸综合征(SARS)-冠状病毒2(SARS-CoV-2)感染。
  3. 根据权利要求1或2所述的方法，其中该冠状病毒表面抗原包括冠状病毒刺突(S)蛋白或其片段或表位。
  4. 根据权利要求1-3中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)胞外域肽或其片段或表位，可选地该S胞外域肽或其片段或表位包括SARS-CoV-2贝塔(beta，B.1.351)变异株S胞外域肽或其片段、变体或突变体，例如含贝塔变异株受体结合域(RBD)及原始型或其它变异株S蛋白肽序列的嵌合序列。
  5. 根据权利要求1-4中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)N-末端结构域(NTD)肽或其片段或表位，可选地该NTD肽是SARS-CoV-2原始型(Hu-1)、阿尔法、贝塔、伽马、德尔塔、或奥密克戎NTD肽或其片段、变体或突变体。
  6. 根据权利要求1-5中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)受体结合结构域(RBD)肽或其片段或表位，可选地该RBD肽是SARS-CoV-2贝塔(beta，B.1.351)变异株RBD肽或其片段、变体或突变体。
  7. 根据权利要求1-6中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S1肽或其片段或表位，可选地该S1肽是SARS-CoV-2贝塔(beta，B.1.351)变异株S1肽或其片段、变体或突变体。
  8. 根据权利要求1-7中任一项所述的方法，其中该冠状病毒表面抗原包括SARS-CoV-2刺突(S)S2肽或其片段或表位，可选地该S2肽是SARS-CoV-2原始型(Hu-1)、阿尔法、贝塔、伽马、德尔塔、或奥密克戎S2肽或其片段、变体或突变体。
  9. 根据权利要求1-8中任一项所述的方法，其中该冠状病毒表面抗原包括具有突变的SARS-CoV-2刺突(S)胞外域肽或其片段或表位。
  10. 根据权利要求9所述的方法，其中该突变包括弗林蛋白酶裂解位点突变，可选地该突变是经缺失、取代或添加一个或几个氨基酸使得弗林蛋白酶切位点不再具有作为弗林蛋白酶切位点的活性，可选地该突变位于682-685中任意一个或几个位点，可选地该突变包括685R→685A。
  11. 根据权利要求9或10所述的方法，其中该突变包括位于或邻近七肽重复序列HR1和中心螺旋交界处的突变，可选地该突变包括脯氨酸替换，例如986K→986P和/或987V→987P。
  12. 根据权利要求9-11中任一项所述的方法，其中该突变包括连续位点氨基酸替换，例如986K→986P和987V→987P。
  13. 根据权利要求1-12中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-95中的任一个所述的序列或与SEQ ID NO:81-95中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  14. 根据权利要求1-13中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:86或与SEQ ID NO:86有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  15. 根据权利要求1-14中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:87或与SEQ ID NO:87有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  16. 根据权利要求1-15中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:88或与SEQ ID NO:88有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  17. 根据权利要求1-16中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:89或与SEQ ID NO:89有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  18. 根据权利要求1-17中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:90或与SEQ ID NO:90有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  19. 根据权利要求1-18中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:91-95中的任一个所述的序列或与SEQ ID NO:91-95中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  20. 根据权利要求1-19中任一项所述的方法，其中该重组亚单位疫苗包括SEQ ID NO:81-85中的任一个所述的第一序列连接到SEQ ID NO:67-80中的任一个所述的第二序列，其中该第一序列的C末端直接或间接连接到该第二序列的N末端。
  21. 根据权利要求1-20中任一项所述的方法，其中该重组亚单位疫苗通过肌肉内注射施用。
  22. 根据权利要求1-21中任一项所述的方法，其中该重组亚单位疫苗通过鼻内喷雾剂施用。
  23. 根据权利要求1-22中任一项所述的方法，其中该重组亚单位疫苗以单剂量施用或以周或月间隔开的一系列剂量施用。
  24. 根据权利要求1-23中任一项所述的方法，其中该重组亚单位疫苗在没有佐剂的情况下施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  25. 根据权利要求1-24中任一项所述的方法，其中该重组亚单位疫苗与佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧 乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
  26. 根据权利要求1-25中任一项所述的方法，其中该重组亚单位疫苗与一种以上佐剂一起施用，可选地该重组亚单位疫苗作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种佐剂组合。
  27. 一种用于检测来自哺乳动物血清的冠状病毒抗体的方法，该方法包括将血清与可溶性冠状病毒SARS-CoV-2贝塔(B.1.351)表面抗原接触的步骤，该可溶性冠状病毒表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白。
  28. 根据权利要求27所述的方法，其中该可溶性冠状病毒表面抗原是S蛋白或肽。
  29. 一种使用包含来自冠状病毒SARS-CoV-2贝塔(B.1.351)的可溶性表面抗原的重组亚单位疫苗的方法，该可溶性表面抗原通过框内融合连接到胶原的C-末端部分以形成二硫键连接的三聚体融合蛋白，该方法包括：免疫哺乳动物，纯化产生的中和抗体，以及使用所述中和抗体通过被动免疫治疗感染所述冠状病毒的患者。
  30. 根据权利要求29所述的方法，其中该中和抗体包括多克隆抗体。
  31. 根据权利要求29所述的方法，其中该中和抗体是单克隆抗体。
  32. 根据权利要求29所述的方法，其中该中和抗体是S蛋白或肽的单克隆抗体。
  33. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2的S蛋白的单克隆抗体。
  34. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2原始型(Hu-1)、阿尔法、贝塔、伽马、德尔塔、奥密克戎和/或其它毒株的S蛋白的单克隆抗体。
  35. 根据权利要求29所述的方法，其中该中和抗体是SARS-CoV-2贝塔(B.1.351)的S蛋白的单克隆抗体。
  36. 一种包括选自由SEQ ID NO:86-95组成的组的重组多肽的复合物。
  37. 一种包含选自由SEQ ID NO:86-95组成的组的重组多肽的三聚体的复合物，其中该重组多肽通过多肽间二硫键三聚化以形成三聚体。
  38. 一种免疫原性组合物，该免疫原性组合物包括重组多肽的三聚体或任何两个或更多个三聚体的组合，该重组多肽包括选自由SEQ ID NO:86-95组成的组的序列。
  39. 根据权利要求38所述的免疫原性组合物，包括具有SEQ ID NO:90or 95中所述的序列的重组多肽的三聚体。
  40. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:86-95组成的组的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  41. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括S蛋白或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:86-95组成的组的重组多肽，可选地该复合物作为作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  42. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括选自由SEQ ID NO:27-66和81-85组成的组的序列，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括选自由SEQ ID NO:86-95组成的组的重组多肽，可选地该复合物作为作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚 单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  43. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括冠状病毒的S蛋白或其抗原片段，并且可选地，该表面抗原包括选自由SEQ ID NO:27-66和81-85组成的组的序列或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物，该复合物包括包含SEQ ID NO:86-95中的任一个所述的序列的重组多肽，可选地该复合物作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  44. 一种用于在受试者中产生对冠状病毒表面抗原的免疫应答的方法，
    其中该表面抗原包括S蛋白或其抗原片段，以及
    该方法包括向受试者施用有效量的复合物或任何两种或更多种复合物的组合，该复合物包括包含选自由SEQ ID NO:86-95组成的组的序列的重组多肽，可选地该复合物或复合物的组合作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  45. 根据权利要求44所述的方法，其中该方法包括向受试者施用有效量的包括重组多肽的复合物，该重组多肽包括SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88、SEQ ID NO:89、和/或SEQ ID NO:90中所述的序列。
  46. 一种包含多个重组多肽的融合蛋白，每个重组多肽从氨基到羧基末端包括：
    a)第一区域，该第一区域包括位于第一冠状病毒的非嵌合冠状病毒刺突蛋白中的冠状病毒刺突蛋白受体结合结构域(RBD)之前的冠状病毒刺突蛋白胞外域的一部分；
    b)第二区域，该第二区域包括不同于所述第一冠状病毒的第二冠状病毒的冠状病毒刺突蛋白受体结合结构域(RBD)；和
    c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，其中第一冠状病毒和第二冠状病毒至少有一个是SARS-CoV-2贝塔(B.1.351)，可选地该融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  47. 根据权利要求46所述的融合蛋白，还包括该第二区域与该胶原的C-末端前肽之间的第三区域。
  48. 根据权利要求47所述的融合蛋白，其中该第三区域包括第三冠状病毒的S1结构域，其中该第三冠状病毒与第一冠状病毒或第二冠状病毒相同或不同。
  49. 根据权利要求47或48所述的融合蛋白，其中该第三区域包括第四冠状病毒的S2结构域，其中该第四冠状病毒与第一、第二或第四冠状病毒相同或不同。
  50. 根据权利要求46-49中任一项所述的融合蛋白，其中该第一区域包括第一冠状病毒的N-末端结构域(NTD)。
  51. 根据权利要求46-50中任一项所述的融合蛋白，其中该第一区域包括与第二冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
  52. 根据权利要求46-51中任一项所述的融合蛋白，其中该第二区域包括与第一冠状病毒中的相应氨基酸残基不同的一个或更多个氨基酸残基。
  53. 根据权利要求46-52中任一项所述的融合蛋白，其中该第一和第二冠状病毒是相同冠状病毒的不同变种或毒株。
  54. 根据权利要求53所述的融合蛋白，其中该第一区域包括第一冠状病毒的NTD，该第二区域包括第二冠状病毒的RBD，并且第一和第二冠状病毒是SARS-CoV-2的不同变种。
  55. 根据权利要求46-54中任一项所述的融合蛋白，其中该第一冠状病毒和第二冠状病毒独立地选自由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和A.23.1谱系的SARS-CoV-2病毒组成的组。
  56. 一种包含三个重组多肽的三聚体融合蛋白，每个重组多肽从氨基到羧基末端包括：
    a)第一区域，该第一区域包括B.1.617.2谱系的SARS-CoV-2的冠状病毒刺突蛋白N-末端结构域(NTD)；
    b)第二区域，该第二区域包括B.1.617.2谱系的SARS-CoV-2的冠状病毒刺突蛋白受体结合结构域(RBD)；和
    c)胶原的C-末端前肽，其中该重组多肽的C-末端前肽形成多肽间二硫键，可选地该融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂 (booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用。
  57. 一种用于预防哺乳动物中冠状病毒感染的方法，该方法包括用有效量的根据权利要求46-56中任一项所述的融合蛋白免疫哺乳动物。
  58. 根据权利要求57所述的方法，其中在哺乳动物中产生针对该第一和第二冠状病毒的中和抗体。
  59. 根据权利要求58所述的方法，其中该第一和第二冠状病毒是SARS-CoV-2的不同变种，并且在哺乳动物中产生的中和抗体中和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和A.23.1谱系的两种或更多种SARS-CoV-2病毒。
  60. 根据权利要求59所述的方法，其中在哺乳动物中产生的中和抗体中和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和A.23.1谱系的三种或更多种SARS-CoV-2病毒。
  61. 根据权利要求57-60中任一项所述的方法，包括用两剂或更多剂的融合蛋白免疫哺乳动物，该两剂或更多剂的融合蛋白中至少有一剂的融合蛋白包含SARS-CoV-2贝塔(B.1.351)刺突蛋白氨基酸序列，可选地该至少一剂融合蛋白包含SEQ ID NO:86-95中的任一个所述的序列或与SEQ ID NO:86-95中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  62. 根据权利要求57-61中任一项所述的方法，其中在一剂或更多剂的免疫原之后以再次加强剂施用融合蛋白，该免疫原包括包含来自相同或不同SARS-CoV-2变种的NTD和RBD的刺突蛋白肽，可选地该一剂或更多剂的免疫原包含SEQ ID NO:27-66和81-85中的任一个所述的序列或与SEQ ID NO:27-66和81-85中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列，可选地该加强剂融合蛋白包含SEQ ID NO:86-95中的任一个所述的序列或与SEQ ID NO:86-95中的任一个所述的序列有至少或约80％、85％、90％、92％、95％、97％、99％的序列同一性的氨基酸序列。
  63. 一种三聚体融合蛋白，包括可溶性冠状病毒SARS-CoV-2贝塔(B.1.351)表面抗原或其片段、变体或突变体，该可溶性冠状病毒表面抗原或其片段、变体或突变体连接到 胶原的C-末端部分，该胶原的C-末端部分形成二硫键连接的三聚体，从而形成该三聚体融合蛋白。
  64. 根据权利要求63所述的三聚体融合蛋白，该三聚体融合蛋白包括选自由SEQ ID NO:86-95组成的组的重组多肽。
  65. 根据权利要求63或64所述的三聚体融合蛋白，用于预防冠状病毒感染，可选地该冠状病毒感染包括SARS-CoV-2原始型(Hu-1)、阿尔法、贝塔、伽马、德尔塔、奥密克戎和其它SARS-CoV-2毒株中的任意一种或几种感染。
  66. 根据权利要求63-65中任一项所述的三聚体融合蛋白，在没有佐剂的情况下施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，
    可选地该三聚体融合蛋白在没有佐剂的情况下作为初始剂使用，该初始剂包括第一针和第二针,
    可选地该三聚体融合蛋白在没有佐剂的情况下作为额外剂使用，该额外剂包括第三针，第四针，和/或更多针,
    可选地该三聚体融合蛋白在没有佐剂的情况下作为同源或异源加强剂使用，该同源或异源加强剂包括第三针，第四针，和/或更多针。
  67. 根据权利要求63-65中任一项所述的三聚体融合蛋白，其中该三聚体融合蛋白与佐剂一起施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种所述佐剂的组合，
    可选地该三聚体融合蛋白与佐剂一起作为初始剂使用，该初始剂包括第一针和第二针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂,
    可选地该三聚体融合蛋白与佐剂一起作为额外剂使用，该额外剂包括第三针，第四针，和/或更多针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂,
    可选地该三聚体融合蛋白与佐剂一起作为同源或异源加强剂使用，该同源或异源加强剂包括第三针，第四针，和/或更多针，该佐剂包括Alum和含CpG寡脱氧核苷酸(CpG-ODN)的佐剂和/或水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂。
  68. 根据权利要求63-65中任一项所述的三聚体融合蛋白，其中该三聚体融合蛋白与一种以上佐剂一起施用，可选地该三聚体融合蛋白作为初始剂(primary series)，额外剂(additional dose)，和/或同源或异源加强剂(booster dose)使用，例如第一剂，第二剂，第三剂，第四剂，和/或更多剂，可选地该初始剂，额外剂，或异源加强剂与其它重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗中的任意一种或几种配合使用，可选地该初始剂，额外剂，和/或同源或异源加强剂中的佐剂可独立地包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂；含TLR9激动剂的佐剂；含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂；或任意一种所述佐剂组合。
  69. 根据权利要求63-65中任一项所述的三聚体融合蛋白，作为初始剂用于免疫哺乳动物，可选地该初始剂包括第一针和第二针。
  70. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针没有佐剂。
  71. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针的佐剂包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；和/或含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂。
  72. 根据权利要求69中所述的三聚体融合蛋白，该第一针和/或第二针的佐剂包括：含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂。
  73. 根据权利要求63-72中任一项所述的三聚体融合蛋白，作为第三针，第四针，和/或更多针额外剂用于免疫哺乳动物。
  74. 根据权利要求63-73中任一项所述的三聚体融合蛋白，作为第三针，第四针，和/或更多针加强剂用于免疫哺乳动物,
    可选地该加强剂没有佐剂，
    可选地该加强剂的佐剂包括：含铝佐剂，例如含明矾和/或氢氧化铝的佐剂；和/或含寡核苷酸的佐剂，例如含CpG寡脱氧核苷酸(CpG-ODN)的佐剂，
    可选地该加强剂的佐剂包括含可代谢油、α-生育酚、和/或聚氧乙烯山梨醇酐单油酸酯(吐温-80)的佐剂，例如水包油乳液形式的含角鲨烯、α-生育酚、和吐温-80的佐剂。
  75. 根据权利要求63-74中任一项所述的三聚体融合蛋白，与其它一种或几种疫苗配合使用，该其它一种或几种疫苗独立选自由重组亚单位疫苗、纳米颗粒疫苗、mRNA疫苗、DNA疫苗、腺病毒载体疫苗、和灭活病毒疫苗所组成的组。
  76. 根据权利要求75中任所述的三聚体融合蛋白，其中该三聚体融合蛋白作为初始剂使用，该其它一种或几种疫苗作为加强剂使用。
  77. 根据权利要求75中任所述的三聚体融合蛋白，其中该其它一种或几种疫苗作为初始剂使用，该三聚体融合蛋白作为加强剂使用。
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