WO2023023940A1 - 一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用 - Google Patents

一种诱导广谱抗冠状病毒的t细胞疫苗免疫原及其应用 Download PDF

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WO2023023940A1
WO2023023940A1 PCT/CN2021/114313 CN2021114313W WO2023023940A1 WO 2023023940 A1 WO2023023940 A1 WO 2023023940A1 CN 2021114313 W CN2021114313 W CN 2021114313W WO 2023023940 A1 WO2023023940 A1 WO 2023023940A1
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seq
cov
coronavirus
virus
peptide
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French (fr)
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徐建青
张晓燕
周东明
曹康丽
王祥
裘天颐
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Fudan University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/08—RNA viruses
    • C07K14/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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

Definitions

  • the invention belongs to the fields of biotechnology and medicine, especially antiviral vaccines. Specifically, the present invention relates to a T cell vaccine immunogen that induces broad-spectrum anti-coronavirus, and its preparation and application.
  • Coronavirus is a type of single-stranded RNA virus that spreads between animals and humans, and can infect mammals and birds, causing digestive tract diseases in cattle and pigs or upper respiratory tract in chickens disease. Coronaviruses are common in nature and are thought to be responsible for 15% to 30% of common colds.
  • Coronaviruses are composed of a single ribonucleic acid (RNA), and this RNA and N protein together make up the virus. Since its genetic material is RNA, it is very prone to mutations. So far, about 45 different strains of coronaviruses have been discovered, which can infect a variety of mammals and birds. As for the coronaviruses that can infect humans, in addition to the 2019 novel coronavirus (2019-nCoV, now officially named SARS-CoV-2 by the International Committee on Taxonomy of Viruses), there are six other species.
  • 2019-nCoV 2019 novel coronavirus
  • SARS-CoV-2 Middle East respiratory syndrome coronavirus
  • CTL cytotoxic T lymphocytes
  • MHC self-molecules
  • the present invention just provides immunogenic peptides that can be used for broad-spectrum anti-coronavirus infection, products, preparation methods and applications thereof.
  • an isolated immunogenic peptide comprising one or more coronavirus protein shared peptides selected from the group consisting of:
  • the immunogenic peptide comprises one or more peptides selected from the group consisting of SEQ ID NO: 11 to SEQ ID NO: 29.
  • T cells activated by the immunogenic peptide specifically bind to a coronavirus epitope selected from the group consisting of: PLPDRWYFYYT (SEQ ID NO: 11); KPISAYAFLMA (SEQ ID NO: 13); LSPRWYFYYL (SEQ ID NO: 30); LAPRWYFYYTG (SEQ ID NO: 31); VPAYSFLPG (SEQ ID NO: 32); and/or APISAMVRMYIFFA (SEQ ID NO: 33).
  • a coronavirus epitope selected from the group consisting of: PLPDRWYFYYT (SEQ ID NO: 11); KPISAYAFLMA (SEQ ID NO: 13); LSPRWYFYYL (SEQ ID NO: 30); LAPRWYFYYTG (SEQ ID NO: 31); VPAYSFLPG (SEQ ID NO: 32); and/or APISAMVRMYIFFA (SEQ ID NO: 33).
  • the shared peptide is derived from a coronavirus selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat- CoV.
  • the shared peptide of ORF1ab comprises the amino acid sequence described in SEQ ID NO: 1, for example, the amino acid sequence of the shared peptide of ORF1ab is shown in SEQ ID NO: 1.
  • the shared peptide of the M protein comprises the amino acid sequence described in SEQ ID NO: 3, for example, the amino acid sequence of the shared peptide of the M protein is shown in SEQ ID NO: 3.
  • the shared peptide of the N protein comprises the amino acid sequence described in SEQ ID NO: 5, for example, the amino acid sequence of the shared sequence of the N protein is shown in SEQ ID NO: 5.
  • the shared peptide of the S protein comprises the amino acid sequence described in SEQ ID NO: 7, for example, the amino acid sequence of the shared peptide of the S protein is shown in SEQ ID NO: 7.
  • the immunogenic peptide further comprises other sequences co-expressed (for example, fusion expression or separate reading frame expression) with the consensus sequence, and the other sequences are used, for example, to expand the antiviral spectrum and improve the ability to induce immune responses, so
  • the other sequences are selected from, for example, immunogens against the following viruses: coronavirus (such as RBD or its modified sequence (such as terminal Cys modified RBD amino acid sequence)), influenza virus (such as HA2), HIV, rabies virus, swine fever virus, PRRS virus, measles virus, Ebola virus, herpes virus, arboviruses (Zika virus, Japanese encephalitis virus, forest encephalitis virus, dengue virus, Hantaan virus, Xinjiang hemorrhagic fever virus) , especially immunogens that respond to activated B cells, to prepare complex vaccines that can simultaneously activate neutralizing antibodies and T cell responses.
  • coronavirus such as RBD or its modified sequence (such as terminal Cys modified R
  • the sequence co-expressed with the immunogenic peptide of the present application is an immunomodulatory sequence selected from the group consisting of IL-2, IL-7, IL-12, IL-18, IL-21, GM- CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibody, CTLA4 antibody, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH and SopE.
  • an immunomodulatory sequence selected from the group consisting of IL-2, IL-7, IL-12, IL-18, IL-21, GM- CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibody, CTLA4 antibody, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera to
  • the immunogenic peptide comprises the amino acid sequence shown in SEQ ID NO: 9, for example, the sequence of the immunogenic peptide is shown in SEQ ID NO: 9; or, it comprises SEQ ID NO: 37 and/or the amino acid sequence shown in 38, for example, the sequence of the immunogenic peptide is shown in SEQ ID NO: 37 and SEQ ID NO: 38.
  • molecules encoding the immunogenic peptides herein, vectors comprising the encoding molecules and/or host cells comprising the encoding molecules or vectors are provided.
  • the encoding molecule comprises one or more nucleotide molecules selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10 and SEQ ID NO: 36;
  • the nucleotide molecule contains or does not contain a linker sequence
  • the coding molecule contains or does not contain a leader sequence
  • the nucleotide molecule comprises or without tags, such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag, HA-tag, Myc-tag, S-tag, SBP-tag, Sof-tag 1, Sof-tag3, Strep-tag, TC tag, V5 tag, T7 tag, VSV tag, XPress tag, 3X FLAG tag, Isopep tag, Spytag, Snoop tag, and PNE tag.
  • tags such as His-tag, AviTag, Calmodulin tag, polyglutamate tag, E-tag, FLAG tag,
  • a product comprising: an immunogenic peptide and/or encoded molecule, vector or cell herein; and optionally, a pharmaceutically or immunologically acceptable carrier, excipient And/or adjuvants (such as one or more adjuvants selected from the group: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant agent, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL-2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21).
  • excipient And/or adjuvants such as one or more adjuvants selected from the group: aluminum adjuvant, cholera toxin and its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adj
  • the product is, for example, a T-cell vaccine or medicament against a coronavirus infection, especially a broad-spectrum T-cell vaccine or medicament against a coronavirus selected from the group consisting of: SARS-CoV-2 , SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, especially SARS-CoV, MERS-CoV and SARS-CoV-2 viruses.
  • a coronavirus infection especially a broad-spectrum T-cell vaccine or medicament against a coronavirus selected from the group consisting of: SARS-CoV-2 , SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, especially SARS-CoV, MERS-CoV and SARS-CoV-2 viruses.
  • the product comprises a conjugate or conjugate of an immunogenic peptide described herein.
  • the product is selected from the group consisting of: nucleic acid vaccines (DNA or RNA vaccines), recombinant protein subunit vaccines, recombinant viruses (such as poxviruses (such as Xuantiantan strain, North American vaccine strain, Wyeth derivative strain, Listeria strains, Ankara derivatives, Copenhagen and New York strains), adenoviruses (such as those selected from Ad5, Ad11, Ad26, Ad35, AdC68), adeno-associated viruses, herpes simplex virus, measles virus, reovirus Virus, Rhabdovirus, Forest Encephalitis Virus, Influenza Virus, Respiratory Syncytial Virus, Poliovirus) vector vaccine, recombinant bacterial vector vaccine, virus-like particle vaccine, nanoparticle vaccine, cell vector vaccine.
  • nucleic acid vaccines DNA or RNA vaccines
  • recombinant protein subunit vaccines such as poxviruses (such as Xuantiantan strain, North American vaccine
  • the form of the product is suitable for an administration mode selected from the group consisting of intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drop, nebulized inhalation, genital tract, rectal, oral or the above-mentioned different inoculation modes. combination.
  • the product is suitable for sequential vaccination with a vaccine prepared from coronavirus S or S1, for example, the source of S or S1 is selected from the following group: SARS-CoV-2, SARS-CoV, MERS- CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV.
  • the product is suitable for sequential vaccination with a vaccine prepared from influenza virus HA or HA2, for example, the source of HA or HA2 is selected from the group consisting of: H1-H18.
  • coronavirus Viral early expression protein polyprotein ORF1ab
  • coronavirus membrane protein M
  • coronavirus nucleocapsid N
  • coronavirus envelope protein E
  • coronavirus spike protein S
  • the application of the immunogenic peptides and/or encoded molecules, vectors or cells herein in the preparation of products against coronavirus infection is also provided.
  • the product is a T cell vaccine or drug against coronavirus infection, especially a broad-spectrum T cell vaccine or drug against coronavirus.
  • the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, especially are SARS-CoV, MERS-CoV and SARS-CoV-2 viruses.
  • the product comprises: a conjugate or conjugate comprising an immunogenic peptide herein.
  • the product comprises: a pharmaceutically or immunologically acceptable carrier, excipient and/or adjuvant (for example, one or more adjuvants selected from the group consisting of aluminum adjuvant, cholera toxin and Its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligands, GM-CSF, IL -2, IL-3, IL-7, IL-11, IL-12, IL-18, IL-21.
  • adjuvants selected from the group consisting of aluminum adjuvant, cholera toxin and Its subunits, oligodeoxynucleotides, manganese ion adjuvant, colloidal manganese adjuvant, Freund's adjuvant, MF59 adjuvant, QS-21 adjuvant, Poly I:C and other TLR ligand
  • the product is selected from the group consisting of nucleic acid vaccines (DNA or RNA vaccines), recombinant protein subunit vaccines, recombinant viruses (such as poxviruses (such as Xuantiantan strain, North American vaccine strain, Wyeth derivative strain, Lister strain, Ankara-derived strains, Copenhagen strains and New York strains of poxviruses), adenoviruses (such as adenoviruses selected from Ad5, Ad11, Ad26, Ad35, AdC68), adeno-associated viruses, herpes simplex virus, measles virus, reovirus, Rhabdovirus, forest encephalitis virus, influenza virus, respiratory syncytial virus, poliovirus) vector vaccine, recombinant bacterial vector vaccine, virus-like particle vaccine, nanoparticle vaccine, cell vector vaccine.
  • recombinant viruses such as poxviruses (such as Xuantiantan strain, North American vaccine strain, Wyeth derivative strain, Lister
  • the form of the product is suitable for an administration mode selected from the group consisting of intramuscular inoculation, intradermal inoculation, subcutaneous inoculation, nasal drop, nebulized inhalation, genital tract, rectal, oral or the above-mentioned different inoculation modes. combination.
  • the product is suitable for sequential vaccination with a vaccine prepared from coronavirus S or S1, for example, the source of S or S1 is selected from the following group: SARS-CoV-2, SARS-CoV, MERS- CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV.
  • the product is suitable for sequential vaccination with a vaccine prepared from influenza virus HA or HA2, for example, the source of HA or HA2 is selected from the following group: H1-H18.
  • the use of the immunogenic peptides herein in the preparation of products for the detection of coronavirus infection for example, the immunogenic peptides alone or with MHC-formed peptide-MHC complex monomers or Polymers (such as dimers, tetramers).
  • Figure 1 Overall genome information of the novel coronavirus.
  • Figure 2A pAdC68XY3-CoV T plasmid construction map
  • Figure 2B Enzyme digestion and identification diagram of adenovirus AdC68-CoV T.
  • mice used in the experiment were 6-8 week old female hACE2 + ICR mice, and the immunogen was adenovirus AdC68-CoV T.
  • Figure 3A ELISPOT method to detect the T cell response of mice at the first week after immunization.
  • the abscissa is the immune group, the ordinate is the number of IFN- ⁇ -secreting cells per million splenocytes, * means p ⁇ 0.05;
  • Figure 3B ELISPOT method to detect the T cell response of mice at the first week after immunization.
  • the abscissa is the name of a single peptide library, and the ordinate is the number of IFN- ⁇ -secreting cells per million splenocytes;
  • Figure 3C ELISPOT method to detect the T cell response of mice at the first week after immunization.
  • the abscissa is the name of the single peptide, and the ordinate is the number of IFN- ⁇ -secreting cells per million splenocytes.
  • R10 in the figure indicates that 10% FBS and 1% double-streptomycin (P.S.) were added to RIPM medium as a negative control.
  • FIG. 4 Responses of splenocytes from mice immunized with AdC68-CoV T to various coronavirus epitopes:
  • mice used in the experiment were 6-8 week old female hACE2 + ICR mice, and the immunogen was adenovirus AdC68-CoV T.
  • the ELISPOT method was used to detect the T cell responses of mouse splenocytes to various coronavirus epitopes in the first week after immunization.
  • the abscissa is the name of the single peptide, and the ordinate is the number of IFN- ⁇ -secreting cells per million splenocytes.
  • Figure 5A Construction map of recombinant plasmid pAdC68XY3-panCoV/Flu;
  • Figure 5B Identification of double enzyme digestion of adenovirus AdC68-panCoV/Flu genome.
  • Figure 6A Comparison of binding antibody titers between AdC68 group and AdC68-panCoV/Flu group
  • Figure 6B Comparison of neutralizing antibody titers between AdC68 group and AdC68-panCoV/Flu group;
  • Figure 6C Comparison of T cell responses between AdC68 group and AdC68-panCoV/Flu group
  • Figure 6D Comparison of body weight changes of mice after challenge between AdC68 group and AdC68-panCoV/Flu group;
  • Figure 6E Comparison of post-challenge mouse survival rates between AdC68 group and AdC68-panCoV/Flu group.
  • the present disclosure relates to the field of vaccines, in particular to the design and verification of a broad-spectrum anti-coronavirus T cell vaccine immunogen.
  • the results of animal experiments prove that the disclosed vaccine is safe, can generate a high level of T cell response, and can be used for the prevention and treatment of coronavirus.
  • the early expression protein polyprotein (ORF1ab) of coronavirus is selected as the main immunogen, combined with the analysis of S, E, M and N protein genes, after collecting 1370 coronavirus sequences through big data, from them Extract shared sequences, predict T cell epitopes, and combine highly immunogenic conserved regions to form T cell vaccine immunogens, which cover almost all current pathogenic coronaviruses.
  • “comprising”, “having” or “comprising” includes “comprising”, “consisting essentially of”, “consisting essentially of”, and “consisting of. 7-8consists of”; “consisting essentially of”, “consisting essentially of” and “consisting of” belong to “contains”, “has “ or the subconcept of "include”.
  • peptide refers to a molecule comprising an amino acid sequence of 2-200 amino acids linked by peptide bonds, but which in particular embodiments may comprise non-amino acid structures (eg, linked organic compounds).
  • the peptides described herein may comprise any of the 20 conventional amino acids or modified forms thereof, or may comprise unnatural amino acids introduced by chemical peptide synthesis or by chemical or enzymatic modification.
  • epitope refers to one or several parts of a protein or factor (which may define a conformational epitope) which is detected by an antibody or part thereof (Fab', Fab2', etc.) or present in B or T Receptors on the cell surface of lymphocytes specifically recognize and bind, and can induce an immune response through the binding.
  • T cell epitope refers to a portion of an antigenic protein or factor that is specifically recognized and bound by a cell surface receptor of a T cell.
  • T cell epitopes can be dominant, subdominant or recessive T cell epitopes, depending on the immune response elicited against the epitope. Dominance depends on the frequency with which the epitope is recognized by T cells and able to activate T cells among all possible T cell epitopes of the protein.
  • T cell epitopes are epitopes bound by MHC class I or MHC class II molecules.
  • T cell epitopes in protein sequences can be identified by functional experiments and/or one or more simulation prediction experiments.
  • the T cell epitopes present in the peptides herein may consist of 8-25 amino acids, 8-16 amino acids, or may consist of 8, 9, 10, 11, 12, 13, 14, 15 or 16 amino acids, for example 10, 11 or 12 amino acids.
  • the T-cell epitope of the immunogenic peptide herein may correspond to the native epitope sequence of the protein, or may be a modified form thereof, provided that the modified T-cell epitope is similar to the native T-cell epitope sequence and retains its ability to bind to MHC .
  • Modified T cell epitopes may have the same binding affinity as the native epitope for the MHC protein, but may also have a lower affinity. In a specific embodiment, the binding affinity of the modified peptide is not less than 10 times lower than that of the original peptide, more preferably not less than 5 times.
  • MHC refers to "major histocompatibility antigen”.
  • HLA human leukocyte antigen
  • HLA-A HLA-B
  • HLA-C HLA-DPA1
  • HLA-DPB HLA-DQA1
  • HLA-DQB HLA-DRA
  • HLA-DRB1 HLA-DRB1
  • MHC class I molecules are expressed on almost all nucleated cells. Peptide fragments presented in MHC class I molecules are recognized by CD8 + T lymphocytes (cytotoxic T lymphocytes or CTLs). CD8 + T lymphocytes often mature into cytotoxic effectors that can lyse cells bearing stimulating antigens. Class II MHC molecules are predominantly expressed on activated lymphocytes and antigen-presenting cells. Activation of CD4 + T lymphocytes (helper T lymphocytes or HTLs) using recognition of unique peptide fragments presented by class II MHC molecules, typically found on antigen presenting cells like macrophages or dendritic cells MHC molecules.
  • CD4 + T lymphocytes proliferate and secrete cytokines that support antibody-mediated responses through the production of IL-4 and IL-10 or cell-mediated responses through the production of IL-2 and IFN- ⁇ .
  • the immunogenic peptides of the present application may bind CD4 + T cells and/or CD8 + T cells.
  • a T cell epitope may consist solely of amino acids that bind to the groove of the major histocompatibility complex (MHC), or may comprise the same amino acids along with flanking amino acid residues. Such flanking residues do not contribute to the binding of the epitope to the MHC but "stick out" of the MHC groove. Flanking residues may be present at the N-terminus and/or C-terminus of the MHC binding portion of the T cell epitope.
  • MHC major histocompatibility complex
  • This article provides an immunogenic peptide, which is selected as the main immunogen by selecting the early expression protein polyprotein (ORF1ab), membrane protein, nucleocapsid protein, envelope protein and spike protein of various types of coronaviruses , after collecting a large number of (such as 1370) coronavirus sequences through big data, extract shared sequences from them, and perform T cell epitope prediction, and combine highly immunogenic conserved regions alone or in combination to form T cell immunogens.
  • the immunogenic peptides herein cover almost all current pathogenic coronaviruses.
  • the epitope prediction can be obtained by analyzing the coronavirus sequence and CD8 + /CD4 + T cells, for example, the epitope sequence herein can be obtained by further performing CD8 + T cell epitope prediction on the shared sequence described herein.
  • CD8 + T cell epitope prediction software is known in the art, including but not limited to the software provided by http://tools.immunepitope.org/main/tcell/ and http://www.syfpeithi.de/, etc. .
  • shared peptide and “shared sequence” are used interchangeably to refer to a peptide molecule comprising an amino acid sequence shared among major proteins of various coronaviruses.
  • Different types, different subtypes, and different strains of coronavirus sequences can be selected for analysis, for example, the sequences of coronaviruses selected from the following group can be analyzed: SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E , HCoV-OC43, HCoV-NL63, HCoV-HKU1, bat-CoV, especially SARS-CoV, MERS-CoV and SARS-CoV-2 viruses.
  • immunogenic peptide includes peptides designed and prepared by the above methods, which have the activity of stimulating T cell responses and have a broad spectrum of activity against various coronaviruses.
  • the immunogenic peptides herein may comprise one or more coronavirus shared epitopes.
  • the shared epitope herein can be one or more epitope peptides selected from the group consisting of: PLPDRWYFYYT (SEQ ID NO: 11, shared by single peptides 6 and 7), VVNKQFGAISS (SEQ ID NO: 12, Monopeptide 31 and 32 are shared), KPISAYAFLMA (SEQ ID NO: 13, single peptide 73 and 74 are shared), SYGPGNTFITD (SEQ ID NO: 14, single peptide 124 and 125 are shared), IKYYSIIPHSIR (SEQ ID NO: 15, single peptide 44 inclusive), IEDLLFDKVET (SEQ ID NO: 16, shared by peptides 13 and 14), SALQKIQDVVN (SEQ ID NO: 17, shared by peptides 13 and 14), ADDEGFITLKN (SEQ ID NO: 18, shared by peptides 80 and 81 ),
  • the immunogenic peptide herein may comprise: n coronavirus epitope sequences targeting T cells, where n is an integer ranging from 1 to 100.
  • the immunogenic peptides herein may comprise shared sequences of one or more coronavirus proteins.
  • the immunogenic peptides herein may comprise ORF1ab shared peptides, M protein shared peptides, N protein shared peptides, E protein shared peptides, and/or S protein shared peptides.
  • the immunogenic peptides herein may also contain other fragments that help to recognize and/or activate T cells and/or enhance T cell activity or enhance anti-coronavirus infection effects, such as immunoregulatory sequences, such as IL-2, IL-7 , IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibody, CTLA4 antibody, chemokine CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH, SopE, etc.
  • immunoregulatory sequences such as IL-2, IL-7 , IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibody, CTLA4 antibody, chemokine CXCL9, CXCL10, CXCL11,
  • the immunogenic peptides herein may be combined with other sequences that are co-expressed (eg, fusion expressed or expressed in separate reading frames) with the consensus sequence in addition to each other.
  • Other sequences can be used, for example but not limited to: expanding the antiviral spectrum and improving the ability to induce immune responses.
  • sequences are selected from, for example, immunogens against the following viruses: coronavirus (such as RBD or its modified sequence (such as terminal Cys modified RBD amino acid sequence)), influenza virus (such as HA2), HIV, rabies virus , swine fever virus, PRRS virus, measles virus, Ebola virus, herpes virus, arboviruses (Zika virus, Japanese encephalitis virus, forest encephalitis virus, dengue virus, Hantaan virus, Xinjiang hemorrhagic fever virus), especially immunogens that respond to activated B cells, to prepare compound vaccines that can simultaneously activate neutralizing antibodies and T cell responses.
  • coronavirus such as RBD or its modified sequence (such as terminal Cys modified RBD amino acid sequence)
  • influenza virus such as HA2
  • HIV such as RBD or its modified sequence (such as terminal Cys modified RBD amino acid sequence)
  • HA2 such as terminal Cys modified RBD amino acid sequence
  • HIV such as RBD or its modified sequence (
  • the co-expressed sequence is an immunomodulatory sequence selected from the group consisting of IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagellin, FimH and SopE.
  • immunomodulatory sequence selected from the group consisting of IL-2, IL-7, IL-12, IL-18, IL-21, GM-CSF, CD40L, CD40 stimulating antibody, PD-1 and PD-L1 antibodies, CTLA4 antibodies, chemokines CXCL9, CXCL10, CXCL11, CXCL12, CXCL3, XCL1, CCL4, CCL20, cholera toxin and its subunits, bacterial flagelli
  • the immunogenic peptides herein may be the product of chemical synthesis, or produced using recombinant techniques from prokaryotic or eukaryotic hosts (eg, bacteria, yeast, higher animal, insect, and mammalian cells).
  • prokaryotic or eukaryotic hosts eg, bacteria, yeast, higher animal, insect, and mammalian cells.
  • the introduction of unnatural amino acids is permitted herein.
  • cysteine residues may be replaced by other amino acids with thiol groups, such as mercaptovaline, homocysteine, or other natural or unnatural amino acids with thiol functionality.
  • cysteine residues should not occur as part of cysteine disulfide bridges.
  • cysteine residues may be modified (eg, by methylation), since methylated cysteines are converted in vivo to cysteines containing free thiol groups.
  • variant forms of the immunogenic peptides herein include (but are not limited to): deletions, insertions of one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acids And/or substitution; adding one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminal and/or N-terminal.
  • substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein or polypeptide.
  • the immunogenic peptides herein may also comprise amino acid sequences (or other organic compounds) that facilitate uptake of the peptide into endosomes for processing and presentation within MHC class II determinants.
  • the immunogenic peptides herein may also comprise, for example, endosomal targeting sequences.
  • endosomal targeting sequences are contained within the cytoplasmic tail of eg gp75 protein, human CD3 ⁇ protein, HLA-BM ⁇ , DEC205 receptor.
  • Further examples of peptides as endosomal sorting signals are disclosed in the review by Bonifacio and Traub (2003) Annu. Rev. Biochem. 72, 395-447.
  • the term "immunogenic peptide encoding molecule” refers to a sequence encoding an immunogenic peptide described herein. In some embodiments, it may comprise, for example, the nucleotide sequence of SEQ ID NO: 2, 4, 6, 8 or 10; molecules that hybridize to these sequences under stringent conditions, or nucleosides that are highly homologous to the above-mentioned molecules Acid molecules, so long as they efficiently encode and express the desired immunogenic peptide. It should be understood that, after obtaining the amino acid sequences of the epitope peptides and immunogenic peptides herein, conventional technical means in the art can be used to obtain their coding sequences and optimize them. Therefore, one or more encoding molecules for the same epitope peptide and/or immunogenic peptide can be provided, as long as the encoding molecule can correctly and effectively express the epitope peptide and/or immunogenic peptide.
  • stringent conditions refers to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60° C.; or (2) hybridization with There are denaturing agents, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, etc.; or (3) only the identity between the two sequences is at least 50%, preferably 55% More than 60%, more than 65%, more than 70%, more than 75%, more than 80%, more than 85% or more than 90%, more preferably more than 95%, hybridization occurs.
  • the encoded molecules herein can usually be obtained by PCR amplification, recombination or artificial synthesis.
  • primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used.
  • the library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.
  • the present invention also relates to vectors comprising molecules encoding immunogenic peptides, and host cells genetically engineered with such vectors.
  • the coding sequences of the present invention can be used to express or recombinantly produce immunogenic peptides by conventional recombinant DNA techniques (Science, 1984; 224:1431). Generally speaking, there are the following steps:
  • vector and "recombinant expression vector” are used interchangeably, and refer to bacterial plasmids, bacteriophages, yeast plasmids, animal cell viruses, mammalian cell viruses or other vectors well known in the art. In short, any plasmid and vector can be used as long as it can be replicated and stabilized in the host.
  • An important feature of expression vectors is that they usually contain an origin of replication, a promoter, marker genes, and translational control elements.
  • expression vectors containing the coding sequence and appropriate transcriptional/translational control signals can be used to construct expression vectors containing the coding sequence and appropriate transcriptional/translational control signals. These methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology and the like. Said DNA sequence can be operably linked to an appropriate promoter in the expression vector to direct mRNA synthesis.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • Vectors containing the above-mentioned appropriate DNA sequences and appropriate promoters or control sequences can be used to transform appropriate host cells so that they can express proteins or polypeptides.
  • the host cell may be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as an animal cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; animal cells and the like.
  • Escherichia coli bacterial cells and mouse dendritic cells are preferably used as host cells.
  • kits comprising the immunogenic peptides of the present invention, their coding sequences, vectors or host cells, such as T cell vaccines, drugs, pharmaceutical compositions or Kits, conjugates, conjugates, etc.
  • active substance or active substance of the invention are used interchangeably to refer to the immunogenic peptide, its coding sequence, vector or host cell herein.
  • a vaccine composition comprising the immunogen
  • the vaccine composition comprises a formulation of epitopes, immunogenic peptides and/or nucleic acid molecules of the present disclosure in a form capable of being administered to a vertebrate, preferably a mammal, and which induces a protective immune response that increases immunity to prevent and/or alleviate the disease and/or at least one symptom thereof.
  • the term "protective immune response” or “protective response” refers to an immune response mediated by an immunogen against an infectious agent or disease, exhibited by a vertebrate such as a human, that prevents or alleviates infection or at least one disease symptom.
  • vertebrate or “subject” or “patient” refers to any member of the subphylum Chordate, including but not limited to: humans and other primates, including non-human primates such as chimpanzees and other apes and monkeys species; domestic animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds including domesticated, wild and game birds such as chickens, turkeys Chickens and other quail birds, ducks, geese.
  • mammals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs
  • birds including domesticated, wild and game birds such as chickens, turkeys Chickens and other quail birds, ducks, geese.
  • mamammal and “animal” are included in this definition and are intended to cover adult, juvenile and newborn individuals.
  • the vaccine herein can be recombinant protein vaccine, recombinant DNA vaccine, recombinant virus vector vaccine (such as adenovirus vector, poxvirus vector, adeno-associated virus vector, herpes simplex virus vector, cytomegalovirus vector), recombinant bacterial vector vaccine, recombinant yeast Vector vaccines or recombinant virus-like particle vaccines.
  • the vaccine herein is selected from recombinant DNA vaccines, recombinant adenovirus vectors, recombinant poxvirus vectors or a combination of one or two or three of them.
  • the vaccine compositions herein comprise an effective amount of an immunogen herein.
  • the immunogen is included in the vaccine compositions of the present disclosure in an amount sufficient to achieve the desired biological effect.
  • effective amount generally refers to an amount of an immunogen that can induce a protective immune response sufficient to induce immunity to prevent and/or alleviate an infection or disease and/or to reduce at least one symptom of an infection or disease.
  • Adjuvants may also be included in the vaccines herein.
  • Adjuvants known to those of ordinary skill in the art may be used, such as those described in Vogel et al., "A Compendium of Vaccine Adjuvants and Excipients” (2nd Ed.), which is hereby incorporated by reference in its entirety.
  • known adjuvants include, but are not limited to: complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum hydroxide adjuvant, lipopolysaccharide (LPS), RIBI adjuvant, MF-59, and the like.
  • the vaccine composition herein may also include pharmaceutically acceptable carriers, diluents, preservatives, solubilizers, emulsifiers and other auxiliary materials.
  • pharmaceutically acceptable carriers include, but are not limited to, water for injection, saline solution, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, and combinations thereof.
  • Pharmaceutically acceptable carriers, diluents and other excipients can be found, for example, in Remington's Pharmaceutcal Sciences.
  • the form of the vaccine composition herein may be suitable for systemic or local (especially intrarespiratory) administration.
  • Methods of administering the vaccine composition include, but are not limited to: parenteral administration (e.g., intradermal, intramuscular, intravenous, and subcutaneous), epidural administration, mucosal administration (e.g., intranasal and oral or administered by the pulmonary route).
  • parenteral administration e.g., intradermal, intramuscular, intravenous, and subcutaneous
  • epidural administration e.g., epidural administration
  • mucosal administration e.g., intranasal and oral or administered by the pulmonary route.
  • the vaccines herein prevent, eliminate or reduce coronavirus infection or at least one symptom thereof in a subject, such as respiratory symptoms (such as nasal congestion, sore throat, hoarseness), headache, cough, sputum, fever, cough Sound, wheezing, dyspnea, pneumonia due to infection, severe acute respiratory syndrome, renal failure, etc.
  • respiratory symptoms such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • headache such as nasal congestion, sore throat, hoarseness
  • cough such as cough, sputum, fever, cough Sound, wheezing, dyspnea
  • pneumonia due to infection, severe acute respiratory syndrome, renal failure, etc.
  • the present invention also relates to an immunoconjugate (also called an immunoconjugate), which comprises the immunogen herein and other substances coupled thereto.
  • the other substances can be targeting substances (such as a part that specifically recognizes a specific target), therapeutic substances (such as drugs, toxins, cytotoxic agents), and labeling substances (such as fluorescent markers, radioactive isotope labels).
  • a combination product which includes the host cell and/or vaccine of the present disclosure, and may also contain one or more vaccines that help to better prevent and/or treat coronavirus infection or its symptoms. function or other substances that enhance the stability of the aforementioned substances.
  • other substances may include other vaccines against coronavirus S or S1, such as those from including but not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV - HKU1, S or S1 vaccines of bat-CoV; other active substances for diseases or disorders benefiting from T cell activation and/or memory immune response with T cells.
  • the immunogenic peptides herein may be alone or form peptide-MHC complex monomers or multimers (eg, dimers, tetramers) with MHC.
  • the immunogenic peptides herein can be complexed with HLA-A2 heavy chain proteins, light chain proteins (such as ⁇ 2m protein) to form peptide-MHC complex monomers, and can optionally be formed from complex monomers. aggregates to form complex polymers.
  • the kit can be used in the detection of coronavirus.
  • Vaccination methods that can be used include but are not limited to: systemic immunization methods, such as intramuscular injection, subcutaneous injection, and intradermal injection; intrarespiratory immunization methods, such as nebulization, nasal drip, etc.
  • systemic immunization methods such as intramuscular injection, subcutaneous injection, and intradermal injection
  • intrarespiratory immunization methods such as nebulization, nasal drip, etc.
  • the initial immunization adopts systemic vaccination or intrarespiratory vaccination, preferably systemic vaccination.
  • the interval between each two vaccinations is at least 1 week, such as 2 weeks, 4 weeks, 2 months, 3 months, 6 months or longer.
  • DNA vaccines are used for primary immunization and cellular vaccines are used for one or more booster immunizations.
  • the immunization method of the present disclosure can adopt the "prime-boost” or “prime-boost-reboost” method, and can adopt a single systemic immunization or respiratory local immunization, or a combination of the two immunization methods.
  • a recombinant DNA vaccine is used for systemic primary immunization to establish a systemic immune response, followed by one or more immune boosts with a cellular vaccine.
  • the vaccine-specific immune response can be effectively established in the local respiratory tract and systemic system by adopting the immunization method herein, which helps to enhance the effectiveness of vaccine protection.
  • Providing a combination product herein in the form of a pharmaceutical pack or kit may, for example, pack one or more of the vaccine compositions herein, or one or more components thereof, in one or more containers, such as in the indicated combination in a hermetically sealed container such as an ampoule or sachet.
  • the vaccine composition can be provided in the form of liquid, sterile lyophilized powder or anhydrous concentrate, which can be diluted, reconstituted and/or formulated with an appropriate liquid (such as water, saline, etc.) to the appropriate concentration and form of the subject.
  • the experimental animals, immunization methods, immunogens and detection methods involved in the experiments of the embodiments are as follows:
  • HFH4-hACE2-C57BL/6 mice 6-8 week-old male HFH4-hACE2-C57BL/6 mice were obtained by transferring the hACE2 gene into mice using the HFH4 pulmonary ciliated epithelial cell-specific promoter (for specific construction methods, refer to Menachery, V.D, etc., SARS-like WIV1- CoV poised for human emergence. Proc. Natl. Acad. Sci. U.S.A. 113, 3048-3053 (2016); Ostrowski LE, Hutchins JR, Zakel K, O′Neal WK. Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter. Mol Ther. 2003; 8(4): 637-645).
  • mice The left and right hind limbs of mice were injected intramuscularly or nasally. Specific dosage see embodiment.
  • MHC class I and class II epitopes are predicted at the same time, a total of 9 MHC class I subtypes, 13 MHC class II subtypes, MHCpan is selected (default parameters) Make predictions.
  • CP(aa n ,...,aa n+m ) represents a chain of shared peptides
  • fre(50%aa)>[0.7(m+1)] represents the number of amino acid preferences of each site exceeding 50% It needs to be greater than 70% of the sequence length
  • num(gap) ⁇ 20% (m+1) means that the number of gaps is less than 20% of the full length.
  • CoV T The final designed shared sequence (i.e. CoV T) was collected from 1370 coronavirus sequences.
  • SEQ ID NO: 9 CoV T amino acid sequence, 1238Aa
  • SEQ ID NO: 10 CoV T coding sequence, 3714 Nt ).
  • Recombinant adenovirus vector vaccines AdC68, AdC68-CoV T, AdC68-panCoV/Flu.
  • Recombinant adenovirus vector vaccine 5E10vp/mouse (intramuscular injection of left and right hind limbs, 50 ⁇ L each), 100 ⁇ L (intramuscular injection); 5E10vp/mouse, 30 ⁇ L (nasal drop).
  • the above method is also used for the preparation of panCoV/Flu.
  • mice were killed by decapitation Two weeks after the last immunization, before the mice were killed by decapitation, the peripheral whole blood of the mice was collected by picking the eyeballs, collected in a 1.5mL EP tube, and allowed to coagulate naturally at room temperature, and the coagulated mice Serum was centrifuged at 7000g for 15min. Transfer the mouse serum to a new 1.5mL EP tube. Before the experiment, the sample needs to be inactivated at 56°C for 30 minutes to destroy the complement activity in the serum. Centrifuge briefly before inactivation to avoid residual sample on the tube wall and bottle cap. The liquid level of the water bath should not exceed the liquid level of the sample, but not exceed the cap of the bottle.
  • Monopeptide 307 and monopeptide 308 have reached the end of SEQ ID NO: 9, and their sequences correspond to SEQ ID NO: 1226-1238 and 1224-1238, respectively.
  • peptide library 31 peptide libraries were established with the above-mentioned single peptides. Each peptide library in peptide libraries 1-30 contained 10 single peptides, and peptide library 31 contained 8 single peptides (ie, single peptides 301-308).
  • Example 1 Construction and identification of pAdC68XY3-CoV T adenovirus expression vector
  • the constructed recombinant plasmid pAdC68XY3-CoV T was linearized with the restriction endonuclease Pac 1 in a water bath at 37°C for 3.5 hours, and the endonuclease was inactivated at 65°C.
  • the adenovirus sample collected above was infected into 293A cells in one T175 culture flask, collected after 24 hours, placed at -80°C for three times, and then infected into 293A cells in six T175 culture flasks, and so on for a large number of Amplify, when the amplification reaches 36 T175 culture flasks, collect the cell pellet, discard the supernatant, resuspend in about 10mL of serum-free and non-resistant DMED medium, freeze and thaw three times at -80°C, and use cesium chloride density
  • the adenovirus was purified by gradient centrifugation, and stored at -80°C after aliquoting.
  • Nanogrop 2000 (Thermo Scientific) was used to measure the OD 260 value of each tube of eluate, and the eluate with OD 260 greater than 2 was combined, 10% sterile glycerin was added, and aliquoted;
  • Nanogrop 2000 was used to measure the OD 260 value of the adenovirus solution after aliquoting, and the OD 260 value ⁇ 1.1 ⁇ 10 12 /mL was the final titer of the purified adenovirus.
  • AdC68 and AdC68-CoV T were used to immunize hACE2 + ICR mice, and both injections were intramuscularly injected, with a dose of 5E10vp/mouse.
  • One week after completing the immunization ie, 4 weeks after the first immunization, the level of T cell response induced by the immunization combination against the CoV T peptide library was evaluated.
  • mice were randomly divided into two groups, which were named AdC68 group and AdC68-CoV T group according to the immunogen.
  • the specific immune combination is shown in Table 2:
  • the total T cell response of the AdC68-CoV T group against the CoV T peptide library is shown in Figure 3A: the average number of IFN- ⁇ -secreting cells per million splenocytes was around 7530, and the highest could reach 13595, which was significantly higher than AdC68.
  • Epitope A PLPDRWYFYYT (SEQ ID NO: 11, shared by monopeptide 6 and 7), N protein from coronavirus;
  • Epitope B KPISAYAFLMA (SEQ ID NO: 13, shared by monopeptide 73 and 74) ORF1ab protein from coronavirus;
  • Both epitope A and epitope B can find corresponding sequences in SARS, MERS and SARS-CoV-2 viral proteins, that is, the vaccine AdC68-CoV T we constructed can induce a broad-spectrum anti-coronavirus T cell response.
  • AdC68-CoV T can induce a broad-spectrum T cell response against coronaviruses, including SARS, MERS, SARS-CoV-2, etc.
  • Example 3 Responses of splenocytes of mice to various coronavirus epitopes after AdC68-CoV T immunization
  • mice immunized with the synthetic natural epitopes of various coronaviruses could also generate T cell responses, which further verified that our T cell vaccines could indeed respond to a variety of coronaviruses .
  • a broad-spectrum T cell response against coronavirus can indicate its broad-spectrum preventive and therapeutic effect on coronavirus.
  • the virus infects the body, it can rapidly activate the antigen-specific T cell response and start the cell killing function.
  • the early gene of the virus which can be killed by the killing effect of CD8 T cells before the virus can form complete virus particles.
  • individual peptides have preventive and therapeutic effects.
  • Example 4 Construction and identification of AdC68-panCoV/Flu adenovirus expression vector
  • CoV T as a T cell vaccine.
  • CoV T gene and RBD-HA2-CD8TM gene Fusion expresses the RBD protein of SARS-CoV-2, the HA2 region of H7N9 and CD8TM, and its nucleotide sequence and encoded amino acid sequence are respectively shown in SEQ ID NO: 34 and 35) Loaded on the vector plasmid pAdC68XY3, constructed Adenovirus AdC68-panCoV/Flu, and identified by enzyme digestion.
  • panCoV/Flu gene (SEQ ID NO: 36), which contains (a) a sequence encoding SopE (which enables T cell immunogens to quickly transfer to the proteasome and enter the degradation process after translation and expression, and better Presenting T cell epitope) and the front part of CoV T (SEQ ID NO: 37), (b) the IRES and KOZAK sequences adjacent to it, and (c) including the encoding HASP signal peptide, the terminal Cys modified RBD sequence, and the HA2 sequence and CD8 hinge region and rear portion of CD8TM (RBD-HA2-CD8TM) (SEQ ID NO: 38).
  • the synthetic fragment was directly loaded on the vector plasmid pAdC68XY3 to obtain the panCoV/Flu adenovirus expression vector pAdC68XY3-panCoV/Flu, and the plasmid construction map is shown in Figure 5A.
  • the constructed recombinant plasmid pAdC68XY3-panCoV/Flu was linearized with the restriction endonuclease Pac 1 in a water bath at 37°C for 3.5 hours, and the endonuclease was inactivated at 65°C.
  • the adenovirus sample collected above was infected into 293A cells in one T175 culture flask, collected after 24 hours, placed at -80°C for three times, and then infected into 293A cells in six T175 culture flasks, and so on for a large number of Amplify, when the amplification reaches 36 T175 culture flasks, collect the cell pellet, discard the supernatant, resuspend in about 10mL of serum-free and non-resistant DMED medium, freeze and thaw three times at -80°C, and use cesium chloride density
  • the adenovirus was purified by gradient centrifugation, and stored at -80°C after aliquoting.
  • Example 5 Immunogenicity of AdC68-panCoV/Flu adenovirus in HFH4-hACE2 mice and bat coronavirus SHC014 challenge protection test
  • mice HFH4-hACE2 mice
  • the mice were randomly divided into two groups, which were named AdC68 group and AdC68-panCoV/Flu group according to the immunogen.
  • the specific immunization combination is shown in Table 4.
  • the immunization method is the first intramuscular injection, the dose is 5E10vp/mouse, the second intramuscular injection (5E10vp/mouse) plus nasal drops (5E10vp/mouse), the total dose is 1E11vp /mouse.
  • Challenge procedure 3 weeks after the end of immunization, the mice were challenged, and the weight change and survival of the mice were continuously observed after the challenge.
  • the binding antibody titer against RBD and the neutralizing antibody titer against SARS-CoV-2 pseudovirus were detected, as shown in Figure 6: the average binding antibody titer of the AdC68-panCoV/Flu group was 10, 160, the highest can reach 25,600, which is significantly different from the AdC68 group (Fig. 6A, P ⁇ 0.01); the average neutralizing antibody titer of the AdC68-panCoV/Flu group is 166, and the highest can reach 528, which is significantly different from the AdC68 group (Fig. 6B, P ⁇ 0.01).
  • the vaccine can induce both T cell response and antibody response.
  • mice were challenged three weeks after the immunization, the selected strain was bat coronavirus S HC014, the dose was 1E5 TCID 50 /mouse, and the method was intranasal drip.
  • the weight and survival of the mice were continuously observed, and it was found that the weight of the mice in the AdC68 group decreased significantly on the fifth day, and this decline continued until all of them died, and the weight of the mice that died on the ninth day all decreased by more than 20%.
  • AdC68-panCoV/Flu showed only slight body weight loss after inoculation, characterized by a later onset (on day 8) and a higher nadir (approximately 8% body weight loss on day 9), And the body weight began to increase on the tenth day (Fig. 6D). Survival data at the end of the observation period also showed a significant difference between the AdC68-panCoV/Flu group and the AdC68 group, with the former having a survival rate of 60% and the latter having no survival (Fig. 6E). These data provide preliminary evidence for the potential of AdC68-panCoV/Flu as a universal coronavirus vaccine.
  • Golden hamster male, purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd.
  • Immunization procedure the golden hamsters were randomly divided into 5 groups, named group 1, group 2, group 3, group 4 and group 5 respectively.
  • group 1, group 2, group 3, group 4 and group 5 The specific immunization combination is shown in Table 5, and the immunization method is intramuscular injection (i.m.) plus intranasal (i.n.), see the table for details.
  • AdC68-panCoV/Flu provided effective protection against coronavirus challenge in golden hamsters.
  • mice SMOC ACE2-transgenic mice (C57BL/6-Tgtn(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)Smoc), female.
  • Immunization procedure the golden hamsters were randomly divided into 3 groups, named group 1, group 2 and group 3 respectively.
  • group 1, group 2 and group 3 The specific immunization combination is shown in Table 6, and the immunization method is intramuscular injection plus nasal drops, see the table for details.
  • mice were challenged with the virus. After the challenge, the mice were continuously observed, and the body weight change and survival were recorded. On the third day after the challenge, 4 mice in each group were sacrificed, and the lung tissues were collected. The left half lung tissue (one large lobe) of each mouse was fixed with 4% paraformaldehyde for 48 hours and then made pathological sections (HE staining and histochemistry); the right half lung tissue (four small lobes) of each mouse was grinded Viral load qPCR RNA copies or viral titer PFU/ml.
  • AdC68-panCoV/Flu provided effective protection against coronavirus challenge in hACE2+C57BL/6 mice.

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Abstract

本发明提供了一种诱导广谱抗冠状病毒的T细胞疫苗免疫原及其应用。具体而言，提供了免疫原性肽，其包含选自各种冠状病毒早期表达蛋白多聚蛋白、膜蛋白、核衣壳蛋白、囊膜蛋白和刺突蛋白的一种或多种共享肽；及其编码分子、载体或宿主细胞、产品。本发明的疫苗可用于各种冠状病毒的预防和治疗。

Description

一种诱导广谱抗冠状病毒的T细胞疫苗免疫原及其应用 技术领域
本发明属于生物技术和医学领域，尤其是抗病毒疫苗。具体而言，本发明涉及一种诱导广谱抗冠状病毒的T细胞疫苗免疫原、及其制备和应用。
背景技术
冠状病毒(Coronavirus)，是一类在动物与人类之间传播的人畜(禽)共患的单链RNA病毒，可感染哺乳动物、鸟类，引起牛和猪的消化道疾病或鸡的上呼吸道疾病。冠状病毒在自然界中很常见，据悉，15％到30％的普通感冒都是由这些病毒导致的。
冠状病毒是由单一的核糖核酸(RNA)构成，这种RNA和N蛋白共同组成病毒。由于其遗传物质是RNA，因此非常容易出现变异。到目前为止，大约有45种不同冠状病毒株被发现，它们能够感染多种哺乳动物和鸟类。至于可感染人的冠状病毒，除2019新型冠状病毒(2019-nCoV，现由国际病毒分类委员会正式命名为SARS-CoV-2)，还有另外六种。其中四种会引起普通的感冒症状，分别为229E、NL63、OC43、HKU1；剩下的两种则是令人闻之丧胆的SARS(非典型肺炎病毒)以及MERS(中东呼吸综合征冠状病毒)，这两种病毒曾入选为人类历史上最可怕的病毒之一。2003年SARS爆发期间，有超过8,000人被感染，死亡率约为10％；而截止2019年12月，中东呼吸综合征(MERS)确诊2,468例，死亡851例，死亡率约为34.5％；至于现在的新型冠状病毒(SARS-CoV-2)则在全球范围内引起了大流行，截止2020年12月14日，已确诊70,829,855例，死亡1,605,091例，死亡率约为2.3％，至今仍未能很好的控制疫情。从冠状病毒相关疾病爆发的规律来看，每隔几年就会有一种冠状病毒出现较大范围内的流行，甚至出现全球范围内的大爆发。因此，设计出广谱抗冠状病毒的疫苗至关重要。
自从Doherty和Zinkernagel发现细胞毒性T淋巴细胞(CTL)能够杀伤外源微生物感染细胞，而且这种杀伤作用依赖于CTL对外源多肽和自身分子(MHC)的双重识别之后，越来越多的研究表明：病毒特异性的CTL介导的细胞免疫 具有清除病毒的功能，是宿主防御病毒感染的主要机制之一。Doherty和Zinkernagel也因他们的发现于1996年获得了生理和医学诺贝尔奖。正因为此，CTL表位及CTL介导的细胞免疫应答的研究受到越来越多的关注，后续则提出了T细胞疫苗的概念。
国际卫生组织(WHO)的《COVID-19全球研究路线图》中指出：冠状病毒疫苗免疫后的动物，再次暴露于活病毒时，可能发生更严重的症状。疫苗免疫产生的非中和抗体或较低的抗体水平可能会引起抗体依赖性增强效应(antibody-dependent enhancement，ADE)，增强病毒致病性。已有的科学研究显示，有些无症状COVID-19感染者虽然没有明显的抗体免疫反应，但是体内存在针对新冠病毒的记忆T细胞。因此，为了减少ADE副作用，同时设计广谱抗冠状病毒的疫苗，T细胞疫苗或许能够作为其发展方向。
发明内容
本发明正是提供了可用于广谱抗冠状病毒感染的免疫原性肽、其产品、制备方法及应用。
在本发明的第一方面中，提供了一种分离的免疫原性肽，其包含选自下组的一种或多种冠状病毒蛋白共享肽：
(a)冠状病毒早期表达蛋白多聚蛋白(ORF1ab)的共享肽；
(b)冠状病毒膜蛋白(M)的共享肽；
(c)冠状病毒核衣壳(N)蛋白的共享肽；
(d)冠状病毒囊膜蛋白(E)蛋白的共享肽；和
(e)冠状病毒刺突蛋白(S)蛋白的共享肽。
在一些实施方式中，免疫原性肽包含选自下组中的一个或多个肽段：SEQ ID NO：11～SEQ ID NO：29。
在一些实施方式中，由所述免疫原性肽激活的T细胞特异性结合于选自下组的冠状病毒表位：PLPDRWYFYYT(SEQ ID NO：11)；KPISAYAFLMA(SEQ ID NO：13)；LSPRWYFYYL(SEQ ID NO：30)；LAPRWYFYYTG(SEQ ID NO： 31)；VPAYSFLPG(SEQ ID NO：32)；和/或APISAMVRMYIFFA(SEQ ID NO：33)。
在一些实施方式中，共享肽来源于选自下组的冠状病毒：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV。
在一些实施方式中，ORF1ab的共享肽包含SEQ ID NO：1所述氨基酸序列，例如，所述ORF1ab的共享肽的氨基酸序列如SEQ ID NO：1所示。
在一些实施方式中，M蛋白的共享肽包含SEQ ID NO：3所述氨基酸序列，例如，所述M蛋白的共享肽的氨基酸序列如SEQ ID NO：3所示。
在一些实施方式中，N蛋白的共享肽包含SEQ ID NO：5所述氨基酸序列，例如，所述N蛋白的共享序列的氨基酸序列如SEQ ID NO：5所示。
在一些实施方式中，S蛋白的共享肽包含SEQ ID NO：7所述氨基酸序列，例如，所述S蛋白的共享肽的氨基酸序列如SEQ ID NO：7所示。
在一些实施方式中，免疫原性肽还包含：两种或以上所述共享肽的连接肽，所述共享肽之间包含或不包含接头序列，例如所述接头序列选自下组：(G4S) n(n＝1～8，例如(G4S) 3、G4S)、GSAGSAAGSGEF、(Gly) 6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly) 8、EGKSSGSGSESKST、IRES、P2A、T2A。
在一些实施方式中，免疫原性肽还包含与共有序列共表达(例如融合表达或单独阅读框表达)的其他序列，所述其他序列用于例如扩大抗病毒谱、提高免疫应答诱导能力，所述其他序列选自例如针对如下病毒的免疫原：冠状病毒(例如RBD或其修饰序列(如末端Cys修饰RBD氨基酸序列))、流感病毒(例如HA2)、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、新疆出血热病毒)，尤其是与活化B细胞应答的免疫原，制备能够同时激活中和抗体与T细胞应答的复合疫苗。
在一些实施方式中，与本申请的免疫原性肽共表达的序列为选自下组的免疫调节序列：IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛 蛋白、FimH及SopE。
在一些实施方式中，免疫原性肽包含SEQ ID NO：9所示的氨基酸序列，例如，所述免疫原性肽的序列如SEQ ID NO：9所示；或者，其包含SEQ ID NO：37和/或38所示的氨基酸序列，例如，所述免疫原性肽的序列如SEQ ID NO：37和SEQ ID NO：38所示。
在本文的一些方面中，提供了本文免疫原性肽的编码分子，包含所述编码分子的载体和/或包含所述编码分子或载体的宿主细胞。
在一些实施方式中，所述编码分子包含选自下组中的一个或多个核苷酸分子：SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：36；可任选地，所述核苷酸分子之间包含或不包含接头序列，所述编码分子包含或不包含前导序列，所述核苷酸分子包含或不包含标签，例如His-tag、AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、Myc-tag、S-tag、SBP-tag、Sof-tag 1、Sof-tag3、Strep-tag、TC tag、V5tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag。
在一些方面中，提供了一种产品，其包含：本文的免疫原性肽和/或编码分子、载体或细胞；和可任选的，药学上或免疫学上可接受的载体、赋形剂和/或佐剂(例如选自下组的一种或多种佐剂：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21)。
在一些实施方式中，所述产品为例如抗冠状病毒感染的T细胞疫苗或药物，尤其是针对冠状病毒的广谱T细胞疫苗或药物，所述冠状病毒选自下组：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，尤其是SARS-CoV、MERS-CoV和SARS-CoV-2病毒。
在一些实施方式中，所述产品包含本文所述的免疫原性肽的缀合物或偶联物。
在一些实施方式中，所述产品选自：核酸疫苗(DNA或RNA疫苗)、重组 蛋白亚单位疫苗、重组病毒(例如痘病毒(如选白天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株的痘病毒)、腺病毒(如选自Ad5、Ad11、Ad26、Ad35、AdC68的腺病毒)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒)载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。
在一些实施方式中，所述产品的形式适于选自下组的给予方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合。
在一些实施方式中，所述产品适于与冠状病毒S或S1所制备的疫苗进行前后序贯接种，例如来源选自下组的S或S1：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV。
在一些实施方式中，所述产品适于与流感病毒HA或HA2所制备的疫苗进行前后序贯接种，例如来源选自下组的HA或HA2：H1-H18。
在本文的一些方面中，提供了一种获得本文的免疫原性肽的方法，所述方法包括：
i)对各种冠状病毒(例如SARS-CoV、MERS-CoV和SARS-CoV-2病毒)的一种或多种选自下组的蛋白质进行大数据分析，提取出其共享肽序列信息：冠状病毒早期表达蛋白多聚蛋白(ORF1ab)、冠状病毒膜蛋白(M)、冠状病毒核衣壳(N)蛋白、冠状病毒囊膜蛋白(E)蛋白和冠状病毒刺突蛋白(S)蛋白；
ii)对所得共享肽序列信息进行T细胞表位预测，获得具有免疫原性的保守区域信息；
iii)根据所述保守区域信息设计并制备免疫原性肽或其编码序列；
iv)可任选地，对所得免疫原性肽或其编码序列进行组合或将其与共表达(例如融合表达或单独阅读框表达)的其他序列进行组合，所述其他序列用于例如扩大抗病毒谱、提高免疫应答诱导能力。
在本文的一些方面中，还提供了本文的免疫原性肽和/或编码分子、载体或细胞在制备抗冠状病毒感染的产品中的应用。
在一些实施方式中，所述产品为抗冠状病毒感染的T细胞疫苗或药物，尤其是针对冠状病毒的广谱T细胞疫苗或药物。在一些实施方式中，所述冠状病毒选自下组：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，尤其是SARS-CoV、MERS-CoV和SARS-CoV-2病毒。在一些实施方式中，所述产品包含：含本文免疫原性肽的缀合物或偶联物。
在一些实施方式中，产品包含：药学上或免疫学上可接受的载体、赋形剂和/或佐剂(例如选自下组的一种或多种佐剂：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21。
在一些实施方式中，产品选自：核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒(例如痘病毒(如选白天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株的痘病毒)、腺病毒(如选自Ad5、Ad11、Ad26、Ad35、AdC68的腺病毒)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒)载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。
在一些实施方式中，所述产品的形式适于选自下组的给予方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合。
在一些实施方式中，所述产品适于与冠状病毒S或S1所制备的疫苗进行前后序贯接种，例如来源选自下组的S或S1：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV。
在一些实施方式中，所述产品适于与流感病毒HA或HA2所制备的疫苗进行前后序贯接种，例如来源选自下组的HA或HA2：H1～H18。
在本文的一些方面中，还提供了本文免疫原性肽在制备检测冠状病毒感染的产品中的应用，例如，所述免疫原性肽单独或与MHC形成的肽-MHC复合 物单聚体或多聚体(如二聚体、四聚体)。
本领域的技术人员可对前述的技术方案和技术特征进行任意组合而不脱离本发明的发明构思和保护范围。本发明的其它方面由于本文的公开内容，对本领域的技术人员而言是显而易见的。
附图说明
下面结合附图对本发明作进一步说明，其中这些显示仅为了图示说明本发明的实施方案，而不是为了局限本发明的范围。
图1：新型冠状病毒整体基因组信息。
图2：CoV T腺病毒的构建及验证：
图2A：pAdC68XY3-CoV T质粒构建图谱；
图2B：腺病毒AdC68-CoV T酶切鉴定图。
图3：AdC68-CoV T在hACE2 +ICR小鼠体内的免疫原性：
实验所用小鼠为6-8周龄雌性hACE2 +ICR小鼠，免疫原为腺病毒AdC68-CoV T。
图3A：ELISPOT方法检测免疫结束后第1周小鼠的T细胞应答。横坐标为免疫组别，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量，*表示p＜0.05；
图3B：ELISPOT方法检测免疫结束后第1周小鼠的T细胞应答。横坐标为单个肽库名称，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量；
图3C：ELISPOT方法检测免疫结束后第1周小鼠的T细胞应答。横坐标为单肽名称，纵坐标为每百万个脾细胞中分泌IFN-γ的细胞数量。
图中R10表示RIPM培养基加入10％FBS及1％双链霉素(P.S.)，作为阴性对照。
图4：AdC68-CoV T免疫后小鼠的脾细胞对多种冠状病毒表位的应答：
实验所用小鼠为6-8周龄雌性hACE2 +ICR小鼠，免疫原为腺病毒AdC68-CoV T。ELISPOT方法检测免疫结束后第1周小鼠脾细胞针对多种冠状病毒表位的T细胞应答。横坐标为单肽名称，纵坐标为每百万个脾细胞中分泌 IFN-γ的细胞数量。
图5：AdC68-panCoV/Flu腺病毒的构建和表征：
图5A：重组质粒pAdC68XY3-panCoV/Flu的构建图谱；
图5B：腺病毒AdC68-panCoV/Flu基因组的双酶切鉴定。
图6：AdC68-panCoV/Flu腺病毒在HFH4-hACE2小鼠体内的免疫原性及蝙蝠冠状病毒SHC014攻毒保护试验：
图6A：AdC68组和AdC68-panCoV/Flu组的结合抗体滴度比较；
图6B：AdC68组和AdC68-panCoV/Flu组的中和抗体滴度比较；
图6C：AdC68组和AdC68-panCoV/Flu组的T细胞应答比较；
图6D：AdC68组和AdC68-panCoV/Flu组的攻毒后小鼠体重变化比较；
图6E：AdC68组和AdC68-panCoV/Flu组的攻毒后小鼠生存率比较。
具体实施方式
本公开涉及疫苗领域，特别涉及一种诱导广谱抗冠状病毒的T细胞疫苗免疫原的设计与验证。动物实验结果证实，本公开的疫苗安全，可产生高水平的T细胞应答，可用于冠状病毒的预防和治疗。
具体而言，本公开中选择冠状病毒早期表达蛋白polyprotein(ORF1ab)等为主要免疫原，并结合对S、E、M和N蛋白基因的分析，通过大数据收集1370条冠状病毒序列后，从中提取共享序列，并进行T细胞表位预测，将免疫原性高的保守区域组合起来，从而形成T细胞疫苗免疫原，该抗原覆盖了目前几乎所有的致病性冠状病毒。
本文中提供的所有数值范围旨在清楚地包括落在范围端点之间的所有数值及它们之间的数值范围。可对本发明提到的特征或实施例提到的特征进行组合。本说明书所揭示的所有特征可与任何组合物形式并用，说明书中所揭示的各个特征，可以任何可提供相同、均等或相似目的的替代性特征取代。因此除有特别说明，所揭示的特征仅为均等或相似特征的一般性例子。
如本文所用，“含有”、“具有”或“包括”包括了“包含”、“主要由......构成”、“基本上由......构成”、和“由......构成”；“主要由......构成”、“基本上由......构 成”和“由......构成”属于“含有”、“具有”或“包括”的下位概念。
定义
本文所用的术语“肽”指某一分子，所述分子包含2-200个氨基酸、由肽键连接的氨基酸序列，但其在具体实施方式中可包含非氨基酸结构(例如，连接有机化合物)。本发明所述的肽可包含任意20个常规氨基酸或其修饰形式，或可包含通过化学肽合成或通过化学或酶修饰引入的非天然氨基酸。
本文所用的术语“表位”指蛋白质或因子的一个或数个部分(其可定义一个构象表位)，所述部分被抗体或其部分(Fab′、Fab2′等)或存在于B或T细胞淋巴细胞的细胞表面的受体特异性识别并结合，并能够通过所述结合来诱导免疫应答。
本文所用术语“T细胞表位”或“T细胞表位”指被T细胞的细胞表面受体特异性识别并结合的抗原性蛋白质或因子的部分。T细胞表位可以是显性、亚显性或隐性T细胞表位，这取决于针对所述表位引起的免疫反应。显性取决于所述表位在蛋白质的所有可能T细胞表位中被T细胞识别以及能够激活T细胞的频率。具体而言，T细胞表位是由MHC I型或MHC II型分子结合的表位。蛋白质序列中的T细胞表位可通过功能性实验和/或一种或多种模拟预测实验来鉴定。本文肽内存在的T细胞表位可由8-25个氨基酸，8-16个氨基酸组成，或可由8、9、10、11、12、13、14、15或16个氨基酸组成，例如10、11或12个氨基酸组成。
本文免疫原性肽的T细胞表位可以对应蛋白质的天然表位序列，或者可以是其修饰形式，前提是修饰的T细胞表位与天然T细胞表位序列相似，保留其与MHC结合的能力。修饰的T细胞表位就MHC蛋白而言可具有与天然表位相同的结合亲和性，但也可以具有较低亲和性。在具体的实施方式中，经修饰肽的结合亲和性相较原始肽降低不小于10倍，更优选降低不小于5倍。
术语“MHC”指“主要组织相容性抗原”。在人类中，MHC基因被称为HLA(“人白细胞抗原”)基因。虽然没有一致遵循的习惯，一些文献使用HLA指代HLA蛋白分子，而使用MHC指代编码HLA蛋白的基因。如此，本文使用 的术语“MHC”和“HLA”可互换使用。在人中的HLA系统在小鼠中具有其等价物，例如，H2系统。最详尽研究的HLA基因是9个所谓的经典MHC基因：HLA-A、HLA-B、HLA-C、HLA-DPA1、HLA-DPB1、HLA-DQA1、HLA-DQB1、HLA-DRA和HLA-DRB1。在人类中，MHC分为3种：I、II和III型。
MHCI型分子在几乎所有的有核细胞上表达。在I型MHC分子中呈递的肽片段由CD8 +T淋巴细胞(细胞毒性T淋巴细胞或CTL)识别。CD8 +T淋巴细胞常常成熟为细胞毒性效应子，其可以裂解带有刺激抗原的细胞。II型MHC分子主要在激活的淋巴细胞和抗原呈递细胞上表达。利用对II型MHC分子呈递的独特的肽片段的识别来激活CD4 +T淋巴细胞(辅助T淋巴细胞或HTL)，通常在像巨噬细胞或树突细胞的抗原呈递细胞上发现这种II型MHC分子。CD4 +T淋巴细胞增殖并分泌细胞因子，其通过产生IL-4和IL-10支持抗体介导的应答或通过产生IL-2和IFN-γ支持细胞介导的应答。本申请的免疫原性肽可结合CD4 +T细胞和/或CD8 +T细胞。
T细胞表位可以仅仅由结合至主要组织相容性复合物(MHC)的槽的氨基酸组成，或可以包含连同侧接氨基酸残基的相同氨基酸。这类侧接残基并非有助于表位与MHC的结合而是“伸出”MHC槽外。侧接残基可以存在于T细胞表位的MHC结合部分的N-末端和/或C-末端。
免疫原性肽及其设计
本文中提供了一种免疫原性肽，其通过选择各种类型冠状病毒的早期表达蛋白多聚蛋白(ORF1ab)、膜蛋白、核衣壳蛋白、囊膜蛋白以及刺突蛋白等为主要免疫原，通过大数据收集大量(如1370条)冠状病毒序列后，从中提取共享序列，并进行T细胞表位预测，将免疫原性高的保守区域单独或组合起来，从而形成T细胞免疫原。本文的免疫原性肽覆盖了目前几乎所有的致病性冠状病毒。表位预测可通过对冠状病毒序列和CD8 +/CD4 +T细胞的分析获得，例如可通过对本文所述的共享序列进一步进行CD8 +T细胞表位预测来获得本文的表位序列。CD8 +T细胞表位预测软件是本领域中已知的，包括但不限于http：//tools.immuneepitope.org/main/tcell/和http：//www.syfpeithi.de/所提供的软件 等。
如本文所用，术语“共享肽”和“共享序列”可互换使用，是指包含各种冠状病毒主要蛋白质中共有的氨基酸序列的肽分子。可选取不同类型、不同亚型、不同株系的冠状病毒序列进行分析，例如可对选自下组的冠状病毒序列进行分析：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，尤其是SARS-CoV、MERS-CoV和SARS-CoV-2病毒。
如本文所用，术语“免疫原性肽”包含了通过上述方法设计并制得的肽，其具有激发T细胞应答的活性，且对各种冠状病毒具有广谱性。
本文的免疫原性肽可包含一个或多个冠状病毒共享表位。在一些实施方式中，本文的共享表位可为选自下组的一个或多个表位肽：PLPDRWYFYYT(SEQ ID NO：11，单肽6和7共有)、VVNKQFGAISS(SEQ ID NO：12，单肽31和32共有)、KPISAYAFLMA(SEQ ID NO：13，单肽73和74共有)、SYGPGNTFITD(SEQ ID NO：14，单肽124和125共有)、IKYYSIIPHSIR(SEQ ID NO：15，单肽44含)、IEDLLFDKVET(SEQ ID NO：16，单肽13和14共有)、SALQKIQDVVN(SEQ ID NO：17，单肽13和14共有)、ADDEGFITLKN(SEQ ID NO：18，单肽80和81共有)、YRVFPYDMDSGVSSF(SEQ ID NO：19，单肽94含)、SVTVEYNIHAVLDTL(SEQ ID NO：20，单肽102含)、QWLVMYGPI(SEQ ID NO：21，单肽141和142共有)、QSGLVKMAQPSGKVE(SEQ ID NO：22，单肽156含)、NGRPQGVFHVTMRSN(SEQ ID NO：23，单肽169含)、VTYGNMTLNGL(SEQ ID NO：24，单肽160和161共有)、SVGNFCYMHQL(SEQ ID NO：25，单肽175和176共有)、LSDNDGLKYAKWEKD(SEQ ID NO：26，单肽246含)、KIKYLYFVKNL(SEQ ID NO：27，单肽251和252共有)、GAVLGTISATVRLQA(SEQ ID NO：28，单肽256含)和TGQAITVKPEA(SEQ ID NO：29，单肽271和272共有)。在一些实施方式中，两个或以上的表位肽可直接连接或通过接头序列连接。
本文的免疫原性肽可包含：n个针对T细胞的冠状病毒表位序列，n为1～100的整数。
本文的免疫原性肽可包含一种或多种冠状病毒蛋白的共享序列。在一些实施方式中，本文的免疫原性肽可包含ORF1ab共享肽、M蛋白共享肽、N蛋白共享肽、E蛋白共享肽和/或S蛋白共享肽。例如，选自SEQ ID NO：1、3、5、7和9的共享肽；或者与前述共享肽具有高同源性或序列相同性(如至少90％，例如至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少98.5％、至少99％、至少99.5％、至少99.8％、至少99.9％)且具有结合和/或激活T细胞活性的肽。
本文的免疫原性肽还可包含有助于识别和/或激活T细胞和/或增强T细胞活性或者增强抗冠状病毒感染效果的其他片段，例如免疫调节序列，如IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH、SopE等。
在一些实施方式中，本文的免疫原性肽除了彼此组合以外，还可与共有序列共表达(例如融合表达或单独阅读框表达)的其他序列组合。其他序列可用于例如但不限于：扩大抗病毒谱、提高免疫应答诱导能力。
在一些实施方式中，其他序列选自例如针对如下病毒的免疫原：冠状病毒(例如RBD或其修饰序列(如末端Cys修饰RBD氨基酸序列))、流感病毒(例如HA2)、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、新疆出血热病毒)，尤其是与活化B细胞应答的免疫原，制备能够同时激活中和抗体与T细胞应答的复合疫苗。例如，本文中还提供了进一步包含冠状病毒RBD区和/或流感病毒HA2区免疫原性肽的序列，如包含SEQ ID NO：34所示的氨基酸序列。
在一些实施方式中，共表达的序列为选自下组的免疫调节序列：IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH及SopE。
本文的免疫原性肽可以是化学合成的产物，或使用重组技术从原核或真核宿主(例如，细菌、酵母、高等动物、昆虫和哺乳动物细胞)中产生。本文可允许引入非天然氨基酸。例如，半胱氨酸残基可由带有硫醇基团的其它氨基酸替代，例如巯基缬氨酸、高半胱氨酸，或具有硫醇功能的其它天然或非天然氨基酸。为了具有还原活性，半胱氨酸残基应不作为半胱氨酸二硫桥的部分出现。不过，半胱氨酸残基可以经(例如通过甲基化)修饰，由于甲基化的半胱氨酸在体内转化为含游离硫醇基团的半胱氨酸。
可对本文的免疫原性肽进行修饰，而不显著影响、不影响或甚至增强其免疫原性和/或反应性。例如，本文免疫原性肽的变异形式包括(但并不限于)：一个或多个(例如1、2、3、4、5、6、7、8、9或10个)氨基酸的缺失、插入和/或取代；在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。例如，在本领域中，用性能相近或相似的氨基酸进行取代时，通常不会改变蛋白质或多肽的功能。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质或多肽的功能。
本文的免疫原性肽还可包含促进所述肽被摄取进入内体以加工并在MHC II型决定簇内呈递的氨基酸序列(或其他有机化合物)。因此，本文的免疫原性肽还可以包含，例如，内体靶向序列。这类内体靶向序列包含在例如gp75蛋白、人CD3γ蛋白、HLA-BMβ、DEC205受体的胞质尾的内部。作为内体分选信号的肽的其它例子公开于Bonifacio和Traub(2003)Annu.Rev.Biochem.72，395-447的综述。
免疫原性肽编码分子、载体和宿主细胞
本文中还提供了编码本文免疫原性肽的分子、包含所述分子的载体和宿主细胞。
如本文所用，术语“免疫原性肽编码分子”是指编码本文所述免疫原性肽的序列。在一些实施方式中，其可包含例如SEQ ID NO：2、4、6、8或10的核苷酸序列；在严格条件下与这些序列杂交的分子、或与上述分子高度同源的核苷酸分子，只要其可有效编码并表达所需的免疫原性肽。应理解，在获得了 本文的表位肽和免疫原性肽的氨基酸序列之后，可采用本领域中的常规技术手段获得其编码序列，并可对其进行优化。因此，可提供针对同一条表位肽和/或免疫原性肽的一种或多种编码分子，只要该编码分子能正确有效地表达该表位肽和/或免疫原性肽。
如本文所用，术语“严格条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在50％，优选55％以上、60％以上、65％以上、70％以上、75％以上、80％以上、85％以上或90％以上，更优选是95％以上时才发生杂交。
本文的编码分子通常可以用PCR扩增法、重组法或人工合成的方法获得。对于PCR扩增法，可根据本发明所公开的有关核苷酸序列，尤其是开放阅读框序列来设计引物，并用市售的cDNA库或按本领域技术人员已知的常规方法所制备的cDNA库作为模板，扩增而得有关序列。当序列较长时，常常需要进行两次或多次PCR扩增，然后再将各次扩增出的片段按正确次序拼接在一起。
本文还涉及包含免疫原性肽编码分子的载体，以及用该载体经基因工程产生的宿主细胞。
通过常规的重组DNA技术(Science，1984；224：1431)，可利用本发明的编码序列可用来表达或重组生产免疫原性肽。一般来说有以下步骤：
(1)用本文的免疫原性肽编码核苷酸分子，或用含有该核苷酸分子的重组表达载体转化或转导合适的宿主细胞；
(2)在合适的培养基中培养的宿主细胞；
(3)从培养基或细胞中分离、纯化蛋白质或多肽。
本发明中，术语“载体”与“重组表达载体”可互换使用，指本领域熟知的细菌质粒、噬菌体、酵母质粒、动物细胞病毒、哺乳动物细胞病毒或其它载体。总之，只要能在宿主体内复制和稳定，任何质粒和载体都可以用。表达载体的一个重要特征是通常含有复制起点、启动子、标记基因和翻译控制元件。
本领域的技术人员熟知的方法能用于构建含编码序列和合适的转录/翻译控制信号的表达载体。这些方法包括体外重组DNA技术、DNA合成技术、体 内重组技术等。所述的DNA序列可有效连接到表达载体中的适当启动子上，以指导mRNA合成。表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
包含上述的适当DNA序列以及适当启动子或者控制序列的载体，可以用于转化适当的宿主细胞，以使其能够表达蛋白质或多肽。宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如动物细胞。代表性例子有：大肠杆菌，链霉菌属、农杆菌；真菌细胞如酵母；动物细胞等。在本发明中，优选采用大肠杆菌细菌细胞、小鼠树突状细胞作为宿主细胞。
产品
本文中还提供了包含本发明所述免疫原性肽、其编码序列、载体或宿主细胞的产品，所述产品可为例如可广谱抗冠状病毒感染的T细胞疫苗、药物、药物组合物或试剂盒、偶联物、缀合物等。如本文所用，术语“活性物质”或“本发明的活性物质”可互换使用，是指本文的免疫原性肽、其编码序列、载体或宿主细胞。
由此，本文中还提供了一种包含所述免疫原的疫苗组合物(或称疫苗)。该疫苗组合物包含其形式能够被给予脊椎动物(优选哺乳动物)的本公开的表位、免疫原性肽和/或核酸分子的配制品，并且其诱导提高免疫力的保护性免疫应答以预防和/或减轻疾病和/或其至少一种症状。术语“保护性免疫应答”或“保护性应答”是指通过免疫原介导的针对传染原或疾病的免疫应答，通过脊椎动物(例如人)展现，预防或减轻感染或减少其至少一种疾病症状。
术语“脊椎动物”或“对象”或“患者”是指脊索动物亚门的任何成员，包括但不限于：人和其他灵长类动物，包括非人灵长类动物诸如黑猩猩和其他猿和猴物种；家畜诸如牛、绵羊、猪、山羊和马；家养哺乳动物诸如狗和猫；实验室动物，包括啮齿动物诸如小鼠、大鼠和豚鼠；鸟包括驯养、野生和猎鸟诸如鸡、火鸡和其他鹑鸡类鸟、鸭、鹅。术语“哺乳动物”和“动物”被包括在这个定义中，旨在涵盖成年、幼年以及新生个体。
本文的疫苗可为重组蛋白疫苗、重组DNA疫苗、重组病毒载体疫苗(例如腺病毒载体、痘病毒载体、腺相关病毒载体、单纯疱疹病毒载体、巨细胞病毒载体)、重组细菌载体疫苗、重组酵母载体疫苗或重组病毒样颗粒疫苗。在一些实施方式中，本文的疫苗选自重组DNA疫苗、重组腺病毒载体、重组痘病毒载体或其中一种或两种或三种的组合。
本文的疫苗组合物中包含有效量的本文免疫原。本公开的疫苗组合物中包含足以实现希望的生物效应的量的免疫原。术语“有效量”通常是指可以诱导足以诱导免疫力的保护性免疫应答以预防和/或减轻感染或疾病和/或以减少感染或疾病的至少一种症状的免疫原的量。
本文的疫苗中还可包含佐剂。可采用本领域普通技术人员已知的佐剂，例如Vogel等人，“A Compendium of Vaccine Adjuvants and Excipients”(第2版)中所记载的佐剂(通过引用以其全文结合在此)。已知佐剂的例子包括但不限于：完全弗氏佐剂、不完全弗氏佐剂、氢氧化铝佐剂、脂多糖(LPS)、RIBI佐剂、MF-59等。
本文的疫苗组合物还可包括药学上可接受的载体、稀释剂、防腐剂、增溶剂、乳化剂等辅料。例如，药学上可接受的载体是已知的，并且包括但不限于注射用水、盐溶液、缓冲盐水、右旋糖、水、甘油、无菌等渗水缓冲液及其组合。药学上可接受的载体、稀释剂和其他赋形剂可例如参见《雷明顿药物科学》(Remngton′s Pharmaceutcal Sciences)中。
本文疫苗组合物的形式可适于系统性或局部(尤其是呼吸道内)给予。给予疫苗组合物的方法包括但不限于：胃肠外给予(例如，真皮内的、肌内的、静脉内的以及皮下的)、硬膜外给予、粘膜给予(例如，鼻内的和口腔或肺的途径给予)。在特定实施例中，
在一些实施方式中，本文的疫苗预防、消除或减轻对象中的冠状病毒感染或其至少一种症状，例如呼吸道症状(如鼻塞、咽喉痛、声嘶)、头痛、咳嗽、痰、发热、啰音、喘息、呼吸困难、因感染引起的肺炎、严重急性呼吸综合症、肾衰竭等。
本文中还涉及了一种免疫偶联物(也可称免疫缀合物)，其包含本文的免疫 原以及与其偶联的其他物质。所述的其他物质可为靶向性物质(如特异性识别特定靶标的部分)、治疗性物质(如药物、毒素、细胞毒剂)、标记性物质(如荧光标记物、放射性同位素标记物)。
在本公开中还提供了一种组合产品，其包括本公开的宿主细胞和/或疫苗，且还可包含一种或多种有助于更好发挥预防和/或治疗冠状病毒感染或其症状的功能或增强前述物质稳定性的其他物质。例如，其他物质可包括针对冠状病毒S或S1的其他疫苗，如来自于包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV的S或S1疫苗；受益于T细胞活化和/或与T细胞的记忆性免疫反应的疾病或病症的其他活性物质。
本公开中还提供了本文所述免疫原性肽、其组合在制备检测试剂盒中的应用。本文的免疫原性肽可单独或与MHC形成肽-MHC复合物单聚体或多聚体(如二聚体、四聚体)。在一些实施方式中，可将本文的免疫原性肽与HLA-A2重链蛋白、轻链蛋白(如β2m蛋白)复合形成肽-MHC复合物单聚体，并可任选地由复合物单聚体形成复合物多聚体。在一些实施方式中，试剂盒可用于冠状病毒检测中。
免疫方法
本文还提供了一种用于预防和/或治疗冠状病毒感染和/或其症状的方法，其包括：至少一次给予预防和/或治疗有效量的本公开的一种或多种疫苗。可采用的接种方式包括但不限于：系统性免疫接种方式，如肌肉注射、皮下注射和皮内注射等；呼吸道内免疫接种方式，如雾化、滴鼻等。在一些实施方式中，初次免疫采用系统性接种或呼吸道内接种，优选系统性接种。
在本公开的一些实施方式中，每两次接种之间的间隔至少为1周，例如2周、4周、2个月、3个月、6个月或更长间隔。
在一些实施方式中，采用DNA疫苗进行初次免疫，并采用细胞疫苗进行一次或多次加强免疫。本公开的免疫方法可采用“初免-加强”或“初免-加强-再加强”的方式，可采用单一的全身系统免疫或呼吸道局部免疫方式，或采用两 种免疫方式的组合。
在一些优选的实施方式中，采用重组DNA疫苗进行系统性初免，从而建立全身系统免疫应答，再用细胞疫苗进行一次或多种免疫加强。
采用本文的免疫方法可在呼吸道局部和全身系统有效建立的疫苗特异性免疫应答，有助于增强疫苗保护的有效性。
以药物包或试剂盒的形式提供本文的组合产品可，例如可将本文的一种或多种疫苗组合物或其一种或多种成分包装在一个或多个容器中，例如包装在指明组合物的量的密封容器诸如安瓿或小药囊中。可以液体、无菌冻干粉或无水浓缩物等形式提供疫苗组合物，可在临用前用适当液体(例如水、盐水等)对其进行稀释、复原和/或配制以获得用于给予至对象的适当浓度和形式。
实施例
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。本领域技术人员可对本发明做出适当的修改、变动，这些修改和变动都在本发明的范围之内。
下列实施例中未注明具体条件的实验方法，可采用本领域中的常规方法，例如参考《分子克隆实验指南》(第三版，纽约，冷泉港实验室出版社，New York：Cold Spring Harbor Laboratory Press，1989)或按照供应商所建议的条件。DNA的测序方法为本领域常规的方法，也可由商业公司提供测试。
除非另外说明，否则百分比和份数按重量计算。除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
动物、材料与方法
实施例的实验中涉及到的实验动物、免疫方式、免疫原及检测方法如下：
I.实验动物
6-8周龄雌性hACE2 +ICR小鼠(即重组表达人ACE2的转基因ICR小鼠)， 购自北京唯尚立德科技有限公司。
6-8周龄雄性HFH4-hACE2-C57BL/6小鼠，使用HFH4肺纤毛上皮细胞特异性启动子将hACE2基因转入小鼠中所得(具体构建方法参考Menachery，V.D等，SARS-like WIV1-CoV poised for human emergence.Proc.Natl.Acad.Sci.U.S.A.113，3048-3053(2016)；Ostrowski LE，Hutchins JR，Zakel K，O′Neal WK.Targeting expression of a transgene to the airway surface epithelium using a ciliated cell-specific promoter.Mol Ther.2003；8(4)：637-645)。
II.免疫方式
对小鼠左、右后肢分别进行肌肉注射，或进行滴鼻。具体剂量见实施例。
III.免疫原的设计
1.背景
新型冠状病毒整体基因组信息如图1所示。
设计时选择了多聚蛋白(polyprotein，orf1ab)、刺突蛋白(spike protein，S)、膜蛋白(Membrane，M)、囊膜蛋白(Envelope，E)、核衣壳蛋白(Nucleocapsid，N)共计5种蛋白质/多肽。可能非冠状病毒共享的其他蛋白质/多肽在此未考虑。
2.数据收集
分别收集NCBI和Uniprot中上述5种蛋白质的序列，数据情况如表1：
表1.蛋白质序列数据
Figure PCTCN2021114313-appb-000001
*：箭头后是100％序列去冗余的结果。
3.运算步骤
3.1多序列比对
分别对于5个蛋白质进行多序列比对，将序列每一个氨基酸的位置对齐。
3.2提取共享序列(consensus sequence)
在每一个位置提取出现频率最高的氨基酸，需要注意的是，冠状病毒蛋白 差异较大，很多位置出现最多的是缺口(gap)。
3.3T细胞表位预测
为了全面模拟共享序列可能引起的T细胞免疫反应，同时进行了MHC I类和II类表位预测，共9个MHC I类亚型，13个MHC II类亚型，选用MHCpan(采用默认参数)进行预测。
3.4搜索保守区域
由于共享序列存在gap或出现频率较低的氨基酸，故在此构建保守区域筛选公式：
Figure PCTCN2021114313-appb-000002
式中，CP(aa n，…，aa n+m)代表一串共享肽；fre(50％aa)＞[0.7(m+1)]代表其中每个位点氨基酸偏好性超过50％的数目需要大于序列长度的70％；num(gap)＜20％(m+1)代表其中gap的数量要小于全长的20％。
找出所有在冠状病毒中保守的区域，并扫描IEDB人源肽段库进行筛选。
3.5将保守区域映射到新冠病毒上
将保守区域映射到新冠病毒上，并按照T细胞表位预测结果推荐免疫原性高的区域。
最终设计出来的共享序列(即CoV T)采自1370条冠状病毒序列，其具体序列见SEQ ID NO：9(CoV T氨基酸序列，1238Aa)和SEQ ID NO：10(CoV T编码序列，3714 Nt)。
重组腺病毒载体疫苗：AdC68、AdC68-CoV T、AdC68-panCoV/Flu。
IV.免疫原制备及免疫剂量
免疫原的制备参见实施例1。
在实施例中所采用的免疫原免疫剂量如下：
重组腺病毒载体疫苗：5E10vp/只小鼠(对小鼠左、右后肢分别进行肌肉注射，左右后肢各50μL)，100μL(肌肉注射)；5E10vp/只小鼠，30μL(滴鼻)。
V.免疫间隔
具体免疫间隔见下文表格。
VI.SARS-CoV2包膜假病毒(Pseudovirus)包装
1.转染前一天准备293T细胞，用于包装质粒的转染与表达。用DMEM完全培养基将细胞稀释至5×10 6个/mL细胞，取1mL稀释好的细胞，铺在10cm的皿中，37℃，5％CO 2，培养过夜；
2.吸取SARS-CoV2膜蛋白质粒pcDNA3.1-S 4μg和pNL4-3Δenv骨架质粒8μg(NIH AIDS Reagent Program，3418)加入500μL双无(无血清、无双抗，双抗为青链霉素混合液)的DMEM中，室温孵育5min；
3.用双无DMEM将24μL TurboFect(Thermo Fisher Scientific)稀释，终体积为500μL/样品，室温孵育5min；
4.将2与3两者混匀，1000μL/样品终体积，室温孵育20min，孵育结束后加到10cm培养皿的293T细胞中。6h后更换新鲜的15mL完全培养基，继续在细胞培养箱中培养48h；
5.培养结束后，收集10cm皿的细胞培养上清，于15mL离心管里，然后4000g，4℃，离心10min，用0.45μm的滤器过滤到新的15mL离心管中，冻存于-80℃保存，滴定后备用。
上述方法也用于panCoV/Flu的制备。
VII.构建稳定表达hACE2受体的293T细胞
1.人工合成人源ACE2(hACE2)序列(Genebank#NCBI_NP_001358344.1)，如SEQ ID NO：13所示，序列5’端带有Age1酶切位点，3’端带有Xba1酶切位点，合成片段与载体质粒pHAGE-MCS-puro使用Age1酶切(Thermo Scientific公司，货号FD1464)与Xba1酶切(Thermo Scientific公司，FD0685)，并通过凝胶电泳后切胶回收，采用Sanprep柱式DNA胶回收试剂盒(Promega公司，货号A9282)回收酶切片段。
2.基因回收产物与酶切线性化载体用T4DNA连接酶的方法连接(Thermo Scientific公司，货号2011A)：将连接产物转化至大肠杆菌E.coli Stable，在含氨苄霉素的培养板上过夜生长。第2天，随机挑取单菌落进行测序，突变位点校正，验证全部序列正确后，成功克隆出hACE2基因的慢病毒表达质粒(pHAGE-hACE2-puro)。
3.取10cm皿，在每个皿中接种约5×10 6个293T细胞，保证第二天转染 时使细胞密度达90％为宜；将pHAGE-hACE2-puro、慢病毒包装质粒psPAX以及VSVG三种质粒，按照质量比1∶2∶1的比例转染293T细胞。
4.37℃，5％CO 2的孵箱培养48小时左右，具体时间根据细胞情况而定，收集细胞上清。将收集的细胞上清用0.45μm的滤器进行过滤，再用PEG 8000进行浓缩，即可得到较为纯化的hACE2慢病毒。
5.提前一天铺5×10 5个的293T细胞于12孔板的一个孔内，次日向铺好的细胞中加入步骤2中浓缩的病毒500μL，1000g，离心2小时。
6.离心感染结束后，继续在37℃，5％CO 2的孵箱培养12小时左右，将培养基换成添加1μg/mL嘌呤霉素(puro)的细胞培养基培养，最后能够存活的细胞便是整合有hACE2基因的293T细胞，并通过流式分选筛选出稳定表达hACE2的293T细胞(能与S蛋白结合)。
VIII.检测方法
采血：
最后一次免疫结束后第2周，将小鼠脱颈处死前，通过摘眼球的方法采集小鼠外周全血，收集于1.5mL EP管中，室温静置使其自然凝血，凝固后的小鼠血清于7000g，离心15min。将小鼠血清转移至新的1.5mL EP管中。实验前需要将样品在56℃灭活30min，来破坏血清内的补体活性。灭活前短暂离心，避免管壁和瓶盖上的样品残存。水浴液面要没过样品液面，但不能超过瓶盖。
ELSIA检测结合抗体
1.用4℃预冷的ELISA包被液稀释检测的抗原蛋白(RBD，购自上海近岸生物科技有限公司)，至终浓度为1μg/mL。在ELISA板的每孔加入100μL包被抗原溶液，4℃过夜；
2.第二天，取出ELISA板，弃掉包被液，用0.05％的PBST缓冲液洗板3次，每次220μL；
3.洗涤完毕后，在吸水纸上拍干，每孔用200μL ELISA封闭液(0.5％脱脂奶粉，PBST溶解)进行封闭，室温封闭2h；
4.封闭结束后，用0.05％的PBST洗板3次，每次220μL；
5.对于血清或者血浆，用ELISA样品稀释液(0.5％脱脂奶粉，PBST溶解)稀释，从1∶100起始，进行2倍比稀释。用未免疫的小鼠血清设置为阴性对照。设置空白孔，只加样品稀释液，每个样品需做2个复孔，每孔终体积为100μL，室温孵育3h；
6.样品孵育结束后，继续用PBST洗板5次，每次220μL；
7.用ELISA封闭液(0.5％脱脂奶粉，PBST溶解)稀释相对应比例的二抗(山羊抗鼠，购自北京中杉金桥生物技术有限公司，货号ZB-2305)，每孔加入100μL，室温孵育1-1.5h；
8.二抗孵育结束后，用0.05％的PBST洗板5次，每次220μL；
9.取一对金银片OPD底物，溶解于20mL去离子水中，随后每孔加入100μL，避光反应5min；
10.显色结束后，用50μL 2nM H 2SO 4进行终止，在酶标仪上读取OD 492～OD 630值；
11.以最后一个稀释度OD 492大于2倍的(negative mean+SD)值对应的血清稀释比的倒数作为抗体滴度。
293T-ACE2细胞检测中和抗体
1.取96孔透明底黑板进行中和实验，第一列设置细胞对照(CC)(150μL)，第二列设置病毒对照(VC)(100μL)，其他均为样品孔，对血清样品进行倍比稀释，最终孔中体积为100μL。
2.除细胞对照组外，每孔加50μL SARS-CoV-2假病毒稀释液，使每孔最终含假病毒为200TCID 50。
3.轻轻震荡混匀，将上述96孔底黑板置于细胞培养箱中，37℃，5％CO 2孵育1h。
4.当孵育时间至20min时，开始准备293T-hACE2靶细胞，并用完全培养基将细胞稀释至10 5个细胞/mL。
5.当孵育时间至1h，向96孔透明底黑板中每孔加100μL靶细胞，使每孔细胞为10 4个。
6.前后左右轻轻晃动96孔透明底黑板，使孔中的细胞均匀分散，再将板 子放入细胞培养箱中，37℃，5％CO 2培养48h。
7.培养48h后，从细胞培养箱中取出96孔透明底黑板，吸掉孔中上清，每孔加入100μL PBS清洗一遍，吸去PBS，每孔加入50μL 1×的裂解缓冲液(购自Promega公司Cat#E153A)，室温在水平摇床上孵育30min使细胞充分裂解；
8.加30μL荧光素酶的底物(购自Promega公司，Cat#E1501)于96孔黑板中，用仪器
Figure PCTCN2021114313-appb-000003
96微孔板发光-检测仪检测荧光素酶活性。
9.导出荧光素读值，计算中和抑制率，结合中和抑制率结果，利用Graphpad Prism 5.0软件计算ID 50。
Figure PCTCN2021114313-appb-000004
ELISPOT检测T细胞应答
小鼠脾脏单细胞分离：
1)将小鼠仰卧，剖开右侧腹部皮肤，打开腹膜，取下小鼠脾脏，放入加有5mL完全RPIM1640培养基的小平皿中；
2)用无菌镊子将脾脏用无菌纱布包裹起来，用小弯镊夹起纱布，轻轻磨碎脾脏，可使脾细胞全部释放到培养基中；
3)随后用5mL移液器将脾细胞悬液经纱布吸到无菌的15mL离心管中，800g，离心5min；
4)弃掉离心后的上清，轻敲15mL离心管重悬细胞沉淀，每个离心管中加入3mL红细胞裂解液裂解红细胞，颠倒混匀后室温静置5min，使红细胞充分裂解又不会损伤脾细胞；
5)裂红结束后，用5mLRPIM1640培养基终止裂红，800g，离心5min；
6)弃掉离心后的上清，用5mL RPIM1640培养基洗1次，800g离心5min；
7)弃离心后上清，脾细胞放于冻存液(90％FBS和10％DMSO)中进行冻存备用。
ELISpot实验操作按照小鼠IFN-γ/猴IFN-γ说明书进行(购自BD，货号551083)：
1)用纯化的IFN-γ抗体包被试剂盒提供的Millipore板，比例1∶250，4℃ 过夜包被；
2)甩掉板中的包被抗体溶液，用200μL RPMI 1640完全培养基洗板一遍，随后用200μL RPMI 1640完全培养基封闭液封闭Millipore板，室温孵育2h；
3)弃掉孔板中的封闭液，根据不同的实验设计，在Millipore板中加入刺激肽库或者单肽(苏州强耀生物科技有限公司合成)，50μL/孔，每条肽的浓度为5μg/mL。在阴性对照孔加入50μL RPMI 1640完全培养基；阳性对照孔中加入50μL佛波醇酯类多克隆刺激剂(PMA，终浓度100ng/mL)和离子霉素(Ionomycin，终浓度2μg/mL)的RPMI 1640完全培养基；
关于CoV T单肽和肽库分别说明如下：
——单肽1～单肽308：每条单肽15个氨基酸(除了单肽307为13个氨基酸)，覆盖整条CoV T序列，共308条单肽(即n＝1～308)，其中：从单肽1～306(即n＝1～306)，其与CoV T(SEQ ID NO：9)相对应的氨基酸位置符合如下规则：起始位置＝n×4-2，终止位置＝n×4+12。例如，单肽1(即n＝1)，其起始位置为SEQ ID NO：9的第2位氨基酸，终止位置为SEQ ID NO：9的第16位氨基酸；单肽2(即n＝2)，为SEQ ID NO：9的第6位至第20位氨基酸；以此类推，单肽306(即n＝306)，为SEQ ID NO：9的第1222位至第1236位氨基酸。单肽307和单肽308因已达SEQ ID NO：9末尾，其序列分别对应于SEQ ID NO：的第1226～1238位和第1224～1238位。
——肽库：以上述单肽建立31个肽库，肽库1～30中每个肽库包含10条单肽，肽库31包含8条单肽(即单肽301～308)。
4)对小鼠脾细胞进行计数，细胞调整为4×10 6个细胞/mL，每孔加入50μL细胞，最终使每孔的细胞数为2×10 5个细胞。将Millipore板放入湿盒，在37℃5％CO 2培养箱中孵育20-22h，期间切勿摇动板子，造成细胞的偏移；
5)培养孵育结束后，从培养箱中取出Millipore板，将板中的液体弃掉，用预冷的去离子水洗两遍，每次220μL，每次清洗孵育3min；
6)用0.05％的PBST(PBS+0.05％Tween-20)洗板3次，每次200μL；
7)用10％FBS的PBS抗体稀释液稀释生物素化检测抗体(Biotinylated Detection antibody，比例1∶200)，每孔加入100μL，室温孵育2h；
8)孵育结束后，再用0.05％的PBST洗板3次，每次220μL；
9)将链霉亲和素-HRP偶联抗体(Streptavidin-HRP Conjugate antibody)用抗体稀释液进行稀释(比例1∶100)，每孔加入100μL，室温孵育1h；
10)孵育结束后，用0.05％的PBST洗板4次，每次220μL；
11)再用PBS清洗板子2次，每次220μL；
12)准备底物溶液(1mL的底物缓冲液加1滴底物溶液)，每孔加入100μL底物溶液。反应5-60min，孵育时间根据斑点形成的情况而定。
13)用去离子水冲洗终止反应，室温晾干后进行计数；
14)利用ChampSpot III型酶联斑点图像分析仪进行斑点形成细胞SFC(spot forming cell)的计数以及QC(Quality Control)。
实施例1：pAdC68XY3-CoV T腺病毒表达载体的构建及酶切鉴定
为了研究CoV T的功能，我们构建了CoV T的腺病毒表达载体，并构建了腺病毒AdC68-CoV T，且进行了酶切鉴定。
首先，我们人工合成了CoV T基因(具体序列如SEQ ID NO：9和10所示)。将合成片段直接加载于载体质粒pAdC68XY3(根据中国专利申请号CN201910777937.2中所记载方法构建，基于黑猩猩型腺病毒AdC68基因组序列，E1/E3删除，E4区域ORF6/7和ORF6替换成AdHu5：AdC68E4序列的删除区域为33518bp-34671bp(对应AC_000011.1)；AdHu5E4序列的插入区域为32914bp-34077bp(对应AC_000008.1)。其中，保留了AdC68的ORF1-4序列。)上，得到CoV T的腺病毒表达载体pAdC68XY3-CoV T，质粒构建图谱如图2A所示。
我们进一步对重组腺病毒进行了包装及扩增。
将构建好的重组质粒pAdC68XY3-CoV T经限制性内切酶Pac 1在37℃水浴中线性化3.5h，65℃灭活内切酶。用293A细胞铺六孔板，将线性化后的重组质粒分别以2.5μg、2μg、1.5μg/孔的量转染至293A细胞(购自ATCC)，培养11天左右噬斑出现，14天左右至细胞均被病毒感染时收样，细胞上清一起收集，置于-80℃反复冻融三次后，取少量感染293A细胞，抽取基因组进行 Bgl II和Mfe I双酶切鉴定(图2B)。结果显示：酶切后条带正确。
将上述收集的腺病毒样品感染至1个T175培养瓶的293A细胞，24h后收样，置于-80℃反复冻融三次后，感染至6个T175培养瓶的293A细胞，以此类推进行大量扩增，扩增至36个T175培养瓶时，收集细胞沉淀，弃上清，用10mL左右无血清无抗性的DMED培养基重悬，-80℃反复冻融三次后，采用氯化铯密度梯度离心法纯化腺病毒，分装后，-80℃保存。
氯化铯密度梯度纯化腺病毒步骤如下：
1)将扩增冻融后的腺病毒4℃，4000g，离心30min，取上清，用0.8μm的滤膜过滤；
2)向超速离心管中加入3mL 1.2M CsCl溶液，将3mL 1.4M CsCl溶液用注射器从底部轻轻缓慢打入，铺好氯化铯梯度；
3)将过滤后的腺病毒溶液轻轻加入超速离心管中，用PBS将液面补满，配平后放入SW41(Beckman)套筒中；
4)选取SW41转子，4℃，25000rpm，离心2.5h，降速调为“no brake”自然降速；
5)待离心结束后，轻轻取出超速离心管，可见两条白色病毒条带，用注射器从侧面戳入超立管中，将下层条带吸出，注意避免吸到上层条带；
6)将Bio-Gel P-6 PG gel脱盐胶注入液相色谱柱(C4169-5，购自Sigma)中，待液体落完，留下2-3cm左右的胶为宜，加入PBS洗涤两遍；
7)将上述吸出的病毒液加入脱盐胶(填胶的柱)，待液体落完后，加入2mL左右PBS洗脱，每个1.5mL EP管收集3-4滴洗脱液；
8)Nanogrop 2000(Thermo Scientific)测定每管洗脱液的OD 260值，将OD 260大于2的洗脱液合并，加入10％无菌甘油，分装；
9)Nanogrop 2000测定分装后腺病毒溶液的OD 260值，OD 260值×1.1×10 12/mL即为纯化后腺病毒的最终滴度。
实施例2：AdC68-CoV T在hACE2 +ICR小鼠体内的免疫原性
采用腺病毒AdC68及AdC68-CoV T免疫hACE2 +ICR小鼠，两针均为肌 肉注射，剂量为5E10vp/小鼠。在完成免疫1周后(即首次免疫后4周时)，评价免疫组合诱导针对CoV T肽库的T细胞应答水平。
将小鼠随机分为2组，根据免疫原分别命名为AdC68组和AdC68-CoV T组。具体免疫组合如表2所示：
表2.AdC68和AdC68-CoV T对hACE2 +ICR小鼠的体内免疫方案
Figure PCTCN2021114313-appb-000005
AdC68-CoV T组产生针对CoV T肽库的总的T细胞应答如图3A所示：每百万个脾细胞里面分泌IFN-γ的细胞数均值在7530左右，最高能到达13595，显著高于AdC68。
同时，我们分析了AdC68-CoV T诱导的针对CoV T各个肽库的应答，如图3B所示：在CoV T的31个肽库中，有19个肽库能够有效或高效刺激小鼠脾细胞分泌IFN-γ，即AdC68-CoV T诱导的T细胞应答在肽库上具有广谱性，可对多个表位有应答，有别于一般腺病毒单表位应答较强的情况。
随后我们对有反应的18个肽库，用其中的单肽刺激小鼠脾细胞，结果如图3C所示：我们发现单肽6、7、13、14、29、30、31、32、43、44、73、74、75、80、81、94、102、124、125、142、156、160、161、169、175、246、251、252、256、272能够刺激小鼠脾细胞分泌IFN-γ。
随后我们分析反应最强的单个肽氨基酸序列的共享序列，得到了两个表位：
表位A：PLPDRWYFYYT(SEQ ID NO：11，单肽6和7共享)，来自冠状病毒的N蛋白；
表位B：KPISAYAFLMA(SEQ ID NO：13，单肽73和74共享)来自冠状病毒的ORF1ab蛋白；
分别与SARS病毒、MERS病毒和SARS-CoV-2病毒进行了以上表位的对应位置匹配，结果如表3所示。
表3.AdC68-CoV T在hACE2 +ICR小鼠体内免疫原性
Figure PCTCN2021114313-appb-000006
Figure PCTCN2021114313-appb-000007
表位A和表位B均可在SARS、MERS和SARS-CoV-2病毒蛋白中找到对应序列，即我们所构建的疫苗AdC68-CoV T能够诱导广谱抗冠状病毒的T细胞应答。
该实验证实，在hACE2 +ICR小鼠中，AdC68-CoV T能够诱导针对冠状病毒的广谱T细胞应答，包括SARS、MERS、SARS-CoV-2等。
实施例3：AdC68-CoV T免疫后小鼠的脾细胞对多种冠状病毒表位的应答
为了验证我们所诱导的反应确实能够对真实的冠状病毒感染起到效果，我们合成了表3中两个表位在三种冠状病毒中的真实的表位肽，用单个的肽来刺激之前免疫后的小鼠的脾细胞，结果如图4所示。
结果表明用合成的各种冠状病毒自身的天然表位来刺激疫苗免疫后的小鼠脾细胞，也能够产生T细胞应答，这进一步验证了我们的T细胞疫苗确实能够针对多种冠状病毒有应答。
有针对冠状病毒的广谱T细胞应答能够表明其对冠状病毒的广谱预防和治疗效果。当病毒感染机体时，能够迅速激活抗原特异性的T细胞应答，启动细胞杀伤功能。并且我们识别的是病毒早期基因，在病毒还未能够形成完整的病毒颗粒时就可以将其通过CD8T细胞的杀伤作用杀死。领域中亦有单独的肽起到预防和治疗效果的文章。
实施例4：AdC68-panCoV/Flu腺病毒表达载体的构建及酶切鉴定
上述研究证明了CoV T作为一款T细胞疫苗的功能，后续为了将T细胞功能和B细胞功能联合起来，同时为了能够应对多种流感病毒，我们将CoV T基因和RBD-HA2-CD8TM基因(融合表达SARS-CoV-2的RBD蛋白、H7N9的HA2区域和CD8TM，其核苷酸序列和所编码的氨基酸序列分别如SEQ ID NO： 34和35所示)加载于载体质粒pAdC68XY3上，构建了腺病毒AdC68-panCoV/Flu，且进行了酶切鉴定。
首先，我们人工合成了panCoV/Flu基因(SEQ ID NO：36)，该基因包含(a)编码SopE序列(使得T细胞免疫原翻译表达后能迅速转移至蛋白酶体中进入降解程序，更好地提呈T细胞表位)和CoV T的前端部分(SEQ ID NO：37)，(b)与其毗连的IRES和KOZAK序列，以及(c)包含编码HASP信号肽、末端Cys修饰RBD序列、HA2序列和CD8铰链区和CD8TM(RBD-HA2-CD8TM)的后端部分(SEQ ID NO：38)。
将合成片段直接加载于载体质粒pAdC68XY3上，得到panCoV/Flu的腺病毒表达载体pAdC68XY3-panCoV/Flu，质粒构建图谱如图5A所示。
我们进一步对重组腺病毒进行了包装及扩增。
将构建好的重组质粒pAdC68XY3-panCoV/Flu经限制性内切酶Pac 1在37℃水浴中线性化3.5h，65℃灭活内切酶。用293A细胞铺六孔板，将线性化后的重组质粒分别以2.5μg、2μg、1.5μg/孔的量转染至293A细胞(购自ATCC)，培养11天左右噬斑出现，14天左右至细胞均被病毒感染时收样，细胞上清一起收集，置于-80℃反复冻融三次后，取少量感染293A细胞，抽取基因组进行Bgl II和Mfe I双酶切鉴定(图5B)。结果显示：酶切后条带正确。
将上述收集的腺病毒样品感染至1个T175培养瓶的293A细胞，24h后收样，置于-80℃反复冻融三次后，感染至6个T175培养瓶的293A细胞，以此类推进行大量扩增，扩增至36个T175培养瓶时，收集细胞沉淀，弃上清，用10mL左右无血清无抗性的DMED培养基重悬，-80℃反复冻融三次后，采用氯化铯密度梯度离心法纯化腺病毒，分装后，-80℃保存。
氯化铯密度梯度纯化腺病毒步骤同实施例1。
实施例5：AdC68-panCoV/Flu腺病毒在HFH4-hACE2小鼠体内的免疫原性及蝙蝠冠状病毒SHC014攻毒保护试验
小鼠：HFH4-hACE2小鼠
毒株：Bat-CoV SHC014株
委托武汉病毒所进行试验。
免疫程序：将小鼠随机分为2组，根据免疫原分别命名为AdC68组和AdC68-panCoV/Flu组。具体免疫组合如表4所示，免疫方式为第一针肌肉注射，剂量为5E10vp/小鼠，第二针肌肉注射(5E10vp/小鼠)加滴鼻(5E10vp/小鼠)，总剂量为1E11vp/小鼠。
攻毒程序：免疫结束后3周对小鼠进行攻毒，攻毒后持续观察小鼠体重变化和生存。
免疫结束后两周检测了针对RBD的结合抗体滴度和针对SARS-CoV-2假病毒的中和抗体滴度，如图6所示：AdC68-panCoV/Flu组结合抗体滴度均值为10，160，最高能到25,600，与AdC68组有显著区别(图6A，P＜0.01)；AdC68-panCoV/Flu组中和抗体滴度均值为166，最高能到528，与AdC68组有显著区别(图6B，P＜0.01)。随后检测了针对CoV T的T细胞应答，AdC68-panCoV/Flu组产生总的T细胞应答如图6C所示：每百万个脾细胞里面分泌IFN-γ的细胞数均值在1220左右，最高能到达4010，显著高于AdC68(P＜0.01)。因此该疫苗既能诱导T细胞应答，又能诱导抗体应答。
随后在免疫结束后三周对小鼠进行了攻毒，选择的毒株为蝙蝠冠状病毒S HC014，剂量为1E5 TCID 50/小鼠，方式为滴鼻。持续观察小鼠体重和生存，发现，AdC68组小鼠体重在第五天出现明显下降，这种下降一直持续到它们全部死亡，第九天死亡的小鼠体重下降均超过20％。相比之下，AdC68-panCoV/Flu接种后仅表现出轻微的体重下降，其特征是起始点较晚(第8天开始)，最低点较高(第九天时体重下降约8％)，并且第十天体重开始上升(图6D)。观察期结束时的生存数据也显示AdC68-panCoV/Flu组与AdC68组之间存在显著差异，前者的存活率为60％，后者均无存活(图6E)。这些数据为AdC68-panCoV/Flu作为一种通用冠状病毒疫苗的潜力提供了初步证据。
表4.AdC68和AdC68-panCoV/Flu在HFH4-hACE2小鼠的体内免疫方案
Figure PCTCN2021114313-appb-000008
实施例6：金黄地鼠新冠病毒攻毒试验
金黄地鼠：雄性，购自上海吉辉实验动物饲养有限公司
毒株：SARS-CoV-2/human/CHN/Shanghai_CH-02/2020
委托第二军医大学进行攻毒试验。
免疫程序：将金黄地鼠随机分为5组，分别命名为组1、组2、组3、组4和组5。具体免疫组合如表5所示，免疫方式为肌肉注射(i.m.)加滴鼻(i.n.)，具体见表。
攻毒程序：免疫结束后2周对小鼠进行攻毒试验，攻毒后持续观察，记录体重变化和生存；攻毒后第三天每组各取4只金黄地鼠处死，取肺组织，每只金黄地鼠的左半边肺组织(一个大叶)用4％多聚甲醛固定48小时后做病理切片(HE染色和组化)；每只金黄地鼠的右半边肺组织(4个小叶)研磨测病毒载量qPCR RNA拷贝或者病毒滴度PFU/ml。
结果显示：AdC68-panCoV/Flu在金黄地鼠体内对冠状病毒攻毒提供有效保护。
表5.AdC68和AdC68-panCoV/Flu在金黄地鼠体内免疫方案
Figure PCTCN2021114313-appb-000009
实施例7：hACE2转基因小鼠新冠病毒攻毒试验
小鼠：南模生物ACE2-转基因小鼠(C57BL/6-Tgtn(CAG-human ACE2-IRES-Luciferase-WPRE-polyA)Smoc)，雌性。
毒株：SARS-CoV-2/human/CHN/Shanghai_CH-02/2020
委托第二军医大学进行攻毒试验。
免疫程序：将金黄地鼠随机分为3组，分别命名为组1、组2和组3。具体免疫组合如表6所示，免疫方式为肌肉注射加滴鼻，具体见表。
攻毒程序：免疫结束后2周对小鼠进行攻毒试验，攻毒后持续观察，记录体重变化和生存；攻毒后第三天每组各取4只小鼠处死，取肺组织，每只小鼠的左半边肺组织(一个大叶)用4％多聚甲醛固定48小时后做病理切片(HE染色 和组化)；每只小鼠的右半边肺组织(4个小叶)研磨测病毒载量qPCR RNA拷贝或者病毒滴度PFU/ml。
结果显示：AdC68-panCoV/Flu在hACE2+C57BL/6小鼠体内对冠状病毒攻毒提供有效保护。
表6.AdC68和AdC68-panCoV/Flu在hACE2+C57BL/6小鼠体内免疫方案
Figure PCTCN2021114313-appb-000010
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。
附录.序列表信息
SEQ ID NO： 序列名称 SEQ ID NO： 序列名称
1 ORF1ab共享氨基酸序列 20 表位肽10
2 ORF1ab共享编码序列 21 表位肽11
3 M蛋白共享氨基酸序列 22 表位肽12
4 M蛋白共享编码序列 23 表位肽13
5 N蛋白共享氨基酸序列 24 表位肽14
6 N蛋白共享编码序列 25 表位肽15
7 S蛋白共享氨基酸序列 26 表位肽16
8 S蛋白共享编码序列 27 表位肽17
9 CoV T共享氨基酸序列 28 表位肽18
10 CoV T共享编码序列 29 表位肽19
11 表位肽1 30 SARS-CoV/CoV-2的表位肽A对应序列
12 表位肽2 31 MERS-CoV的表位肽A对应序列
13 表位肽3 32 SARS-CoV的表位肽B对应序列
14 表位肽4 33 SARS-CoV-2的表位肽B对应序列
15 表位肽5 34 RBD-HA2-CD8TM核苷酸序列
16 表位肽6 35 RBD-HA2-CD8TM氨基酸序列
17 表位肽7 36 panCoV/Flu核苷酸序列
18 表位肽8 37 panCoV/Flu的前端部分氨基酸序列
19 表位肽9 38 panCoV/Flu的后端部分氨基酸序列

Claims (16)

  1. 一种分离的免疫原性肽，其包含选自下组的一种或多种冠状病毒蛋白共享肽：
    (a)冠状病毒早期表达蛋白多聚蛋白(ORF1ab)的共享肽；
    (b)冠状病毒膜蛋白(M)的共享肽；
    (c)冠状病毒核衣壳(N)蛋白的共享肽；
    (d)冠状病毒囊膜蛋白(E)蛋白的共享肽；和
    (e)冠状病毒刺突蛋白(S)蛋白的共享肽。
  2. 如权利要求1所述的免疫原性肽，其包含选自下组中的一个或多个肽段：
    PLPDRWYFYYT(SEQ ID NO：11)；
    VVNKQFGAISS(SEQ ID NO：12)；
    KPISAYAFLMA(SEQ ID NO：13)；
    SYGPGNTFITD(SEQ ID NO：14)；
    IKYYSIIPHSI(SEQ ID NO：15)；
    IEDLLFDKVET(SEQ ID NO：16)；
    SALQKIQDVVN(SEQ ID NO：17)；
    ADDEGFITLKN(SEQ ID NO：18)；
    YRVFPYDMDSGVSSF(SEQ ID NO：19)；
    SVTVEYNIHAVLDTL(SEQ ID NO：20)；
    QWLVMYGPI(SEQ ID NO：21)；
    QSGLVKMAQPSGKVE(SEQ ID NO：22)；
    NGRPQGVFHVTMRSN(SEQ ID NO：23)；
    VTYGNMTLNGL(SEQ ID NO：24)；
    SVGNFCYMHQL(SEQ ID NO：25)；
    LSDNDGLKYAKWEKD(SEQ ID NO：26)；
    KIKYLYFVKNL(SEQ ID NO：27)；
    GAVLGTISATVRLQA(SEQ ID NO：28)；和
    TGQAITVKPEA(SEQ ID NO：29)。
  3. 如权利要求1所述的免疫原性肽，由所述免疫原性肽激活的T细胞特异性结合于选自下组的冠状病毒表位：PLPDRWYFYYT(SEQ ID NO：11)； KPISAYAFLMA(SEQ ID NO：13)；LSPRWYFYYL(SEQ ID NO：30)；LAPRWYFYYTG(SEQ ID NO：31)；VPAYSFLPG(SEQ ID NO：32)；和/或APISAMVRMYIFFA(SEQ ID NO：33)；和/或
    所述共享肽来源于选自下组的冠状病毒：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV。
  4. 如权利要求1所述的免疫原性肽，其中，
    所述ORF1ab的共享肽包含SEQ ID NO：1所述氨基酸序列，例如，所述ORF1ab的共享肽的氨基酸序列如SEQ ID NO：1所示；和/或
    所述M蛋白的共享肽包含SEQ ID NO：3所述氨基酸序列，例如，所述M蛋白的共享肽的氨基酸序列如SEQ ID NO：3所示；和/或
    所述N蛋白的共享肽包含SEQ ID NO：5所述氨基酸序列，例如，所述N蛋白的共享序列的氨基酸序列如SEQ ID NO：5所示；和/或
    所述S蛋白的共享肽包含SEQ ID NO：7所述氨基酸序列，例如，所述M蛋白的共享肽的氨基酸序列如SEQ ID NO：7所示。
  5. 如权利要求1所述的免疫原性肽，其还包含：
    两种或以上所述共享肽的连接肽，所述共享肽之间包含或不包含接头序列，例如所述接头序列选自下组：(G4S) n(n＝1～8，例如(G4S) 3、G4S)、GSAGSAAGSGEF、(Gly) 6、EFPKPSTPPGSSGGAP、KESGSVSSEQLAQFRSLD、(Gly) 8、EGKSSGSGSESKST、IRES、P2A、T2A；和/或
    与共有序列共表达(例如融合表达或单独阅读框表达)的其他序列，所述其他序列用于例如扩大抗病毒谱、提高免疫应答诱导能力，所述其他序列选自例如针对如下病毒的免疫原：冠状病毒(例如RBD或其修饰序列(如末端Cys修饰RBD氨基酸序列))、流感病毒(例如HA2)、艾滋病毒、狂犬病毒、猪瘟病毒、蓝耳病病毒、麻疹病毒、埃博拉病毒、疱疹病毒、虫媒病毒(寨卡病毒、流行性乙型脑炎病毒、森林脑炎病毒、登革病毒、汉坦病毒、新疆出血热病毒)，尤其是与活化B细胞应答的免疫原，制备能够同时激活中和抗体与T细胞应答的复合疫苗；
    例如所述共表达的序列为选自下组的免疫调节序列：IL-2、IL-7、IL-12、IL-18、IL-21、GM-CSF、CD40L、CD40刺激抗体、PD-1与PD-L1抗体、CTLA4抗体、趋化因子CXCL9、CXCL10、CXCL11、CXCL12、CXCL3、XCL1、CCL4、CCL20、霍乱毒素及其亚单位、细菌鞭毛蛋白、FimH及SopE。
  6. 如权利要求1所述的免疫原性肽，其包含SEQ ID NO：9所示的氨基酸序列，例如，所述免疫原性肽的序列如SEQ ID NO：9所示；或者，其包含SEQ ID NO：37和/或38所示的氨基酸序列，例如，所述免疫原性肽的序列如SEQ ID NO：37和38所示。
  7. 如权利要求1～6中任一项所述免疫原性肽的编码分子，包含所述编码分子的载体和/或包含所述编码分子或载体的宿主细胞。
  8. 如权利要求7所述的编码分子、载体或宿主细胞，其中，所述编码分子包含选自下组中的一个或多个核苷酸分子：SEQ ID NO：2、SEQ ID NO：4、SEQ ID NO：6、SEQ ID NO：8、SEQ ID NO：10和SEQ ID NO：36；
    可任选地，所述核苷酸分子之间包含或不包含接头序列，所述编码分子包含或不包含前导序列，所述核苷酸分子包含或不包含标签，例如His-tag、AviTag、Calmodulin tag、polyglutamate tag、E-tag、FLAG tag、HA-tag、Myc-tag、S-tag、SBP-tag、Sof-tag1、Sof-tag3、Strep-tag、TC tag、V5 tag、T7 tag、VSV tag、Xpress tag、3X FLAG tag、Isopep tag、Spytag、Snoop tag和PNE tag。
  9. 一种产品，其包含：
    如权利要求1～6中任一项所述的免疫原性肽和/或如权利要求7～8中任一项所述的编码分子、载体或细胞；和
    可任选的，药学上或免疫学上可接受的载体、赋形剂和/或佐剂(例如选自下组的一种或多种佐剂：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21)。
  10. 如权利要求9所述的产品，所述产品为例如抗冠状病毒感染的T细胞疫苗或药物，尤其是针对冠状病毒的广谱T细胞疫苗或药物，所述冠状病毒选自下组：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，尤其是SARS-CoV、MERS-CoV和SARS-CoV-2病毒；和/或
    所述产品包含：含如权利要求1～6中任一项所述的免疫原性肽的缀合物或偶联物。
  11. 如权利要求9所述的产品，所述产品选自：核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒(例如痘病毒(如选白天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株的痘病毒)、腺 病毒(如选自Ad5、Ad11、Ad26、Ad35、AdC68的腺病毒)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒)载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗。
  12. 如权利要求9所述的产品，所述产品的形式适于选自下组的给予方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合；和/或
    所述产品适于与冠状病毒S或S1所制备的疫苗进行前后序贯接种，例如来源选自下组的S或S1：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV；和/或
    所述产品适于与流感病毒HA或HA2所制备的疫苗进行前后序贯接种，例如来源选自下组的HA或HA2：H1-H18。
  13. 一种获得如权利要求1～6中任一项所述的免疫原性肽的方法，所述方法包括：
    i)对各种冠状病毒(例如SARS-CoV、MERS-CoV和SARS-CoV-2病毒)的一种或多种选自下组的蛋白质进行大数据分析，提取出其共享肽序列信息：冠状病毒早期表达蛋白多聚蛋白(ORF1ab)、冠状病毒膜蛋白(M)、冠状病毒核衣壳(N)蛋白、冠状病毒囊膜蛋白(E)蛋白和冠状病毒刺突蛋白(S)蛋白；
    ii)对所得共享肽序列信息进行T细胞表位预测，获得具有免疫原性的保守区域信息；
    iii)根据所述保守区域信息设计并制备免疫原性肽或其编码序列；
    iv)可任选地，对所得免疫原性肽或其编码序列进行组合或将其与共表达(例如融合表达或单独阅读框表达)的其他序列进行组合，所述其他序列用于例如扩大抗病毒谱、提高免疫应答诱导能力。
  14. 如权利要求1～6中任一项所述的免疫原性肽和/或如权利要求7～8中任一项所述的编码分子、载体或细胞在制备抗冠状病毒感染的产品中的应用。
  15. 如权利要求14所述的应用，其中，所述产品为抗冠状病毒感染的T细胞疫苗或药物，尤其是针对冠状病毒的广谱T细胞疫苗或药物；和/或
    所述冠状病毒选自下组：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV，尤其是SARS-CoV、MERS-CoV和SARS-CoV-2病毒；和/或
    所述产品包含：含如权利要求1～6中任一项所述的免疫原性肽的缀合物或偶联物；和/或
    所述产品包含：药学上或免疫学上可接受的载体、赋形剂和/或佐剂(例如选自下组的一种或多种佐剂：铝佐剂、霍乱毒素及其亚单位、寡脱氧核苷酸、锰离子佐剂、胶体锰佐剂、弗氏佐剂、MF59佐剂、QS-21佐剂、Poly I：C及其他TLR配体、GM-CSF、IL-2、IL-3、IL-7、IL-11、IL-12、IL-18、IL-21；和/或
    所述产品选自：核酸疫苗(DNA或RNA疫苗)、重组蛋白亚单位疫苗、重组病毒(例如痘病毒(如选自天坛株、北美疫苗株、惠氏衍生株、李斯特株、安卡拉衍生株、哥本哈根株和纽约株的痘病毒)、腺病毒(如选自Ad5、Ad11、Ad26、Ad35、AdC68的腺病毒)、腺相关病毒、单纯疱疹病毒、麻疹病毒、呼肠弧病毒、弹状病毒、森林脑炎病毒、流感病毒、呼吸道合胞病毒、脊髓灰质炎病毒)载体疫苗、重组细菌载体疫苗、病毒样颗粒疫苗、纳米颗粒疫苗、细胞载体疫苗；和/或
    所述产品的形式适于选自下组的给予方式：肌肉接种、皮内接种、皮下接种、滴鼻、雾化吸入、生殖道、直肠、口服或上述不同接种方式的组合；和/或
    所述产品适于与冠状病毒S或S1所制备的疫苗进行前后序贯接种，例如来源选自下组的S或S1：SARS-CoV-2、SARS-CoV、MERS-CoV、HCoV-229E、HCoV-OC43、HCoV-NL63、HCoV-HKU1、bat-CoV；和/或
    所述产品适于与流感病毒HA或HA2所制备的疫苗进行前后序贯接种，例如来源选自下组的HA或HA2：H1-H18。
  16. 如权利要求1～6中任一项所述的免疫原性肽在制备检测冠状病毒感染的产品中的应用，例如，所述免疫原性肽单独或与MHC形成的肽-MHC复合物单聚体或多聚体(如二聚体、四聚体)。
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