WO2023023940A1 - Immunogène destiné à induire un vaccin à lymphocytes t anti-coronavirus à large spectre et son utilisation - Google Patents

Immunogène destiné à induire un vaccin à lymphocytes t anti-coronavirus à large spectre et son utilisation 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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徐建青
张晓燕
周东明
曹康丽
王祥
裘天颐
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复旦大学
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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

La présente invention concerne un immunogène destiné à induire un vaccin à lymphocytes T anti-coronavirus à large spectre et son utilisation. Plus précisément, l'invention porte sur un peptide immunogène, qui comprend un ou plusieurs peptides partagés choisis parmi différentes polyprotéines à protéines d'expression précoce, protéines membranaires, protéines de nucléocapside, protéines d'enveloppe et protéines de spicule de coronavirus, et une molécule destinée à les coder, un vecteur ou une cellule hôte de ce peptide, et un produit. Le vaccin de la présente invention peut être utilisé pour la prévention et le traitement de différents coronavirus.
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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116410271A (zh) * 2023-03-13 2023-07-11 华南农业大学 H5n1亚型aiv mhc b1限制性t细胞表位肽及其应用
CN117143206A (zh) * 2023-08-03 2023-12-01 华南农业大学 Alv-j mhc-b21限制性表位肽及其筛选方法和应用

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1829736A (zh) * 2003-04-10 2006-09-06 希龙公司 严重急性呼吸道综合征冠状病毒
CN102227442A (zh) * 2008-11-28 2011-10-26 日油株式会社 Sars冠状病毒的细胞毒性t细胞表位肽及其用途
WO2016138160A1 (fr) * 2015-02-24 2016-09-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunogènes du coronavirus du syndrome respiratoire du moyen-orient, anticorps et leur utilisation
CN112557645A (zh) * 2020-03-13 2021-03-26 珠海碳云智能科技有限公司 抗原表位多肽的筛选方法及装置
CN112592390A (zh) * 2020-04-21 2021-04-02 苏州系统医学研究所 新型冠状病毒特异性抗原肽及其用途
WO2021155323A1 (fr) * 2020-01-31 2021-08-05 Beth Israel Deaconess Medical Center, Inc. Compositions et procédés de prévention et de traitement d'une infection par le coronavirus - vaccins contre le sars-cov-2

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1829736A (zh) * 2003-04-10 2006-09-06 希龙公司 严重急性呼吸道综合征冠状病毒
CN102227442A (zh) * 2008-11-28 2011-10-26 日油株式会社 Sars冠状病毒的细胞毒性t细胞表位肽及其用途
WO2016138160A1 (fr) * 2015-02-24 2016-09-01 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Immunogènes du coronavirus du syndrome respiratoire du moyen-orient, anticorps et leur utilisation
WO2021155323A1 (fr) * 2020-01-31 2021-08-05 Beth Israel Deaconess Medical Center, Inc. Compositions et procédés de prévention et de traitement d'une infection par le coronavirus - vaccins contre le sars-cov-2
CN112557645A (zh) * 2020-03-13 2021-03-26 珠海碳云智能科技有限公司 抗原表位多肽的筛选方法及装置
CN112592390A (zh) * 2020-04-21 2021-04-02 苏州系统医学研究所 新型冠状病毒特异性抗原肽及其用途

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* Cited by examiner, † Cited by third party
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CN116410271A (zh) * 2023-03-13 2023-07-11 华南农业大学 H5n1亚型aiv mhc b1限制性t细胞表位肽及其应用
CN117143206A (zh) * 2023-08-03 2023-12-01 华南农业大学 Alv-j mhc-b21限制性表位肽及其筛选方法和应用

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