CN116444631A - Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine - Google Patents
Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine Download PDFInfo
- Publication number
- CN116444631A CN116444631A CN202310446178.8A CN202310446178A CN116444631A CN 116444631 A CN116444631 A CN 116444631A CN 202310446178 A CN202310446178 A CN 202310446178A CN 116444631 A CN116444631 A CN 116444631A
- Authority
- CN
- China
- Prior art keywords
- novel coronavirus
- cell
- peptide
- antigen
- epitope peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 207
- 210000001744 T-lymphocyte Anatomy 0.000 title claims abstract description 159
- 241000711573 Coronaviridae Species 0.000 title claims abstract description 120
- 229960005486 vaccine Drugs 0.000 title claims abstract description 28
- 238000002360 preparation method Methods 0.000 title claims description 11
- 239000000427 antigen Substances 0.000 claims abstract description 101
- 108091007433 antigens Proteins 0.000 claims abstract description 88
- 102000036639 antigens Human genes 0.000 claims abstract description 88
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 6
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 72
- 210000004027 cell Anatomy 0.000 claims description 44
- 108090000623 proteins and genes Proteins 0.000 claims description 16
- 102000025850 HLA-A2 Antigen Human genes 0.000 claims description 15
- 108010074032 HLA-A2 Antigen Proteins 0.000 claims description 15
- 239000000203 mixture Substances 0.000 claims description 13
- 210000000612 antigen-presenting cell Anatomy 0.000 claims description 10
- 239000003814 drug Substances 0.000 claims description 5
- 230000000890 antigenic effect Effects 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 238000002474 experimental method Methods 0.000 abstract description 25
- 239000000178 monomer Substances 0.000 abstract description 10
- 230000006044 T cell activation Effects 0.000 abstract description 4
- 208000015181 infectious disease Diseases 0.000 abstract description 4
- 210000005259 peripheral blood Anatomy 0.000 abstract description 4
- 239000011886 peripheral blood Substances 0.000 abstract description 4
- 229940126585 therapeutic drug Drugs 0.000 abstract description 4
- 238000000338 in vitro Methods 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- 238000009509 drug development Methods 0.000 abstract 1
- 229920000642 polymer Polymers 0.000 abstract 1
- 102100034922 T-cell surface glycoprotein CD8 alpha chain Human genes 0.000 description 31
- 238000001514 detection method Methods 0.000 description 23
- 230000030741 antigen processing and presentation Effects 0.000 description 16
- 229920001184 polypeptide Polymers 0.000 description 14
- 241000700605 Viruses Species 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 12
- 238000002835 absorbance Methods 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 238000002965 ELISA Methods 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 210000001266 CD8-positive T-lymphocyte Anatomy 0.000 description 7
- 238000012216 screening Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 102100030385 Granzyme B Human genes 0.000 description 5
- 101001009603 Homo sapiens Granzyme B Proteins 0.000 description 5
- 241000701044 Human gammaherpesvirus 4 Species 0.000 description 5
- 102100037850 Interferon gamma Human genes 0.000 description 5
- 108010074328 Interferon-gamma Proteins 0.000 description 5
- 238000003501 co-culture Methods 0.000 description 5
- 238000000684 flow cytometry Methods 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 206010022000 influenza Diseases 0.000 description 5
- 102000004169 proteins and genes Human genes 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000002255 vaccination Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000004913 activation Effects 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 230000036737 immune function Effects 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 238000012827 research and development Methods 0.000 description 4
- 230000001413 cellular effect Effects 0.000 description 3
- 230000009918 complex formation Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000017188 evasion or tolerance of host immune response Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000005342 ion exchange Methods 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 238000004153 renaturation Methods 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- BFSVOASYOCHEOV-UHFFFAOYSA-N 2-diethylaminoethanol Chemical compound CCN(CC)CCO BFSVOASYOCHEOV-UHFFFAOYSA-N 0.000 description 2
- 102000000412 Annexin Human genes 0.000 description 2
- 108050008874 Annexin Proteins 0.000 description 2
- 208000001528 Coronaviridae Infections Diseases 0.000 description 2
- 108010002350 Interleukin-2 Proteins 0.000 description 2
- 108010090804 Streptavidin Proteins 0.000 description 2
- 108091008874 T cell receptors Proteins 0.000 description 2
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000007969 cellular immunity Effects 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 238000009169 immunotherapy Methods 0.000 description 2
- 208000037797 influenza A Diseases 0.000 description 2
- 108010061181 influenza matrix peptide (58-66) Proteins 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000002808 molecular sieve Substances 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 239000011534 wash buffer Substances 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 108700028369 Alleles Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000025721 COVID-19 Diseases 0.000 description 1
- 241001678559 COVID-19 virus Species 0.000 description 1
- YASYEJJMZJALEJ-UHFFFAOYSA-N Citric acid monohydrate Chemical compound O.OC(=O)CC(O)(C(O)=O)CC(O)=O YASYEJJMZJALEJ-UHFFFAOYSA-N 0.000 description 1
- 208000034657 Convalescence Diseases 0.000 description 1
- GUBGYTABKSRVRQ-WFVLMXAXSA-N DEAE-cellulose Chemical compound OC1C(O)C(O)C(CO)O[C@H]1O[C@@H]1C(CO)OC(O)C(O)C1O GUBGYTABKSRVRQ-WFVLMXAXSA-N 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000011786 HLA-A Antigens Human genes 0.000 description 1
- 108010075704 HLA-A Antigens Proteins 0.000 description 1
- 102000008949 Histocompatibility Antigens Class I Human genes 0.000 description 1
- 108010088652 Histocompatibility Antigens Class I Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 1
- 241000985627 Lota Species 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 230000010782 T cell mediated cytotoxicity Effects 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- ZTOJFFHGPLIVKC-CLFAGFIQSA-N abts Chemical compound S/1C2=CC(S(O)(=O)=O)=CC=C2N(CC)C\1=N\N=C1/SC2=CC(S(O)(=O)=O)=CC=C2N1CC ZTOJFFHGPLIVKC-CLFAGFIQSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000008485 antagonism Effects 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960002303 citric acid monohydrate Drugs 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- OOTFVKOQINZBBF-UHFFFAOYSA-N cystamine Chemical compound CCSSCCN OOTFVKOQINZBBF-UHFFFAOYSA-N 0.000 description 1
- 229940099500 cystamine Drugs 0.000 description 1
- UFULAYFCSOUIOV-UHFFFAOYSA-N cysteamine Chemical compound NCCS UFULAYFCSOUIOV-UHFFFAOYSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000005860 defense response to virus Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000013024 dilution buffer Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 230000005965 immune activity Effects 0.000 description 1
- 230000007124 immune defense Effects 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 229940031551 inactivated vaccine Drugs 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 229960003151 mercaptamine Drugs 0.000 description 1
- 229960004857 mitomycin Drugs 0.000 description 1
- GEVPUGOOGXGPIO-UHFFFAOYSA-N oxalic acid;dihydrate Chemical compound O.O.OC(=O)C(O)=O GEVPUGOOGXGPIO-UHFFFAOYSA-N 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000004393 prognosis Methods 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
- C07K14/70539—MHC-molecules, e.g. HLA-molecules
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5154—Antigen presenting cells [APCs], e.g. dendritic cells or macrophages
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
- A61K2039/515—Animal cells
- A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/20011—Coronaviridae
- C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- Molecular Biology (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Immunology (AREA)
- Biophysics (AREA)
- Animal Behavior & Ethology (AREA)
- Pharmacology & Pharmacy (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Gastroenterology & Hepatology (AREA)
- Mycology (AREA)
- Cell Biology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a novel coronavirus T cell epitope peptide and application thereof in preparing vaccines, wherein the amino acid sequence of the novel coronavirus T cell epitope peptide is shown as any one of SEQ ID NO. 1-9. The pMHC complex monomer is used for preparing the pMHC complex monomer, and the pMHC complex polymer is further prepared, so that the pMHC complex monomer can be used for detecting antigen-specific T cells in peripheral blood of novel coronavirus vaccinators and infection rehabilitators and is used for in-vitro T cell activation experiments, and the novel coronavirus T cell epitope peptide can be used for preparing general vaccines aiming at various novel coronavirus mutant strains, novel coronavirus-related immunodetection and broad-spectrum therapeutic drug development, and is worthy of intensive research and popularization.
Description
The application is a divisional application of an invention patent application of which the application date is 2022, 2, 24 and the application number is 2022101769472, and the invention is named as a novel coronavirus T cell epitope peptide and application of the novel coronavirus T cell epitope peptide in preparation of vaccines.
Technical Field
The invention relates to the technical field of biology, in particular to a novel coronavirus T cell epitope polypeptide and application thereof in preparing vaccines.
Background
Currently, the global inoculation of novel coronavirus vaccine injection reaches 92 hundred million doses. Currently, vaccination remains the best and only tool to control epidemic spread. However, as new coronavirus variants continue to emerge, a number of new coronavirus variants can escape through immunization and continue to infect people who have been vaccinated with new coronavaccines, establishing an effective population immune barrier through vaccine immunization being the central means.
The universal vaccine is a broad-spectrum vaccine which can effectively protect the immune crowd even after the virus antigen is mutated. Therefore, there is an urgent need to develop a general vaccine and a broad-spectrum therapeutic drug that can still provide effective immune protection to vaccine immunized populations after various variants of viruses occur.
According to the research on coronaviruses such as SARS with higher homology, the T cell immune response plays an important role in the antiviral defense of the organism after virus infection and in the pathological damage process of the organism immunity, in particular to CD8 + T cells, which remained after 11 years of antigen-specific immune activity, demonstrated CD8 + T cell immunityThe important role of epidemic response in the immune defenses against coronaviruses and its important role in vaccine development. CD8 + The first step in the immune response of T cells is the specific recognition of epitope peptides presented by the virus-infected cells by T cells through their surface antigen recognition receptors. Therefore, the epitope peptide is an important key molecule for specifically recognizing viruses and playing an immune protection role by T cells, and is a key targeting molecule for immunodetection, immunotherapy and vaccine development.
CD8 + T cells recognize pMHC activation through T Cell Receptors (TCRs), kill virus-infected cells, and eliminate viruses, thereby exerting an antiviral cellular immune effect. Therefore, the identification of the epitope peptide capable of effectively activating the T cells and inducing effective T cell immune protection after the virus antigen is mutated is one of the keys for developing novel crown general vaccine and broad-spectrum therapeutic drugs.
Immune escape is the antagonism, blocking and suppression of the immune response of the body by immunosuppressive pathogens through their structural and non-structural products. The novel coronavirus, because of frequent and sustained mutation, produces a large number of variants, which seriously threatens the immune barrier effect of the vaccine.
Disclosure of Invention
The invention aims to overcome the defect that a T cell epitope peptide for a novel coronavirus general vaccine and application thereof are not developed in the field of novel coronaviruses in the prior art, and provides a novel coronavirus T cell epitope peptide and application thereof in preparing vaccines; it can assemble into pMHC complex with HLA-A2 heavy chain and HLA-A2 light chain beta 2m protein; or directly loaded to antigen presenting cells, can activate T cells, effectively induce T cell immunity, avoid immune escape of mutant strains, and can detect specific CD8+ T cells in both new coronavirus vaccinators and rehabilitators, thereby being applicable to research and development of novel coronavirus general vaccines, preparation, drug research and development and clinical treatment.
The first object of the invention is to provide a novel coronavirus T cell epitope peptide.
The second object of the invention is to provide a novel coding gene of coronavirus T cell epitope peptide.
A third object of the invention is to provide a novel coronavirus T cell epitope peptide composition.
A fourth object of the present invention is to provide a pMHC complex.
It is a fifth object of the present invention to provide an antigenic peptide-antigen presenting cell complex.
The sixth object of the present invention is to provide the application of one or more of the antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide encoding gene, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex, and the antigen peptide-antigen presenting cell complex in preparing novel coronavirus vaccine.
The seventh object of the present invention is to provide the antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex, and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus drugs.
In order to achieve the above object, the present invention is realized by the following means:
T2-A2 is an antigen presenting cell line expressing human MHC class I molecule HLa-A2 by recombinant genetic engineering techniques. Only effective epitope peptides can be presented, so that a stable pMHC complex is formed on the cell surface, and therefore, the antigen peptide can be used as artificial antigen presenting cells for stimulating T cells.
T cell epitope peptides alone cannot work and must be activated in the manner of pMHC complexes or antigen peptide-antigen presenting cell complexes. The invention utilizes MHC monomer and identified novel coronavirus T cell epitope to carry out joint renaturation, thus preparing the pMHC complex. The identified novel coronavirus T cell epitope peptide is loaded on the surface of antigen presenting cells (T2-A2 cells), antigen peptide-antigen presenting cell complexes are prepared, then the prepared pMHC complexes are used for labeling T cells, the antigen epitope peptide can be found to effectively activate T cells in peripheral blood of healthy people, and compared with the antigen epitope peptide of an original virus strain, the variant strain can increase the activation capacity of the T cells and can also effectively kill target cells carrying novel coronavirus antigens. The pMHC complex with the PE fluorescent channel assembled by the T cell epitope peptide can be detected in both new crown vaccine inoculators and rehabilitators. The novel coronavirus T cell epitope peptide found newly can effectively induce T cell immunity, avoid immune escape of mutant strain, and can be used as general vaccine or applied to immunotherapy and the like.
Therefore, the invention requires a novel coronavirus T cell epitope peptide, and the amino acid sequence of the novel coronavirus T cell epitope peptide is shown in any one of SEQ ID NO. 1-9.
SEQ ID NO:1：VTWFHAIHV，
SEQ ID NO:2：VTWFHAISG，
SEQ ID NO:3：FKLKDCVMYA，
SEQ ID NO:4：FKLKECVMYA，
SEQ ID NO:5：YHDVRVVLDFI，
SEQ ID NO:6：YHDVRVVLI，
SEQ ID NO:7：ASLPFGWLI，
SEQ ID NO:8：ASLLFGWLI，
SEQ ID NO:9：VTWFHVIHV。
A coding gene of a novel coronavirus T cell epitope peptide, wherein the coding gene codes the novel coronavirus T cell epitope peptide.
A novel coronavirus T cell antigen epitope peptide composition comprises a polypeptide with an amino acid sequence shown in SEQ ID NO: 1-9, and any several of novel coronavirus T cell epitope peptides.
A pMHC complex comprising said novel coronavirus T cell epitope peptide or said novel coronavirus T cell epitope peptide composition.
Preferably, the preparation method of the pMHC complex is that HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the novel coronavirus T cell epitope peptide are renatured.
An antigen peptide-antigen presenting cell complex, and the surface of the antigen presenting cell is provided with the novel coronavirus T cell antigen epitope peptide or the novel coronavirus T cell antigen epitope peptide composition.
Preferably, the antigen presenting cells are T2-A2 cells.
More preferably, the T2-A2 cells are T2 cells over-expressing HLa-A2.
The antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus vaccines.
The antigen epitope peptide, the encoding gene of the novel coronavirus T cell antigen epitope peptide, the novel coronavirus T cell antigen epitope peptide composition, the pMHC complex and the application of one or more of the antigen peptide-antigen presenting cell complexes in preparing novel coronavirus medicaments.
Compared with the prior art, the invention has the following beneficial effects:
the invention discovers 9 novel coronavirus T cell epitope peptides, which can be used for detecting antigen-specific T cells in peripheral blood of novel coronavirus vaccinators and infection healers and used for in vitro T cell activation experiments after being prepared into pMHC complexes with PE fluorescent channels, and can be used for preparing general vaccines aiming at various novel coronavirus mutant strains, novel coronavirus-related immunodetection and broad-spectrum therapeutic drugs.
The novel coronavirus T cell epitope peptide is prepared into a pMHC complex with a PE fluorescent channel, or directly loaded to antigen presenting cells, so that T cells are activated, and the novel coronavirus T cell epitope peptide can be used for research and development of novel coronavirus vaccines, preparation, drug research and development and clinical treatment, and can be applied to:
1) Development and preparation of novel crown vaccines: after the novel coronavirus is mutated, the plurality of T cell epitopes can induce the organism to generate immune response to generate antigen-specific T cells. Therefore, the T cell epitope is a candidate antigen epitope peptide of the novel coronavirus universal vaccine.
2) Detecting whether it has cellular immune function against infection by the novel coronavirus: the novel coronavirus antigen specific T cells are detected in the body of a person to be detected, which represents that the body has generated T cell immune function, and the T cell immune function of the body and the possibility of infection of the novel coronavirus can be evaluated according to the proportion of the antigen specific CD8T marked by the pMHC complex prepared into the fluorescent channel by the epitope peptide.
3) The effect after vaccination was evaluated: the detection of novel coronavirus antigen-specific T cells in the inoculant, which represent that the organism has developed T cell immune function, can be used to assess the likelihood of the organism re-infecting the novel coronavirus.
4) Monitoring the condition: can be used for monitoring the change of the illness state of the close contact person, the medical observer, the suspected and the diagnosed patient.
5) And (3) prognosis judgment: poor prognosis is predicted if the body fails to mount a T cell immune response, or the proportion of antigen-specific T cells continues to decrease.
Drawings
FIG. 1 is an identification of T2-A2 antigen presentation of 33 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 33 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of the formation of pMHC complex by 33 antigen peptides, ELISA method is used to screen 33 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the above 33 peptides are used to conduct peptide exchange analysis, and enzyme label instrument is used to measure absorbance (OD value) at 450nm wavelength.
FIG. 2 is an identification of T2-A2 antigen presentation of 10 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 10 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 10 antigen peptides C, screening 10 novel coronavirus T cell epitope peptides by ELISA, peptide exchange analysis with uv-sensitive peptide/MHC complex and the 10 peptides described above, and absorbance (OD value) measurement with a microplate reader at 450nm wavelength.
FIG. 3 is an identification of T2-A2 antigen presentation of 28 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 28 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 28 antigen peptides C28 novel coronavirus T cell epitope peptides were screened by ELISA, peptide exchange analysis was performed with uv-sensitive peptide/MHC complex and the 28 peptides described above, and absorbance (OD value) was measured with a microplate reader at a wavelength of 450 nm.
FIG. 4 is an identification of T2-A2 antigen presentation of 6 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 6 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 6 antigen peptides, ELISA method is used for screening 6 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 6 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 5 is an identification of T2-A2 antigen presentation of 30 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 30 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 30 antigen peptides, ELISA method is used for screening 30 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the above 30 peptides are used for peptide exchange analysis, and enzyme-labeled instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 6 is an identification of T2-A2 antigen presentation of 32 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 32 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; detection of pMHC complex formation by 32 antigen peptides C, screening of 32 novel coronavirus T cell epitope peptides by ELISA, peptide exchange analysis with uv-sensitive peptide/MHC complex and the above 32 peptides, and absorbance (OD value) measurement with a microplate reader at 450nm wavelength.
FIG. 7 is an identification of T2-A2 antigen presentation of 22 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: identification experiments of T2-A2 antigen presentation of 22 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is the detection of 22 antigen peptides forming pMHC complex, ELISA method is used for screening 22 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 22 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 8 is an identification of T2-A2 antigen presentation of 6 epitope peptides of novel coronavirus T cells and detection of antigen peptide formation into pMHC complexes; a: an identification experiment of T2-A2 antigen presentation of 6 novel coronavirus antigen polypeptides; b is a repeated nub of three experiments, and is a statistical graph of A; c is detection of formation of pMHC complex by 6 antigen peptides, ELISA method is used for screening 6 novel coronavirus T cell antigen epitope peptides, ultraviolet sensitive peptide/MHC complex and the 6 peptides are used for peptide exchange analysis, and enzyme label instrument is used for measuring absorbance (OD value) at 450nm wavelength.
FIG. 9 shows the purification results of pMHC complexes after assembly; a: purifying the result of the DEAE cellulose ion exchange column after assembling the pMHC complex; b: purification results of the assembled pMHC complex with molecular sieves (GE Superdex75 pg).
FIG. 10 evaluation of 167 antigen peptides versus CD8 + Activation of T cells. Induction of specific CD8 after 7 days of antigenic peptide stimulation + T cell production, NC: positive-ctrl (T2-A2 contains no peptide and CD 8) + T cell co-culture) as a negative control, PC: positive-ctrl (influenza A M1 peptide GILGFVFTL) as Positive control, and flow cytometry for detecting antigen-specific CD8 in peripheral blood of healthy volunteers activated by the above antigen peptide + T cell。
FIG. 11 is a summary of results of evaluating activation of CD8+ T cells by 167 antigen peptides; a: the results summarize show that antigen-specific CD8 can be detected by the pMHC complex with fluorescent channels formed by these mutated epitopes + T, the corresponding T cell epitope peptide of the variant strain specifically activates CD8 as compared with the T cell epitope peptide of the original strain + The proportion of T cells is unchanged or increased; b: the result summarization shows that the antigen-specific CD8 can be detected by the pMHC complex with fluorescent channel formed by the T cell epitope peptide of the novel coronavirus variant virus strain and the T cell epitope peptide of the corresponding original virus strain + T, the corresponding T cell epitope peptide of the variant strain specifically activates CD8 as compared with the T cell epitope peptide of the original strain + The proportion of T cells decreases; c: the result summarization shows that the antigen-specific CD8 can be detected by the pMHC complex with fluorescent channel formed by the T cell epitope peptide of the novel coronavirus variant virus strain and the T cell epitope peptide of the corresponding original virus strain + T, the corresponding T cell epitope peptide of the variant strain specifically activates CD8 as compared with the T cell epitope peptide of the original strain + The proportion of T cells increases or decreases.
FIG. 12 evaluation of 167 antigen peptides versus CD8 + T cell toxic effects; a (upstream): antigenic peptide induced specificity CD8 + T cell mediated killing of cells, CFSE labeled T2-A2 cells as viable target cells; day 0-ctrl: dyeing before stimulation; T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture); neg-ctrl (epstein barr virus IVTDFSVIK); pos-ctrl (influenza a M1 peptide gilgfveftl) was a positive control; b (left): the results show that the antigen peptide can kill target cells (T2-A2 cells); a (downstream): specific CD8 + T cell mediated apoptosis of target cells, T2-A2-ctrl (T2-A2 peptide-free co-culture with CD8+ T cells); neg-ctrl (EB virus IVTDFSVIK), pos-ctrl (influenza A M1 peptide GILGFVFTL), flow cytometry detects the proportion of apoptosis in T2-A2 cells labeled with CFSE and apoptosis markers Annexin V-APC; b (right): the results collectively show that the antigen peptide can induce target cells (T2-A2 cells) apoptosis; c (upstream): specific CD8 + T cell IFN-gamma + Is released. T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture), neg-ctrl (epstein barr virus IVTDFSVIK), pos-ctrl (influenza a M1 peptide gilgfftl). Flow cytometer detection markers with CD8 + IFN-γ + Proportion of the components; d (left): the result summarization shows that the antigen peptide can induce CD8 + T release IFN-gamma + The method comprises the steps of carrying out a first treatment on the surface of the C (downstream): specific CD8 + T cell GZMB + Is released. T2-A2-ctrl (T2-A2 is peptide-free and CD 8) + T cell co-culture), neg-ctrl (epstein barr virus IVTDFSVIK), pos-ctrl (influenza a M1 peptide gilgfftl); flow cytometer detection markers with CD8 + GZMB + Proportion of the components; d (right): the result summarization shows that the antigen peptide can induce CD8 + T release GZMB + 。
FIG. 13 evaluates the proportion of specific CD8+ T cells in vivo 14 days after new crown recovery and 7 days after vaccination with the second needle; detection of HLA-A2+COVID-19 by A flow cytometry the above 9 patients (T cell epitope peptide of novel coronavirus variant strain compared with T cell epitope peptide of corresponding original strain upon T cell activation) produced specific CD8 + T cell proportion is increased or unchanged) of the peptide; b: the result summarization shows that 10 prepared pMHC complexes with fluorescent channels can detect memory CD8 in novel coronavirus infection rehabilitation person + T cells; c flow cytometry detection of specific CD8 of the above 9 (T cell activation, T cell epitope peptide of novel coronavirus variant strain, produced specific CD8+ ratio increased or unchanged compared to T cell epitope peptide of corresponding original strain) peptides of HLA-A2+ volunteers 7 days after vaccination of the second needle + T cells; d: the results summarised show that the 9 prepared pMHC complexes with fluorescent channels can detect memory cd8+ T cells in the vaccinations.
Detailed Description
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 prediction of novel coronavirus HLA-A2 restriction epitope peptides
1. Experimental method
CD8T cell epitope prediction was performed on SARS-CoV-2 original virus strain (NC_ 045512.2) spike (S), membrane (M), nucleocapsid (N), envelope (E) and ORF protein sequences using the "MHC I Binding" tool (http:// tools. Iedb. Org/mhci). The prediction method used was IEDB Recommended 2.22 (NetMHCpan EL), MHC allele selection was HLA-A 02:01, which is the most common class of HLa genotypes in the chinese population.
The T cell epitope peptides of all novel coronavirus variant strains containing the same species as b.1.1.7 (Alpha type mutant) (id=1), b.1.351 (Beta type mutant) (id=2), p.1 (Gamma type mutant) (id=3), p.2 (id=4), P.3 (id=5), b.1.429 (Epsilon type mutant) (id=6), b.1.526.1 (Lota type mutant) (id=7), b.1.526.2 (id=8), b.1.618 (id=9), b.1.617.1 (id=10), b.1.617.2 (Delta type mutant) (id=11), b.1.617.3 (id=12), and b.1.1.529 (omacron type mutant) (id=13) were studied with the T cell epitope peptides of the corresponding original strains. The variant sequence of each epitope (GISAID. Org website) was extracted from the GISAID database for further analysis.
2. Experimental results
167 high-efficiency specific candidate T cell epitope peptides are screened out. The information is shown in Table 1.
Table 1 novel 167 epitope peptides of coronavirus T cells:
/>
/>
/>
EXAMPLE 2 identification of novel coronavirus HLA-A2 restriction epitope peptides
1. Experimental method
Candidate T cell epitope peptides predicted in synthetic example 1 were prepared at a concentration of 20. Mu.M. Taking logarithmic growth state T2-A2 cells, planting into 96-well plates, 10 each 5 The blank wells, negative control peptide (epstein barr virus, IVTDFSVIK), positive control peptide (influenza a M1 polypeptide, gilgfftl) and each synthetic candidate T cell epitope peptide were distributed, 3 wells per group, and a final volume of 200 μl. After incubation for 4 hours at 37℃the cells were washed twice by centrifugation, labeled with FITC anti-human HLA-A2 (. Beta.2m) antibodies, incubated at 4℃for 30 minutes in the absence of light and then detected by flow cytometry. Experiments were performed 3 times in total.
2. Experimental results
The results are shown in figures 1 to 8, a and B, which show that 167 antigen polypeptides can be efficiently presented by antigen presenting cells to T cells, indicating that these peptides are T cell epitope peptides.
Example 3 detection of antigen Polypeptides to form pMHC complexes
1. Experimental method
ELISA method detects 167 novel coronavirus T cell epitope peptides screened in the example 2.
100. Mu.L of 0.5. Mu.g mL was used at room temperature -1 Streptavidin was incubated on 96U-plates for 16-18 hours, washed 3 times with wash buffer and blocked with dilution buffer (0.5M Tris pH 8.0,1M NaCl,1% BSA,0.2% Tween 20) for 30 min at room temperature. pMHC complex of photopeptide (kilgffjv) and MHC ((BioLegend, cat#280003, us)) was used as HLA blank, influenza a M1 peptide (M58-66 gilgfvffl) was used as positive control, and epstein barr virus peptide (IVTDFSVIK) was used as negative control. Then, 20. Mu.L of the dilutions (400. Mu.M) of 167 novel coronavirus T cell epitope peptides of example 1 and 20. Mu.L of Flex-T were each added, which were replaced with a 365nm three-way ultraviolet analyzer (Qian Bell, cat# 1903274) TM Dilutions of the monomer (200. Mu.g/mL) were made in 100. Mu.L to 96-well plates and incubated for 1 hour in a cell incubator at 37 ℃. After three washes with wash buffer, 100 μl of diluted HRP-binding antibody (BioLegend, cat#280303, us) was added and incubation was continued for 1 hour at 37 ℃ before washing. 100 μl of substrate solution (10.34 mL deionized water, 1.2mL 0.1m citric acid monohydrate/trisodium citrate dihydrate, pH 4.0, 240 μl 40mm abts,120 μl hydrogen peroxide solution) was added and incubated at room temperature in the dark for 8min. The reaction was quenched with 50. Mu.L of a termination solution (2% w/v oxalic acid dihydrate). Absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader over 30 minutes.
2. Experimental results
The results are shown in fig. 1 to 8C, which show that 167 antigen polypeptides can form pMHC complexes.
EXAMPLE 4 preparation of epitope peptide pMHC Complex monomer
1. Experimental method
HLA-A2 heavy chain, HLA-A2 light chain β2m and 167 epitope peptides in table 1 of example 1 were each followed by 1:2: the molar ratio of 10 was gradually added dropwise to a renaturation solution (5M urea, 0.4M arginine, 100mM Tris, 3.7mM cystamine, 6.3mM cysteamine and 2mM EDTA) for renaturation to obtain the pMHC complex monomer of the epitope peptide.
The pMHC complex monomer was further purified by passing through DEAE ion exchange column, eluting with 0.5M NaCl, and collecting proteins according to the OD280nm uv absorbance peak. The proteins purified by DEAE ion exchange column were then purified by Superdex75pg molecular sieve, eluted with PBS, and the different molecular weight proteins were collected according to the OD280nm UV absorbance peak.
2. Experimental results
After purification, pMHC complex monomers of the epitope peptide were obtained (fig. 9).
EXAMPLE 5 novel coronavirus HLA-A2 restricted epitope peptide-activated T cells
1. Experimental method
T2 cells activate T cells by expressing HLa-A2 molecules (T2-A2). Mononuclear lymphocytes (PBMCs) in peripheral venous blood of healthy volunteers were isolated and cd8+ T cells were further isolated. T2-A2 cells were labeled with CFSE and incubated with 167 different antigen peptides from example 1 after 20 min treatment with 20. Mu.g/mL mitomycin.
The specific method comprises the following steps:
will be 0.5X10 6 CD8+ T cells and different epitope peptides were loaded 0.5X10 6 T2-A2 cells were co-cultured and co-stimulated with 1. Mu.g/mL anti-human CD28 antibody and 50IU/mL IL-2. 50IU/mL IL-2 and 20 mu M epitope peptide were supplemented every two days.
The pMHC complex monomer of 30 μl of the epitope peptide obtained in example 4 was mixed with 3.3 μl of PE streptavidin (BioLegend cat#405203, US) in a 96-well plate, and after incubation at 4 ℃ for 30 minutes in the dark, 2.4 μl of blocking solution (1.6 μl of 50mM biotin (Thermo Fisher, cat#b20656, US)) and 198.4 μl of LPBS were added to stop the reaction, and incubated overnight at 4-8 ℃ to obtain pMHC complex with PE fluorescence channel.
After 7 days of culture, the percentage of survival of T2-A2, the proportion of specific CD8+ T cells labeled with pMHC complex with PE fluorescent channels and the percentage of apoptosis marker Annexin V-APC on T2-A2 cells were calculated and the release of IFN-gamma and GZMB from specific CD8+ T cells was examined.
2. Experimental results
As shown in FIGS. 10 and 11, 167 novel coronavirus T cell epitope peptides selected in example 2 were all able to activate T cells.
Wherein, for the T cell epitope peptide VTWFHAISG (SEQ ID NO: 2) of the B.1.1.7 mutant strain, it corresponds to the T cell epitope peptide VTWFHAIHV (SEQ ID NO: 1) of the original virus strain, CD8 is activated + The cytocapacity of T is relatively stable before and after mutation.
Whereas for the P.3 mutant, T cell epitope peptide FKLKECVMYA (SEQ ID NO: 4), which corresponds to T cell epitope peptide FKLKDCVMYA (SEQ ID NO: 3) of the original strain; t cell epitope peptide YHDVRVVLI (SEQ ID NO: 6) for the B.1.526.1 mutant strain, which corresponds to T cell epitope peptide YHDVRVVLDFI (SEQ ID NO: 5) for the original strain; t cell epitope peptide ASLLFGWLI (SEQ ID NO: 8) for the B.1.526.2 mutant strain, which corresponds to T cell epitope peptide ASLPFGWLI (SEQ ID NO: 7) for the original strain; for the T cell epitope peptide VTWFHVIHV (SEQ ID NO: 9) of the B.1.1.529 mutant strain, which corresponds to the T cell epitope peptide VTWFHAIHV (SEQ ID NO: 1) of the original strain, the T cell epitope peptide of the above original strain activates CD8 as compared with the T cell epitope peptide of the mutant strain + T's cellular capacity is enhanced.
Specific CD8 activated by the above peptides + T kills target cells and is specific for CD8 + T cells release IFN-gamma and GZMB (as shown in FIG. 12).
Example 6 novel coronavirus HLA-A2 restricted epitope peptide activated T cells
1. Experimental method
Isolation of PBMCs in peripheral venous blood of new coronavirus original strain (NC 045512.2) infected convalescence persons for 14 days and 7 days after the second needle of beijing ke xing inactivated vaccine and identification of HLA subtype thereof, HLA-a2 positive PBMCs samples were further stained with pMHC complex with PE fluorescent channel and CD8-PerCP antibody of example 5 and then subjected to on-stream detection.
2. Experimental results
The results are shown in FIG. 13, which shows that the pMH with fluorescent channels has the amino acid sequences shown in SEQ ID NO. 1-9 for 9 epitope peptidesThe C complex can recognize antigen-specific CD8 generated in novel coronavirus infection convalescents and vaccinators + T cells.
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. The novel coronavirus T cell epitope peptide is characterized in that the amino acid sequence is shown as any one of SEQ ID NO. 3.
2. A novel coronavirus T cell epitope peptide encoding gene, wherein said encoding gene encodes the novel coronavirus T cell epitope peptide of claim 1.
3. A novel coronavirus T cell epitope peptide composition, which is characterized by comprising an amino acid sequence shown in SEQ ID NO:3, and any several of the novel coronavirus T cell epitope peptides.
4. A pMHC complex comprising the novel coronavirus T cell epitope peptide of claim 1 or the novel coronavirus T cell epitope peptide composition of claim 3.
5. The pMHC complex according to claim 4, wherein the preparation method of the pMHC complex is that HLA-A2 heavy chain, HLA-A2 light chain beta 2m and novel coronavirus T cell epitope peptide renaturing is obtained.
6. An antigen peptide-antigen presenting cell complex, characterized in that the antigen presenting cell has the novel coronavirus T cell epitope peptide of claim 1 or the novel coronavirus T cell epitope peptide composition of claim 3 on the surface.
7. The antigen peptide-antigen presenting cell complex of claim 6, wherein the antigen presenting cell is a T2-A2 cell.
8. The antigenic peptide-antigen presenting cell complex of claim 7 wherein the T2-A2 cells are T2 cells over-expressing HLa-A2.
9. Use of one or more of the epitope peptide of claim 1, the gene encoding the novel coronavirus T cell epitope peptide of claim 2, the novel coronavirus T cell epitope peptide composition of claim 3, the pMHC complex of claim 4 or 5, and the antigen peptide-antigen presenting cell complex of claim 6 in the preparation of a novel coronavirus vaccine.
10. Use of one or more of the epitope peptide of claim 1, the gene encoding the novel coronavirus T cell epitope peptide of claim 2, the novel coronavirus T cell epitope peptide composition of claim 3, the pMHC complex of claim 4 or 5, and the antigen peptide-antigen presenting cell complex of claim 6 in the preparation of novel coronavirus drugs.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310446178.8A CN116444631A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210176947.2A CN114751965B (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446178.8A CN116444631A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
Related Parent Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210176947.2A Division CN114751965B (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116444631A true CN116444631A (en) | 2023-07-18 |
Family
ID=82325844
Family Applications (5)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310446205.1A Pending CN116574159A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446186.2A Pending CN116396366A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446209.XA Pending CN116375821A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202210176947.2A Active CN114751965B (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446178.8A Pending CN116444631A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
Family Applications Before (4)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310446205.1A Pending CN116574159A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446186.2A Pending CN116396366A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202310446209.XA Pending CN116375821A (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
| CN202210176947.2A Active CN114751965B (en) | 2022-02-24 | 2022-02-24 | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine |
Country Status (1)
| Country | Link |
|---|---|
| CN (5) | CN116574159A (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN117487000A (en) * | 2022-08-30 | 2024-02-02 | 暨南大学 | Tumor T cell epitope peptide, pMHC, and preparation and application thereof |
| CN117430664B (en) * | 2023-10-24 | 2024-04-09 | 暨南大学附属第六医院(东莞市东部中心医院) | Influenza A virus T cell epitope peptide and application thereof |
| CN117430665B (en) * | 2023-10-24 | 2024-06-04 | 暨南大学附属第六医院(东莞市东部中心医院) | Influenza A virus T cell epitope peptide and application thereof |
| CN117402214B (en) * | 2023-10-24 | 2024-03-19 | 暨南大学附属第六医院(东莞市东部中心医院) | Influenza A virus CD8+ T cell epitope peptide and application thereof |
| CN117402213B (en) * | 2023-10-24 | 2024-04-05 | 暨南大学附属第六医院(东莞市东部中心医院) | Influenza A virus CD8+ T cell epitope peptide and application thereof |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20230324402A1 (en) * | 2020-03-23 | 2023-10-12 | The Regents Of The University Of California | Devices for detecting antibodies to coronavirus antigens and methods for using them |
| CN111548396B (en) * | 2020-04-17 | 2021-07-20 | 暨南大学 | Novel coronavirus T cell epitope peptide, pMHC, preparation and application thereof |
| KR102425492B1 (en) * | 2020-04-27 | 2022-07-26 | 한림대학교 산학협력단 | Development method of epitope-base peptide vaccine against sars-cov-2 virus |
| WO2022109751A1 (en) * | 2020-11-27 | 2022-06-02 | The University Of Western Ontario | Point-of-care testing for sars-cov antibodies |
| CN112876542B (en) * | 2021-02-08 | 2021-10-29 | 暨南大学 | Novel epitope peptide of coronavirus T cell and application thereof |
| CN113735947A (en) * | 2021-09-22 | 2021-12-03 | 四川大学 | Specific T cell epitope peptide P48 screened by novel coronavirus S protein holoproteome and application thereof |
-
2022
- 2022-02-24 CN CN202310446205.1A patent/CN116574159A/en active Pending
- 2022-02-24 CN CN202310446186.2A patent/CN116396366A/en active Pending
- 2022-02-24 CN CN202310446209.XA patent/CN116375821A/en active Pending
- 2022-02-24 CN CN202210176947.2A patent/CN114751965B/en active Active
- 2022-02-24 CN CN202310446178.8A patent/CN116444631A/en active Pending
Also Published As
| Publication number | Publication date |
|---|---|
| CN114751965A (en) | 2022-07-15 |
| CN114751965B (en) | 2023-04-28 |
| CN116375821A (en) | 2023-07-04 |
| CN116396366A (en) | 2023-07-07 |
| CN116574159A (en) | 2023-08-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN114751965B (en) | Novel coronavirus T cell epitope peptide and application thereof in preparation of vaccine | |
| CN114656529B (en) | Novel antigen epitope peptide of coronavirus T cell and application thereof | |
| De Groot et al. | Immuno‐informatics: Mining genomes for vaccine components | |
| Grifoni et al. | Global assessment of dengue virus-specific CD4+ T cell responses in dengue-endemic areas | |
| CN111548396B (en) | Novel coronavirus T cell epitope peptide, pMHC, preparation and application thereof | |
| ZA200502273B (en) | T-cell epitopes in staphylococcal enterotoxin B. | |
| Ott et al. | CD28 costimulation enhances the sensitivity of the ELISPOT assay for detection of antigen-specific memory effector CD4 and CD8 cell populations in human diseases | |
| CN105848677A (en) | Peptide combinations and uses thereof in treating dust mite allergy | |
| Cello et al. | A study of the cellular immune response to enteroviruses in humans: identification of cross-reactive T cell epitopes on the structural proteins of enteroviruses | |
| CN117486999A (en) | Tumor T cell epitope peptide, pMHC, and preparation and application thereof | |
| RU2202613C2 (en) | Foot and mouth virus immunogenic peptides | |
| CN102662054A (en) | Method for synchronously detecting quantity and functions of specific thymus dependent lymph cells | |
| CN101370517B (en) | Peptide derived from hepatitis C virus | |
| CN117402214B (en) | Influenza A virus CD8+ T cell epitope peptide and application thereof | |
| CN117430665B (en) | Influenza A virus T cell epitope peptide and application thereof | |
| CN117402213B (en) | Influenza A virus CD8+ T cell epitope peptide and application thereof | |
| CN117430664B (en) | Influenza A virus T cell epitope peptide and application thereof | |
| CN107141355B (en) | HTNV (human immunodeficiency Virus) antigen epitope linear tandem polypeptide, epitope peptide-compound tetramer and application | |
| CA2379618A1 (en) | Viral protein binding compositions and methods | |
| Hayney | Pharmacogenomics and infectious diseases: impact on drug response and applications to disease management | |
| EP4142780A2 (en) | Methods of generating vaccines against novel coronavirus, named sars-cov-2 comprising variable epitope libraries (vels) as immunogens | |
| Haberer et al. | Leishmania pifanoi amastigote antigen P-4: epitopes involved in T-cell responsiveness in human cutaneous leishmaniasis | |
| US20240216507A1 (en) | Sars-cov-2-specific t cells and methods of treatment using them | |
| WO2022244891A1 (en) | T CELL EPITOPE PEPTIDE DERIVED FROM SARS-CoV-2 | |
| Elko et al. | COVID-19 vaccination recruits and matures cross-reactive antibodies to conserved epitopes in endemic coronavirus Spike proteins |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20240401 Address after: 510632 No. 601, Whampoa Avenue, Guangzhou, Guangdong Applicant after: Jinan University Country or region after: China Applicant after: Zhuhai Pengsheng Biotechnology Co.,Ltd. Address before: 510632 No. 601, Whampoa Avenue, Tianhe District, Guangdong, Guangzhou Applicant before: Jinan University Country or region before: China |
|
| TA01 | Transfer of patent application right |