CN112961223A

CN112961223A - SARS-CoV-2 lymphocyte antigen epitope peptide and its application

Info

Publication number: CN112961223A
Application number: CN202110209065.7A
Authority: CN
Inventors: 沈传来; 金萧萧; 丁艳; 孙世慧; 赵光宇; 何玉先
Original assignee: Southeast University
Current assignee: Southeast University; Academy of Military Medical Sciences AMMS of PLA; Institute of Pathogen Biology of CAMS
Priority date: 2021-02-24
Filing date: 2021-02-24
Publication date: 2021-06-15

Abstract

The invention discloses a lymphocyte epitope peptide and application thereof, belongs to the field of medical immunology and infectious pathology, and particularly relates to 164 thymus-dependent lymphocyte epitope peptides of 5 proteins of SARS-CoV-2 and application thereof. The epitope peptide can be presented by HLA-A molecules, and stimulates the activation, proliferation and differentiation of SARS-CoV-2 specific thymus dependent lymphocyte, thereby playing the role of immune effect for resisting SARS-CoV-2 infection; the antigen peptides can be used for preparing mixed polypeptide vaccines of SARS-CoV-2, recombinant protein vaccines and DNA and RNA vaccines which are connected with a plurality of epitope peptides in series, can be used for preparing detection kits for detecting SARS-CoV-2 specific thymus-dependent lymphocytes, can also be used for preparing effector thymus-dependent lymphocytes or medicaments for treating SARS-CoV-2 infection, and have potential application values in the prevention, treatment and diagnosis of SARS-CoV-2 infection and COVID-19.

Description

SARS-CoV-2 lymphocyte antigen epitope peptide and its application

Technical Field

The invention relates to the field of medical immunology and infectious pathology, in particular to a SARS-CoV-2 lymphocyte epitope peptide and application thereof.

Background

SARS-CoV-2 is an enveloped, single-stranded, plus-strand RNA, β -coronavirus of the family Coronaviridae, with a genome length of 30000 bases. Comprises 14 Open Reading Frames (ORF), 16 nonstructural proteins (Nsp1-16) are coded by Orf1ab at the 5' end, wherein Nsp7, Nsp8 and Nsp12 form RNA-dependent RNA polymerase (RdRp) of SARS-CoV-2 virus, and the total length is 932 amino acids. The 13 Orf at the 3' end encode 4 structural proteins and 9 putative cofactors, respectively. Wherein the 4 virus structural proteins are respectively: the S protein (spike protein), 1273 amino acids in total length; the E protein (envelope protein) has 75 amino acids in total length; m protein (membrane protein), 222 amino acids in total length; n protein (nucleocapsid protein) with 419 amino acids in total length.

The SARS-CoV-2 epitope peptide which can stimulate the T cell response reaction is still few at present, thereby limiting the monitoring analysis of the specific T cell immune function of the infectors carrying SARS-CoV-2, also limiting the immunopathology and immunoprotection mechanism research of the specific T cell immune response in the interaction between SARS-CoV-2 and the host, and further limiting the development of vaccines and therapeutic preparations for inducing the organism to generate the virus specific T cell response.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a SARS-CoV-2 lymphocyte epitope peptide and the application thereof.

The purpose of the invention can be realized by the following technical scheme:

an epitope peptide, the amino acid sequence of which is shown in any one of SEQ ID NO 1-164. An epitope peptide, the amino acid sequence of which is obtained by substituting, deleting, inserting and/or adding 1 or more amino acid sequences in SEQ ID NO. 1-164.

A vaccine comprises the following effective components: the epitope peptide described above or a nucleic acid encoding the epitope peptide described above.

Effector T cells obtained by stimulating peripheral blood lymphocytes with the above-mentioned epitope peptide or antigen-presenting cells that present the epitope peptide to HLA molecules.

An adoptive immune therapeutic cell, the effective component of which comprises the effector T cell.

In addition, the invention also discloses:

the epitope peptide is applied to the preparation of SARS-CoV-2 vaccine or effector T cell.

The epitope peptide is applied to the preparation of a reagent or a kit for detecting SARS-CoV-2 specific T cells.

The epitope peptide is applied to the preparation of drugs for treating SARS-CoV-2 infection.

Optionally, the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining assay reagent, a chemiluminescence assay reagent, an enzyme-linked immunosorbent assay reagent, and a human leukocyte antigen polymer fluorescent staining flow cytometry analysis reagent.

The epitope peptide sequence can be used for preparing a new coronavirus pneumonia (COVID-19) mixed polypeptide vaccine, a recombinant protein vaccine or a recombinant gene vaccine for inducing an organism to generate specific T cell immune response. Preparing a mixed polypeptide vaccine: the antigen epitope peptide sequence of the invention is artificially synthesized into a plurality of antigen peptides, which are mixed with an adjuvant to prepare a soluble preparation, or biological nano materials are loaded to prepare a nano polypeptide vaccine, and the nano polypeptide vaccine is injected into a healthy person, a SARS-CoV-2 infected person or a pneumonia patient body caused by SARS-CoV-2 interference to stimulate the activation, proliferation and differentiation of specific T cells in the body into effector T cells, thereby enhancing the immune protection capability of specific cells in the body, preventing the healthy person from infecting SARS-CoV-2 or enhancing the capability of an infected person for inhibiting virus replication and proliferation and resisting reinfection, and further preparing the T cell epitope mixed polypeptide vaccine of SARS-CoV-2. Preparing a recombinant protein vaccine: according to the polypeptide sequence of the present invention, a recombinant plasmid which is connected with a plurality of epitope peptides in series is constructed, prokaryotic or eukaryotic expression is carried out, then the recombinant protein is purified and mixed with an adjuvant to prepare a soluble preparation, and the soluble preparation is injected into a healthy person, a SARS-CoV-2 infected person or a pneumonia patient body caused by SARS-CoV-2 infection to stimulate the activation, proliferation and differentiation of specific T cells in an organism to effector T cells, thereby enhancing the immune protection capability of specific cells of the organism, preventing the healthy person from infecting SARS-CoV-2 or enhancing the capability of an infected person for inhibiting virus replication proliferation and resisting reinfection, and further preparing the recombinant protein vaccine of SARS-CoV-2. Preparing a recombinant gene vaccine: according to the polypeptide sequence of the present invention, recombinant DNA gene fragment, recombinant plasmid, recombinant virus vector or recombinant mRNA of serial connection of various epitope sequences is constructed, and injected into a healthy person, a SARS-CoV-2 infected person or a pneumonia patient caused by SARS-CoV-2 infection, specific T cells in an organism are stimulated to activate, proliferate and differentiate into effector T cells, so that the specific cellular immune protection capability of the organism is enhanced, the infection of the healthy person with SARS-CoV-2 is prevented, or the capability of an infected person for inhibiting virus replication proliferation and resisting reinfection is enhanced, and the recombinant gene vaccine of SARS-CoV-2 is prepared.

In addition, the lymphocyte epitope peptide sequence can be used for preparing a detection preparation or a kit for detecting SARS-CoV-2 antigen specific T cells: according to the antigen epitope peptide sequence of the invention, a plurality of antigen peptides are artificially synthesized, and are used as antigen stimulating agents in an enzyme-linked immunosorbent assay, an intracellular cytokine fluorescent staining method, a chemiluminescence method and an enzyme-linked immunosorbent assay, and are mixed with Peripheral Blood Mononuclear Cells (PBMC) of a patient for culture, so that the activation, proliferation and cell factor secretion of SARS-CoV-2 antigen specific T cells are stimulated, and the secretion amount of the cell factor is detected by other combined reagents, thereby reflecting the number and the reactivity of the specific T cells; the polypeptide sequence may be also used in preparing human leucocyte antigen-polypeptide complex and its polymer through gene engineering and protein engineering technology, preparing fluorescein or metal substance labeled preparation, and flow cytometry analysis to detect the amount of SARS-CoV-2 antigen specific T cell in patient's peripheral blood PBMC cell population. The related kit is a SARS-CoV-2 specific T cell detection kit which is assembled by the preparation and other conventional reagents in different detection methods.

The epitope peptide sequence can be used for preparing medicines for treating SARS-CoV-2: the mixed polypeptide vaccine, recombinant protein vaccine or gene vaccine of the epitope peptide sequence is combined with other immunotherapy preparations or chemotherapy preparations to prepare the clinical medicine for treating SARS-CoV-2.

The invention has the beneficial effects that:

a group of SARS-CoV-2 virus protein T cell epitope peptides obtained by online virtual prediction and functional experimental verification has not been reported previously. Therefore, the new epitope peptide sequence provides the necessary key antigen component, namely the epitope peptide sequence, for developing preventive and therapeutic polypeptide vaccines, recombinant protein vaccines and gene vaccines aiming at SARS-CoV-2 virus, designing and detecting reagents and methods of SARS-CoV-2 virus antigen specific T cells and the like.

Drawings

The invention will be further described with reference to the accompanying drawings.

Fig. 1 is a technical experimental roadmap for immunogenicity validation of epitope peptides to be identified in some examples of the invention.

FIGS. 2-16 are flow analysis diagrams of DC-polypeptide-PBL coculture experiments for 144T-cell epitope peptides of the SARS-CoV-2 viral protein having immunogenicity in some examples of the invention; co-culturing T cell epitope polypeptide with DC cell of blood donor and PBL of blood donor for 14 days, co-staining with CD3 and CD8 fluorescent monoclonal antibody, intracellular staining with IFN-gamma fluorescent monoclonal antibody, and detecting and analyzing IFN-gamma by flow cytometry⁺/CD8⁺T cells in CD8⁺Frequency in T cell populations.

FIGS. 17-21 are flow analysis plots of DC-polypeptide-PBL coculture experiments for 40T-cell epitope peptides of the SARS-CoV-2 viral protein having immunogenicity in some examples of the invention; t cell epitope polypeptides co-stained with donor DC cells and CFSE-prestained PBLsAfter 14 days of culture, CD3 and CD8 fluorescent monoclonal antibodies were co-stained, and then detected by flow cytometry for analysis of CD8⁺The proliferation rate of the T cell population;

FIG. 22 shows that the mixed polypeptide vaccine of T cell epitope peptide of 31 SARS-CoV-2 virus proteins in some examples of the present invention can effectively stimulate specific CD8 in transgenic mice⁺Statistical plots of results for T cell responses: three mixed polypeptide vaccines are respectively used for immunizing and inoculating HLA-A2/DR1 transgenic mice, spleen cells of the immunized mice are taken, 31T cell epitope peptides (9-mer/species) form 8 peptide libraries, the peptide libraries are respectively mixed with the spleen cells for culturing for 20 hours, and the number of the T cells secreting IFN-gamma is detected by an ELISPOT method; (A) the total number of spots for all 5 SARS-CoV-2 virus proteins in each mouse splenocyte population; (B) number of spots in each mouse splenocyte population for a single SARS-CoV-2 virus protein;

FIG. 23 is a photograph of CD8 from 3 mice in the control group of FIG. 22⁺ELISPOT spot plots of T cell responses: inoculating HLA-A2/DR1 transgenic mice with unloaded PLGA-NP or normal saline, and taking splenocytes for detection;

FIG. 24 is a graph showing CD8 of mice in Vaccine A vaccinated group 3 of FIG. 22⁺ELISPOT spot plots of T cell responses: PLGA-NP/polypeptide vaccine is inoculated to HLA-A2/DR1 transgenic mice, and then splenocytes are taken for detection;

FIG. 25 is a set of 3 mice in the Vaccine B vaccinated group of FIG. 22 showing CD8⁺ELISPOT spot plots of T cell responses: the R848/polypeptide vaccine is inoculated to an HLA-A2/DR1 transgenic mouse, and then splenocytes are taken for detection;

FIG. 26 is a graph showing CD8 of mice in Vaccine C vaccinated group 3 of FIG. 22⁺ELISPOT spot plots of T cell responses: c/polypeptide vaccine is inoculated on HLA-A2/DR1 transgenic mice, and then splenocytes are taken for detection;

FIG. 27 shows that the mixed polypeptide vaccine of T cell epitope peptide of 31 SARS-CoV-2 virus proteins in some examples of the present invention can effectively stimulate specific CD8 in transgenic mice⁺Statistical plots of results for T cell responses: three mixed polypeptide vaccines are respectively immunized with HLA-A2/DR1 transgenic mice, spleen cells of the immunized mice are taken, 31T cell epitope peptides (9-mer/species) are combined into a plurality of peptide libraries according to antigen types, and the peptide libraries are respectively mixed with the spleen cells for culture for 20 hoursDetecting IFN-gamma-secreting CD8 using flow cytometry⁺T cell frequency; (A) IFN-gamma against all 5 SARS-CoV-2 virus proteins per mouse splenocyte population⁺/CD8⁺The total frequency of T cells; (B) IFN-gamma against a single SARS-CoV-2 virus protein in each mouse splenocyte population⁺/CD8⁺The total frequency of T cells;

FIG. 28 is a CD8 of 3 mice from the control group in FIG. 27⁺Flow assay scatter plot of T cell response: inoculating HLA-A2/DR1 transgenic mice with unloaded PLGA-NP or normal saline, and taking splenocytes for detection;

FIG. 29 is a CD8 sample from 3 mice in the Vaccine A vaccinated group of FIG. 27⁺Flow assay scatter plot of T cell response: PLGA-NP/polypeptide vaccine is inoculated to HLA-A2/DR1 transgenic mice, and then splenocytes are taken for detection;

FIG. 30 is a CD8 sample from 3 mice in the Vaccine B vaccinated group of FIG. 27⁺Flow assay scatter plot of T cell response: the R848/polypeptide vaccine is inoculated to an HLA-A2/DR1 transgenic mouse, and then splenocytes are taken for detection;

FIG. 31 is a CD8 sample from 3 mice in the Vaccine C vaccinated group of FIG. 27⁺Flow assay scatter plot of T cell response: c/polypeptide vaccine is inoculated on HLA-A2/DR1 transgenic mice, and then splenocytes are taken for detection;

FIG. 32 is a spot diagram of the ELISPOT method for detecting specific T cells cross-reacting with SARS-CoV-2 in PBMCs of blood donors according to some examples of the present invention using the above-described T cell epitope peptide library of SARS-CoV-2: combining 164T cell epitopes of the SARS-CoV-2 verified into 10 peptide libraries, establishing an ELISPOT detection method in a 96-hole PVDF membrane plate, and analyzing the number of specific T cells which are in cross reaction with SARS-CoV-2 in PBMC of healthy people (blood donors);

FIG. 33 is a timeline of immunization of HLA-A2/DR1 transgenic mice in some examples of the invention.

Detailed Description

The technical solutions in the examples of the present invention will be clearly and completely described below with reference to the drawings in the examples of the present invention, and it is obvious that the described examples are only a part of examples of the present invention, and not all examples. All other embodiments, which can be derived by a person skilled in the art from the examples given herein without making any inventive step, are within the scope of the present invention.

Example 1: in some examples of the present invention, a method for predicting and identifying the amino acid sequence of T cell epitope peptide of SARS-CoV-2 protein is provided, which comprises the following steps:

1. virtual prediction of T-cell epitope peptides from 5 SARS-CoV-2 proteins restricted by HLA-A molecules

5 SARS-CoV-2 proteins are selected, which may be, for example, E protein (envelope protein), M protein (membrane protein), N protein (nucleocapesid protein), S protein (spike protein) and RdRp protein (RNA-dependent RNA polymerase). Furthermore, T cell epitope peptides of each of the above proteins, which are molecularly restricted, such as HLA-A0201, A1101, A2402, A3101, A0206, A0207, A3303, A3001, A0203, A1102, A0301, A0101 and A2601, are virtually predicted by epitope peptide prediction tools such as SYFPEITHI, EPIJEN, ConvMHC, IEDB (including ANN, Consensus, NetMHCpan, SMM and SMMPMBEC).

The HLA class I molecule antigen binding groove is sealed at two ends, the length of the received antigen peptide is 8-11 amino acid residues, wherein 9 and 10 amino acids are the most common, therefore, in some examples of the invention, polypeptides with the length of 9 and 10 amino acids are selected as a research object, the amino acid sequence of each SARS-CoV-2 virus protein is respectively input into a corresponding amino acid sequence input box of a prediction database website, the length of the epitope peptide is respectively selected to be 9 and 10 amino acids, then a specific HLA-A molecule is selected, and the T cell epitope peptide of the SARS-CoV-2 virus protein is subjected to online virtual prediction.

Aiming at each HLA-A molecule and each SARS-CoV-2 virus protein, 9 peptides and 10 peptides predicted by different databases are respectively arranged according to the scores from high to low, and epitope peptides meeting at least more than two prediction method score standards are selected as candidate epitope peptides. For each HLA-A molecule, aiming at each new crown protein, 1-6 polypeptides with highest score or highest affinity are selected from candidate epitope peptides as epitope peptides to be identified.

2. Separating peripheral blood PBMC of blood donors:

1) collecting peripheral blood sampling discs (400mL leukocyte filtering discs used in peripheral anticoagulation sorting of erythrocytes) of blood donation volunteers in a blood center, spraying alcohol, putting into a super clean bench, carefully sterilizing an inlet pipe and an outlet pipe by using an alcohol cotton ball, cutting the inlet pipe and the outlet pipe, sucking PBS by using a 50mL injector, taking the outlet of the sampling disc as an injector inlet, collecting PBS flushing fluid at the inlet of the sampling disc by using 6 50mL centrifuge tubes, and collecting about 300mL cell suspensions;

2) collecting 50mL of blood flushed out firstly by the 1 st centrifugal tube without centrifugation; uniformly collecting the rest 5 centrifugal tubes, centrifuging at room temperature of 1500rpm for 12min, transferring the turbid supernatant into a 50mL centrifugal tube, centrifuging again at room temperature of 1500rpm for 12min, and collecting all cell precipitates; collecting the cell sediment in the next 5 centrifugal tubes into 150mL centrifugal tube, filling PBS to 50mL, and fully mixing with 50mL blood in the 1 st tube;

3) 5mL of human lymphocyte separation fluid (Dake is biological, Shenzhen) is added into 10 15mL centrifuge tubes in advance; slowly adding the mixed 100mL of leukocyte suspension along the tube wall, flatly paving the mixture above the liquid level of the separation liquid, keeping two interfaces clear, centrifuging the mixture for 20min at 20 ℃ and 2000rpm in 10mL of leukocyte suspension/tube, wherein the acceleration is 1, and the deceleration is 0;

4) after the centrifugation is finished, a suction pipe is used for moving on the same plane, all opalescent PBMC layers are sucked to a 50mL sterile centrifuge tube (about 30mL), plasma layers above a leucocyte layer are not sucked as much as possible, then the leucocyte layer cell suspension is evenly divided into 4 centrifuge tubes with 50mL, each tube is supplemented with PBS to 50mL, the mixture is fully mixed, 1300rpm is carried out, and centrifugation is carried out for 12 min; discarding the supernatant, bouncing the cell sediment, mixing and merging the sediment and equally dividing the sediment into two of the 50mL centrifuge tubes, supplementing PBS to 50 mL/tube, blowing and evenly mixing the sediment, centrifuging the mixture twice at 1000rpm for 12min, discarding the supernatant, bouncing the cell sediment by using fingers, adding a small amount of serum-free 1640 culture solution (Dake is biological, Shenzhen) for resuspension, collecting and merging the cell sediment into 150mL centrifuge tube, and supplementing the serum-free 1640 culture solution to about 20 mL;

5) cell counting: taking an EP tube, adding 380 mu L of leukocyte diluent, then adding 20 mu L of cell suspension, fully and uniformly mixing, filling a cell counting plate, counting under a microscope, and calculating the cell concentration according to the following formula:

total number of 4 large cells/4 × 104 × dilution factor 20 ═ total cells/mL.

3. HLA-A allelic typing can then be performed, for example, as follows.

Taking PBMC obtained by separation in the above example, extracting genomic DNA using a human whole blood genomic DNA extraction kit (Tiangen organism, Beijing); the DNA sequences of exon 2, intron 2, exon 3 and partial introns l and 3 of the A site were amplified by PCR using HLA-A site specific primers A1 and A3, and the product size was 985 bp. The amplification conditions were: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15 s; annealing at 62 ℃ for 15 s; extension at 72 ℃ for 90 s; 35 cycles; extension at 72 ℃ for 5 min. The amplified product was size-characterized by 1% agarose gel electrophoresis, purified and double-sequenced. PCR reagents were purchased from Biotech, Nanjing Novozam, see Table 1.

TABLE 1

Splicing sequencing results of the exon 2 and the exon 3 into a complete HLA-A overlapping sequence (Contig) by Seqman software of a Lasergene program, carefully checking whether bases subjected to bidirectional sequencing are completely consistent, finding out bases of heterozygote and replacing the bases with merged bases, wherein M represents A and C, R represents A and G, W represents A and T, S represents C and G, Y represents C and T, and K represents G and T, and finally determining the sequence fragment of the amplified HLA-A allele. And (3) comparing the spliced HLA-A base sequence with exon 2 and exon 3 sequences of all HLA-A alleles in a database by utilizing a Nucleotide BLAST tool until a completely matched gene combination is obtained, thereby determining the HLA-A alleles.

4. Subsequently, DC induction and polypeptide-DC-PBL coculture experiments can be performed to verify the immunogenicity of epitope peptides:

1) the above PBMC resuspended in 20mL serum-free 1640 medium was inoculated into the cellsIn cell culture flasks (1-1.5X 10)⁸cells/20 mL/medium size bottle, 1.5-3X 10⁸cells/30 mL/large size vial), incubating at 37 ℃ for about 4 h;

2) shaking the culture bottles, blowing the culture bottles once by using a suction pipe, transferring the non-adherent cells and the culture solution into 1 medium-sized culture bottle for PBL culture, supplementing common serum to the final concentration of 10%, culturing overnight, and freezing and storing the PBL for later use the next day;

3) adding 10mL of serum-free 1640 culture solution into the adherent cells, blowing and beating once by using a straw, shaking and washing the culture bottle, discarding the culture solution, repeatedly washing once again, discarding the culture solution, and supplementing 10% of Australian fetal serum-1640 culture solution (15 mL/medium bottle and 20 mL/large bottle) with the corresponding volume;

4) d0 adding human GM-CSF (Prepotech) and human rIL-4 (Prepotech) at final concentration of 1000IU/mL and 500IU/mL, culturing at 37 deg.C; d3 evening, half amount of liquid is changed, GM-CSF and rIL-4 are supplemented at the same time, and the culture is continued at 37 ℃; d5 evening, adding LPS to stimulate the maturation of DC, the final concentration is 1 mug/mL, simultaneously supplementing GM-CSF and rIL-4, and continuing culturing at 37 ℃; d6 reviving the frozen PBLs of the blood donated volunteers at night for use; d7 harvest mature dcs (mdcs): beating and blowing the culture bottle, sucking the culture solution into a 50mL centrifuge tube, washing the culture bottle with a small amount of serum-free culture solution, and then counting cells (counting with stock solution, not counting very small lymphocytes);

5) analysis of mDC phenotype: take 3X 10⁵The cells are evenly divided into 6 branch-flow centrifuge tubes, after centrifugation, 5 fluorescent monoclonal antibodies (CD83\ CD80\ CD86\ DR \ ABC) are respectively used for single staining, and the positive percentage of each mark is analyzed in a flow mode;

6) centrifuging the rest mDC at 1200rpm for 10min, discarding supernatant, resuspending the cell pellet in serum-free 1640 culture medium, and adjusting the concentration of mDC to 0.5 × 10⁵cells/mL, temporarily placing in an incubator for storage;

7) observing the flow detection result and the mDC proportion, and inoculating the mDC into a 24-hole plate, 50000cells/1.0 mL/hole; then, 10. mu.L (final concentration: 20. mu.g/mL) of a single epitope peptide corresponding to the HLA-A molecule of the blood-donating volunteers was added to each well. Approximately 10 or so epitope peptides were validated per blood sample, while three control wells were set: DC-T negative control wells (no peptide added), PBL positive wells (small amount of PHA added), PBL negative wells (no stimulant added);

8) incubation was performed for 4h, then according to PBL: DC 20: 1 ratio of PBL (1X 10) from this blood-donating volunteer resuscitated overnight and either pre-stained with CFSE or not⁶cells/mL, 1.0 mL/well);

9) d11, adding IL-2 with the final concentration of 20 IU/mL; d14, if the color of the culture solution turns yellow, changing the culture solution by half; each well was supplemented with 20. mu.L each of the corresponding single peptides (final concentration 20. mu.g/mL); d17, observing the color of the culture solution, and supplementing half amount of IL-2; if the colony is large, blowing off with a suction pipe;

10) d21, collecting cells from each DC-polypeptide-PBL co-culture well without CFSE pre-staining, centrifuging, resuspending the cells in serum-free culture medium, transferring to 48-well plate, supplementing each well with corresponding single epitope peptide, co-culturing in cell culture box for 16 hr, preparing for IFN-gamma intracellular staining and flow analysis, detecting CD3⁺/CD8⁺Cell population and CD3⁺/CD8^-IFN-gamma in cell populations⁺The proportion of cells, the specific steps are discussed in detail later;

11) d22, collecting cells from each CFSE-prestained DC-polypeptide-PBL coculture well, centrifuging, and collecting 8X 10 cells⁵PBL is co-stained with anti-human CD3 and anti-human CD8 fluorescent monoclonal antibody, and CD3 is detected by flow cytometry⁺/CD8⁺Cell population and CD3⁺/CD8^-CFSE in cell populations⁺The proliferation rate of the cells.

12) CD3 was compared to negative control well (DC-PBL coculture well) cells⁺/CD8⁺IFN-gamma in cell populations⁺Increase in the proportion of cells by 100% or CFSE⁺DC-polypeptide-PBL coculture wells with a 20% increase in cell proliferation rate were used as experimental positive wells, i.e., wells in which the polypeptide stimulated CD8⁺T cells activate and proliferate, and have immunogenicity.

5. Then, intracellular IFN-gamma fluorescent staining is carried out, and the operation steps are as follows:

1) d21, collecting cells of each DC-polypeptide-PBL co-culture well which is not pre-stained by CFSE into a sterile EP tube, supplementing common serum-free culture solution to about 1.5mL, centrifuging at 1200rpm for 10min, discarding supernatant at one time, bouncing cell sediment, re-suspending the cells by 0.5mL of serum-free culture solution (Dake, David), and transferring the cells into a 48-well plate;

2) adding corresponding single polypeptide 10 μ L (final concentration 20 μ g/mL) into each well, and setting non-polypeptide stimulation well and PHA stimulation well to respectively serve as negative control and positive stimulation control of the cell sample; placing the cell culture box for incubation for 16 h;

3) adding 2 mu L of BFA/Monensin (250X combined organism) into each hole, and continuously incubating for 6 h;

4) collecting each group of cells to a flow detection test tube, adding 1 μ L Fc Block (anti-CD16/CD32) into each tube, mixing uniformly, and mixing uniformly once at 4 ℃ for 20 min;

5) each experimental tube was co-stained with FITC-anti-CD3 and APC-anti-CD8 fluorescent monoclonal antibodies (eBiosciences), respectively; two tubes of blank PBL are reserved, one tube is not dyed, and the other tube is singly dyed and compensated by FITC-anti-CD 3; placing each tube at 4 deg.C for 30min, adding PBS to 3mL, 1200rpm for 10min, removing supernatant, and bouncing cell precipitate;

6) adding 100 μ L of membrane-breaking agent MEDIUM A (Union biological) into each tube, and incubating at room temperature for 15 min; supplementing 3mL of 5% FBS-PBS, uniformly mixing, centrifuging for 5min at 350g of 300-;

7) adding 100 μ L of membrane breaking agent MEDIUM B (Union biology) into each tube, adding 20 μ L of PE-anti-IFN- γ (eBiosciences) into each experimental tube, and adding no PE-anti-IFN- γ into non-dyeing tubes; incubating each tube at 4 deg.C for 30min, and mixing uniformly;

8) adding 5% FBS-PBS to 3mL per tube, mixing, centrifuging for 5min at 300-350g, discarding supernatant, bouncing cell precipitate, resuspending with 0.5mL 5% FBS-PBS, detecting CD3 with flow cytometer⁺/CD8⁺Or CD3⁺/CD8^-IFN-gamma in cell populations⁺The proportion of cells.

6. The PBL cryopreservation, resuscitation and CFSE prestaining steps used in some examples of the invention

1) D0, transferring all non-adherent PBLs after separating the DCs into 1 medium bottle, supplementing 10% common serum-1640 culture solution, and incubating overnight; d1 collecting PBL, centrifuging, placing the cell sediment in a cell freezing tube, adding cell freezing solution (Xinsaimei) (4 × 10)⁷cells/mL) at-80 deg.C; recovering after 5 days, and culturing with 10% common serum-1640 culture solution overnight;

2) d7, collecting PBL at 1200rpm for 12min, discarding supernatant, resuspending PBL in PBS sufficiently, adjusting cell concentration to 3 × 10⁷cells/mL, 1 mL/tube (here leave part of PBL without CFSE pre-staining, flow regulation compensation use);

3) adding 99 μ L PBS into one piece of CFSE (storage concentration 10mM, 1 μ L/branch) solution, mixing, adding 15 μ L diluted CFSE (final concentration 1.5 μ M) into each PBL tube, mixing gently, incubating at 37 deg.C for 20min, shaking once every 5min to ensure CFSE staining uniformity;

4) adding 5 times volume of precooled 10% FBS-1640 culture solution into each tube, continuously incubating for 5min to stop CFSE staining, carrying out 1200rpm for 12min, and carrying out centrifugal washing for 2 times to obtain CFSE pre-stained PBL;

5) resuspending in 10% Aurea placenta-1640 culture medium, adjusting cell concentration to 2 × 10⁶cells/mL, ready for use.

The PBMC samples of 156 blood-donating volunteers are collected, and the DC-polypeptide-PBL coculture experiment is repeatedly carried out on the candidate epitope peptide, and IFN-gamma-secreting CD8 is detected⁺T cell ratio or CD8⁺Proliferation ratio of T cells, the immunogenicity of each single peptide was verified. The results show that: 144 epitope peptides can stimulate CD8 of blood donor volunteers⁺T cells activated and secreted IFN- γ, exhibiting CTL positive responses (table 3, fig. 2-16); 40 epitope peptides can stimulate CD8 of blood donor volunteers⁺T cells activated and proliferated and showed CTL positive responses (table 3, fig. 17 to fig. 21). The T epitope peptides of 164 novel immunogenic coronavirus proteins are finally verified to be obtained by two detection methods, as shown in Table 2.

TABLE 2

Table 3 is a summary table of the positive results of the validation experiments for the T-cell epitope peptides of the new coronavirus obtained in the above example: in DC-polypeptide-PBL co-culture experiments, the T-cell epitope peptides can stimulate static CD8 in PBMC of blood donors⁺Activation and secretion of IFN-gamma (IFN-gamma) by T cells⁺/CD8⁺T cell frequency increased by more than 100% compared with negative control group) or proliferation (CD 8)⁺The proliferation ratio of the T cells is improved by more than 20 percent compared with that of a negative control group).

TABLE 3

For the experiments in table 3, it should be noted that "CFSE" in the validation method refers to the proliferation rate of CD 8T cell population analyzed by flow cytometry after co-staining CD3 and CD8 fluorescent mabs after 14 days of co-culture of the T cell epitope polypeptide with donor DC cells and CFSE-preincubated PBMCs of the donor; "IFN-gamma" refers to the T cell epitope polypeptide and donor DC cells and donor PBMC after 14 days of co-culture, CD3 and CD8 fluorescent monoclonal antibody co-staining, IFN-gamma fluorescent monoclonal antibody intracellular staining, and then flow cytometry detection analysis of IFN-gamma +/CD8+ T cell frequency in CD 8T cell population.

In this example, the "CD 8T improvement rate" may be selected for evaluation, specifically: in the CFSE validation method, the proliferation rate of CD 8T cells is improved by a percentage compared with the proliferation rate of CD 8T cells in a negative control group (without polypeptide stimulation). The T cell epitope peptide with the improvement rate of more than 20 percent is considered to have immunogenicity and can stimulate the static CD 8T cells in PBMCs of blood donors to obviously activate and proliferate; in the IFN-gamma validation method, the frequency of IFN-gamma + CD 8T cells in the CD 8T cell population is improved by a percentage compared with that of the negative control group. An increase of more than 100% is considered to be immunogenic, stimulating significant activation and secretion of IFN- γ by resting CD 8T cells in donor PBMCs.

Example 2: in some examples of the present invention, a mixed polypeptide vaccine can be prepared by synthesizing some epitope peptides from the 164 amino acid sequences, i.e., T cell epitope peptide sequences shown in SEQ ID Nos. 1-164. For example, the preparation steps are as follows:

1. preparing mixed peptide pool and adjuvant

1) 1-164T cell epitope peptide sequences are utilized to synthesize 31 epitope peptides which can be presented by HLA-A2 molecules. The polypeptide can be synthesized by Suzhou Qiangyao biotechnology company, the purity is more than 95%, 4 mg/peptide, each tube is separately packaged with 1mg, and the polypeptide is stored at the temperature of-800 ℃ for later use;

2) polypeptide solubilization: 1 tube (1mg) of each polypeptide was stored to room temperature for pre-warming, dissolved in 20. mu.L DMSO, and then supplemented with 180. mu.L PBS to achieve a polypeptide stock concentration of 5. mu.g/. mu.L, and stored at-80 ℃ for further use.

3) In this example, for example, the following 31 polypeptides can be grouped and mixed to make 4 peptide pools:

peptide pool 1: a1, A3, A4, A5, B1, B2, B3, B4, 200 muL/short peptide, and 1600 muL in total

Peptide pool 2: b6, C1, C2, C3, D2, D5, D6, D7 and 200 muL/short peptide, and mixing to obtain 1600 muL

Peptide pool 3: d11, D12, D13, R3, R4, R5, R6, R8, 200 muL/short peptide, and 1600 muL in total

Peptide pool 4: r9, R10, R11, R12, R13, R14, R15, 200 muL/short peptide, 1400 muL in total after mixing

4) Dissolution adjuvants R848 and Poly (I: C): adding 5mg R848(InvivoGen corporation) into 5mL physiological saline (1mg/mL), mixing, packaging, and storing at-20 deg.C; 10mg of Poly (I: C) (InvivoGen company) was added to 10mL of physiological saline (1mg/mL) and mixed, heated at 65 ℃ to 70 ℃ for 10min, cooled at room temperature for 1h, and stored at-20 ℃ after being dispensed.

2. Then preparing PLGA-NP/polypeptide vaccine

Polylactic-co-glycolic acid (PLGA) is a degradable, biocompatible, non-toxic biomacromolecule, often produced as nano-or microparticles (NP or MP) for use in humans for nearly thirty years as a drug or vaccine delivery material. In some examples of the invention, the PLGA-NP can be used for loading a mixed peptide pool to prepare a mixed polypeptide nano vaccine. The amount of PLGA-NP inoculated per peptide pool was set at 20 mg/mouse, and three mice per group, 60mg of PLGA-NPs loaded with 1 peptide pool was prepared for each inoculation. When 60mg PLGA-NP is prepared, the dosage of the peptide pool should be 230 μ L, about 1150 μ g polypeptide and the actual loading amount is about 1150 × 0.21 ═ 241.5 μ g, calculated as the protein loading rate of PLGA-NP is about 21%. Each mouse was injected with about 20mg of PLGA-NP loaded with each peptide pool, and the actual injected peptide pools were about 80. mu.g, and each polypeptide was about 10. mu.g.

1) 60mg of PLGA powder was weighed and dissolved in 15mL of dichloromethane, and 115. mu.L (575. mu.g) of single peptide pool liquid, sonicated on ice for 30s, amplitude 40% (1400J) (sonicated for 2s, 3s pause once, 15 cycles total) was added to prepare colostrum;

2) the colostrum was added in its entirety to 75mL of a 1% PVA solution with a dropper, sonicated on ice for 90s, amplitude 40% (1400 joules) (sonication for 2s, 3s pause, 45 cycles total) to produce a multiple emulsion;

3) placing 150mL of 0.5% PVA solution in a magnetic stirrer, continuously stirring at room temperature at 600rpm, dropwise adding the multiple emulsion by using a dropper, continuously stirring for 4 hours to volatilize dichloromethane, and solidifying the nanoparticles;

4) transferring all the solutions to an ultracentrifuge centrifuge tube, firstly centrifuging at 6000rpm for 5min, transferring the supernatant to a new 50mL ultracentrifuge tube, discarding the micron particle precipitate, then centrifuging the supernatant at 12000rpm for 10min at high speed to obtain PLGA-NPs precipitate, centrifuging and washing with deionized water for 2 times, and collecting the PLGA-NPs.

5) After centrifugation, the pellet was resuspended in 10mL of PBS, and then 0.1917g of EDC and 0.05754g of NHS were added, mixed well, and the reaction was stirred at room temperature for 1h (during which 5mL of a 2% PEI solution was prepared).

6) The PLGA-NPs were collected by centrifugation at 12000rpm for 12min, washed once with sterile deionized water, resuspended in 5mL sterile deionized water, and the pellet was stirred with a magnetic stirrer at room temperature, followed by 5mL of 2% PEI solution and stirred at room temperature for 4 h.

7) All solutions were collected and all washes were collected after washing the beaker with sterile deionized water, centrifuged and washed 1 time with sterile deionized water at 12000rpm for 15min, resuspended pellet with 1mL PBS, transferred all to 2mL lep tubes, added to a single pool of 115 μ L (575 μ g) of peptide solution and incubated overnight at 4 ℃ on a rotating disk.

8) Centrifuging at 4 deg.C, 12000rpm, 15min, resuspending the precipitate with sterile physiological saline, and storing at 4 deg.C.

3. C/polypeptide vaccine and R848/polypeptide vaccine are prepared, three vaccination schemes of the vaccine are established, immunization is carried out, and the efficacy of the vaccine is verified, and the specific steps are as follows:

the inoculum size of each polypeptide was set as: 10 ug/time/mouse, the amount of each peptide pool was 70-80 ug/time/mouse, and each peptide pool was inoculated with 1 injection site. Each mouse was inoculated 3 times, 3 mice per group.

The amount of adjuvant R848 or Poly (I: C) was set as: 25 μ L/injection site/time/mouse, 4 injection sites/mouse, 3/group.

The amount of PLGA-NP inoculated per peptide pool was set as: 20 mg/injection site/time/mouse, about 70-80. mu.g of actual injected peptide pool, about 10. mu.g of each polypeptide.

12 HLA-A2/DR1 transgenic C57BL/6 mice, female, 10 weeks old were selected and divided into 4 groups and 3 groups. Each mouse and each peptide pool was inoculated with 1 injection site for a total of 4 subcutaneous injections (caudal root subcutaneous, cervical dorsal subcutaneous), and the specific vaccine configuration and group vaccination protocols are shown in table 4 below:

TABLE 4

The time axis for immunization of HLA-A2/DR1 transgenic mice can be configured as shown in FIG. 33.

5. After inoculation is finished, the effect of the vaccine can be verified by detecting the specific T cell reaction in the mouse body

In some examples of the invention, specific T cell responses in vaccinated mice can be detected, for example, using the ELISPOT assay. The method comprises the following specific steps:

1) the 31 immunized polypeptides were grouped into 8 peptide libraries for detection according to the following table 5:

peptide library 1	3 neutral peptides +1 basic peptides	A1 A3 A4+A5
			Peptide library
2	2 neutral peptides +2 acidic peptides	B1 B2+B4 B6
			Peptide library
3	1 strip of basic peptides	B3
			Peptide library
4	2 neutral peptides +1 basic peptides	C1 C2+C3
			Peptide library
5	4 neutral peptides +2 acidic peptides	D2 D7 D12 D13+D5 D6
			Peptide library
6	1 strip of basic peptides	D11
			Peptide library
7	7 acidic peptides	R5 R6 R8 R11 R12 R14 R15
			Peptide library 8	2 neutral peptides +3 basic peptides	R3 R10+R4 R9 R13

TABLE 5

2) Preparation of spleen cell suspension: dislocating and killing each group of mice at Day28, taking spleen aseptically, adding PBS and grinding, collecting single cell suspension after passing through 200 mesh steel net, transferring to 15mL centrifuge tube, centrifuging at 1200rpm for 10min, and discardingCleaning, adding 5 times volume of erythrocyte lysate into the precipitate, mixing, standing at room temperature for 5min-8min, adding PBS to stop lysis, centrifuging at 1200rpm for 10min, discarding supernatant, re-suspending the cell precipitate with serum-free cell culture solution (Dake corporation), counting cells, and adjusting cell concentration to 2 × 10 with serum-free cell culture solution⁶And (4) placing the cells in an incubator for later use.

3) The spleen cell suspension of each mouse was inoculated into 100. mu.l (2X 10) per well of a PVDF membrane strip specially used for ELISPOT which was pre-coated with anti-mouse IFN-. gamma.⁵One/hole), 10 holes are inoculated on spleen cells of each mouse, peptide libraries 1 to 8 for detection are respectively added into the 1 st to 8 th holes, and the adding amount of single peptide in each peptide library is 2 mu g/peptide/hole; the 9 th hole is a negative control hole, and no polypeptide is added; as a positive control well, 5. mu.g of PHA was added to the 10 th well. Each well was supplemented with serum-free medium to 120. mu.l, and cultured in a cell culture chamber for 18 hours.

4) Cell lysis: taking out the ELISPOT strip plate from the cell incubator, throwing off the cell suspension, adding 200 mu L of deionized water into each hole, standing at 4 ℃ for 10min, throwing off the deionized water, and patting dry on absorbent paper. Add 200. mu.L PBS per well, stand for 1min, remove liquid, repeat the washing 5 times, and dry on absorbent paper after removing liquid for the last time.

5) Adding a detection antibody: add 100. mu.L of detection antibody (biotin-IFN-gamma detection antibody working solution) to each well and incubate for 2 hours at room temperature in the dark. And (3) throwing off liquid, adding 200 mu L of PBS into each hole, standing for 1min, throwing off the liquid, repeatedly washing the plate for 5 times, and patting dry on absorbent paper after the liquid is thrown off for the last time.

6) Adding HRP-streptavidin: add 100. mu.L of HRP-streptavidin working solution to each well and incubate for 1 hour at room temperature in the dark. Spin off the liquid, add 200 μ L PBS per well), rest for 1min, spin off the liquid, repeat the wash of the plate 5 times in this way, spin off the liquid for the last time and then pat dry on absorbent paper.

7) Color development: add 100. mu.L AEC color developing solution into each well (ready to use), and keep standing at room temperature in dark place for 25 min. And (3) throwing off the liquid, unloading the base of the plate, washing the front side and the back side of the PVDF membrane and the base for 3-5 times by using deionized water to stop color development, then placing the plate in a room-temperature dark place, and loading the plate on the base after natural drying.

8) And (3) counting the spots: spots were counted by themselves under an upright optical microscope with a low power lens. Photographing and spot counting can also be performed by an enzyme-linked immunospot analyzer. 2X 10 of each mouse⁵In individual splenocyte population, the SARS-CoV-2 protein CD8⁺The number of IFN-. gamma.secreting cells after stimulation with the T cell epitope peptide was the sum of the number of spots per well of each of the 1 st to 8 th wells minus the number of spots of the negative control well, and represents 2X 10⁵The number of responding memory and active T cells with 31T cell epitope peptides specific for the new coronavirus in each spleen cell population.

The results show that: compared with three mice in a control group, the three forms of the mixed polypeptide vaccine can stimulate HLA-A2/DR1 transgenic mice to cause strong specificity CD8⁺T cell response, IFN-. gamma.secreting cells were 400-fold more abundant than control mice. FIG. 22 is a statistical chart showing the number of spots in the spleen cell population of each group of mice; fig. 23 to 26 are spot scans of ELISPOT experiments in various groups of mice. This result confirmed that SEQ ID NO: 1-164T cell epitope peptide of SARS-CoV-2 protein has the potential of inducing specific T cell reaction for preparing SARS-CoV-2 vaccine.

6. After inoculation is finished, the effect of the vaccine can be verified by detecting the specific T cell reaction in the mouse body

In other embodiments of the present invention, intracellular cytokine staining and flow cytometry can be used to detect specific T cell responses in vaccinated mice, for example, by:

1) spleen cell suspension from each mouse was inoculated into 48-well cell culture plates at 500. mu.L (1X 10) per well⁶One/hole), forming a plurality of peptide libraries by 31 kinds of epitope peptides of the inoculated mice according to the types of antigens, and respectively adding the peptide libraries into different holes, wherein the adding amount of each polypeptide in each peptide library is 8 mu g/peptide/hole; additionally arranging a negative control hole without adding any polypeptide; adding 5 mu g of PHA into the positive control hole; placing the mixture in a cell culture box for culturing for 16 h;

2) adding 2 mu L of BFA/Monensin Mixture combined organism into each hole), and continuously incubating for 6 h; collecting cells in a flow analysis tube, centrifuging at 1600rpm for 5min, and removing supernatant; add 0.5. mu.g anti-mouse CD16/CD32 antibody to each tube to block FC receptor, 20min at 4 ℃;

3) adding 0.25 mu g of PE-anti-CD8a and 1 mu g of FITC-anti-CD3e into each tube, and keeping the temperature at 4 ℃ for 30 min; then adding 3ml LPBS, mixing, centrifuging at 1600rpm for 5min, discarding supernatant, bouncing cell for resuspension, adding 100 μ L MEDI μm M A into each tube, and incubating at room temperature for 15 min; supplementing 3mL of 5% FBS-PBS, uniformly mixing, centrifuging at 1600rpm for 5min, removing supernatant, and bouncing cell sediment; adding 100 μ L MEDI μm M B and 0.125 μ g APC-anti-IFN- γ (eBiosciences) monoclonal antibody into each tube, mixing, incubating at 4 deg.C for 30min, and mixing once;

4) adding 5% FBS-PBS to 3mL, mixing, centrifuging at 1600rpm for 5min, discarding supernatant, bouncing cell precipitate, resuspending with 5% FBS-PBS 500 μ L, detecting with BDCallibur flow cell, and analyzing CD3⁺/CD8⁺IFN-gamma in cell populations⁺The frequency of the cells.

The results show that: compared with three mice in a control group, the three forms of the mixed polypeptide vaccine can stimulate HLA-A2/DR1 transgenic mice to cause strong specificity CD8⁺T cell response, IFN-gamma secreting CD8⁺The T cell frequency is about 20-30 times that of the control mice. FIG. 27 shows CD3 in the spleen cell population of mice in each group⁺/CD8⁺IFN-gamma in cell populations⁺A statistical plot of the results of the cell frequency; FIGS. 28 to 31 show IFN-. gamma.secreting CD8 from the splenic cell populations of mice in each group⁺Flow scatter plots of T cells. The results again confirm that: SEQ ID NO: 1-164T cell epitope peptide of SARS-CoV-2 protein can be used for preparing SARS-CoV-2 vaccine and inducing specific T cell reaction.

Example 3: in some examples of the invention, the nucleic acid sequence is encoded by SEQ ID NO: 1-164, synthesizing epitope peptides to form 10 peptide libraries, establishing an ELISPOT method, detecting the number of specific T cells which are in cross reaction with the T cell epitope peptide of SARS-CoV-2 protein in healthy blood donor groups, and verifying the feasibility of detecting the immune function of the SARS-CoV-2 specific T cells by using the reagent prepared from the epitope peptides. The specific steps may be as follows:

1) the 164 kinds of epitope peptides described above were divided into 10 peptide pools according to the kinds of source proteins, wherein the epitope peptide of each protein was divided into 2 peptide pools (23 kinds of epitope peptides of E protein, 36 kinds of epitope peptides of M protein, 25 kinds of epitope peptides of N protein, 41 kinds of epitope peptides of S protein, 39 kinds of epitope peptides of RdRp), each peptide pool contained 12 to 22 kinds of polypeptides. Each polypeptide was dissolved in 1mg to 500. mu.L of serum-free 1640 medium (2. mu.g/. mu.L), and then each polypeptide was mixed in the same mass ratio to a peptide library.

2) The PBMC of the donor frozen at the early stage of recovery was inoculated into a 24-well plate, 10% FCS-1640 cell culture medium was added thereto, and the mixture was cultured overnight in a cell culture chamber.

3) Donor PBMC were collected in 24-well plates, washed by PBS centrifugation, centrifuged at 1200rpm for 10min, the supernatant discarded, the pellet resuspended in PBS, and washed once in the same centrifugation. Resuspending the cell pellet with serum-free cell culture medium (Dake, Inc.), counting the cells, and adjusting the cell concentration to 2X 10 with serum-free cell culture medium⁶And (4) placing the cells in an incubator for later use.

4) Donor PBMC suspension was inoculated into 100. mu.l (2X 10) per well of ELISPOT-specific PVDF membrane strip precoated with anti-human IFN-. gamma.⁵One/well), 12 wells were inoculated per donor's PBMC, and peptide pools 1 to 10 were added to wells 1 to 10, respectively, with each polypeptide added at 2 μ g/peptide/well in each peptide pool; the 11 th hole is a negative control hole, and no polypeptide is added; the 12 th well is a positive control well, to which phytohemagglutinin PHA 5. mu.L (25. mu.g/mL) was added. Each well was supplemented with serum-free medium to 150. mu.l, and cultured in a cell culture chamber for 18 hours.

5) Cell lysis: the ELISPOT strip plate is taken out of the cell incubator, the cell suspension is thrown off, 200 mu L of deionized water is added into each hole, the mixture is placed at 4 ℃ for 10 minutes, the deionized water is thrown off, and the mixture is patted dry on absorbent paper. Add 200. mu.L PBS per well, rest for 1min, remove liquid, repeat the wash 5 times, and dry on absorbent paper after the last liquid removal.

6) Adding a detection antibody: add 100. mu.L of detection antibody (biotin-IFN-gamma detection antibody working solution) to each well and incubate for 2 hours at room temperature in the dark. And (3) throwing off liquid, adding 200 mu L of PBS into each hole, standing for 1 minute, throwing off liquid, repeatedly washing the plate for 5 times, and beating dry on absorbent paper after the liquid is thrown off for the last time.

7) Adding HRP-streptavidin: add 100. mu.L of HRP-streptavidin working solution to each well and incubate for 1 hour at room temperature in the dark. And (3) throwing off liquid, adding 200 mu L of PBS into each hole, standing for 1 minute, throwing off liquid, repeatedly washing the plate for 5 times, and beating dry on absorbent paper after the liquid is thrown off for the last time.

8) Color development: add 100. mu.L of AEC color developing solution (ready to use) to each well, and keep it at room temperature for 25 minutes in the dark. And (3) throwing off the liquid, unloading the base of the plate, washing the front side and the back side of the PVDF membrane and the base for 3-5 times by using deionized water to stop color development, then placing the plate in a room-temperature dark place, and loading the plate on the base after natural drying.

9) And (3) counting the spots: spots were counted by themselves under an upright optical microscope with a low power lens. Photographing and spot counting can also be performed by an enzyme-linked immunospot analyzer. 2X 10 of each mouse⁵In individual splenocyte population, the SARS-CoV-2 protein CD8⁺The number of IFN-. gamma.secreting cells after stimulation with the T cell epitope peptide was the sum of the number of spots per well minus the number of spots in the negative control well in the 1 st to 10 th wells, and represents 2X 10⁵The number of T cells in individual splenocytes with memory and activity cross-reacting with T cell epitope peptide of SARS-CoV-2 protein.

The results show that memory T cells that cross-react with the new coronavirus are also present in PBMCs of healthy subjects. FIG. 32 is a schematic representation of a nucleic acid sequence utilizing the above-described SEQ ID NO: 1-164 ELISPOT dot-plots for detecting SARS-CoV-2 specific T cells by the T cell epitope peptide and partial donor PBMC method. The results confirm that the T cell epitope peptide of SARS-CoV-2 listed in SEQ ID NO 1-164 and the ELISPOT method can be used to establish a detection method and a reagent for SARS-CoV-2 specific T cells.

By way of example above, SEQ ID NO: the epitope peptide 1-164 can be applied to the preparation of a reagent or a kit for detecting SARS-CoV-2 specific T cells, and can also be applied to the preparation of SARS-CoV-2 vaccines or effector T cells.

In the above example, SEQ ID NO: 1-164 can be used for preparing SARS-CoV-2 vaccine and inducing specific T cell reaction. Therefore, the effector T cells obtained by stimulating peripheral blood lymphocytes with the above-mentioned epitope peptide or antigen-presenting cells presenting the epitope peptide to HLA can exert a specific immune effect against SARS-CoV-2, and therefore, adoptive immune therapeutic cell preparations against SARS-CoV-2 infection can be prepared using the effector T cells and applied to the treatment and prevention of SARS-CoV-2 infection. It is noted that the epitope peptide can be presented by HLA-A molecules on an antigen presenting cell membrane, and stimulates activation, proliferation and differentiation of SARS-CoV-2 specific thymus dependent lymphocyte, thereby playing a role in immune effect against SARS-CoV-2 infection; the antigen peptides can be used for preparing mixed polypeptide vaccines of SARS-CoV-2, recombinant protein vaccines of tandem connection of a plurality of epitope peptides, DNA and RNA vaccines, and can also be used for preparing detection kits for detecting specific thymus-dependent lymphocytes of SARS-CoV-2, and have potential application value in the prevention, treatment and diagnosis of SARS-CoV-2.

It is understood that SEQ ID NO: 1-164 can be used as an effective component of a vaccine, and the nucleic acid encoding the epitope peptide can also be used as an effective component of a vaccine, and the epitope peptide can also be applied to the preparation of a medicament for treating SARS-CoV-2.

The detection reagent can be enzyme-linked immunosorbent assay reagent, intracellular cytokine fluorescent staining reagent, chemiluminescence reagent, enzyme-linked immunosorbent assay reagent, human leukocyte antigen polymer fluorescent staining or flow cytometry analysis reagent.

The amino acid sequence of SARS-CoV-2 antigen used to predict epitope in this example can be obtained by UniProt Global protein resources database search, and the specific sequences are as follows:

e Protein (envelope Protein) (Protein _ ID — YP _ 009724392.1):

molecular types of sequences: PRT

Scientifically named biological species: coronaviridae family novel Coronaviridae species

M Protein (membrane Protein) (Protein _ ID — YP _ 009724393.1):

molecular types of sequences: PRT

N Protein (nucleocapsid Protein) (Protein _ ID — YP _ 009724397.2):

molecular types of sequences: PRT

S Protein (spike Protein) (Protein _ ID — YP _ 009724390.1):

molecular types of sequences: PRT

RdRp Protein (RNA-dependent RNA polymerase) (Protein-ID — YP _ 009725307.1): molecular types of sequences: PRT

It should be noted that the above list is only the amino acid sequence of SARS-CoV-2 antigen used in the present example for predicting epitope, and is not intended to limit the whole invention, and the amino acid sequence of SARS-CoV-2 antigen obtained by those skilled in the art based on other variants of SARS-CoV-2 can also be applied in the technical scheme of the present invention and examples thereof.

The epitope peptide prediction tool sources in the above examples are shown in table 6, for example.

TABLE 6

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited by the foregoing examples, which are provided to illustrate the principles of the invention, and that various changes and modifications may be made without departing from the spirit and scope of the invention, which is also intended to be covered by the appended claims.

Claims

1. An epitope peptide is characterized in that the amino acid sequence of the epitope peptide is shown in any one of SEQ ID NO 1-164.

2. An epitope peptide characterized by having an amino acid sequence obtained by substituting, deleting, inserting and/or adding 1 or more amino acid sequences in SEQ ID NO. 1 to 164.

3. A vaccine, characterized in that the effective components thereof comprise: the epitope peptide according to claim 1 or 2 or a nucleic acid encoding the epitope peptide according to claim 1 or 2.

4. Effector T cells obtained by stimulating peripheral blood lymphocytes with the epitope peptide of claim 1 or 2 or an antigen-presenting cell that presents said epitope peptide to an HLA molecule.

5. An adoptive immune therapeutic cell characterized in that its effective ingredient comprises the effector T cell according to claim 4.

6. Use of the epitope peptide according to claim 1 or 2 for the preparation of a SARS-CoV-2 vaccine or an effector T cell.

7. Use of the epitope peptide according to claim 1 or 2 for the preparation of a reagent or kit for the detection of T cells specific for SARS-CoV-2.

8. The use of claim 7, wherein the detection reagent is an ELISA reagent, an intracellular cytokine fluorescent staining reagent, a chemiluminescent reagent, an ELISA reagent, a human leukocyte antigen multimer fluorescent staining reagent, or a flow cytometry reagent.

9. Use of the epitope peptide according to claim 1 or 2 for the preparation of a medicament for the treatment of SARS-CoV-2 infection.