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

Abstract

The invention discloses an SARS-CoV-2 lymphocyte antigen epitope peptide and application thereof, belongs to the fields of medical immunology and infectious pathology, and particularly relates to three thymus dependent lymphocyte antigen epitope peptides of SARS-CoV-2. The epitope peptide is characterized in that the amino acid sequence of the epitope peptide is shown as any one of SEQ ID NO: 1-3; or an amino acid sequence obtained by substituting, deleting, inserting and / or adding one or more amino acid sequences in SEQ ID NO: 1-3. The antigen epitope peptide can be presented by HLA-DR molecules, and stimulates activation, proliferation and differentiation of SARS-CoV-2 specific thymus dependent lymphocytes, so that the immune effect of resisting SARS-CoV-2 infection is exerted; the antigen peptides can be used for preparing mixed polypeptide vaccines of SARS-CoV-2, recombinant protein vaccines connected with a plurality of epitope peptides in series and DNA and RNA vaccines, can be used for preparing a detection kit for detecting SARS-CoV-2 specific thymus dependent lymphocytes, and can also be used for preparing effector thymus dependent lymphocytes or drugs for treating SARS-CoV-2 infection. The SARS-CoV-2 antibody has a potential application value in the prevention, treatment and diagnosis of SARS-CoV-2 infection and COVID-19.

Description

Technical Field

[0001] The present invention relates to the fields of medical immunology and infectious diseases, and in particular to a SARS-CoV-2 lymphocyte antigen epitope peptide and an application thereof. Background Art

[0002] SARS-CoV-2 is an enveloped, single-stranded positive-strand RNA beta coronavirus of the Coronaviridae family, with a genome length of 30,000 bases. It includes 14 open reading frames (ORFs), and the 5'-end Orf1ab encodes 16 non-structural proteins (Nsp1-16), of which Nsp7, Nsp8 and Nsp12 constitute the RNA-dependent RNA polymerase (RdRp) of the SARS-CoV-2 virus, with a total length of 932 amino acids. The 13 Orfs at the 3' end encode 4 structural proteins and 9 presumed auxiliary factors. The 4 viral structural proteins are: S protein (spike protein), with a total length of 1273 amino acids; E protein (envelope protein), with a total length of 75 amino acids; M protein (membrane protein), with a total length of 222 amino acids; N protein (nucleocapsid protein), with a total length of 419 amino acids.

[0003] Currently, there are still very few SARS-CoV-2 antigen epitope peptides that can stimulate T cell responses, which limits the monitoring and analysis of specific T cell immune function in SARS-CoV-2 infected persons, the research on the immunopathology and immunoprotection mechanism of specific T cell immune responses in the interaction between SARS-CoV-2 and the host, and the development of vaccines and therapeutic preparations that induce the body to produce virus-specific T cell responses. Summary of the invention

[0004] In view of the deficiencies in the prior art, the present invention proposes a SARS-CoV-2 lymphocyte antigen epitope peptide and application thereof.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The first aspect of the present invention relates to an epitope peptide, the amino acid sequence of which is:

[0007] Any one of SEQ ID NOs: 1 to 3;

[0008] Alternatively, an amino acid sequence obtained by replacing, deleting, inserting and / or adding one or more amino acid sequences in SEQ ID NOs: 1 to 3.

[0009] The second aspect of the present invention relates to a nucleic acid molecule capable of encoding the above-mentioned epitope peptide.

[0010] The third aspect of the present invention relates to a recombinant vector containing the above-mentioned nucleic acid molecule.

[0011] Optionally, the recombinant vector includes: a recombinant DNA gene segment, a recombinant plasmid, a recombinant viral vector or a recombinant mRNA.

[0012] The fourth aspect of the present invention relates to a vaccine, the active ingredients of which include: the above-mentioned epitope peptide or the nucleic acid molecule encoding the above-mentioned or the above-mentioned recombinant vector.

[0013] The fifth aspect of the present invention relates to an adoptive immunotherapy cell capable of recognizing the above-mentioned epitope peptide and being activated thereby.

[0014] Optionally, the method comprises stimulating effector T cells obtained from peripheral blood lymphocytes using the above-mentioned epitope peptides or antigen presenting cells that present the epitope peptides to HLA molecules.

[0015] A sixth aspect of the present invention relates to a method for obtaining effector T cells, comprising the following steps:

[0016] PBMCs were isolated by density gradient centrifugation;

[0017] The above-mentioned epitope peptide is co-cultured with PBMCs, or the above-mentioned epitope peptide is added to the antigen presenting cell culture medium to present the epitope peptide to HLA, and then co-cultured with PBMCs to obtain the effector T cells.

[0018] A seventh aspect of the present invention relates to an application, comprising any of the following aspects:

[0019] Use of the above epitope peptides in the preparation of SARS-CoV-2 vaccines or effector T cells;

[0020] Use of the above epitope peptide in the preparation of a reagent or kit for detecting SARS-CoV-2 specific T cells;

[0021] The use of the above-mentioned adoptive immunotherapy cells in the preparation of drugs for treating SARS-CoV-2.

[0022] Optionally, the detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, a chemiluminescent assay reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining reagent, or a flow cytometry reagent.

[0023] The above-mentioned epitope peptide sequence can be used to prepare a mixed polypeptide vaccine, recombinant protein vaccine or recombinant gene vaccine for pneumonia caused by new coronavirus infection (COVID-19) that induces the body to produce a specific T cell immune response.

[0024] Preparation of mixed polypeptide vaccines: According to the antigen epitope peptide sequence of the present invention, a variety of antigen peptides are artificially synthesized, mixed with adjuvants to form a soluble preparation, or loaded with biological nanomaterials to form a nano polypeptide vaccine, which is injected into healthy people, SARS-CoV-2 infected people or patients with pneumonia 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 body's specific cellular immune protection ability, preventing healthy people from being infected with SARS-CoV-2, or enhancing the ability of infected people to inhibit viral replication and proliferation and resist reinfection, thereby preparing a SARS-CoV-2 T cell epitope mixed polypeptide vaccine.

[0025] Preparation of recombinant protein vaccine: According to the antigen epitope peptide sequence of the present invention, a recombinant plasmid with multiple epitope peptides in series is constructed, and prokaryotic or eukaryotic expression is performed. The recombinant protein is then purified and mixed with an adjuvant to form a soluble preparation, which is injected into healthy people, people infected with SARS-CoV-2, or patients with pneumonia caused by SARS-CoV-2 infection to stimulate the activation, proliferation and differentiation of specific T cells in the body into effector T cells, thereby enhancing the body's specific cellular immune protection ability, preventing healthy people from being infected with SARS-CoV-2, or enhancing the ability of infected people to inhibit viral replication and proliferation and resist reinfection, thereby preparing a recombinant protein vaccine for SARS-CoV-2.

[0026] Preparation of recombinant gene vaccines: According to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, a recombinant plasmid, a recombinant viral vector or a recombinant mRNA with multiple epitope sequences in series is constructed and injected into healthy people, people infected with SARS-CoV-2 or people with pneumonia caused by SARS-CoV-2 infection, to stimulate the activation, proliferation and differentiation of specific T cells in the body into effector T cells, thereby enhancing the body's specific cellular immune protection ability, preventing healthy people from being infected with SARS-CoV-2, or enhancing the ability of infected people to inhibit viral replication and proliferation and resist reinfection, thereby preparing a recombinant gene vaccine for SARS-CoV-2.

[0027] In addition, the above-mentioned lymphocyte antigen epitope peptide sequence can be used to prepare a detection preparation or kit for detecting SARS-CoV-2 antigen-specific T cells: according to the antigen epitope peptide sequence of the present invention, a variety of antigen peptides are artificially synthesized, and in the enzyme-linked immunospot method, intracellular cytokine fluorescence staining method, chemiluminescence method and enzyme-linked immunosorbent assay, as antigen stimulants, mixed cultured with the patient's peripheral blood mononuclear cells (PBMC), stimulate SARS-CoV-2 antigen-specific T cells to activate, proliferate and secrete cytokines, and then detect the secretion of the cytokine through other combined reagents, thereby reflecting the number and reactivity of specific T cells; these polypeptide sequences can also be used to prepare human leukocyte antigen and polypeptide complexes (peptide-HLA complex) and their polymers through genetic engineering technology and protein engineering technology, and then further prepared into preparations labeled with fluorescent or metal substances, and the number of SARS-CoV-2 antigen-specific T cells in the patient's peripheral blood PBMC cell population is detected by flow cytometry. The relevant kit refers to a SARS-CoV-2 specific T cell detection kit assembled from the above-mentioned preparations and other conventional reagents in different detection methods.

[0028] The epitope peptide sequence can be used to prepare drugs for treating SARS-CoV-2: the mixed polypeptide vaccine, recombinant protein vaccine or gene vaccine of the above-mentioned antigen epitope peptide sequence is combined with other immunotherapy preparations or chemotherapeutic preparations to prepare clinical drugs for treating SARS-CoV-2.

[0029] Beneficial effects of the present invention:

[0030] The present invention obtains a group of SARS-CoV-2 virus protein T cell epitope peptides through online virtual prediction and functional experimental verification. These new antigen epitope peptide sequences will provide the required key antigen components, i.e., antigen epitope peptide sequences, for the development of preventive and therapeutic polypeptide vaccines, recombinant protein vaccines and gene vaccines against SARS-CoV-2 virus, and for the design of reagents and methods for detecting SARS-CoV-2 virus antigen-specific T cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The present invention will be further described below in conjunction with the accompanying drawings.

[0032] Figure 1 The present invention provides an experimental technical roadmap for validating the immunogenicity of epitope peptides to be identified in some examples of the present invention.

[0033] Figure 2The flow cytometric analysis diagram of the DC-peptide-PBL co-culture experiment of three immunogenic SARS-CoV-2 virus protein T cell epitope peptides in the present invention; after the T cell epitope peptides were co-cultured with the DC cells and PBL of the blood donor for 14 days, they were co-stained with CD3 and CD4 fluorescent monoclonal antibodies, and intracellular staining was performed with IFN-γ fluorescent monoclonal antibodies, and then IFN-γ was detected and analyzed by flow cytometry. + / CD4 + T cells in CD4 + Frequency in T cell population.

[0034] Figure 3 Epitope peptides induced specific CD4 in HLA-A2 / DR1 transgenic mice + T cell response

[0035] A mixed polypeptide vaccine was prepared by combining 20 HLA-A0201-restricted positive epitope peptides with the adjuvant poly(I:C) and immunizing HLA-A2 / DR1 transgenic mice on days 0, 7, and 21, respectively. Seven days after the last immunization, the mouse spleen cells were taken and restimulated with PBS and each positive epitope peptide for 10 h, and then BFA / Monesion (to block cytokine release) was added to continue culturing for 6 h. Then, IFN-γ intracellular staining and flow cytometry analysis were performed to detect IFN-γ. + / CD3 + / CD4 + The frequency of T cells. Figure 3 A is a representative flow cytometry analysis; B is the IFN-γ + / CD3 + / CD4 + T cell frequency statistics. DETAILED DESCRIPTION

[0036] The following will be combined with the drawings in each example of the present invention to clearly and completely describe the technical solutions in the examples of the present invention. Obviously, the described examples are only part of the examples of the present invention, not all of them. Based on the various examples in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention. The project involved in this application is funded by the key project "Research on Pathogens and Epidemic Prevention Technology System" of the National Key R&D Program (2022YFC2304103).

[0037] Example 1: In some examples of the present invention, a method for predicting and identifying the amino acid sequence of a SARS-CoV-2 viral protein T cell epitope peptide is provided, and the specific steps are as follows:

[0038] 1. Virtual prediction of T cell epitope peptides restricted by HLA-DRB1 molecules from multiple SARS-CoV-2 proteins

[0039] The S, E, M, N, and R proteins of SARS-CoV-2 were selected, and then the T cell epitope peptides targeting the S protein restricted by molecules such as DRB1:0901, 1501, 0701, 1202, 0803, 1101, 0301, 0405, 1302, 1502, 1602, and 0406 were virtually predicted using epitope peptide prediction tools such as SYFPEITHI, EPIJEN, ConvMHC, and IEDB (including ANN, Consensus, NetMHCpan, SMM, and SMMPMBEC).

[0040] The antigen binding groove of the HLA class II molecule is open at both ends, and the accepted antigen peptide length is 13-25 amino acid residues, among which 16 amino acids are the most common. Therefore, in some examples of the present invention, a polypeptide with a length of 16 amino acids is selected as the research object, and the amino acid sequence of each SARS-CoV-2 virus S protein is input into the corresponding amino acid sequence input box of the prediction database website, and then the length of the epitope peptide is selected as 16 amino acids, and then a specific HLA-DRB1 molecule is selected to perform online virtual prediction of the T cell epitope peptide of the SARS-CoV-2 virus S protein.

[0041] For each HLA-DRB1 molecule and SARS-CoV-2 virus S protein, the 16 peptides predicted by different databases are arranged from high to low according to the scores, and then the epitope peptides that meet the scoring criteria of at least two or more prediction methods are selected as candidate epitope peptides. For each HLA-DRB1 molecule, 1-6 peptides with the highest scores or the highest affinity are selected from the candidate epitope peptides as epitope peptides to be identified. The sources of epitope peptide prediction tools in the above examples are shown in Table 1.

[0042]

[0043]

[0044] Table 1

[0045] 2. Isolation of PBMC from peripheral blood of blood donors:

[0046] 1) Collect peripheral blood collection trays (leukocyte filter trays used for 400mL peripheral anticoagulant blood sorting of red blood cells) from blood donors at the blood center, spray them with alcohol and place them in a clean bench, carefully disinfect the inlet and outlet tubes with alcohol cotton balls, cut the inlet and outlet tubes, draw PBS with a 50mL syringe, use the outlet of the blood collection tray as the syringe access port, and use 6 50mL centrifuge tubes to collect the PBS flushing solution at the inlet of the blood collection tray, and collect about 300mL of cell suspension in total;

[0047] 2) The first 50 mL of blood flushed out was collected in the first centrifuge tube without centrifugation; the remaining 5 centrifuge tubes were evenly collected with the remaining 250 mL of PBS, centrifuged at room temperature, 1500 rpm, for 12 min, and the turbid supernatant after centrifugation was transferred to a 50 mL centrifuge tube and centrifuged again at room temperature, 1500 rpm, for 12 min to collect all the cell precipitates; the cell precipitates in the last 5 centrifuge tubes were collected in a 50 mL centrifuge tube, filled with PBS to 50 mL, and fully mixed with the 50 mL of blood in the first tube;

[0048] 3) Pre-add 5 mL of human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen) to 10 15 mL centrifuge tubes; slowly add 100 mL of the mixed leukocyte suspension along the tube wall and spread it above the separation solution surface to keep the two interfaces clear, 10 mL of leukocyte suspension / tube, 20°C, 2000 rpm, centrifuge for 20 min, acceleration of 1, deceleration of 0;

[0049] 4) After the centrifugation, use a pipette to move in the same plane to suck all the milky white PBMC layers into a 50mL sterile centrifuge tube (about 30mL), try not to suck the plasma layer above the white film layer, and then divide the cell suspension of the white film layer evenly into 4 50mL centrifuge tubes, add PBS to 50mL to each tube, mix thoroughly, and centrifuge at 1300rpm for 12min; discard the supernatant, flick up the cell pellet, mix and combine the pellets and divide them equally into two of the 50mL centrifuge tubes, add PBS to 50mL / tube, mix by blowing and centrifuging again twice at 1000rpm for 12min, discard the supernatant, flick the cell pellet with your fingers, add a small amount of serum-free 1640 culture medium (Dakoway Bio, Shenzhen) to resuspend, collect and combine in 1 50mL centrifuge tube, and add serum-free 1640 culture medium to about 20mL;

[0050] 5) Cell counting: Take an EP tube, add 380 μL of leukocyte diluent, then add 20 μL of cell suspension, mix thoroughly and pour into a cell counting plate. Count under a microscope and calculate the cell concentration according to the following formula: total number of cells in 4 large grids / 4×104×dilution factor 20=total cells / mL.

[0051] 3. HLA-DRB1 allele typing can then be performed, for example, the steps are as follows.

[0052] The PBMC isolated in the above example was taken, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Bio, Beijing); HLA-DRB1 site-specific primers DRB1-F and DRB1-R were used for PCR to amplify the DNA sequence of exon 2 and part of introns 2 and 3 of the HLA-DRB1 locus, and the product size was 285 bp. The amplification conditions were: 95°C pre-denaturation for 3 min; 95°C denaturation for 15 s; 62°C annealing for 15 s; 72°C extension for 90 s; 35 cycles; 72°C extension for 5 min. The amplified product was identified by 1% agarose gel electrophoresis, purified and bidirectionally sequenced. PCR reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd., see Table 2.

[0053]

[0054] Table 2

[0055] The sequencing results were assembled into a complete HLA-DRB1 overlapping sequence (Contig) using the Lasergene program's Seqman software. The bases of the bidirectional sequencing were carefully checked to see if they were complete and consistent. The bases of the heterozygotes were found and replaced with annexed bases, such as M for A and C, R for A and G, W for A and T, S for C and G, Y for C and T, and K for G and T. Finally, the sequence fragments of the amplified HLA-DRB1 alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA-DRB1 base sequence with the sequences of all HLA-DRB1 alleles in the database until a completely matching gene combination was obtained, thereby determining the HLA-DRB1 alleles.

[0056] 4. Subsequently, DCs were induced and peptide-DC-PBL co-culture experiments were performed to verify the immunogenicity of the epitope peptides:

[0057] 1) Inoculate the PBMCs resuspended in 20 mL of serum-free 1640 medium into a cell culture flask (1-1.5×10 8 cells / 20mL / medium bottle, 1.5-3×10 8 cells / 30mL / large bottle), incubated at 37°C for about 4h;

[0058] 2) Shake the culture flask and pipette it once, then transfer the non-adherent cells and culture medium into a medium-sized culture flask for PBL culture, add normal serum to a final concentration of 10%, culture overnight, and freeze the PBL for future use the next day;

[0059] 3) Add 10 mL of serum-free 1640 culture medium to the adherent cells, pipette once, shake and wash the culture bottle, discard the culture medium, repeat the washing once, discard the culture medium, and add the corresponding volume of 10% Australian fetal serum-1640 culture medium (15 mL / medium bottle, 20 mL / large bottle);

[0060] 4) On day 0, add human GM-CSF (PrepoTech) with a final concentration of 1000 IU / mL and human rIL-4 (PrepoTech) with a final concentration of 500 IU / mL, and culture at 37°C; on the evening of the third day, replace half of the medium, and add GM-CSF and rIL-4 at the same time, and continue to culture at 37°C; on the evening of the fifth day, add LPS to stimulate DC maturation with a final concentration of 1 μg / mL, and add GM-CSF and rIL-4 at the same time, and continue to culture at 37°C; on the evening of the sixth day, thaw the frozen PBL of the blood donor for use; on the seventh day, harvest mature DC (mDC): knock and blow the culture bottle, aspirate the culture medium into a 50 mL centrifuge tube, wash the culture bottle with a small amount of serum-free culture medium, and then count the cells (count with the original solution, do not count very small lymphocytes);

[0061] 5) Analysis of mDC phenotype: 3×10 5 The cells were evenly divided into 6 flow cytometry centrifuge tubes, and after centrifugation, they were stained with 5 fluorescent monoclonal antibodies (CD83\CD80\CD86\DR\ABC), and the positive percentage of each marker was analyzed by flow cytometry.

[0062] 6) The remaining mDCs were centrifuged at 1200 rpm for 10 min, the supernatant was discarded, and the cell pellet was resuspended in serum-free 1640 culture medium to adjust the mDC cell concentration to 0.5×10 5 cells / mL, temporarily stored in an incubator;

[0063] 7) Observe the flow cytometry results and the ratio of mDC, inoculate mDC in a 24-well plate, 50,000 cells / 1.0 mL / well; then add 10 μL of a single epitope peptide corresponding to the HLA-A molecule of the blood donor to each well (final concentration is 20 μg / mL). About 10 epitope peptides are verified for each blood sample, and three control wells are set up at the same time: DC-T negative control well (no peptide), PBL positive well (add a small amount of PHA), PBL negative well (no stimulant is added);

[0064] 5. Then, intracellular IFN-γ fluorescence staining is performed, and the operation steps are, for example:

[0065] 1) On day 21, collect cells from each DC-polypeptide-PBL co-culture well into a sterile EP tube, add about 1.5 mL of ordinary serum-free culture medium, centrifuge at 1200 rpm for 10 min, discard the supernatant once, flick off the cell pellet, resuspend the cells with 0.5 mL of serum-free culture medium (Dakoway), and transfer to a 48-well plate;

[0066] 2) Add 10 μL of the corresponding single polypeptide to each well (final concentration 20 μg / mL), and set up a non-peptide stimulation well and a PHA stimulation well as the negative control and positive stimulation control of the cell sample, respectively; incubate in a cell culture incubator for 16 hours;

[0067] 3) Add 2 μL of BFA / Monensin (250X Lianke Biotechnology) to each well and continue incubation for 6 h;

[0068] 4) Collect cells from each group into flow cytometry test tubes, add 1 μL Fc Block (anti-CD16 / CD32) to each tube, mix well, and incubate at 4°C for 20 min, mixing once during the process;

[0069] 5) Each experimental tube was co-stained with FITC-anti-CD3 and APC-anti-CD4 fluorescent monoclonal antibodies (eBiosciences) respectively; two blank PBL tubes were reserved, one of which was not stained, and the other was stained with FITC-anti-CD3 for compensation; each tube was placed at 4°C for 30 minutes, PBS was added to 3 mL, and the mixture was incubated at 1200 rpm for 10 minutes, the supernatant was discarded, and the cell pellet was flicked off;

[0070] 6) Add 100 μL of membrane permeabilization agent MEDIUM A (Lianke Biotechnology) to each tube and incubate at room temperature for 15 min; add 3 mL of 5% FBS-PBS, mix well, centrifuge at 300-350 g for 5 min, discard the supernatant, and flick the cell pellet;

[0071] 7) Add 100 μL of membrane permeabilization agent MEDIUM B (Lianke Biotechnology) to each tube, add 20 μL of PE-anti-IFN-γ (eBiosciences) to each experimental tube, and do not add PE-anti-IFN-γ to the unstained tube; incubate each tube at 4°C for 30 minutes, mixing once during the period;

[0072] 8) Add 5% FBS-PBS to each tube to make up to 3 mL, mix well, centrifuge at 300-350g for 5 min, discard the supernatant, flick off the cell pellet, resuspend with 0.5 mL of 5% FBS-PBS, and detect CD3 by flow cytometry. + / CD4 + or CD3 + / CD4 - IFN-γ + The proportion of cells.

[0073] 6. PBL cryopreservation and thawing steps used in some examples of the present invention

[0074] 1) D0, all the PBL that did not adhere to the wall after separating DC were transferred into a medium-sized bottle, supplemented with 10% normal serum-1640 culture medium and incubated overnight; D1, PBL were collected, centrifuged, and the cell pellet was placed in a cell freezing tube, and cell freezing medium (New Cymer) (4×10 7 cells / mL) were frozen at -80°C; thawed after 5 days and cultured overnight in 10% normal serum-1640 culture medium;

[0075] 2) D7, collect PBL, spin at 1200 rpm for 12 min, discard the supernatant, resuspend PBL in PBS, and adjust the cell concentration to 3×10 7 cells / mL, 1mL / tube (some PBLs are kept here without CFSE pre-staining and used for flow cytometry compensation);

[0076] By collecting PBMC samples from 30 blood donation volunteers, DC-peptide-PBL co-culture experiments were repeatedly performed on candidate epitope peptides, and the secretion of IFN-γ by CD4 + T cell ratio, verify the immunogenicity of each single peptide. Through the above steps, this example mainly involves 3 verified positive epitope peptides.

[0077]

[0078] Table 3

[0079] Table 3 shows the sequence information of the three novel coronavirus HLA-DRB1 restricted T cell epitope peptides obtained in the above examples: In the DC-peptide-PBL co-culture experiment, these T cell epitope peptides can stimulate the resting CD4 + T cells activate and secrete IFN-γ (IFN-γ + / CD4 + The T cell frequency was increased by more than 100% compared with the negative control group).

[0080]

[0081] Table 4

[0082] Table 4 shows the restriction of HLA molecules, comprehensive database predictions and DC-T experimental results of the three new coronavirus T cell epitope peptides obtained in the above examples.

[0083] 7. Immunize HLA transgenic mice with epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0084] 20 epitope peptides restricted by HLA-A0201 molecules (including epitope peptides No. 1, No. 2 and No. 3 in this example) were made into mixed polypeptides and then mixed with adjuvant poly I:C. HLA-A0201 / DR0101 transgenic mice were randomly divided into 2 groups, 3 mice in each group. The experimental group was vaccinated with peptide cocktail / poly(I:C) (epitope peptide: 10μg / single peptide, poly(I:C): 100μg / mouse) on day 0, day 7, and day 21, respectively; the adjuvant group was also vaccinated with poly(I:C) (100μg /

[0085] Subcutaneous injections were given at four points (neck back, tail base, and bilateral groins).

[0086] On the 28th day, mice were killed and spleen cell suspension was routinely prepared. The cell concentration was adjusted to 1×10 7 / mL, inoculated into a 48-well cell culture plate (0.5mL / well), each mouse spleen cell inoculation well was divided into 21 groups, and PBS (negative control) and each positive epitope peptide (including the three epitope peptides in this example, 20μg / mL / peptide) were added respectively; after culturing for 10 hours, BFA and Monensin were added to each well and incubated for another 6h; then the cells in each well were collected, blocked with FcR Blocking Reagent mouse for 15 minutes, and then co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30min; after washing, each tube was treated with a membrane permeabilizing agent, and finally APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30min; after washing, CD3 was analyzed by flow cytometry + / CD8 + / IFN-γ+T cell frequency, observe the specific CD8 induced by epitope peptide + The strength of the T cell response.

[0087] The results showed that the three positive epitope peptides (peptide cocktail / polyI:C group) in this example all induced strong epitope peptide-specific CD8 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these three epitope peptides + / CD8 + The T cell frequency was 12.9-34.7 times that of the adjuvant group.

[0088] The sequences involved in this application include:

[0089] Seq1

[0090] Gln Ser Leu Leu Ile Val Asn Asn Ala Thr Asn Val Val Ile Lys Val

[0091] QSLLIVNNATNVVIKV Seq1

[0092] Asp Gly Val Pro Phe Val Val Ser Thr Gly Tyr His Phe Arg Glu Leu

[0093] DGVPFVVSTGYHFREL Seq3

[0094] Pro Val Thr Leu Ala Cys Phe Val Leu Ala Ala Val Tyr Arg Ile Asn

[0095] PVTLACFVLAAVYRI

[0096] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0097] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. An epitope peptide, characterized in that: Its amino acid sequence is: Any one of SEQ ID NOs: 1 to 3; Alternatively, an amino acid sequence obtained by replacing, deleting, inserting and / or adding one or more amino acid sequences in SEQ ID NOs: 1 to 3.

2. A nucleic acid molecule, characterized in that Capable of encoding the epitope peptide according to claim 1.

3. A recombinant vector, characterized in that: Containing the nucleic acid molecule according to claim 2.

4. The recombinant vector according to claim 1, characterized in that The recombinant vector includes: a recombinant DNA gene segment, a recombinant plasmid, a recombinant virus vector or a recombinant mRNA.

5. A vaccine, characterized in that Its effective ingredients include: the epitope peptide according to claim 1 or the nucleic acid molecule encoding the nucleic acid according to claim 2 or the recombinant vector according to claim 3 or 4.

6. An adoptive immunotherapy cell, characterized in that: Capable of recognizing the epitope peptide described in claim 1 and being activated thereby.

7. The adoptive immunotherapy cell according to claim 6, characterized in that The method comprises stimulating effector T cells obtained by peripheral blood lymphocytes using the epitope peptide according to claim 1 or antigen presenting cells that present the epitope peptide to HLA molecules.

8. A method for obtaining effector T cells, characterized in that: The following steps are involved: PBMCs were isolated by density gradient centrifugation; The epitope peptide according to claim 1 is co-cultured with PBMCs, or the epitope peptide according to claim 1 is added to an antigen presenting cell culture medium to present the epitope peptide to HLA, and then co-cultured with PBMCs to obtain the effector T cells.

9. An application, characterized in that, Including any of the following: Use of the epitope peptide according to claim 1 in the preparation of SARS-CoV-2 vaccines or effector T cells; Use of the epitope peptide according to claim 1 in the preparation of a reagent or kit for detecting SARS-CoV-2 specific T cells; Use of the adoptive immunotherapy cells described in claim 6 or 7 in the preparation of drugs for treating SARS-CoV-2.

10. The use according to claim 9, characterized in that: The detection reagent is an enzyme-linked immunosorbent assay reagent, an intracellular cytokine fluorescent staining reagent, a chemiluminescent assay reagent, an enzyme-linked immunosorbent assay reagent, a human leukocyte antigen polymer fluorescent staining or a flow cytometric analysis reagent.

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