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

Abstract

The application discloses SARS-CoV-2 lymphocyte antigen epitope peptides and application thereof, and belongs to the field of medical immunology and infectious diseases, and particularly relates to three kinds of thymus-dependent lymphocyte antigen epitope peptides of SARS-CoV-2. An epitope peptide is characterized in that an amino acid sequence thereof is 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 an HLA-DR molecule, stimulate SARS-CoV-2-specific thymus-dependent lymphocyte activation, proliferation and differentiation, thereby exerting an immune effect on SARS-CoV-2 infection; the antigen peptides can be used to prepare a mixed polypeptide vaccine, a recombinant protein vaccine with multiple epitope peptides in series, and DNA and RNA vaccines of SARS-CoV-2, can be used to prepare a detection kit for detecting SARS-CoV-2-specific thymus-dependent lymphocytes, and can be used to prepare effector thymus-dependent lymphocytes or drugs 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

Technical Field

[0001] This invention relates to the fields of medical immunology and infectious diseases, specifically to a SARS-CoV-2 lymphocyte antigen epitope peptide and its application. Background Technology

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

[0003] Currently, there are still very few SARS-CoV-2 antigenic epitope peptides that can stimulate T cell responses, which limits the monitoring and analysis of specific T cell immune function in infected individuals carrying SARS-CoV-2. It also limits the study of immunopathology and immune protection mechanisms of specific T cell immune responses in the interaction between SARS-CoV-2 and the host, and further limits the development of vaccines and therapeutic agents that induce virus-specific T cell responses in the body. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention proposes a SARS-CoV-2 lymphocyte antigen epitope peptide and its application.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A first aspect of the present invention relates to an epitope peptide having the following amino acid sequence:

[0007] As shown in any of SEQ ID NO: 1 to 3;

[0008] Alternatively, an amino acid sequence obtained by substituting, deleting, inserting, and / or adding one or more amino acid sequences in SEQ ID NO:1–3.

[0009] A second aspect of the invention relates to a nucleic acid molecule capable of encoding the aforementioned epitope peptide.

[0010] A third aspect of the present invention relates to a recombinant vector containing the aforementioned nucleic acid molecules.

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

[0012] A fourth aspect of the present invention relates to a vaccine whose active ingredient comprises: the epitope peptide described above or encoding the nucleic acid molecule described above or the recombinant vector described above.

[0013] A fifth aspect of the invention relates to an adoptive immunotherapy cell capable of recognizing and being activated by the aforementioned epitope peptide.

[0014] Optionally, this includes effector T cells obtained by stimulating peripheral blood lymphocytes with the epitope peptide described above or by antigen-presenting cells that present the epitope peptide 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 separated using density gradient centrifugation.

[0017] The above-mentioned epitope peptides are co-cultured with PBMCs, or the above-mentioned epitope peptides are added to the antigen-presenting cell culture medium to present the epitope peptides 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] The application of the above-mentioned epitope peptides in the preparation of SARS-CoV-2 vaccines or effector T cells;

[0020] The application of the above-mentioned epitope peptides in the preparation of reagents or kits for detecting SARS-CoV-2 specific T cells;

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

[0022] Optionally, the detection reagents are enzyme-linked immunospot assay reagents, intracellular cytokine fluorescence staining reagents, chemiluminescence assay reagents, enzyme-linked immunosorbent assay reagents, human leukocyte antigen multimer fluorescence staining reagents, or flow cytometry reagents.

[0023] The epitope peptide sequences described above can be used to prepare mixed polypeptide vaccines, recombinant protein vaccines, or recombinant gene vaccines for COVID-19 pneumonia that induce specific T-cell immune responses in the body.

[0024] Preparation of mixed peptide vaccines: Multiple antigenic peptides are artificially synthesized according to the antigenic epitope peptide sequence of the present invention, and mixed with adjuvants to form a soluble preparation, or loaded with bio-nanomaterials to form a nanopeptide vaccine. This vaccine is injected into healthy individuals, SARS-CoV-2 infected individuals, or patients with pneumonia caused by SARS-CoV-2 interference. It stimulates 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 individuals from being infected with SARS-CoV-2, or enhancing the ability of infected individuals to inhibit viral replication and proliferation and resist reinfection, thus preparing a SARS-CoV-2 T cell epitope mixed peptide vaccine.

[0025] Preparation of recombinant protein vaccine: According to the antigenic epitope peptide sequence of the present invention, a recombinant plasmid with multiple epitope peptides in series is constructed, expressed in prokaryotes or eukaryotes, and then the recombinant protein is purified and mixed with adjuvant to prepare a soluble formulation. This formulation is injected into healthy individuals, SARS-CoV-2 infected individuals, or patients with pneumonia caused by SARS-CoV-2 infection. It stimulates 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 individuals from being infected with SARS-CoV-2, or enhancing the ability of infected individuals to inhibit viral replication and proliferation and resist reinfection, thus preparing a recombinant protein vaccine for SARS-CoV-2.

[0026] Preparation of recombinant gene vaccine: According to the polypeptide sequence of the present invention, a recombinant DNA gene fragment, recombinant plasmid, recombinant viral vector or recombinant mRNA with multiple epitope sequences in tandem is constructed and injected into healthy individuals, SARS-CoV-2 infected individuals or patients with pneumonia caused by SARS-CoV-2 infection. This stimulates 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 individuals from being infected with SARS-CoV-2 or enhancing the ability of infected individuals to inhibit viral replication and proliferation and resist reinfection, thereby preparing a recombinant gene vaccine for SARS-CoV-2.

[0027] Furthermore, the aforementioned lymphocyte antigenic epitope peptide sequences can be used to prepare detection agents or kits for detecting SARS-CoV-2 antigen-specific T cells. According to the antigenic epitope peptide sequences of this invention, various antigenic peptides are artificially synthesized and used as antigen stimulants in enzyme-linked immunospot assays, intracellular cytokine fluorescence staining, chemiluminescence assays, and enzyme-linked immunosorbent assays. These peptides are co-cultured with peripheral blood mononuclear cells (PBMCs) from patients to stimulate the activation, proliferation, and secretion of cytokines by SARS-CoV-2 antigen-specific T cells. The amount of cytokine secretion is then detected using other combined reagents, thereby reflecting the number and activity of specific T cells. Alternatively, these peptide sequences can be used to prepare human leukocyte antigen-peptide complexes and their polymers using genetic engineering and protein engineering techniques. These polymers can then be further prepared into fluorescein- or metal-labeled preparations, and the number of SARS-CoV-2 antigen-specific T cells in the peripheral blood PBMC cell population can be analyzed using flow cytometry. Related kits refer to SARS-CoV-2 specific T cell detection kits assembled from the aforementioned preparations and other conventional reagents used in different detection methods.

[0028] The epitope peptide sequences described above can be used to prepare drugs for treating SARS-CoV-2: by combining the above-mentioned antigen epitope peptide sequences with other immunotherapeutic agents or chemotherapeutic agents, a clinical drug for treating SARS-CoV-2 can be prepared.

[0029] The beneficial effects of this invention are:

[0030] This invention presents a set of SARS-CoV-2 viral protein T-cell epitope peptides obtained through online virtual prediction and functional experimental verification. These novel antigenic epitope peptide sequences will provide key antigenic components, namely antigenic epitope peptide sequences, for the development of prophylactic and therapeutic peptide vaccines, recombinant protein vaccines, and gene vaccines against SARS-CoV-2 virus, as well as for the design of reagents and methods for detecting SARS-CoV-2 virus antigen-specific T cells. Attached Figure Description

[0031] The invention will now be further described with reference to the accompanying drawings.

[0032] Figure 1 This is a flowchart illustrating the experimental technique for verifying the immunogenicity of epitope peptides to be identified in some examples of this invention.

[0033] Figure 2This is a flow cytometry analysis of the DC-peptide-PBL co-culture experiment of three immunogenic SARS-CoV-2 viral protein T cell epitope peptides in this invention. After co-culturing the T cell epitope peptides with donor DC cells and donor PBLs for 14 days, the cells were co-stained with CD3 and CD4 fluorescent monoclonal antibodies, and intracellularly stained with IFN-γ fluorescent monoclonal antibody. Then, IFN-γ was detected and analyzed by flow cytometry. + / CD4 + T cells in CD4 + Frequency in the T cell population.

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

[0035] A mixed peptide vaccine was prepared by using 20 HLA-A0201-restricted positive epitope peptides and combined with the adjuvant poly(I:C). HLA-A2 / DR1 transgenic mice were immunized on days 0, 7, and 21. Seven days after the last immunization, mouse spleen cells were harvested and cultured with PBS and each positive epitope peptide for 10 hours, followed by BFA / Monesion (cytokine release inhibitor) for another 6 hours. IFN-γ intracellular staining and flow cytometry were then performed to detect IFN-γ. + / CD3 + / CD4 + The frequency of T cells. Figure 3 A in the graph represents a representative flow cytometry analysis plot; Figure 3 In this context, B represents the IFN-γ of each group of mice. + / CD3 + / CD4 + Frequency statistics of T cells. Detailed Implementation

[0036] The technical solutions of the various examples of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described examples are only a part of the examples of this invention, and not all of them. Based on the various examples of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention. The project involved in this application is supported by the National Key Research and Development Program of China, "Research on Pathogen and Epidemic Prevention Technology System" (2022YFC2304103).

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

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

[0039] The S, E, M, N, and R proteins of SARS-CoV-2 were selected, and then epitope peptide prediction tools such as SYFPEITHI, EPIJEN, ConvMHC, and IEBB (including ANN, Consensus, NetMHCpan, SMM, and SMMPMBEC) were used to virtually predict T-cell epitope peptides targeting the S protein, which are molecularly restricted, such as DRB1:0901,1501,0701,1202,0803,1101,0301,0405,1302,1502,1602, and0406.

[0040] HLA class II molecules have open antigen-binding grooves at both ends, accepting antigen peptides with lengths of 13-25 amino acid residues, with 16 amino acids being the most common. Therefore, in some examples of this invention, peptides with a length of 16 amino acids are selected as research objects. 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. Then, the length of the epitope peptide is selected as 16 amino acids. Finally, 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 the SARS-CoV-2 viral S protein, the 16 peptides predicted by different databases were ranked from highest to lowest score. Epitope peptides that met the scoring criteria of at least two prediction methods were then selected as candidate epitope peptides. For each HLA-DRB1 molecule, the 1-6 peptides with the highest scores or the highest affinity were selected from the candidate epitope peptides as the epitope peptides to be identified. The sources of the epitope peptide prediction tools in the above examples are shown in Table 1.

[0042]

[0043]

[0044] Table 1

[0045] 2. Separation of peripheral blood mononuclear cells (PBMCs) from blood donors:

[0046] 1) Collect peripheral blood from blood donors at the blood center using a blood collection tray (a leukocyte filter tray used for sorting red blood cells in 400mL peripheral anticoagulated blood). Spray with alcohol and place in a clean bench. Carefully disinfect the inlet and outlet tubes with alcohol swabs. Cut open the inlet and outlet tubes. Draw PBS with a 50mL syringe and use the outlet of the blood collection tray as the syringe inlet. Collect the PBS rinsing solution at the inlet of the blood collection tray using 6 50mL centrifuge tubes. Collect a total of about 300mL of cell suspension.

[0047] 2) Collect the first 50 mL of blood flushed out in the first centrifuge tube without centrifuging; collect the remaining 250 mL of PBS evenly in the other 5 centrifuge tubes, centrifuge at 1500 rpm for 12 min at room temperature, transfer the turbid supernatant to a 50 mL centrifuge tube and centrifuge again at 1500 rpm for 12 min at room temperature, and collect all cell pellets; collect the cell pellets from the last 5 centrifuge tubes into a 50 mL centrifuge tube, add PBS to make up to 50 mL, and mix thoroughly with the 50 mL of blood from the first tube;

[0048] 3) Add 5 mL of human lymphocyte separation medium (Dake Biotech, Shenzhen) to each of 10 15 mL centrifuge tubes; slowly add 100 mL of the mixed leukocyte suspension along the tube wall, spreading it evenly above the surface of the separation medium, keeping the two interfaces clear; centrifuge at 20℃, 2000 rpm for 20 min, with an acceleration of 1 and a deceleration of 0;

[0049] 4) After centrifugation, use a pipette to move in the same plane and aspirate the entire milky white PBMC layer into a 50mL sterile centrifuge tube (about 30mL). Try not to aspirate the plasma layer above the white membrane layer. Then, divide the white membrane cell suspension equally into four 50mL centrifuge tubes. Add PBS to each tube to 50mL, mix thoroughly, and centrifuge at 1300rpm for 12min. Discard the supernatant, tap the cell pellet, mix the pellets together and divide them equally into two 50mL centrifuge tubes. Add PBS to each tube to 50mL, mix well, and centrifuge twice more at 1000rpm for 12min. Discard the supernatant, tap the cell pellet with your finger, add a small amount of serum-free 1640 culture medium (Dake Biotech, Shenzhen) to resuspend, collect and combine them into one 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 white blood cell diluent, then add 20 μL of cell suspension, mix thoroughly, and then perfuse the cell counting chamber. Count the cells under a microscope and calculate the cell concentration using the following formula: Total number of cells in 4 large squares / 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] Genomic DNA was extracted from the PBMCs isolated in the above examples using a human whole blood genomic DNA extraction kit (Tiangen Biotech, Beijing). PCR was performed using HLA-DRB1 site-specific primers DRB1-F and DRB1-R to amplify the DNA sequences of exon 2 and some introns 2 and 3 at the HLA-DRB1 site, with a product size of 285 bp. The amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 62℃ annealing for 15 s; 72℃ extension for 90 s; 35 cycles; 72℃ extension for 5 min. The amplified products were identified by 1% agarose gel electrophoresis, purified, and subjected to bidirectional sequencing. PCR reagents were purchased from Nanjing Novizan Biotechnology Co., Ltd. (see Table 2).

[0053]

[0054] Table 2

[0055] The sequencing results were assembled into a complete HLA-DRB1 contig using the Seqman software in the Lasergene program. The bidirectional sequencing bases were carefully checked for completeness and consistency. Heterozygous bases were identified and replaced with degenerate bases; for example, 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. This process ultimately determined the amplified HLA-DRB1 allele sequence. The Nucleotide BLAST tool was then used to align the assembled HLA-DRB1 base sequence with all HLA-DRB1 allele sequences in the database until a perfectly matching gene combination was found, thus identifying the HLA-DRB1 allele.

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

[0057] 1) The PBMCs resuspended in 20 mL of serum-free 1640 medium were seeded into cell culture flasks (1-1.5 × 10⁻⁶). 8 Cells / 20mL / medium-sized bottle, 1.5-3×10 8 (cells / 30mL / large bottle), incubate at 37℃ for about 4 hours;

[0058] 2) Shake the culture flask and blow it once with a pipette. Then transfer the non-adherent cells and culture medium into a medium-sized culture flask for PBL culture. Add ordinary serum to the final concentration of 10%, culture overnight, and freeze the PBL the next day for later use.

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

[0060] 4) On day 0, add human GM-CSF (PrepoTech) at a final concentration of 1000 IU / mL and human rIL-4 (PrepoTech) at 500 IU / mL, and culture at 37°C. On the evening of day 3, change 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 day 5, add LPS to stimulate DC maturation at 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 day 6, thaw the frozen PBL of the blood donor for later use. On day 7, harvest mature DCs (mDC): tap and pipette the culture flask, aspirate the culture medium into a 50 mL centrifuge tube, wash the culture flask with a small amount of serum-free culture medium, and then count the cells (count using the original solution, do not count very small lymphocytes).

[0061] 5) Analyze mDC phenotypes: Take 3×10 5 Cells were evenly divided into 6 flow cytometry tubes. After centrifugation, each tube was stained with one of 5 fluorescent monoclonal antibodies (CD83, CD80, CD86, DR, and ABC). The positive percentage of each marker was analyzed by flow cytometry.

[0062] 6) Centrifuge the remaining mDCs at 1200 rpm for 10 min, discard the supernatant, resuspend the cell pellet in serum-free 1640 medium, and adjust the mDC cell concentration to 0.5 × 10⁻⁶. 5 cells / mL, temporarily stored in an incubator;

[0063] 7) Observe the flow cytometry results and mDC ratio. Seed mDCs into 24-well plates at 50,000 cells / 1.0 mL / well. Then, add 10 μL of the single epitope peptide corresponding to the HLA-A molecule of the blood donor to each well (final concentration 20 μg / mL). Approximately 10 epitope peptides are verified from each blood sample. Three control wells are also set up: DC-T negative control well (no peptide added), PBL positive well (a small amount of PHA added), and PBL negative well (no stimulant added).

[0064] 5. Then perform intracellular IFN-γ fluorescence staining, the procedure of which is as follows:

[0065] 1) On day 21, cells from each DC-peptide-PBL co-culture well were collected into a sterile EP tube, and serum-free culture medium was added to about 1.5 mL. The tube was centrifuged at 1200 rpm for 10 min, and the supernatant was discarded. The cell pellet was sponged up and resuspended in 0.5 mL of serum-free culture medium (Dakow) and seeded into 48-well plates.

[0066] 2) Add 10 μL of the corresponding single peptide to each well (final concentration 20 μg / mL), and set up wells without peptide stimulation and wells with PHA stimulation to serve as negative and positive stimulation controls for the cell samples, respectively; incubate in a cell culture incubator for 16 h;

[0067] 3) Add 2 μL each of BFA and 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, incubate at 4℃ for 20 min, mixing once during the incubation period;

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

[0070] 6) Add 100 μL of 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 300-350g for 5 min, discard the supernatant, and bounce the cell pellet.

[0071] 7) Add 100 μL of MEDIUM B (eBiosciences) to each tube and 20 μL of PE-anti-IFN-γ (eBiosciences) to each experimental tube. Do not add PE-anti-IFN-γ to unstained tubes. Incubate each tube at 4℃ for 30 min, mixing once during the incubation period.

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

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

[0074] 1) D0: Transfer all non-adherent PBLs after DC separation to a medium-sized flask, add 10% ordinary serum-1640 culture medium, and incubate overnight; D1: Collect PBLs, centrifuge, place the cell pellet in a cell cryopreservation tube, and add cell cryopreservation medium (New Serum) (4×10⁻⁶). 7 (cells / mL) were stored at -80°C; after 5 days, they were thawed and cultured overnight in 10% ordinary serum-1640 medium.

[0075] 2) On day 7, collect PBL at 1200 rpm for 12 min, discard the supernatant, resuspend PBL thoroughly in PBS, and adjust the cell concentration to 3 × 10⁻⁶ cells / mL. 7 cells / mL, 1mL / tube (a portion of PBL is reserved here without CFSE pre-staining, to be used for flow cytometry adjustment and compensation);

[0076] By collecting PBMC samples from 30 blood donors, and repeatedly performing DC-peptide-PBL co-culture experiments on candidate epitope peptides, the secretion of IFN-γ by CD4 was detected. + The proportion of T cells was measured, and the immunogenicity of each single peptide was verified. Through the above steps, this embodiment mainly involves three verified positive epitope peptides.

[0077]

[0078] Table 3

[0079] Table 3 shows the sequence information of three SARS-CoV-2 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 could stimulate quiescent CD4+ in donor PBMCs. + T cells are activated and secrete IFN-γ (IFN-γ). + / CD4 + T cell frequency was more than 100% higher than in the negative control group.

[0080]

[0081] Table 4

[0082] Table 4 shows the HLA molecular restrictions of the three SARS-CoV-2 T-cell epitope peptides obtained in the above examples, based on comprehensive database predictions and DC-T experimental results.

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

[0084] Twenty HLA-A0201-restricted epitope peptides (including epitope peptides 1, 2, and 3 in this embodiment) were prepared into a mixed polypeptide and then mixed with the adjuvant poly I:C. HLA-A0201 / DR0101 transgenic mice were randomly divided into two groups of three mice each. The experimental group was inoculated with peptide cocktail / poly(I:C) (epitaxe: 10 μg / peptide, poly(I:C): 100 μg / mouse) on days 0, 7, and 21, respectively; the adjuvant group was similarly inoculated with poly(I:C) (100 μg / mouse) on days 0, 7, and 21, respectively.

[0085] (Only one). Administer subcutaneously at four points (back of the neck, base of the tail, and bilateral groins).

[0086] On day 28, the mice were euthanized, and a spleen cell suspension was prepared using standard procedures. The cell concentration was adjusted to 1×10⁶ cells / mL using 10% FBS-1640 medium. 7 0.5 mL / well was seeded into 48-well cell culture plates. Each mouse spleen cell seeding well was divided into 21 groups, each containing PBS (negative control) and 20 μg / mL / peptide of each positive epitope peptide (including the three epitope peptides in this example). After 10 hours of culture, BFA and Monensin were added to each well, and the cells were incubated for another 6 hours. Cells from each well were then collected, blocked with FcR Blocking Reagent mouse for 15 minutes, and co-stained with FITC-anti-mouse CD3 and PE-anti-mouse CD8a for 30 minutes. After washing, a cell membrane-permeable agent was added to each tube, and finally, APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30 minutes. After washing, CD3 was analyzed by flow cytometry. + / CD8 + / IFN-γ+ T cell frequency, observation of epitope peptide-induced specific CD8 + The strength of the T cell response.

[0087] The results showed that all three positive epitope peptides (peptide cocktail / polyI:C group) in this embodiment induced robust epitope peptide-specific CD8 in mice. + T cell response. Compared to the adjuvant group, these three epitope peptides induced IFN-γ... + / 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, references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0097] The foregoing has shown and described 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. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention.

Claims

1. A combination of epitope peptides, characterized in that, include: The amino acid sequences are epitope peptides of SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:

3.

2. A nucleic acid molecule, characterized in that, It is capable of encoding the epitope peptide combination of claim 1.

3. A recombinant vector, characterized in that, It contains the nucleic acid molecule as described in claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector includes: recombinant DNA gene fragments, recombinant plasmids, recombinant viral vectors, or recombinant mRNA.

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

6. An adoptive immunotherapy cell, characterized in that, The epitope peptide combination of claim 1 is identified and activated thereon.

7. The adoptive immunotherapy cells according to claim 6, characterized in that, This includes effector T cells obtained by stimulating peripheral blood lymphocytes with the epitope peptide combination of claim 1 or by presenting the epitope peptide combination to an HLA molecule.

8. An application characterized in that, Including any of the following aspects: The epitope peptide combination of claim 1 is used in the preparation of SARS-CoV-2 vaccines or effector T cells, wherein the epitope peptide combination can simultaneously induce in vitro CD4+ T cell responses and in vivo CD8+ T cell responses. The use of the epitope peptide combination described in claim 1 in the preparation of detection reagents or kits for detecting SARS-CoV-2 specific T cells.

9. The application according to claim 8, characterized in that, The detection reagents are enzyme-linked immunospot assay reagents, intracellular cytokine fluorescence staining reagents, chemiluminescence assay reagents, enzyme-linked immunosorbent assay reagents, human leukocyte antigen multimer fluorescence staining reagents, or flow cytometry assay reagents.

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