HLA-B restrictive SARS-CoV-2 lymphocyte antigen epitope peptide and application thereof

Abstract

The invention discloses an HLA-B restrictive 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 amino acid sequence of the epitope peptide is shown as any one of SEQ ID NO: 1-3, and the amino acid sequence of the epitope peptide is shown as any one of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6. Or an amino acid sequence obtained by substituting, deleting, inserting and / or adding one or more amino acid sequences in any sequence of SEQ ID NO: 1-3. The antigen epitope peptide can be presented by an HLA-B4601 molecule and an HLA-B4001 molecule, and is used for stimulating the 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 an HLA-B restricted 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 any one of 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, 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 novel 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 polypeptide 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] A group of SARS-CoV-2 virus protein T cell epitope peptides obtained through online virtual prediction and functional experimental verification have not been reported before. Therefore, these new antigen epitope peptide sequences will provide the key antigen components, namely, antigen epitope peptide sequences, required for the development of preventive and therapeutic peptide vaccines, recombinant protein vaccines and gene vaccines against SARS-CoV-2 virus, and 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 2 Epitope peptides stimulate CD8 in peptide-PBMCs co-culture experiments + Flow cytometry detection of T cell activation; Figure 1 In the table, A is the CD3 of each recovered COVID-19 patient.+ / CD8 + The flow cytometry scatter plot of IFN-γ secretion in the T cell population after being stimulated by a specific epitope peptide; B is the CD3 + / CD8 + Statistical graph of the proportion of cells in the T cell population that secrete IFN-γ after being stimulated by specific epitope peptides.

[0034] Figure 3 Peptide competition binding assay to analyze the binding affinity of three SAES-CoV-2 T cell epitopes to specific HLA-B molecules. NC, negative control; Epitope peptide 1; Epitope peptide 2; Epitope peptide 3; The blue solid line and red solid line show the cell fluorescence peaks at 5μM and 15μM concentrations of the tested peptide, respectively, while the gray filled line represents the maximum fluorescence intensity of the cells when there is only APC-reference peptide. The light gray solid line in the NC graph represents the negative control background fluorescence of the cells without peptide wells. DETAILED DESCRIPTION

[0035] The project involved in the technical solution of this application is funded by the key project "Research on Pathogens and Epidemic Prevention Technology System" of the National Key R&D Program (2022YFC2304103).

[0036] 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:

[0037] 1 Virtual prediction of T cell epitope peptides restricted by HLA-B molecules from multiple proteins of SARS-CoV-2

[0038] The S, E, M, N, and R proteins of SARS-CoV-2 were selected, and then the T cell epitope peptides targeting S, E, M, N, and R proteins with molecular restrictions such as HLA-B: 4601, 4001, 5801, 1502, 5101, 1301, 1302, 1501, 3501, 4403, 5201, 4801, 0702 were virtually predicted using epitope peptide prediction tools such as SYFPEITHI, EPIJEN, ConvMHC, and IEDB (including ANN, Consensus, NetMHCpan, SMM, and SMMPMBEC).

[0039] The antigen binding groove of the HLA class I molecule is closed at both ends, and the accepted antigen peptide length is 8-11 amino acid residues, among which 9 and 10 amino acids are the most common. Therefore, in some examples of the present invention, polypeptides with a length of 9 and 10 amino acids are selected as research objects, and the amino acid sequence of each SARS-CoV-2 virus S, E, M, N, and R 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 9 and 10 amino acids, respectively, and then a specific HLA-B molecule is selected to perform online virtual prediction of the T cell epitope peptides of the SARS-CoV-2 virus S, E, M, N, and R proteins.

[0040] For each HLA-B molecule and SARS-CoV-2 virus S, E, M, N, R protein, the 9 and 10 peptides predicted by different databases were ranked from high to low according to the scores, and then the epitope peptides that met the scoring criteria of at least two prediction methods were selected as candidate epitope peptides. For each HLA-B molecule, 1-6 peptides with the highest scores or the highest affinity were selected from the candidate epitope peptides as epitope peptides to be identified.

[0041] Table 1 Epitope peptide prediction database website

[0042]

[0043] 2Isolation of PBMC from peripheral blood of recovered COVID-19 patients

[0044] Take fresh anticoagulated whole blood stored at room temperature and dilute it appropriately with sterile PBS; add 1 times the volume of original blood human lymphocyte separation solution (Dakoway Biotechnology, Shenzhen) to a 15 mL centrifuge tube; slowly spread the diluted blood on the separation solution and centrifuge at room temperature for 20 min at 2000 rpm; aspirate the mononuclear cell (PBMCs) layer and wash it twice by centrifugation; resuspend it with serum-free medium (Dakoway Biotechnology, Shenzhen), count the cells, and adjust the cell concentration to 3×10 6 / mL for future use.

[0045] 3 Identification of the immunogenicity of SARS-CoV-2 T cell epitope peptides using IFN-γICS method

[0046] Technical route such as Figure 1 As shown:

[0047] 4HLA-B allele typing

[0048] 200 μL of anticoagulated blood was collected from the patient, and genomic DNA was extracted using a human whole blood genomic DNA extraction kit (Tiangen Biotechnology, Beijing). HLA-B locus-specific primers B1 (SEQ ID NO.4) and B3 (SEQ ID NO.5) were used for PCR to amplify the DNA sequences of exon 2, intron 2, exon 3, and part of introns 1 and 3 of the B locus, with a product size of 985 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 and sent to Shanghai Sunny Biotechnology Co., Ltd. for purification and bidirectional sequencing. PCR reagents were purchased from Nanjing Novozyme Biotechnology Co., Ltd.

[0049] Table 2 HLA-B locus-specific PCR amplification primers

[0050]

[0051] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-B overlapping sequence (Contig) by using the Seqman software of the Lasergene program. 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-B alleles were determined. The Nucleotide BLAST tool was used to compare the assembled HLA-B base sequence with the exon 2 and exon 3 sequences of all HLA-B alleles in the database until a completely matching gene combination was obtained, thereby determining the HLA-B alleles.

[0052] 5. Peptide-PBMCs co-culture and IFN-γ intracellular staining experiment

[0053] PBMCs were separated by density gradient centrifugation and the concentration of PBMCs was adjusted to 3 × 10 6 / mL, inoculated into a U-bottom 96-well plate, 100 μL / well, and placed at 37°C, 5% CO 2 The constant temperature incubator was set aside. According to the HLA-B allele typing results of the recovered COVID-19 patients, the corresponding candidate epitope peptides were co-cultured with the PBMCs in the above 96-well plate for 6 hours, and the Brefeldin A Solution / Monensin Solution (1000×) blocker was added for co-culture, and then IFN-γ intracellular fluorescence staining was performed, and flow cytometry was used to detect the CD8 secretion of IFN-γ. +T cell frequency; at the same time, a single culture well of PBMCs without peptide stimulation was set up as a negative control group. Compared with the negative control, IFN-γ + / CD8 + The candidate epitope peptides with a T cell frequency increased by more than 2 times were set as positive epitope peptides, that is, the epitope peptides could stimulate the memory CD8 in PBMCs of recovered COVID-19 patients. + T cells become activated and secrete cytokines.

[0054] Through the IFN-γICS experiment, PBMCs from 30 recovered COVID-19 patients were collected and co-cultured with related peptides from 95 candidate epitope peptides. Among them, three candidate epitope peptides (sequences were FVLAAVYRI, FSKQLQQSM, and AESHVDTDL) induced strong CD8 + T cell response caused IFN-γ to appear in PBMCs of at least 8 recovered COVID-19 patients + / CD8 + The T cell frequency increased by more than 10 times, so the three candidate epitope peptides were determined to be positive epitopes and dominant epitope peptides with strong immunogenicity ( Figure 2 ), through HLA-B genotype sequencing of positive population, it was found that the three epitope peptides were all presented by HLA-B4601 and HLA-B4001 molecules.

[0055] 6. Peptide competition binding assay to analyze the affinity of candidate epitope peptides to corresponding HLA-B molecules

[0056] The 67 candidate epitopes, including the three positive epitope peptides No. 1, No. 2, and No. 3 screened in the above steps and verified by the peptide-PBMCs costimulation experiment, were assigned to the relevant epitope peptide queues of each HLA-B molecule according to the predicted HLA-B restriction and the HLA-B allele type of the patient with positive T cell response. As the peptides to be tested, the peptide competition binding experiment was carried out with the fluorescent reference peptide of the HLA-B molecule. Finally, the three positive peptides all shifted the cell fluorescence peak to the left ( Figure 2 ), indicating that these positive epitope peptides can compete with the fluorescent reference peptides for binding to the relevant HLA-B molecules on the HMy2.CIR cell membrane. These three positive epitope peptides all showed high or medium affinity binding to the corresponding HLA-B molecules, especially HLA-B4601 and HLA-B4001 molecules (Table 3).

[0057] Table 3 Peptide competition binding assay to analyze the binding affinity of three positive T cell epitopes to 10 dominant HLA-B molecules

[0058]

[0059]

[0060] The sequences involved in the examples of this application include:

[0061] SEQ ID NO.1

[0062] Phe Val Leu Ala Ala Val Tyr Arg Ile

[0063] SEQ ID NO.2

[0064] Phe Ser Lys Gln Leu Gln Gln Ser Met

[0065] SEQ ID NO.3

[0066] Ala Glu Ser His Val Asp Thr Asp Leu

[0067] 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 by the above examples, and the above examples and descriptions are only for illustrating 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 claimed for protection.

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 any one of 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, the nucleic acid molecule encoding the nucleic acid molecule 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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