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

Abstract

The invention particularly relates to an SARS-CoV-2 lymphocyte antigen epitope peptide and application thereof, and belongs to the field of medical immunology and infectious pathology. The amino acid sequence of the epitope peptide is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 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-DRB1 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 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 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 vaccine: According to the epitope peptide 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 a healthy person, a SARS-CoV-2 infected person or a patient 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 2 Epitope peptides stimulate CD4 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. + / CD4 + 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 + / CD4 + 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 Epitope peptides induced specific CD4 in HLA-A2 / DR1 transgenic mice + T cell response A mixed peptide vaccine was prepared by combining 20 HLA-DRB10101 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

[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments 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).

[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 disclosed, and the specific steps are as follows:

[0037] 1. Virtual prediction of T cell epitope peptides restricted by HLA-DRB1 molecules from multiple SARS-CoV-2 proteins

[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 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).

[0039] 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, 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 16 amino acids, and then a specific HLA-DRB1 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-DRB1 molecule and SARS-CoV-2 virus S, E, M, N, R protein, the 16 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-DRB1 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-DRB1 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-DRB1 locus-specific primers DRB1-F and DRB1-R 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 DRB1 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-DRB1 locus-specific PCR amplification primers

[0050]

[0051] The sequencing results of exon 2 and exon 3 were assembled into a complete HLA-DRB1 overlapping sequence (Contig) by using the Seqman software of the Lasergene program. The bases of the bidirectional sequencing were carefully checked to be 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 exon 2 and exon 3 sequences of all HLA-DRB1 alleles in the database until a completely matched gene combination was obtained, thereby determining the HLA-DRB1 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 in a 37°C, 5% CO2 constant temperature incubator for standby use. According to the HLA-DRB1 allele typing results of recovered COVID-19 patients, the corresponding candidate epitope peptides were co-cultured with PBMCs in the above 96-well plate for 6 hours, and 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 CD4 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 + / CD4 + 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 CD4 in PBMCs of recovered COVID-19 patients. + T cells become activated and secrete cytokines.

[0054] Through the IFN-γICS experiment, PBMCs from 60 recovered COVID-19 patients were collected and co-cultured with related peptides from 95 candidate epitope peptides. Among them, three candidate epitope peptides (denoted as: No. 1, No. 2, No. 3, with sequences of KPSFYVYSRVKNLNSS, MWSFNPETNILLNVPL, and SASAFFGMSRIGMEVT, respectively) induced strong CD4 + T cell response caused IFN-γ to appear in PBMCs of at least 8 recovered COVID-19 patients + / CD4 + 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 ).

[0055] 6. Immunize HLA transgenic mice with epitope peptides to verify their immunogenicity in inducing specific T cell responses in vivo

[0056] 20 epitope peptides restricted by HLA-DRB10101 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 / mouse) on day 0, day 7, and day 21, respectively. Four subcutaneous injections were made (neck back, tail base, bilateral groins).

[0057] 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 CD4 for 30min; after washing, each tube was treated with a membrane permeabilizer, and finally APC-anti-mouse IFN-γ monoclonal antibody was added for intracellular staining for 30min; after washing, CD3 was analyzed by flow cytometry + / CD4 + / IFN-γ+T cell frequency, observe the specific CD4 + The strength of the T cell response.

[0058] The results showed that the three positive epitope peptides (peptide cocktail / polyI:C group) in this example all induced strong epitope peptide-specific CD4 + T cell response. Compared with the adjuvant group, the IFN-γ induced by these three epitope peptides + / CD4 + The T cell frequency was 12.9-34.7 times that of the adjuvant group.

[0059] The sequences involved in this application include:

[0060] SEQ ID NO:1

[0061] Lys Pro Ser Phe Tyr Val Tyr Ser Arg Val Lys Asn Leu Asn Ser Ser

[0062] KPSFYVYSRVKNLNSS

[0063] SEQ ID NO:2

[0064] Met Trp Ser Phe Asn Pro Glu Thr Asn Ile Leu Leu Asn Val Pro Leu

[0065] MWSFNPETNILLNVPL

[0066] SEQ ID NO:3

[0067] Ser Ala Ser Ala Phe Phe Gly Met Ser Arg Ile Gly Met Glu Val Thr

[0068] SASAFFGMSRIGMEVT

[0069] 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 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.

Images