Novel coronavirus broad-spectrum immunodominance CD4 + T cell epitope peptide and application thereof

Abstract

The invention relates to the technical field of biomedicine, and discloses a novel coronavirus broad-spectrum immunodominance CD4 + T cell epitope peptide, which comprises a common Th epitope peptide aiming at a novel coronavirus original strain and a variant thereof, and also comprises a specific immunodominance mutation Th epitope peptide aiming at an Orsecret flannel variant. The invention also discloses a Th epitope peptide-MHC tetramer and application thereof. The epitope peptide obtained by the invention accords with the rule that HLA-II molecular binding peptides are presented in a natural state, the detection rate of Th epitopes is high, and the economic cost can be reduced by 1 / 4; the Th epitope peptide-MHC tetramer prepared by utilizing the epitope peptide disclosed by the invention has a strong effect of stimulating CD4 + T cell activation; the epitope peptide and the Th epitope peptide-MHC tetramer provided by the invention can be made into a kit.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology and relates to a novel coronavirus with broad-spectrum immune advantage CD4 + T cell epitope peptides and their applications. Background Art

[0002] The epidemic (COVID-19) caused by the new coronavirus (SARS-CoV-2) infection is still raging around the world. Under the pressure of host immunity, SARS-CoV-2 continues to mutate, and the Omicron variant is currently prevalent and dominant in most countries around the world. The transmission rate, severity and mortality of these new variants in the elderly population are enhanced compared to previous mutants. As of February 2024, the new coronavirus infection has caused more than 773 million cases of COVID-19 and more than 6 million deaths. Most people in the world have been vaccinated and experienced the first wave of infection to form "herd immunity", but the Omicron variant is still repeatedly infected. At present, its immune escape mechanism is not clear. In-depth exploration of its mechanism can provide new means for the design of new vaccines.

[0003] It has been confirmed that CD4 + T cells can accelerate cytotoxic CD8 + T cell proliferation, maintaining CD8 + T cell memory can interact and interact with innate immune cells. They can also promote B cells to differentiate into plasma cells, produce neutralizing antibodies and promote memory B cells to produce rapid recall responses. + T cells can kill infected cells directly or act as antitoxins by secreting cytokines such as IFN-γ and TNF-α. + T cells play an important role in antiviral immunity. Human HLA is a class of immune molecules whose main function is to present specific epitope peptides to TCR and activate T cells. The human MHC locus is also called human leukocyte antigens (HLA), which mainly encodes three HLA-I class molecules (HLA-A, HLA-B, HLA-C) and three HLA-II class molecules (HLA-DR, HLA-DP, HLA-DQ), CD4 + The T cell receptor (TCR) of T cells recognizes the peptide presented by HLA-II class molecules. However, due to the change in the length of the side chain of HLA-II class molecules, it is different from CD4 +The length of T cell epitope binding ranges from 11 amino acid residues to more than 20 amino acid residues, and some can even reach more than 30 amino acids. The bound peptide is buried in the antigen binding groove, leaving some side chains to bind to TCR. Studies have shown that in the antigen binding groove, only 9 amino acid residues from the peptide bind to MHC class II molecules, of which the sites binding to the HLA-DR region are mainly located at positions 1, 4, 6, and 9 of the binding peptide. Due to the high polymorphism of HLA molecules, 986 HLA-II allele sequences have been discovered so far. This polymorphism is manifested in the peptide binding groove, indicating that a given protein sequence can recognize different epitope sequences for different individuals.

[0004] Studies on COVID-19 have observed that HLA-DRB1*15:01, DQB1*06:02, B*27:07, and HLA-C*12:02 are susceptible to SARS-CoV-2 infection and are significantly associated with severe / critical COVID-19. The diversity of human HLA molecules is significantly associated with the effectiveness of the new coronavirus vaccine. People with HLA-DQA1*03:03, DRB1*12:01, DRB1*03:01, or HLA-DRB1*07:01, which carry HLA-II molecules, can enhance the level of serum neutralizing antibodies after vaccination with the new coronavirus BNT162b2 mRNA vaccine. It was also found that people carrying HLA-DQB1*06 had significantly enhanced anti-RBD antibodies compared to other people after vaccination with the new coronavirus ChAdOx1, BNT162b2, or mRNA-1273 vaccine. In addition, DRB1*04:01 can bind tightly to antigenic peptides, and it is significantly positively correlated with the production of neutralizing antibodies immunized by the new coronavirus vaccine. Therefore, people carrying this HLA supertype can antagonize SARS-CoV-2 infection. However, some studies have found that specific HLA supertypes are significantly negatively correlated with vaccine response. Among the recovered patients who have been infected with SARS-CoV-2 and received the BNT162b2 vaccine again, those carrying the HLA-DRB1*04:04 allele had the lowest anti-RBD antibodies. In addition, HLA-II molecules such as DRB1*03:01, DRB1*07:01 and DRB1*12:01 can activate CD4 by presenting viral antigenic peptides. + T cells, thereby promoting the production of neutralizing antibodies in COVID-19 mRNA vaccines to antagonize SARS-CoV-2 infection. Despite these findings, how to identify the novel coronavirus epitope peptides presented by MHC-II remains a problem.

[0005] In recent years, with the application and development of bioinformatics technology, CD4 +The efficiency of T cell epitope prediction is gradually improving. At present, there are many online prediction tools for MHC-II class molecule affinity epitopes, and these software have their own advantages and disadvantages. Recently, more and more mathematical models have introduced factors that affect the binding affinity of MHC molecules, including the polymorphism of MHC molecules, the nine-peptide core, and the flanking residues at both ends, so that the experimental data has been updated, expanded and improved, and the accuracy and practicality of epitope prediction have been improved. Human HLA-II molecules have higher polymorphism than HLA-I molecules in the population. In addition, HLA-II molecules are composed of two chains, α and β, and their combination further increases their polymorphism. At present, the "stepping method" is usually used to synthesize peptides for identification when identifying the Th epitopes of new antigens, which can identify more and more effective Th epitopes. Summary of the invention

[0006] In view of this, the inventors initially clustered the HLA-II alleles according to gene loci based on the structural basis of the binding of the Th epitope to the HLA-II molecule - 9 peptides as the core, by calculating the characteristics and distances of the 9-peptide cores of different HLA-II molecules to merge their polymorphisms; a "one-step" prediction tool for Th epitopes was constructed using a site scoring matrix algorithm, which achieves the purpose of predicting Th candidate epitopes while ignoring HLA restriction; the software system was used to predict the Th epitopes of the new coronavirus and its variants of the present invention, providing new epitope peptide sequences for the research and development of new coronavirus vaccines.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] Novel coronavirus broad-spectrum immune advantage CD4 + T cell epitope peptides, including Th epitope peptides common to the original strain of the new coronavirus and its variants, wherein the Th epitope peptides include one or more of the following sequences: SEQ ID NO.1: APAICHDGKAHFPRE; SEQ ID NO.2: EGVFVSNGTHWFVTQ; SEQ ID NO.3: AGLIAIVMVTIMLCCM; SEQID NO.4: KFNGLTVLPPLLTDE; SEQ ID NO.5: GTHWFVTQRNFYEPQI; SEQ ID NO.6: EDLLFNKVTLADAG; SEQ ID NO.7: TFEYVSQPFLMDLEG.

[0009] Furthermore, it also includes an immunodominant mutant Th epitope peptide specific to the mysterious Kronobacterium variant, and the Th epitope peptide includes one or more of the following sequences: SEQ ID NO.8: DLFLPFFSNVTWFHVI; SEQ ID NO.9: DYSVLYNLAPFFTFK; SEQ ID NO.10: DSKVSGNYNYLYRLFRK; SEQ ID NO.11: AVLYQGVNCTEVP.

[0010] To achieve the above object, the present invention also provides a method for utilizing CD4 + Th epitope peptide-MHC tetramer prepared from T cell epitope peptide.

[0011] Furthermore, the Th epitope peptide-MHC tetramer is obtained by hybridizing and incubating any Th epitope peptide with the HLA-DRB1*07:01 molecule.

[0012] To achieve the above object, the present invention also provides CD4 + Application of T cell epitope peptides in the preparation of a kit for detecting immune responses of people infected or after infection with the new coronavirus.

[0013] To achieve the above object, the present invention also provides CD4 + The application of T cell epitope peptides in the preparation of novel coronavirus preventive or therapeutic polypeptide vaccines for all populations.

[0014] To achieve the above objectives, the present invention also provides the use of Th epitope peptide-MHC tetramer in the preparation of a kit for detecting immune responses of people infected or after infection with the new coronavirus.

[0015] To achieve the above objectives, the present invention also provides the use of Th epitope peptide-MHC tetramer in the preparation of a novel coronavirus preventive or therapeutic polypeptide vaccine for all populations.

[0016] The beneficial effects of the present invention are as follows: the present invention obtains Th epitope peptides for the original strain of the novel coronavirus and its variants, and also obtains the immunodominant mutant Th epitope peptides specific to the Kronos variant. The in silico evaluation results show that the epitope peptides obtained by the present invention are more in line with the law of presentation of HLA-II class molecule binding peptides in the natural state, have a high detection rate for Th epitopes, and can reduce the economic cost by one quarter; the Th epitope peptide-MHC tetramer prepared by the epitope peptide of the present invention has a strong stimulation effect on CD4 + The epitope peptide and Th epitope peptide-MHC tetramer of the present invention can be made into a kit for detecting CD4 +The frequency and functional status of T cells (IL-2 and IFN-γ secretion) are measured. The prepared kit can be used for vaccination of people with HLA-DRB1*07:01 genetic background, infection with the original strain of the new coronavirus, infection with the variant strain, and CD4 + The detection kit for T cell immune response, the obtained Th cell epitope peptide library can be used to develop new coronavirus vaccines for all populations. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below in conjunction with the accompanying drawings, wherein:

[0018] Figure 1 CD4 before and after the Ad5-nCoV booster injection in the embodiment + Results of T cell immunity change trends;

[0019] Figure 2 The results of HLA-B ultrasonography of some volunteers in the examples;

[0020] Figure 3 The result graph of stimulating PBMCs of four volunteers one by one using the synthesized Th epitope peptides common to the original strain and variant strains of the novel coronavirus in the embodiment;

[0021] Figure 4 The result diagram of stimulating PBMCs of four volunteers one by one using the synthesized immunodominant Th epitope peptides of the novel coronavirus secret Kron mutant in the embodiment;

[0022] Figure 5 The result of stimulating PBMCs of HLA-DRB1*07:01 volunteers with Th epitope peptide-tetramers of the novel coronavirus and mutant strains in the examples; DETAILED DESCRIPTION

[0023] The following describes the embodiments of the present invention by specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments only illustrate the basic concept of the present invention in a schematic manner, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0024] Experimental methods for which specific conditions are not specified in the examples are generally performed under conventional conditions, such as those described in Molecular Cloning Laboratory Manual (3rd edition, by J. Sambrook et al.), or under conditions recommended by manufacturers.

[0025] In addition, the content related to volunteers in this embodiment has obtained the clinical research approval ethics demonstration of the Scientific Research Project Review Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University, and the ethics number is: 2021-78.

[0026] Example:

[0027] In this example, the immunodominant Th epitope of the S antigen of the SARS-CoV-2 virus was predicted, and the specific method was as follows: entering the NCBI system, obtaining relevant information of the new coronavirus SARS-CoV-2 gene, and the results showed that the NCBI registration number of SARS-CoV-2 was: GenBank: NC_045512; and the amino acid sequence of the SARS-CoV-2S protein was obtained, including the S protein amino acid sequence of the new coronavirus original strain (Wuhan-1), the S protein amino acid sequence of the new coronavirus mysterious Kron variant-BA.4.5, the S protein amino acid sequence of the new coronavirus mysterious Kron variant-XBB.1.16, and the S protein amino acid sequence of the new coronavirus mysterious Kron variant-EG5.1.

[0028] The amino acid sequence of the S protein of the original strain of the new coronavirus (Wuhan-1):

[0029] >sp|P0DTC2|SPIKE_SARS2 Spike glycoprotein OS=Severe acuterespiratory syndrome coronavirus 2OX=2697049GN=S PE=1SV=1

[0030] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFSNVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPGDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCALDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEHVNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQNAQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0031] Amino acid sequence of the S protein of the novel coronavirus Omicron variant - BA.4.5:

[0032] >UTZ81033.1_BA.4.5

[0033]

[0034] The amino acid sequence of the S protein of the mysterious Kron variant of the new coronavirus - XBB.1.16:

[0035] >WNV39505.1_XBB.1.16

[0036]

[0037] The amino acid sequence of the S protein of the novel coronavirus mysterious Kron variant-EG5.1:

[0038] >WNV39449.1_EG.5.1

[0039] MFVFLVLLPLVSSQCVNLITRTQSYTNSFTRGVYYPDKVFRSSVLHSTHDLFPFFSNVTWFHAIHVSGTNGTKRFDNPALPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIVNNATNVVIKVCEFQFCNDPFLDVYQKNNKSWMESEFRVYSSANNCTFEYVSQPFLMDLEGKEGNFKNLREFVFKNIDGYFKIYSKHTPINLERDLPQGFSALEPLVDLPIGINITRFQTLLALHRSYLTPVDSSSGWTAGAAAYYVGYLQPRTFLLKYNENGTITDAVDCLDPLSETKCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFHEVFNATTFASVYAWNRKRISNCVADYSVIYNFAPFFAFKCYGVSPTKLNDLCFTNVYADSFVIRGNEVSQIAPGQTGNIADYNYKLPDDFTGCVIAWNSNKLDSKPSGNYNYLYRLLRKSKLKPFERDISTEIYQAGNKPCNGVAGPNCYSPLQSYGFRPTYGVGHQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQGVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEYVNNSYECDIPIGAGICASYQTQTKSHRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPTNFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLKRALTGIAVEQDKNTQEVFAQVKQIYKTPPIKYFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQYGDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIPFAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNHNAQALNTLVKQLSSKFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAAEIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKNFTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVNNTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLNESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCCKFDEDDSEPVLKGVKLHYT

[0040] The Th epitope "one-step" prediction tool independently developed by our research team was then used to predict the possible Th epitopes of the Spike protein of the original SARS-COV-2 strain and various Omicron variants. The name of the "one-step" prediction tool is: ThoStepred, and its website is: https: / / www.immunoinformatics.net / ThoStepred / . The homepage of the website is attached. Figure 3 .

[0041] At the same time, we compared the identified Th epitopes in the IEDB database to screen the unidentified candidate Th epitopes, and selected some identified epitopes to optimize their length. We also focused on retaining the candidate CD4 + Based on this, we conducted a CD4 + The results of T cell epitope scanning are shown in Table 1, where the bold sequences represent newly emerged Th epitopes from the mutation.

[0042] At the same time, we also used other common software systems, including the TEPITOPE database (http: / / www.vaccinome.com) and the MULTIPRED neural network (http: / / antigen.i2r.a-star.edu.sg / multipred1) and the EpiMatrix matrix (http: / / www.epivax.com), to predict the Th epitopes of the new coronavirus, as shown in Table 2, where the black bold epitopes are Th epitopes that have been reported in the literature and have been functionally verified.

[0043] Table 1 New Th epitopes due to mutations in the three Omicron variants currently prevalent in China

[0044]

[0045]

[0046] Table 2 uses other commonly used software systems to predict the Th epitopes of the original strain and variant strain of the new coronavirus.

[0047]

[0048]

[0049] After screening the immune dominant Th epitopes shared by the original strain and variants of the new coronavirus and unique to the Kronovirus variant, the Th epitope peptide sequences shared by the original strain of the new coronavirus and variants such as Kronovirus were obtained, as follows: ①APAICHDGKAHFPRE; ②EGVFVSNGTHWFVTQ; ③AGLIAIVMVTIMLCCM; ④KFNGLTVLPPLLTDE; ⑤GTHWFVTQRNFYEPQI; ⑥EDLLFNKVTLADAG; ⑦TFEYVSQPFLMDLEG.

[0050] We also obtained the immunodominant mutant Th epitope peptide sequences specific to the mystical Kronobacterium variant: ①DLFLPFFSNVTWFHVI; ②DYSVLYNLAPFFTFK; ③DSKVSGNYNYLYRLFRK; ④AVLYQGVNCTEVP.

[0051] Next, the inventors synthesized the Th epitope peptides screened above, and hybridized and incubated the synthesized multiple Th epitope peptides with HLA-DRB1*07:01 molecules to obtain Th epitope peptide-MHC tetramers. The Th epitope peptide-MHC tetramers were used as peptide library stimulation vaccines to stimulate PBMCs of vaccine recipients and analyze their immunogenicity and reactivity.

[0052] See attached Figure 1 The common CTL peptide library of the predicted novel coronavirus original strain and variant strains such as Cronus vulnerabilities (stimulation group: NC and Ag peptide library (CTL epitope peptide library)) was used to stimulate the PBMC of the vaccine recipients, and CD4 was detected by flow cytometry 12 hours later. + T cell immune response, demonstrated by induction of CD4 + The optimal peptide library concentration required for Th activation is between 100 and 350 nM.

[0053] Attached Figure 1 and attached Figure 5 The results shown here show that the peptide library can activate peripheral blood CD4 + T cells secrete TNF-α and IFN-γ, indicating that the Th epitope peptide obtained in this example has immunogenicity and immunoreactivity.

[0054] This example also performed HLA-B supertype sequencing analysis on 250 volunteers.

[0055] At the level of PBMC cell suspension or extracted RNA, full-length cDNA is obtained under the action of efficient reverse transcriptase, and the targeted gene is captured by combining with a highly specific capture panel (purchased from Nanjing Visualization Biotechnology Co., Ltd.). After adding sequencing primers to the obtained product, a library that can be used for sequencing is obtained.

[0056] After the library is constructed, Qubit3.0 is used for quantification, and then Qsp100 is used to detect the insert size of the library. When the insert size meets expectations, PE150 sequencing is performed using the Illumina X10 or NovaSeq6000 platform.

[0057] After the original data were obtained by sequencing, the data filtering software fastp (v0.21.0) was used to filter and quality control the data. The high-quality sequencing data obtained after filtering were then analyzed for HLA typing using the HLA typing software arcasHLA.

[0058] The raw data needs to be filtered and quality controlled. The software fastp (v 0.21.0) was used for data filtering and quality control.

[0059] The fastp filtering conditions are as follows: 1) truncate the adapter sequence; 2) filter out sequences less than 15 bp in length; 3) filter out reads with more than 40% base quality values ​​less than 15; 4) filter out reads containing more than 6 N bases; 5) truncate exogenous sequences incorporated into the library construction.

[0060] The filtered high-quality sequencing data was analyzed by the genotype module of the software arcasHLA to obtain the HLA analysis results of each volunteer sample. The HLA-B ultrasonography test results of some volunteers are attached. Figure 2 .

[0061] The synthetic Th epitope peptides were used to stimulate the PBMCs of the vaccinated volunteers one by one, and then the common immunodominant Th epitopes of the three mutant strains and the original strain of the new coronavirus were screened and obtained. See the attached Figure 4 .

[0062] It can be seen that the Th epitope peptide sequences shared by the original strain of the new coronavirus and variants such as Kronos are: ①APAICHDGKAHFPRE; ②EGVFVSNGTHWFVTQ; ③AGLIAIVMVTIMLCCM; ④KFNGLTVLPPLLTDE; ⑤GTHWFVTQRNFYEPQI; ⑥EDLLFNKVTLADAG; ⑦TFEYVSQPFLMDLEG.

[0063] The Th epitope peptide sequence specific to the immune-dominant mutation of the mysterious Kronobacterium variant is: ①DLFLPFFSNVTWFHVI; ②DYSVLYNLAPFFTFK; ③DSKVSGNYNYLYRLFRK; ④AVLYQGVNCTEVP.

[0064] Since the selected Th epitope peptides include the common Th epitope peptides for the original strain and variant strain of the new coronavirus, and the specific immune advantage mutant Th epitope peptides for Omicron, the Th epitope peptides obtained in this example can detect whether a person is infected with the new coronavirus and whether there is CD4 + T cell response, the specific detection steps are as follows:

[0065] 1. Prepare PBMC cells from volunteers vaccinated with the novel coronavirus vaccine, patients infected with Kronovirus, and those who are not infected;

[0066] 2. Centrifuge the cells at 2500×g for 5 min and store the prepared Tetramer on ice;

[0067] 3. Add 2×10^ 6 Place cells in a 96-well U-shaped plate or a 12x75mm tube, adjust the volume to 200ul with cell staining buffer, and add 2μl Flex-T to each sample. TM Tetramer complex, mix well, and incubate on ice in the dark for 30 min;

[0068] 4. No need to wash. If co-stained with other surface antibodies, prepare the antibody mixture according to the optimal antibody concentration; incubate on ice in the dark for 30 minutes;

[0069] 5. Wash the cells twice with Staining Buffer and resuspend the cells in Staining Buffer;

[0070] 6. Analyze Tetramers by Flow Cytometry + CD8 + The frequency of T cells.

[0071] This example shows that Tetramer + CD4 + Further analysis of T cells revealed that they can secrete IFN-r+, TNF-α+, IFN-γ+, and IL-2+. The results are shown in the attached Figure 5 .

[0072] It can be seen that the epitope peptide-HLA-DRB1*07:01 tetramer of this embodiment can be prepared into a kit, and the peptide-MHC tetramer can be directly used to stimulate peripheral blood PBMC, so that CD4 + The frequency and functional status of T cells. Therefore, this kit can be used as a means of detecting novel coronavirus infection and cellular immune response after infection, and the epitope peptide library obtained can also be used for the subsequent development of novel coronavirus vaccines.

[0073] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution, which should be included in the scope of the claims of the present invention.

Claims

1. The novel coronavirus has a broad spectrum of immune advantages, CD4 + The T cell epitope peptide is characterized in that It includes Th epitope peptides common to the original strain of the new coronavirus and its variants, and the Th epitope peptides include one or more of the following sequences: SEQID NO.1: APAICHDGKAHFPRE; SEQ ID NO.2: EGVFVSNGTHWFVTQ; SEQ ID NO.3: AGLIAIVMVTIMLCCM; SEQ ID NO.4: KFNGLTVLPPLLTDE; SEQ ID NO.5: GTHWFVTQRNFYEPQI; SEQID NO.6: EDLLFNKVTLADAG; SEQ ID NO.7: TFEYVSQPFLMDLEG.

2. The novel coronavirus broad-spectrum immunodominant CD4 + The T cell epitope peptide is characterized in that It also includes an immune-dominant mutant Th epitope peptide specific to the mysterious Kronobson mutant, and the Th epitope peptide includes one or more of the following sequences: SEQ ID NO.8: DLFLPFFSNVTWFHVI; SEQ ID NO.9: DYSVLYNLAPFFTFK; SEQ ID NO.10: DSKVSGNYNYLYRLFRK; SEQ ID NO.11: AVLYQGVNCTEVP.

3. Using the CD4 of any one of claims 1-2 + Th epitope peptide-MHC tetramer prepared from T cell epitope peptide.

4. The Th epitope peptide-MHC tetramer according to claim 3, characterized in that: The Th epitope peptide-MHC tetramer is obtained by hybridizing and incubating any one of the Th epitope peptides in any one of claims 1 to 2 with the HLA-DRB1*07:01 molecule.

5. The CD4 according to any one of claims 1 to 2 + Application of T cell epitope peptides in the preparation of a kit for detecting immune responses of people infected or after infection with the new coronavirus.

6. The CD4 according to any one of claims 1 to 2 + The application of T cell epitope peptides in the preparation of novel coronavirus preventive or therapeutic polypeptide vaccines for all populations.

7. Use of the Th epitope peptide-MHC tetramer according to any one of claims 3-4 in the preparation of a kit for detecting immune responses of people infected or after infection with the new coronavirus.

8. Use of the Th epitope peptide-MHC tetramer described in any one of claims 3-4 in the preparation of a novel coronavirus preventive or therapeutic polypeptide vaccine for all populations.

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