Novel specific T cell epitope peptide for screening coronavirus S1, N and M protein holoproteome and application thereof

Abstract

The invention discloses a specific T cell epitope peptide for screening a whole proteome of S1, N and M proteins of a novel coronavirus and application, relates to the technical field of biological medicines, and has the technical key points that the scheme of the invention provides a plurality of novel coronavirus CD4 + T and CD8 + T cell epitope polypeptides, the invention also relates to a gene for coding the epitope polypeptide, a recombinant protein or a compound containing the epitope polypeptide, a sensitization antigen presenting cell and a specific immune effector cell aiming at the epitope polypeptide in research and development of novel coronavirus vaccines and disease treatment. The polypeptide is screened on the basis of a peripheral blood sample of a convalescent patient infected with Omicro BA.5 after enhanced inoculation of an inactivated vaccine, CD4 + T and CD8 + T cellular immune response aiming at novel coronavirus protein can be safely and effectively induced, and the polypeptide has important guiding significance on research and development of novel vaccines of novel coronavirus.

Description

[0001] This application is a divisional application of the invention patent application with application number CN2023114666337, invention name “Specific T cell epitope peptides and applications screened from the whole protein group of new coronavirus S1, N and M proteins”, and application date November 7, 2023. Technical Field

[0002] The present invention relates to the field of biomedicine technology, and specifically to specific T cell epitope peptides screened from the whole proteome of the novel coronavirus S1, N and M proteins and their applications. Background Art

[0003] The novel coronavirus (SARS-CoV-2) has not completely disappeared. It has coexisted with humans for a long time and continues to mutate, still posing a potential threat to human health. Therefore, the research and development of effective vaccines and therapeutic drugs must continue. Specific T cell responses play a vital role in the body's resistance to viral infection. Therefore, studying the T cell immune response induced by viral infection, especially the characteristics of T cell epitopes, is crucial for understanding immune defense mechanisms and is a prerequisite for the development of vaccines and immunotherapies.

[0004] The basis for T cell recognition and activation is the recognition of viral antigen peptides presented by HLA - T cell epitopes. Therefore, studying the characteristics of viral T cell epitopes is crucial for understanding immune defense mechanisms and is a prerequisite for developing vaccines and immunotherapies. At the same time, due to the high polymorphism of HLA, the identification of SARS-CoV-2 T cell epitopes and their corresponding HLA has the following important significance:

[0005] (1) It can help predict the T cell response induced by natural infection or vaccination with SARS-CoV-2 in a population of a certain race or geographic region, which is related to the HLA allele composition prevalent in that population.

[0006] (2) It can guide the development of vaccines and use specific dominant epitopes to enhance T cell responses and increase the effectiveness of vaccines.

[0007] (3) It can also help monitor whether potential viral mutations can escape T cell responses.

[0008] (4) It can promote the development of memory T cell-based diagnostics to distinguish recovered individuals from uninfected individuals.

[0009] The SARS-CoV-2 genome consists of structural and non-structural protein coding regions. The structural protein coding region primarily encodes the spike protein (S protein), membrane glycoprotein (M protein), nucleocapsid protein (N protein), and envelope protein (E protein). The S protein consists of two subunits, S1 and S2. S1 primarily contains the receptor binding domain (RBD), responsible for recognizing cellular receptors, while S2 contains essential components required for membrane fusion. The non-structural protein coding region primarily comprises open reading frame (ORF) 1a and ORF1b genes, encoding 16 non-structural proteins (NSP proteins), namely NSP1 to 16. In addition, SARS-CoV-2 contains nine accessory proteins, ORFs 3a, 3b, 6, 7a, 7b, 8, 9b, 9c, and 10. These accessory proteins facilitate viral infection of human hosts, replication, and ultimately human-to-human transmission.

[0010] Currently, most studies on T cell epitopes rely on computer software to predict epitopes based on given HLA genotypes and protein sequences. Due to the complex structure of MHC molecules, significant bias can occur when predicting certain epitope peptides, particularly for CD4+ T cell epitopes. Unlike CD8+ T cell epitopes, which cannot accurately predict the binding epitope peptide based on a given HLA genotype, studies on CD4+ T cell epitopes currently primarily rely on in vitro stimulation with peptide libraries. However, the limited availability of human PBMC samples significantly limits our understanding of CD4+ T cell epitopes.

[0011] Identifying potentially dominant CD4+ T and CD8+ T cell epitopes in the Chinese population will provide a more comprehensive understanding of T cell responses in humans following booster vaccination and natural infection. Currently, there are no reports of novel coronavirus (COVID-19) S1, N, and M protein-specific CD4+ T and CD8+ T cell epitope peptides screened from a whole-protein peptide library. Therefore, the present invention aims to provide specific T cell epitope peptides screened from the whole-protein panel of novel coronavirus S1, N, and M proteins and their applications to address the aforementioned issues. Summary of the Invention

[0012] The purpose of the present invention is to solve the above problems, provide specific T cell epitope peptides screened from the whole protein group of novel coronavirus S1, N and M proteins, and provide human HLA allele information that can recognize the epitope peptides.

[0013] In order to achieve the above object, the technical solution of the present invention is as follows:

[0014] The present invention provides novel coronavirus CD4+T and CD8+T epitope polypeptides, characterized in that the amino acid sequence of the epitope polypeptide is shown in any one of SEQ ID NO.1-11.

[0015] SEQ ID NO.1-11:

[0016]

[0017]

[0018] The present invention also provides a nucleic acid molecule encoding the epitope polypeptide as shown in any one of SEQ ID NOs. 1-11.

[0019] The present invention also provides a vector comprising a nucleic acid molecule encoding the epitope polypeptide as shown in any one of SEQ ID NOs. 1-11.

[0020] The present invention also provides a delivery system, comprising a nucleic acid molecule encoding the epitope polypeptide as shown in any one of SEQ ID NOs. 1-11, and the delivery system comprises a virus, a virus-like particle, a plasmid or a nanoparticle.

[0021] The present invention also provides a presenting cell, which comprises the epitope polypeptide described above, or the vector described above, or the delivery system described above.

[0022] Furthermore, the presenting cells are antigen presenting cells.

[0023] The present invention also provides a pharmaceutical composition, which comprises the epitope polypeptide described above, or the vector described above, or the vector described above, or the delivery system described above, or the presenting cell described above, and optionally includes a pharmaceutically acceptable carrier or adjuvant.

[0024] The present invention also provides the use of the above-mentioned epitope polypeptide, or the above-mentioned nucleic acid molecule, or the above-mentioned vector, or the above-mentioned delivery system, or the above-mentioned cell in the preparation of a drug or vaccine for preventing or treating the new coronavirus.

[0025] The present invention also provides a detection reagent, which includes the epitope polypeptide described above.

[0026] The present invention also provides the use of the epitope polypeptide in the preparation of a novel coronavirus infection screening detection reagent or a reagent for detecting the specific T cell immune response level of a novel coronavirus infection.

[0027] In the above-mentioned embodiments of the present invention, the specific T cell epitope peptides of the present invention also provide detection antigens for evaluating the long-term immune protection of the new coronavirus vaccine. The specific T cell epitope peptides of the present invention also provide detection antigens for evaluating the immune response induced by virus variants. The specific T cell epitope peptides of the present invention also provide detection antigens for detecting the level of specific T cell immune response in patients infected with the new coronavirus. The specific T cell epitope peptides of the present invention also provide antigens for inducing specific T cells in vitro. The present invention also provides a variety of nucleic acids encoding the above-mentioned specific T cell epitope peptides.

[0028] Compared with the existing technology, this solution has the following beneficial effects:

[0029] The epitope polypeptides in the scheme of the present invention are screened based on the whole protein peptide library of S1, M and N proteins, which can effectively induce immune responses against the new coronavirus S1, M and N proteins, and have important guiding significance for the development of its vaccine. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 This is the screening strategy in Example 1 of the present invention;

[0031] Figure 2 This is the screening process of the S26 single peptide (NO.1) in Example 1 of the present invention;

[0032] Figure 3 This is the screening process of the S10 single peptide (NO.2) in Example 1 of the present invention;

[0033] Figure 4 This is the screening process of the S30 single peptide (NO.3) in Example 1 of the present invention;

[0034] Figure 5 This is the screening process of single peptides M10 (NO.4) and M42 (NO.8) in Example 1 of the present invention;

[0035] Figure 6 This is the screening process of the M20 (NO.5) single peptide in Example 1 of the present invention;

[0036] Figure 7 This is the screening process of single peptides M27 (NO.6) and M28 (NO.7) in Example 1 of the present invention;

[0037] Figure 8 This is the screening process of single peptides N25 (NO.9) and N27 (NO.10) in Example 1 of the present invention;

[0038] Figure 9 This is the screening process of the single peptide N54 (NO.11) in Example 1 of the present invention;

[0039] Figure 10 This is the verification result of the 9mer epitope in the S30 (NO.3) single peptide in Example 4 of the present invention. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be further described in detail below in conjunction with the embodiments of the present invention and the accompanying drawings. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts should fall within the scope of protection of the present invention.

[0041] It should be noted that, in the absence of conflict, the embodiments and features of the embodiments of the present invention can be combined with each other. The present invention will be described in detail below with reference to the embodiments.

[0042] The solution provided in the embodiments of the present invention is as described above, providing novel coronavirus CD4+T and CD8+T cell epitope polypeptides, the amino acid sequences of the epitope polypeptides are shown in SEQ ID NO.1-11.

[0043] SEQ ID NO.1-11:

[0044]

[0045]

[0046] Also provided is a nucleic acid molecule encoding the aforementioned epitope polypeptide. Also provided is a vector comprising the aforementioned nucleic acid molecule. Also provided is a delivery system comprising the aforementioned nucleic acid molecule, comprising a virus, virus-like particle, plasmid, or nanoparticle. Also provided is a presenting cell comprising the aforementioned epitope polypeptide, or the aforementioned vector, or the aforementioned delivery system. The presenting cell is an antigen-presenting cell. Also provided is a pharmaceutical composition comprising the aforementioned epitope polypeptide, or the aforementioned vector, or the aforementioned vector, or the aforementioned delivery system, or the aforementioned presenting cell, and optionally a pharmaceutically acceptable carrier or adjuvant. Also provided is the use of the aforementioned epitope polypeptide, nucleic acid molecule, vector, delivery system, or cell in the preparation of a drug or vaccine for preventing or treating the novel coronavirus. Also provided is a detection reagent comprising the aforementioned epitope polypeptide. Also provided is the use of the epitope polypeptide in the preparation of a detection reagent for screening novel coronavirus infection or a reagent for detecting the level of specific T cell immune response in novel coronavirus infection.

[0047] Example 1: Screening of CD4+ T cell and CD8+ T cell epitopes of S1, M and N proteins

[0048] 1.1 Peptide library information

[0049] Peptide library synthesis plan: 15AA, 5 steps. The S1 protein (1-681AA) peptide library contains 135 peptides, S1-S135, and the S2 protein (682-1273AA) peptide library contains 117 peptides: S137-S253, S136 is a peptide library for both S1 and S2. The N protein peptide library contains 82 peptides, and the M protein peptide library contains 43 peptides.

[0050] Table 1S1 protein peptide library sequence

[0051]

[0052] Table 2 M protein and N protein peptide library sequences

[0053]

[0054] 1.2 Screening strategies (such as Figure 1 shown)

[0055] First round of screening: After specific stimulation with S1, M, and N proteins, the cells were expanded for 9 days, 200,000 cells per well were placed in a 96-well round-bottom plate, and the peptide library was added for stimulation for 6 hours (after 1 hour of stimulation, BFA was added to block the transport of cytokines), and CD4 was detected by flow cytometry. + ,CD8 + IFNγ expression in T cells. We divided the S1 peptide library into five subpools: S1-1, S1-2, S1-3, S1-4, and S1-5. We divided the M protein into five subpools: M-1, M-2, M-3, M-4, and M-5; and we divided the N protein into five subpools: N-1, N-2, N-3, N-4, and N-5.

[0056] Second round of screening: The five S1 libraries were divided into six secondary sublibraries, A, B, C, D, E, and F. Similarly, the five N protein sublibraries were divided into six secondary sublibraries. After the second round of screening, single peptide results were obtained for the M protein.

[0057] The third round of screening: S1 protein and N protein need to be screened for the third round. Samples with positive results in the secondary sub-library are selected. 200,000 cells per well are placed in a 96-well round-bottom plate. Single peptide is added. After stimulation for 6 hours, intracellular staining is performed to detect CD4 + T cells and CD8 + IFNγ expression in T cells.

[0058] 1.3 Screening results

[0059] like Figure 2The results of the screening of the S26 peptide (NO.1) are shown in Figure 1. The samples used in the screening of Example 1 of the present invention were I-37 samples that were specifically stimulated and amplified by the S1 peptide library. The ratio of IFNγ+CD4+T cells to total CD4+T cells was detected (see Figure 1). Figure 2 A. Results of the second round of screening of the S1-1 protein peptide library. B. Results of the third round of screening of the S1-1F protein peptide library. The results showed that the proportion of IFNγ+CD4+ T cells in the S26-stimulated group was 1.61% of total CD4+ T cells, significantly higher than in the other single-peptide-stimulated groups in the S1-1F protein peptide library. Therefore, this sample responded to the S26 peptide alone. Among the 51 samples we screened from recovered individuals who had experienced breakthrough infection with Omicron BA.5 after inactivated vaccination, four responded to S26: I-37, I-47, I-389, and I502.

[0060] like Figure 3 The results of the screening of S10 single peptide (NO.2) in Example 1 of the present invention are as follows: the sample used is the I-389 sample that has been specifically stimulated and amplified by the S1 peptide library, and the proportion of IFNγ+CD8+T cells in the total CD8+T cells is detected. The results of the second round of screening of the S1-1 protein peptide library show that the S1-1B stimulation group has a response ( Figure 3 A). Therefore, the S1-1B protein peptide library was screened for the third round, and the results showed that the proportion of IFNγ+CD8+T cells in the S10 stimulation group accounted for 4.52% of the total CD8+T cells, while that in the control group was 0.22%. Therefore, the sample responded to the S10 single peptide ( Figure 3 B) Among the 51 recovered individuals who experienced breakthrough infection with Omicron BA.5 after vaccination with the inactivated vaccine, two individuals, I-13 and I-389, responded to S10.

[0061] like Figure 4 As shown, this is the screening process for the S30 single peptide (NO.3). The screening results in Example 1 of the present invention were obtained from the I-229 sample, which had been specifically stimulated and amplified by the S1 peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (A. Second round screening results of the S1-1 protein peptide library B. Third round screening results of the S1-1F protein peptide library, which showed that this sample responded to the S30 single peptide). The results showed that the proportion of IFNγ+CD8+T cells in the S30-stimulated group was 4.71%, while that in the control group was 0.19%, indicating that this sample responded to the S30 single peptide. Among the 51 samples of recovered patients who had breakthrough infection with Omicron BA.5 after vaccination with an inactivated vaccine that we screened, 6 responded to S30: I-229, I-57, I-62, I-54, I-73, and I-34.

[0062] like Figure 5As shown, it is the screening process of M10 (NO.4) and M42 (NO.8) single peptides. The screening results in Example 1 of the present invention are based on the I-212 sample that has been specifically stimulated and amplified by the M peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (the first round screening results of the AM protein peptide library; the second round screening results of the BM-3 protein peptide library showed that the sample responded to both M10 and M42 single peptides). The results showed that the proportion of IFNγ+CD8+T cells in the M10 and M42 stimulation groups was 6.21% and 9.90% of the total CD8+T cells, respectively, while that in the control group was 1.01%. Therefore, the sample responded to M10 and M42 single peptides. Among the 51 samples of recovered patients who had breakthrough infection with Omicron BA.5 after receiving inactivated vaccine that we screened, 6 responded to M10: I-212, I-47, I-71I-72, I-77 and I-7; only I-212 responded to M42.

[0063] like Figure 6 As shown, it is the screening process of M20 (NO.5) single peptide. The screening results in Example 1 of the present invention, the sample used is the I-31 sample that has been specifically stimulated and amplified by the M peptide library, and the proportion of IFNγ+CD8+T in the total CD8+T cells is detected (the first round screening results of the AM protein peptide library and the second round screening results of the BM-3 protein peptide library, and it was found that the sample responded to both M20 and M21 single peptides). The results showed that the proportion of IFNγ+CD8+T in the total CD8+T cells in the M20 stimulation group was 2.27% and 2.22%, respectively, which was significantly higher than that of other single peptide stimulation groups in the M-3 protein peptide library. Therefore, the sample responded to M20 and M21 single peptides. Among the 51 samples of recovered patients who broke through the infection of Omicron BA.5 after being vaccinated with inactivated vaccines that we screened, there were 2 responders to M20: I-7 and I-31;

[0064] like Figure 7As shown, it is the screening process of M27 (NO.6) and M28 (NO.7) single peptides. The screening results in Example 1 of the present invention are based on the I-20 sample that has been specifically stimulated and amplified by the M peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells is detected (the first round screening results of the AM protein peptide library; the second round screening results of the BM-3 protein peptide library found that the sample responded to the M27 single peptide; the second round screening results of the CM-4 protein peptide library found that the sample responded to the M28 single peptide). The results showed that the proportion of IFNγ+CD8+T cells in the M27 and M28 stimulation groups was 2.19% and 1.98% of the total CD8+T cells, respectively, which was significantly higher than that of other single peptide stimulation groups in the M-3 and M-4 protein peptide libraries. Therefore, the sample responded to the M27 and M28 single peptides. Among the 51 samples of recovered patients who had breakthrough infection with Omicron BA.5 after receiving inactivated vaccine that we screened, there were two who responded to M27: I-20 and I-502; and only one, I-20, responded to M28.

[0065] like Figure 8 As shown, it is the screening process of N25 (NO.9) and N27 (NO.10) single peptides. The screening results in Example 1 of the present invention are based on the I-502 sample that has been specifically stimulated and amplified by the N peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (the first round screening results of the AN protein peptide library; the second round screening results of the BN-2 protein peptide library; the third round screening results of the CN-2C protein peptide library, which found that the sample responded to the N25 single peptide; the third round screening results of the DN-2D protein peptide library, which found that the sample responded to the N27 single peptide). The results showed that the proportion of IFNγ+CD8+T cells in the N25 and N27 stimulation groups was 1.07% and 10.4% of the total CD8+T cells, respectively, which was significantly higher than that of the other single peptide stimulation groups in the N-2C and N-2D protein peptide libraries. Therefore, the sample responded to the N25 and N27 single peptides. Among the 51 samples of recovered patients who had breakthrough infection with Omicron BA.5 after vaccination with inactivated vaccine that we screened, there were 5 responders to N25: I-31, I-145, I-71, I-502, I-71 and I-72; there were 10 responders to N27: I-389, I-46, I-54, I-62, I-65, I-66, I-502, I-77, I-79 and I-51.

[0066] like Figure 9The figure shows the screening process for the single peptide N54 (No. 11). The screening results in Example 1 of the present invention were obtained from sample I-149, which had been specifically stimulated and amplified using the N peptide library. The proportion of IFNγ+CD8+ T cells in total CD8+ T cells was measured (results from the first round of screening using the AN protein peptide library; the second round of screening using the BN-4 protein peptide library; and the third round of screening using the CN-4B protein peptide library. This sample responded to both the N53 and N54 single peptides). The results showed that the proportions of IFNγ+CD8+ T cells in the N53 and N54 stimulation groups were 10.1% and 12.2%, respectively, significantly higher than those in the other single peptide stimulation groups from the N-4B protein peptide library. Therefore, this sample responded to both the N53 and N54 single peptides. Among the 51 samples of recovered individuals who had experienced breakthrough infection with Omicron BA.5 after inactivated vaccination, four responded to N54: I-149, I-65, I-66, and I-366.

[0067] Example 2: Comprehensive Analysis of HLA Restriction of CD4+ T Cell and CD8+ T Cell Epitopes of S1, M, and N Proteins

[0068] 2.1 Predominant CD4+ T cell epitopes and HLA restriction in the S1 protein: Four dominant epitopes were identified in the S1 protein: S6, S16, S26, and S34. All four responders to the S26 (No. 1) epitope had HLA-DRB*15:01:01.

[0069] Table 3 HLA genotype results of CD4+ T cell epitope responders to S1 protein

[0070]

[0071] 2.2 Predominant CD4+ T cell epitopes and HLA restriction in M ​​and N proteins

[0072] Screening revealed three dominant epitopes in the M protein: M30, M34, and M36. Screening also revealed three dominant epitopes in the N protein: N22, N53, and N65. Most N22 responders harbored HLA-DPB1*05:01:01. Analysis of the dominant CD4+ T cell epitope in the M protein revealed that M36 is present on a wide range of HLA types.

[0073] Table 4 HLA genotype results of CD4+ T cell epitope responders to M and N proteins

[0074]

[0075] 2.3 Analysis of CD8+ T cell dominant epitopes and HLA restriction in S1 and M proteins

[0076] The dominant CD8+ T cell epitopes in the S1 protein are S8, S10, S30, S62, and S72. The six individuals who responded to S30- all harbored HLA-B*15. The dominant CD8+ T cell epitopes in the M protein are M10, M20, M27, M34, and M35. Samples that responded to M10 included I-212, I-47, I-71, I-72, I-77, and I-7. Except for I-72, all of these individuals harbored HLA-A*24:01:01.

[0077] Table 5 HLA genotype results of CD8+ T cell epitope responders to S1 and M proteins

[0078]

[0079] 2.4. Predominant CD8+ T Cell Epitopes and HLA Restriction in the N Protein: The N protein contains numerous predominant CD8+ T cell epitopes (Table 5). N72 / 73-HLA-A*11:01:01 and N65-HLA-A*11:01:01 have been reported and validated in the literature. Except for I-51, all individuals responding to the N27 epitope harbored HLA-A*11:01:01. Furthermore, sample I-149 responded to both N54 and N53, which harbors both CD8+ and CD4+ T cell epitopes.

[0080] Table 6 HLA genotype results of the CD8+ T cell epitope responders to N protein

[0081]

[0082] Case Study 3: Analysis of S30-HLA-B15:02 Binding Ability

[0083] Bioinformatic prediction of MHC binding capacity is a key component of epitope identification. We further analyzed the binding capacity of any nine peptides from S30 (HKNNKSWMESEFRVY) to HLA-B15:02 using https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.0 / . The results showed strong binding between WMESEFRVY and HLA-B15:02 (see Table 7 below).

[0084] Table 7: Analysis of the binding ability of any nine peptides in S30 to HLA-B15:02

[0085]

[0086] Case Study 4: Validation Experiment of WMESEFRVY (9mer) Epitope Peptide

[0087] After 9 days of stimulation with the S1 protein peptide library, I_229 PBMC were stimulated with S30 and WMESEFRVY (9mer) single peptide in S30, and the proportion of IFNγ+ T cells in CD8+ T cells was detected. Figure 10 As shown, the 9mer single peptide can effectively activate specific CD8+ T cells. Therefore, the inventors of the present application further clarified that the epitope sequence in the S30 single peptide is WMESEFRVY.

[0088] In summary, the epitope polypeptides in the above embodiments of the present invention are screened based on the whole protein peptide library of S1, M and N proteins, which can effectively induce immune responses against the new coronavirus S1, M and N proteins, and have important guiding significance for the development of vaccines therefor.

[0089] The above specific embodiments are merely explanations of the present invention and are not limitations of the present invention. After reading this specification, those skilled in the art may make non-creative modifications to the embodiments as needed. However, as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. CD8 of a novel coronavirus + The T cell epitope polypeptide-HLA complex is characterized by The compound comprises: Epitope polypeptide, the amino acid sequence of which is shown in SEQ ID NO. 9 or RPQGLPNNTASWFTA; and The HLA molecule is HLA-B*54:

01.

2. A novel coronavirus epitope polypeptide, characterized in that: The epitope polypeptide is derived from the amino acid sequence shown in SEQ ID NO. 3, and the amino acid sequence is shown in WMESEFRVY.

3. CD8 of a novel coronavirus + The T cell epitope polypeptide-HLA complex is characterized by The complex comprises the epitope polypeptide according to claim 2, preferably, the HLA is HLA-B*15, more preferably HLA-B15:

02.

4. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the complex according to claim 1 , the epitope polypeptide according to claim 2 or the complex according to claim 3 .

5. A carrier, characterized in that The vector comprises the nucleic acid molecule according to claim 4.

6. A delivery system, characterized in that The delivery system comprises the nucleic acid molecule of claim 4, wherein the delivery system comprises a virus, a virus-like particle, a plasmid or a nanoparticle.

7. A presenting cell, characterized in that The presenting cell comprises the complex of claim 1, the epitope polypeptide of claim 2, the complex of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, or the delivery system of claim 6, Preferably, the presenting cells are antigen presenting cells.

8. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the complex of claim 1, the epitope polypeptide of claim 2, the complex of claim 3, the nucleic acid molecule of claim 4, the vector of claim 5, the delivery system of claim 6, or the presenting cell of claim 7, and optionally includes a pharmaceutically acceptable carrier or adjuvant.

9. Use of the complex according to claim 1, the epitope polypeptide according to claim 2, the complex according to claim 3, the nucleic acid molecule according to claim 4, the vector according to claim 5, the delivery system according to claim 6, or the presenting cell according to claim 7, comprising one or more of the following: 1) Preparation of drugs or vaccines for the prevention or treatment of novel coronavirus; 2) Preparation of detection reagents for screening of patients infected with the new coronavirus; 3) Prepare reagents for detecting the level of specific T cell immune response in patients infected with the new coronavirus.

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