Specific t cell epitope peptide screened by novel coronavirus s1, n and m protein proteome and application thereof

Abstract

The application discloses a specific T cell epitope peptide screened by a whole proteome of S1, N and M proteins of a novel coronavirus and application, relates to the technical field of biological medicine, and its technical points are: the application provides a plurality of CD4+ T and CD8+ T cell epitope polypeptides of the novel coronavirus, and the genes, recombinant proteins or complexes containing the epitope polypeptides, sensitized antigen presenting cells and specific immune effector cells for the epitope polypeptides are used in the development of a novel coronavirus vaccine and the treatment of diseases.The polypeptide provided by the application is screened from the peripheral blood sample of a recovered person who breaks through infection of Omicron BA.5 after inactivated vaccine booster vaccination, and can safely and effectively induce CD4+ T and CD8+ T cell immune responses to the novel coronavirus protein, and has important guiding significance for the development of a novel coronavirus vaccine.

Description

[0001] This application is a divisional application of the invention patent application with application number CN2023114666337, entitled "Specific T cell epitope peptides for screening the whole proteome of novel coronavirus S1, N and M proteins and their applications", filed on November 7, 2023. Technical Field

[0002] This invention relates to the field of biomedical technology, specifically to specific T-cell epitope peptides obtained from the screening of the complete proteome of the S1, N, and M proteins of the novel coronavirus and their applications. Background Technology

[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 development of highly effective vaccines and therapeutic drugs must continue. Specific T-cell responses play a crucial role in the body's fight against viral infections. Therefore, studying the T-cell immune responses induced by viral infection, especially the characteristics of T-cell epitopes, is essential for understanding immune defense mechanisms and is a prerequisite for developing vaccines and immunotherapies.

[0004] The basis of T cell recognition and activation lies in the recognition of viral antigenic 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 vaccine and immunotherapy development. Furthermore, due to the high polymorphism of HLA, identifying SARS-CoV-2 T cell epitopes and their corresponding HLA types has the following important implications:

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

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

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

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

[0009] The SARS-CoV-2 genome includes structural protein-coding and non-structural protein-coding regions. The structural protein-coding regions primarily encode the Spike protein (S protein), Membrane glycoprotein (M protein), Nucleocapsid protein (N protein), and Envelope protein (E protein). The S protein comprises two subunits, S1 and S2. S1 mainly contains the receptor-binding domain (RBD), responsible for recognizing cellular receptors, while S2 contains essential elements required for membrane fusion. The non-structural protein-coding regions primarily include the open reading frame (ORF) 1a and ORF1b genes, encoding 16 non-structural proteins (NSP proteins), namely NSP1–16. In addition, SARS-CoV-2 contains nine accessory proteins: ORF 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] Current research on T-cell epitopes largely relies on predicting epitopes using computer software based on given HLA genotypes and protein sequences. Due to the complexity of MHC molecules, significant prediction biases occur when predicting certain epitope peptides, especially for CD4+ T-cell epitopes. Unlike CD8+ T-cell epitopes, which can be predicted with relatively high accuracy based on a given HLA genotype, research on CD4+ T-cell epitopes currently primarily relies on in vitro stimulation using peptide libraries. However, the limited availability of human PBMC samples severely restricts our research on CD4+ T-cell epitopes.

[0011] Identifying potential dominant CD4+ and CD8+ T cell epitopes in the Chinese population will contribute to a more comprehensive understanding of T cell responses in the human body after booster vaccination and natural infection. Currently, there are no reports of obtaining specific CD4+ and CD8+ T cell epitope peptides for the S1, N, and M proteins of the novel coronavirus through screening of a complete protein peptide library. Therefore, this invention aims to provide specific T cell epitope peptides for the S1, N, and M proteins of the novel coronavirus through complete proteome screening and their applications, in order to address the aforementioned problems. Summary of the Invention

[0012] The purpose of this invention is to solve the above-mentioned problems by providing specific T-cell epitope peptides for screening the whole proteome of the S1, N and M proteins of the novel coronavirus, and providing human HLA allele information that can recognize the epitope peptides.

[0013] To achieve the above objectives, the technical solution of the present invention is as follows:

[0014] The present invention provides novel coronavirus CD4+T and CD8+T epitope peptides, characterized in that the amino acid sequences of the epitope peptides are as 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 that encodes an epitope polypeptide as shown in any one of SEQ ID NO. 1-11.

[0019] The present invention also provides a vector comprising a nucleic acid molecule encoding an epitope polypeptide as shown in any one of SEQ ID NO. 1-11.

[0020] The present invention also provides a delivery system comprising a nucleic acid molecule encoding an epitope polypeptide as shown in any one of SEQ ID NO. 1-11, the delivery system comprising a virus, virus-like particle, plasmid, or nanoparticle.

[0021] The present invention also provides a presenting cell, the presenting cell comprising the epitope polypeptide described above, or the carrier 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 comprising the epitope polypeptide described above, or the carrier described above, or the delivery system described above, or the presenting cell described above, and optionally including a pharmaceutically acceptable carrier or adjuvant.

[0024] The present invention also provides the use of the epitope polypeptide, or the nucleic acid molecule, or the carrier, or the delivery system, or the cell described above in the preparation of drugs or vaccines for the prevention or treatment of novel coronavirus.

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

[0026] The present invention also provides the application of the epitope peptide in the preparation of screening and detection reagents for novel coronavirus infection or reagents for detecting the level of specific T-cell immune response in novel coronavirus infection.

[0027] In the above-described embodiments of the present invention, the specific T-cell epitope peptides also provide detection antigens for assessing the duration of immune protection against the novel coronavirus vaccine. Furthermore, the specific T-cell epitope peptides also provide detection antigens for evaluating immune responses induced by viral variants. Additionally, the specific T-cell epitope peptides also provide detection antigens for detecting the level of specific T-cell immune responses in patients infected with the novel coronavirus. Finally, the specific T-cell epitope peptides also provide antigens for in vitro induction of specific T cells. The present invention also provides various nucleic acids encoding the aforementioned specific T-cell epitope peptides.

[0028] Compared with existing technologies, the beneficial effects of this solution are:

[0029] The epitope peptides in this invention are derived from a full-protein peptide library of S1, M, and N proteins and can effectively induce an immune response against the S1, M, and N proteins of the novel coronavirus, which has important guiding significance for the development of its vaccine. Attached Figure Description

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

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

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

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

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

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

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

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

[0038] Figure 9 This refers to the screening process of N54(NO.11) single peptide in Example 1 of this 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 Implementation

[0040] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be described in further detail below with reference to the embodiments and accompanying drawings. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0041] It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other. The present invention will now be described in detail with reference to the embodiments.

[0042] The present invention provides a solution as described above, which provides novel coronavirus CD4+T and CD8+T cell epitope peptides, the amino acid sequences of which are shown in SEQ ID NO.1-11.

[0043] SEQ ID NO.1-11:

[0044]

[0045]

[0046] A nucleic acid molecule is also provided, which encodes the aforementioned epitope polypeptide. A carrier is also provided, comprising the aforementioned nucleic acid molecule. A delivery system is also provided, comprising the aforementioned nucleic acid molecule, including a virus, virus-like particles, plasmid, or nanoparticle. A presenting cell is also provided, comprising the aforementioned epitope polypeptide, or the aforementioned carrier, or the aforementioned delivery system. The presenting cell is an antigen-presenting cell. A pharmaceutical composition is also provided, comprising the aforementioned epitope polypeptide, or the aforementioned carrier, or the aforementioned delivery system, or the aforementioned presenting cell, and optionally including a pharmaceutically acceptable carrier or adjuvant. The use of the aforementioned epitope polypeptide, nucleic acid molecule, carrier, delivery system, or cell in the preparation of drugs or vaccines for the prevention or treatment of novel coronavirus is also provided. A detection reagent is also provided, comprising the aforementioned epitope polypeptide. The use of the provided epitope polypeptide in the preparation of a screening test reagent for novel coronavirus infection or a reagent for detecting the level of specific T-cell immune response in novel coronavirus infected individuals is also provided.

[0047] Example 1: Screening of S1, M, and N protein epitopes on CD4+ T cells and CD8+ T cells

[0048] 1.1 Peptide Library Information

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

[0050] Table 1. S1 protein peptide library sequences

[0051]

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

[0053]

[0054] 1.2 Screening strategies (e.g.) Figure 1 (As shown)

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

[0056] The second round of screening involved dividing the five sub-libraries of S1 into six secondary sub-libraries: A, B, C, D, E, and F. Similarly, the five sub-libraries of N protein were divided into six secondary sub-libraries. After the second round of screening, the M protein yielded single peptide results.

[0057] Third round of screening: S1 and N proteins require a third round of screening. Samples positive for secondary subcells are selected. 200,000 cells per well are placed in a 96-well circular plate, a single peptide is added, and after 6 hours of stimulation, intracellular staining is performed, and CD4 count is detected. + T cells and CD8 + Expression of IFNγ in T cells.

[0058] 1.3 Results of the screening

[0059] like Figure 2The image shows the screening process for S26 single peptide (NO.1). The screening results in Example 1 of this invention used I-37 samples that had undergone specific stimulation and amplification by the S1 peptide library. The proportion of IFNγ+CD4+ T cells in the total CD4+ T cells was detected (see...). Figure 2 A. Second round screening results of the S1-1 protein peptide library; B. Third round screening results 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 the total CD4+ T cells, significantly higher than other single-peptide stimulation groups in the S1-1F protein peptide library. Therefore, this sample responded to the S26 single peptide. Among the 51 recovered patients who broke through Omicron BA.5 infection after receiving an inactivated vaccine, four responded to S26: I-37, I-47, I-389, and I502.

[0060] like Figure 3 The image shows the screening process for S10 single peptide (NO.2). The screening results in Example 1 of this invention used I-389 samples that had undergone specific stimulation and amplification of the S1 peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected. The second round of screening results for the S1-1 protein peptide library showed a response in the S1-1B stimulation group. Figure 3 A). Therefore, a third round of screening was conducted on the S1-1B protein peptide library. The results showed that the proportion of IFNγ+CD8+T cells in the S10 stimulation group was 4.52% of the total CD8+T cells, while that in the control group was 0.22%. Therefore, this sample responded to the S10 single peptide (A). Figure 3 B). Among the 51 recovered patients who broke through Omicron BA.5 infection after receiving the inactivated vaccine, two responded to S10: I-13 and I-389.

[0061] like Figure 4 The image shows the screening process for S30 single peptide (NO.3). The screening results in Example 1 of this invention used sample I-229, which was 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 S30 single peptide). The results showed that the proportion of IFNγ+CD8+T cells in the S30-stimulated group was 4.71% of the total CD8+T cells, while it was 0.19% in the control group. Therefore, this sample responded to S30 single peptide. Among the 51 recovered patients who broke through OmicronBA.5 infection after receiving an inactivated vaccine, 6 responded to S30: I-229, I-57, I-62, I-54, I-73, and I-34.

[0062] like Figure 5The image shows the screening process for M10 (NO.4) and M42 (NO.8) peptides. The screening results in Example 1 of this invention used I-212 samples that had undergone specific stimulation and amplification by the M peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (first round screening results from the AM protein peptide library; second round screening results from the BM-3 protein peptide library, which showed that the sample responded to both M10 and M42 peptides). The results showed that the proportions of IFNγ+CD8+T cells in the M10 and M42 stimulation groups were 6.21% and 9.90% of the total CD8+T cells, respectively, while the control group was 1.01%. Therefore, this sample responded to both M10 and M42 peptides. Of the 51 recovered patients who broke through Omicron BA.5 infection after receiving the inactivated vaccine, six responded to M10: I-212, I-47, I-71, I-72, I-77, and I-7; only one responded to M42: I-212.

[0063] like Figure 6 The image shows the screening process for the M20 (NO.5) single peptide. The screening results in Example 1 of this invention used sample I-31, which was amplified by specific stimulation of the M peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (first round screening results of the AM protein peptide library and second round screening results of the BM-3 protein peptide library, showing that this sample responded to both M20 and M21 single peptides). The results showed that the proportion of IFNγ+CD8+T cells in the M20 stimulation group was 2.27% and 2.22% of the total CD8+T cells, respectively, significantly higher than other single peptide stimulation groups in the M-3 protein peptide library. Therefore, this sample responded to both M20 and M21 single peptides. Among the 51 recovered patients who broke through Omicron BA.5 infection after receiving an inactivated vaccine, two responded to M20: I-7 and I-31.

[0064] like Figure 7The diagram shows the screening process for M27 (NO.6) and M28 (NO.7) peptides. The screening results in Example 1 of this invention used I-20 samples that had undergone specific stimulation and amplification by the M peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (first round screening results from the AM protein peptide library; second round screening results from the BM-3 protein peptide library, showing that the sample responded to the M27 peptide; second round screening results from the CM-4 protein peptide library, showing that the sample responded to the M28 peptide). The results showed that the proportions of IFNγ+CD8+T cells in the M27 and M28 stimulation groups were 2.19% and 1.98% of the total CD8+T cells, respectively, significantly higher than other peptide stimulation groups in the M-3 and M-4 protein peptide libraries. Therefore, this sample responded to the M27 and M28 peptides. Of the 51 recovered patients who broke through Omicron BA.5 infection after receiving the inactivated vaccine, two responded to M27: I-20 and I-502; only one responded to M28: I-20.

[0065] like Figure 8 The diagram shows the screening process for N25 (NO.9) and N27 (NO.10) single peptides. The screening results in Example 1 of this invention used I-502 samples that had undergone specific stimulation and amplification by the N-peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (first round screening results from the AN protein peptide library; second round screening results from the BN-2 protein peptide library; third round screening results from the CN-2C protein peptide library, showing that the sample responded to the N25 single peptide; third round screening results from the DN-2D protein peptide library, showing that the sample responded to the N27 single peptide). The results showed that the proportions of IFNγ+CD8+T cells in the N25 and N27 stimulation groups were 1.07% and 10.4% of the total CD8+T cells, respectively, significantly higher than other single peptide stimulation groups in the N-2C and N-2D protein peptide libraries. Therefore, this sample responded to the N25 and N27 single peptides. Of the 51 recovered patients who broke through Omicron BA.5 infection after receiving the inactivated vaccine, 5 responded to N25: I-31, I-145, I-71, I-502, I-71, and I-72; and 10 responded 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 image shows the screening process for the N54 (NO.11) single peptide. The screening results in Example 1 of this invention used sample I-149, which was amplified by specific stimulation of the N-peptide library. The proportion of IFNγ+CD8+T cells in the total CD8+T cells was detected (results of the first round of screening from the AN protein peptide library; the second round of screening from the BN-4 protein peptide library; and the third round of screening from the CN-4B protein peptide library, which showed that this sample responded to both 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% of the total CD8+T cells, respectively, significantly higher than other single peptide stimulation groups in the N-4B protein peptide library. Therefore, this sample responded to both N53 and N54 single peptides. Among the 51 recovered patients who broke through OmicronBA.5 infection after receiving an inactivated vaccine, four responded to N54: I-149, I-65, I-66, and I-366.

[0067] Example 2: Pooled analysis of HLA-restricted epitopes of S1, M, and N proteins on CD4+ T cells and CD8+ T cells.

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

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

[0070]

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

[0072] Three dominant epitopes, M30, M34, and M36, were identified in the M protein, while three dominant epitopes, N22, N53, and N65, were identified in the N protein. Most N22 responders contained HLA-DPB1*05:01:01. Analysis of the dominant CD4+ T cell epitopes in the M protein revealed that many HLA subtypes could present M36.

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

[0074]

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

[0076] The dominant CD8+ T cell epitopes in the S1 protein are S8, S10, S30, S62, and S72. All six responders to S30- contained HLA-B*15. The dominant CD8+ T cell epitopes in the M protein are M10, M20, M27, M34, and M35. Samples of M10 responders included I-212, I-47, I-71, I-72, I-77, and I-7; all except I-72 contained 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. Dominant CD8+ T cell epitopes and HLA restriction in the N protein: The N protein contains a number of dominant 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 relevant literature. Except for I-51, all respondents to the N27 epitope contained HLA-A*11:01:01. Furthermore, sample I-149 responded to both N54 and N53; N53 contains both CD8+ T cell epitopes and CD4+ T cell epitopes.

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

[0081]

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

[0083] Bioinformatics prediction of MHC binding affinity is a key component of epitope identification methods. The binding affinity of any 9 peptides from S30 (HKNNKSWMESEFRVY) to HLA-B15:02 was further analyzed using https: / / services.healthtech.dtu.dk / services / NetMHCpan-4.0 / . The results showed that WMESEFRVY and HLA-B15:02 have strong binding affinity (as shown in Table 7 below).

[0084] Table 7: Analysis of the binding affinity between any 9 peptides in S30 and HLA-B15:02

[0085]

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

[0087] After I_229PBMCs were amplified for 9 days by stimulation with the S1 protein peptide library, they were stimulated with S30 and the WMESEFRVY (9mer) single peptide from S30, respectively, and the proportion of IFNγ+ T cells in CD8+ T cells was detected. Figure 10 As shown, the 9mer peptide can effectively activate specific CD8+ T cells. Therefore, the inventors of this application further clarified that the epitope sequence in the S30 peptide is WMESEFRVY.

[0088] In summary, the epitope peptides in the above embodiments of the present invention are screened from a full-protein peptide library of S1, M and N proteins, and can effectively induce an immune response against the S1, M and N proteins of the novel coronavirus, which has important guiding significance for the development of its vaccine.

[0089] The above specific embodiments are merely explanations of the present invention and are not intended to limit the present invention. After reading this specification, those skilled in the art can make modifications to these embodiments without contributing any inventive step, but as long as they are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. 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.

2. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the epitope polypeptide of claim 1.

3. A carrier, characterized in that, The carrier includes the nucleic acid molecule as described in claim 2.

4. The application of the epitope polypeptide of claim 1, the nucleic acid molecule of claim 2, or the vector of claim 3 in the preparation of a vaccine against the novel coronavirus.

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