T cell epitope peptide derived from influenza virus np protein and application thereof

Abstract

This invention discloses a group of T-cell epitope peptides derived from influenza virus NP proteins and their applications. The amino acid sequences of the T-cell epitope peptides are shown in SEQ ID NO:1-8, including four CD4⁺ T-cell epitope peptides and four CD8⁺ T-cell epitope peptides. These epitope peptides exhibit high conservation in the NP proteins of different influenza virus strains, and have been verified by in vitro stimulation experiments on mouse spleen cells infected with influenza virus to induce a significant IFN-γ positive response. This invention also provides epitope peptide compositions containing the epitope peptides, immunogenic compositions, and the application of the epitope peptides in the preparation of products for detecting, evaluating, or inducing anti-influenza T-cell immune responses. The T-cell epitope peptides provided by this invention have well-defined sequences, are easy to synthesize, and are low in cost, and can be used for evaluating anti-influenza cellular immune responses, detecting vaccine immunization efficacy, and designing multi-epitope vaccine antigens.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to T-cell epitope peptides derived from influenza virus NP protein and their applications. Background Technology

[0002] Influenza viruses, due to their frequent antigenic mutations, can cause seasonal epidemics and potential pandemics. Current seasonal influenza vaccines primarily rely on inducing a humoral immune response against the viral surface antigen hemagglutinin (HA) to provide protection. However, HA is prone to antigenic drift; when the vaccine strain does not match the circulating strain, the neutralizing effect of the vaccine-induced antibodies decreases, leading to fluctuations in protective efficacy.

[0003] Besides humoral immunity, T-cell immunity also plays a crucial role in combating influenza virus infection. In particular, CD8⁺ T cells can recognize target cells infected by the influenza virus and participate in the clearance of infected cells by secreting IFN-γ and releasing cytotoxic granules. Compared to HA, influenza virus nucleoprotein (NP) is relatively conserved among different influenza A virus strains and is an important target protein for screening anti-influenza T-cell epitopes.

[0004] Currently, some influenza virus T-cell epitopes have been included in databases such as IEDB, and some studies have used full-length NP proteins to stimulate immune cells and detect cytokine responses such as IFN-γ to evaluate anti-influenza cellular immune responses. However, existing technologies mostly focus on reported epitopes, and systematic validation of novel T-cell reactive epitopes in certain conserved regions of NP proteins is still lacking. In particular, NP-derived T-cell epitope peptides that simultaneously possess high sequence conservation, MHC-I binding potential, and have been shown to induce IFN-γ positive responses in mouse spleen cell infection experiments still await further discovery and application.

[0005] The applicant searched the IEDB database, PubMed literature database, and public patent database and found no publicly available reports of epitope peptide sequences identical to those described in this invention and having the same IFN-γ positive T cell response function.

[0006] The existing technology has the following main drawbacks: (1) The number of existing influenza virus T cell epitopes is limited and cannot cover all potential conserved T cell epitopes in NP proteins, which limits the development of novel anti-influenza cell immune evaluation epitopes and candidate vaccine epitopes.

[0007] (2) Candidate epitopes predicted solely by bioinformatics cannot directly prove that the epitope can be recognized by T cells produced by the body after infection, nor can they prove that it has the ability to induce functional cellular immune responses such as IFN-γ.

[0008] (3) When using full-length NP protein to stimulate immune cells, although the overall T cell response can be detected, it is difficult to identify specific functional epitopes. Moreover, the experimental cost is high and the sample requirement is large, which is not conducive to establishing an in vitro T cell immune evaluation system with clear sequence, low cost and standardization.

[0009] (4) The existing technology lacks a set of NP protein-derived T cell epitope peptides that have been verified by systematic conservation analysis, database search and IFN-γ response of infected mouse spleen cells, which makes it difficult to meet the needs of anti-influenza cell immune response detection, vaccine immune evaluation and multi-epitope antigen design.

[0010] (5) The prior art lacks experimentally validated multi-epitope peptide compositions derived from NP proteins, especially multi-epitope peptide compositions that can significantly induce CD8⁺ T cell activation in vitro and achieve activation levels superior to those stimulated by a single epitope peptide. Therefore, there is still a need for an NP-derived epitope peptide composition that has a well-defined sequence, is easy to synthesize, is easy to standardize and apply, and can be used for comprehensive evaluation of anti-influenza T cell immune responses in vitro. Summary of the Invention

[0011] The purpose of this invention is to overcome the shortcomings of the prior art and provide T-cell epitope peptides derived from influenza virus NP protein and their applications. These epitope peptides include eight NP protein-derived T-cell epitope peptides with no previously reported equivalent functions. They exhibit high conservation among NP proteins of different influenza virus strains and, as verified by in vitro stimulation experiments on infected mouse spleen cells, can induce an IFN-γ positive response.

[0012] To achieve the above objectives, the technical solution adopted by the present invention includes: In a first aspect, the present invention provides a T-cell epitope peptide derived from influenza virus NP protein, wherein the T-cell epitope peptide comprises any one of the polypeptides with amino acid sequences as shown in SEQ ID NO:1-8.

[0013] This invention provides a group of novel, highly conserved epitope peptides derived from influenza virus NP protein with a clear T-cell activation function. Each epitope peptide is derived from a different region of the influenza virus NP protein. Among them, the T-cell epitope peptides with amino acid sequences as shown in SEQ ID NO:1-4 are CD4⁺ T-cell epitopes (HTL epitopes) with a length of 15 amino acids, and the T-cell epitope peptides with amino acid sequences as shown in SEQ ID NO:5-8 are CD8⁺ T-cell epitopes (CTL epitopes) with a length of 9-10 amino acids.

[0014] Preferably, the T-cell epitope peptide further includes functionally equivalent variants of any polypeptide with an amino acid sequence as shown in SEQ ID NO:1-10, amino acid-modified peptides, or pharmaceutically acceptable salts.

[0015] Preferably, the functionally equivalent variant is a polypeptide that undergoes only conserved amino acid substitution, terminal truncation, and retains the activity of inducing T cell secretion of IFN-γ; the modified peptide includes N-terminal acetylated peptide, C-terminal amidated peptide, biotin-labeled peptide, and fluorescently labeled peptide.

[0016] In a second aspect, the present invention provides an influenza T-cell immune-stimulating epitope peptide composition, the composition comprising at least one of the T-cell epitope peptides.

[0017] Preferably, the composition comprises all of the T-cell epitope peptides; the T-cell epitope peptides are mixed in equal mass ratios.

[0018] The composition prepared by mixing the above eight epitope peptides in equal proportions can significantly induce CD8⁺ T cell CD69⁺ activation. Moreover, its T cell activation effect is not only significantly better than that of the PBS control group, but also significantly stronger than the stimulation effect of any single epitope peptide. This indicates that the composition has clear T cell immunostimulatory activity and can be used for in vitro detection or evaluation of anti-influenza T cell immune response.

[0019] Preferably, the composition is used to stimulate mammalian immune cells in vitro, detect at least one T cell activation marker among IFN-γ and CD69, and evaluate the level of anti-influenza T cell immune response; the immune cells are human peripheral blood mononuclear cells, mouse spleen cells, non-human primate spleen cells, or lymph node cells.

[0020] Thirdly, the present invention provides an immunogenic composition, wherein the active ingredient of the immunogenic composition is the single T cell epitope peptide, or the epitope peptide composition; and further comprises at least one of a pharmaceutically acceptable carrier, an immune adjuvant, a carrier protein, a nanodelivery carrier, a liposome, and a polymer particle.

[0021] Fourthly, the present invention provides a multi-epitope antigen construct comprising at least one T-cell epitope peptide, wherein the multi-epitope antigen construct is selected from: tandem fusion polypeptides, DNA / mRNA nucleic acid molecules encoding the epitope peptides, recombinant expression plasmid vectors, viral vectors, and prokaryotic / eukaryotic recombinant expression proteins.

[0022] Fifthly, the present invention provides the use of the single T cell epitope peptide and the epitope peptide composition in the preparation of reagents for in vitro detection or evaluation of anti-influenza T cell immune responses.

[0023] Preferably, the reagent is used to detect the secretion of IFN-γ by immune cells in the sample or to detect the proportion of CD69-positive activated T cells.

[0024] In a sixth aspect, the present invention provides the application of the single T-cell epitope peptide and the epitope peptide composition in the preparation of candidate epitope materials for anti-influenza multi-epitope antigen design.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) This invention provides a new set of T-cell epitope peptides derived from influenza virus NP protein. A search of IEDB, PubMed, and publicly available patent databases revealed no publicly available reports with the same epitope peptide sequences or the same IFN-γ positive function verification as those in this invention, indicating a strong basis for novelty.

[0026] (2) The epitope peptides of the present invention are derived from the relatively conserved NP protein region of influenza virus. They are highly conserved in H1N1, H3N2 and representative strains from different eras, and can be used for evaluation of anti-influenza cellular immune response and design of broad-spectrum multi-epitope antigens.

[0027] (3) The epitope peptides of the present invention have been functionally verified by an infection model. After the above-mentioned epitope peptides were used to stimulate mouse spleen cells infected with influenza virus in vitro, a significant IFN-γ response was generated, indicating that the epitope peptides can be recognized by the T cell immune response formed after infection and have a clear immunoreactivity.

[0028] (4) The epitope peptides of the present invention have well-defined sequences, are easy to synthesize chemically, have low cost, and are easy to standardize. Compared with full-length NP proteins or overlapping peptide libraries, the epitope peptides or epitope peptide compositions provided by the present invention can reduce detection complexity and are more suitable for in vitro detection of anti-influenza T-cell immune responses, evaluation of vaccine immunization effects, and screening of candidate epitopes.

[0029] (5) This invention provides an experimentally validated composition of NP protein-derived T cell epitope peptides. This composition consists of eight specific epitope peptides mixed in equal mass ratios, which can significantly induce the activation of CD8⁺ T cells and CD69⁺ cells in the spleen cells of influenza virus-infected mice, with an activation effect higher than that of the PBS control group and the groups stimulated by each single epitope peptide. Compared with single epitope peptides, this composition can integrate the immunostimulatory activity of multiple NP protein-derived T cell epitope peptides, exhibiting a stronger overall T cell activation effect, and can be used for comprehensive in vitro evaluation of anti-influenza T cell immune responses. Attached Figure Description

[0030] Figure 1 This is a diagram showing the conservation of epitope peptides for each candidate epitope peptide.

[0031] Figure 2 The figure shows the ELISPOT assay results for IFN-γ production in mouse spleen cells stimulated by candidate epitope peptides.

[0032] Figure 3The graphs show the evaluation of the activation effects of epitope peptides and their combinations on CD8⁺ T cells; (A) is a scatter plot from flow cytometry; (B) shows the activation levels of CD8⁺ T cells induced by each single epitope peptide; and (C) compares the activation levels of CD8⁺ T cells induced by an 8-peptide mixture with those induced by each single epitope peptide. Data are expressed as Mean ± SD, n = 3 / group, and analyzed using one-way ANOVA. * P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Detailed Implementation

[0033] To better illustrate the objectives, technical solutions, and advantages of this invention, the invention will be further described below with reference to specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0034] Unless otherwise specified, all raw materials used in the following examples are commercially available and commonly used materials; and unless otherwise specified, all experimental methods used are conventional experimental methods in the field.

[0035] Example 1. Screening and conservation verification of candidate T cell epitope peptides derived from NP protein Representative influenza virus NP protein sequences were selected as the analysis objects, and bioinformatics methods were used to predict potential CD4⁺ and CD8⁺ T cell epitopes. Based on the predicted results of the specific amino acid sequences of each epitope peptide, the corresponding virus strains of the NP protein, HLA sites, antigenicity, and IFN-γ inducibility, a group of candidate NP protein-derived T cell epitope peptides were screened, and their specific sequences are shown in Table 1.

[0036] Table 1. Information on T cell epitope peptides from which NP proteins originate. Further, NP protein sequences of representative influenza virus strains from different eras (H1N1, H3N2, BV, and BY) were collected. Multiple sequence alignment was performed on the regions containing candidate epitope peptides to analyze their conservation across different subtypes and eras of influenza virus strains. The results showed ( Figure 1 As shown in Table 1, the eight epitope peptides exhibit high conservation in the analyzed influenza virus NP protein sequences, suggesting their potential as broad-spectrum anti-influenza T-cell immune evaluation epitopes.

[0037] Example 2. Validation of IFN-γ secretion induced by a candidate single epitope peptide in infected mouse spleen cells A mouse model of influenza virus infection was established. Spleens were collected from mice on day 4 after infection with H1N1, H3N2, and BV, respectively. Single-cell suspensions of spleen cells were prepared, and the spleen cells were co-incubated with the eight candidate epitope peptides listed in Table 1. The IFN-γ response was detected by ELISPOT. The experimental setup included a candidate peptide stimulation group for infected mouse spleen cells, a peptide-free control group, a negative control peptide group, and a positive stimulation control group.

[0038] The results show that ( Figure 2 All eight candidate epitope peptides can induce IFN-γ responses in infected mouse spleen cells, indicating that these epitope peptides can be recognized by the T cell immune response formed after influenza virus infection and have functional immunostimulatory activity.

[0039] Example 3. Evaluation of the effect of epitope peptides and their compositions on inducing CD8⁺ T cell activation To further evaluate the T-cell stimulatory activity of the NP protein-derived T-cell epitope peptides shown in Table 1, mouse spleen cells were stimulated with influenza virus using each of the single epitope peptides shown in Table 1 and an octapeptide composition formed by mixing eight epitope peptides in equal mass ratios. The proportion of CD69⁺ cells in CD8⁺ T cells was detected by flow cytometry. In this experiment, the single epitope peptide stimulation group and the octapeptide composition stimulation group used the same total peptide concentration; the epitope peptides in the octapeptide composition were mixed in equal mass ratios. The experimental setup included a PBS control group, single epitope peptide stimulation groups, octapeptide composition stimulation groups, and a positive stimulation control group (PMA + ionomycin).

[0040] The results show that ( Figure 3 Compared with the PBS control group, each single epitope peptide shown in Table 1 induced CD69⁺ activation of CD8⁺ T cells to varying degrees, indicating that the above-mentioned single epitope peptides have definite T cell stimulating activity and can be used as single epitope stimulating antigens for in vitro evaluation of anti-influenza T cell immune responses. Further comparison showed that the 8-peptide equal-mass mixed composition significantly increased the proportion of CD69⁺CD8⁺ T cells, and its average activation level was significantly higher than that of the single epitope peptide stimulation groups.

[0041] The above results fully demonstrate that the eight NP protein-derived T cell epitope peptides provided by the present invention all have in vitro T cell stimulating activity; the epitope peptide composition formed by mixing the eight epitope peptides in equal mass ratios can integrate the immunoreactivity of multiple NP protein-derived T cell epitope peptides, induce a stronger overall CD8⁺ T cell activation effect, and is suitable for comprehensive evaluation of anti-influenza T cell immune responses in vitro.

[0042] Finally, it should be noted that the above embodiments are used to illustrate the technical solutions of the present invention and not to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A T-cell epitope peptide derived from the NP protein of influenza virus, characterized in that, The T-cell epitope peptide includes any one of the polypeptides with amino acid sequences as shown in SEQ ID NO:1-8.

2. The T-cell epitope peptide as described in claim 1, characterized in that, The T-cell epitope peptides also include functionally equivalent variants of any polypeptide with an amino acid sequence as shown in SEQ ID NO:1-8, amino acid-modified peptides, or pharmaceutically acceptable salts.

3. A composition of influenza T-cell immunostimulatory epitope peptides, characterized in that, The composition comprises at least one of the T-cell epitope peptides of claim 1 or 2.

4. The epitope peptide composition according to claim 3, characterized in that, The composition comprises all of the T-cell epitope peptides of claim 1.

5. The epitope peptide composition according to claim 4, characterized in that, All T-cell epitope peptides were mixed in equal mass ratios.

6. An immunogenic composition, characterized in that, The active ingredient of the immunogenic composition is the single T-cell epitope peptide of claim 1 or 2, or the epitope peptide composition of any one of claims 3-5; it also contains at least one of a pharmaceutically acceptable carrier, an immune adjuvant, a carrier protein, a nanodelivery carrier, a liposome, or a polymer particle.

7. A multi-epitope antigen construct, characterized in that, The construct comprises at least one T-cell epitope peptide as described in claim 1 or 2, wherein the multi-epitope antigen construct is selected from: tandem fusion polypeptides, nucleic acid molecules encoding the epitope peptides, recombinant expression plasmid vectors, viral vectors, and prokaryotic / eukaryotic recombinant expression proteins.

8. The use of the single T cell epitope peptide of claim 1 or 2, or the epitope peptide composition of any one of claims 3-5, in the preparation of reagents for in vitro detection or evaluation of anti-influenza T cell immune responses.

9. The application as described in claim 8, characterized in that, The reagents are used to detect the secretion of IFN-γ by immune cells in a sample or to detect the proportion of CD69-positive activated T cells.

10. The use of the single T-cell epitope peptide of claim 1 or 2, or the epitope peptide composition of any one of claims 3-5, in the preparation of candidate epitope materials for anti-influenza multi-epitope antigen design.

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