CN117430664B - Influenza A virus T cell epitope peptide and application thereof - Google Patents

Abstract

The invention belongs to the technical field of immunotherapy, and particularly relates to an influenza A virus T cell epitope peptide and application thereof. The technical problem to be solved by the invention is that a T cell epitope peptide for a universal influenza A virus vaccine is not developed in the field of influenza A virus at present. The technical scheme of the invention is that the amino acid sequence of the T cell epitope peptide of the influenza A virus is shown as SEQ ID No. 6. The epitope peptide provided by the invention has strong immunogenicity and can induce antigen-specific CD8+T cells; it can assemble into pMHC complex with HLA-A2 heavy chain and HLA-A2 light chain beta 2m protein; or directly loaded to antigen presenting cells, can activate T cells, effectively induce T cell immunity, and can be used for research and development of influenza A virus universal vaccines, preparation and drug research and development and clinical treatment.

Description

Influenza A virus T cell antigen epitope peptide and its application

Technical Field

The present invention belongs to the technical field of immunotherapy, and in particular relates to an influenza A virus T cell antigen epitope peptide and an application thereof.

Background technique

Seasonal influenza and its complications caused by influenza A virus (IAV) are still a major threat to human health. According to statistics from the World Health Organization, the annual infection rate of seasonal influenza is as high as 5-15% worldwide, about 340 million to 1 billion people are infected with influenza, and up to 250,000 to 500,000 people die from influenza, with a mortality rate of 0.1%. Although influenza outbreaks occur in winter in most areas, influenza may occur at any time of the year in warm climates. Therefore, influenza is still a serious threat to human health, especially an important disease for children, the elderly, pregnant women and people with weakened immune function. Strengthening research on influenza still has great scientific and social significance.

Due to the lack of effective antiviral drugs, the treatment of influenza and its complications is mainly symptomatic treatment. Vaccination is currently a relatively effective preventive measure, which mainly induces the body to actively produce protective antibodies. However, since influenza A virus is a single-stranded negative sense RNA virus, it is very easy to mutate, resulting in antigenic drift and antigenic shift, which makes neutralizing antibodies lose their effectiveness, thus causing the vaccine to fail. Correspondingly, cellular immune responses recognize highly conserved presented antigens, and therefore show efficient cross-protection between different influenza A virus subtypes. For example, CD8+T cell immune responses specific to GILGFVFTL (M158-66 GIL, referred to as GIL) at positions 58 to 66 of influenza A virus (IAV) matrix protein 1 (Matrix Protein 1, M1) were found to account for up to 80% of anti-influenza A virus immune responses. Therefore, studying the cellular immune response against influenza A virus is expected to better understand the mechanism of antiviral immunity and provide a theoretical and experimental basis for the development of a broad-spectrum and efficient anti-influenza A virus vaccine.

Given the continuous emergence of influenza A virus mutations, it provides an opportunity for the future development of universal influenza A virus vaccines and broad-spectrum therapeutic drugs. Universal vaccines are a type of broad-spectrum vaccine that can effectively protect the immune population even after the virus antigens mutate. Therefore, it is urgent to develop universal vaccines and broad-spectrum therapeutic drugs that can still provide effective immune protection to the vaccine-immunized population after various mutations of influenza A virus.

Studies have shown that T cell immune response plays an important role in the body's antiviral defense and immune pathological damage after viral infection, especially CD8+T cells, whose antigen-specific immune activity still exists 11 years later, which shows the important role of CD8+T cell immune response in anti-influenza A virus immune defense and its important position in vaccine development. The first step of CD8+T cell immune response is that T cells specifically recognize antigen epitope peptides presented by virus-infected cells through antigen recognition receptors on their surface. Therefore, antigen epitope peptides are important key molecules for T cells to specifically recognize viruses and exert immune protection, and are key targeting molecules for immune detection, immunotherapy and vaccine development.

CD8+T cells recognize pMHC activation through T cell receptors (TCR), kill virus-infected cells, and clear viruses, thereby exerting antiviral cellular immunity. Therefore, identifying peptides that can effectively activate T cell antigen epitopes and still induce effective T cell immune protection after influenza A virus antigens mutate is one of the keys to developing universal vaccines and broad-spectrum therapeutic drugs for influenza A virus.

Immune escape is the process by which immunosuppressive pathogens antagonize, block and inhibit the body's immune response through their structural and non-structural products. Influenza A virus frequently and continuously mutates, producing a large number of variants, which seriously threaten the immune barrier effect of the vaccine.

Summary of the invention

The technical problem to be solved by the present invention is that currently in the field of influenza A virus, a T cell antigen epitope peptide for a universal influenza A virus vaccine has not yet been developed.

The technical solution of the present invention is an influenza A virus T cell antigen epitope peptide, the amino acid sequence of which is shown in SEQ ID No.6.

Furthermore, the present invention also provides a nucleic acid molecule encoding the antigen epitope peptide.

The present invention also provides a pMHC complex containing the antigen epitope peptide.

Furthermore, the pMHC complex is obtained by renaturing the HLA-A2 heavy chain, the HLA-A2 light chain β2m and the antigen epitope peptide.

Wherein, the molar ratio of HLA-A2 heavy chain, HLA-A2 light chain β2m and the antigen epitope peptide is 1:2:10.

The present invention further provides an antigen peptide-antigen presenting cell complex, which is an antigen presenting cell with the antigen epitope peptide loaded on its surface.

Wherein, the antigen presenting cells are CD8+T cells.

Preferably, the CD8+T cells are T2-A2 cells.

The present invention also provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and/or the antigen peptide-antigen presenting cell complex in the preparation of influenza A virus drugs.

The present invention also provides the use of the antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and/or the antigen peptide-antigen presenting cell complex in screening influenza A virus drugs.

The present invention further provides the use of the above antigen epitope peptide, the nucleic acid molecule encoding the antigen epitope peptide, the pMHC complex and/or the antigen peptide-antigen presenting cell complex in the preparation of influenza A virus vaccine.

The present invention further provides the use of the above antigen epitope peptide, nucleic acid molecule encoding the antigen epitope peptide, pMHC complex and/or antigen peptide-antigen presenting cell complex in the preparation of a drug for evaluating the effect of influenza A virus vaccination.

The beneficial effects of the invention are as follows: the invention provides an influenza A virus T cell antigen epitope peptide, which has strong immunogenicity and can induce antigen-specific CD8+T cells; it can be assembled into a pMHC complex with HLA-A2 heavy chain and HLA-A2 light chain β2m protein; or it can be directly loaded into antigen presenting cells, which can activate T cells, effectively induce T cell immunity, avoid immune escape of influenza A virus mutant strains, and specific CD8+T cells can be detected in influenza vaccine recipients and recovered patients, and can be used for the research and development, preparation and drug development and clinical treatment of influenza A virus universal vaccine.

The present invention also uses the influenza A virus CD8+T cell antigen epitope peptide to prepare a pMHC complex with a PE fluorescent channel, which can be used for the detection of antigen-specific T cells in the peripheral blood of influenza A virus vaccine recipients and infected patients, and for in vitro T cell activation experiments. These influenza A virus CD8+T cell antigen epitope peptides can be used to prepare universal vaccines for various influenza A virus mutants, influenza A virus-related immune detection and broad-spectrum therapeutic drugs. These influenza A virus CD8+T cell antigen epitope peptides are prepared into pMHC complexes with a PE fluorescent channel, or directly loaded into antigen presenting cells to activate T cells, which can be used for influenza A virus vaccine research and development, preparation and drug development, clinical treatment, and can be applied to:

1) Research and development and preparation of influenza A virus vaccine: After influenza A virus mutates, these multiple T cell epitopes can induce the body to produce immune responses and generate antigen-specific T cells. Therefore, these T cell epitopes are candidate antigen epitope peptides for universal influenza A virus vaccines.

2) Detection of cellular immune function against influenza A virus infection: The detection of influenza A virus antigen-specific T cells in the body of the subject indicates that the body has developed T cell immune function. The proportion of antigen-specific CD8+T labeled with pMHC complexes prepared into fluorescent channels based on the antigen epitope peptide can be used to evaluate the strength of the body's T cell immune function and the possibility of influenza A virus infection.

3) Evaluate the effect of vaccination: The detection of influenza A virus antigen-specific T cells in the vaccine recipient indicates that the body has developed T cell immune function. Based on the proportion of T cells, the possibility of reinfection with influenza A virus can be evaluated.

4) Monitoring of condition: It can be used to monitor changes in the condition of close contacts, medical observers, suspected and confirmed patients.

5) Prognosis: If the body cannot produce a T cell immune response, or the proportion of antigen-specific T cells continues to decrease, it indicates a poor prognosis.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Screening and identification of influenza A virus (IAV) T cell antigen epitopes. A and B are T2 stability experiments. A is the flow cytometric detection of anti-β2m, and B is the statistical graph of A. C: Results of photosensitive peptide displacement ELISA. Blank: no peptides; Negative ctrl: EBV virus peptide IVTDFSVIK; Positive ctrl: influenza A M1 peptide GILGFVFTL (the same below).

Figure 2. Preparation of pMHC complex of HLA-A2. A is the result of purification of pMHC complex by molecular sieve (Sephacryl S-200) with four different samples (numbered 1, 2, 3, 4 in the figure), and numbers 1-4 represent the first to fourth peaks collected. B is the result of 15% SDS-PAGE stained with Coomassie blue; M: protein molecular weight marker, numbers 1-4 in the figure correspond to peaks 1-4 in Figure A, where the red marked 2 represents the target peak, which is the heavy chain (a) and light chain (β2m) of HLA.

Figure 3. Identification of immunogenicity of influenza A virus T cell antigen epitopes. A and B: Detection of CD69 (upper row) and CD137 (lower row) molecules after 16 hours, B is the statistical graph of A.

Figure 4. The generation of antigen-specific T cells was detected after mixed stimulation of antigen epitopes; A shows the generation of antigen-specific T cells detected by the pMHC complex with a fluorescence channel 0 days (upper row) and 7 days (lower row) after mixed stimulation of healthy human CD8+T cells with antigen epitopes, and B is a statistical graph of A.

Figure 5. The percentage of T2 cell apoptosis mediated by epitope-stimulated T cells. A-B (upper row): the percentage of apoptosis of T2A2 cells after stimulation and culture for 7 days; (lower row) the percentage of apoptosis of T2A2 cells at the end of the 7th day.

Figure 6. Detection of the expression of IFN-γ (upper row) and Granzyme B (lower row) in activated CD8+ T cells (A). B is a statistical graph of A.

Figure 7. Evaluation of the proportion of specific CD8+T cells in the body 14 days after inoculation with inactivated influenza vaccine and 7 days after recovery from influenza A virus infection; A-B: flow cytometry detection of CD8+T cells specific to the above 4 peptides (antigen-specific CD8+T epitopes that can be produced when T cells are activated) in HLA-A2+ inoculated influenza vaccine recipients; C-D: flow cytometry detection of CD8+T cells specific to the above 4 peptides (antigen-specific CD8+T epitopes that can be produced when T cells are activated) in HLA-A2+ recovered from influenza A virus infection.

Detailed ways

T2-A2 cells are an antigen presenting cell line that expresses human MHC class I molecule HLA-A2 through recombinant genetic engineering technology. Only effective antigen epitope peptides can be presented by them, thereby forming a stable pMHC complex on their cell surface, so they can be used as artificial antigen presenting cells to stimulate T cells.

T cell antigen epitope peptides cannot work alone, and T cell activation must be performed in the form of pMHC complexes or antigen peptide-antigen presenting cell complexes. The present invention utilizes MHC monomers and identified influenza A virus T cell epitopes for joint renaturation to prepare pMHC complexes. The identified influenza A virus T cell antigen epitope peptides are loaded on the surface of antigen presenting cells (T2-A2 cells), antigen peptide-antigen presenting cell complexes are prepared, and then the prepared pMHC complexes are used to mark CD8+T cells, and it is found that the antigen epitope peptides can effectively activate T cells in the peripheral blood of healthy people, and have strong immunogenicity, and can also effectively kill target cells carrying influenza A virus antigens. The pMHC complex with PE fluorescence channel assembled by the T cell antigen epitope peptide can be detected in both influenza A virus vaccine recipients and recovered patients. It is proved that the newly discovered influenza A virus CD8+T cell antigen epitope peptide can effectively induce T cell immunity, avoid immune escape of virus mutations, and can be used as a universal vaccine or applied to immunotherapy, etc.

The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

Example 1 Prediction of HLA-A2 restricted antigen epitope peptides of influenza A virus

HLA-A2 restricted antigen epitopes were predicted using the MHC-I class molecule prediction tool (http://tools.iedb.org/mhci/) provided by the US NIH Antigen Epitope Database (IEDB). The recruited volunteers were vaccinated with influenza A virus split vaccine, which used the epidemic strains or similar strains (Northern Hemisphere) of influenza A virus (2021/2022) of type A1, A3 and B recommended by WHO and the European Union. The main components of the vaccine are antigens containing the following strains (2021/2022) (an A/Victoria/2570/2019(H1N1)pdm09; an A/Cambodia/e0826360/2020(H3N2); aB/Washington/02/2019(B/Victoria lineage)-like virus). The most common H1N1 and H3N2 were selected to find the corresponding sequence numbers (EPI_ISL_417210; EPI_ISL_806547) in the Gisaid database for antigen epitope protein prediction. Finally, 11 candidate influenza A virus-specific HLA-A2+ CD8 ⁺ T cell antigen epitope peptides were obtained, as shown in Table 1.

Table 1 Eleven antigen epitope peptides of influenza A virus T cells

serial number Starting position End position length sequence serial number F1 413 421 9 NMLSTVLGV SEQ ID No.1 F2 46 54 9 FMYSDFHFI SEQ ID No.2 F3 18 26 9 STICFFMQI SEQ ID No.3 F4 275 283 9 CLPACVYGL SEQ ID No.4 F5 373 381 9 AMDSNTLEL SEQ ID No.5 F6 twenty four 32 9 MQIAILITT SEQ ID No.6 F7 458 466 9 FQ SEQ ID No.7 F8 122 130 9 AIMDKNIIL SEQ ID No.8 F9 218 226 9 CVNGSCFTV SEQ ID No.9 F10 249 257 9 KADTKILFI SEQ ID No.10 F11 128 136 9 IVLEANFSV SEQ ID No.11

Example 2 Identification of HLA-A2 restricted antigen epitope peptides of influenza A virus

The candidate T cell antigen epitope peptides predicted in Example 1 were artificially synthesized (Nanjing GenScript Biotechnology Co., Ltd.) and configured to a concentration of 20 μM. T2-A2 cells in logarithmic growth state (T2-A2 was a gift from Dr. Anna Gil of the University of Massachusetts Medical School) were taken and planted in a 96-well plate, 10 ⁵ cells per well, and blank wells, negative control peptides (Epstein-Barr virus, IVTDFSVIK), positive control peptides (influenza A M1 peptide, SEQ ID No. 12: GILGFVFTL) and each synthetic candidate T cell antigen epitope peptide were distributed, with 3 replicate wells in each group, and the final volume was 200 μL. After incubation at 37°C for 4 hours, centrifugation and washing were performed twice, and the cells were labeled with FITC anti-human HLA-A2 (β2m) antibody, incubated at 4°C in the dark for 30 minutes, and then detected by flow cytometry. The experiment was performed 3 times in total.

The results are shown in Figure 1A and B, which show that all 11 antigen peptides can be effectively presented to T cells by antigen presenting cells, indicating that these peptides are T cell antigen epitope peptides.

Example 3 Detection of pMHC complex formation by antigenic polypeptides

The 11 influenza A virus T cell antigen epitope peptides screened in Example 2 were detected using the ELISA method. The specific operation is as follows:

At room temperature, 100 μL of 0.5 μg mL ^-1 streptavidin was incubated on 96 U-shaped plates for 16-18 hours, washed three times with washing buffer (BioLegend, Cat#420201, US), and blocked with dilution buffer (0.5 M Tris pH 8.0, 1 M NaCl, 1% BSA, 0.2% Tween 20) for 30 minutes at room temperature. The pMHC complex formed by the photosensitive peptide (SEQ ID No. 13: KILGFVFJV) and MHC ((BioLegend, Cat#280003, US)) was set as the HLA blank control, the influenza A M1 peptide (GILGFVFTL) was set as the positive control, and the Epstein-Barr virus peptide (SEQ ID No. 14: IVTDFSVIK) was set as the negative control. Then, 20 μL of the 11 influenza virus T cell epitope peptide dilutions (400 μM) and 20 μL of the Flex-T ^TM monomer (200 μg/mL) diluted with a 365 nm triple UV analyzer (Qilin Bell, Cat#1903274) were added to a 96-well plate and incubated in a cell culture incubator at 37°C for 1 hour. After washing 3 times with washing buffer, 100 μL of diluted HRP-conjugated antibody (BioLegend, Cat#280303, US) was added and incubated at 37°C for 1 hour, followed by washing. 100 μL of substrate solution (10.34 mL of deionized water, 1.2 mL of 0.1 M citric acid monohydrate/trisodium citrate dihydrate, pH 4.0, 240 μL of 40 mM ABTS, 120 μL of hydrogen peroxide solution) was added and incubated at room temperature in the dark for 8 min. The reaction was terminated using 50 μL of stop solution (2% w/v oxalic acid dihydrate), and the absorbance (OD value) was measured at a wavelength of 450 nm using a microplate reader within 30 minutes.

The results are shown in FIG1C , which show that all 11 antigen polypeptides can form pMHC complexes.

Example 4 Preparation of pMHC complex monomers of antigen epitope peptides

The nucleotide sequences of HLA-A2 α chain and β2m were respectively constructed into pET28a expression vectors and transformed into Escherichia coli BL21 (DE3) for protein expression. HLA-A2 protein (HLA-A2 heavy chain α chain, HLA-A2 light chain β2m) was purified using nickel column (Biorad, Cat#7324610, US) affinity chromatography. The purified protein HLA-A2 α chain protein, β2m protein and 11 antigen epitope peptides in Table 1 in Example 1 were gradually added dropwise to 40 mL of renaturation solution (5 M urea, 0.4 M arginine, 100 mM Tris, 3.7 mM cystamine, 6.3 mM cysteamine and 2 mM EDTA) at a molar ratio of 1:2:10 for renaturation to obtain pMHC complex monomers of antigen epitope peptides.

The pMHC complex monomers were further purified by DEAE ion exchange column, eluted with 0.5M NaCl, and the proteins were collected according to the OD280nm ultraviolet absorption peak. Subsequently, the proteins purified by DEAE ion exchange column were purified by Superdex 75pg molecular sieve according to the molecular weight, eluted with PBS, and different molecular weight proteins were collected according to the OD280 nm ultraviolet absorption peak. As shown in Figures 2A and 2B, after purification, the pMHC complex trimer of the antigen epitope peptide (trimer of HLA-A2 heavy chain a chain + HLA-A2 light chain β2m + antigen peptide) was obtained.

Example 5 Activation of T cells by HLA-A2 restricted antigen epitope peptides of influenza A virus

T2 cells activate T cells by expressing HLA-A2 molecules (T2-A2, PMID: 34414379; PMID: 35194575; PMID: 35116022; PMID: 37117789). Mononuclear lymphocytes (PBMCs) were isolated from the peripheral venous blood of healthy volunteers, and CD8+ T cells were further isolated. T2-A2 cells were labeled with CFSE, treated with 20 μg/mL mitomycin for 20 minutes, and then incubated with 11 different antigen peptides in Example 1.

The specific steps are:

0.5×10 ⁶ CD8+T cells were co-cultured with 0.5×10 ⁶ T2-A2 cells loaded with 11 epitope peptides in Table 1 (11 wells in total) in each well of a 96-well plate, and co-stimulated with 1 μg/mL anti-human CD28 antibody and 50 IU/mL IL-2. 50 IU/mL IL-2 and 20 μM epitope peptides were supplemented every two days.

CD8+T cells were isolated from the peripheral venous blood of healthy volunteers, co-cultured with T2 cells loaded with antigen peptides, and co-stimulated with 1 μg/mL anti-human CD28 antibody and 50 IU/mL IL-2. The expression of T cell activation molecules CD69 and CD137 was detected after 16 hours of culture.

30 μL of the pMHC complex monomer of the antigen epitope peptide obtained in Example 4 was mixed with 3.3 μL of PE streptavidin (BioLegend Cat#405203, US) in a 96-well plate, and incubated at 4°C in the dark for 30 minutes, then 2.4 μL of blocking solution (1.6 μL of 50 mM biotin (Thermo Fisher, Cat#B20656, US)) and 198.4 μL of PBS were added to stop the reaction, and the plate was incubated at 4-8°C overnight to obtain a pMHC complex with a PE fluorescent channel.

After 7 days of culture, the percentage of T2-A2 survival, the proportion of specific CD8+T cells labeled with pMHC complexes with PE fluorescence channels, and the percentage of apoptosis marker Annexin V-APC on T2-A2 cells were calculated, and the release of IFN-γ and GZMB by specific CD8+T cells was detected. After 7 days of culture with CD8+T cells, the percentage of IAV-mediated T2 apoptosis was determined: CFSE-labeled T2A2 was used as the target cell, and the number of residual target cells was detected, which was calculated by subtracting the percentage of surviving cells from 50% of T2 cells.

As shown in Figures 3A and 3B, the 11 influenza A virus T cell antigen epitope peptides screened in Example 2 can all activate T cells. Among them, the T cell antigen epitope peptide STICFFMQI corresponding to F3, the T cell antigen epitope peptide MQIAILITT corresponding to F6, the T cell antigen epitope peptide KADTKILFI corresponding to F10, and the T cell antigen epitope peptide IVLEANFSV corresponding to F11 have strong ability to activate CD8+T cells, as shown in Figures 4A and 4B. At the same time, the specific CD8+T activated by the above 11 peptides can kill target cells, as shown in Figures 5A and 5B; the specific CD8+T cells release IFN-γ and GZMB, as shown in Figures 6A and 6B.

Example 6 Activation of T cells by HLA-A2 restricted antigen epitope peptides of influenza A virus

PBMCs were isolated from the peripheral venous blood of volunteers 14 days after the second dose of inactivated influenza A virus vaccine and 7 days after recovery from influenza A virus infection, and their HLA subtypes were identified. HLA-A2 positive PBMCs samples were further stained with the pMHC complex with PE fluorescent channel and CD8-APC antibody in Example 5, and then detected by flow cytometry.

The results are shown in Figure 7, which show that the pMHC complexes with fluorescent channels of the four antigen epitope peptides F3, F6, F10 and F11 can recognize antigen-specific CD8+ T cells produced in recipients of influenza A virus vaccine (Figures 7A and 7B) and in patients who have recovered from influenza A virus infection (Figures 7C and 7D).

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims (7)

1. An influenza A virus T cell antigen epitope peptide, characterized in that: its amino acid sequence is shown in SEQ ID No.6. 2. A nucleic acid molecule encoding the antigenic epitope peptide according to claim 1. 3. A pMHC complex containing the antigen epitope peptide according to claim 1, characterized in that it is obtained by renaturing HLA-A2 heavy chain, HLA-A2 light chain β2m and the antigen epitope peptide. 4. The pMHC complex according to claim 3, characterized in that the molar ratio of HLA-A2 heavy chain, HLA-A2 light chain β2m and the antigen epitope peptide is 1:2:10. 5. An antigen peptide-antigen presenting cell complex, characterized in that it is an antigen presenting cell with the antigen epitope peptide according to claim 1 loaded on its surface; the antigen presenting cell is a T2-A2 cell. 6. Use of the antigenic epitope peptide according to claim 1, the nucleic acid molecule encoding the antigenic epitope peptide according to claim 2, the pMHC complex according to claim 3 or 4, and/or the antigenic peptide-antigen presenting cell complex according to claim 5 in the preparation of influenza A virus drugs. 7. Use of the antigenic epitope peptide according to claim 1, the nucleic acid molecule encoding the antigenic epitope peptide according to claim 2, the pMHC complex according to claim 3 or 4, and/or the antigenic peptide-antigen presenting cell complex according to claim 5 in the preparation of influenza A virus vaccines or in the preparation of drugs for evaluating the efficacy of influenza A virus vaccination.