CN117430664B - Influenza A virus T cell epitope peptide and application thereof - Google Patents
Influenza A virus T cell epitope peptide and application thereof Download PDFInfo
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- CN117430664B CN117430664B CN202311379346.2A CN202311379346A CN117430664B CN 117430664 B CN117430664 B CN 117430664B CN 202311379346 A CN202311379346 A CN 202311379346A CN 117430664 B CN117430664 B CN 117430664B
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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
Technical Field
The invention belongs to the technical field of immunotherapy, and particularly relates to an influenza A virus T cell epitope peptide and application thereof.
Background
Seasonal influenza and its complications caused by influenza a virus (influenza A virus, IAV, hereinafter referred to as influenza a virus) remain a major threat to human health. According to statistics of world health organization, the annual seasonal influenza infection rate of the world is up to 5-15%, about 3 hundred million to 10 hundred million people are infected with influenza, and up to 25-50 ten thousand people die from influenza, and the death rate is up to 0.1%. Although most areas have influenza outbreaks in winter, for warm climates, there may be influenza at any time of the year. Therefore, influenza is still an important disease seriously threatening human health, especially for children, old people, pregnant women and immunocompromised people, and the enhancement of the research related to influenza still has great scientific and social significance.
Because of the lack of effective antiviral drugs, the treatment of influenza and its complications is mainly symptomatic. Vaccination is a relatively effective prophylactic measure at present, mostly inducing the active production of protective antibodies by the body. However, since influenza a virus is a single-stranded antisense RNA virus, it is highly susceptible to mutation, resulting in antigen drift (antigenic drift) and antigen shift (antigenic shift), disabling neutralizing antibodies, and thus rendering the vaccine ineffective. Correspondingly, the cellular immune response recognizes highly conserved presentation antigens, thus exhibiting a high cross-protective capacity between different influenza a virus subtypes. For example, the cd8+ T cell immune response specific for Influenza A Virus (IAV) Matrix Protein 1 (M1) 58 to gilfveftl at position 66 (M158-66 GIL, abbreviated GIL) was found to occupy up to 80% of the anti-influenza a virus immune response. Therefore, the research on the cellular immune response of the anti-influenza A virus is expected to better understand the mechanism of antiviral immunity, and provides theoretical and experimental basis for developing broad-spectrum efficient anti-influenza A virus vaccine.
In view of the continuous occurrence of current influenza A virus variation, the method provides a trigger for developing universal influenza A virus vaccines and broad-spectrum therapeutic drugs in the future. The universal vaccine is a broad-spectrum vaccine which can effectively protect the immune crowd even after the virus antigen is mutated. Therefore, development of a general vaccine and a broad-spectrum therapeutic drug that can still provide effective immune protection for vaccine immunized people after various variants of influenza a virus occur is urgent.
Studies show that the T cell immune response plays an important role in the antiviral defense of organisms after virus infection and in the immune pathological injury process of organisms, in particular CD8+ T cells, the antigen specific immune activity of which still exists after 11 years, and the important role of the CD8+ T cell immune response in the immune defense against influenza A virus and the important role of the CD8+ T cell immune response in vaccine development are demonstrated. The first step in the cd8+ T cell immune response is the specific recognition of epitope peptides presented by the virus-infected cells by T cells through their surface antigen recognition receptors. Therefore, the epitope peptide is an important key molecule for specifically recognizing viruses and playing an immune protection role by T cells, and is a key targeting molecule for immunodetection, immunotherapy and vaccine development.
CD8+ T cells recognize pMHC activation through T Cell Receptor (TCR), kill virus infected cells, clear virus, thereby exerting antiviral cellular immunity. Therefore, the identification of the epitope peptide capable of effectively activating the T cells can still induce effective T cell immune protection after the influenza A virus antigen is mutated, and is one of the keys for developing universal influenza A virus vaccines and broad-spectrum therapeutic medicaments.
Immune escape is the antagonism, blocking and suppression of the immune response of the body by immunosuppressive pathogens through their structural and non-structural products. Influenza a viruses, because of frequent and persistent mutations, produce a large number of variants, which severely threatens the immune barrier effect of the vaccine.
Disclosure of Invention
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.
Furthermore, the invention also provides a nucleic acid molecule encoding the epitope peptide.
The invention also provides a pMHC complex containing the epitope peptide.
Further, the pMHC complex is obtained by renaturation of HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the antigen epitope peptide.
Wherein the molar ratio of HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the epitope peptide is 1:2:10.
The invention further provides an antigen peptide-antigen presenting cell complex, which is an antigen presenting cell with the surface loaded with the antigen epitope peptide.
Wherein the antigen presenting cells are cd8+ T cells.
Preferably, the CD8+ T cells are T2-A2 cells.
The invention also provides application of the antigen epitope peptide, a nucleic acid molecule for encoding the antigen epitope peptide, a pMHC complex and/or an antigen peptide-antigen presenting cell complex in preparation of influenza A virus medicaments.
The invention also provides application of the antigen epitope peptide, a nucleic acid molecule for encoding the antigen epitope peptide, a pMHC complex and/or an antigen peptide-antigen presenting cell complex in screening influenza A virus drugs.
The invention further provides application of the antigen epitope peptide, a nucleic acid molecule for encoding the antigen epitope peptide, a pMHC complex and/or an antigen peptide-antigen presenting cell complex in preparation of influenza A virus vaccines.
The invention further provides application of the antigen epitope peptide, a nucleic acid molecule for encoding the antigen epitope peptide, a pMHC complex and/or an antigen peptide-antigen presenting cell complex in preparing a medicament for evaluating the vaccination effect of influenza A virus.
The invention has the beneficial effects that: the invention provides an influenza A virus T cell epitope peptide which 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, avoid immune escape of influenza A virus mutant strains, and can detect specific CD8+ T cells in influenza vaccinators and rehabilitators, thereby being applicable to research and development of influenza A virus universal vaccines, preparation, drug research and development and clinical treatment.
The invention also utilizes the influenza A virus CD8+ T cell epitope peptide to prepare a pMHC compound with a PE fluorescent channel, can be used for detecting antigen-specific T cells in peripheral blood of influenza A virus vaccinators and infection rehabilitators and is used for in-vitro T cell activation experiments, and the influenza A virus CD8+ T cell epitope peptide can be used for preparing general vaccines aiming at various influenza A virus mutant strains, immune detection related to influenza A viruses and broad-spectrum therapeutic medicaments. The influenza A virus CD8+ T cell epitope peptide is prepared into a pMHC complex with a PE fluorescent channel, or directly loaded to antigen presenting cells, so that T cells are activated, and the antigen presenting cell can be used for research and development of influenza A virus vaccines, preparation and drug research and development, and clinical treatment, and can be applied to:
1) Development and preparation of influenza a virus vaccine: after influenza A virus is mutated, the plurality of T cell epitopes can induce the body to generate immune response to generate antigen-specific T cells. Therefore, the T cell epitope is a candidate antigen epitope peptide of the influenza A virus universal vaccine.
2) Detecting whether it has cellular immune function against influenza a virus infection: the antigen-specific T cells of the influenza A virus are detected in the body of a person to be detected, which represents that the body has generated T cell immune function, and the intensity of the T cell immune function of the body and the possibility of infection to the influenza A virus can be evaluated according to the proportion of the antigen-specific CD8+T marked by the pMHC complex prepared into the fluorescent channel by the epitope peptide.
3) The effect after vaccination was evaluated: the detection of influenza a virus antigen specific T cells in the vaccinator, which represents that the body has developed T cell immune function, can be used to assess the likelihood of the body re-infecting influenza a virus.
4) Monitoring the condition: can be used for monitoring the change of the illness state of the close contact person, the medical observer, the suspected and the diagnosed patient.
5) And (3) prognosis judgment: poor prognosis is predicted if the body fails to mount a T cell immune response, or the proportion of antigen-specific T cells continues to decrease.
Drawings
FIG. 1, screening and identification of Influenza A Virus (IAV) T cell epitopes, A and B are T2 stability experiments. A is flow cytometry detection against β2m, and B is a statistical plot of A. C: results of the light-sensitive peptide displacement assay ELISA. Blank (Blank): no peptides; negative ctrl (Negative control): EBV virus peptide IVTDFSVIK; positive ctrl (Positive control), influenza a M1 peptide GILGFVFTL (below).
FIG. 2, preparation of pMHC complexes of HLA-A 2. A is the result of purifying the pMHC complex molecular sieve (Sephacryl S-200) in four different samples (numbered 1,2,3,4 in the figure), and numbers 1-4 represent the first through fourth peaks collected. B is a 15% SDS-PAGE result of Coomassie blue staining; m: protein molecular weight markers, numbers 1-4 in the figure correspond to peaks 1-4 in panel A, where red labeled 2 represents the peak of interest, heavy (a) and light (β2m) chains of HLA.
FIG. 3, immunogenicity identification of T cell epitopes of influenza A virus. A and B: detection of CD69 (upper row) and CD137 (lower row) molecules after 16 hours, B is a statistical plot of a.
FIG. 4, antigen-specific T cell production detected after mixed stimulation of epitopes; antigen-specific T cell production was detected by pMHC complexes with fluorescent channels after 0 day (top row) and 7 days (bottom row) of epitope mixed stimulation of healthy human cd8+ T cells, and B was a statistical plot of a.
Figure 5, epitope-stimulated T cell-mediated T2 apoptosis ratio. a-B (upper row): percentage of apoptosis of T2A2 cells stimulated for 7 days of culture; percent T2A2 apoptosis at day 7 (bottom row).
FIG. 6, detection of IFN-. Gamma. (upper row) and Granzyme B (lower row) expression in activated CD8+ T cells (A). B is a statistical graph of A.
FIG. 7, evaluation of the proportion of specific CD8+ T cells in vivo 14 days after influenza inactivated vaccination and 7 days after influenza A virus infection in convalescence patients; a-B: flow cytometry detected specific cd8+ T cells of the above 4 (antigen-specific cd8+ T epitopes that can be generated upon T cell activation) peptides from the HLA-A2+ influenza inactivated vaccine; C-D: flow cytometry examined the specific cd8+ T cells of the 4 above (antigen-specific cd8+ T epitopes that can be produced upon T cell activation) peptides from the HLA-A2+ influenza a virus infection healer.
Detailed Description
T2-A2 cells are an antigen presenting cell line expressing human MHC class I molecule HLa-A2 by recombinant genetic engineering techniques. Only effective epitope peptides can be presented, so that a stable pMHC complex is formed on the cell surface, and therefore, the antigen peptide can be used as artificial antigen presenting cells for stimulating T cells.
T cell epitope peptides alone cannot work and must be activated in the manner of pMHC complexes or antigen peptide-antigen presenting cell complexes. The invention utilizes MHC monomer and identified influenza A virus T cell epitope to carry out joint renaturation, thus preparing the pMHC complex. The identified antigen epitope peptide of the influenza A virus T cell is loaded on the surface of an antigen presenting cell (T2-A2 cell) to prepare an antigen peptide-antigen presenting cell complex, and then the prepared pMHC complex is used for labeling CD8+ T cells, so that the antigen epitope peptide can effectively activate T cells in peripheral blood of healthy people, has strong immunogenicity and can also effectively kill target cells carrying influenza A virus antigens. The pMHC complex with PE fluorescent channel assembled by the T cell epitope peptide can be detected in influenza A virus vaccinators and rehabilitators. The newly discovered antigen epitope peptide of the influenza A virus CD8+T cell can effectively induce T cell immunity, avoid immune escape of virus mutation, and can be used as a general vaccine or applied to immunotherapy and the like.
The invention will be further described in detail with reference to the drawings and specific examples, which are given solely for the purpose of illustration and are not intended to limit the scope of the invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.
Example 1 prediction of influenza A Virus HLA-A2 restriction epitope peptide
HLA-A2 restriction epitope prediction was performed using MHC class I molecular prediction tools (http:// tools. IEDB. Org/mhci /) provided by the United states NIH epitope database (IEDB). The volunteers recruited were vaccinated with influenza a split vaccines using epidemic strains or similar strains (northern hemisphere) of influenza a type 1, A3 and B (2021/2022) recommended by WHO and the european union. The main components of the vaccine are antigens (an A/Victoria/2570/2019 (H1N 1) pdm09; an A/Cambodia/e0826360/2020 (H3N 2) containing the following strains (2021/2022); a B/Washington/02/2019 (B/Victoria line) -like viruses). The most common H1N1 and H3N2 are selected to find the corresponding sequence numbers (EPI_ISL_417210; EPI_ISL_806547) in the Gisaid database for predicting the epitope protein. Finally obtaining 11 candidate CD8 of the influenza A virus specificity HLA-A2+ + T cell epitope peptides are shown in Table 1.
TABLE 1 influenza A virus T cell 11 epitope peptides
| Numbering device | Start position | End position | Length of | Sequence(s) | Sequence 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 | 24 | 32 | 9 | MQIAILITT | SEQ ID No.6 |
| F7 | 458 | 466 | 9 | FQGRGVFEL | 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 influenza A Virus HLA-A2 restriction epitope peptide
Candidate T cell epitope peptides predicted in example 1 (Nanjing gold sry biotechnology Co., ltd.) were prepared at a concentration of 20. Mu.M. Logarithmic growth state T2-A2 cells (T2-A2 is given by Anna Gil doctor of university of Massachusetts medical college (University of Massachusetts Medical School)) were seeded into 96-well plates, 10 per well 5 Blank wells, negative control peptide (EB virus, IVTDFSVIK), positive control peptide (influenza A M1 peptide, SEQ ID No.12: GILGFVFTL) and each synthetic candidate T cell epitope peptide were distributed, 3 duplicate wells per group, and a final volume of 200. Mu.L. After incubation for 4 hours at 37℃the cells were washed twice by centrifugation, labeled with FITC anti-human HLA-A2 (. Beta.2m) antibodies, incubated at 4℃for 30 minutes in the absence of light and then detected by flow cytometry. Experiments were performed 3 times in total.
The results are shown in FIGS. 1A and B, which show that 11 antigen polypeptides can be efficiently presented by antigen presenting cells to T cells, indicating that these peptides are T cell epitope peptides.
Example 3 detection of antigen Polypeptides to form pMHC complexes
The 11 influenza a virus T cell epitope peptides screened in example 2 were detected by ELISA. The specific operation is as follows:
100. Mu.L of 0.5. Mu.g mL was used at room temperature -1 Streptavidin was incubated on 96U-plates for 16-18 hours, washed 3 times with wash buffer (BioLegend, cat #420201, U.S.), and blocked with dilution buffer (0.5M Tris pH 8.0,1M NaCl,1% BSA,0.2% Tween 20) for 30 minutes at room temperature. The pMHC complex formed by the photoactive peptide (SEQ ID No.13: KILGFVFJV) and MHC ((BioLegend, cat #280003, US)) was used as HLA blank control, influenza A M1 peptide (GILGFVFTL) was used as positive control, and the EB virus peptide (SEQ ID No.14: IVTDFSVIK) was used as negative control. Then, 20. Mu.L of dilutions (400. Mu.M) of the 11 influenza virus T cell epitope peptides of example 1 and 20. Mu.L of Flex-T were each added, which were replaced with a 365nm three-way ultraviolet analyzer (Qian Bell, cat # 1903274) TM Dilutions of the monomer (200. Mu.g/mL) were made in 100. Mu.L to 96-well plates and incubated for 1 hour in a cell incubator at 37 ℃. After 3 washes with wash buffer, 100 μl of diluted HRP-binding antibody (BioLegend, cat#280303, us) was added and incubation was continued for 1 hour at 37 ℃ before washing. 100 μl of substrate solution (10.34 mL deionized water, 1.2mL 0.1m citric acid monohydrate/trisodium citrate dihydrate, pH 4.0, 240 μl 40mm abts,120 μl hydrogen peroxide solution) was added and incubated at room temperature in the dark for 8min. The reaction was quenched with 50. Mu.L of a termination solution (2% w/v oxalic acid dihydrate). Absorbance (OD value) was measured at a wavelength of 450nm with a microplate reader over 30 minutes.
The results are shown in FIG. 1C, which shows that all 11 antigen polypeptides can form pMHC complexes.
EXAMPLE 4 preparation of epitope peptide pMHC Complex monomer
The nucleotide sequences of HLA-A2 alpha chain and beta 2M are respectively constructed on a pET28a expression vector, transferred into escherichia coli BL21 (DE 3) for protein expression, nickel column (Biorad, cat#7324610, US) is used for purifying HLA-A2 protein (HLA-A 2 heavy chain a chain, HLA-A2 light chain beta 2M), the purified proteins HLA-A2 alpha chain protein, beta 2M protein and 11 epitope peptides in the table 1 in the example 1 are gradually dripped into 40mL of renaturation solution (5M urea, 0.4M arginine, 100mM Tris, 3.7mM cystamine, 6.3mM cysteamine and 2mM EDTA) according to the molar ratio of 1:2:10 respectively for renaturation, and the pMHC complex monomer of the epitope peptide is obtained.
The pMHC complex monomer was further purified by passing through DEAE ion exchange column, eluting with 0.5M NaCl, and collecting proteins according to the OD280nm uv absorbance peak. The proteins purified by DEAE ion exchange column were then purified by Superdex 75pg molecular sieve, eluted with PBS, and the different molecular weight proteins were collected according to the OD280nm UV absorbance peak. FIGS. 2A and 2B show that the antigen epitope peptide pMHC complex trimer (trimer of HLA-A2 heavy chain a chain+HLA-A 2 light chain β2m+antigen peptide) was obtained after purification.
Example 5 activation of T cells by influenza A Virus HLA-A2 restriction epitope peptide
T2 cells activate T cells by expressing HLa-A2 molecules (T2-A2, PMID:34414379; PMID:35194575; PMID:35116022; PMID: 37117789). Mononuclear lymphocytes (PBMCs) in peripheral venous blood of healthy volunteers were isolated and cd8+ T cells were further isolated. T2-A2 cells were labeled with CFSE and incubated with 11 different antigenic peptides from example 1 after 20 min treatment with 20. Mu.g/mL mitomycin.
The specific method comprises the following steps:
0.5X10 each well of a 96-well plate was seeded 6 0.5X10 of each CD8+ T cell was loaded with 11 epitope peptides of Table 1 6 T2-A2 cells were co-cultured (11 wells total) and co-stimulated with 1. Mu.g/mL anti-human CD28 antibody and 50IU/mL IL-2. 50IU/mL IL-2 and 20 mu M epitope peptide were supplemented every two days.
CD8+ T cells in peripheral venous blood of healthy volunteers were isolated and co-cultured with antigen polypeptide-loaded T2 cells, co-stimulated with 1. Mu.g/mL of anti-human CD28 antibody and 50IU/mL of IL-2, and the expression of T cell activating molecules CD69 and CD137 was detected after 16 hours of culture.
The pMHC complex monomer of 30 μl of the 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 after incubation at 4 ℃ for 30 minutes in the dark, 2.4 μl of blocking solution (1.6 μl of 50mM biotin (Thermo Fisher, cat#b20656, US)) and 198.4 μl of PBS were added to stop the reaction, and incubated overnight at 4-8 ℃ to obtain pMHC complex with PE fluorescence channel.
After 7 days of culture, the percentage of survival of T2-A2, the proportion of specific CD8+ T cells labeled with pMHC complex with PE fluorescent channels and the percentage of apoptosis marker Annexin V-APC on T2-A2 cells were calculated and the release of IFN-gamma and GZMB from specific CD8+ T cells was examined. Percentage of IAV-mediated T2 apoptosis after 7 days in culture with cd8+ T cells: CFSE labeled T2A2 as target cells, the number of residual target cells was detected and calculated as 50% of T2 cells minus the percentage of surviving cells.
As shown in FIGS. 3A and 3B, the 11 influenza A virus T cell epitope peptides selected in example 2 were all capable of activating T cells. Wherein, the T cell epitope peptide KADTKILFI corresponding to the T cell epitope peptide STICFFMQI, F corresponding to the T cell epitope peptide MQIAILITT, F corresponding to the F3 and the T cell epitope peptide IVLEANFSV corresponding to the F11 have strong ability to activate cd8+ T cells, as shown in fig. 4A and 4B. Meanwhile, the specific CD8+T activated by the 11 peptides can kill target cells, as shown in FIGS. 5A and 5B; specific cd8+ T cells released IFN- γ and GZMB as shown in fig. 6A and 6B.
Example 6 activation of T cells by influenza A Virus HLA-A2 restriction epitope peptide
PBMCs in peripheral venous blood of volunteers 14 days after the second needle of the influenza a virus-vaccinated inactivated vaccine and 7 days after the influenza a virus-infected healed were isolated and HLA subtypes thereof were identified, and HLA-a2 positive PBMCs samples were further stained with pMHC complex with PE fluorescent channel and CD8-APC antibody in example 5, and then subjected to on-stream detection.
The results are shown in fig. 7, which shows that pMHC complexes with fluorescent channels of F3, F6, F10 and F114 epitope peptides can recognize antigen-specific cd8+ T cells generated in influenza a virus vaccinators (fig. 7A and 7B) and influenza a virus infection healers (fig. 7C and 7D).
It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the scope of the present invention, and that other various changes and modifications can be made by one skilled in the art based on the above description and the idea, and it is not necessary or exhaustive to all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (7)
1. An influenza a virus T cell epitope peptide, characterized in that: the amino acid sequence is shown as SEQ ID No. 6.
2. A nucleic acid molecule encoding the epitope peptide of claim 1.
3. A pMHC complex containing the epitope peptide of claim 1, characterized in that: is obtained by renaturation of HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the antigen epitope peptide.
4. A pMHC complex according to claim 3, characterised in that: the molar ratio of HLA-A2 heavy chain, HLA-A2 light chain beta 2m and the epitope peptide is 1:2:10.
5. An antigenic peptide-antigen presenting cell complex, characterized by: an antigen presenting cell surface-loaded with the epitope peptide of claim 1; the antigen presenting cells are T2-A2 cells.
6. Use of an epitope peptide according to claim 1, a nucleic acid molecule encoding an epitope peptide according to claim 2, a pMHC complex according to claim 3 or 4 and/or an antigen peptide-antigen presenting cell complex according to claim 5 for the preparation of an influenza a virus medicament.
7. Use of an epitope peptide according to claim 1, a nucleic acid molecule encoding an epitope peptide according to claim 2, a pMHC complex according to claim 3 or 4 and/or an antigen peptide-antigen presenting cell complex according to claim 5 for the preparation of an influenza a virus vaccine or for the preparation of a medicament for assessing the vaccination effect of an influenza a virus.
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