H5n1 subtype aiv mhc b4-restricted t cell epitope peptide, screening method and application thereof

Abstract

The application belongs to the field of biology, and discloses three H5N1 subtype AIV MHC B4 restrictive T cell epitope peptides, the amino acid sequences of the epitope peptides are shown as SEQ ID NO. 1-SEQ ID NO. 3. The three epitope peptides are M 173‑181 , PB1 601‑609 and NS1 82‑90 , and the three polypeptides can significantly stimulate CD8 low+ and CD8 high+ cells to secrete IFN-γ. Meanwhile, the application also discloses the application of the three epitope peptides and a method for screening the three epitope peptides, and the screening method has the advantage of high accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biology, specifically relating to various H5N1 subtype AIV MHC B4-restricted T cell epitope peptides, their screening methods, and applications. Background Technology

[0002] The H5N1 subtype of highly pathogenic avian influenza virus (HPAIV) remains prevalent in several countries and continues to pose a threat to global public health. Ducks are a major host for AIV, and H5N1 AIV exhibits extremely high adaptability and transmissibility in ducks. To control the spread of H5N1 AIV, it is essential to reduce the risk of duck infection. Vaccination is a crucial strategy for preventing AIV infection. Currently, commercial vaccines are mainly inactivated vaccines, which stimulate the body to produce neutralizing antibodies against AIV. However, AIV can mutate through genetic recombination or antigenic drift, thereby evading antibody neutralization and reducing the protective effect of vaccines. Therefore, there is an urgent need to develop more effective avian influenza vaccines.

[0003] Numerous studies have already demonstrated that CD8 + T-cell responses can provide broader and more durable cross-immune protection against different strains. Dai et al. also found this in duck infection experiments using duck CD8... + T cell responses play a crucial role in clearing H5N1 infection. Therefore, based on CD8... + Avian influenza vaccines based on T-cell responses may become effective universal vaccines. CD8 + T-cell responses are mediated by antigenic epitopes presented by major histocompatibility complex (MHC) class I molecules to T-cell receptors (TCRs). MHC-I molecules are highly polymorphic and can encode different peptide-binding motifs, thus presenting a variety of peptide epitopes. Breeding MHC haplotypes can mitigate the interference of MHC polymorphism. For common MHC haplotypes in chickens, many studies have successfully identified restricted CD8+ T-cell epitopes targeting H5N1 AIV.

[0004] The applicant previously filed a patent ZL202310239432.7, H5N1 subtype AIV MHC B1 restricted T cell epitope peptide and its application.

[0005] They screened B1 haplotype duck MHC class I molecule-restricted T cell epitopes using H5N1AIV-specific duck memory PBMCs, and ultimately screened 12 epitope peptides;

[0006] There is currently no published literature on epitope peptides related to B4 haplotype ducks. Summary of the Invention

[0007] Based on this, the first objective of the present invention is to provide three H5N1 subtype AIV MHC B4-restricted T cell epitope peptides, the three epitope peptides being M... 173-181 PB1 601-609 and NS1 82-90 Three peptides can significantly stimulate CD8 low+ and CD8 high+ Cells secrete IFN-γ.

[0008] This invention also discloses the application of these three epitope peptides and methods for screening them. Specifically, this invention first infects B4 haplotype SPF ducks with H5N1 AIV (A / Duck / Guangdong / 383 / 2008). By detecting changes in cloacal and laryngeal viral shedding, serum antibody levels, T cell subtypes in PBMCs, and immune-related genes in PBMCs, it is confirmed that B4 haplotype ducks have been successfully infected with H5N1 to serve as subsequent experimental materials. Then, H5N1 subtype AIV-specific CD8 cells are successfully cultured and amplified in vitro. + T cells. MHC I-restricted T cell epitopes were predicted using the NetMHCpan database for predicting MHC class I restricted binding peptides. These epitopes were then co-cultured with duck memory PBMCs, and immunogenic peptide epitopes in H5N1 AIV were screened using qRT-PCR and ICS experiments. This method offers the advantage of high screening accuracy.

[0009] Terminology Explanation:

[0010] AIV: Avian influenza virus; MHC I: Major histocompatibility complex type I; PBMC: Peripheral blood mononuclear cells; APC: Antigen-presenting cells; SPF duck: Specific pathogen-free duck; CTL: Cytotoxic T cells; IFN-γ: Interferon-gamma; DPI: Days after challenge; EID50: Chicken embryo half-maximal infectious dose; FBS: Fetal bovine serum; ICS: Intracellular cytokine staining.

[0011] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0012] Three H5N1 subtype AIV MHC B4-restricted T cell epitope peptides, the amino acid sequences of which are shown in SEQ ID NO.1 to SEQ ID NO.3.

[0013] In addition, the present invention also discloses a nucleic acid molecule that encodes the epitope peptide as described above, as well as an expression cassette, recombinant vector or recombinant cell line containing the nucleic acid molecule as described above.

[0014] The use of the above-mentioned epitope peptide or the nucleic acid molecule or the expression cassette, recombinant vector or recombinant cell line in at least one of the following:

[0015] (1) Preparation of H5N1 subtype AIV vaccine;

[0016] (2) Preparation of H5N1 subtype AIV MHC B4 restricted T cells;

[0017] (3) Prepare products for monitoring H5N1 subtype AIV;

[0018] (4) Prepare kits for inducing and / or expanding T cells;

[0019] (5) Application in the preparation of antibodies against H5N1 subtype AIV;

[0020] (6) Prepare a kit for detecting H5N1 subtype AIV infection.

[0021] In addition, the present invention also discloses a vaccine comprising the epitope peptide as described above, or the nucleic acid molecule as described above, or the expression cassette, recombinant vector, or recombinant cell line as described above.

[0022] In the aforementioned vaccines, the vaccines also contain pharmaceutically acceptable carriers and / or excipients.

[0023] In addition, the present invention also discloses a kit comprising the epitope peptide as described above, or the nucleic acid molecule as described above, or the expression cassette, recombinant vector, or recombinant cell line as described above.

[0024] Another important aspect of this invention is that it provides a method for screening epitope peptides as described above, characterized by comprising the following steps:

[0025] Step 1: MHC I-restricted T cell epitopes were predicted from the H5N1 AIV protein sequence, yielding multiple peptides;

[0026] Step 2: Stimulate B4 haplotype duck memory PBMC cells with the peptides predicted in Step 1, and use real-time quantitative PCR to screen the mRNA expression level of IFN-γ in PBMC cells; peptides that can upregulate mRNA expression levels are screened out.

[0027] Step 3: Stimulate haplotype duck memory PBMC cells with the peptide B4 obtained in Step 2, and detect CD8+ using the ICS assay. + The expression of IFN-γ protein in T cells, if the peptide can stimulate CD8... + If the expression of IFN-γ protein in T cells is upregulated, then the polypeptide is the epitope peptide obtained through screening.

[0028] Compared to the applicant's earlier application, step 3 has been added: detecting CD8 through ICS experiments. + The expression of IFN-γ protein in T cells can be further screened, and the peptides screened in step 2 can be further functionally verified, which can significantly improve the screening accuracy.

[0029] In the above screening method, in step 3, the CD8 + T cells are CD8 low+ Cells and CD8 high+ cell.

[0030] The beneficial effects of this invention are as follows:

[0031] The three epitope peptides obtained by screening in this invention are M 173-181 PB1 601-609 and NS1 82-90 Three peptides can significantly stimulate CD8 low+ and CD8 high+ Cells secrete IFN-γ.

[0032] The epitope peptides obtained from the above screening, as well as the nucleotide molecules that edit these epitope peptides, can be used for various purposes, such as vaccines.

[0033] This invention also discloses the application of these three epitope peptides and methods for screening them. Specifically, this invention first infects B4 haplotype SPF ducks with H5N1 AIV (A / Duck / Guangdong / 383 / 2008). By detecting changes in cloacal and laryngeal viral shedding, serum antibody levels, T cell subtypes in PBMCs, and immune-related genes in PBMCs, it is confirmed that B4 haplotype ducks have been successfully infected with H5N1 to serve as subsequent experimental materials. Then, H5N1 subtype AIV-specific CD8 cells are successfully cultured and amplified in vitro. + T cells. MHC I-restricted T cell epitopes were predicted using the NetMHCpan database for predicting MHC class I restricted binding peptides. These epitopes were then co-cultured with duck memory PBMCs, and immunogenic peptide epitopes in H5N1 AIV were screened using qRT-PCR and ICS experiments. This method offers the advantage of high screening accuracy. Attached Figure Description

[0034] Figure 1A A trend graph showing the change in viral titer over time in pharyngeal swabs from ducks infected with H5N1 AIV haplotype B4.

[0035] Figure 1B A trend graph showing the change in viral titer over time in cloacal swabs from ducks infected with H5N1 AIV haplotype B4.

[0036] Figure 1A and Figure 1B The data came from three biological samples in each group; one-way ANOVA was used for statistical analysis.

[0037] Figure 2 This is a chart showing the level of HI antibodies in serum.

[0038] Figure 2 The data came from three biological samples in each group; one-way ANOVA was used for statistical analysis.

[0039] Figure 3A CD4 + T-cell sphere gate strategy;

[0040] Figure 3B CD8 + T-cell sphere gate strategy;

[0041] Figure 3C CD4 + T-ratio change chart;

[0042] Figure 3D CD8 + Chart showing changes in the proportion of T cells;

[0043] Figures 3A to 3D The data came from four biological samples in each group; statistical analysis was performed using the unpaired-t test.

[0044] Figure 4 Morphological changes of B4 haplotype duck PBMCs in vitro.

[0045] Figure 5A For flow cytometry staining zone gate strategy;

[0046] Figure 5B CD8 after H5N1 AIV stimulation + Chart showing changes in T cell proportion and cell number.

[0047] Figure 5B Statistical analysis was performed using the unpaired-t test.

[0048] Figure 6A A flow cytometry gating strategy for H5N1 AIV-stimulated proliferation of CFSE-labeled PBMCs;

[0049] Figure 6B A graph showing the proliferation of CFSE-labeled PBMCs stimulated by H5N1 AIV.

[0050] Figure 6BIn the middle, red represents unstimulated CFSE-labeled PBMC cells, blue represents CFSE-labeled PBMC cells cultured for 3 days after H5N1 stimulation, and orange represents CFSE-labeled PBMC cells cultured for 5 days after H5N1 stimulation. The sample numbers from left to right are #44, #47, and #1.

[0051] Figure 7 A graph showing the expression of IFN-γ gene mRNA after peptide pool stimulation of duck memory PBMCs for qRT-PCR analysis;

[0052] Figure 7 The data came from three biological samples, each in triplicate, for qPCR testing. Statistical analysis was performed using an unpaired t-test.

[0053] Figure 8 A graph showing the expression of IFN-γ gene mRNA after stimulation of duck memory PBMCs with a single 9-peptide from the positive peptide pool by qRT-PCR analysis.

[0054] Figure 8 The data came from three biological samples, each in triplicate, for qPCR testing. Statistical analysis was performed using an unpaired t-test.

[0055] Figure 9A To generate CD8 for IFN-γ + Gating strategies for T cell subsets;

[0056] Figure 9B For peptide-stimulated CD8 + T cell IFN-γ + The expressed flow cytometry detection plot;

[0057] Figure 9C To statistically analyze the CD8+ levels after peptide stimulation of B4 haplotype duck PBMCs low+ IFN-γ in cells + Presentation charts;

[0058] Figure 9D To statistically analyze the CD8+ levels after peptide stimulation of B4 haplotype duck PBMCs high+ IFN-γ in cells + A chart to illustrate the situation.

[0059] Figure 9C and Figure 9D Statistical analysis was performed using the unpaired-t test. Detailed Implementation

[0060] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0061] 1. Experimental Materials

[0062] 1.1 Background of the strain

[0063] A / Duck / Guangdong / 383 / 2008 (H5N1 subtype AIV) was isolated and preserved by the Infectious Disease Research Laboratory of the College of Veterinary Medicine, South China Agricultural University.

[0064] 1.2 Chicken embryos and laboratory animals

[0065] Two-week-old B4 haplotype SPF ducks were purchased from the National Poultry Laboratory Animal Resource Bank, and 9-11-day-old SPF chicken embryos were provided by Guangdong Xinxing Dahua Agricultural Poultry Egg Co., Ltd.

[0066] 1.3 Main Reagents

[0067] RPMI-1640 medium, L-glutamine (200 mM), 10000 UI of penicillin-dextrin antibiotics, FBS Australian fetal bovine serum, 0.25% Trypsin-EDTA trypsin, sodium pyruvate (100 mM), and non-essential amino acids (100×) were purchased from Gibco, USA; duck peripheral blood lymphocyte isolation kit and erythrocyte lysis buffer were purchased from Tianjin Haoyang Biotechnology Co., Ltd.; 2-mercaptoethanol (55 μM), concanavalin A (ConA), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich, USA; RNA extraction kit and ChamQ SYRBR qPCR Master Mix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; reverse transcriptase was purchased from Takara.

[0068] 1.4 Flow Cytometry Antibody

[0069] Mouse anti-Duck CD8 flow cytometry antibody was purchased from GeneTex; Mouse anti-Duck CD4 flow cytometry antibody was purchased from Bio-Rad; Goat anti-Mouse IgG2b-FITC flow cytometry antibody and Goat anti-Mouse IgG3-PE flow cytometry antibody were purchased from Southern Biotech. Goat anti-Mouse 488 flow cytometry antibody and CFSE-Labeling kit were purchased from Abcam. Mouse anti-Duck IFN-γ flow cytometry antibody was obtained from our laboratory.

[0070] 1.5 Preparation of Main Reagents

[0071] (1) 1% chicken red blood cells: 1% chicken red blood cells + 99% PBS, after preparation, store at 4℃ for later use.

[0072] (2) Flow cytometry buffer: 2% FBS + 98% PBS, after preparation, store at 4℃ for later use.

[0073] (3) Cell cryopreservation solution: 10% DMSO + 90% FBS, after preparation, store at 4℃ for later use.

[0074] (4) 10% double antibody PBS: 10% double antibody + 90% PBS, after preparation, store at 4℃ for later use.

[0075] (5) 1640 complete medium (RP-10): 10% FBS + 90% RPMI-1640, after preparation, store at 4℃.

[0076] (6) DMEM complete medium: 1% double antibiotics + 10% FBS + 89% DMEM medium, after preparation, store at 4℃.

[0077] (7) T cell culture medium: 10% FBS + 1% non-essential amino acids, 1% glutamine, 1% sodium pyruvate, 1% penicillin and antibiotics, β-mercaptoethanol + 90% RMPI 1640. After thorough mixing, the culture medium is divided into portions and stored at 4°C for use.

[0078] 2 Experimental Methods

[0079] 2.1 Median lethal dose (EID) of the virus in chicken embryos 50 Measurement

[0080] EID 50 Determination: Use PBS containing double antibodies at a ratio of 10... -1 Serial dilution to 10 -10 Take 10 -5 ~10 -10 Inoculation was performed using diluted virus solution. SPF chicken embryos aged 9–11 days were sterilized with alcohol, and each embryo was inoculated with 100 μL of diluted virus solution. The embryos were sealed with paraffin and incubated at 37°C. After 24–48 hours, the dead embryos were placed in a 4°C refrigerator for 6 hours to allow the blood vessels to constrict. Upon virus harvesting, the embryos were sterilized with alcohol, the air cell was opened, and 25 μL of allantoic fluid was pipetted to determine the hemagglutination titer. The EID was calculated using the Reed-Muench method. 50 .

[0081] Determination of hemagglutination titer (HA): Take a 96-well V-type reaction plate, add 25 μL of sterile PBS to wells 1-12 using a multi-channel pipette, add 25 μL of allantoic fluid to well 1, pipette 20 times, and then add 25 μL to well 2. Serially dilute up to well 11, discarding the 25 μL from well 11. Well 12 is used as a negative control. Add 25 μL of 1% chicken red blood cells to each well, vortex to mix, and incubate at room temperature for 30 min.

[0082] 2.2 Haplotype Duck Background Parameter Detection

[0083] Three days before the challenge, 2 mL of non-anticoagulated blood was collected from all haplotype ducks. After standing at room temperature, the serum was separated and the AIV antibody level was detected by hemagglutination inhibition test according to the latest version of the national standard (GB / T 18936-2020).

[0084] 2.3 H5N1 subtype AIV infection of B4 haplotype ducks

[0085] 2.3.1 H5N1 AIV infection of B4 haplotype ducks

[0086] The virus solution was diluted with sterile PBS containing antibiotics. B4 haplotype ducks were divided into two groups: an experimental group and a control group, with six ducks in each group. Inoculation was performed via eye drops or nasal drops. The experimental group ducks were inoculated with 200 μL of virus, representing a challenge dose of 10... 2.5 EID 50 The virus solution was divided into 200 μL and the control group was inoculated with 200 μL of sterile PBS. Pharyngeal and cloacal swabs were collected from haplotype ducks on days 3, 5, 7 and 9 after challenge, and peripheral anticoagulated and non-anticoagulated blood were tested.

[0087] 2.3.2 Detection of B4 haplotype duck viral shedding and antibody levels

[0088] A throat and cloacal swab was collected from each duck and placed in sterile PBS containing antibiotics and 30% glycerol, then stored at -80°C for later use. The EID was determined according to step 1. 50 This was used to assess the detoxification status of B4 haplotype ducks.

[0089] 2.3.3 Serum antibody level detection

[0090] Collect 0.5 mL of non-anticoagulated blood, let it stand at room temperature for 6 h, collect the separated serum into a 1.5 mL sterile EP tube, centrifuge at 2000 rpm at 4℃ for 10 min, then aspirate the supernatant and store at -20℃ for later use.

[0091] 2.3.4 Detection of peripheral blood T cell subtype changes in B4 haplotype ducks

[0092] Two mL of haplotyped duck anticoagulated blood was collected from the jugular vein. Peripheral blood lymphocytes (PBMCs) were isolated according to the instructions of the duck peripheral blood lymphocyte isolation kit. An appropriate amount of cells was then used for flow cytometry staining. The specific procedure is as follows: 2 × 10⁶ cells were collected from each duck. 6 Each cell line was incubated with mouse anti-duck CD4 monoclonal antibody or mouse anti-duck CD8 monoclonal antibody at the concentration recommended in the manufacturer's instructions, and incubated at 4°C in the dark for 30 min. After washing with PBS, FITC-labeled goat anti-mouse IgG antibody was added, and the cells were incubated at 4°C in the dark for 30 min. After staining, the cells were washed twice with PBS, and 200 μL of flow cytometry buffer was added to each tube for resuspending. Flow cytometry analysis was performed using a flow cytometer, and the results were analyzed using FlowJo software.

[0093] 2.4H5N1 subtype AIV-specific CD8 + Establishment of an in vitro T cell expansion and culture method

[0094] 2.4.1 In vitro stimulation of H5N1 subtype AIV with B4 haplotype duck CD8 + T cell expansion

[0095] (a) H5N1 AIV infection of antigen-presenting cells

[0096] The virus was inoculated into PBMCs at an MOI of 5, incubated for 1 hour, and then the T cell culture medium was replaced. The cells were incubated at 39°C for 5 hours. PBMCs infected with H5N1 AIV were used as antigen-presenting cells (APCs) in the experiment.

[0097] (b) APC stimulation of B4 haplotype duck PBMCs

[0098] The isolated PBMCs were divided into three groups and seeded in 48-well plates. After 6 hours of culture, the experimental groups were stimulated with APC, the positive control group was stimulated with ConA, and the negative control group received no treatment. The plates were incubated at 39°C. Cell morphology was observed daily, and cells were collected for trypan blue staining and counting to record changes in cell number.

[0099] (c) Flow cytometry analysis of changes in T cell proportion after H5N1 AIV stimulation

[0100] After culturing B4 haplotype duck PBMCs with H5N1 AIV for 7 days, cells were collected for counting. 1×10⁻⁶ cells were collected from both the experimental and control groups. 6 Transfer the cells to flow cytometry tubes for later use. After washing and centrifugation, perform flow cytometry staining with anti-Duck CD4 and CD8 antibodies to analyze CD8 levels in duck PBMCs after H5N1 AIV stimulation. + Pattern of T cell proportion changes.

[0101] 2.4.2 CFSE marker detection of B4 haplotype duck T cell proliferation

[0102] (a) CFSE-marked B4 haplotype duck PBMC

[0103] Before the experiment, sterile PBS and 1640 complete culture medium were preheated in a 37°C water bath. After washing the cells with PBS, they were resuspended in PBS containing CFSE and incubated in a 37°C water bath in the dark for 10 min. After incubation, the cells were centrifuged at 400g for 5 min, the supernatant was discarded, and the cells were washed with preheated 1640 complete culture medium. After centrifugation and discarding the supernatant, the cells were resuspended in T cell culture medium.

[0104] (b) APCs after H5N1 AIV infection and CSFE marker

[0105] H5N1 AIV was used to infect CFSE-labeled duck PBMCs with MOI=5. After incubation for 1 hour, the medium was replaced with T cell culture medium and incubated at 39°C for another 5 hours.

[0106] (c) CFSE-APC stimulation of CFSE-PBMC

[0107] CFSE-labeled duck PBMCs were divided into three groups and seeded in 48-well plates. After 6 hours, the experimental groups were stimulated with CFSE-APC, the positive control group was stimulated with ConA, and the negative control group received no treatment. The plates were incubated at 39°C, and cell morphology was observed daily. Cells were also harvested for flow cytometry analysis to record cell proliferation changes.

[0108] 2.5H5N1 subtype AIV specific B4 haplotype duck CD8 + Screening for T cell epitopes

[0109] 2.5.1 Synthesis, preservation and dissolution of 9 peptides

[0110] (a) Prediction and synthesis of 9-mer amino acid polypeptide sequences

[0111] The MHC I-restricted binding peptide prediction database (NetMHCpan-4.0-Services-DTUHealth Tech) was used to predict B4 haplotype duck MHC I-restricted T cell epitopes. A total of 64 high-scoring amino acid peptides were predicted, derived from influenza virus PB1, NS1, NS2, NP, M, and NA proteins. These peptides were synthesized by Genscript Biotech Ltd., with a purity >95%.

[0112] (b) Dissolution and preservation of 9mer amino acid peptides

[0113] The synthesized amino acid peptide powder was stored at -20°C. Before dissolution, the peptides were removed from the refrigerator and centrifuged at 500g for 5 minutes. According to the dissolution instructions, most of the peptides were dissolved in 100μL of DMSO and a small portion was dissolved in 100μL of sterile water. Each peptide was diluted to a storage concentration of 10μg / μL and frozen at -80°C for long-term storage.

[0114] 2.5.2 Screening for immunogenic 9mer peptides

[0115] After resuscitating B4 haplotype duck memory PBMCs, they were seeded into 48-well plates and incubated at 39°C in a 5% CO2 incubator for 6 hours. After the peptides thawed, peptide libraries were prepared by mixing 5-6 peptides from the same protein, resulting in 12 peptide libraries. 10 μL of the mixed peptide libraries was added to the cells cultured in the 48-well plates. Different control groups were set up: a negative control group was given 10 μL of dimethyl sulfoxide solution, a blank control group received no treatment, and a positive control group was stimulated with ConA. Simultaneously, BFA was added to block IFN-γ secretion. The mixture was then incubated at 39°C, and cell morphological changes were observed, with medium changes performed as needed. Cells stimulated for 6 hours were collected for later use.

[0116] 2.5.3 Detection of IFN-γ gene expression

[0117] The collected cells were centrifuged at 440g for 5 min, the supernatant was discarded, and RNA was extracted from the cells according to the RNA extraction kit instructions. RNA concentration was measured using a micro spectrophotometer, followed by quantification and reverse reversal. The reaction mixture was prepared and run on a 7500 Real Time PCR system, following the Vazyme qPCR enzyme program instructions. Experimental results were statistically analyzed using GraphPad Prism8 software.

[0118] Based on the expression of IFN-γ, a peptide library that can significantly stimulate T cells to produce IFN-γ was selected, and each peptide in the peptide library was further screened using the same method.

[0119] 2.5.4 ICS assay for IFN-γ protein expression

[0120] Take 1×10 6Cells stimulated with the peptide were placed in flow cytometry tubes, and mouse anti-duck CD8 antibody was added at the recommended concentration according to the manufacturer's instructions. The cells were incubated at 4°C in the dark for 30 min. After washing with PBS, FITC-labeled goat anti-mouse IgG2b antibody was added, and the cells were incubated at 4°C in the dark for 30 min. After washing once with flow cytometry buffer, 100 μL of fixative was added, and the cells were permeabilized at 4°C for 20 min. After permeabilization, the cells were washed once with flow cytometry buffer, stained with mouse anti-duck IFN-γ monoclonal antibody, and then stained with PE-labeled goat anti-mouse IgG3 antibody. After staining, the cells were washed twice with PBS, resuspended in 200 μL of flow cytometry buffer, and analyzed using a flow cytometer. The results were analyzed using FlowJo software.

[0121] 3 Results

[0122] 3.1 Detection of viral shedding in the laryngeal cloaca of ducks infected with H5N1 AIV haplotype B4

[0123] With 10 2.5 EID 50 After H5N1 subtype AIV infection of haplotype B4 ducks, pharyngeal and cloacal swabs were collected from three ducks at 3, 5, 7, and 9 days post-challenge to detect viral shedding. Figure 1A and Figure 1B As shown, in haplotype B4 ducks, the viral load was highest in laryngeal and cloacal swabs at 5 DPI after challenge, and began to decline at 7 DPI. The control group tested negative for viral shedding.

[0124] Figure 1A A trend graph showing the change in viral load in laryngeal swabs over time;

[0125] Figure 1B This is a graph showing the trend of viral load in cloacal swabs over time.

[0126] 3.2 Serum antibody level detection

[0127] Non-anticoagulated blood samples were collected from haplotyped ducks on days 3, 5, 7, 9, 14, and 28 post-infection to detect serum antibody levels. The results were as follows: Figure 2 As shown, serum antibody levels significantly increased at 5 DPI (P<0.001); continued to increase at 9 DPI (P<0.001); and remained stable after 14 DPI. Antibody tests in the control group were all negative. These results indicate that the decrease in viral load may be due to the clearance effect of increased antibody levels in haplotype ducks.

[0128] 3.4 Changes in the proportion of T cells in PBMCs of B4 haplotype ducks infected with H5N1 AIV

[0129] Figures 3A to 3DTo detect the immune response of duck B4 haplotype T cells after challenge, peripheral blood was collected from ducks, PBMCs were isolated, and staining was performed using flow cytometry.

[0130] Figure 3A CD4 + T-cell sphere gate strategy;

[0131] Figure 3B CD8 + The T-cell gating strategy was used, and the flow cytometry results were analyzed using FlowJo software.

[0132] like Figures 3C-3D As shown, after H5N1 AIV infection of haplotype B4 ducks, the experimental group's CD4 count was significantly lower at 5–9 DPI. + T cells (P<0.01 or P<0.05) and CD8 + The proportion of T cells (P<0.05, P<0.01, or P<0.001) in the experimental group was significantly higher than that in the control group. Specifically, at 7 DPI, the CD4+ level in the experimental group was significantly higher. + T cells and CD8 + The proportion of T cells was highest. The results indicate that the decrease in viral load in swabs at 7 DPI may be due to CD4+. + T cells and CD8 + The significant increase in the proportion of T cells played a role in virus clearance.

[0133] 3.5H5N1 AIV memory T cell in vitro culture

[0134] 3.5.1 In vitro stimulation of B4 haplotype duck PBMCs with H5N1 AIV for in vitro amplification and culture

[0135] (1) Morphological changes of B4 haplotype duck PBMCs after H5N1 AIV stimulation for proliferation

[0136] Duck PBMCs were cultured with H5N1 AIV-stimulated proliferation agent and their morphology was observed under a microscope at different time points. Figure 4 As shown, compared with the unstimulated group, the cells in the H5N1 subtype AIV stimulation group showed cluster growth on the 3rd day of culture, and the aggregation was obvious on the 5th day, gradually growing into cell clusters; while the control group always grew individually and did not show clustering, the number of dead cells increased, and finally only a small number of adherent cells survived.

[0137] (2) Flow cytometry analysis of CD8 values ​​after H5N1 AIV stimulation of B4 haplotype duck PBMCs for proliferation. + Changes in the proportion of T cells

[0138] On day 7 after H5N1 AIV stimulation, three samples were taken from each of the stimulated and unstimulated PBMCs for flow cytometry staining. Figure 5AThe streaming gating strategy shown was analyzed using FlowJo software, and the results are as follows. Figure 5B As shown, H5N1 AIV can stimulate memory CD8 in B4 haplotype duck PBMCs. + T cell proliferation, and CD8 in the H5N1 AIV stimulation group + The proportion of T cells (P<0.001) and the number of T cells (P<0.001) were both significantly higher in the group than in the unstimulated group.

[0139] 3.5.2 CFSE labeling detection of PBMC proliferation

[0140] To further verify the effect of H5N1 subtype AIV on T cell proliferation in B4 haplotype duck PBMCs, CFSE was used to label and detect B4 haplotype duck PBMCs by flow cytometry. Figure 6A The gate strategy shown was analyzed using FlowJo software. The results are as follows: Figure 6B As shown, compared with the control group, the experimental group showed a proliferation peak on the 3rd day after stimulation, and the fluorescence intensity decreased with the increase of proliferation number, further confirming that H5N1 subtype AIV can stimulate the proliferation of T cells in B4 haplotype duck PBMCs.

[0141] 3.6H5N1 AIV-specific CD8 + Screening for T cell epitopes

[0142] 3.6.1 Preparation of 9-mer amino acid peptides

[0143] The MHCⅠ-restricted binding peptide prediction database (NetMHCpan-4.0-Services-DTUHeaLth Tech) was used to predict MHCⅠ-restricted T cell epitopes in the H5N1 AIV protein sequence. The predicted peptides are shown in Table 1.

[0144] Table 1. Possible immunogenic peptides predicted from the database.

[0145]

[0146]

[0147]

[0148] 3.6.2 Detection of IFN-γ mRNA expression by real-time PCR

[0149] The synthesized 9 peptides were grouped into groups of 5-6 peptides on the same protein and mixed to prepare a peptide library. B4 haplotype duck memory PBMCs were stimulated separately. After 6 hours, cells were collected for RNA extraction and reverse transfection. IFN-γ expression was detected by qRT-PCR. Experimental results are as follows: Figure 7 Statistical analysis revealed that the mRNA expression of IFN-γ in cells stimulated by the five peptide pools pool-4, pool-5, pool-7, pool-9 and pool-10 was significantly upregulated (P<0.05 or P<0.01), indicating that there are immunogenic epitopes in the above peptides.

[0150] The peptide library that significantly stimulates IFN-γ expression in cells was selected, and each peptide in the library was further screened using the same method. Figure 8 As shown, M 173-181 ,PB1 635-643 ,PB1 601-609 ,PB1 389-397 ,NP 291-299 ,NP 25-33 NS2 50-58 NS1 153-161 (P<0.05) and PB1 583-591 NS1 134-142 NS1 82-90 (P<0.01) The expression levels of IFN-γ mRNA of 11 peptides were significantly upregulated after stimulating B4 haplotype duck PBMCs containing memory T cells, which initially showed that these 11 peptides were immunogenic.

[0151] 3.6.3 ICS verification of IFN-γ protein expression after peptide stimulation

[0152] B4 haplotype duck memory PBMCs were stimulated with the 11 peptides that tested positive by qRT-PCR described above. Cells were then subjected to ICS assays 6 hours after stimulation to detect CD8+ levels after peptide stimulation. + The expression of IFN-γ protein in T cells. Results are as follows: Figures 9A to 9D As shown, compared with the control group, 3 out of 11 peptides significantly stimulated CD8. low+ and CD8 high+ Cells secreted IFN-γ (P<0.01 or P<0.001), which were M 173-181 PB1 601-609 and NS1 82-90 (Table 2)

[0153] Table 2. Three subtypes of AIV CD8 targeting the H5N1 subtype of B4 haplotype ducks. + T cell epitope information

[0154]

[0155] Summarize:

[0156] 1. This invention studies the cellular immune response of B4 haplotype ducks to H5N1 AIV and identifies H5N1-specific CD8+. + The three epitope peptides obtained from T cell epitope screening are M 173-181 PB1 601-609 and NS1 82-90 Three peptides can significantly stimulate CD8 low+ and CD8 high+ Cells secrete IFN-γ.

[0157] 2. The epitope peptides obtained from the above screening and the nucleotide molecules that edit the epitope peptides can be used for various purposes, such as vaccines.

[0158] 3. The epitope peptide screening method of this invention has higher accuracy. This invention uses the MHC class I molecule restriction binding peptide prediction database (NetMHCpan) to predict MHC I-restricted T cell epitopes, then co-cultures them with duck memory PBMCs, and uses qRT-PCR and ICS experiments to screen for immunogenic peptide epitopes in H5N1 AIV. This method has the advantage of high screening accuracy.

[0159] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. The use of an epitope peptide with an amino acid sequence as shown in SEQ ID NO.2, or a nucleic acid molecule encoding the epitope peptide, or an expression cassette, recombinant vector, or recombinant cell line containing the nucleic acid molecule in at least one of the following: (1) Preparation of H5N1 subtype AIV vaccine; (2) Preparation of H5N1 subtype AIV MHC B4 restricted T cells; (3) Prepare products for monitoring H5N1 subtype AIV; (4) Prepare a kit for in vitro induction and / or expansion of T cells; (5) Prepare a kit for detecting H5N1 subtype AIV infection.

2. A vaccine, characterized in that, The vaccine comprises an epitope peptide with an amino acid sequence as shown in SEQ ID NO.2, or a nucleic acid molecule encoding the epitope peptide, or an expression cassette, recombinant vector, or recombinant cell line containing the nucleic acid molecule; the vaccine is an H5N1 subtype AIV vaccine.

3. The vaccine according to claim 2, characterized in that, The vaccine also contains pharmaceutically acceptable carriers and / or excipients.

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