Broad-spectrum epitope of H9N2 subtype avian influenza virus as well as construction method and application of broad-spectrum epitope

Abstract

The invention belongs to the technical field of biology, and relates to an antigen optimization method based on H9N2 subtype avian influenza virus hemagglutinin protein epitope modification. According to the invention, immune response effects of different key antigen regions of H9N2 subtype AIV HA protein are systematically evaluated through monoclonal antibody scheduling and antigen mapping analysis, and antigen regions A (sequences of S145D, K149T, D153G and H9 HA with signal peptides) having an optimization effect on current recombinant viruses are screened out. Experimental results show that the modified recombinant vaccine candidate strain R118-A has broad-spectrum neutralization titer and a protection effect on the group 1 virus strain and the group 2 virus strain, and the recombinant vaccine candidate strain R118-A has the broad-spectrum neutralization titer and the protection effect on the group 1 virus strain and the group 2 virus strain.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, particularly immunology and vaccinology. Specifically, it relates to an antigen optimization method based on the modification of hemagglutinin (HA) protein epitopes of H9N2 subtype avian influenza virus (AIV). This method systematically evaluates the immune response effects of different antigenic regions of the HA protein through monoclonal antibody (mAb) hemagglutination inhibition (HI) sequencing experiments and antigen mapping analysis. This allows for the selection of antigenic region A (S145D, K149T, D153G, the sequence of H9 HA with a signal peptide) for modification, thereby optimizing recombinant viral vaccine candidate strains and enhancing their broad-spectrum protective efficacy. This method can be widely applied to the antigen engineering design of AIV vaccines, providing new strategies and technical support for improving the broad-spectrum protective effect of AIV vaccines. Background Technology

[0002] The H9N2 subtype AIV is a widely circulating low pathogenic avian influenza virus (LPAIV) that often co-infects with other pathogens, leading to high mortality rates and severe economic losses in poultry. Multiple studies have found that the internal genes of human-infected H7N9 subtype AIV, the novel H5N2 HPAIV, and the novel H10N8 AIV all originate from the H9N2 subtype AIV. This indicates that the H9N2 subtype AIV serves as a "gene pool" for various novel reassortant AIVs, playing a significant role in AIV evolution and cross-species transmission. The H9N2 subtype AIV itself also has the potential for cross-species transmission; infection with H9N2 AIV has been detected in mammals such as pigs, dogs, raccoon dogs, and minks. Simultaneously, the H9N2 subtype AIV also has the potential for zoonotic transmission; not only have a few cases of human infection with H9N2 AIV been discovered, but the antibody positivity rate for H9N2 subtype AIV is significantly elevated in healthy blood donors and poultry workers. This indicates that the H9N2 subtype of AIV has expanded its host range, increasing the risk of zoonotic transmission and viral reassortment.

[0003] Whole-virus inactivated vaccines have become an important means of controlling H9N2 AIV due to their good safety, immunogenicity, and broad antigenic coverage. Currently, China has approved more than 20 inactivated vaccines related to the H9N2 subtype AIV. The efficacy of inactivated vaccines is highly dependent on the HA protein, which is the core of AIV antigenicity and vaccine efficacy. Amino acid mutations in the HA protein head are the main driving factor for antigenic drift and also promote the emergence of novel HA branches. Research by Zhang et al. shows that the H9N2 subtype AIV prevalent in recent years can be divided into two antigenic groups (branches): group 1 and group 2. Their antigenicity is dominated by mutations at 12 antigenic sites in the HA protein head, and in some regions, both antigenic groups coexist. Therefore, improving the broad-spectrum protective effect of vaccines is a significant challenge.

[0004] Researchers have developed various HA protein modification strategies to enhance vaccine efficacy. For example, Eggink et al. enhanced stem immunogenicity by glycosylation to shield dominant epitopes of the HA protein; Wang et al. achieved broad-spectrum protection by removing glycosylation sites in the HA protein to expose key epitopes; Broecker et al. optimized the antigenicity of the HA protein by designing a mosaic HA antigen by replacing the immunodominant region of the vaccine strain's HA protein with other subtype HA sequences. Poh et al. significantly enhanced the antigenicity of the HA protein through multi-point mutations and enabled the vaccine strain to achieve cross-strain protection in mice. Furthermore, Carter and Giles et al. modified the HA sequence using computer-optimized broad-response antigen technology and combined it with virus-like particle technology to develop HA protein vaccines against H1N1 and H5N1, inducing broadly neutralizing antibodies. Reverse genetics has also improved vaccine yield and immunogenicity through high-yield viral backbones (such as PR8).

[0005] Previous studies have shown that AIV antigenicity is mainly determined by key antigenic regions in the HA head, primarily including antigenic regions A, B, C, D, and E (using H3 HA as a reference). We found that the two major antigenic groups (group 1 and group 2) of the H9N2 subtype AIV are mainly determined by mutations in antigenic regions A (S145D, K149T, D153G, the sequence of the H9 HA with the signal peptide), region B1 (Q164R, N166D, N167G, A168N), region B2 (T197D, V198T, T200R, N201S), and region E (G90E). Currently, widely used H9N2 inactivated vaccines are mainly based on whole-virus inactivation technology, but due to antigenic drift of the HA protein, their immunoprotective effect is limited, making it difficult to provide broad-spectrum cross-protection. Therefore, optimizing the antigenic epitopes of the HA protein to improve the broad-spectrum protective ability of vaccines has become an urgent problem to be solved. Summary of the Invention

[0006] This invention aims to provide an antigen optimization method based on monoclonal antibody sequencing and antigen mapping to optimize the modification of H9N2 subtype AIV HA protein epitopes, thereby enhancing the broad-spectrum protective efficacy of H9N2 subtype AIV vaccine candidate strains and providing a new technical direction for the optimization of H9N2 subtype AIV vaccines.

[0007] The technical solution provided by this invention is as follows:

[0008] A broad-spectrum antigenic epitope of H9N2 subtype avian influenza virus, the amino acid sequence of which is shown in SEQ ID NO.4.

[0009] The present invention also provides the application of the above-mentioned broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus in the preparation of H9N2 subtype avian influenza vaccine.

[0010] The present invention also provides the application of the above-mentioned broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus in the preparation of H9N2 subtype avian influenza virus detection reagents.

[0011] The present invention also provides a gene fragment for constructing a recombinant vaccine strain of H9N2 subtype avian influenza, the nucleotide sequence of which is shown in SEQ ID NO.3.

[0012] Furthermore, the modification sites of the gene fragment relative to the HA plasmid are D145S, T149K, and G153D.

[0013] The present invention also provides a method for constructing the above-mentioned broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus, and uses monoclonal antibody spectral analysis and recombinant viral immune serum antigen mapping to screen the broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus.

[0014] Furthermore, using the pHW2000-W120118-HA plasmid as a template, site-directed mutagenesis of the target antigen region was performed via polymerase chain reaction.

[0015] Furthermore, the primers used are shown in SEQ ID NO.5 and 6.

[0016] Furthermore, recombinant viral antigens with smaller standard deviations and coefficients of variation of the Euclidean distances between coordinate points in the recombinant viral immune serum antigen mapping, and with the smallest enclosing circle radius, were selected.

[0017] Furthermore, the antigenicity changes of the antigenic epitopes and the immunoprotective effect were verified through MN and SPF chicken challenge protection experiments.

[0018] Beneficial effects

[0019] This invention provides a method for optimizing the HA protein epitope of H9N2 subtype AIV based on monoclonal antibody sequencing and antigen mapping, comprising the following steps: First, screening representative strains of H9N2 subtype AIV, and constructing vaccine candidate strains with single antigenic determinants or multiple antigenic determinants combined mutations using reverse genetics technology on the maternal strain 120118; determining vaccine candidate strains using monoclonal antibody sequencing analysis containing representative strains and antigen mapping analysis of recombinant virus immune serum; verifying the antigenic changes and immunoprotective effects of vaccine candidate strains through MN and SPF chicken challenge protection experiments; finally, screening out vaccine candidate strains with broad-spectrum protective efficacy.

[0020] To enhance the broad-spectrum protective efficacy of the vaccine, this invention constructs vaccine candidate strains by introducing multiple point mutations into the HA protein antigen region. Antigen mapping analysis is then used to determine the antigen distance by analyzing the reactivity of the candidate strains with different H9N AIV2 sera. Simultaneously, the binding affinity of the candidate strains to various monoclonal antibodies (mAbs) is evaluated, and the optimal candidate strain is selected. Finally, the broad-spectrum protective effect of the recombinant vaccine candidate strain R118-A is verified through serological experiments and SPF chicken immune challenge experiments.

[0021] This invention systematically evaluated the immune response effects of different key antigenic regions of the H9N2 subtype AIV HA protein through monoclonal antibody sequencing and antigen mapping analysis, and screened out antigenic region A (S145D, K149T, D153G, and the sequence of H9 HA with a signal peptide) that has an optimizing effect on the current recombinant virus. Experimental results show that the modified recombinant vaccine candidate strain R118-A exhibits broad-spectrum neutralizing titer and protective effect against both group 1 and group 2 strains. Attached Figure Description

[0022] Figure 1 The cross-HI reaction between R120118 and its recombinant virus and H9N2 monoclonal antibody;

[0023] Figure 2 The titer of homologous HI antibodies was measured from day 7 to day 35 after immunization with R120118 and its recombinant virus.

[0024] Figure 3Serum HI titers 14 to 35 days after immunization with R120118 and its recombinant virus: F98 (A), RTX (B), W100318 (C), W120118 (D), W616G2 (E), W110704 (F), and W05091 (G); Statistical significance analysis was performed using one-way analysis of variance (ANOVA) software with GraphPad Prism 7. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

[0025] Figure 4 Protective range of R120118 and its recombinant viruses in antigen mapping; (AE) seroprotective range 21 days after immunization with R120118, R118-A, R118-B1, R118-AE, and R118-AB1E; (FJ) seroprotective range 28 days after immunization with R120118, R118-A, R118-B1, R118-AE, and R118-AB1E.

[0026] Figure 5 The MN titer of mixed immune serum against H9N2 subtype AIV on day 21 (A) and day 28 (B) after immunization with R120118 and its recombinant virus;

[0027] Figure 6 MN titers of individual serum samples 28 days after immunization with R120118 and its recombinant virus against RTX (A), W100318 (B), W120118 (C), W616G2 (D), W110704 (E), and W05091 (F); ns: No experiment performed;

[0028] Figure 7 This study investigated the viral shedding of strains W100318(A), W120118(B), and W05091(C) in pharyngeal and anal swabs taken on days 3, 5, and 7 after immunization and challenge. Detailed Implementation

[0029] Example 1

[0030] Selection of representative H9N2 strains

[0031] In Zhang et al.'s study, H9N2 subtype AIV RTX and W100318 were classified into group 1 antigenic group, while W120118, W616G2, W110704, and W05091 were classified into group 2 antigenic group. These representative H9N2 strains will be used to evaluate the serological protective effect of subsequent recombinant virus immune sera. The W120118 strain belongs to the predominantly circulating group 2 antigenic group. Its HA gene can successfully rescue the virus together with other gene fragments in the reverse genetics system, exhibiting good stability. Therefore, it was selected as the maternal HA gene of the vaccine candidate strain for subsequent research. In the subsequent SPF chicken immunization protection experiment, W100318 (group 1) and W120118 (group 2) were used for challenge. Furthermore, to evaluate the protective ability of the vaccine against newly emerging strains, we also used W05091 (group 2), isolated in 2023, as the challenge strain. ).

[0032]

[0033] Example 2

[0034] Construction and monoclonal antibody profile analysis of recombinant H9N2 subtype AIVs containing different antigenic determinants

[0035] 2.1 Construction of Recombinant Viral Reverse Genetics Plasmid

[0036] Using the mutation primers in Table 2, and with plasmid pHW2000-W120118-HA as a template, site-directed mutagenesis of the target antigen region was performed via polymerase chain reaction (PCR). After homologous recombination, the PCR product was transformed into Trans1-T1 competent cells and inoculated onto LB (Luria-Bertani) solid medium plates containing ampicillin. Single colonies were then picked and sent to Shanghai Sangon Biotech Co., Ltd. for sequencing verification (sequencing was performed using primers on the vector: upstream primer T7: 5'-TAATACGACTCACTATAGG-3', downstream primer BGH: 5'-CAGGGTCAAGGAAGGCAC-3'). Positive clones with confirmed mutation sequences were preserved, and plasmids were extracted for subsequent recombinant virus reverse genetic rescue experiments. For plasmids containing mutations in two or more antigen regions, a stepwise construction strategy was adopted, introducing one mutation region at a time and sequentially stacking them to complete the construction. The HA plasmids of recombinant viruses R118-A, R118-B1, and R118-B2 were mutated using the corresponding primers listed in the table below. R118-AE, R118-AB1E, and R118-AB1B2E required multiple mutations using the corresponding primers. After transformation into Trans1-T1 E. coli competent cells, positive clones were selected and plasmids were extracted. A total of six mutant plasmids were ultimately constructed: pHW2000-W120118-HA-A, pHW2000-W120118-HA-B1, pHW2000-W120118-HA-B2, pHW2000-W120118-HA-AE, pHW2000-W120118-HA-AB1E, and pHW2000-W120118-HA-AB1B2E.

[0037]

[0038] H9N2 recombinant vaccine strain R120118:

[0039] HA nucleotide sequence:

[0040]

[0041] HA amino acid sequence:

[0042] METISLITILLAATVSYADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGMLCATSLGQPLILDTCTIEGLIYGNPSCDLSLEEKEWSYIVERPSAVNGLCYPGNVENLEELRSLFSSARSFQRIQIFPDTIWNVSYDGTSTACSGSFYRSMRWLTRKDGNYPTQDAQYTNNQGKNILFMWGINQPPTDDTQRSLYTKTDTTTSVATEEINRIFKPLIGPRPRVNGLMGRIDYYWSVLKPGQTLRIKSDGNLIAPWYGYILSGESHGRILKTDLKRGSCTVQCQTEKGGLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSGLVAGWYGFQHSNDQGVGMAVDRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVETRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGTNAAEDGKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYSTVASSLVIAMGFAAFLFWAMSNGSCRCNICI (SEQ ID NO.2).

[0043] H9N2 recombinant vaccine strain R118-A:

[0044] HA modification sites: D145S, T149K and G153D (H9 with signal peptide sequence)

[0045]

[0046] Modified amino acid sequence:

[0047] (SEQ ID NO.4).

[0048] H9N2 recombinant vaccine strain R118-B1:

[0049] HA modification sites: R164Q, D166N, G167N and N168Q (H9 signal peptide sequence). The modified nucleotide sequences relative to SEQ ID NO.1 have the following changes: position 491 is changed from G to A, position 496 is changed from G to A, positions 499 and 500 are changed from GG to AA, position 502 is changed from A to G, and position 503 is changed from A to C.

[0050] H9N2 recombinant vaccine strain R118-B2:

[0051] HA modification sites: D197T, T198V, R200T and S201N (H9 signal peptide sequence). The modified nucleotide sequences relative to SEQ ID NO.1 have the following changes: positions 589 and 590 are changed from GA to AC, positions 592 and 593 are changed from AC to GT, position 599 is changed from G to C, and position 602 is changed from G to A.

[0052] H9N2 recombinant vaccine strain R118-AE:

[0053] HA modification sites: E90G, D145S, T149K and G153D (H9 signal peptide sequence). The modified nucleotide sequence relative to SEQ ID NO.1 has the following changes: position 269 is changed from A to G, positions 433 and 434 are changed from GA to AG, position 446 is changed from C to A, and position 458 is changed from G to A.

[0054] H9N2 recombinant vaccine strain R118-AB1E:

[0055] HA modification sites: E90G, D145S, T149K, G153D, R164Q, D166N, G167N and N168Q (H9 signal peptide sequence). The modified nucleotide sequences relative to SEQ ID NO.1 are as follows: position 269 is changed from A to G, positions 433 and 434 are changed from GA to AG, position 446 is changed from C to A, position 458 is changed from G to A, position 491 is changed from G to A, position 496 is changed from G to A, positions 499 and 500 are changed from GG to AA, and positions 502 and 503 are changed from AA to GC.

[0056] H9N2 recombinant vaccine strain R118-AB1B2E:

[0057] HA modification sites: E90G, D145S, T149K, G153D, R164Q, D166N, G167N, N168Q, D197T, T198V, R200T, and S201N (H9 signal peptide sequence). Position 269 is mutated from A to G; positions 433 and 434 are mutated from GA to AG; position 446 is mutated from C to A; position 458 is mutated from G to A; position 491 is mutated from G to A; position 496 is mutated from G to A; positions 499 and 500 are mutated from GG to AA; positions 502 and 503 are mutated from AA to GC; positions 589 and 590 are mutated from GA to AC; positions 592 and 593 are mutated from AC to GT; position 599 is mutated from G to C; and position 602 is mutated from G to A.

[0058] 2.2 Rescue of Recombinant Viruses

[0059] 293T cells were fused with MDCK cells and cultured in six-well cell culture plates. Following the transfection reagent instructions, eight viral gene fragment plasmids linked to pHW2000 (including the HA gene plasmid, pHW2000-PR8-PB1, PB2, PA, NP, NS, M, and pHW2000-TX-NA) were diluted in DMEM medium. 600 ng of the polymerase gene plasmid and 300 ng of each of the other plasmids were used. After incubation for 15 min with appropriate transfection reagent, the plasmid-transfection reagent mixture was added dropwise to the cell culture plates and incubated at 37°C. Twelve h after transfection, the medium was changed to DMEM medium containing 2 μg / mL TPCK trypsin. Seventy-two h post-transfection, the supernatant from transfected cells was collected and inoculated into 9-11 day old SPF chicken embryos. Seventy-two h later, the allantoic fluid from the chicken embryos was collected, and the HA titer was determined to verify successful virus rescue. For HA-positive allantoic fluid, total viral RNA was extracted and reverse transcribed into cDNA. The resulting cDNA was used for AIV gene amplification, and various AIV gene fragments were amplified using Hoffman primers. The amplified products were subjected to 1.5% agarose gel electrophoresis, gel excision, and sequencing verification by Shanghai Sangon Biotech Co., Ltd. Results showed that the parent virus and seven recombinant viruses were successfully rescued. Their biological characteristics are shown in Table 3. The HA titer of the first-generation recombinant virus was between 7 and 10 log2, and it showed a TCID value in MDCK cells. 50 In 10 3.67 -10 5.5 between.

[0060]

[0061] NA nucleotides from A / chicken / Taixing / 10 / 2010 (GenBank: JN653652.1)

[0062] PB1 gene nucleotides from A / Puerto Rico / 8 / 1934 (GenBank: MH785017.1)

[0063] PB2 gene nucleotides from A / Puerto Rico / 8 / 1934 (GenBank: MH785018.1)

[0064] The PA gene nucleotide sequence from A / Puerto Rico / 8 / 1934 is indexed in GenBank as: MH785016.1

[0065] The NP nucleotide sequence for A / Puerto Rico / 8 / 1934 is available in GenBank: MH785014.1

[0066] NS gene nucleotides from A / Puerto Rico / 8 / 1934 (GenBank: MH785015.1)

[0067] The M gene nucleotide sequence for A / Puerto Rico / 8 / 1934 is MH785012.12.3 (GenBank).

[0068] 2.3 Monoclonal antibody profile of recombinant viruses

[0069] Monoclonal antibodies targeting strains from antigen groups 1 and 2 were selected and cross-HI assays were performed with recombinant viruses. The experimental results were visualized using a heatmap generated using OmicStudio (https: / / www.omicstudio.cn / tool). The preparation method of the monoclonal antibodies included: purifying the viral allantoic fluid using sucrose density gradient centrifugation; the resulting concentrated and purified protein served as the immunogen for immunizing mice. BALB / c mice were immunized at 200 ng / mouse, administered subcutaneously every two weeks. The first immunization used Freund's complete adjuvant, while subsequent immunizations used Freund's incomplete adjuvant. After four immunizations, whole blood was collected from the immunized mice, and serum was analyzed. Mice with high HI titers were selected for fusion. Three days before cell fusion, mice were immunized again via intraperitoneal injection with inactivated and purified virus. Subsequently, hybridoma cells were subcloned using limiting dilution, and monoclonal antibodies were screened using the HI assay.

[0070] The results showed that the R118-A mutation further increased the HI titer of group 1 monoclonal antibodies 2D6, 2E4, and 3E6, but also decreased the HI titer of group 2 monoclonal antibodies 4D7, 5D7, 1B10, and 5E5. Figure 1 The R118-B1 mutation increased the HI titers of group 1 monoclonal antibodies 2F7, 1B9, and 10G8, and slightly increased those of 3E6, 5D3, and 1C10, but decreased the titer of group 2 monoclonal antibody 1B10. The R118-B2 mutation slightly increased the HI titers of group 1 monoclonal antibodies 3E6 and 3F9, while decreasing the HI titers of group 2 monoclonal antibodies 1B10, 6C2, and 1C11. Notably, the R118-AB1E and R118-AB1B2E mutations had a more significant impact on the HI titer of monoclonal antibody 5E5 than the R118-A or R118-B1 mutations, possibly due to the combined effect of site A and B1 mutations. Similarly, the R118-AB1B2E mutation significantly decreased the HI titer of monoclonal antibody 7G4, while significantly increasing it for monoclonal antibodies 1C10 and 3F9. Figure 1 ).

[0071] In summary, the combined effects of different antigenic sites have a more significant impact on HI epitopes. However, in order to select antigens that are protective against both group 1 and group 2, we excluded recombinant viruses R118-AB1B2E and R118-B2, which only recognize monoclonal antibodies with a single antigenic group in the monoclonal antibody screening profile.

[0072] Example 3

[0073] Cross-HI assay and antigen mapping analysis of recombinant viral immune serum

[0074] 3.1 Preparation of recombinant viral immune serum

[0075] After passage of the recombinant virus, the allantoic fluid was titrated to 8 log2 HA titer with sterile PBS. Formaldehyde was then pre-diluted to a 2% concentration and thoroughly mixed with the virus solution to be inactivated, resulting in a final formaldehyde concentration of 0.15%. The resulting mixture was placed at 4°C and inactivated using a shaker for 24 h. The HA titer of the inactivated allantoic fluid was determined (Table 4). 4% Tween 80 was added and mixed thoroughly to form the aqueous phase. White spp. was used as the oil phase. The aqueous and oil phases were emulsified using a tissue homogenizer at a 1:3 ratio to prepare an oil-based vaccine. Three-week-old SPF chickens were immunized subcutaneously in the neck with a dose of 0.3 mL per chicken. Whole blood was collected from SPF chickens on days 7, 14, 21, 28, and 35 post-immunization. The blood was incubated at 37°C for 3 h, and the serum was separated and collected. The serum was then stored at -80°C for later use.

[0076]

[0077] 3.2 HI test of recombinant viral immune serum

[0078] On day 7 post-immunization, the HI titers of all recombinant viral immune sera against the homologous immunogens were below 5 log2. On day 14, the titers increased significantly, peaking between days 21 and 28, and slightly decreasing on day 35. R120118, R118-B1, and R118-AE showed the highest mean HI titers on day 21, at 10.17±0.41 log2, 10.00±1.10 log2, and 10.50±0.55 log2, respectively. The HI titers of R118-A and R118-AB1E immune sera peaked on day 28, at 10.50±1.05 log2 and 10.33±0.52 log2, respectively. Figure 2 ).

[0079] Cross-HI assays showed that for the F98, RTX, and W100318 strains in group 1, the antibody titer was highest after immunization with R118-AB1E at all time points, followed by R118-A, and then R118-AE. R120118 and R118-B1 consistently showed the lowest titers at all four time points. Figure 3 A, B, and C). For the W120118, W616G2, and W110704 strains in group 2, R120118, R118-A, and R118-AE had higher HI titers at all time points. Among the four time points, R118-B1 and R118-AB1E had the lowest titers. Figure 3 D, E, and F). For the W05091 strain in group 2, R118-A consistently showed the highest titer from day 14 to day 35, followed by R118-AB1E, while R118-B1 showed the lowest titer ( Figure 3 G).

[0080] Overall, R118-AB1E immune serum showed the highest cross-HI titer against group 1 antigens, followed by R118-A. For group 2 antigens, R120118 and R118-A immune serums showed higher HI titers.

[0081] 3.3 Protective range of recombinant viral immune serum in antigen mapping

[0082] The HI test results of the above recombinant viruses and the representative H9N2 strain were used to plot antigens using the R package Racmacs (http: / / www.antigenic-cartography.org), and the protective range of serum titers exceeding 6 log2 for each individual on days 21 and 28 was plotted using the ggplot package. The results showed that the immune sera of R120118 and R118-AE on day 28 failed to cover the F98+ group 1 antigen, while 4-5 sera of R118-A covered the F98+ group 1 antigen, and the serum of R118-AB1E almost covered every antigen of F98+ group 1. The group 2+W05091 antigen was located at the edge of the protective range of R118-AB1E, and other recombinant viruses could cover the group 2 strain antigens well. Figure 4 ).

[0083] Furthermore, we calculated the standard deviation (SD) and coefficient of variation (CV) of the Euclidean distance between the coordinates of each serum point group, as well as the radius of the minimum enclosing circle formed by these coordinates, to assess the protective characteristics of the serums. On day 21, the SD and CV of the Euclidean distance between the coordinates of R118-A and R118-AE serums were relatively small (0.12 and 30.37%, 0.23 and 42.12%, respectively). On day 28, the SD of R118 and R118-A were relatively small (0.23 and 0.27, respectively), while the CV of R118 and R118-AB1E were relatively low (23.70% and 45.79%, respectively). R118-A exhibited the smallest radius of the minimum enclosing circle (day 21: 0.60, day 28: 0.81). Furthermore, R118-A had a relatively large protective radius (day 21: 4.33±0.82, day 28: 4.50±1.05). This indicates that immune responses to the same immunogen can vary among different individuals (Tables 5 and 6).

[0084] Overall, R118-A serum showed good coverage of antigens from group 1 and group 2 strains, and the immune response to the antigens was relatively consistent among different individuals.

[0085]

[0086]

[0087] Example 4

[0088] MN titer determination of recombinant viral immune serum

[0089] 4.1 MN Experiment

[0090] MDCK cells were seeded into 96-well plates and allowed to adhere and grow into a monolayer. The viral load was diluted with DMEM to 2000 TCID. 50 / mL of viral working solution. Antibodies were serially diluted fourfold in new 96-well plates, with three replicates for each antibody. An equal volume of viral working solution was added, and the plates were incubated at 37°C for 1 h. After washing the culture plates containing MDCK cells with sterile PBS, the virus-antibody mixture was seeded at 100 μL / well and incubated for 1 h. Cells were then washed with PBS, and the medium was replaced with DMEM containing 2 μg / mL TPCK trypsin. The plates were then incubated for another 72 h. The culture supernatant was analyzed for HA (hypoallergenicity) to determine positive or negative results, and the half-maximal protective dose (50% protective dose, PD) of the serum was calculated using the Reed-Muench method. 50 (Regarding PD)50 Statistical significance analysis of the bar charts was performed using ANOVA and GraphPad Prism 7 software. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001).

[0091] 4.2 Crossover MN experiment between recombinant viral immune serum and H9N2 representative strain

[0092] Based on the changes in HI titer, mixed sera (from 6 individuals) from days 21 and 28, when the titer peak occurred, were selected for the MN experiment. The MN experiment results were consistent with the HI experiment results. For RTX and W100318 strains (group 1), the MN titers of R118-AB1E and R118-A immune sera were the highest, followed by R118-B1 and R118-AE, with the latter two generally showing lower titers. For W120118, W05091, and W110704 strains (group 2), the R118-A and R118-AE immune sera showed the highest neutralizing titers, followed by R120118; the titers of R118-B1 and R118-AB1E were low at all time points. ).

[0093] To further evaluate the MN titer of individual sera, supplementary experiments were conducted using two recombinant viruses with the best performance 28 days post-immunization, as well as maternal R120118 serum. For the RTX and W100318 strains (group 1), the neutralizing titers of R118-AB1E serum were 2.83±0.72 log4 and 4.38±0.14 log4, respectively, while the titers of R118-A serum were 3.10±0.52 log4 and 3.65±0.72 log4. The titers of both recombinant viruses were significantly higher than those of R120118, but there was no statistically significant difference between R118-AB1E and R118-A. For strains W120118, W616G2, W110704, and W05091 (group 2), the titers of R118-A serum were 5.92±0.96 log4, 5.79±0.58 log4, 5.67±0.98 log4, and 4.21±0.78 log4, respectively, while the titers of R118-AE serum were 5.21±0.49 log4, 5.29±0.56 log4, 5.75±1.03 log4, and 3.79±1.21 log4, respectively. However, no significant differences were observed between the two recombinant viruses and the parent strain for these antigens. ).

[0094] In summary, R118-A exhibited the broadest MN titer in both group 1 and group 2 strains.

[0095] Example 5

[0096] Recombinant virus immune challenge protection experiment

[0097] 5.1 Animal grouping and testing protocol

[0098] The vaccine was prepared using R120118, R118-A, and PBS. Three-week-old SPF chickens were vaccinated according to the above method. Three weeks post-immunization, the animals were challenged with the virus according to the groups listed in Table 7. The challenge strains included W100318 (group 1), W120118 (group 2), and W05091 (group 2). Chickens were intranasally inoculated with 200 μL of a vaccine containing 10... 6 EID 50 Viral fluid was collected, and throat swabs and cloacal swabs were collected on days 3, 5, and 7 after the challenge.

[0099]

[0100] 5.2 Swab testing methods

[0101] Laryngeal and cloacal swabs were collected on days 3, 5, and 7 after viral challenge, and the nucleic acid copy number was quantitatively determined by RT-qPCR of the M gene to count viral shedding.

[0102] 5.3 Protective effect of recombinant virus on SPF chickens after challenge with the virus and subsequent viral shedding

[0103] Following challenge with W120118, viral shedding in throat swabs decreased significantly in both the R120118 and R118-A groups compared to the PBS group on days 3 and 5. On day 3, the number of shedding animals (10 / 10) and the average viral shedding amount (9.52E+02 copies) in the R118-A group were slightly higher than in the R120118 group (6 / 10, 3.57E+02 copies). On day 5, the number of shedding animals in the R118-A group (7 / 10) was one more than in the R120118 group (6 / 10), but the average viral shedding amount was lower (R118-A group: 1.54E+02 copies, R120118: 2.32E+02 copies). By day 7, no animals in the R118-A group were shedding viral, while one animal in the R120118 group had an extremely low viral shedding amount. Therefore, although R118-A provides slightly weaker protection on day 3, by day 7, its protection against W120118 is comparable to that of R120118. A).

[0104] For W100318 challenge, throat swab results showed that on days 3 and 5, 10 out of 10 animals in both the R120118 and R118-A groups shed the virus. However, the average viral load in the R118-A group (day 3: 2.55E+03 copies, day 5: 1.55E+03 copies) was lower than that in the R120118 group (day 3: 2.46E+04 copies, day 5: 2.43E+03 copies). On day 7, 5 out of 10 animals in the R120118 group shed the virus, while only 2 out of 10 animals in the R118-A group shed the virus. Anal swab results showed that on days 3 and 5, the challenge control group shed only a small amount of virus, while no viral shedding was detected in the other immunized groups. These results indicate that R118-A is superior to R120118 in protecting against W100318. B).

[0105] For W05091 challenge, throat swab results showed that on days 3 and 5, 10 out of 10 animals in both the R120118 and R118-A groups shed the virus. However, the mean viral load in the R118-A group (day 3: 7.56E+03 copies, day 5: 6.18E+02 copies) was lower than that in the R120118 group (day 3: 1.72E+04 copies, day 5: 1.45E+03 copies). On day 7, 4 out of 10 animals in the R120118 group shed the virus, while 2 out of 10 animals in the R118-A group shed the virus. Anal swab results showed that on days 3 and 5, a few animals in the challenge control group shed the virus, while no viral shedding was detected in the other immunized groups. These results indicate that R118-A provides better protection against W05091 than R120118. C).

[0106] In summary, R118-A provides comparable protection against W120118 to R120118. For W100318 and W05091, R118-A reduces the amount of virus shed and the number of virus-shedding animals. These results indicate that the recombinant vaccine candidate strain R118-A provides good cross-protection against different strains.

[0107] Recombinant vaccine candidate strain R118-A was selected through a combination of monomeric spectral analysis and antigenic mapping. Its immune serum showed high MN titers against representative strains of H9N2 subtype AIV group 1 and group 2. In SPF chicken immunoprotection assays, R118-A provided good protection against both antigenic groups, indicating that this strain has broad-spectrum cross-protection potential and is a promising vaccine candidate strain for disease control.

Claims

1. A broad-spectrum antigenic epitope of H9N2 subtype avian influenza virus, characterized in that, The amino acid sequence of the broad-spectrum antigenic epitope of the H9N2 subtype avian influenza virus is shown in SEQ ID NO.

4.

2. The application of the broad-spectrum antigenic epitope of the H9N2 subtype avian influenza virus as described in claim 1 in the preparation of H9N2 subtype avian influenza vaccine.

3. The application of the broad-spectrum antigenic epitope of H9N2 subtype avian influenza virus as described in claim 1 in the preparation of H9N2 subtype avian influenza virus detection reagent.

4. A gene fragment for constructing a recombinant vaccine strain for the H9N2 subtype avian influenza, characterized in that, The nucleotide sequence of the gene fragment is shown in SEQ ID NO.

3.

5. The gene fragment for constructing a recombinant vaccine strain for H9N2 subtype avian influenza according to claim 4, characterized in that, The modified sites of the gene fragment relative to the HA plasmid are D145S, T149K, and G153D.

6. The method for constructing broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus according to claim 1, characterized in that, We used monoclonal antibody spectral analysis and recombinant viral immune serum antigen mapping to screen for broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus.

7. The method for constructing broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus according to claim 6, characterized in that, Using the pHW2000-W120118-HA plasmid as a template, site-directed mutagenesis of the target antigen region was performed by polymerase chain reaction.

8. The method for constructing broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus according to claim 6, characterized in that, The primers used are shown in SEQ ID NO.5 and 6.

9. The method for constructing broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus according to claim 7, characterized in that, The antigenicity changes of antigenic epitopes and the immunoprotective effect were verified through MN and SPF chicken challenge protection experiments.

10. The method for constructing broad-spectrum antigenic epitopes of H9N2 subtype avian influenza virus according to claim 7, characterized in that, Choose recombinant viral antigens with smaller standard deviations and coefficients of variation of the Euclidean distances between coordinate points in the recombinant viral immune serum antigen plot, and with the smallest enclosing circle radius.

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