A monoclonal antibody that broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus and its application

Abstract

The present invention discloses a monoclonal antibody that broadly binds to the HA protein of the multi-branched H5Nx subtype avian influenza virus and its application. The monoclonal antibody is named 1G10, belongs to the IgG1 subclass, comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises amino acid sequences as shown in SEQ ID NO.5-7. H CDR1, V H CDR2 and V H CDR3, the light chain variable region comprises amino acid sequences as shown in SEQ ID NO.8-10 L CDR1, V L CDR2 and V L The monoclonal antibody specifically recognized the HA protein in indirect immunofluorescence assays, protein immunoblotting, and neutralization tests. Furthermore, the monoclonal antibody was able to undergo specific hemagglutination inhibition with H5Nx subtype avian influenza viruses, including H5N1, H5N6, and H5N8, from different HA gene evolutionary branches, such as 2.3.2.1d, 2.3.4.4b, and 2.3.4.4d, indicating that the antigenic epitope recognized by the 1G10 monoclonal antibody is a broad-spectrum neutralizing epitope for H5 subtype avian influenza viruses. Chicken embryo immune escape assays and sequence alignment revealed that the neutralizing epitope recognized by the 1G10 monoclonal antibody includes amino acid positions 170 and 231 of the HA protein, and that mutations significantly affect antigenicity.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a monoclonal antibody capable of broadly binding to HA protein of multi-branched H5Nx subtype avian influenza virus, and the identification and application of neutralizing epitopes. Background Art

[0002] The H5 subtype highly pathogenic avian influenza virus (AIV) is distributed worldwide, seriously endangering the healthy development of the poultry industry and can cause infection and disease in a variety of mammals, including humans. Therefore, it also has important public health significance. Statistics show that from January 2005 to November 2022, 8,534 H5 subtype highly pathogenic avian influenza outbreaks have been reported worldwide, with as many as 389 million poultry dying or being culled (Shi J, Zeng X, Cui P, et al. Alarming situation of emerging H5 and H7 avian influenza and effective control strategies [J]. Emerg Microbes Infect, 2023, 12(1):2155072). As of July 19, 2024, the World Health Organization reported that the H5N1 subtype AIV has caused a total of 896 confirmed human cases since 2003, of which 463 have died, with a mortality rate of over 50% (https: / / www.who.int / ). In addition, H5N2, H5N6 and H5N8 subtypes of AIV can also cause infection and disease in humans.

[0003] Hemagglutinin (HA) is the primary antigenic glycoprotein on the surface of AIV, capable of agglutinating red blood cells. It is a core target for vaccines, neutralizing antibodies, and diagnostics. However, the HA gene is also the most variable gene in the AIV genome, directly influencing viral antigenicity. The highly pathogenic H5 subtype of AIV is known to have given rise to numerous HA gene clades, including the primary clades 0-9 and their multiple subclades. Among them, highly pathogenic H5 subtypes of AIV from clades 2.3.2 and 2.3.4 have long been predominant in my country, and significant antigenic variation exists between the two clades, necessitating the development of separate vaccine strains to address their respective epidemics. Furthermore, strains within the 2.3.2 and 2.3.4 clades continue to evolve and mutate, resulting in antigenic differences. In response to the further derivation of epidemic branches such as 2.3.2.1d and 2.3.2.1e from the 2.3.2 branch, the recombinant inactivated vaccine of H5 subtype avian influenza virus approved for use in China was updated from Re-6 to Re-12; and in response to the continuous evolution of mainstream sub-branches such as 2.3.4.4g, 2.3.4.4h, and 2.3.4.4b from the 2.3.4 branch, the vaccine strains have at least gone through Re-5, Re-8, Re-11 to the currently used Re-13 and Re-14 (Zeng Xianying, Tian Guobin, Chen Hualan. Progress in the development and application of H5 / H7 subtype avian influenza vaccines in China [J]. Science China: Life Sciences, 2023, 53(12): 1700-1712).

[0004] Because the degree of antigenic match between circulating strains and vaccine strains is crucial for vaccine protection, monitoring and selecting antigenically consistent circulating strains as vaccine candidates is crucial. Furthermore, the development of commercial vaccines for newly emerged H5 subtype AIV variants often lags behind the emergence of circulating strains, potentially exacerbating the spread of the epidemic by escaping poorly antigenically matched vaccines. Therefore, developing a universal vaccine with broad protection is of paramount practical importance for the effective prevention and control of highly pathogenic avian influenza (HPAI) H5 subtypes. However, the design of a universal vaccine relies on the discovery of conserved, broad-spectrum epitopes, and one of the primary approaches to discovering these epitopes is by identifying the epitopes targeted by monoclonal antibodies with broad-spectrum recognition. Because antibodies targeting the globular head domain of the HA protein can specifically neutralize the virus and can be detected by hemagglutination inhibition assays, many universal influenza vaccine designs focus on this region of the HA protein.

[0005] In recent years, monoclonal antibodies against the HA protein have also been widely used to study the antigenicity of influenza viruses. For example, Zheng Qingbing et al. used multiple monoclonal antibodies against the HA protein of the 2009 influenza A (H1N1) vaccine strain A / Califomia / 07 / 2009 to analyze the antigenic diversity of virus isolates. They identified key amino acid mutations at positions 128 and 158 in the Sa antigenic region, position 225 in the Ca2 antigenic region, and position 188 in the Sb antigenic region as significantly affecting the antigenicity of the virus (Zheng Qingbing, Chen Yixin, Zeng Fen, et al. Monitoring Antigenic Variation of Influenza A (H1N1) Virus Using Hemagglutinin Monoclonal Antibodies [C] / / Chinese Medical Association, Chinese Society of Microbiology and Immunology. Proceedings of the 2012 National Symposium on Clinical Microbiology and Infectious Immunity. National Engineering Research Center for Infectious Disease Diagnostic Reagents and Vaccines, School of Public Health, Xiamen University, 2012: 1). Zhang et al. identified positions 120, 126, 141, 156, 185, and 189 as important antigenic sites by binding a group of monoclonal antibodies against the HA protein of the H5 subtype AIV vaccine strain Re-8, which is of great significance for the mapping of the antigenic map of the 2.3.4.4 branch H5 influenza virus (Zhang Y, Cui P, Shi J, et al. Key amino acid residues thatdetermine the antigenic properties ofhighly pathogenic H5 influenza virusesbearing the clade 2.3.4.4 hemagglutinin gene [J]. Viruses. 2023, 15(11):2249).

[0006] However, since monoclonal antibodies target a single antigenic epitope, their response spectrum is usually limited and they can often only recognize viruses belonging to the same HA branch. Therefore, the identification of broad-spectrum neutralizing monoclonal antibodies that can simultaneously recognize the HA proteins of multiple-branch H5 viruses will provide important biological materials for H5 subtype AIV antigen monitoring and vaccine development. Summary of the Invention

[0007] The present invention discloses a monoclonal antibody 1G10 that broadly binds to the HA protein of multiple-branched H5Nx subtype avian influenza viruses. This monoclonal antibody targets the HA protein and exhibits specific immunological binding in both indirect immunofluorescence and Western blot assays. It also exhibits specific hemagglutination inhibition and neutralizing activity against H5Nx subtype avian influenza viruses, including H5N1, H5N6, and H5N8, from multiple HA evolutionary branches, including 2.3.2.1d, 2.3.4.4b, and 2.3.4.4d.

[0008] The present invention relates to a monoclonal antibody that broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, light chain variable region contains V L CDR1, V L CDR2 and V L CDR3;

[0009] The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5;

[0010] The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6;

[0011] The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7;

[0012] The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8;

[0013] The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 9;

[0014] The V L The amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0015] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO. 4.

[0016] In the present invention, the nucleotide sequence of the heavy chain variable region of the monoclonal antibody that broadly binds to the HA protein of the multi-branched H5Nx subtype avian influenza virus is shown in SEQ ID NO. 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO. 3. The antibody can be expressed using conventional genetic engineering or protein engineering methods, avoiding the loss of antibody genes during long-term cryopreservation of hybridoma cells. This also facilitates antibody optimization at the gene and protein levels, improving antibody specificity and affinity.

[0017] The present invention also discloses an expression vector, which contains the above-mentioned heavy chain variable region nucleotide sequence and light chain variable region nucleotide sequence.

[0018] The present invention also discloses a host cell, which contains the above-mentioned expression vector.

[0019] The invention also discloses the application of the monoclonal antibody in influenza virus antigenic variation analysis.

[0020] Furthermore, the application of the monoclonal antibody in preparing a detection reagent for H5 subtype avian influenza virus is disclosed.

[0021] The present invention also provides the identification of the neutralizing antigenic epitope recognized by the monoclonal antibody. The wild-type H5 subtype avian influenza virus liquid is mixed and incubated with the HA protein monoclonal antibody with neutralizing properties of the present invention, and the escape mutant strain with significantly reduced hemagglutination inhibition titer is prepared by inoculating SPF chicken embryos. The HA gene sequence of the escape mutant strain is compared with the wild type, and the 170th and 231st differential amino acid sites are determined to be the antigenic epitopes recognized by the monoclonal antibody. Big data analysis shows that 170N and 231T are well conserved in H5 subtype influenza viruses of different evolutionary branches.

[0022] The present invention also provides the use of the monoclonal antibody in studying antigenic variation of H5 subtype avian influenza virus. By screening escape mutants, it is identified that HA protein N170D and T231N mutations can significantly affect antigenicity.

[0023] The characteristics and advantages of the present invention are as follows: the present invention discloses a neutralizing monoclonal antibody 1G10 that broadly binds to the HA protein of multi-branch H5Nx subtype avian influenza virus, can produce hemagglutination inhibition reaction with multiple strains of 2.3.2.1 and 2.3.4.4 epidemic branches H5N1, H5N6 and H5N8 subtype avian influenza viruses, and can specifically recognize the HA protein of H5 subtype avian influenza virus in indirect immunofluorescence and Western blot tests. The antibody can be further applied to the preparation of H5 subtype avian influenza virus detection reagents, the design of universal vaccines and the study of antigenic variation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1Figures show the results of an indirect immunofluorescence assay (IFA) using the monoclonal antibody 1G10 with 293T cells transfected with pCAGGS-171033-HA. A shows bright green fluorescence when IFA was performed using mouse ascites fluid 1G10 as the primary antibody against 293T cells transfected with pCAGGS-171033-HA. B shows bright green fluorescence when IFA was performed using mouse positive serum as the primary antibody against 293T cells transfected with pCAGGS-171033-HA, serving as a positive control. C shows no green fluorescence when IFA was performed using mouse ascites fluid 1G10 as the primary antibody against 293T cells transfected with the empty pCAGGS vector, serving as a negative control. D shows no green fluorescence when IFA was performed using mouse ascites fluid 1G10 as the primary antibody against normal 293T cells not transfected with the plasmid, serving as a negative control.

[0025] Figure 2 Figure 2 shows the results of Western blot assay of monoclonal antibody 1G10 and 293T cells transfected with pCAGGS-171033-HA.

[0026] Figure 3 This is the phylogenetic tree of the HA gene of different H5Nx subtype avian influenza viruses used to determine the broad-spectrum reactivity of monoclonal antibody 1G10. The strains marked with red triangles are the test strains, and the others are reference strains.

[0027] Figure 4 Spatial structural location of the key antigenic site of the H5 subtype avian influenza virus escape mutant strain screened for monoclonal antibody 1G10 in the HA protein.

[0028] Figure 5 Figure 1 shows the amino acid conservation of the epitope targeting the HA protein by monoclonal antibody 1G10. (A) shows the conservation of amino acids 170 and 231 in the HA gene of influenza virus subtype H5, clade 2.3.2.1; (B) shows the conservation of amino acids 170 and 231 in the HA gene of influenza virus subtype H5, clade 2.3.4.4. Different colors represent different amino acids: A, Ala (alanine); D, Asp (aspartic acid); E, Glu (glutamic acid); G, Gly (glycine); H, His (histidine); L, Leu (leucine); K, Lys (lysine); I, Ile (isoleucine); N, Asn (aspartic acid); P, Pro (proline); T, Thr (threonine); S, Ser (serine). DETAILED DESCRIPTION

[0029] The present invention will be further described below with reference to the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and implement it. However, the examples are not intended to limit the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used can be obtained from commercial sources unless otherwise specified. The materials and reagents used are as follows:

[0030] Dimethyl sulfoxide (DMSO), Freund's complete adjuvant, Freund's incomplete adjuvant, polyethylene glycol 1500 (PEG1500), HAT, and HT were products of Sigma; DMEM medium and fetal bovine serum (FBS) were products of Gibco; goat anti-mouse IgG FITC and monoclonal antibody isotyping kit were purchased from ThermoFisher Scientific.

[0031] Example 1: Acquisition of hybridoma cell line 1G10

[0032] Preparation of immunogen

[0033] The laboratory-stored Clade 2.3.2.1d H5N1 subtype avian influenza virus strain A / duck / China / 1033 / 2017 (Ge Z, Gu M, Cai T, et al. Phylogenetic tracing and biological characterization of a novel clade 2.3.2.1reassortant of H5N6 subtype avianinfluenza virus in China [J]. TransboundEmerg Dis, 2021, 68(2): 730-741) was inactivated with β-propiolactone. The allantoic fluid of the completely inactivated virus (abbreviated as 171033) was centrifuged at 10,000 r / min and 4°C for 30 min, and the supernatant was aspirated. A 20% sucrose solution was placed at the bottom, and the supernatant was slowly added along the wall of the tube with a syringe. The tube was ultracentrifuged at 30,000 r / min and 4°C for 2 h, and the supernatant was discarded. After ultracentrifugation, resuspend the pellet in sterile PBS buffer stored at 4°C and fill the ultracentrifuge tube. Ultracentrifuge again at 30,000 rpm at 4°C for 2 hours, discard the supernatant to remove sucrose from the viral solution, and resuspend the pellet in sterile PBS buffer stored at 4°C. The amount of PBS used is approximately 1 / 50 of the volume of allantoic fluid. After resuspending the pellet, determine the HA titer, filter through a 0.45 μm filter, aliquot, and store at -70°C.

[0034] Animal Immunization

[0035] Mix an appropriate amount of the purified virus with Freund's complete adjuvant in equal proportions and homogenize at 5500 rpm for 15 seconds each time for a total of six times to achieve complete emulsification. Immunize 6-week-old BALB / c mice with a dose of 0.2 mL / mouse via multiple subcutaneous injections. Repeat the initial immunization 2-3 weeks after the initial immunization. For the second and third immunizations, use incomplete Freund's adjuvant for antigen emulsification. Ten days after the third immunization, collect tail vein blood from the immunized mice to obtain triple-immune serum, and determine the antibody titer using the hemagglutination inhibition (HI) assay.

[0036] Cell fusion

[0037] Myeloma cell preparation: One week before cell fusion, resuscitate Sp2 / 0 myeloma cells and passage them approximately three times to stabilize the cells before expanding the culture to an appropriate volume for future use. Before fusion, gently pipette the cells with 10 mL of incomplete DMEM medium and collect them in a 50 mL fusion tube for later use.

[0038] Preparation of splenic lymphocytes 3 days before fusion: Mice with high titers of triple-immune serum antibodies were selected for shock immunization, i.e., 0.2 mL of purified virus was injected intraperitoneally; on the day of fusion, positive serum was collected by blood sampling from the eyeballs, and the sacrificed mice to be fused were placed in 75% alcohol for 10 minutes and then fixed in a supine position on a dissecting board in a clean bench; the spleen of the mouse was aseptically removed and placed in incomplete DMEM medium for washing to remove excess connective tissue and fat; the spleen cells were then transferred to a cell sieve and ground in another dish containing incomplete DMEM medium. The ground spleen cell suspension was collected in a centrifuge tube and placed in a cell culture incubator with 5%-10% CO2 and 37°C for 10-15 minutes.

[0039] Preparation of feeder cells One day before fusion, 8-week-old negative ICR mice were taken and killed by eye bleeding. The serum was collected as a negative control and mixed with 15% FBS DMEM HAT medium. The cells were plated in a 96-well plate at a volume of 100 μL per well. After being placed in 75% alcohol for 10 minutes, they were fixed in a supine position on the dissection plate in the clean bench; the skin was cut to expose the peritoneum; 10 mL of HAT medium was drawn up with a 10 mL syringe and carefully injected into the abdominal cavity. The abdomen was gently massaged with an alcohol cotton ball to withdraw the fluid in the abdominal cavity. After dilution, it was divided into 96-well cell culture plates and placed in a saturated humidity incubator containing 5%-10% CO2 and 37°C for culture until use.

[0040] Fusion: Aspirate the spleen cells prepared above, leaving the orange-red debris at the bottom of the centrifuge tube, and transfer them to the same fusion tube with the Sp2 / 0 cells. Centrifuge at 100 rpm for 10 minutes, remove the supernatant, gently scrape the bottom of the fusion tube to disperse the precipitated cells, and slowly add 1 mL of preheated PEG-1500 in a 37°C water bath to complete fusion. Terminate the fusion reaction with antibody-free, serum-free DMEM medium. Let stand at 37°C for 10 minutes, then centrifuge. Resuspend the cells in DMEM HAT medium supplemented with 15% FBS and evenly distribute 100 µL / well to a 96-well plate containing feeder cells. Incubate in a 5%-10% CO2, 37°C incubator. After 5 days, perform a half-change of medium with fresh HAT medium. After 7-10 days, perform a full change of medium with HT medium. Observe the growth of the hybridoma cells. When the cell culture supernatant turns yellow or the cells occupy more than 1 / 10 of the well bottom area, hybridoma screening can be performed. Aspirate the cell supernatant and test for antibodies.

[0041] Hybridoma screening: Hybridoma supernatant from a 96-well plate was used as a test antibody. A 4-unit antigen solution was prepared using the immunogenic virus strain 171033 and subjected to a HI assay. Wells showing no red blood cell agglutination were considered positive for HI activity, while those showing no HI activity were considered negative. Wells that tested positive in all three assays were selected for subcloning, and hybridoma cells were cloned using limiting dilution. After 3-5 cloning cycles, cell lines that consistently secreted antibodies were selected for expansion and culture. Finally, a positive hybridoma cell line, 1G10, was obtained.

[0042] Example 2:

[0043] Large-scale production of monoclonal antibodies

[0044] Take the BALB / c female mice that have given birth, and inject sterilized liquid paraffin 0.3-0.5mL / mouse into the abdominal cavity. After 7-10 days, the hybridoma cells expanded above were cultured at a rate of about 1×10 6 Inject the mouse intraperitoneally. After 5 days, observe the abdomen of the female mouse daily. When the abdomen becomes obviously swollen, collect the ascites with a 16# needle. After centrifugation at 5000 rpm for 5 minutes, aspirate the supernatant and store it in aliquots at -70°C.

[0045] Subclass identification and HI titer determination

[0046] Subclass identification of the mAb was performed according to the instructions for the Mouse Monoclonal Antibody (MAB) Subclass Identification Kit. Hybridoma cell supernatant and mouse ascites fluid titers were tested using the HI assay, with the maximum dilution factor for positive wells representing the HI titer. Results showed that 1G10 was an IgG1 class antibody, with HI titers of 4 log2 in hybridoma cell supernatant and 13 log2 in mouse ascites fluid.

[0047] Example 3: Monoclonal Antibody Variable Region Gene Sequencing

[0048] Take 1×10 6 Hybridoma cells were collected and total cellular RNA was extracted using Trizol reagent (purchased from Life Technologies, catalog number 15596026), and RNA bands were detected by 1% agarose gel electrophoresis. Subsequently, the extracted RNA was reverse transcribed into cDNA using cDNASynthesis Kit (purchased from Nanjing Novozyme Biotechnology Co., Ltd., catalog number R312-02). Next, the heavy chain variable region (V H ) and light chain variable region (V L ) and the PCR products were detected by 1.5% agarose gel electrophoresis. The PCR products were then ligated into the pMD19-T vector, transformed into DH5α competent cells, and plated onto ampicillin-resistant LB plates. Single colonies formed after overnight culture were selected and inoculated into LB liquid medium containing ampicillin. After incubation at 37°C for 12 hours, the culture suspension was analyzed by PCR and electrophoresis using the M13+ and M13- primer pairs. Finally, plasmids were extracted from positive bacterial suspensions, and DNA sequencing was performed using the M13+ and M13- primers to deduced the corresponding amino acid sequence.

[0049] After sequencing, the V H IgG1 subtype, V L It is a κ subtype. H The nucleotide sequence is shown in SEQ ID NO. 1, V H The amino acid sequence is shown in SEQ ID NO. 2, V L The nucleotide sequence is shown in SEQ ID NO. 3, V L The amino acid sequence is shown in SEQ ID NO. 4. H and V L The complementarity-determining region (CDR) of the protein contains three subregions: CDR1, CDR2 and CDR3. H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5, V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6, V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7; V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8, V LThe amino acid sequence of CDR2 is shown in SEQ ID NO. 9, V L The amino acid sequence of CDR3 is shown in SEQ ID NO. 10.

[0050] Example 4: Identification by indirect immunofluorescence assay (IFA)

[0051] First, the HA gene of the 171033 virus strain (GenBank accession number MK554842.1) was connected to the eukaryotic expression vector pCAGGS (purchased from Miaoling Biotechnology, product number P0165) to construct the eukaryotic expression plasmid pCAGGS-171033-HA expressing the HA protein. The construction steps are as follows: 171033 virus RNA was extracted using a conventional commercial RNA extraction kit according to the instructions. After completion, the AIV universal reverse transcription primer Uni12 (Hoffmann E, Stech J, Guan Y, et al. Universal primer set for the full-length amplificationof all influenza A viruses [J]. Arch Virol, 2001, 146(12): 2275-2289) was added for reverse transcription to obtain cDNA; the design carried Sac I restriction enzyme site upstream primer 5'-GGCAAAGAATTCGAGCTCATGGAGAAAATAGTTCTTTTCTTTG-3' (SEQ ID NO. 11) and Nhe The full-length HA gene was amplified by PCR using the downstream primer 5'-GGGAAAAAGATCTGCTAGCTTAAATGCAAATTCTGCACTG-3' (SEQ ID NO. 12) of the restriction enzyme cleavage site I. The PCR product was recovered by 1% agarose gel electrophoresis using a conventional commercial DNA gel recovery kit. Nhe I and SacI. When the empty vector is used, AGGS is double-enzyme digested and linearized by electrophoresis and gel recovery. Then, the full-length HA recovered from the gel is ligated with the linearized empty vector pCAGGS according to the instructions using a homologous recombination kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd., product number CU201-02). The ligation product is used to transform DH5α competent cells, spread on LB plates containing ampicillin and cultured at 37°C. A single colony is picked and inoculated into ampicillin-resistant LB liquid medium with shaking culture for 12 hours, and then the bacterial solution is identified by PCR. Positive bacterial solution is used to extract plasmids using a conventional commercial plasmid extraction kit. The plasmid that is finally verified to be correct by sequencing is the successfully constructed eukaryotic expression plasmid pCAGGS-171033-HA.

[0052] The pCAGGS-171033-HA eukaryotic expression plasmid was transfected into 293T cells, and the pCAGGS empty vector was transfected as a negative control. After 36 hours, the cell supernatant was discarded, and the cells were washed three times with 4°C pre-cooled PBS, and then fixed with pre-cooled 4% paraformaldehyde at 4°C for 20 minutes. The fixative was discarded, and after it evaporated naturally, 5% skim milk was prepared with PBST and allowed to stand at room temperature for 1 hour for blocking. After blocking, the cells were washed three times with PBST, each for 5 minutes. The mouse ascites to be tested was added to the cell wells as the primary antibody, incubated overnight at 4°C, and washed three times as described above. A FITC-labeled goat anti-mouse IgG fluorescent secondary antibody diluted 1:500 with PBST was added, and the cells were incubated on a shaker at room temperature for 1 hour in the dark. After washing three times as above, the cells were observed under a microscope for green fluorescence. The results are shown in the attached figure. Figure 1 As shown, green fluorescence reaction was observed in 293T cells transfected with mouse ascites 1G10, mouse positive serum and pCAGGS-171033-HA plasmid ( Figure 1 A, B); 293T cells transfected with mouse ascites 1G10 and pCAGGS empty vector and normal 293T cells showed no fluorescence reaction ( Figure 1 (C, D).

[0053] Example 5: Western blot identification

[0054] 293T cells were transfected with the pCAGGS-171033-HA eukaryotic expression plasmid. After 36 hours, the cell culture medium was discarded, the cells were briefly washed with PBS, and the excess water was removed by pipette tip. The cells were lysed on ice. The lysate was centrifuged at 10,000 rpm for 10 minutes, and the supernatant was collected as the protein sample. The protein sample prepared above was mixed with a predetermined amount of loading buffer and boiled for 5 minutes. 10 μL of sample was loaded per well and subjected to SDS-PAGE. The cells were then transferred to a PVDF membrane using a standard wet transfer method. The membrane was blocked with 5% skim milk at room temperature for 2 hours. The membrane was then incubated with mouse ascites diluted 1:2000 in 5% skim milk as the primary antibody at 4°C on a shaker overnight. The membrane was washed three times with TBST for 5 minutes each time, and then incubated with goat anti-mouse IgG-HRP diluted 1:5000 in TBST as the secondary antibody at room temperature on a shaker for 1 hour. After three washes as above, the membrane was visualized using high-sensitivity ECL and imaged. The results are as attached Figure 2 As shown in the figure, 1G10 can react specifically with the HA protein produced by pCAGGS-171033-HA plasmid transfection, with a clear band at around 70 kDa; the cell samples transfected with the empty vector pCAGGS had no reaction band.

[0055] Example 6: Neutralization test identification

[0056] Primary CEF cells were cultured at 5 × 10 5 Cells were plated onto 96-well plates at a density of 100 cells / mL. 171033 allantoic fluid was diluted 10-fold with maintenance solution (V solution). Five wells of cells were inoculated at each dilution. After 1 hour of adsorption, the cells were rehydrated and incubated in a 5%-10% CO2, 37°C cell culture incubator. After 72 hours, the TCID was calculated based on the hemagglutination (HA) titer of the cell supernatant using the Reed-Muench method. 50 . TCID 50 The 171033 strain was diluted with V solution to 200 times TCID 50 For later use, dilute to 100 times TCID 50 , 10 times TCID 50 , 1 times TCID 50 , 0.1 times TCID 50 The virus solution was used for TCID 50 Regression test. Take the sterilized 1G10 mouse ascites, pre-dilute 10 times with V solution, and then dilute it 2 times in a new 96-well plate with 50μL of the system, and make 4 replicates for each dilution. Next, add 200 times TCID 50Add 50 μL / well of the diluted monoclonal antibody to a 96-well plate and gently pipette to mix the virus solution and monoclonal antibody evenly. At this time, the mixed virus solution contains 100 times the TCID per 100 μL. 50 At the same time, set up the regression test wells for the virus, with 4 wells for each dilution, and incubate them in a 37°C incubator for 1 hour. Discard the CEF cell culture medium cultured in the 96-well plate, transfer the mixture of virus and monoclonal antibody that has been neutralized for 1 hour in the previous step to a 96-well cell plate covered with a monolayer of CEF cells, and then add 150 μL of V solution to each well, place it in a 5%~10% CO2, 37°C incubator for culture. Take out the cell plate at 72 hours, detect the HA in the supernatant of each well, and calculate the neutralization titer of the monoclonal antibody by the Reed-Muench method. The results are as follows: TCID of 171033 virus on CEF cells 50 is 10 7.5 10 times the TCID in the virus regression assay for cell neutralization assay 50 The supernatant of the inoculated cell wells can all agglutinate red blood cells, and 0.1 times TCID 50 The supernatants from the inoculated wells were all non-agglutinated red blood cells, indicating that the virus solution dilution factor was accurate and the neutralization test results were reliable. Monoclonal antibody 1G10 was determined to have neutralizing properties, with a neutralization titer of 1:8129.

[0057] Example 7: Broad spectrum analysis

[0058] The HA gene genetic tree of H5Nx subtype AIV viruses from different HA evolutionary branches is shown in the attached figure. Figure 3 As shown, the red markers represent the strains tested, which are distributed in the 2.3.2.1d, 2.3.2.1e, 2.3.4.4b, and 2.3.4.4d clades and include the H5N1, H5N2, H5N6, and H5N8 subtypes. The HI titer of 1G10 monoclonal antibody ascites was measured against various virus strains to assess its broad-spectrum reactivity. The results are shown in Table 1 (HI titers were measured only to 10 log2; therefore, readings of 10 are indicated as "≥10" and non-reactivity is indicated as "-"). Antibody 1G10 exhibited good reactivity with the corresponding immunogen strain, 171033, and several H5N1 strains within the same subclade, 2.3.2.1d. Although it did not react with the two tested H5 strains within the 2.3.2.1e subclade, 1G10 exhibited strong cross-reactivity with some H5N6 and H5N8 strains within the 2.3.4.4b and 2.3.4.4d subclades. This indicates that antibody 1G10 exhibits a relatively broad spectrum of reactivity, specifically eliciting HI reactions with H5Nx viruses from different HA clades, and that the recognized epitope may be a conserved, broadly neutralizing epitope shared by multiple clades.

[0059] Table 1 HI reactivity of mAb 1G10 with clade 2.3.2.1 and 2.3.4.4 H5Nx viruses

[0060]

[0061] Note: - indicates no specific reactivity in the HI test.

[0062] Example 8: Identification of antigenic epitopes

[0063] Take 200 μL of 1G10 monoclonal antibody ascites after filtration and sterilize, and mix with 200 μL of 10 5 TCID 50 The 171033 or 181123 strain allantoic fluid was mixed evenly and incubated in a 37°C incubator for 1 h. Subsequently, the mixture of the above monoclonal antibody and the virus was diluted to 1 mL with sterile PBS and inoculated into 10-day-old SPF chicken embryos via the allantoic cavity route, with 0.2 mL inoculated into each embryo. 72 h after inoculation, the chicken embryo allantoic fluid was collected for HA test, and the allantoic fluid with HA titer was subjected to HI test with 1G10. The allantoic fluid with significantly reduced HI titer was considered positive, and the virus was further purified by limiting dilution method. In brief, 200 μL of positive allantoic fluid was mixed with an equal volume of 1G10 monoclonal antibody, and after acting at 37°C for 1 h, the mixture was diluted 10 times continuously, and 10 -5 , 10 -6 and 10 -7 The dilutions were repeated at 0.2 mL / embryo for each dilution, with two 10-day-old SPF chicken embryos inoculated. After 72 hours, allantoic fluids were collected. The allantoic fluids with the highest dilution ratio and a significantly reduced HI titer that reacted with 1G10 were selected as the MAb escape strains. The HA gene sequences of the escape strains were further sequenced and compared with the original sequence for mutation analysis to identify the key amino acids in the epitope recognized by MAb 1G10. As shown in Table 2, MAb 1G10 screened for one escape mutant each against strains 171033 and 181123. Both strains lost HI reactivity with MAb 1G10 and had HI titers of 0. HA gene sequencing identified key amino acid mutations in the escape mutants. The escape mutant against strain 171033 harbored a double-site mutation, N170D and T231N, while the escape mutant against strain 181123 harbored a single-site mutation, N170K. The results showed that the antigen epitope recognized by monoclonal antibody 1G10 included amino acid sites 170 and 231 of HA protein, among which 170N may be more critical.

[0064] Table 2 Amino acid site mutations in the HA gene of the escape mutants screened by mAb 1G10

[0065]

[0066] Note: - indicates no specific reactivity in the HI test; mutation site numbers are based on the H5 subtype HA gene coding region.

[0067] On the one hand, the spatial structure positioning analysis of the key antigenic sites identified above was performed. Using the Swiss-Model homology modeling website (https: / / swissmodel.expasy.org / ), the H5N1 subtype avian influenza virus HA protein monomer structure numbered 6PCX in the PDB protein structure database (https: / / www.rcsb.org / ) was used as a template to perform homology modeling on the HA protein of the 171033 virus strain, and the three-dimensional molecular modeling software PyMOL (https: / / pymol.org / ) was used to display the protein spatial structure and key amino acid sites. As shown in the attached figure. Figure 4 The side view of the spatial structure simulation diagram of HA protein shown in the figure shows that the two sites 170N (red mark) and 231T (green mark) are both located in the globular head region of HA protein, which is the main structural domain of HA protein for antigen recognition function.

[0068] On the other hand, conservation analysis was performed on the identified key antigenic sites. The amino acid sequences of the HA genes of H5 subtype influenza viruses of the 2.3.2.1 clade (involving 2.3.2.1a, 2.3.2.1b, 2.3.2.1c, and 2.3.2.1 without further subclades, n=3082) and 2.3.4.4 clade (involving 2.3.4.4b, 2.3.4.4c, 2.3.4.4e, 2.3.4.4g, 2.3.4.4h, and 2.3.4.4 without further subclades, n=21775) that can be directly retrieved were downloaded from the GISAID database (https: / / gisaid.org / ). The amino acids at positions 170 and 231 of the HA gene were aligned and analyzed, and the results are shown in the attached figure. Figure 5 As shown, although position 170 is mainly D in the 2.3.2.1 branch, 170N still accounts for a high proportion (1026 / 3082, 33.29%), indicating that 170N is somewhat conservative in the 2.3.2.1 branch ( Figure 5 In the 2.3.4.4 branch, 170N is highly conserved (21130 / 21775, 97.04%) ( Figure 5 Middle (B). For 231T, both in the 2.3.2.1 branch (3050 / 3082, 98.96%) ( Figure 5 (A)) and 2.3.4.4 branch (21723 / 21775, 99.76%) are highly conserved ( Figure 5Middle (B). This indicates that the identified antigenic epitopes are well conserved in the HA genes of viruses from different epidemic branches and can be further applied in the development of universal vaccines against influenza viruses.

Claims

1. A monoclonal antibody that broadly binds to the HA protein of multi-branched H5Nx subtype avian influenza virus, characterized in that: The monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises V H CDR1, V H CDR2 and V H CDR3, light chain variable region contains V L CDR1, V L CDR2 and V L CDR3; The V H The amino acid sequence of CDR1 is shown in SEQ ID NO. 5; The V H The amino acid sequence of CDR2 is shown in SEQ ID NO. 6; The V H The amino acid sequence of CDR3 is shown in SEQ ID NO. 7; The V L The amino acid sequence of CDR1 is shown in SEQ ID NO. 8; The V L The amino acid sequence of CDR2 is shown in SEQ ID NO. 9; The V L The amino acid sequence of CDR3 is shown in SEQ ID NO.

10.

2. The monoclonal antibody that broadly binds to the multi-branched H5Nx subtype avian influenza virus HA protein according to claim 1, characterized in that: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO. 2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

3. The monoclonal antibody that broadly binds to the multi-branched H5Nx subtype avian influenza virus HA protein according to claim 1, characterized in that: The nucleotide sequence of the heavy chain variable region is shown in SEQ ID NO. 1, and the nucleotide sequence of the light chain variable region is shown in SEQ ID NO.

3.

4. An expression vector, characterized in that The expression vector contains the heavy chain variable region nucleotide sequence and the light chain variable region nucleotide sequence according to claim 3.

5. A host cell, characterized in that The host cell contains the expression vector according to claim 4.

6. Use of the monoclonal antibody according to any one of claims 1 to 3 in the preparation of a detection reagent for H5 subtype avian influenza virus, wherein the H5 subtype avian influenza virus is an H5N1, H5N6 or H5N8 subtype avian influenza virus.

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