Monoclonal antibody against NA protein of H3N8 subtype avian influenza virus and application thereof

Abstract

The application discloses a monoclonal antibody against H3N8 subtype avian influenza virus neuraminidase (NA) protein and application thereof. The application immunizes BALB / c mice with H3N8 whole virus, and obtains a hybridoma cell strain stably secreting monoclonal antibody by using hybridoma technology, which is named 4B9. The heavy chain subtype of the monoclonal antibody 4B9 is IgG1, can specifically recognize H3N8 virus NA protein, and has cross-reaction with Eurasian lineage N8 protein and canine N8. The antibody has high virus neutralization activity and neuraminidase inhibition activity. Antibody pressure screening identifies that a key antigen epitope is threonine at the 295th position of the NA protein. In-vivo mouse test shows that, under a certain dose, 4B9 has 100% prevention and treatment protection effect on H3N8 virus infection. The application can be used for detection, diagnosis, prevention and development of drugs for H3N8 avian influenza virus.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology, specifically relating to a monoclonal antibody against the NA protein of H3N8 subtype avian influenza virus and its application. Background Technology

[0002] Avian influenza virus (AIV) belongs to the genus *Avian influenza virus*, type A, family Orthomyxoviridae. Based on the antigenicity of its surface glycoproteins hemagglutinin (HA) and neuraminidase (NA), AIV is classified into 16 HA subtypes and 9 NA subtypes. Among them, the H3N8 subtype is a low-pathogenic AIV and one of the most widely distributed avian influenza viruses. H3N8 infects not only wild birds and poultry but also mammals, exhibiting strong transmissibility. In recent years, outbreaks of H3N8 in chicken flocks have caused severe economic losses to the poultry farming industry. Notably, the H3N8 virus has the ability to infect humans. Three cases of H3N8 virus infection in humans have been reported to date, posing a serious threat to public health security.

[0003] Neuraminidase (NA) is a key glycoprotein on the surface of influenza viruses. It exists as a homotetramer and possesses sialidase activity, capable of cleaving sialic acid receptors on the host cell surface, mediating the release and spread of progeny viruses. The NA protein head contains antigenic determinants and an enzyme active site, which can induce the host to produce specific antibodies. Antibodies targeting the NA active site can effectively inhibit viral release and are important targets for anti-influenza drugs (such as oseltamivir). However, with the widespread use of anti-influenza drugs, drug-resistant strains continue to emerge, necessitating the development of new antiviral drugs. Currently, research on monoclonal antibodies against the NA protein of the novel H3N8 virus remains limited, and its key antigenic epitopes have not been fully elucidated, which restricts the development of related diagnostic reagents and therapeutic drugs.

[0004] Therefore, the preparation of monoclonal antibodies against the H3N8 avian influenza virus NA protein with high specificity, high neutralizing activity, and broad-spectrum reactivity is of great significance for identifying key N8 antigenic epitopes, establishing rapid detection methods, elucidating the viral immune escape mechanism, and developing novel antiviral products. Summary of the Invention

[0005] Objective: To address the shortcomings of existing technologies in the identification of key epitopes of the H3N8 avian influenza virus NA protein and the lack of highly effective in vivo protective antibodies, this invention provides a monoclonal antibody 4B9 against the H3N8 subtype avian influenza virus NA protein and its applications. The monoclonal antibody 4B9 prepared by this invention exhibits good specificity and reactivity, specifically recognizing the H3N8 virus NA protein and showing cross-reactivity with Eurasian N8 protein and canine N8 protein, demonstrating broad-spectrum recognition capability. This antibody possesses highly efficient virus neutralizing activity and neuraminidase inhibitory activity, directly acting on the NA enzyme active site. Antibody pressure screening identified the key antigenic epitope as threonine at position 295 of the NA protein. In vivo mouse experiments show that 4B9 at a dose of 10 mg / kg provides complete preventive and therapeutic protection against H3N8 virus infection. This invention can be used for the detection, diagnosis, prevention, and treatment of H3N8 avian influenza virus, thereby solving the problem of the lack of broad-spectrum, highly effective, and epitope-defined monoclonal antibodies against the H3N8 NA protein in existing technologies.

[0006] The present invention also provides the antigenic epitopes and key amino acids recognized by the monoclonal antibody, as well as their applications.

[0007] Technical Solution: To achieve the above objectives, the present invention provides a monoclonal antibody 4B9 against the NA protein (neuraminidase protein) of H3N8 subtype avian influenza virus, which is secreted by hybridoma cell line 4B9. The hybridoma cells are used to immunize mice with whole H3N8 virus as an immunogen, and a hybridoma cell bank is prepared through cell fusion. A two-round indirect immunofluorescence screening process is adopted. In the first round, H3N8-infected cells are used to screen for positive wells of antiviral antibody, and in the second round, cells expressing NA alone are used to screen for positive wells of anti-NA specific antibody, ensuring that the obtained monoclonal antibody targets the NA protein. The positive hybridoma cells are subjected to two rounds of limiting dilution subcloning to obtain hybridoma cells 4B9 with a single genotype and stable secretion.

[0008] The monoclonal antibody 4B9 specifically binds to the NA protein of H3N8 avian influenza virus, and can also specifically bind to Eurasian lineage N8 and canine lineage N8.

[0009] The heavy chain subtype of the monoclonal antibody 4B9 is IgG1.

[0010] The antigenic epitope recognized by the monoclonal antibody 4B9 against the H3N8 subtype avian influenza virus NA protein of the present invention is characterized in that the key amino acid residue of the antigenic epitope is threonine at position 295 of the NA protein.

[0011] Specifically, when the 295th threonine of the NA protein is mutated to lysine, the virus escape monoclonal antibody 4B9 can recognize it.

[0012] The application of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus described in this invention in the preparation of drugs or reagents that neutralize the activity of H3N8 virus.

[0013] The application of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus described in this invention in the preparation of an inhibitor of H3N8 virus neuraminidase (NA) activity.

[0014] The application of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus described in this invention in the preparation of products for the detection, prevention or treatment of H3N8 subtype avian influenza virus.

[0015] The kit for detecting, identifying, preventing and treating H3N8 subtype avian influenza virus according to the present invention is characterized in that the kit includes the monoclonal antibody 4B9 and auxiliary materials that make up the kit.

[0016] The monoclonal antibody 4B9 described in this invention specifically binds to the NA protein of H3N8 avian influenza virus and exhibits cross-reactivity with the Eurasian N8 protein and the canine N8 protein.

[0017] Furthermore, the present invention also provides a method for preparing the monoclonal antibody 4B9, wherein the hybridoma cell line is used to secrete and produce the anti-H3N8 monoclonal antibody 4B9.

[0018] Furthermore, the hybridoma cell line was used to secrete and proliferate in the peritoneal cavity of mice, and the ascites fluid was separated and purified to produce the monoclonal antibody 4B9 against H3N8.

[0019] The antigenic epitope recognized by the monoclonal antibody 4B9 described in this invention has a key amino acid residue at threonine position 295 (T295) of the NA protein. Through antibody stress screening of escaped strains and sequencing identification, it was found that when this site is mutated to lysine (T295K), the virus escapes 4B9 recognition, confirming that T295 is a key site for 4B9 binding.

[0020] The method for preparing monoclonal antibody 4B9 based on the NA protein of H3N8 subtype avian influenza virus according to the present invention includes the following steps:

[0021] (1) Using the whole virus of H3N8 subtype avian influenza as an immunogen, BALB / c mice were immunized three times at 14-day intervals. Spleen cells of the immunized mice were then fused with SP2 / 0 myeloma cells.

[0022] (2) Hybridoma cells were screened using HAT selective medium, and then MDCK cells infected with H3N8 virus were used as antigens for the first round of indirect immunofluorescence screening. Then, 293T cells transfected with pCAGGS-N8 plasmid were used as antigens for the second round of indirect immunofluorescence screening to obtain positive hybridoma cells that secrete anti-NA protein monoclonal antibodies.

[0023] (3) The positive hybridoma cells were subcloned using the limiting dilution method to obtain a hybridoma cell line that stably secretes monoclonal antibodies, which was named 4B9;

[0024] (4) Hybridoma cell line 4B9 was injected into the peritoneal cavity of BALB / c mice sensitized with paraffin oil to collect ascites fluid, which was purified by Protein G affinity chromatography column to obtain monoclonal antibody 4B9 against H3N8 subtype avian influenza virus NA protein.

[0025] The present invention relates to the application of the monoclonal antibody 4B9 based on the NA protein of H3N8 subtype avian influenza virus in the preparation of products targeting the NA protein of H3N8 virus.

[0026] This invention constructs a monoclonal antibody, 4B9, against the NA protein of H3N8 subtype avian influenza virus. Using whole H3N8 subtype avian influenza virus as an immunogen, a hybridoma cell line, 4B9, capable of stably secreting a monoclonal antibody against the NA protein was obtained through hybridoma technology screening. The heavy chain subtype of this monoclonal antibody, 4B9, is IgG1, which specifically recognizes the H3N8 virus NA protein and exhibits cross-reactivity with Eurasian N8 protein and canine N8 protein, demonstrating broad-spectrum recognition capability. This antibody possesses highly efficient virus neutralizing activity and neuraminidase inhibitory activity, and can directly act on the NA enzyme active site.

[0027] This invention evaluated the in vivo protective effect of monoclonal antibody 4B9 in SPF-grade BALB / c mice. The results showed that a single intraperitoneal injection of 4B9 at a dose of 10 mg / kg achieved 100% preventive and therapeutic protection against H3N8 virus infection, and significantly reduced viral load in the lungs of infected mice. These results demonstrate that passive immunization of mice with the monoclonal antibody 4B9 of this invention can produce good preventive and therapeutic effects.

[0028] This invention utilizes hybridoma technology to obtain cell lines that stably secrete specific antibodies, and prepares and purifies high-purity monoclonal antibodies using mouse ascites fluid. Regarding specificity, 4B9 accurately recognizes the conserved epitope (threonine at position 295) of the N8 subtype NA protein; regarding broad spectrum, 4B9 can recognize N8 viruses of North American, Eurasian, and mammalian lineages; regarding in vivo protection, a single low-dose injection can achieve 100% preventive and therapeutic protection. Therefore, this invention provides a highly efficient, broad-spectrum antibody solution with clearly defined epitopes for the rapid detection, diagnosis, and clinical treatment of H3N8 avian influenza virus.

[0029] This invention uses H3N8 whole virus as an immunogen to immunize BALB / c mice, and prepares a hybridoma cell bank through cell fusion. A two-round indirect immunofluorescence (IFA) screening process is employed: the first round uses H3N8-infected cells to screen for positive wells of antiviral antibodies, and the second round uses NA-expressing cells to screen for positive wells of anti-NA-specific antibodies, ensuring that the obtained monoclonal antibodies target the NA protein. Positive hybridoma cells are subjected to two rounds of limiting dilution subcloning to obtain monoclonal cell lines with a single genotype and stable secretion. In functional identification, HI activity detection, neutralization assays, and NA enzyme activity inhibition assays are performed simultaneously to systematically evaluate the antibody's mechanism of action and its stage of action. Escape mutations are induced through viral passage under antibody stress, combined with NA gene sequencing, to identify key amino acid sites for antibody binding. A mouse challenge protection model is established, and prophylactic and therapeutic administration experiments are conducted to evaluate the in vivo protective effect of the antibody.

[0030] The antibody preparation method of this invention has the following advantages:

[0031] 1. Precise targeting and high specificity: The 4B9 monoclonal antibody has undergone two rounds of IFA screening and is specifically targeted at the NA protein. It does not recognize other viral proteins such as HA and NP, and the target is clearly defined.

[0032] 2. Clear mechanism of action: 4B9 does not block viral adsorption (no HI activity), but directly inhibits the active site of NA enzyme, blocking the release of progeny viruses from the surface of infected cells and disrupting the viral transmission chain. Dual experiments with ELLA and MU-NANA confirmed that it possesses both steric hindrance and direct enzyme activity inhibition functions.

[0033] 3. Clear epitope identification: The key binding epitope was located at threonine 295 (T295) of the NA protein through escaped strain sequencing. This site is located near the active site of the NA head, providing a structural basis for antibody optimization and drug resistance monitoring.

[0034] 4. Broad-spectrum reactivity: 4B9 can not only neutralize the immunogen strain (North American lineage H3N8), but also cross-recognize and inhibit the NA activity of Eurasian lineage avian H1N8 and canine H1N8, indicating that its recognition epitope is highly conserved in the N8 subtype.

[0035] 5. Filling existing technological gaps: It solves common problems such as poor broad-spectrum activity, unclear epitopes, and limited in vivo protective effects of existing anti-NA antibodies, and provides biomaterials with practical value for the diagnosis, prevention and control of H3N8 avian influenza and the response to cross-species transmission risks.

[0036] This invention targets the enzyme active site of the NA protein and uses direct inhibition of NA enzyme activity as the core screening indicator for antibodies. It prioritizes both neutralizing and enzyme-inhibiting activity as the main criteria for judging antibody functional value, rather than solely relying on antigen binding as the traditional standard. The invention identifies T295 as a key antigenic epitope of the H3N8 virus NA protein, obtains N8 monoclonal antibodies with cross-reactivity across three lineages (North American, Eurasian, and canine), and achieves a dual NA enzyme activity inhibition mechanism and 100% in vivo protective effect in a single H3N8 NA antibody.

[0037] Currently, existing anti-N8 antibodies are mainly used as detection reagents. 4B9 represents a leap from "detection-oriented" to "functional," possessing highly efficient neutralizing activity (3.9 µg / mL) and the ability to directly inhibit the NA enzyme active site (ELLA IC50 = 0.0187 µg / mL). While existing N8 antibodies mostly target single strains, 4B9 has been experimentally verified to simultaneously recognize and inhibit the NA activity of three key lineages of N8 viruses: North American avian, Eurasian avian, and canine. In mouse models, a single dose of 4B9 at 10 mg / kg achieved 100% protection rates for both prevention and treatment; no N8 or H3N8 subtype monoclonal antibodies have been previously reported.

[0038] This invention provides a monoclonal antibody 4B9 against H3N8 NA protein. This antibody exerts a highly efficient neutralizing effect (3.9 µg / mL) by directly inhibiting the active site of the NA enzyme. The key epitope T295 is identified for the first time. A single dose of 10 mg / kg can achieve 100% preventive and therapeutic protection in mice, filling the gap in existing anti-NA antibodies that do not have in vivo protective effects.

[0039] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0040] 1) The monoclonal antibody 4B9 of the present invention is produced by a single hybridoma cell line. The antibody has characteristics such as high specificity and uniformity. The monoclonal antibody has high purity, strong specificity and can be continuously and infinitely supplied. It has a wider range of research application value and commercial use value in immune detection and passive immunotherapy.

[0041] 2) The monoclonal antibody 4B9 of the present invention has strong reactivity. It specifically binds to the NA protein of H3N8 virus in indirect immunofluorescence (IFA) and reacts positively with cells infected with Eurasian lineage avian H1N8 virus and canine H1N8 virus, indicating that it recognizes conserved epitopes of N8 subtypes and has broad-spectrum cross-reactivity, and can be used for the detection of various N8 subtype avian influenza viruses.

[0042] 3) The monoclonal antibody 4B9 prepared in this invention has no hemagglutination inhibition (HI) activity, but it has highly efficient virus neutralization activity (neutralization titer 3.9 μg / mL) and neuraminidase inhibition (NI) activity. The IC50 of the ELLA assay is 0.0187 μg / mL and the IC50 of the MU-NANA assay is 7.339 μg / mL, indicating that the antibody can directly act on the active site of NA enzyme and effectively block virus release. It can be used as a candidate molecule for the development of antiviral drugs.

[0043] 4) The antigenic epitope recognized by the monoclonal antibody 4B9 of this invention is located at threonine 295 (T295) of the NA protein of the H3N8 subtype avian influenza virus. When this site is mutated to lysine (T295K), the virus escapes antibody recognition. The identification of this epitope provides an important molecular target for further research on the biological function of the NA protein, the mechanism of antigenic variation, and the design of antiviral drugs.

[0044] 5) The monoclonal antibody 4B9 of this invention exhibits excellent protective effects in animals. In vivo experiments in mice showed that a single intraperitoneal injection at a dose of 10 mg / kg achieved 100% protection against H3N8 virus infection in both prevention and treatment, and significantly reduced viral load in the lungs, providing a new biological agent for the emergency prevention and clinical treatment of H3N8 avian influenza.

[0045] In summary, this invention not only provides specific monoclonal antibodies for establishing detection methods against H3N8 and N8 subtype avian influenza viruses, but also provides valuable biomaterials and technical support for conducting molecular epidemiology and pathogenic mechanism studies of H3N8 virus, as well as the development of antiviral drugs and passive immunization agents. Attached Figure Description

[0046] Figure 1 The results are from indirect immunofluorescence (IFA) of monoclonal antibody 4B9 and 293T cells transfected with pCAGGS-N8 plasmid. Figure 1 Group A is the experimental group. Figure 1 B is the control group;

[0047] Figure 2The results show the IFA reactivity of monoclonal antibody 4B9 with MDCK cells infected with different lineages of H1N8 virus. In the sample, 2A: 4B9 with Eurasian lineage H1N8 virus, 2B: positive control (Eurasian lineage), 2C: 4B9 with canine H1N8 virus, and 2D: positive control (canine lineage).

[0048] Figure 3 The results of the plaque inhibition assay of monoclonal antibody 4B9 against H3N8 virus are shown, where 3A: 5 μg / mL 4B9 treatment group, and 3B: virus control group;

[0049] Figure 4 The inhibitory activity of monoclonal antibody 4B9 against neuraminidase of H3N8 virus is shown, with 4A: inhibition curve of ELLA assay and 4B: inhibition curve of MU-NANA assay.

[0050] Figure 5 The results show the inhibitory activity of monoclonal antibody 4B9 against neuraminidase of Eurasian avian H1N8 virus and canine H1N8 virus, with 5A representing the results of the ELLA assay and 5B representing the results of the MU-NANA assay.

[0051] Figure 6 This is a schematic diagram of the location of the T295 site in the three-dimensional structure of the NA protein; the green dot indicates the position of threonine 295 in the NA protein, which is the key antigenic epitope recognized by the monoclonal antibody 4B9.

[0052] Figure 7 The results show the preventive and therapeutic effects of monoclonal antibody 4B9 on mice infected with H3N8 virus, including: 7A: mouse body weight change curve (including prevention and treatment groups); 7B: mouse survival rate curve (including prevention and treatment groups); and 7C: results of viral load detection in the lungs. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0054] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. Experimental methods not specifically described in the examples are generally performed under standard conditions or as recommended by the manufacturer.

[0055] The H3N8 subtype avian influenza virus (A / chicken / Anhui / 081(2023) (AH081)) was isolated, preserved, and provided by Yangzhou.

[0056] The H3N8 M8 strain was isolated, preserved, and provided by Yangzhou.

[0057] The eukaryotic expression plasmid pCAGGS-AH081N8, which expresses H3N8 virus NA, was constructed and preserved by Yangzhou University.

[0058] Mouse myeloma cells (SP2 / 0), MDCK cells, and 293T cells were purchased from the American Type Culture Collection (ATCC) and provided by Yangzhou University.

[0059] Protein G affinity chromatography column was purchased from Cytiva.

[0060] Fetoglobulin, DMEM medium, Opti-MEM medium, 96-well black microplates and MU-NANA substrate were all purchased from Thermo Fisher Scientific.

[0061] SPF-grade BALB / c mice and ICR mice were purchased from the Comparative Medicine Experimental Center of Yangzhou University, and SPF-grade chicken embryos were purchased from Zhejiang Lihua Agricultural Technology Co., Ltd.

[0062] FITC-labeled goat anti-mouse IgG and HRP-labeled goat anti-mouse IgG were purchased from Sigma.

[0063] Mouse monoclonal antibody subclass identification kit, Bio-Long BF16002.

[0064] Example 1

[0065] Establishment of hybridoma cell line 4B9 and preparation of monoclonal antibodies

[0066] 1. Animal immunization and serum titer detection

[0067] Using whole H3N8 subtype avian influenza virus as an immunogen, 0.2 mL of the virus was injected intraperitoneally into 6-week-old SPF-grade BALB / c mice. A booster immunization was administered 14 days later, for a total of 3 immunizations. Serum IFA titers were measured 10 days after the last immunization. Mice with the highest titers were selected for a pulse immunization 3 days before fusion.

[0068] 2. Cell fusion and selective culture of hybridomas

[0069] Spleens were harvested from mice after shock immunization, and spleen cell suspensions were prepared. These suspensions were then fused with SP2 / 0 myeloma cells in logarithmic growth phase at a ratio of 5:1 using PEG fusion agent. The fusion was terminated by adding DMEM medium. The supernatant was discarded after centrifugation, and the cell pellet was resuspended in HAT selective medium. The pellets were then aliquoted into 96-well plates containing feeder cells and cultured at 37°C with 5% CO2.

[0070] 3. Initial screening of positive wells in the first round of IFA hybridoma screening

[0071] On day 12 post-fusion, hybridoma supernatant was collected. The first round of IFA screening was performed using H3N8 virus-infected MDCK cells as antigens: 50 μL of supernatant was added to each well, and the cells were incubated at 37°C for 45 min. After washing, FITC-labeled goat anti-mouse IgG (1:150 dilution) was added, and the cells were incubated in the dark for 45 min. The cells were observed under a fluorescence microscope, and those showing green fluorescence were considered positive wells.

[0072] 4. Secondary IFA-targeted NA protein-specific screening

[0073] The supernatant from the first round of positive wells was used for a second round of IFA screening using 293T cells transfected with pCAGGS-N8 plasmid (expressing NA protein) as the antigen. Wells that reacted only with pCAGGS-N8 cells and not with empty vector cells were identified as positive wells that secreted monoclonal antibodies against NA protein. Figure 1 The results of indirect immunofluorescence (IFA) of monoclonal antibody 4B9 and 293T cells transfected with pCAGGS-N8 plasmid are shown. 1A is the experimental group and 1B is the control group.

[0074] 5. Limiting dilution method for subcloning and cell line establishment

[0075] Positive hybridoma cells were subcloned using limiting dilution (to 5-10 cells per milliliter, seeded in 96-well plates). Subcloning was performed twice consecutively, with IFA assays performed after 7-10 days of culture each time, until positive monoclonal hybridoma cells were obtained. A hybridoma cell line that stably secretes monoclonal antibodies was obtained and named 4B9.

[0076] 6. Ascites preparation and monoclonal antibody purification and identification

[0077] After expanding the culture of hybridoma cell line 4B9, it was intraperitoneally injected into BALB / c multiparous female mice sensitized with paraffin oil. Ascites fluid was collected 7-10 days later, and the supernatant was collected after centrifugation at 3000 rpm for 10 min. Purification was performed using Protein G affinity chromatography: the ascites fluid was diluted with buffer, passed through the column, washed, and the eluent was dialyzed against PBS for 48 h to obtain the purified monoclonal antibody 4B9. SDS-PAGE verification showed that the purified sample exhibited two characteristic protein bands under reducing conditions, corresponding to the antibody heavy and light chains, respectively.

[0078] Example 2

[0079] Isotype identification, reaction profile and hemagglutination inhibition activity assay of monoclonal antibody 4B9

[0080] 1. Subtype identification

[0081] The results, obtained using a mouse monoclonal antibody subclass identification kit, showed that the heavy chain subtype of 4B9 was IgG1.

[0082] 2. Broad-spectrum reactivity assay (IFA)

[0083] MDCK cells were infected with Eurasian avian H1N8 virus and canine H1N8 virus, respectively. After fixation, IFA was performed using purified 4B9 antibody as the primary antibody, and positive control serum was used as a control.

[0084] Virus source: Rescued using plasmids provided by the Harbin Veterinary Research Institute and fragments of PR8. MDCK cells were seeded into 96-well cell culture plates and cultured at 37°C in a 5% CO2 incubator until a monolayer was formed (approximately 16 h). The culture supernatant was discarded, and the cells were washed twice with PBS. Eurasian avian H1N8 virus and canine H1N8 virus were serially diluted 10-fold with Opti-MEM medium containing 1 μg / mL TPCK trypsin and 1% penicillin antibiotics. The appropriate dilution of virus solution (determined based on preliminary experiments, approximately 10⁻³~10⁻⁻⁴) was collected. 4 Add 100 μL of MDCK cells (dilution) to each well, with three replicates per sample. Uninfected cells serve as a negative control. Incubate at 37°C, 5% CO2 for 2 h. Discard the virus solution, wash twice with PBS, and replace with Opti-MEM medium containing 1 μg / mL TPCK trypsin and 1% penicillin antibody. Continue culturing for 48 h. Fix the cells when more than 80% show obvious cytopathic effects. Discard the medium, wash twice with PBS, and add 100 μL of pre-chilled acetone-ethanol fixative (acetone:anhydrous ethanol = 3:2) to each well. Fix at room temperature for 5 min, discard the fixative, and air dry at room temperature for later use.

[0085] Purified 4B9 monoclonal antibody was used as the primary antibody (working concentration: 5 μg / mL, determined through preliminary experiments). A positive control (1:200 dilution of H3N8-immunized mouse serum) and a negative control (uninfected cells and PBS as a substitute for the primary antibody) were also set up. 50 μL of the primary antibody working solution was added to each well, and the wells were incubated in a humidified chamber at 37°C for 45 min. The primary antibody was discarded, and the cells were washed three times with PBST for 5 min each time. Under light-protected conditions, 50 μL of FITC-labeled goat anti-mouse IgG (1:150 dilution, Sigma) was added to each well, and the cells were incubated at 37°C for 45 min. The cells were washed three times with PBST for 5 min each time. 50 μL of PBS was added to each well, and the cells were observed and photographed under an inverted fluorescence microscope. The results are as follows: Figure 2 As shown: Cells infected with 4B9 and Eurasian lineage H1N8 virus produce specific green fluorescence ( Figure 2 A), positive control serum and cells infected with Eurasian H1N8 virus also showed positive reactions ( Figure 2 B), the negative control showed no fluorescence reaction; 4B9 and cells infected with canine H1N8 virus produced specific green fluorescence ( Figure 2C), positive control serum and cells infected with canine H1N8 virus also showed positive reactions ( Figure 2 D). Negative controls (uninfected cells) showed no fluorescence. These results indicate that 4B9 can recognize both Eurasian and canine N8 subtype viruses and exhibits broad-spectrum cross-reactivity.

[0086] 4B9 ascites fluid was serially diluted twice with PBS (1:100, 1:200, 1:400, 1:800, 1:1600, 1:3200) and added as primary antibodies to fixed infected cells. The remaining steps were the same as for IFA detection. The highest dilution showing specific green fluorescence was taken as the IFA titer of the antibody. Serial dilution assays showed that the IFA titer of 4B9 ascites fluid against Eurasian H1N8 and canine H1N8 was 1:800, consistent with the IFA titer of 4B9 against H3N8 virus.

[0087] Example 3

[0088] Detection of neutralizing activity and neuraminidase inhibitory activity of monoclonal antibody 4B9

[0089] 1. Micro-neutralization test

[0090] The 4B9 antibody was serially diluted from 1 mg / mL to an equal volume of 100 TCID50. 50 The H3N8 virus was mixed and incubated at 37°C for 45 min, then an equal volume of MDCK cells was added. After culturing for 72 h, the supernatant was collected for the HA assay. Under conditions of no medium change throughout the process, the neutralizing titer of 4B9 was 3.9 μg / mL.

[0091] 2. Plaque Inhibition Test

[0092] H3N8 virus was mixed with 4B9 in an equal volume ratio and then used to infect MDCK cells. After adsorption for 2 hours, the cells were cultured in 1% agarose medium. After 72 hours of culture, the cells were fixed with formaldehyde and stained with crystal violet. The results are as follows: Figure 3 As shown: Compared with the virus control group ( Figure 3 Compared to group B), the 4B9 treatment group (B) Figure 3 A) The number of vacuoles was significantly reduced.

[0093] 3. ELLA test

[0094] The ELISA plate was coated with fetal globulin, and an equal volume of serially diluted 4B9 and inactivated H3N8 virus (equal volume ratio) was added. The plate was incubated at 37°C for 16 h, and then 100 μL of HRP-PNA (Sigma-Aldrich, catalog number L7759) was added for colorimetric determination of OD. 450 ELLAIC 4B9 50 It is 0.0187 μg / mL ( Figure 4 A).

[0095] 4. MU-NANA test

[0096] Add serially diluted 4B9 and inactivated virus (1:1) to a black 96-well plate, add 50 μM MU-NANA substrate, and react at 37°C in the dark for 1 h. Measure fluorescence. MU-NANA IC50 of 4B9. 50 It was 7.339 μg / mL. Figure 4 B) indicates that it can directly act on the active site of NA enzyme.

[0097] 5. NI activity against different lineages of N8 virus

[0098] The inhibitory activity of 4B9 against Eurasian-derived H1N8 and canine H1N8 viruses was tested according to steps 3 (ELLA test) and 4 (MU-NANA test). The results are as follows: Figure 5 As shown: Eurasian lineage H1N8-ELLA IC 50 =0.07263 μg / mL, MU-NANA IC 50 =14.51 μg / mL; canine H1N8-ELLA IC 50 =0.09634 μg / mL, MU-NANA IC 50 =1.267 μg / mL. This indicates that 4B9 has a good inhibitory effect on three different lineages of N8 virus: North American lineage, Eurasian lineage, and canine lineage.

[0099] Example 4

[0100] Epitope identification and in vivo protective effect of monoclonal antibody 4B9 in mice

[0101] 1. Preparation and epitope identification of escaped strains

[0102] 100μL 10 5 TCID 50 H3N8 virus was mixed with 500 μL of 4B9 ascites fluid and incubated at 37°C for 30 min. The mixture was then inoculated into SPF chicken embryos, and allantoic fluid was collected after 72 h. This fluid was then incubated with an equal volume of 4B9 antibody and passaged until the virus stably multiplied. RNA was extracted from the escaped strain, and the NA gene was amplified by RT-PCR and sequenced. The results showed that the NA protein in the escaped strain underwent a mutation at amino acid position 295, changing from threonine (T) to lysine (K) (T295K). The NA protein was modeled using PyMol software in three dimensions, as shown in the figure. Figure 6 As shown, T295 is located in the top region of the NA protein head, adjacent to the enzyme's active site.

[0103] 2. Evaluation of preventive and therapeutic effects in mice

[0104] To evaluate the in vivo protective effect of monoclonal antibody 4B9 against H3N8 virus infection, a mouse infection model was established using the mouse-adapted strain H3N8 M8.

[0105] Prevention test: 6-week-old BALB / c mice (n=8) were intraperitoneally injected with 10 mg / kg 4B9, and 4 hours later were intranasally inoculated with 2 MLD. 50 H3N8 M8 virus; the virus control group was inoculated with an equal amount of virus. Patients were weighed and their survival status was recorded daily. Lung tissue was harvested on day 5 post-challenge to detect viral load.

[0106] Treatment trial: 6-week-old BALB / c mice were first intranasally inoculated with 2 MLD 50 The virus was administered via intraperitoneal injection at a dose of 10 mg / kg 4B9 (n=8) 8 hours later; the virus control group was inoculated with an equal amount of virus. Observation indicators were the same as in the prevention trial.

[0107] The results are as follows Figure 7 As shown. Weight change ( Figure 7 A): In the prevention group, the body weight of mice reached its lowest point on day 7 after challenge, and then gradually recovered; in the treatment group, the body weight remained stable with no significant decrease; in the virus control group, the body weight of mice continued to decrease, and by day 7, the decrease exceeded 25%. Survival rate ( Figure 7 B): The survival rate of mice in both the prevention and treatment groups was 100%, while all mice in the virus control group died on day 7. Lung viral load ( Figure 7 C): On day 5 after challenge, the viral load in the lungs of mice in both the prevention and treatment groups was significantly lower than that in the virus control group.

[0108] The above results indicate that monoclonal antibody 4B9 has 100% protective efficacy against H3N8 virus infection at a dose of 10 mg / kg, and the key antigenic epitope is threonine at position 295 of the NA protein (T295K mutation leads to escape).

[0109] In summary, to obtain a specific monoclonal antibody against the NA protein of the H3N8 subtype avian influenza virus, this invention successfully prepared a monoclonal antibody that recognizes the H3N8 virus NA protein using whole H3N8 virus as an immunogen through monoclonal antibody techniques such as mouse immunization and cell fusion, named 4B9. Subsequent IFA experiments demonstrated that 4B9 not only reacts with the immunogen H3N8 virus but also specifically reacts with cells infected with Eurasian lineage avian H1N8 virus and canine H1N8 virus, indicating that it recognizes the conserved epitope of the N8 subtype and has broad-spectrum cross-reactivity. To understand the mechanism by which 4B9 recognizes only the N8 subtype, escaped strains were screened using antibody pressure and sequenced. It was found that the mutation of threonine at position 295 of the NA protein to lysine (T295K) is key to the virus escaping 4B9 recognition. Further three-dimensional structural modeling of the NA protein using PyMol software showed that T295 is located in the top region of the NA head, adjacent to the enzyme active site, indicating high antibody accessibility.

[0110] Currently, detection and prevention methods for the novel H3N8 avian influenza virus remain relatively limited, especially lacking highly efficient monoclonal antibodies targeting conserved epitopes of the NA protein. Using the monoclonal antibody 4B9 prepared in this invention, along with its recognized epitopes and key amino acid sites, serological detection methods, antigen capture kits, and passive immunization agents against H3N8 and N8 subtype avian influenza viruses can be developed, which is beneficial for controlling the spread of H3N8 virus. Therefore, this monoclonal antibody and the identified epitopes hold promise as important biomaterials and molecular tools for the detection, diagnosis, prevention, and treatment of H3N8 avian influenza virus.

[0111] Existing commercially available monoclonal and polyclonal antibodies against H3N8 subtype NA are limited to laboratory detection methods such as ELISA and Western blot, with no reports of viral neutralizing activity or in vivo protective efficacy. The 4B9 monoclonal antibody of this invention represents a leap from "detection-oriented" to "functional," exhibiting highly efficient neutralizing activity (3.9 μg / mL) and NA enzyme activity inhibition (ELLA IC50 = 0.0187 μg / mL), achieving 100% preventive and therapeutic protection with a single 10 mg / kg dose in a mouse model. In summary, 4B9 represents a fundamental difference and significant advancement over existing technologies in terms of functional characteristics and in vivo protective efficacy, filling the gap in functional antibodies against H3N8 NA.

Claims

1. A monoclonal antibody 4B9 against the NA protein (neuraminidase protein) of H3N8 subtype avian influenza virus, characterized in that, The hybridoma cells were secreted by the hybridoma cell line 4B9. The hybridoma cells were used to immunize mice with H3N8 whole virus as an immunogen, and a hybridoma cell bank was prepared by cell fusion. A two-round indirect immunofluorescence screening process was adopted. In the first round, H3N8-infected cells were used to screen for positive wells of antiviral antibody. In the second round, cells expressing NA alone were used to screen for positive wells of anti-NA specific antibody to ensure that the obtained monoclonal antibody targets NA protein. The positive hybridoma cells were subjected to two rounds of limiting dilution subcloning to obtain hybridoma cells 4B9 with a single genotype and stable secretion.

2. The monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus according to claim 1, characterized in that, The monoclonal antibody 4B9 specifically binds to the NA protein of H3N8 avian influenza virus, and can also specifically bind to Eurasian lineage N8 and canine lineage N8.

3. The monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus according to claim 1, characterized in that, The heavy chain subtype of the monoclonal antibody 4B9 is IgG1.

4. An antigenic epitope recognized by the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus as described in claim 1, characterized in that, The key amino acid residue of the antigen epitope is threonine at position 295 of the NA protein.

5. The antigenic epitope recognized by the monoclonal antibody 4B9 against the H3N8 subtype avian influenza virus NA protein according to claim 1, characterized in that, When the 295th threonine of the NA protein is mutated to lysine, the virus escapes recognition by the monoclonal antibody 4B9.

6. The use of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus as described in claim 1 in the preparation of a drug or reagent that neutralizes the activity of H3N8 virus.

7. The use of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus as described in claim 1 in the preparation of an inhibitor of H3N8 virus neuraminidase (NA) activity.

8. The use of the monoclonal antibody 4B9 against the NA protein of H3N8 subtype avian influenza virus as described in claim 1 in the preparation of products for detecting, preventing or treating H3N8 subtype avian influenza virus.

9. A kit for detecting, identifying, preventing, and treating H3N8 subtype avian influenza virus, characterized in that, The kit includes the monoclonal antibody 4B9 as described in claim 1 and auxiliary materials used in the kit composition.

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