A neutralizing antibody against influenza A virus and its application

Abstract

This invention discloses a neutralizing antibody against influenza A virus and its applications. The neutralizing antibody has a heavy chain and a light chain. The heavy chain comprises heavy chain CDR1 (amino acid sequence as shown in SEQ ID NO:5), heavy chain CDR2 (amino acid sequence as shown in SEQ ID NO:6), and heavy chain CDR3 (amino acid sequence as shown in SEQ ID NO:7); the light chain comprises light chain CDR1 (amino acid sequence as shown in SEQ ID NO:8), light chain CDR2 (amino acid sequence as shown in SEQ ID NO:9), and light chain CDR3 (amino acid sequence as shown in SEQ ID NO:10). The antibody of this invention exhibits highly efficient and broad-spectrum neutralizing activity against influenza A virus, and can bind to multiple subtypes of hemagglutinin, inhibiting the cleavage of hemagglutinin precursor proteins and conformational changes of hemagglutinin in acidic environments. This invention has significant implications and promising applications for the prevention and treatment of diseases caused by influenza A virus.

Description

Technical Field

[0001] This invention relates to the fields of biomedicine and virology, and in particular to a neutralizing antibody against influenza A virus and its applications. Background Technology

[0002] For the past three centuries, influenza viruses have swept the globe every few decades, each outbreak causing widespread infection and even death. Statistics show approximately 1 billion cases globally each year, with 3 to 5 million severe cases and 290,000 to 650,000 deaths. Children, the elderly, pregnant women, and those with weakened immune systems are particularly high-risk groups for influenza. The rapid spread and continued global impact of influenza viruses pose a significant challenge to global public health, thus urgently requiring innovative treatments to combat the virus and protect human health.

[0003] Influenza viruses are classified into four types: A, B, C, and D. Types A and B are the most prevalent and cause seasonal epidemics. Influenza A virus is the most common type and the most likely to trigger large-scale outbreaks. Hemagglutinin (HA) and neuraminidase (NA) are two important proteins on the surface of influenza viruses. HA is responsible for recognizing and binding to receptors on the surface of host cells, allowing the virus to enter the cell and begin replication. NA is responsible for cleaving acetylneuraminic acid between the cell and the virus, releasing progeny influenza viruses from the host cell surface to infect more cells. HA protein is the most abundant protein on the surface of influenza virus particles, a key component mediating viral infection of host cells, and the target of most influenza neutralizing antibodies and vaccines.

[0004] This invention focuses on the development of novel antiviral agents. Antibodies have always been important in antiviral therapy and passive immunization, especially broad-spectrum and highly effective neutralizing antibodies against influenza A virus, which will be effective for emergency prevention and treatment of influenza A virus-related diseases. Summary of the Invention

[0005] In view of this, the main objective of this invention is to provide a neutralizing antibody against influenza A virus. The neutralizing antibody described in this invention is named M18.1.2.2. This antibody specifically binds to the stem region of HA (hypoallergenic alpha) and has a very high affinity for HA, thus effectively neutralizing various influenza A viruses. This invention provides the amino acid sequence of the above-mentioned antibody, verifies its neutralizing activity, and finally conducts a preliminary investigation into its mechanism of action.

[0006] Specifically, the present invention relates to the following:

[0007] A neutralizing antibody against influenza A virus, the antibody having a heavy chain and a light chain, the heavy chain comprising heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:5, heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:6, and heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO:7; the light chain comprising light chain CDR1 with an amino acid sequence as shown in SEQ ID NO:8, light chain CDR2 with an amino acid sequence as shown in SEQ ID NO:9, and light chain CDR3 with an amino acid sequence as shown in SEQ ID NO:10.

[0008] The heavy chain CDR1-3 and light chain CDR1-3 regions are the core sites for the interaction between the antibody and the antigen (in this invention, the HA stem region of influenza A virus); the unique combination endows the antibody with high specificity and affinity for the HA stem region of influenza A virus.

[0009] Furthermore, the variable region amino acid sequence of the heavy chain is shown in SEQ ID NO: 3.

[0010] Furthermore, the amino acid sequence of the heavy chain is as shown in SEQ ID NO: 1; and / or a sequence that is at least 80% homologous to SEQ ID NO: 1 and has the same function.

[0011] It should be noted that differences leading to amino acid sequence homology include modifications to one or more amino acids in the antibody based on the amino acid sequence provided in this invention. It should be pointed out that, in this invention, the amino acid modification refers to an amino acid modification that does not significantly alter the antibody's binding characteristics. The modification includes amino acid substitution, addition, or deletion. Amino acid modification can be introduced into the antibody using conventional techniques in the art. Specifically, substitution includes replacing a certain amino acid with an amino acid having a similar side chain. The similar side chain is defined based on conventional understanding in the art, including having similar basic side chains, such as lysine, arginine, and histidine; similar acidic side chains, such as aspartic acid and glutamic acid; and similar nonpolar or polar side chains. Amino acid substitution can also be based on the hydrophilicity / hydrophobicity of the amino acid, for example, substitution to improve hydrophilicity / hydrophobicity; or amino acid substitution to improve the stability of the antibody protein sequence. The above-mentioned changes in the amino acid sequence will also lead to corresponding changes in the nucleotide sequence expressing the amino acid sequence.

[0012] Furthermore, the variable region amino acid sequence of the light chain is shown in SEQ ID NO: 4.

[0013] Furthermore, the amino acid sequence of the light chain is as shown in SEQ ID NO: 2; and / or a sequence that is at least 80% homologous to SEQ ID NO: 2 and has the same function.

[0014] In this invention, the antibody is characterized not only by its precise heavy chain (SEQ ID NO:1) and light chain (SEQ ID NO:2) amino acid sequences, but also by other amino acid sequences that have at least 80% homology with these sequences and are able to maintain the same function (i.e., specific binding to the HA stem region of influenza A virus). This characteristic broadens the application range of the antibody, allowing for the optimization of antibody stability, affinity, or other biological properties through slight sequence variations to meet different therapeutic or diagnostic needs.

[0015] A second objective of this invention is to provide a nucleotide molecule encoding any of the neutralizing antibodies described above or a functional fragment thereof. Whether the standard sequences shown in SEQ ID NO:1 and SEQ ID NO:2, or highly homologous functional variants thereof, these nucleotide molecules are the cornerstone of antibody genetic engineering and can be used to express and produce desired antibodies in in vitro or in vivo systems.

[0016] A third objective of this invention is to provide an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the aforementioned nucleotide molecules.

[0017] To convert nucleic acid molecules encoding antibodies into actual antibody proteins, this invention also covers expression cassettes, recombinant vectors, recombinant microorganisms (such as bacteria, yeast, or mammalian cell lines), or transgenic cell lines containing these nucleic acid molecules. These expression systems allow for the efficient and stable production of antibodies on a laboratory or industrial scale, providing a solid foundation for the research and development and production of antibody drugs.

[0018] The present invention also aims to provide the use of any of the above-described neutralizing antibodies, nucleotide molecules, or expression cassettes, recombinant vectors, recombinant bacteria, or transgenic cell lines in the preparation of any of the following products:

[0019] (1) Drugs for treating influenza A virus infection;

[0020] (2) Drugs that inhibit influenza A virus infection;

[0021] (3) Reagents for detecting the HA protein of influenza A virus;

[0022] (4) Reagents that bind to the HA protein of influenza A virus.

[0023] The antibody involved in this invention can be directly used to treat diseases caused by influenza A virus infection by neutralizing viral particles, reducing their replication and spread in the body. It can also be used as part of antiviral therapy to inhibit influenza A virus infection, reducing the severity and duration of the disease. Simultaneously, in the scientific research field, this antibody can be used to study the structure and function of the influenza A virus HA protein, as well as the interaction mechanism between the virus and host cells. Furthermore, as a diagnostic tool, it can be used to develop rapid and accurate methods for detecting influenza A virus, which is particularly crucial for the rapid identification and isolation of infected individuals during outbreaks.

[0024] Furthermore, the drug is an injectable form.

[0025] The present invention also provides a pharmaceutical composition comprising the above-described antibody, wherein the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier to ensure the stability, safety, and efficacy of the antibody. This pharmaceutical composition is suitable for direct use in the treatment or prevention of influenza A virus infection via intravenous injection, intramuscular injection, or other suitable routes of administration.

[0026] The variable region-specific amino acid or nucleotide sequence of the anti-influenza A neutralizing antibody M18.1.2.2 in this invention, the same nucleotide sequence or the nucleotide sequence encoding the same amino acid can be artificially synthesized in vitro. The synthesized antibody gene is ligated into a eukaryotic cell expression vector to obtain the antibody gene. The antibody gene is then transferred into eukaryotic cells to obtain anti-influenza A virus neutralizing antibody or related protein products.

[0027] Based on the antibody sequence of this invention, other variants with improved affinity or neutralizing activity can be obtained by many methods in the art, all of which are included within the scope of protection of this invention. For example, substitution, addition, or deletion of amino acids to obtain antibodies with the same function, or optimization of the antibody nucleotide sequence according to the codon preference of the expression host to improve antibody expression efficiency, are also within the scope of this invention.

[0028] The beneficial effects of this invention include at least the following:

[0029] (1) The neutralizing antibody of the present invention can specifically bind to the stem region of HA and has high neutralizing activity against influenza A virus, and has great development potential.

[0030] (2) The neutralizing antibody of the present invention can effectively neutralize a variety of influenza A viruses and can prevent and treat a variety of influenza A viruses;

[0031] (3) The neutralizing antibody of the present invention can effectively prevent the cleavage process of multiple subtypes of HA precursor protein, and at the same time inhibit the conformational change of HA under acidic conditions.

[0032] (4) This invention has significant scientific value and application potential in the prevention of diseases caused by influenza A virus, the development of clinical treatment plans, and the research of diagnostic reagents. Attached Figure Description

[0033] Figure 1 The figure shows the results of neutralization tests for multiple influenza A viruses to detect the activity of antibody M18.1.2.2.

[0034] Figure 2 The graph shows the results of the binding affinity determination between M18.1.2.2 and HA (H1N1 CA / 2009).

[0035] Figure 3 The figure shows the results of M18.1.2.2 inhibiting HA0 enzyme cleavage into HA1 and HA2.

[0036] Figure 4 The figure shows the results of M18.1.2.2 inhibiting low pH-induced HA conformational changes. Detailed Implementation

[0037] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0038] Furthermore, the technical solutions of the various embodiments of the present invention can be combined with each other, but only if they are feasible for those skilled in the art. If the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention.

[0039] The following specific embodiments illustrate the solution proposed in this invention:

[0040] Example 1: Expression and purification of antibody M18.1.2.2

[0041] The invention mentions that various methods known in the art can be used to obtain the antibody. Specifically, in an embodiment of the invention, the light chain and heavy chain genes of M18.1.2.2 are cloned into different sites of the pFUSE-hIgG1-Fc2 full antibody expression vector, respectively, and transfected into FreeStyle 293-F cells. Secretory expression of the full antibody is achieved using a suspension cell culture system to obtain the full antibody M18.1.2.2. The detailed steps are as follows:

[0042] 1. Antibody gene design and vector construction

[0043] This invention targets the hemagglutinin stem region of influenza virus and obtains the light and heavy chain amino acid sequences of antibody M18.1.2.2 through computational design. The amino acid sequence of the heavy chain is shown in SEQ ID NO: 1, which includes the heavy chain variable region shown in SEQ ID NO: 3; the amino acid sequence of the light chain is shown in SEQ ID NO: 2, which includes the light chain variable region shown in SEQ ID NO: 4.

[0044] The amino acid sequence of heavy chain CDR1 is GDSVSSYNAV (SEQ ID NO: 5); the amino acid sequence of heavy chain CDR2 is TYFRDGWYQ (SEQ ID NO: 6); the amino acid sequence of heavy chain CDR3 is ARSGHITVWGVNVDAFDM (SEQ ID NO: 7); the amino acid sequence of light chain CDR1 is QSLRSY (SEQ ID NO: 8); the amino acid sequence of light chain CDR2 is AGT (SEQ ID NO: 9); and the amino acid sequence of light chain CDR3 is AQSNV (SEQ ID NO: 10).

[0045] The nucleotide sequence encoding the above antibody was synthesized by Beijing Qingke Biotechnology Co., Ltd. (Beijing, China). The nucleotide sequence of the heavy chain is shown in SEQ ID NO: 11; the nucleotide sequence of the light chain is shown in SEQ ID NO: 12.

[0046] Subsequently, the antibody heavy chain gene was inserted between the EcoRI and BglII restriction sites of the expression vector pFuse-hIgG1-Fc2 (invivoGen, catalog number #pfuse-hg1fc2). The sequence encoding the M18.1.2.2 antibody light chain was then subcloned into pFUSE-hCL (the Fc segment of the vector pFuse-hIgG1-Fc2 was removed). The recombinant plasmid was extracted using a plasmid extraction kit, and the N-terminal interleukin-2 signal peptide was integrated into the construct to promote secretion.

[0047] 2. Antibody expression

[0048] The day before transfection, at 1×10 6 Freestyle 293F cells were seeded and cultured at a density of cells / mL. Before transfection, all reagents were allowed to equilibrate at room temperature for 10 min. The following procedures use a 30 mL cell culture system as an example:

[0049] Plasmids encoding the light and heavy chains were co-transfected at a molar ratio of 1:1. In one centrifuge tube, 15 μg of recombinant plasmid DNA was diluted to 3 mL of serum-free expi 293 medium (Gibco) and gently pipetted 3-4 times. In another centrifuge tube, 22.5 μL of transfection reagent FectroPro (Polyplus) was added. The diluted transfection reagent was then added to the recombinant plasmid DNA solution in one go, mixed, and allowed to stand for 10 min. The transfection mixture was then evenly added dropwise to a cell culture flask and gently shaken to disperse the transfection complex. Subsequently, 15 μL of booster was added within 0-4 h to enhance protein expression efficiency. Cells were cultured in a shaker at 37°C with 8% CO2.

[0050] 3. Antibody purification

[0051] Five days after cell culture, Freestyle 293F cell culture medium was collected and centrifuged at 500g, 4℃ for 15 min. The supernatant was then filtered through a 0.45μm filter membrane and set aside. Antibody purification was performed using Protein GAgarose packing material. A gravity centrifuge column was loaded with Protein GAgarose packing material and pretreated with 3 column volumes of 20% ethanol. The column was then equilibrated with 5 column volumes of binding buffer (1×PBS, 0.1% Tween 20, pH 7.0). The sample was then loaded into the column, and the column was washed again with 10 column volumes of binding buffer. Finally, the antibody was eluted from the column with 3 column volumes of elution buffer (0.05M citrate, pH 3.5). Neutralization buffer (1M Tris, pH 9.0) was added to the elution buffer to adjust the pH to approximately 7.5. Finally, the antibody solution was dialyzed three times in 5L of 1×PBS solution. The antibody was then concentrated, aliquoted, and stored at -80℃ for later use.

[0052] Example 2: Neutralization activity assay of antibody M18.1.2.2

[0053] 1. Virus TCID 50 Measurement

[0054] First, prepare the virus dilution solution: DMEM + 0.35% BSA (bovine serum albumin) + 0.12% NaHCO3 + antibiotics, specifically 458 mL DMEM culture medium, 25 mL 7% BSA (bovine serum albumin), 12 mL 5% NaHCO3, and 5 mL 100× penicillin-streptomycin stock solution.

[0055] MDCK cells were seeded into 96-well plates and grown to 90% confluence. The virus was serially diluted 10-fold with viral dilution buffer, with four replicates for each dilution. The cell control (NC) consisted of 100 μL of viral dilution buffer. The culture medium was aspirated from the MDCK cells, and each well was washed twice with 100 μL of PBS. 100 μL of each viral dilution was transferred to the corresponding well and incubated at 37°C and 5% CO2 for 2 h. The supernatant was aspirated, and each well was washed once with 100 μL of PBS. 100 μL of fresh viral dilution buffer was added to each well (TPCK-trypsin may be added depending on the virus characteristics), and the cells were incubated at 37°C and 5% CO2 for 18-22 h. The supernatant was aspirated, and the cells were washed once with PBS. The cells were fixed with 100 μL / well of pre-chilled tissue cell fixative (4% paraformaldehyde) for 30 min. The absorbance was measured by ELISA using anti-NP antibody, and the data were analyzed.

[0056] 2. Micro-neutralization experiment

[0057] One day in advance, seed MDCK cells into 96-well plates and allow them to grow to 90% confluence. Dilute antibody M18.1.2.2 starting at 50 μg / mL, then serially dilute 2-fold with viral dilution buffer to a final volume of 50 μL / well. Add 50 μL of a solution containing 100 TCID45 molecules to each well. 50 The virus (pre-diluted to 100 TCID with virus diluent) 50 / 50μL); perform 3-4 replicates per antibody concentration. Select 8 wells in column 12 for virus back titration, and perform serial dilutions of the virus to obtain 100 TCID. 50 50TCID 50 25TCID 50 12.5 TCID 50 6.25 TCID 50 3.125 TCID 50 1.5625 TCID 50 0.78125 TCID 50The virus control (PC) consisted of 50 μL of virus dilution + 50 μL of virus (11 columns AD); the cell control (NC) consisted of 100 μL of virus dilution (11 columns EH). Both were incubated at 37°C and 5% CO2 for 1 h. MDCK cells were aspirated of culture medium, washed twice with 100 μL PBS in each well, and the antibody-virus incubation mixture was transferred to each well. Incubation was performed at 37°C and 5% CO2 for 2 h. The supernatant was aspirated, and cells were washed once with 100 μL PBS in each well. 100 μL of fresh virus dilution was added (TPCK-trypsin may be added depending on the virus characteristics), and the cells were cultured at 37°C and 5% CO2 for 18-22 h. The supernatant was aspirated, cells were washed once with PBS, and fixed with 100 μL / well of pre-chilled tissue cell fixative (4% paraformaldehyde) for 30 min. Absorbance was measured by ELISA using anti-NP antibody, and the data were analyzed.

[0058] 3. ELISA testing

[0059] The washing buffer was PBS + 0.05% TWEEN-20; the blocking buffer was PBS + 0.1% bovine serum albumin + 0.1% TWEEN-20. First, pour PBS into the sample well, adjust the multichannel micropipette to 200 μL, insert and secure the pipette tip, and then pipette 200 μL of PBS into each well of the 96-well plate. Wash the plate three times, each time for 3 minutes. After discarding the PBS, pat the remaining liquid dry on absorbent paper to remove residual paraformaldehyde. Dilute the primary antibody (anti-avian influenza virus nucleoprotein NP monoclonal antibody) with blocking buffer at a ratio of 1:5000 (or the optimal dilution), add 50 μL of the diluted primary antibody to each well, and incubate at room temperature for 1 hour. Pour the washing buffer into the sample well. Adjust the pipette (multi-channel micropipette) to 200 μL. After inserting and securing the pipette tip, pipette 200 μL of washing buffer into each well of the 96-well plate. Wash the plate four times, for 3 minutes each time. Discard the washing buffer and pat the remaining liquid dry on absorbent paper to remove unbound primary antibody. Dilute the secondary antibody (HRP-labeled goat anti-mouse IgG secondary antibody) with blocking buffer at a ratio of 1:10000 (or the optimal dilution). Add 50 μL of the diluted secondary antibody to each well and incubate at room temperature for 1 hour. Pour the washing buffer into the sample well. Adjust the pipette (multi-channel micropipette) to 200 μL. After inserting and securing the pipette tip, pipette 200 μL of washing buffer into each well of the 96-well plate. Wash the plate six times, for 3 minutes each time. Discard the washing buffer and pat the remaining liquid dry on absorbent paper to remove unbound secondary antibody. Add 50 μL of TMB single-component chromogenic solution to each well and incubate at room temperature for approximately 10 minutes until the positive control wells turn orange-yellow while the negative control wells remain unchanged. Then, add 50 μL of stop solution to each well to terminate the reaction. Read the OD value of each well using a microplate reader (450 nm) for result analysis.

[0060] The results are as follows Figure 1 As shown, the antibody obtained in this invention has excellent ability to inhibit influenza A virus infection. M18.1.2.2 showed strong neutralizing efficacy against all tested strains, and in some strains, its IC50 value was significantly higher than that of other strains. 50 It reached the ng / ml range, demonstrating extremely strong neutralizing ability and showcasing the potential for highly efficient neutralization against a wide range of IAV strains.

[0061] Example 3: Antibody-HA Binding Affinity Detection Experiment

[0062] This experiment used a Biacore T200 instrument to evaluate the binding affinity of HA (H1N1 CA / 2009) protein to antibody M18.1.2.2 at 25°C. First, the CM5 chip was correctly mounted into the Biacore T200, ensuring the chip surface was free of contamination. An EDC / NHS mixed solution (1:1 ratio, freshly prepared) was prepared and injected into the chip channels to activate the carboxyl groups on the chip surface, forming active esters. The antigen HA protein was dissolved in 10mM acetate buffer, and the antigen solution was injected into the activated chip channels. The antigen was covalently coupled to the chip surface through the reaction of amine groups with the activated carboxyl groups. 1M ethanolamine (pH 8.5) was injected to passivate uncoupled active ester sites, preventing non-specific binding. The chip was repeatedly rinsed with running buffer (PBS, 0.5% P20) to remove unbound antigen and impurities. Baseline stability was tested to ensure the chip surface was suitable for subsequent experiments. During this period, the antibody was diluted using a 3-fold serial dilution sequence: 18.5 nM - 6.2 nM - 2.1 nM - 0.7 nM - 0.2 nM. The program was set up and started to collect data. BIAevaluation software was used to perform global analysis of the data and calculate the antigen-antibody affinity K. D value.

[0063] The results are as follows Figure 2 As shown, the antibody M18.1.2.2 obtained in this invention has extremely high affinity for the H1N1 CA / 2009HA protein, K D The value is 0.27nM.

[0064] Example 4: Antibody Inhibition of HAO Enzyme Digestion Experiment

[0065] To comprehensively evaluate the inhibitory effect of the antibody on HA0 protein hydrolysis, the required HA protein and antibody protein M18.1.2.2 were prepared first. The HA protein used was a recombinant protein from Sinocare, with a 6×His tag at its C-terminus for subsequent detection. The HA protein and antibody M18.1.2.2 were mixed at a molar ratio of 20:1 (Ab:HA) and co-incubated at 37°C for 40 min to ensure sufficient binding between the antibody and HA protein. PBS buffer was used as a control to exclude the influence of non-specific reactions. After incubation, to simulate the protease reaction environment, TPCK-treated trypsin at a final concentration of 2.5 μg / mL was added to the mixture, and incubation continued at 37°C. To dynamically monitor the inhibitory effect of the antibody on HA protein hydrolysis, samples were taken at 5, 10, 20, and 40 min. The protein hydrolysis reaction was rapidly terminated by adding a loading buffer containing sodium dodecyl sulfate (SDS) and dithiothreitol (DTT) to ensure the accuracy of subsequent analysis. Subsequently, the sample after the reaction was terminated was heated to 100°C and held for 10 minutes to fully denature the proteins for subsequent electrophoretic analysis.

[0066] After processing, protein samples were loaded onto 10% SDS-PAGE gels for electrophoretic separation. Western blotting was used to assess the degree of HA protein hydrolysis. Following the transfer step, HRP-conjugated 6×His-tagged antibody (provided by Proteintech, Chicago, USA) was used to visualize the His-tagged HA protein on the membrane. The integrity and intensity of the protein bands were observed to determine the extent of trypsin cleavage of the HA protein and the inhibitory effect of antibody M18.1.2.2 on its hydrolysis at different time points.

[0067] The results are as follows Figure 3 As shown, M18.1.2.2 can inhibit the cleavage of multiple HAO subtypes, including H1, H3, H4, H5, H7, H9, H10 and H16, and all of them show good inhibition effects.

[0068] Example 5: Antibody Inhibition of HA Conformation Change Experiment

[0069] To evaluate the inhibitory effect of antibody M18.1.2.2 on conformational changes of HA protein under acidic pH conditions, a detailed experimental protocol was designed and implemented. Histidine-labeled and pre-cleaved HA protein was used to ensure the accuracy of subsequent analyses. HA protein and antibody M18.1.2.2 were incubated at 37°C for 1 hour, with a series of different pH conditions introduced to simulate an acidic environment. The acidic condition was established by adding 0.05M citrate buffer (adjusted to pH 3.5) to ensure that the inhibitory effect on HA conformational changes was evaluated under different acidic conditions. In the control group, no antibody was added to the HA protein, and incubation was performed at pH 5.0 and pH 8.0 to verify the specificity of the antibody's inhibitory effect on HA conformational changes. After the incubation period, to avoid the influence of the acidic environment on subsequent reactions, all samples were neutralized with 1M Tris-HCl buffer (pH 8.0) to restore the reaction environment to neutral conditions. After neutralization, to further investigate the antibody's inhibitory effect on HA protein hydrolysis, TPCK-treated trypsin (added at a ratio of 20:1 based on the amount of HA protein) was added to the sample, and the mixture was incubated at 37°C for 30 min to promote the hydrolysis reaction of HA protein. After the hydrolysis reaction was completed, the reaction was terminated by adding sample buffer containing SDS.

[0070] All samples were separated by SDS-PAGE and then transferred to PVDF membranes for Western blotting analysis. Immunoassay was performed using an antibody targeting the His tag to specifically identify and visualize His-tagged HA proteins. Changes in protein bands were observed, and the inhibitory effect of antibody M18.1.2.2 on HA conformational changes and its protective effect against HA protein hydrolysis under different acidic pH conditions were analyzed.

[0071] The results are as follows Figure 4 As shown, at pH 5.0, M18.1.2.2 exhibited a strong ability to inhibit conformational changes for all detected HA protein subtypes.

[0072] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0073] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A neutralizing antibody against influenza A virus, characterized in that, The neutralizing antibody has a heavy chain and a light chain. The heavy chain includes heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO:5, heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO:6, and heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO:

7. The light chain includes light chain CDR1 with an amino acid sequence as shown in SEQ ID NO:8, light chain CDR2 with an amino acid sequence as shown in SEQ ID NO:9, and light chain CDR3 with an amino acid sequence as shown in SEQ ID NO:

10.

2. The influenza A virus neutralizing antibody according to claim 1, characterized in that, The variable region amino acid sequence of the heavy chain is shown in SEQ ID NO:

3.

3. The influenza A virus neutralizing antibody according to claim 2, characterized in that, The amino acid sequence of the heavy chain is shown in SEQ ID NO: 1; and / or a sequence that is at least 80% homologous to SEQ ID NO: 1 and has the same function.

4. The influenza A virus neutralizing antibody according to claim 1, characterized in that, The variable region amino acid sequence of the light chain is shown in SEQ ID NO:

4.

5. The influenza A virus neutralizing antibody according to claim 4, characterized in that, The amino acid sequence of the light chain is shown in SEQ ID NO: 2; and / or a sequence that is at least 80% homologous to SEQ ID NO: 2 and has the same function.

6. A nucleotide molecule encoding the neutralizing antibody or a functional fragment thereof as described in any one of claims 1-5.

7. An expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the nucleotide molecule of claim 6.

8. The use of a neutralizing antibody according to any one of claims 1-5, or a nucleotide molecule according to claim 6, or an expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line according to claim 7 in the preparation of any of the following products: (1) Drugs for treating influenza A virus infection; (2) Reagents for detecting the HA protein of influenza A virus.

9. The application according to claim 8, characterized in that, The drug is an injectable form.

10. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the antibody as described in any one of claims 1-5, and a pharmaceutically acceptable excipient, diluent, or carrier.

Images