Influenza A virus neutralizing antibody and application thereof

Abstract

The invention discloses an influenza A virus neutralizing antibody and application thereof, the neutralizing antibody comprises a heavy chain and a light chain, the heavy chain comprises a heavy chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 5, a heavy chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 6 and a heavy chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 7; the light chain comprises a light chain CDR1 with an amino acid sequence as shown in SEQ ID NO: 8, a light chain CDR2 with an amino acid sequence as shown in SEQ ID NO: 9 and a light chain CDR3 with an amino acid sequence as shown in SEQ ID NO: 10. The antibody disclosed by the invention has high-efficiency broad-spectrum neutralizing activity in influenza A virus, and can be combined with various subtypes of hemagglutinin and inhibit splitting of hemagglutinin precursor protein and conformational change of the hemagglutinin in an acid environment. The invention has important significance and application prospect for preventing and treating diseases caused by influenza A virus.

Description

Technical Field

[0001] The present invention relates to the fields of biomedicine and virology, and in particular to an influenza A virus neutralizing antibody and an application thereof. Background Art

[0002] Over the past three hundred years, influenza viruses have wreaked havoc around the world every few decades. Each outbreak has caused a large number of infections and even deaths. According to statistics, there are about 1 billion related cases worldwide each year, of which 3 million to 5 million are severe cases and 290,000 to 650,000 die. Children, the elderly, pregnant women, and people with weak immune systems are at high risk of influenza. The rapid spread of influenza viruses and their continued global spread have brought huge challenges to the world's public health. Therefore, innovative treatment solutions are urgently needed to fight the virus and protect human health.

[0003] Influenza viruses are divided into four types: A, B, C, and D. The types A and B are the most common among the population and cause seasonal epidemics. Influenza A virus is the most common type of influenza virus and the type most likely to cause a large-scale influenza outbreak. HA (hemagglutinin) and NA (neuraminidase) 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 to replicate; NA is responsible for cutting acetylneuraminic acid between the cell and the virus, thereby releasing progeny influenza viruses from the surface of the host cell and infecting more cells. Among them, the HA protein is the most abundant protein on the surface of influenza virus particles. It is a key component that mediates virus infection of host cells and is also the target of most influenza neutralizing antibodies and vaccines.

[0004] The present invention focuses on the research and development of new antiviral infection preparations, and antibodies have always been important in antiviral treatment and passive immune prevention, especially broad-spectrum and highly effective neutralizing antibodies against influenza A virus, which will be effectively used for emergency prevention and treatment of influenza A virus-related diseases. Summary of the invention

[0005] In view of this, the main purpose of the present invention is to provide an influenza A virus neutralizing antibody, the neutralizing antibody of the present invention is named M18.1.2.2, the antibody specifically binds to the stem region of HA, has a very high affinity with HA, and can effectively neutralize a variety of influenza A viruses. The present invention provides the amino acid sequence of the above antibody, verifies its neutralizing activity, and finally conducts a preliminary exploration of its mechanism of action.

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

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

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

[0009] Furthermore, the amino acid sequence of the variable region 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 the differences that lead to the homology of the amino acid sequence include the modification of one or some amino acids in the antibody based on the antibody of the amino acid sequence provided by the present invention. It should be noted that in the present invention, the amino acid modification refers to the amino acid modification that does not significantly change the binding characteristics of the antibody. The modification includes amino acid replacement, addition, or deletion. Amino acid modification can be introduced into the antibody by conventional technical means in the art. Specifically, the replacement includes replacing a certain amino acid with an amino acid with a similar side chain, and the similar side chain is defined based on the conventional cognition of those skilled in the art, including having similar basic side chains, such as lysine, arginine, histidine, similar acidic side chains, such as aspartic acid, glutamic acid, similar non-polar side chains or polar side chains, etc. Amino acid replacement can also be based on amino acid affinity / hydrophobicity replacement, such as replacement of amino acids to improve affinity / hydrophobicity; or amino acid replacement 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 amino acid sequence of the variable region 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 the present invention, the characteristics of the antibody are not limited to its precise heavy chain (SEQ ID NO: 1) and light chain (SEQ ID NO: 2) amino acid sequences, but also include other amino acid sequences that have at least 80% homology with these sequences and can maintain the same function (i.e., specific binding to the HA stem region of influenza A virus). This feature broadens the application range of antibodies and allows the stability, affinity or other biological properties of antibodies to be optimized through slight sequence variations to meet different therapeutic or diagnostic needs.

[0015] The second object of the present invention is to provide a nucleotide molecule encoding any of the above-mentioned neutralizing antibodies or their functional fragments. Whether it is the standard sequence shown in SEQ ID NO: 1 and SEQ ID NO: 2, or a highly homologous functional variant thereof, these nucleotide molecules are the cornerstone of antibody genetic engineering and can be used to express and produce the desired antibodies in an in vitro or in vivo system.

[0016] The third object of the present invention is to provide an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the above-mentioned nucleotide molecule.

[0017] In order to convert the nucleic acid molecules encoding antibodies into actual antibody proteins, the present 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 efficient and stable production of antibodies on a laboratory or industrial scale, providing a solid foundation for the development and production of antibody drugs.

[0018] The present invention also aims to provide the use of any of the above-mentioned 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 the treatment of influenza A virus infection;

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

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

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

[0023] The antibodies involved in the present invention can be directly used to treat diseases caused by influenza A virus infection, by neutralizing virus particles and reducing their replication and spread in the body. They can also be used as part of antiviral therapy to inhibit influenza A virus infection and reduce the severity and duration of the disease. At the same time, in the field of scientific research, the antibodies can be used to study the structure and function of influenza A virus HA protein and the interaction mechanism between the virus and host cells. On the other hand, as a diagnostic tool, it can be used to develop a rapid and accurate method for detecting influenza A virus, which is particularly important for quickly identifying and isolating infected people during an outbreak.

[0024] Furthermore, the medicine is an injection.

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

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

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

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

[0029] (1) The neutralizing antibodies of the present invention can specifically bind to the stem region of HA and have high neutralizing activity against influenza A virus, thus having great development potential;

[0030] (2) The neutralizing antibodies 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 antibodies of the present invention can effectively prevent the cleavage process of multiple subtypes of HA precursor proteins and inhibit the conformational changes of HA under acidic conditions;

[0032] (4) The present invention has great 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a graph showing the activity results of antibody M18.1.2.2 in a neutralization test against multiple influenza A viruses.

[0034] Figure 2 This is a graph showing the results of the binding affinity test between M18.1.2.2 and HA (H1N1 CA / 2009).

[0035] Figure 3 This is a graph showing the result of M18.1.2.2 inhibiting the cleavage of HA0 into HA1 and HA2.

[0036] Figure 4 This is a graph showing the results of M18.1.2.2 inhibiting low pH-induced HA conformational changes. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] In addition, the technical solutions between the various embodiments of the present invention can be combined with each other, but it must be based on the fact that ordinary technicians in the field can implement it. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0039] The solution proposed by the present invention is specifically described below through specific embodiments:

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

[0041] It is mentioned in the invention that various methods known in the art can be used to obtain the antibody. Specifically, in the embodiment of the present 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, transfected into FreeStyle 293-F cells, and the 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] The present invention uses the stem region of influenza virus hemagglutinin as a target, and obtains the light chain and heavy chain amino acid sequences of antibody M18.1.2.2 by 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 the heavy chain CDR1 is GDSVSSYNAV (SEQ ID NO: 5); the amino acid sequence of the heavy chain CDR2 is TYFRDGWYQ (SEQ ID NO: 6); the amino acid sequence of the heavy chain CDR3 is ARSGHITVWGVNVDAFDM (SEQ ID NO: 7); the amino acid sequence of the light chain CDR1 is QSLRSY (SEQ ID NO: 8); the amino acid sequence of the light chain CDR2 is AGT (SEQ ID NO: 9); and the amino acid sequence of the 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), and 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), and the sequence encoding the M18.1.2.2 antibody light chain was subcloned into pFUSE-hCL (the vector pFuse-hIgG1-Fc2 removed the Fc segment), and 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] One day before transfection, 1×10 6 Freestyle 293F cells were plated and cultured at a density of 100 / mL. During transfection, all reagents were placed at room temperature for 10 minutes. The following operation is based on a 30mL cell culture system as an example:

[0049] The plasmids encoding the light chain and heavy chain were co-transfected at a molar ratio of 1:1. In a centrifuge tube, a total of 15 μg of recombinant plasmid DNA was diluted to 3 mL of serum-free expi293 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 added to the recombinant plasmid DNA solution at one time, mixed and allowed to stand for 10 minutes; the transfection mixture was evenly dropped into the cell culture bottle and gently shaken to disperse the transfection complex. Subsequently, 15 μL of booster was added within 0-4 hours to improve protein expression efficiency. Cells were cultured in a constant temperature shaker containing 8% CO2 and 37°C.

[0050] 3. Antibody Purification

[0051] After 5 days of cell culture, the Freestyle 293F cell culture fluid was collected, centrifuged at 500g and 4°C for 15 minutes, and the supernatant was filtered through a 0.45μm filter membrane for standby use. Protein GAgarose filler was used to purify the antibody, and a gravity centrifugal column was used to fill the Protein GAgarose filler, and the filler was pretreated with 3 column volumes of 20% ethanol. Subsequently, the column was equilibrated with 5 column volumes of binding buffer (1×PBS, 0.1% Tween20, pH 7.0). Subsequently, the sample was loaded into the column, and the column was washed again with 10 column volumes of binding buffer, and finally the antibody was eluted from the column with 3 column volumes of elution buffer (0.05M citric acid, pH3.5). Neutralization buffer (1MTris, pH 9.0) was added to the eluate to adjust its pH to about 7.5. Finally, the antibody solution was dialyzed three times in 5 L of 1×PBS solution, and the antibody was concentrated and stored in aliquots at -80°C for later use.

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

[0053] 1. Virus TCID 50 Determination

[0054] First, prepare the virus diluent: 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 plated on 96-well plates and grown to 90% for standby use. The virus was diluted 10 times with virus diluent, and 4 replicate wells were made for each dilution, where the cell control (NC) was added with 100 μL virus diluent. The culture medium of MDCK cells was aspirated, and 100 μL PBS was added to each well to wash the cells twice. 100 μL of virus solution of different dilutions were all transferred to MDCK cells, and incubated at 37°C and 5% CO2 for 2h; the supernatant was aspirated, and 100 μL PBS was added to each well to wash the cells once, and 100 μL fresh virus diluent was added (whether TPCK-trypsin was added was selected according to the characteristics of the virus), and the cells were cultured at 37°C and 5% CO2 for 18-22h; the supernatant was aspirated, and the cells were washed once with PBS, and 100 μL / well of pre-cooled tissue cell fixative (4% polyformaldehyde) was fixed for 30min; the absorbance value was measured by ELISA with anti-NP antibody, and the data was analyzed.

[0056] 2. Micro-neutralization experiment

[0057] One day in advance, MDCK cells were plated on a 96-well plate and grown to 90% for later use. Antibody M18.1.2.2 was diluted from 50 μg / mL and diluted 2-fold with virus diluent to a final volume of 50 μL / well. 50 μL of 100 TCID 50 Virus (previously diluted to 100 TCID 50 / 50μL); 3-4 replicate wells were made for each antibody concentration. The 8 wells in the 12th column were selected for virus back titration, and the virus was serially diluted to 100TCID 50 , 50TCID 50 , 25TCID 50 , 12.5TCID 50 , 6.25TCID 50 , 3.125TCID 50 , 1.5625TCID 50 , 0.78125TCID 50, where the virus control (PC) is (11 columns AD) 50 μL virus dilution + 50 μL virus; the cell control (NC) is (11 columns EH) 100 μL virus dilution, incubated at 37°C, 5% CO2 for 1 hour. The culture medium of MDCK cells was aspirated, and 100 μL PBS was added to each well to wash the cells twice. The antibody-virus incubation mixture was transferred to the MDCK cells accordingly, and incubated at 37°C, 5% CO2 for 2 hours; the supernatant was aspirated, and 100 μL PBS was added to each well to wash the cells once, and 100 μL fresh virus dilution was added (whether TPCK-trypsin was added was selected according to the characteristics of the virus), and the cells were incubated at 37°C, 5% CO2 for 18-22 hours; the supernatant was aspirated, and the cells were washed once with PBS, and the cells were fixed with 100 μL / well of pre-cooled tissue cell fixative (4% paraformaldehyde) for 30 minutes, and the absorbance was measured by ELISA with anti-NP antibody, and the data was analyzed.

[0058] 3.ELISA test

[0059] The washing solution is PBS + 0.05% TWEEN-20; the blocking solution is PBS + 0.1% bovine serum albumin + 0.1% TWEEN-20. First, pour PBS into the sample tank, adjust the sample range of the multi-channel micropipette to 200μL, insert and fix the gun head, and then draw 200μL PBS into each well of the 96-well plate. Wash the culture plate 3 times, each time for 3min. After discarding PBS, pat the remaining liquid on absorbent paper as much as possible to remove residual paraformaldehyde. Dilute the primary antibody (anti-avian influenza virus nucleoprotein NP monoclonal antibody) with the blocking solution at a ratio of 1:5000 (or the optimal dilution), add 50μL of the diluted primary antibody to each well, and incubate at room temperature for 1h. Pour the washing solution into the sample loading tank, adjust the sample loading range of the shotgun (multi-channel micro-pipettor) to 200μL, insert and fix the gun head, draw 200μL of washing solution to each well of the 96-well plate, wash the culture plate 4 times, each time for 3min, discard the washing solution, and pat the remaining liquid on absorbent paper as much as possible to remove the unbound primary antibody. Dilute the secondary antibody (HRP-labeled goat anti-mouse IgG secondary antibody) with blocking solution at a ratio of 1:10000 (or the best dilution), add 50μL of diluted secondary antibody to each well, and incubate at room temperature for 1h. Pour the washing solution into the sample loading tank, adjust the sample loading range of the shotgun (multi-channel micro-pipettor) to 200μL, insert and fix the gun head, draw 200μL of washing solution to each well of the 96-well plate, wash the culture plate 6 times, each time for 3min, discard the washing solution, and pat the remaining liquid on absorbent paper as much as possible to remove the unbound secondary antibody. Add 50 μL of TMB single-component colorimetric solution to each well and place at room temperature for about 10 minutes to develop color until the cell positive control well turns orange-yellow, while the cell negative control well has not yet changed color. Then add 50 μL of stop solution to each well to terminate the reaction. Use an ELISA reader (450 nm) to read the OD value of each well and analyze the results.

[0060] The results are as follows Figure 1 As shown, the antibodies obtained by the present invention have excellent ability to inhibit influenza A virus infection. M18.1.2.2 showed strong neutralizing efficacy against all tested strains. In some strains, its IC 50 It reached the ng / ml range, showing extremely strong neutralizing ability and demonstrating high neutralizing potential against a wide range of IAV strains.

[0061] Example 3: Antibody-HA binding affinity test

[0062] This experiment uses the Biacore T200 instrument to evaluate the binding affinity of HA (H1N1 CA / 2009) protein and antibody M18.1.2.2 at 25°C. First, the CM5 chip was properly installed in the Biacore T200 and the chip surface was ensured to be free of contamination; the EDC / NHS mixed solution (mixed in a 1:1 ratio, prepared and used immediately) was prepared, and the EDC / NHS solution was injected into the chip channel to activate the carboxyl groups on the chip surface to form active esters; the antigen HA protein was dissolved in 10mM acetate buffer, and the antigen solution was injected into the activated chip channel. The antigen was covalently coupled to the chip surface through the reaction of the amine group with the activated carboxyl group; 1M ethanolamine (pH8.5) was injected to passivate the uncoupled active ester sites to avoid nonspecific binding; the chip was repeatedly rinsed with running buffer (PBS, 0.5% P20) to remove unbound antigens and impurities; the baseline stability was tested to ensure that the chip surface was suitable for subsequent experiments. During this period, dilute the antibody and set the antibody concentration gradient, 3-fold gradient dilution: 18.5nM-6.2nM-2.1nM-0.7nM-0.2nM, set the program and start running, collect data, use BIAevaluation software 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 by the present invention has a very high affinity with the H1N1 CA / 2009 HA protein, K D The value is 0.27nM.

[0064] Example 4: Antibody inhibition of HA0 enzyme cleavage experiment

[0065] In order to comprehensively evaluate the inhibitory effect of the antibody on HA0 proteolysis, the HA protein and antibody protein M18.1.2.2 required for the experiment were first prepared. The HA protein was selected from the recombinant protein from Sino Biological Shenzhou, and its C-terminus was tagged with a 6×His tag for subsequent detection. The HA protein and antibody M18.1.2.2 were mixed at a molar ratio of 20:1 (Ab:HA) and incubated at 37°C for 40 minutes to ensure that the antibody and HA protein were fully bound. PBS buffer was used as the control group to exclude the influence of nonspecific reactions. After the incubation, TPCK-treated trypsin with a final concentration of 2.5 μg / mL was added to the above mixture to simulate the protease action environment, and the mixture was continued to be incubated at 37°C. In order to dynamically monitor the inhibitory effect of the antibody on HA proteolysis, samples were taken after 5, 10, 20 and 40 minutes. The proteolysis reaction was quickly 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 maintained for 10 min to fully denature the protein for subsequent electrophoresis analysis.

[0066] After the protein samples were processed, they were loaded onto a 10% SDS-PAGE gel for electrophoresis separation. In order to evaluate the degree of hydrolysis of HA protein, Western Blot technology was used for detection. After the transfer step was completed, the His-tagged HA protein on the membrane was visualized using an HRP-coupled 6×His tag antibody (provided by Proteintech, Chicago, USA). By observing the integrity and intensity of the protein bands, the degree of cleavage of HA protein by trypsin and the inhibitory effect of antibody M18.1.2.2 on its hydrolysis at different time points were determined.

[0067] The results are as follows Figure 3 As shown, M18.1.2.2 can inhibit the cleavage of multiple subtypes of HA0, including H1, H3, H4, H5, H7, H9, H10 and H16, and all show good inhibitory effects.

[0068] Example 5: Antibody inhibition of HA conformational change experiment

[0069] In order to evaluate the inhibitory effect of antibody M18.1.2.2 on HA protein conformational changes under acidic pH conditions, a detailed experimental protocol was designed and implemented. The experiment used histidine-tagged and pre-cleaved HA proteins to ensure the accuracy of subsequent analysis. The HA protein was incubated with antibody M18.1.2.2 at 37°C for 1h, and a series of different pH conditions were introduced to simulate an acidic environment. Among them, the acidic conditions were set by adding 0.05M citrate buffer (adjusted to pH3.5) to ensure that the inhibitory effect on HA conformational changes was evaluated under different acidic environments. In the control experimental group, no antibody was added to the HA protein, and it was incubated at pH5.0 and pH8.0, respectively, to verify the specificity of the antibody's inhibitory effect on HA conformational changes. After the incubation period, in order to avoid the influence of the acidic environment on subsequent reactions, all samples were neutralized with 1M Tris-HCl buffer (pH8.0) to restore the reaction environment to neutral conditions. After neutralization, in order to further study the inhibitory ability of the antibody on HA protein hydrolysis, TPCK-treated trypsin was added to the sample (based on the amount of HA protein, added at a ratio of 20:1), and incubated at 37°C for 30 minutes to promote the hydrolysis 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 transferred to PVDF membrane for Western Blot analysis. Immunodetection was performed using antibodies against the His tag to specifically identify and visualize the His-tagged HA protein. The changes in protein bands were observed, and the inhibitory effect of antibody M18.1.2.2 on HA conformational changes and its protective effect on HA proteolysis were analyzed under different acidic pH environments.

[0071] The results are as follows Figure 4 As shown, at pH 5.0, M18.1.2.2 showed a strong ability to inhibit conformational changes for all HA proteins of the tested subtypes.

[0072] It should be noted that, in this article, the terms "include", "comprises" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, an element defined by the sentence "comprises a ..." does not exclude the existence of other identical elements in the process, method, article or device including the element.

[0073] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0074] The above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. An influenza A virus neutralizing antibody, characterized in that: The neutralizing antibody has a heavy chain and a light chain, the heavy chain includes a heavy chain CDR1 with an amino acid sequence such as SEQ ID NO:5, a heavy chain CDR2 with an amino acid sequence such as SEQ ID NO:6, and a heavy chain CDR3 with an amino acid sequence such as SEQ ID NO:7; the light chain includes a light chain CDR1 with an amino acid sequence such as SEQ ID NO:8, a light chain CDR2 with an amino acid sequence such as SEQ ID NO:9, and a light chain CDR3 with an amino acid sequence such as SEQ ID NO:

10.

2. The influenza A virus neutralizing antibody according to claim 1, characterized in that The amino acid sequence of the variable region 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 amino acid sequence of the variable region 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 according to any one of claims 1 to 5.

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

8. Use of the neutralizing antibody according to any one of claims 1 to 5, or the nucleotide molecule according to claim 6, or the 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 the treatment of influenza A virus infection; (2) Drugs that inhibit influenza A virus infection; (3) Reagents for detecting influenza A virus HA protein; (4) Reagents that bind to influenza A virus HA protein.

9. The use according to claim 8, characterized in that: The medicine is an injection.

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

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