A fully human anti-h3n2 virus neutralizing antibody iav-2 and uses thereof

Abstract

This invention provides a fully human anti-H3N2 virus neutralizing antibody IAV-2 and its applications, belonging to the field of viral antibody preparation technology. The heavy chain variable region of the neutralizing antibody IAV-2 includes CDR1, CDR2, and CDR3 regions; the light chain variable region also includes CDR1, CDR2, and CDR3 regions. The neutralizing antibody IAV-2 binds to the Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021 antigen proteins (ECGs). 50 The values ​​were 33.14 ng / mL, 38.20 ng / mL, and 34.96 ng / mL, respectively, and the IC50 values ​​against the Darwin9-2021 strain were... 50 The value is 0.03285 μg / mL, which has the characteristics of high expression, fully human origin, and good stability, and has broad application prospects.

Description

Technical Field

[0001] This invention belongs to the field of viral antibody preparation technology, and in particular relates to a fully human anti-H3N2 virus neutralizing antibody IAV-2 and its application. Background Technology

[0002] Influenza A, an acute respiratory infectious disease caused by the influenza A virus, exhibits significant seasonal characteristics. Clinically, patients often present with sudden onset of high fever, dry cough, severe muscle pain, headache, and severe fatigue; in severe cases, it can progress to viral pneumonia and even respiratory failure. The elderly, infants, young children, patients with chronic diseases, and immunocompromised individuals are considered high-risk groups for developing severe illness.

[0003] Influenza A virus (IAV) is the causative agent of this disease. Its transmission primarily relies on respiratory droplets or direct contact, and the source of infection includes both symptomatic infected individuals and asymptomatic carriers. IAV has a very broad host spectrum, infecting multiple species including humans, birds, and pigs. Due to its high rate of antigenic mutation, different subtypes such as H1N1 and H3N2 have emerged, forming the virological basis for past global pandemics. For example, the H3N2 subtype, since its emergence in 1968, has rapidly evolved into the dominant strain of seasonal influenza. With its widespread transmissibility, strong pathogenicity, and extremely high rate of genetic mutation, H3N2 can easily breach existing immune barriers, making it difficult for antibodies acquired through natural infection or vaccination to provide lasting protection, thus causing repeated infections in the population.

[0004] IAVs belong to the Orthomyxoviridae family. Their genome is a single-stranded negative-sense RNA composed of eight independent segments, encoding at least eleven viral proteins. Two key glycoproteins—hemagglutinin (HA) and neuraminidase (NA)—are embedded on the viral envelope surface, serving as the core basis for classifying viral subtypes. To date, 18 HA subtypes (H1-H18) and 11 NA subtypes (N1-N11) have been identified. Functionally, HA mediates viral adsorption to sialic acid receptors on the host cell surface and triggers membrane fusion; while NA releases and spreads newly generated viral particles by cleaving sialic acid residues on the cell surface.

[0005] Current clinical antiviral treatment for IAV infection primarily targets three core stages of the viral replication cycle: M2 ion channels (inhibitors such as amantadine), neuraminidase (inhibitors such as oseltamivir and zanamivir), and RNA-dependent RNA polymerase (inhibitors such as favipiravir). It is important to emphasize that the efficacy of these drugs is highly dependent on the timing of administration; treatment should typically be initiated within 48 hours of symptom onset. This stage is when viral replication is most vigorous, and drug intervention can maximally block replication. Delaying treatment not only significantly reduces drug efficacy but also increases the probability of complications and the risk of viral transmission.

[0006] Neutralizing antibodies can specifically recognize and bind to viral surface antigens, thereby blocking the protein molecules of the virus from entering host cells. Compared with conventional antiviral drugs, neutralizing antibodies have multiple advantages: neutralizing antibodies precisely block the viral infection process by targeting specific antigenic epitopes on the viral surface, with minimal impact on the normal function of host cells; a single dose can provide protection lasting for several weeks to months, and can be used for both pre-exposure prophylaxis and post-exposure treatment; neutralizing antibodies targeting conserved epitopes of viral proteins can effectively inhibit viral strains that have developed resistance to conventional drugs.

[0007] Currently, although neutralizing antibodies have shown great potential in influenza treatment, most candidate antibody drugs are still in the early stages of development or preclinical translation. The H3N2 influenza A epidemic continues to pose a threat, and existing prevention and control measures remain limited. Developing new, broad-spectrum, and highly effective neutralizing antibodies is an urgent clinical need. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a fully human anti-H3N2 virus neutralizing antibody IAV-2 and its application; the neutralizing antibody IAV-2 provided by this invention has high neutralizing activity, is suitable for industrial production, and has great application value.

[0009] This invention provides a fully human neutralizing antibody IAV-2 against H3N2 virus. The amino acid sequence of the heavy chain variable region of the neutralizing antibody IAV-2 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the neutralizing antibody IAV-2 is shown in SEQ ID NO: 2.

[0010] Preferably, the amino acid sequence of the heavy chain constant region of the neutralizing antibody IAV-2 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:4.

[0011] The present invention provides a recombinant vector for expressing the neutralizing antibody IAV-2, comprising an initial vector, a nucleic acid sequence encoding the heavy chain variable region of the neutralizing antibody IAV-2, and a nucleic acid sequence encoding the light chain variable region of the neutralizing antibody IAV-2.

[0012] Preferably, the initial vector is a eukaryotic expression vector containing nucleic acid sequences of the light chain constant region and the heavy chain constant region of the neutralizing antibody IAV-2.

[0013] The present invention provides a recombinant cell for producing the neutralizing antibody IAV-2, obtained by transferring the recombinant vector into a host cell.

[0014] Preferably, the host cells include Expi 293F cells or CHO-S cells.

[0015] This invention provides the use of the neutralizing antibody IAV-2, the recombinant expression vector, and the recombinant cells in the preparation of drugs for the prevention and / or treatment of H3N2 virus infection.

[0016] Preferably, the H3N2 virus includes H3N2 subtypes Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The fully human anti-H3N2 virus neutralizing antibody IAV-2 provided by the present invention has a heavy chain variable region including CDR1, CDR2 and CDR3 regions; and a light chain variable region including CDR1, CDR2 and CDR3 regions; the present invention uses flow cytometry to sort memory B cells from H3N2 infected individuals, and then determines the sequences of the heavy chain variable region and the light chain variable region by nested PCR amplification.

[0018] The neutralizing antibody IAV-2 described in this invention has binding activity against multiple H3N2 HA antigen proteins, including those of Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021. 50 The values ​​were 33.14 ng / mL, 38.20 ng / mL, and 34.96 ng / mL, respectively. The antiviral efficacy was assessed by detecting the inhibition of viral luciferase expression. The IC50 of the neutralizing antibody IAV-2 against the Darwin9-2021 strain was... 50 The value is 0.03285 μg / mL, indicating neutralizing activity. Attached Figure Description

[0019] Figure 1 This is a graph showing the OD values ​​of H3N2-binding antibodies screened by ELISA in Example 1. Figure 2 This is a graph showing the SDS-PAGE results of the neutralizing antibody IAV-2 in Example 1. Figure 3The results show the neutralizing activity of the neutralizing antibody IAV-2 in the H3N2 virus detection in Example 2. Detailed Implementation

[0020] This invention provides a fully human neutralizing antibody IAV-2 against H3N2 virus. The amino acid sequence of the heavy chain variable region of the neutralizing antibody IAV-2 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO: 2.

[0021] This invention utilizes single-cell PCR technology to develop fully human neutralizing antibodies. The principle is that recovered viral infections possess protective monoclonal antibodies against the virus. The gene encoding these antibodies is located within single lymphocytes in human peripheral blood. These single cells are obtained through flow cytometry sorting, and the encoding gene for the neutralizing antibody is amplified using single-cell PCR technology. Subsequently, through genetic engineering, the neutralizing antibodies are prepared on a large scale in vitro. Compared to traditional human-mouse chimeric antibodies or humanized antibodies, fully human antibodies represent a significant trend in the development of therapeutic antibody drugs due to their low immunogenicity and high safety.

[0022] The sequence of the fully human anti-H3N2 virus neutralizing antibody IAV-2 described in this invention is as follows: Heavy chain variable region: EVQLVESGGGLVKPGESLRLSCAAS GFTFSGCA MNWVRQAPGKGLEWVSS ITTDGRYI YYADSVRGRFTVSRDNAKNSLYLQMNSLRAEDTAVYYC ARDYDVDTETKINFDS WGQGTLVTVSS (SEQ ID NO:1) The heavy chain variable region includes CDR1 region, CDR2 region and CDR3 region; The amino acid sequence of the CDR1 region of the heavy chain variable region is shown as positions 26-33 of SEQ ID NO:1; The amino acid sequence of the CDR2 region of the heavy chain variable region is shown as positions 51-58 of SEQ ID NO:1; The amino acid sequence of the CDR3 region of the heavy chain variable region is shown as positions 97-112 of SEQ ID NO:1; Light chain variable region: SYELTQPPSVSVSPGQTARITCSGD ALPKQY AYWYQQRPGQAPVLVIY KDS ERPSGIPERFSGSSSGTTVTLTISGVQAEDEADYYC QSADSSGPYR VFGGGTRLTVL (SEQ ID NO:2) The variable region of the light chain includes CDR1 region, CDR2 region and CDR3 region; The amino acid sequence of the CDR1 region of the light chain variable region is shown as positions 26-31 of SEQ ID NO:2; The amino acid sequence of the CDR2 region of the light chain variable region is shown as positions 49-51 of SEQ ID NO:2; The amino acid sequence of the CDR3 region of the light chain variable region is shown in positions 88-97 of SEQ ID NO:2.

[0023] In this invention, the amino acid sequence of the heavy chain constant region of the neutralizing antibody IAV-2 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:4; specifically as follows: Heavy chain constant region: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO:4) Light chain constant region: RQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO:5) This invention provides the encoding gene of the neutralizing antibody IAV-2, wherein the nucleotide sequence encoding the heavy chain variable region is shown in SEQ ID NO:5, and the nucleotide sequence encoding the light chain variable region is shown in SEQ ID NO:6; specifically as follows: Nucleotide sequence encoding the variable region of the heavy chain: GAAGTGCAGCTGGTGGAGTCTGGGGGAGGCCTGGTCAAGCCTGGGGAGTCCCTGAGACTCTCCTGTGCAGCGTCTGGATTCACCTTCAGTGGCTGTGCCATGAACTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCGTCCATTACTACTGATGGTCGTTACATATACTACGCAGACT CAGTGAGGGGCCGATTCACCGTCTCCAGAGACAACGCCAAGAACTCACTATATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCTGCTATTACTGTGCGAGAGACTATGACGTGGATACAGAAACAAAGATCAACTTTGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA (SEQID NO:5) Nucleotide sequence encoding the variable region of the light chain: TCCTATGAGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAGACGGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAACAATATGCTTATTGGTACCAGCAGAGGCCAGGCCAGGCCCCTGTGTTGGTGATATATAAAGACAGTGAGAGGCCCTC AGGGATCCCTGAGCGATTCTCTGGCTCCAGTTCAGGGACAACAGTCACGTTGACCATCAGTGGAGTCCAGGCAGAAGACGAGGCTGACTATTACTGTCAATCAGCAGACAGCAGTGGTCCTTATCGGGTGTTCGGCGGAGGGACCCGGCTGACCGTCCTA (SEQ ID NO:6) In this invention, the nucleotide sequence encoding the heavy chain constant region is shown in SEQ ID NO:7, and the nucleotide sequence encoding the light chain constant region is shown in SEQ ID NO:8.

[0024] Nucleotide sequence encoding the constant region of the heavy chain: GCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAAGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAGGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCAGGTAAA(SEQ IDNO:7) Nucleotide sequence encoding the light chain constant region: CGTCAGCCCAAGGCTGCCCCCTCGGTCACTCTGTTCCCACCCTCGAGTGAGGAGCTTCAAGCCAACAAGGCCACACTGGTGTGTCTCATAAGTGACTTCTACCCGGGAGCCGTGACAGTGGCCTGGAAGGCAGATAGCAGCCCCGTCAAGGCGGGAGTGGA GACCACCACACCCTCCAAACAAAGCAACAACAAGTACGCGGCCAGCAGCTACCTGAGCCTGACGCCTGAGCAGTGGAAGTCCCACAAAAGCTACAGCTGCCAGGTCACGCATGAAGGGAGCACCGTGGAGAAGACAGTGGCCCCTACAGAATGTTCA (SEQ ID NO:8) This invention provides a recombinant vector for expressing the neutralizing antibody IAV-2, comprising a heavy chain recombinant expression vector and a light chain recombinant expression vector. In this invention, the heavy chain recombinant expression vector includes nucleotide sequences encoding the heavy chain variable region and the heavy chain constant region, and an initial eukaryotic expression vector; the light chain recombinant expression vector includes nucleotide sequences encoding the light chain variable region and the light chain constant region, and an initial eukaryotic expression vector. This invention does not specifically limit the type of the initial eukaryotic expression vector; any eukaryotic cell expression vector known in the art can be used.

[0025] This invention provides a recombinant cell for producing the neutralizing antibody IAV-2, obtained by transfecting the recombinant vector into host cells. In this invention, the host cells include Expi 293F cells or CHO-S cells.

[0026] This invention also provides the use of the neutralizing antibody IAV-2, the encoding gene, the recombinant expression vector, or the recombinant cells in the preparation of drugs for the prevention and / or treatment of H3N2 virus infection. In this invention, the H3N2 virus includes H3N2 subtypes Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021. The neutralizing antibody IAV-2 is characterized by high expression, fully human origin, and good stability, and can be used in the preparation of drugs for the prevention and / or treatment of H3N2 virus infection.

[0027] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0028] Unless otherwise specified, all reagents, antigen proteins, etc. used in this invention are commercially available products.

[0029] Example 1

[0030] Screening and preparation of human monoclonal antibodies

[0031] 1. Blood sample collection and PBMC separation

[0032] After obtaining informed consent, 20 mL of blood samples were collected from recovered H3N2 virus patients 14 days after infection. PBMCs were separated using Ficoll density gradient centrifugation and the lymphocytes were resuspended in PBS buffer for later use.

[0033] 2. Flow cytometry sorting of antigen-specific memory B cells

[0034] Lymphocytes were counted and stained with fluorescent dyes (PE-Anti Human IgG, Alexa Fluor 700-Anti Human CD19, PerCP-Anti Human CD3, PE Cy7-Anti Human CD27) and biotin-labeled HA antigen (Darwin9-2021), and incubated at 4°C in the dark for 1 h. After washing twice with FPBS, cells were stained with streptavidin conjugated with Alexa Fluor 488 and incubated at 4°C in the dark for 30 min. After washing twice with FPBS, cells were resuspended in FPBS and antigen-specific single memory B cells were sorted using a cell sorter (SONY, MA900). The sorting strategy was CD3- / CD19+ / IgG+ / CD27+ / HA antigen+. Single memory B cells were directly sorted into 96-well plates, each well containing 20 U RNase inhibitor and 20 μL of RNase-free water, and stored at -80°C.

[0035] 3. Amplification of antibody variable region genes

[0036] (1) Reverse transcription PCR

[0037] Add reverse transcription PCR reagent to a 96-well PCR plate containing a single B cell. The reagent consists of: 6 μL 5× buffer, 1.2 μL dNTPs, 1.2 μL reverse transcriptase, and 0.2 μM specific primers for each subtype of the heavy chain (H), κ light chain, and λ light chain (see Table 3, paragraph 0032 of CN115850465A for primers). Add water to a final volume of 30 μL. The PCR reaction conditions are: reverse transcription at 50℃ for 30 min, pre-denaturation at 95℃ for 15 min, followed by 40 cycles of 95℃ for 40 s, 55℃ for 30 s, and 72℃ for 1 min, with a final extension at 72℃ for 10 min. The amplified cDNA is stored at -20℃.

[0038] (2) Nested PCR

[0039] Using the reverse transcription product as a template, nested PCR was performed to amplify the H, κ, and λ chains of the antibody (primers are listed in Table 4, section 0035 of CN115850465A). The PCR reaction system contained: 2.5 μL of 10× buffer, 0.5 μL of 10 mM dNTPs, 0.25 μL of DNA polymerase, 0.2 μM primers for each of the H, κ, and λ chains, 1 μL of the reverse transcription product as template, and water to a final volume of 25 μL. The PCR reaction conditions were: 94℃ pre-denaturation for 4 min, followed by 40 cycles of 94℃ for 30 s, 57℃ for 30 s, and 72℃ for 45 s, with a final extension at 72℃ for 10 min. Positive clones were identified by nucleic acid electrophoresis, and the amplification products of positive clones were sequenced.

[0040] 3. Preliminary screening of binding antibodies using linear expression cassettes

[0041] Antibodies were rapidly screened by constructing a linear expression cassette from the antibody variable region gene. The designed linear expression cassette contained all the elements for monoclonal antibody expression in mammalian cells. The linear expression cassette, from the 5' end, sequentially contained the CMV promoter sequence (Genbank accession number: X03922.1), the coding sequence of the antibody leader peptide, the antibody variable region (ablated from single cells, nucleotide sequence SEQ ID NO: 5~6), the antibody constant region (synthesized by bioengineering, the DNA coding sequence of the heavy chain constant region is shown in SEQ ID NO: 7, and the DNA coding sequence of the Lambda-type light chain constant region is shown in SEQ ID NO: 8), and the poly-A tail (Genbank accession number: X03896.1) linked together (for specific construction methods, see paragraph 0039 of CN115947838A).

[0042] The linear form of DNA was transfected into cells for antibody expression. Antibody binding activity was measured in the cell supernatant containing the antibody: One day prior to the experiment, 2 µg / mL of HA antigen protein was coated onto each well of a 96-well ELISA plate at 4°C overnight (100 µL per well). On the day of the experiment, the plate was washed 5 times, and 100 µL of blocking buffer was added to each well, blocking at 37°C for 1 hour. After washing 5 times, the cell supernatant was added to a microplate and incubated at 37°C for 1 hour. After washing 5 times, HPR-labeled goat anti-human IgG secondary antibody was added to the microplate and incubated at 37°C for 1 hour. After washing 5 times, 100 µL of TMB single-component chromogenic solution was added to each well, and the plate was incubated at 37°C in the dark for 3 minutes. Then, 50 µL of stop solution was added to each well to terminate the reaction. The absorbance at 450 nm was measured using a microplate reader with 630 nm as a reference. Wells without the test sample were used as negative controls, and wells with OD450-630 greater than the negative control by more than 2.1 times were considered positive.

[0043] 4. Constructing antibody expression vectors

[0044] The variable region gene binding to the antibody was amplified by PCR, and homologous arms were added at the 5' and 3' ends. The variable region was then ligated into a eukaryotic expression vector containing the light and heavy chain constant region genes via homologous recombination (the homologous arm sequence and vector are shown in patent CN 114480501A, paragraph 0030). The homologous recombination product was transformed into TOP10 competent cells and cultured overnight on plates containing ampicillin. Single clones were picked and sequenced, and clones whose sequences matched the nested product sequencing results were selected for plasmid extraction.

[0045] 5. Antibody Expression and Purification

[0046] Take 15 μg each of the antibody light and heavy chain expression plasmids and transfect them into 30 mL of Expi293F cells according to the transfection instructions. After culturing at 125 rpm and 5% CO2 for 120 h, the culture product is centrifuged at 3000 g for 10 min to collect the expression supernatant. After filtering through a 0.22 μm syringe filter, the antibody is purified by rProtein A affinity. The collected antibody is concentrated and replaced with PBS, and the antibody concentration is determined by spectrophotometry.

[0047] 6. Results

[0048] The results are as follows Figure 1 As shown, ELISA detection revealed that antibody IAV-2, from the amplified paired antibodies, possesses binding activity against multiple H3N2 HA antigen proteins, including those of Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021. 50 The values ​​were 33.14 ng / mL, 38.20 ng / mL, and 34.96 ng / mL, respectively. Expression plasmids were further constructed, expressed, and purified. The purity of the target antibody protein was confirmed by SDS-PAGE, as shown below. Figure 2 As shown, the light and heavy chains of antibody IAV-2 after unwinding can be clearly observed.

[0049] Example 2

[0050] Neutralizing activity assay of antibody IAV-2

[0051] Antiviral efficacy was assessed by detecting the inhibition of viral luciferase expression. Genetically modified MDCK cells, integrating a gene fragment that can be specifically transcribed by influenza A virus RNA-dependent RNA polymerase, were used, capable of translating into a secretible Gaussian luciferase reporter gene. This system effectively induced reporter gene expression by directly providing influenza virus polymerase or by providing RdRp through viral infection. After co-incubating MDCK-Gluc cells with serially diluted IAV-2 antibody and influenza virus (Darwin9-2021, MOI=0.1), fluorescence intensity was quantified using a microplate chemiluminescence analyzer. The half-maximal inhibitory concentration (IC50) of the antibody was calculated using GraphPad Prism 9.5 software based on the dose-response curve. 50 ) value. Result as follows Figure 3 As shown.

[0052] Depend on Figure 3 It can be seen that the neutralizing antibody IAV-2 provided by this invention has an IC50 value against Darwin9-2021. 50 The value was 0.03285 μg / mL. The antibody exhibited neutralizing activity against the H3N2 subtype Darwin9-2021 strain of influenza A virus.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A fully human neutralizing antibody against H3N2 virus, IAV-2, characterized in that, The amino acid sequence of the heavy chain variable region of the neutralizing antibody IAV-2 is shown in SEQ ID NO: 1, and the amino acid sequence of the light chain variable region of the neutralizing antibody IAV-2 is shown in SEQ ID NO:

2.

2. The neutralizing antibody IAV-2 according to claim 1, characterized in that, The amino acid sequence of the heavy chain constant region of the neutralizing antibody IAV-2 is shown in SEQ ID NO:3, and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:

4.

3. A recombinant vector expressing the neutralizing antibody IAV-2 as described in claim 1 or 2, characterized in that, This includes the initial vector, the nucleic acid sequence encoding the heavy chain variable region of the neutralizing antibody IAV-2, and the nucleic acid sequence encoding the light chain variable region of the neutralizing antibody IAV-2.

4. The recombinant vector according to claim 3, characterized in that, The initial vector is a eukaryotic expression vector containing the nucleic acid sequence of the light chain constant region and the nucleic acid sequence of the heavy chain constant region of the neutralizing antibody IAV-2.

5. A recombinant cell for producing the neutralizing antibody IAV-2 as described in claim 1 or 2, characterized in that, The recombinant vector described in claim 3 or 4 is obtained by transferring it into a host cell.

6. The recombinant cell according to claim 5, characterized in that, The host cells include Expi 293F cells or CHO-S cells.

7. The use of the neutralizing antibody IAV-2 according to claim 1 or 2, the recombinant expression vector according to claim 3 or 4, and the recombinant cells according to claim 5 or 6 in the preparation of medicaments for the prevention and / or treatment of H3N2 virus infection.

8. The application according to claim 7, characterized in that, The H3N2 virus includes H3N2 subtypes Victoria / 361 / 2011, Kansas / 14 / 2017, and Darwin9 / 2021.

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