Neutralizing monoclonal antibodies against HA protein of Eurasian avian and Pdm / 09 lineage H1 subtype swine influenza virus and their applications

Abstract

The present invention discloses a neutralizing monoclonal antibody against the HA protein of the Eurasian avian and Pdm / 09 evolutionary lineage H1 subtype swine influenza virus and its application. The monoclonal antibody is named 5F3 and the deposit number is CCTCC NO: C2022145. The monoclonal antibody targets the HA protein and can not only specifically recognize Eurasian avian H1N1 subtype swine influenza viruses of various genotypes including G4, but also has good reactivity to the 2009 pandemic (Pdm / 09) H1N1 subtype swine influenza virus, and can bind to human and avian H1N1 subtype influenza viruses, showing broad-spectrum action characteristics against H1 subtype influenza viruses. The monoclonal antibody of the present invention has been shown to have strong neutralizing activity through chicken embryo neutralization tests and mouse preventive protection tests, and is expected to be used in the diagnosis and prevention of diseases of pigs caused by H1N1 subtype swine influenza viruses and possible human infections.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and mainly relates to a hybridoma cell line that can stably secrete neutralizing monoclonal antibodies against the HA protein of Eurasian avian and Pdm / 09 evolutionary lineage H1 subtype swine influenza viruses, as well as the monoclonal antibodies secreted by the hybridoma cell line and their applications. Background Art

[0002] Swine influenza virus belongs to the influenza A virus genus of the Orthomyxoviridae family. Its genome consists of a single-stranded negative-sense RNA segment consisting of eight segments, in descending order of sequence length: PB2, PB1, PA, HA, NP, NA, M, and NS. The hemagglutinin protein, encoded by the fourth RNA segment, is the primary antigenic glycoprotein on the virus's surface and a key target for the development of vaccines, neutralizing antibodies, and diagnostic reagents.

[0003] Swine influenza virus infection of pigs can cause swine flu, an acute, febrile, highly contagious respiratory infection common in swine herds. It occurs and spreads worldwide, seriously threatening the healthy development of the swine industry. Furthermore, swine influenza has significant public health implications. Because the pig's respiratory epithelium harbors both α-2,6 receptors for human influenza viruses and α-2,3 receptors for avian influenza viruses, it acts as a genetic "mixing vessel" and is considered a key site for genetic reassortment between human and avian influenza viruses. Furthermore, some swine influenza viruses can cross the species barrier, spreading directly from pigs to humans and even effectively between humans.

[0004] Swine influenza viruses circulating globally include subtypes such as H1N1, H1N2, and H3N2. The most widespread H1N1 subtype is further divided into lineages based on genetic differences in the HA gene: classical swine (CS) H1N1, Eurasian avian-like (EA) H1N1, and the 2009 pandemic (Pdm / 09) H1N1. In my country, the H1N1 subtype has gradually become dominant in the circulation of swine influenza viruses since 2001. Although EAH1N1's dominance was briefly replaced by Pdm / 09H1N1 in the years following 2009, it remains the predominant swine influenza virus circulating in domestic pig populations. Of particular note, EAH1N1 continues to generate numerous genotypes (G) through reassortment of internal genes within Pdm / 09H1N1. Among them, genotype 4 (G4) EA H1N1 has become dominant in my country's pig population since 2016 and has a strong potential for cross-host transmission, posing a serious threat to the pig industry and public health security (Huanliang Yang, Yan Chen, Chuanling Qiao, et al. Prevalence, genetics, and transmissibility in ferrets of Eurasian avian-like H1N1 swine influenza viruses[J]. Proc Natl Acad Sci USA, 2016, 113(2): 392-397; Honglei Sun, Yihong Xiao, Jiyu Liu et al. Prevalent Eurasian avian-like H1N1 swine influenza virus with 2009 pandemic viral genes facilitating human infection[J]. Proc Natl Acad Sci USA, 2020, 117(29): 17204-17210; Fei Meng, Yan Chen, Zuchen Song, et al. Continued evolution of the Eurasian avian-like H1N1 swine influenzaviruses in China [J]. Sci China Life Sci, 2023, 66(2): 269-282).Relevant investigations have shown that cases of human infection with the G4 genotype EA H1N1 swine influenza virus have occurred in Yunnan Province and Shaanxi Province in my country in 2020 and 2022, respectively (Li Zi, Zhao Xiaonan, Huang Weijuan, et al. Pathogenic characteristics of the first case of human infection with the G4 genotype Eurasian avian H1N1 swine influenza virus in Yunnan Province [J]. Acta Virologica Sinica, 2022, 38(2): 290-297; Qin Long, Zhang Junjun, Chen Bin, et al. Genetic characteristics of the first case of human infection with the G4 genotype Eurasian avian H1N1 swine influenza virus in Shaanxi Province [J]. Chinese Journal of Preventive Medicine, 2023, 57(9): 1434-1439). Therefore, monitoring and preventing the EA H1N1 swine influenza virus, especially the G4 genotype, and warning of the possible recurrence of the Pdm / 09H1N1 virus are of great practical significance to both pig farming and human health.

[0005] Monoclonal antibodies are highly homogeneous, specific antibodies directed against a single, specific antigenic epitope and are widely used in the diagnosis and prevention of infectious diseases. Neutralizing monoclonal antibodies, in particular, offer advantages in disease prevention and treatment because they can block the binding of pathogens to target cells. Currently, there is a lack of neutralizing monoclonal antibodies that can simultaneously target both the EA H1N1 and Pdm / 09 H1N1 swine influenza viruses. Summary of the Invention

[0006] To address the challenges of the existing technology, the present invention has produced a monoclonal antibody using hybridoma technology that is capable of antagonizing both EA (H1N1) and Pdm / 09 (H1N1) subtype swine influenza viruses, and also recognizes H1N1 subtype human influenza viruses and H1N1 subtype avian influenza viruses. This monoclonal antibody specifically targets the HA protein, exhibits hemagglutination inhibition and neutralization activity, and belongs to the IgG2b subclass. The successful development of this monoclonal antibody not only lays a foundation for the rapid detection of H1 subtype swine influenza viruses of different evolutionary lineages, as well as H1 subtype human and avian influenza viruses, but also provides a potential candidate antiviral agent for preventing potential human infection with H1 subtype swine influenza viruses.

[0007] The present invention provides a hybridoma cell line capable of stably secreting neutralizing monoclonal antibodies against HA protein of Eurasian avian and Pdm / 09 evolutionary lineage H1 subtype swine influenza virus.

[0008] The monoclonal antibody 5F3 described in the present invention is produced by hybridoma cell line CCTCC NO: C2022145.

[0009] The monoclonal antibody of the present invention is of IgG2b and κ subtypes, and in addition to being able to recognize different genotypes of EA H1N1 and Pdm / 09 H1N1 swine influenza viruses, it can also bind to H1N1 subtype human influenza virus and avian influenza virus.

[0010] The present invention also provides the use of the monoclonal antibody 5F3 in the preparation of a preparation for resisting zoonotic G4 genotype H1N1 swine influenza virus infection.

[0011] The monoclonal antibody of the present invention specifically targets HA protein and has hemagglutination inhibition activity and neutralization activity.

[0012] The monoclonal antibody of the present invention can provide preventive protection against EA H1N1 subtype swine influenza virus infection in mammalian model BALB / c mice.

[0013] The characteristics and advantages of the present invention are as follows: the present invention discloses a monoclonal antibody 5F3 that can recognize H1N1 subtype swine influenza virus (taking into account the evolutionary lineages of EA H1N1 and Pdm / 09H1N1), human influenza virus and avian influenza virus. The monoclonal antibody 5F3 is secreted by hybridoma cell 5F3 (deposit number CCTCC NO: C2022145), targets the surface antigen HA protein of the virus, has hemagglutination inhibition activity and broad binding spectrum of H1 subtype, and can be used for further development of universal rapid detection products for H1 subtype animal influenza virus; the monoclonal antibody is tested by chicken embryo neutralization test and has high neutralization activity against both EA H1N1 and Pdm / 09H1N1 and good in vivo neutralization activity, can provide a preventive effect on BALB / c mice infected with G4 genotype EA H1N1 swine influenza virus, and is expected to be further used in the development of antiviral preparations for preventing and treating H1N1 subtype swine influenza virus infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The HA gene evolution tree is shown in Figure 1, where ▲ represents the virus strain corresponding to the immunogen used to prepare the HA protein monoclonal antibody.

[0015] Figure 2 Mouse immunization program for the preparation of monoclonal antibodies against the HA protein of H1 subtype swine influenza virus.

[0016] Figure 3 This is the IFA identification image of monoclonal antibody 5F3 on 293T cells transfected with HA protein eukaryotic expression plasmid.

[0017] Figure 4 This is a Western blot analysis of the monoclonal antibody 5F3 against 293T cells transfected with the HA protein eukaryotic expression plasmid.

[0018] Figure 5 The neutralization titer of monoclonal antibody 5F3 against H1 subtype swine influenza viruses of different evolutionary lineages.

[0019] Figure 6HI titers of monoclonal antibody 5F3 and different H1 subtype influenza viruses.

[0020] Figure 7 This is the IFA reaction diagram of monoclonal antibody 5F3 and MDCK cells infected with different H1 subtype influenza viruses.

[0021] Figure 8 This is the SDS-PAGE verification image of the purified monoclonal antibody 5F3.

[0022] Figure 9 This is the weight change curve of mice.

[0023] Figure 10 This is the mouse survival curve.

[0024] Figure 11 is the viral load in mouse lungs.

[0025] Figure 12 The figure shows the pathological histological changes of mouse lung.

[0026] The hybridoma cell line 5F3 in the present invention was deposited in the China Center for Type Culture Collection (address: Wuhan University, China) on May 25, 2022, and was classified and named hybridoma cell line 5F3 with a deposit number of CCTCC NO: C2022145. DETAILED DESCRIPTION

[0027] The present invention will be further described below in conjunction with the accompanying drawings and specific examples so that those skilled in the art can better understand the present invention and be able to implement it, but the examples are not intended to limit the present invention. The experimental methods used in the following examples are all conventional methods unless otherwise specified. The materials and reagents used are all commercially available unless otherwise specified. The following materials are all disclosed and stored in the applicant's laboratory and are promised to be made available to the public.

[0028] H1N1 subtype swine influenza virus strains A / swine / Shandong / SG03 / 2019(SG03), A / Swine / Jiangsu / 48 / 2010(JS48): Kaibiao Chen, Ming Kong, Jiao Liu, et al. Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay[J]. Arch Virol, 2021, 166(8): 2217-2224.

[0029] Eurasian avian-like H1N1 subtype swine influenza G4 genotype virus strain A / swine / Jiangsu / HD11 / 2020(HD11): Min Gu, Kaibiao Chen, Zhichuang Ge, et al. Zoonotic Threat of G4 Genotype Eurasian Avian-Like Swine Influenza A(H1N1)Viruses, China, 2020[J]. Emerg Infect Dis, 2022, 28(8): 1664-1668.

[0030] H1N1 subtype swine influenza virus strain A / swine / Jiangsu / zg14 / 2011(ZG14): Kaibiao Chen, Ming Kong, Jiao Liu, et al. Rapid differential detection of subtype H1 and H3 swine influenza viruses using a TaqMan-MGB-based duplex one-step real-time RT-PCR assay[J]. Arch Virol, 2021, 166(8): 2217-2224.

[0031] H1N1 subtype swine influenza virus strain A / swine / Jiangsu / 01 / 2016 (JS01): Shi Liwei. Study on the enhanced pathogenicity of recombinant H1N1 swine influenza virus containing the internal gene cassette of H9N2 avian influenza virus in mice[D]. Yangzhou: Yangzhou University, 2021.

[0032] H1N1 subtype human influenza virus strain A / California / 04 / 2009 (CA04), H1N1 subtype avian influenza virus strain A / duck / Shandong / SDd11 / 2013 (SDd11): Min Gu, Jun Jiao, Suhan Liu, et al. Monoclonal antibody targeting a novel linear epitope on nucleoprotein confers pan-reactivity to influenza A virus[J]. Appl Microbiol Biotechnol, 2023, 107(7-8): 2437-2450.

[0033] H1N2 subtype avian influenza virus strain A / duck / Huaian / 0151 / 2018(180151): Zhang Xinyu, Cai Tianyu, Zhao Ying, et al. Isolation, identification and whole genome genetic evolution analysis of a duck-derived H1 subtype avian influenza virus[J]. Chinese Journal of Veterinary Science, 2021, 41(12): 2325-2332.

[0034] The materials and reagents used are as follows:

[0035] Example 1

[0036] Obtaining hybridoma cell line 5F3:

[0037] Preparation of immunogens: H1N1 subtype swine influenza virus strains A / swine / Shandong / SG03 / 2019 (SG03) and A / Swine / Jiangsu / 48 / 2010 (JS48) were selected as immunogens. The HA gene nucleotide identity between the two strains is only 70.3%, and the amino acid similarity is only 78.3%. SG03 and JS48 belong to different genetic evolutionary lineages and are respectively divided into EA H1N1 and Pdm / 09H1N1 branches on the HA gene evolutionary tree. Figure 1 SG03 and JS48 were serially diluted 10-fold, and 10 -6 A dilution of 0.2 mL / embryo was inoculated into 9-day-old SPF chicken embryos. After 96 hours, allantoic fluid with an HA titer of ≥7 was collected. Subsequently, the allantoic fluid was centrifuged at 8,000 rpm and 4°C for 10 minutes, and the supernatant was collected. The centrifuged allantoic fluid was then mixed with β-propiolactone at a ratio of 3,000:1 and inactivated by vertical shaking at 4°C for 24 hours. The β-propiolactone was then inactivated by inoculation into chicken embryos to confirm full viral inactivation. The inactivated virus was ultracentrifuged at 30,000 rpm and 4°C for 2 hours, and the supernatant was discarded. The ultracentrifuged pellet was resuspended in 1.5 mL of sterile PBS buffer stored at 4°C. The purified virus was then filtered through a 0.22 μm filter and stored at -70°C until ready for use.

[0038] Animal Immunization: Inactivated, purified whole virus was mixed with Freund's complete adjuvant in a 1:1 ratio and homogenized five times at 6500 rpm for 16 seconds to achieve full emulsification. Six-week-old BALB / c mice were immunized subcutaneously at multiple sites with a dose of 150 μL / mouse. Three immunizations were performed at a 2-week interval. For the second and third immunizations, equal volumes of virus were mixed with Freund's incomplete adjuvant. After uniform emulsification, mice were again immunized by subcutaneous multiple sites. Three days before fusion, mice were boosted with an intraperitoneal injection of whole virus. Seven days after the third immunization, serum was collected from the tail vein of the mice to obtain triple-immune serum. The antibody titer in the serum was determined using a hemagglutination inhibition (HI) assay. Cell fusion was permitted if the HI titer exceeded 6 log2. In order to obtain monoclonal antibodies against swine influenza viruses of both EA H1N1 and Pdm / 09H1N1 evolutionary lineages, the present invention optimizes the animal immunization procedure and uses swine influenza virus strains SG03 and JS48 belonging to the EAH1N1 and Pdm / 09H1N1 evolutionary lineages, respectively, for alternating immunization. Figure 2 shown.

[0039] Cell fusion: After the third immunization of mice, mouse myeloma cells SP2 / 0 were revived and expanded to a suitable number after the cell state stabilized; 3 days before fusion, 300 μL of purified virus was injected into the mouse peritoneal cavity for shock immunization; on the day of fusion, positive serum was collected by eye sampling of immunized mice, and the mice were sacrificed by dislocating the neck, immersed in 75% alcohol for 10 minutes, and then fixed on the dissecting table; the spleen of the mouse was aseptically collected, the connective tissue and fat were removed, and the spleen was placed on a cell sieve and fully ground using the piston of a 2.5 mL syringe. The resulting spleen cell suspension was transferred to a 50 mL fusion tube and placed in a 5-10% CO2, 37°C incubator for 10-15 minutes; at the same time, the SP2 / 0 cells were gently blown off with HT culture medium, collected into a centrifuge tube containing spleen cells, and gently mixed thoroughly. After low-speed centrifugation at 000r / min for 10 minutes, remove the supernatant, gently rub the bottom of the fusion tube with the palm of your hand to disperse the cells, and slowly add 1mL of PEG1500 preheated to 37℃ in a 37℃ water bath environment to complete the fusion of the two. Add antibiotic-free and serum-free DMEM medium from slow to fast speed to terminate the fusion reaction; let the fusion tube stand at 37℃ for 10 minutes, then centrifuge and discard the supernatant, resuspend the cell pellet with HAT medium, evenly add 100μL / well to a 96-well cell plate, and culture in a 5-10% CO2, 37℃ incubator; after 5 days, half of the medium is replaced with fresh HAT medium; after 7-10 days, the medium is fully replaced with HT medium; when the cell culture supernatant turns yellow or the cells are distributed to more than 1 / 10 of the bottom area of ​​the well, the hybridoma cells can be screened by aspirating the cell supernatant to detect antibodies.

[0040] Hybridoma Screening: Since the original design of this invention aimed to obtain neutralizing monoclonal antibodies against both EA (H1N1) and Pdm / 09 (H1N1) swine influenza viruses, and neutralizing antibodies generally target the viral HA protein and may also exhibit hemagglutination inhibition activity, this example screened for positive hybridomas using an HI assay, selecting two virus strains, SG03 (EA (H1N1)) and JS48 (Pdm / 09 (H1N1)), for simultaneous testing. When hybridoma supernatants showed high HI titers against both SG03 (EA (H1N1)) and JS48 (Pdm / 09 (H1N1)), positive hybridomas were identified that secreted monoclonal antibodies that recognized both the EA (H1N1) and Pdm / 09 (H1N1) swine influenza virus HA proteins. However, whether the secreted antibodies possessed neutralizing activity required further testing.

[0041] The HI assay screening process is briefly described as follows: Approximately 10 days after fusion, when hybridoma cells have spread to more than 1 / 10 of the well bottom area, hybridoma screening is ready. A 1% red blood cell suspension is prepared to measure the HA titer of the immunization virus strains SG03 (EA H1N1) and JS48 (Pdm / 09 H1N1). Four units of virus are then prepared based on the HA titer. 25 μL of cell supernatant from a 96-well cell plate is transferred to each well of a 96-well hemagglutination plate. An equal volume of four units of virus is then added to each well. The plates are shaken to mix thoroughly and then placed in a 37°C incubator for 15 minutes. After removal, 25 μL of the red blood cell suspension is added to each well, mixed thoroughly, and allowed to stand at room temperature for 10 minutes for observation. Wells in which red blood cells showed no agglutination and only a single, well-growing cell clump in tests with both SG03 (EA H1N1) and JS48 (Pdm / 09H1N1) viruses were considered primary screening positive wells for HI titer determination. A 96-well hemagglutination plate pre-filled with 25 μL of PBS buffer was removed and 25 μL of the supernatant from the primary screening positive well was aspirated and added to well 1. This was then serially diluted to well 10. 25 μL of four units of antigen was then added to each well until well 11. The cells were then shaken to mix and incubated at 37°C for 15 minutes. Afterward, 25 μL of a 1% red blood cell suspension was added to each well, and the specific HI titer was measured. After screening, a positive hybridoma cell line, 5F3, was successfully isolated, capable of simultaneously recognizing both SG03 (EA H1N1) and JS48 (Pdm / 09H1N1) and exhibiting a high HI titer. The HI titer of the hybridoma cell 5F3 supernatant in reaction with SG03 (EA H1N1) virus can reach 6 log2, and the HI titer in reaction with JS48 (Pdm / 09H1N1) virus can reach 5 log2.

[0042] Limiting dilution subcloning of positive hybridoma cells: Gently remove cells from well 5F3 of the positive hybridoma cell line with an appropriate amount of HT medium. Pipette 100 μL of the cell suspension into well 1 of column 1 of a new 96-well plate and serially dilute the suspension two-fold with HT medium from top to bottom. Place the plate in a 37°C incubator for a short period of time and count the cells. Select a well with 60-100 cells and remove the cells from the well. Transfer the well to a centrifuge tube containing 4 mL of HT medium and gently mix thoroughly. Plate the prepared hybridoma cell suspension at 100 μL / well into columns 2-3 of the 96-well plate. Fill the centrifuge tube to 4 mL of medium and mix thoroughly again. Plate the prepared hybridoma cell suspension into columns 4-5 of the 96-well plate at 100 μL / well. Repeat this process and plate the hybridoma cell suspension into columns 6-7, 8-9, and 10-11, respectively. Finally, incubate the plate in a 37°C cell culture incubator with 5-10% CO2 for incubation. After about 5 days, the cells were observed for growth and wells containing only single cell colonies were marked. Around 10 days later, the supernatant from each well was assayed for HI antibody levels. The single cell wells with the highest antibody titers were selected and cloned again using limiting dilution. After three repetitions of this subcloning procedure, the supernatant from the fusion-positive hybridoma cells, 5F3, showed 100% HI antibody positivity against both the SG03 (EA H1N1) and JS48 (Pdm / 09 H1N1) viruses.

[0043] Example 2

[0044] Preparation of ascites:

[0045] Sterile liquid paraffin (300 μL / mouse) was injected into parous BALB / c female mice via intraperitoneal injection. Around 7 days later, the well-growing hybridoma 5F3 cells were blown off and centrifuged at 1000 rpm for 10 min, after which the supernatant was discarded. Sterile PBS buffer was added to resuspend the cells, and the cells were centrifuged at 1000 rpm for 10 min again, after which the supernatant was discarded. An appropriate amount of sterile PBS was added to resuspend the cells and count the cells. Approximately 1×10 6 Inject hybridoma cells into the peritoneal cavity of mice. Observe the mice daily for approximately 5 days. When the abdomen becomes noticeably distended, collect ascites with a #16 syringe needle. Centrifuge at 4000 rpm for 10 minutes, then collect the supernatant. Determine the HI titer, aliquot, and store in a -70°C freezer until needed.

[0046] Example 3

[0047] Subclass identification of monoclonal antibody 5F3: The identification was performed using the mouse monoclonal antibody Ig class / subclass identification ELISA kit (Cat. No.: BF16001) from Suzhou Biolong Technology Co., Ltd. According to the kit instructions, the following operations were performed: 50 μL of hybridoma cell supernatant was diluted 1:1 with specimen diluent, added to the detection wells of the ELISA plate, and negative and positive controls were set up simultaneously, and incubated at 37°C for 30 min; the liquid in the plate was discarded, the ELISA plate was washed 5 times, 100 μL each of enzyme-labeled secondary antibodies of IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, Kappa (κ), and Lambda (λ) were added to the detection wells, and the cells were incubated at 37°C for 30 min; the liquid in the plate was discarded, the ELISA plate was washed 5 times again, 50 μL each of color developer A solution and B solution were added, and the cells were incubated at 37°C in the dark for 20 min; finally, 50 μL of reaction stop solution was added to each well, and the OD value of each well at a wavelength of 450 nm was read using a microplate reader.

[0048] The test results are shown in Table 1. The monoclonal antibody 5F3 secreted by the hybridoma cell line 5F3 has a heavy chain of the IgG2b subclass and a light chain of the κ type.

[0049] Table 1 Subclass identification of monoclonal antibody 5F3

[0050]

[0051] Example 4

[0052] IFA detection of monoclonal antibody 5F3:

[0053] First, the Eurasian avian H1N1 subtype swine influenza G4 genotype virus strain A / swine / Jiangsu / HD11 / 2020 (HD11) was selected, and its HA gene (GenBank accession number OL744689.1) was connected to the eukaryotic expression vector pCAGGS (purchased from Miaoling Bio, product number P0165) to construct the eukaryotic expression plasmid pCAGGS-HA expressing HA protein. The operation steps are briefly described as follows: HD11 viral RNA was extracted using the tissue / cell RNA rapid extraction kit (product number 2103) of Beijing Junno Biotechnology Co., Ltd. according to the instructions, and then reverse transcription was performed by adding the reverse transcription primer 5'-AGCAAAAGCAGG-3' (SEQ ID No. 1). The cDNA obtained by reverse transcription was further PCR amplified using the upstream primer 5'-GTCTCATCATTTTGGCAAAGAATTCATGGAAGCAAGATTATTTGTATTATTCTGTGC-3' (SEQ ID No. 2) and the downstream primer 5'-AGGGAAAAAGATCTGCTAGCTCGAGTTAAATGCATACTCTGCACTGCAATGAC-3' (SEQ ID No. 3) to amplify the full length of the HA gene. The PCR amplification product was subjected to 1% agarose gel electrophoresis and then gel-recovered using the DNA gel extraction kit (AxyPrep DNA Gel Extraction Kit, product number AP-GX-50G) of Axygen. At the same time, the empty vector pCAGGS was cleaved using the restriction endonuclease EcoR I and Xho I were digested at 37°C for 20 minutes, and the digestion products were recovered by gel electrophoresis. Next, the recovered full-length HA was ligated with the linearized empty vector pCAGGS using the pEASY-Basic Seamless Cloning and Assembly Kit (Cat. No. CU201-02) from Beijing Quanshijin Biotechnology Co., Ltd. according to the manufacturer's instructions. After the ligation reaction, the ligation product was transformed into Escherichia coli DH5α competent cells. The transformed plates were incubated at 37°C until single colonies appeared. Well-growing colonies were selected and incubated in LB broth containing ampicillin for 12 hours. Cultures that tested positive for PCR were then plasmid-extracted using the Axygen Plasmid Miniprep Kit (Cat. No. AP-MN-P-50) according to the manufacturer's instructions. The extracted plasmids were then sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing verification. The eukaryotic expression plasmids with the correct sequence were designated pCAGGS-HA.

[0054] The constructed eukaryotic expression plasmid pCAGGS-HA and empty vector pCAGGS were further transfected into 293T cells respectively. After 36 hours, the cell supernatant was discarded and washed three times with 4°C pre-cooled PBS, and then fixed with 4% paraformaldehyde at 4°C for 20 minutes; the fixative was discarded, and after it evaporated naturally, a 3% bovine serum albumin (BSA) solution prepared with PBST was added and allowed to stand at room temperature for 1 hour for blocking; the blocking solution was discarded and washed three times with PBST, each for 5 minutes; a 1:300-fold diluted monoclonal antibody 5F3 mouse ascites was used as the primary antibody, and the mouse positive serum was diluted 1:300-fold as the positive control, and incubated in a 4°C refrigerator overnight; after washing with PBST three times, a 1:500-fold diluted FITC-labeled goat anti-mouse IgG fluorescent secondary antibody was added, and the cells were incubated in a light-proof shaker at room temperature for 1 hour. After washing with PBST three times as above, the cells were observed under a microscope. The results are shown in the attached figure. Figure 3 As shown, 5F3 mouse ascites (see Figure 3 A) and mouse positive serum (see Figure 3 C) can make 293T cells transfected with HA plasmid show bright green fluorescence; while 293T cells transfected with empty vector pCAGGS and incubated with 5F3 mouse ascites showed no visible fluorescence (see Figure 3 B) Therefore, monoclonal antibody 5F3 targets HA protein and has IFA properties and can be used in IFA assays.

[0055] Example 5

[0056] Western Blot Detection of Monoclonal Antibody 5F3:

[0057] The pCAGGS-HA eukaryotic expression plasmid and the pCAGGS empty vector stored in the laboratory were transfected into 293T cells respectively. Cell lysates were collected after 36 hours, and the cell sample transfected with the pCAGGS empty vector was used as a negative control. The collected cell lysates were mixed with 5×SDS-PAGE loading buffer, heated and denatured, and then electrophoresed and transferred to the membrane. After the transfer, the membrane was blocked with 5% skim milk solution and incubated at 37°C for 2 hours. The membrane was washed with PBST three times, 5 minutes each time. The ascites of 5F3 mice was diluted 1:1000 times as the primary antibody and placed on a shaker at 37°C for 1 hour. After washing the membrane with PBST three times as above, HRP-labeled goat anti-mouse IgG diluted 1:5000 times with TBST was incubated as the secondary antibody and incubated at 37°C in the dark for 1 hour. The membrane was washed with PBST three times as above, and developed by ultrasensitive ECL and observed by imaging. The results are shown in the attached figure. Figure 4As shown, the 293T cell sample transfected with the empty pCAGGS vector showed no band, while the 293T cell sample transfected with the pCAGGS-HA plasmid produced a distinct single reactive band around 70 kDa, the same size as the HA protein, indicating that the monoclonal antibody 5F3 is specific for the HA protein. Therefore, 5F3 has Western blot reactivity and can be used in Western blot experiments.

[0058] Example 6

[0059] Neutralizing activity assay of monoclonal antibody 5F3:

[0060] Neutralization activity of antibodies against H1 subtype swine influenza viruses of different evolutionary lineages was determined by chicken embryo neutralization test. Two viruses, HD11 (EA H1N1) and JS48 (Pdm / 09H1N1), were selected and their chicken embryo half infectious dose (EID) was determined. 50 5F3 ascites was diluted 100 times with PBS and then serially diluted to 1:100, 1:200, 1:400...1:102400, respectively, with an equal volume of 200 EID 50 The virus was mixed evenly and incubated at 37°C for 1 hour before inoculating the mixture of the monoclonal antibody and the virus into 9-day-old SPF chicken embryos. Three embryos were inoculated at each dilution. Chicken embryos inoculated with only the virus solution but no monoclonal antibody were used as positive controls, while chicken embryos inoculated with only PBS were used as negative controls. Dead embryos within 24 hours were discarded, and after 72 hours, the number of hemagglutination-positive chicken embryos at each dilution was determined. Based on the results, the maximum dilution of ascites that could protect 50% of the chicken embryos was calculated using the Reed-Muench method as the half-life protective dose (PD). 50 ), which is the neutralizing titer of the monoclonal antibody. The results are shown in the attached Figure 5 As shown, the neutralization titer of 5F3 ascites against HD11 (EA H1N1) virus was as high as 10 -4.56 , that is, a 1:30368 dilution of ascites fluid can still protect 50% of the inoculated chicken embryos from HD11 (EA H1N1) virus infection; the neutralization titer of JS48 (Pdm / 09H1N1) virus also reached 10 -4.19 That is, a 1:15488 dilution of ascites can still protect 50% of the inoculated chicken embryos from JS48 (Pdm / 09H1N1) virus infection, indicating that the monoclonal antibody has high neutralizing activity against H1 subtype swine influenza virus strains of the Eurasian avian and Pdm / 09 evolutionary lineages.

[0061] Example 7

[0062] Broad spectrum detection of monoclonal antibody 5F3:

[0063] HI titer determination of 5F3 ascites and H1 subtype influenza viruses from different host sources and different HA evolutionary lineages:

[0064] Four units of different H1 subtype animal influenza viruses (as shown in Table 2) stored in the laboratory were prepared. Monoclonal antibody 5F3 was diluted 2-fold in a 96-well hemagglutination plate. The mouse ascites dilutions were reacted with the four units of the prepared virus in a 37°C incubator for 15 minutes. 1% chicken red blood cell suspension was added to each well and the plate was left to stand at room temperature for 15 minutes before observing the HI results. The results are shown in the attached figure. Figure 6 As shown, the three strains of EAH1N1 swine influenza viruses of different genotypes and two strains of Pdm / 09H1N1 viruses of swine or human origin all reacted well with 5F3 ascites. Furthermore, one strain of H1N1 and H1N2 subtype influenza viruses of avian origin also produced high HI titers with monoclonal antibody 5F3. Therefore, the HI test with different H1 subtype animal influenza viruses demonstrates that monoclonal antibody 5F3 not only has the ability to simultaneously recognize EAH1N1 and Pdm / 09H1N1 swine influenza viruses, but also exhibits good broad-spectrum reactivity against H1 subtype influenza viruses of swine, humans, and avian origin.

[0065] Table 2 H1 subtype influenza virus strains tested with monoclonal antibody 5F3

[0066]

[0067] IFA reactivity of 5F3 ascites with different H1 subtype influenza viruses:

[0068] In addition to testing the broad spectrum of monoclonal antibody 5F3 through the above-mentioned HI test, an IFA test was also performed to test the ability of 5F3 to recognize different H1 subtype influenza viruses. The HD11 (swine EA H1N1), JS48 (swine Pdm / 09H1N1), CA04 (human Pdm / 09H1N1) and 180151 (avian H1N2) viruses in Table 2 were selected to infect MDCK cells, and MDCK cells that were not infected with the virus were set up as negative controls. After the cells were fixed and blocked according to the method in the above-mentioned IFA test, 5F3 ascites diluted 1:300 was used as the primary antibody, incubated at 4°C overnight, and washed 3 times with PBST, each time for 5 minutes; then FITC-labeled goat anti-mouse IgG fluorescent secondary antibody diluted 1:500 with PBST was added, incubated on a shaker at room temperature in the dark for 1 hour, washed 3 times with PBST, and observed under a fluorescence microscope. The results are shown in the attached figure. Figure 7As shown, bright green fluorescence was observed in MDCK cells infected with the four viruses HD11 (swine EAH1N1), JS48 (swine Pdm / 09H1N1), CA04 (human Pdm / 09H1N1), and 180151 (avian H1N2), while no specific fluorescence was observed in the negative control cells. Therefore, monoclonal antibody 5F3 was able to produce good IFA reactivity with the H1 subtype animal influenza viruses from these different host sources, indicating that 5F3 has a broad spectrum of recognition of H1 subtype influenza viruses.

[0069] Example 8

[0070] Purification of monoclonal antibody 5F3:

[0071] The obtained mouse monoclonal antibody ascites was purified using Protein A+G Agarose (Cat. No.: P2019-2mL) reagent from Shanghai Biyuntian Biotechnology Co., Ltd. The following steps were performed according to the reagent instructions: (1) 1 mL of Protein A+G Agarose was loaded onto the purification column, and then the column was washed and equilibrated with 10 column volumes of PBS; (2) The mouse ascites to be purified was centrifuged at 8,000 rpm for 10 min, the supernatant was taken and diluted 10 times with PBS before loading onto the purification column; (3) After the purified antibody was completely passed through the column, it was washed with 10 column volumes of PBS to remove unbound and non-specifically bound proteins; (4) After washing, 100 μL of neutralizing solution (1 M Tris-HCl, pH 8.8) was added per mL of eluent, and an appropriate amount of neutralizing solution was added to the collection tube in advance, and then 10 mL of 50 mM glycine, pH 2.7 was used as the eluent to elute the bound antibody; (5) The concentration of the purified antibody was further determined using the BCA protein concentration assay kit (Cat. No. P0012) from Shanghai Biyuntian Biotechnology Co., Ltd., and the purification effect was detected by SDS-PAGE.

[0072] The concentration of the purified monoclonal antibody 5F3 was 1.892 mg / ml as determined by the BCA method. 8% (Tris-Glycine gel) SDS-PAGE results (attached Figure 8 ) showed that the monoclonal antibody 5F3 purified by Protein A+G Agarose had a clear band at about 175KD in the non-reduced state, which was basically equivalent to the relative molecular mass of mouse IgG and had almost no mixed bands, indicating that the purity was good and could be used for the next in vivo animal test.

[0073] Example 9

[0074] Prophylactic protective efficacy test of monoclonal antibody 5F3 in mouse model:

[0075] In order to evaluate whether the monoclonal antibody 5F3 can provide a certain in vivo protective effect, BALB / c mice were selected as the experimental mammalian model. Thirty-eight 6-week-old BALB / c mice were randomly divided into 4 groups with 5 mice / group, namely the first group of antibody administration with a dose of 5 mg / kg, the second group of antibody administration with a dose of 10 mg / kg, the third group of virus control group without antibody administration, and the fourth group of blank control group without antibody administration and virus inoculation. Except for the blank control group of 5 mice, the other 3 groups included 11 mice (5 for weighing and 6 for autopsy to collect lungs). The experimental operation steps are briefly described as follows: the initial weight of each mouse was recorded before the administration of the antibody; the monoclonal antibody 5F3 was injected intraperitoneally into the mice in groups 1 and 2 at doses of 5 mg / kg and 10 mg / kg, respectively; 2h later, the mice in groups 1, 2 and 3 were inoculated intranasally with 100 μL containing 2×10 6 EID 50 The G4 genotype EAH1N1 swine influenza virus HD11 was used for the treatment. The mice in group 4 were not inoculated with the virus but were inoculated with 100 μL PBS intranasally. After the inoculation, the experimental mice were weighed every day and observed for 14 days. Among them, 3 mice in groups 1, 2, and 3 were killed 3 days and 5 days after the infection, respectively, and their lungs were collected. Part of the lungs were used to detect the viral load, and part of the lungs were used to prepare pathological tissue sections.

[0076] The results are as attached Figure 9-12 As shown, the body weight of mice in group 3, which were not given antibodies but only inoculated with HD11 virus, continued to decrease within 5 days after infection ( Figure 9 ), all 5 mice died ( Figure 10 ); The mice in group 4, which were neither administered with antibodies nor challenged with toxic substances, gained weight during the entire 14-day observation period ( Figure 9 ) and all survive ( Figure 10 ); The above results indicate that the experimental control group was established. In the antibody administration group, the body weight of mice in the 5mg / kg dose injection group showed a continuous downward trend after infection ( Figure 9 ), 3 mice died within 7-9 days, and the final survival rate was only 40% ( Figure 10 ), indicating that the 5mg / kg dose of monoclonal antibody 5F3 could not provide ideal preventive protection against viral infection; the mice in the 10mg / kg injection group showed a transient decrease in body weight 1-6 days after infection, but continued to gain weight 7-14 days later ( Figure 9 ), none of the 5 mice died, and the protection rate was 100% ( Figure 9-10 ), indicating that 10mg / kg dose of monoclonal antibody 5F3 can provide good preventive protection against viral infection. The viral load results of the lungs of mice on days 3 and 5 of the challenge are shown in the attached figure. Figure 11As shown in Figure 2, both 5mg / kg and 10mg / kg doses of monoclonal antibody 5F3 were able to significantly inhibit viral replication in the lungs, and the inhibitory effect of the 10mg / kg dose group was more obvious. In addition, the pathological section results of the lungs (see Figure 12 ) showed that compared with the challenge control group, the 5 mg / kg dose group alleviated the lung damage caused by viral infection to a certain extent, while the 10 mg / kg dose group significantly reduced the destruction of the lung tissue structure and inflammatory response caused by viral infection, indicating that the monoclonal antibody 5F3 can provide preventive protection against the G4 genotype EA H1N1 swine influenza virus and is expected to be further developed into an antiviral preparation.

Claims

1. Hybridoma cell line 5F3, deposited with CCTCC NO: C2022145.

2. A neutralizing monoclonal antibody 5F3 against the HA protein of Eurasian avian and Pdm / 09 lineage H1 subtype swine influenza virus, characterized in that The monoclonal antibody produced by the hybridoma cell line 5F3 according to claim 1 is of IgG2b, κ type.

3. Use of the monoclonal antibody 5F3 according to claim 2 in the preparation of an anti-zoonotic G4 genotype H1N1 swine influenza virus infection preparation.

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