A neutralizing monoclonal antibody targeting Gn glycoprotein of fever with thrombocytopenia syndrome virus and its application

Abstract

The present invention provides a neutralizing monoclonal antibody B1G11 targeting the Gn glycoprotein of fever with thrombocytopenia syndrome virus and its application, which belongs to the field of medical technology. The present invention immunizes mice with SFTSV Gn protein as an immunogen, and obtains a highly efficient neutralizing monoclonal antibody B1G11 that specifically targets Gn through flow sorting and single B cell PCR. The monoclonal antibody B1G11 has a very high in vitro neutralizing activity against SFTSV (the IC50 for fake virus can be as low as 0.04 μg / mL, and the IC50 for real virus can be as low as 0.4 μg / mL). The monoclonal antibody B1G11 has application value in preventing and treating SFTSV infection, and can be used to prepare drugs for preventing and treating SFTSV infection and SFTSV detection products.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a highly neutralizing monoclonal antibody B1G11 targeting the Gn protein of the fever with thrombocytopenia syndrome virus and its application. Background Art

[0002] Fever with thrombocytopenia syndrome virus (SFTSV) belongs to the Bunyavirales order, Leucoviridae family, and Buntavirus genus. The virus can infect both wild and domestic animals, as well as humans, causing acute fever, thrombocytopenia, leukopenia, vomiting, and diarrhea. A small number of severe cases can die from multiple organ failure, with a mortality rate as high as 10%-30%. Current prevention and treatment for SFTSV infection primarily relies on supportive care for symptoms, and there is still a lack of safe and effective vaccines and antiviral drugs available.

[0003] Antibody-mediated immune response is one of the body's important means of fighting viral infections and is also one of the determining factors for the preventive and protective effects of vaccines. In addition, highly effective neutralizing protective antibodies can be used for emergency treatment of sudden viral infections and to control the spread of epidemics. They can also help susceptible and high-risk populations prevent viral infections. At the same time, the research and development of antibodies also contributes to the rapid development of serological diagnostic kits, facilitating the rapid diagnosis of infectious diseases. Studies on various viruses have found that isolated and purified monoclonal antibodies can effectively inhibit viral replication, while passive transfer of polyclonal serum and monoclonal antibodies to experimental animals can effectively prevent and protect animals from infection with the corresponding viruses. There are already antibody drugs on the market for the treatment of viral infections (such as Palivizumab for respiratory syncytial virus infection and Ibalizumab for HIV, etc.), and there are also many antibody drugs for different viral infections in the research and clinical trial stages.

[0004] The M segment of SFTSV encodes two envelope proteins, Gn and Gc. The heteromeric Gn and Gc complex covers the entire surface of the virion and is crucial for viral attachment and entry into host cells. Studies on Rift Valley fever virus (RVFV) and SFTSV, both members of the Phlebovirus family, have shown that Gn and Gc are important target antigens for inducing specific immune protection, and that vaccine efficacy is positively correlated with the concentration of antibodies reactive to the viral surface glycoproteins. However, no monoclonal antibodies targeting SFTSV have yet entered clinical trials or are commercially available for treatment.

[0005] Therefore, there is an urgent need to develop a highly neutralizing monoclonal antibody against SFTSV. Summary of the Invention

[0006] The object of the present invention is to provide a monoclonal antibody targeting Gn that is highly effective in neutralizing fever with thrombocytopenia syndrome virus and its application. The present invention first immunizes BALB / c mice with SFTSV Gn extracellular domain protein, removes the mouse spleen and lymph nodes, grinds them into a single cell suspension, uses the expressed Gn protein as bait protein, and screens single B cells that specifically bind to SFTSV Gn by flow sorting. The screened single B cells are then subjected to reverse transcription PCR and nested PCR to obtain the antibody's heavy chain variable region and light chain variable region nucleotide fragments, which are cloned into an antibody expression vector containing a constant region. After expression and purification in Expi293 cells, the antigen binding ability and virus neutralization ability are verified by ELISA, WB, and indirect immunofluorescence experiments, thereby obtaining a monoclonal antibody B1G11 with high neutralizing activity against SFTSV virus.

[0007] To achieve the above objectives, the present invention adopts the following technical solutions.

[0008] In one aspect, the present invention provides a monoclonal antibody B1G11 or an antigen-binding fragment thereof, which targets the SFTSV Gn protein, wherein the heavy chain variable region thereof comprises three complementarity determining regions: a CDR1 (GFTFSDYY) with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 (IRNKAKGYTT) with an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 (ARDYRYDDEYYYAMDY) with an amino acid sequence as shown in SEQ ID NO: 3; and the light chain variable region thereof comprises three complementarity determining regions: a CDR1 (QGISNY) with an amino acid sequence as shown in SEQ ID NO: 6, a CDR2 (YTS) with an amino acid sequence as shown in SEQ ID NO: 7, and a CDR3 (QQYSKFPRT) with an amino acid sequence as shown in SEQ ID NO: 8.

[0009] In one embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody B1G11 or its antigen-binding fragment is shown in SEQ ID NO: 4; the amino acid sequence of the light chain variable region of the monoclonal antibody B1G11 or its antigen-binding fragment is shown in SEQ ID NO: 9.

[0010] In one embodiment, the amino acid sequence of the heavy chain of the monoclonal antibody B1G11 or its antigen-binding fragment is shown in SEQ ID NO: 5; the amino acid sequence of the light chain of the monoclonal antibody B1G11 or its antigen-binding fragment is shown in SEQ ID NO: 10.

[0011] In one embodiment, the monoclonal antibody further comprises: an antibody with the same function obtained by substituting, deleting and / or adding one or more amino acids in the amino acid sequence of the monoclonal antibody; or an antibody with the same or similar function obtained by humanizing the variable region of a murine antibody.

[0012] In one embodiment, the antigen binding fragment is selected from Fab, Fab', Fab'-SH, scFv, F(ab')2.

[0013] In another aspect, the present invention provides a polypeptide comprising an amino acid sequence selected from SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 9 or SEQ ID NO: 10.

[0014] In another aspect, the present invention provides a polynucleotide encoding any of the aforementioned monoclonal antibodies or antigen-binding fragments or polypeptides, including a sequence encoding the heavy chain variable region (such as SEQ ID NO: 12) and / or the light chain variable region (such as SEQ ID NO: 14) of the B1G11 antibody, and a sequence encoding the heavy chain (such as SEQ ID NO: 13) and / or the light chain (such as SEQ ID NO: 15) of the B1G11 antibody.

[0015] In another aspect, the present invention provides an expression vector comprising the above polynucleotide, capable of expressing the above polynucleotide in a prokaryotic or eukaryotic host cell.

[0016] The expression vector may specifically be, but is not limited to, a prokaryotic expression vector, a phage vector, a viral vector or a mammalian expression vector. The present invention specifically uses a mammalian expression vector.

[0017] In another aspect, the present invention provides a host cell containing the above expression vector, wherein the host cell includes a prokaryotic or eukaryotic expression cell, which is capable of expressing the above expression vector.

[0018] In another aspect, the present invention provides the use of the monoclonal antibody B1G11 or its antigen-binding fragment in the preparation of a drug for treating or preventing FETV infection or a product for detecting FETV or its Gn protein.

[0019] A drug or drug combination for treating or preventing fever with thrombocytopenia syndrome virus infection, comprising the monoclonal antibody B1G11 or an antigen-binding fragment thereof.

[0020] A reagent or kit for detecting HFTS virus or its Gn protein, comprising the monoclonal antibody B1G11 or its antigen-binding fragment.

[0021] The monoclonal antibody B1G11 provided by the present invention targets the key surface antigen Gn of SFTSV and shows excellent neutralization effect. The half inhibitory concentration (IC 50 ) is 0.04μg / mL, for real virus IC 50 The concentration of B1G11 can be as low as 0.4 μg / mL. Furthermore, B1G11 neutralized both pseudoviruses and authentic strains of SFTSV, demonstrating its broad-spectrum neutralizing activity against different strains of SFTSV. The results of the present invention demonstrate that the monoclonal antibody B1G11 has broad application prospects in the preparation of therapeutic drugs or detection products for fever with thrombocytopenia syndrome. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Molecular sieve purification and SDS-PAGE identification of SFTSV Gn extracellular domain protein.

[0023] Figure 2 : SDS-PAGE image of the purified B1G11 monoclonal antibody.

[0024] Figure 3 : ELISA test of the binding curve of monoclonal antibody B1G11 and Gn antigen.

[0025] Figure 4 :Monoclonal antibody B1G11 detects Gn antigen of SFTSV pseudovirus by WB.

[0026] Figure 5 :Indirect immunofluorescence detection of SFTSV-infected cells using monoclonal antibody B1G11.

[0027] Figure 6 : Neutralization of SFTSV pseudovirus by monoclonal antibody B1G11 at the cellular level.

[0028] Figure 7 : Neutralization of SFTSV virulence by monoclonal antibody B1G11 at the cellular level. DETAILED DESCRIPTION

[0029] The present invention will be further described below in conjunction with specific examples to make the purpose, technical solutions and advantages of the present invention more clearly understood. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0030] Unless otherwise specified, all terms and techniques used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In the event of any conflict, the present specification shall prevail.

[0031] Unless otherwise specified, all raw materials, reagents, instruments, consumables, etc. used in the present invention can be purchased from the market or prepared by existing methods.

[0032] The following will describe in detail the monoclonal antibody of the present invention, its preparation method and application effects in combination with specific examples and experimental data.

[0033] Example 1 Expression and purification of the extracellular domain of SFTSV Gn protein

[0034] 1. The Gn sequence of the SFTSV strain WCH / 97 / HN / China / 2011 (abbreviated as WCH97) (SEQ ID NO: 11) was selected and an HRV3C protease cleavage site and a six-histidine tag were attached to its 3' end. Total RNA from the WCH97 strain was extracted with Trizol. Reverse transcription PCR was performed according to the manufacturer's instructions (Novozymes, R211-02) to obtain viral cDNA. The reverse transcription PCR system consisted of 10 μL of 2× RT Mix, 2 μL of HiScript III Enzyme Mix, 1 μL of Random Hexamers, 3 μL of RNA, and 4 μL of water. The PCR protocol was as follows: 25°C for 5 minutes, reverse transcription at 50°C for 45 minutes, followed by reaction at 85°C for 2 minutes. 2 μL of cDNA was used as a template to generate the Gn fragment sequence of the WCH97 strain by PCR (upstream primer 5'-GCGGAATTCGATACTGGACCGATCATTTGC-3', downstream primer 5'-CCAAGGTCGACCCGCTTACCTCCAATGTTGC-3'). The fragment was constructed into the pFastBac1 vector using EcoRI and SalI endonucleases and transformed into the DH10Bac competent cell. Correct clones were screened by blue-white staining and bacmids were extracted. Baculovirus was then transfected into SF9 cells for packaging. After virus amplification, the virus was inoculated into Hi5 cells to express the SFTSV Gn ectodomain protein.

[0035] 2. The Hi5 cell supernatant was collected and centrifuged at 10,000 g for 20 minutes at 4°C to remove cells and cell debris. The target protein was enriched by nickel-charged resin FF chromatography (GenScript). The highly purified SFTSV Gn extracellular domain protein was obtained by gel filtration chromatography (Superdex200Increase10 / 300GL, Cytiva). The SDS-PAGE gel image showed a size of approximately 38 kDa, which was consistent with the expected size. Figure 1 .

[0036] Example 2 Isolation of SFTSV Gn-specific single B cells

[0037] 1. Immunization of Mice

[0038] BALB / c mice were immunized four times with 10 μg of the Gn protein purified in Example 1 (Addvax as adjuvant, 1:1 mixture, Invivogen) injected intramuscularly every three weeks. Finally, 12.5 μg of Gn protein was injected intraperitoneally and intravenously every three weeks for booster immunization. The mice were sacrificed 5 days later, and the spleens were removed for flow cytometry sorting of Gn-specific single B cells.

[0039] 2. Isolation of SFTSV Gn Protein-specific Single B Cells

[0040] 1) In a biosafety cabinet, place a grinding mesh on a plate containing 1640 (2% FBS) medium. After blood collection, sacrifice and dissect the mouse. Remove the spleen and lymph nodes (remove as much fat and other tissue as possible), grind them in the grinding mesh, and collect the suspension. After treating with red blood cell lysis buffer, wash twice with 1640 (Mona, 2% FBS) and count the cells using a cell counter.

[0041] 2) Prepare cells according to the following requirements:

[0042] A. Single-staining tube cells: Prepare 8 equal amounts of cells for single staining with each fluorescent antibody to adjust compensation, with approximately 5 × 10 cells per aliquot. 5 cells, and resuspended in 50 μL staining buffer (PBS, 2% FBS, 1 mM EDTA);

[0043] B. Blank tube cells: Prepare another set of cells of the same size as the single-stained tube as a non-stained control group, i.e. 5×10 5 Resuspend cells in 50 μL staining buffer;

[0044] C. Cells to be sorted: 3 to 10 × 10 6 Resuspend cells in 100 μL staining buffer to prepare samples for sorting.

[0045] 3) Primary antibody staining:

[0046] A. Single-stained cells: Select highly expressed CD marker antibodies with FVS-780, CD3 / 4 / 8-BV510, CD19-PE-Cy7, IgD-PerCp-Cy5.5, CD138-BB515, CD95-PE, CD38-Pacific Blue, and APC fluorophores (all purchased from BD). Add each antibody to the single-stained cells at the recommended dilution ratio in the manufacturer's instructions and mix thoroughly.

[0047] B. Blank tube cells: not stained, but treated the same way as other samples.

[0048] C. Cells to be sorted: Add 0.5 μg / mL Biotin-SFTSV-Gn (EZ-Link NHS-PEG4-Biotin, Thermo Scientific, biotinylate the Gn protein purified in Example 1 according to the manufacturer's instructions) and mix well. Incubate at 4°C for 30 minutes.

[0049] 4) Wash cells twice with 100 μL staining buffer.

[0050] 5) Secondary Antibody Staining: Add the following antibodies to the cells to be sorted according to the ratio specified in the instructions: CD3 / 4 / 8-BV510, CD19-PE-Cy7, IgD-PerCp-Cy5.5, CD138-BB515, CD95-PE, CD38-Pacific Blue, Streptavidin-APC, and incubate at 4°C in the dark for 30 minutes.

[0051] 6) Wash the cells twice more. Resuspend the cells in wash buffer and transfer them to flow cytometer tubes. Store at 4°C in the dark until ready for loading.

[0052] 7) Flow cytometry was used to collect CD19+, CD3 / 4 / 8-, IgD-, and APC+ single B cells into a pre-prepared 96-well plate containing RNase inhibitor (Promega).

[0053] Example 3 Construction of Monoclonal Antibody Vectors from Single B Cell Clone

[0054] 1. Reverse Transcription PCR: Follow the manufacturer's instructions (Novozyme, R211-02) with the following procedure: Transfer the plate containing 7 μL of the sorted single B cell suspension per well from -80°C to ice, let it rest for 5 minutes, and centrifuge at 400 × g for 30 seconds at 4°C. Incubate at 65°C for 5 minutes, then quickly chill on ice for 2 minutes. Prepare the first-strand cDNA synthesis reaction mixture. Reverse transcription PCR system: 10 μL of 2× RT Mix, 2 μL of HiScript III Enzyme Mix, and 1 μL of Random Hexamers. PCR protocol: 25°C for 5 minutes, reverse transcription at 50°C for 45 minutes, followed by incubation at 85°C for 2 minutes.

[0055] 2. Nested PCR:

[0056] 1 μL of the reverse transcription product was used as a template for the first round of PCR amplification of the variable regions of antibodies H, κ, and λ. The amplification primers are shown in Table 1 below.

[0057] Table 1. First round PCR primers

[0058]

[0059]

[0060] The PCR reaction system was prepared according to the manufacturer's instructions (Novozymes, P505-d3), including: 10 μL of 2× Phanta Buffer, 0.4 μL of 10 mM dNTPs, 0.15 μL of upstream primer mix (5 μM each), 1 μL of downstream primer (10 μM), 0.5 μL of Phanta polymerase, and the total volume was made up to 20 μL with double-distilled water. The PCR program was as follows: initial denaturation at 95°C for 30 seconds, followed by 50 cycles of denaturation at 95°C for 15 seconds, annealing at 46°C for 15 seconds, and extension at 72°C for 1 minute, with a final extension at 72°C for 10 minutes. Nested PCR was performed using 1.5 μL of the first-round PCR product as template. Amplification primers are listed in Table 2.

[0061] Table 2. Nested PCR primers

[0062]

[0063] The PCR reaction system was prepared according to the manufacturer's instructions (EsTaq, Kangwei Century), consisting of 10 μL of 2× EsTaq Mix, 1 μL of upstream primer (10 μM), 1 μL of downstream primer (10 μM), and 1 μL of double-distilled water to a volume of 20 μL. The PCR program was as follows: initial denaturation at 94°C for 2 minutes, followed by 40 cycles of denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 20 seconds, followed by a final extension at 72°C for 10 minutes.

[0064] PCR products were separated by 1.2% agarose gel electrophoresis, and a 400-500 bp band was sequenced. The heavy chain variable region sequencing result is shown as SEQ ID NO: 12, and the light chain variable region sequencing result is shown as SEQ ID NO: 14. The corresponding monoclonal antibody was named B1G11. Immunogenetics (IMGT) online software was used to align and analyze the germline gene information of the antibody sequences.

[0065] The results of sequence alignment of the B1G11 monoclonal antibody with germline genes are shown in Tables 3 and 4 below:

[0066] Table 3. Comparison of B1G11 antibody heavy chain and germline genes

[0067] VH alleles DH allele JH allele B1G11 IGHV7-3*02 IGHD2-14*01 IGHJ4*01

[0068] Table 4. Comparison of B1G11 antibody light chain and germline genes

[0069] VK allele JK allele B1G11 IGKV10-94*01 IGKJ1*01

[0070] 3. Construction of Antibody Expression Vector

[0071] Using the above-mentioned first-round PCR product as a template, the specific primers are shown in Table 5 (the upstream 5'-end primers are mixed into a primer mix and used, and the downstream 3'-end primers are specific to the germline). By PCR, a signal peptide sequence and a restriction enzyme cleavage site AgeI (5'-ACCGGT) were added before the heavy chain variable region and the light chain variable region, a restriction enzyme cleavage site SalI (5'-GTCGAC) was added to the tail of the heavy chain variable region, and a restriction enzyme cleavage site BsiWI (5'-CGTACG) was added to the tail of the kappa chain variable region. The vector AbVec2.0-IGHG1 (Addgene) containing the human IgG1 heavy chain constant region was double-digested (AgeI, SalI), and the vector AbVec1.1-IgKC (Addgene) containing the light chain κ chain constant region was double-digested (AgeI, BsiWI). Then, the heavy and light chain variable regions of the B1G11 antibody were constructed separately into the aforementioned expression vectors containing the constant regions through homologous recombination (Novozymes, C112-02), thus obtaining the heavy and light chain expression vectors of the B1G11 antibody.

[0072] The amino acid sequence of the heavy chain variable region of the B1G11 antibody is shown in SEQ ID NO:4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:9. The heavy chain variable region includes three complementarity determining regions: CDR1 with an amino acid sequence shown in SEQ ID NO:1, CDR2 with an amino acid sequence shown in SEQ ID NO:2, and CDR3 with an amino acid sequence shown in SEQ ID NO:3. The light chain variable region also includes three complementarity determining regions: CDR1 with an amino acid sequence shown in SEQ ID NO:6, CDR2 with an amino acid sequence shown in SEQ ID NO:7, and CDR3 with an amino acid sequence shown in SEQ ID NO:8. The heavy chain amino acid sequence of the antibody B1G11 is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:10.

[0073] Table 5. Cloning PCR primers

[0074]

[0075]

[0076]

[0077] Example 4 Expression and Purification of B1G11 Antibody

[0078] Using the Expi293 mammalian expression system, 200 mL of cells were passaged to 1.0 × 10 6 / mL, when the density reaches 2.0×10 6 / mL, 150μg heavy chain and 150μg light chain plasmids were transfected using PEI MAX (Polysciences) according to the instructions. After 6 days, the supernatant was collected and centrifuged at 10000g for 20 minutes at 4 degrees. After filtration through a 0.22μm filter membrane, it was purified using a Protein A affinity column (Tiandirenhe). The target antibody was eluted with 0.1M glycine at pH 2.7. It was concentrated by ultrafiltration tube (Millipore) and the protein was identified by SDS-PAGE. The results are as follows. Figure 2 As shown, a highly pure B1G11 antibody was obtained.

[0079] Example 5 Analysis of the Binding Activity of B1G11 Antibody to Gn

[0080] The SFTSV Gn protein purified in Example 1 was diluted to 3 μg / mL with coating buffer, and 50 μL was coated on each well of the ELISA plate at 4 degrees overnight. After washing the ELISA plate with PBST using a plate washer (BioTek), it was blocked with blocking solution (PBST+1% BSA) for 2 hours. The B1G11 antibody purified in Example 4 was then diluted 10-fold from 10 μg / mL to 8 gradients with blocking solution, added to the ELISA plate and incubated at 37 degrees for 2 hours. After washing with PBST again, HRP-coupled goat anti-human IgG (H+L) (1:20000, ABclonal) secondary antibody was added, incubated at 37 degrees for 1 hour, and then washed. TMB (New Saimei) was added for color development, and then terminated with 1M hydrochloric acid. The absorbance value at a wavelength of 450nm was detected by an enzyme-linked microplate reader. The results are as follows Figure 3 As shown, the B1G11 antibody bound to SFTSV Gn specifically and in a dose-dependent manner, indicating that B1G11 targeted SFTSV Gn.

[0081] Example 6 WB experiment of specific binding of B1G11 to SFTSV Gn protein

[0082] Take the SFTSV QD02 pseudovirus packaged in Example 8 (the supernatant when the 293T packaged virus is used), 1 μL or 0.5 μL, the 293T control supernatant that is not transfected with any plasmid, and take 20 ng, 60 ng, 100 ng, 140 ng, and 180 ng of the SFTSV Gn protein purified in Example 1, respectively, and dissolved in 5× reducing loading buffer, boiled at 95°C for 10 minutes to completely denature the protein, and subjected to SDS-PAGE at 160V for 55 minutes, and then transferred to a PVDF membrane. After blocking with blocking solution (TBST + 5% skim milk powder) for 2 hours, the B1G11 antibody purified in Example 4 was diluted to 1 μg / mL with blocking solution, incubated for 2 hours, washed 3 times with TBST, and then HRP-labeled goat anti-human IgG (H+L) secondary antibody (ABclonal) was added at a dilution of 1:20,000 and incubated for 1 hour. After washing 3 times with TBST, the membrane was developed. The results are as follows. Figure 4 As shown, the binding of B1G11 to Gn is specific and dose-dependent.

[0083] Example 7B1G11 Indirect immunofluorescence test for SFTSV infection

[0084] The day before the experiment, 1.5×10 5 Vero E6 cells were seeded at a density of 100 / mL in a 96-well plate and infected with 200 TCID / mL per well the next day. 50 The SFTSV QD02 pseudovirus packaged in Example 8 was cultured at 37 degrees for 36 hours, the culture medium was discarded, 4% paraformaldehyde was added for fixation, PBS was washed three times, and the cells were permeabilized with 0.5% Triton X-100 for 20 minutes, and PBST + 2% BSA was added for 2 hours. The cells were incubated with the B1G11 antibody purified in Example 4 at a concentration of 1 μg / mL for 1 hour, washed three times with PBST, and then FITC-conjugated rabbit anti-human secondary antibody (Solarbo) was added. After washing three times with PBST, fluorescence images were taken with a fluorescence microscope. The results are shown in Figure 2. Figure 5 Host cells infected with SFTSV can be specifically labeled by the B1G11 antibody and emit green fluorescence.

[0085] Example 8B1G11 neutralizes SFTSV pseudovirus infection

[0086] Construction of the SFTSV M segment expression vector: Total RNA was extracted from authentic SFTSV QD02 and WCH97 strains using Trizol. Reverse transcription PCR was performed according to the manufacturer's instructions (Novozymes, R211-02) to obtain viral cDNA. The reverse transcription PCR system consisted of 10 μL of 2× RT Mix, 2 μL of HiScript III Enzyme Mix, 1 μL of Random Hexamers, 3 μL of RNA, and 4 μL of water. The PCR protocol was as follows: 25°C for 5 minutes, reverse transcription at 50°C for 45 minutes, followed by reaction at 85°C for 2 minutes. 2 μL cDNA was used as a template to obtain the M segment sequences of SFTSV QD02 and WCH97 strains by PCR (QD02 strain primers: upstream primer 5′-CATTTTGGCAAAGAATTCACGCGTG CCACCATGATGAAAGTC-3′, downstream primer 5′-CAGAGGGAAAAAGATCTTTATGCGGCCGCGAGCTCCTAAGCCAGCTTCGTCCTTG-3′; WCH97 strain primers: upstream primer 5′-CATTTTGGCAAAGAATTCACGCGTGCCA CCATGATGAAAGTCGATCTGG-3′, downstream primer 5′-TAGCTCGAGTTATCCGGCCAGCT TTGTCCGGGA CCGGAAGATC TGTTTGGTGCCCAGC-3'), and then digested with MluI and NotI to construct into pCAGGS vector, respectively, to obtain pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97-M vectors, which can express the full-length Gc and Gn envelope glycoproteins of SFTSV.

[0087] To package VSV-ΔG-eGFP, BHK21 cells were plated at 5×10^ 5 / mL density was plated in 12-well plates one day in advance. The next day, the cells were 90% confluent, the supernatant was discarded, 100 μL vTF7-3 (vaccinia virus expressing T7 RNA polymerase) and 100 μL DMEM (no FBS / no antibiotics) were added to infect the cells, and cultured at 37°C for 1 hour. The infection solution was removed, and Genetwin (Bomed) transfected the plasmid (VSV-N: P: G: L: pVSV-ΔG-eGFP = 3: 5: 8: 1: 5, VSV-N, P, G, L, pVSV-ΔG-eGFP can all be purchased from Kerafast) for a total of 2.75 μg. After 6 hours of transfection, the medium was replaced with complete culture medium (DMEM, 4% FBS). The supernatant was collected for about 48-54 hours and filtered with a 0.22 μm filter to remove vTF7-3 to obtain VSV-ΔG-eGFP P0 generation virus.

[0088] BHK21 cells were plated in 24-well plates and transfected with 0.75 μg of VSV-G 24 hours in advance. VSV-ΔG-eGFP P0 (50 μL virus plus 150 μL DMEM) was added at a 1:10 ratio. Approximately 8 hours later, 300 μL of complete medium was added and the cells were cultured at 34°C. 24 hours after infection, the supernatant was harvested as VSV-ΔG-eGFP P1 virus. Viral titer was calculated by scanning with ImmunoSpot for green fluorescent spots.

[0089] The pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97--M plasmids were transfected into 293T cells using Genetwin (Biomed). 24 hours later, the cells were transfected with VSV-ΔG-eGFP (1×10 6 TCID 50 The cells were infected with 1 μg / mL of VSV-G monoclonal antibody (VSV-SFTSV-QD02 and VSV-SFTSV-WCH97) for 5 hours. After three washes with PBS, the cells were supplemented with complete medium (DMEM, 4% FBS) containing 1 μg / mL of VSV-G monoclonal antibody (purified from the I1 hybridoma). After 24 hours, the supernatants (VSV-SFTSV-QD02 and VSV-SFTSV-WCH97) were collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and stored frozen at -80°C. The pseudoviruses were serially diluted and titrated on Vero E6 cells. Viral titers were calculated by scanning for green fluorescent spots using ImmunoSpot.

[0090] The antibody B1G11 purified in Example 4 was serially diluted with DMEM (2% FBS) (the pseudo-antibody for the QD02 strain was diluted as follows: starting from 8 μg / mL, 4-fold serial dilutions for 9 steps; the pseudo-antibody for the WCH97 strain was diluted as follows: starting from 32 μg / mL, 4-fold serial dilutions for 10 steps). The antibody was mixed with 300 SFTSV pseudo-viruses obtained by packaging above, incubated at 37°C for 1 hour, and then added to a 96-well plate pre-seeded with Vero E6 cells. After incubation for 24 hours, the supernatant was discarded, and the fluorescent spots were scanned with ImmunoSpot to plot the infection inhibition curve and calculate the half inhibitory concentration (IC). 50 , the results are as follows Figure 6 As shown. IC of B1G11 neutralizing QD02 pseudovirus 50 =0.04μg / mL, neutralizes the IC of WCH97 pseudovirus 50 =0.4μg / mL.

[0091] Example 9B1G11 neutralizes SFTSV wild-type virus infection

[0092] The antibody B1G11 purified in Example 4 was serially diluted with DMEM (2% FBS) (the pseudo-antibody of the QD02 strain was diluted as follows: starting from 100 μg / mL, 5-fold serial dilution for 9 steps; the pseudo-antibody of the WCH97 strain was diluted as follows: starting from 167 μg / mL, 4-fold serial dilution for 10 steps) and mixed with 200 TCID 50 SFTSV QD02 or WCH97 strains were mixed and incubated at 37 degrees for 1 hour and then added to a 96-well plate pre-seeded with Vero E6 cells. After incubation for 36 hours, the culture medium was discarded, 4% paraformaldehyde was added for fixation, PBS was washed 3 times, and the cells were permeabilized with 0.5% Triton X-100 for 20 minutes, and PBST+2% BSA was added for blocking for 2 hours. After incubation for 1 hour with the antibody B1G11 purified in Example 4, the cells were washed 3 times with PBST, and then FITC-coupled rabbit anti-human secondary antibody (Solerbo) was added. After washing 3 times with PBST, the green fluorescence was scanned by ImmunoSpot, the inhibition infection curve was drawn, and the half inhibition concentration IC was calculated. 50 , the results are as follows Figure 7 As shown. IC of B1G11 against authentic SFTSV QD02 50 =1.7μg / mL, neutralizes the IC of SFTSV WCH97 virulence 50 =0.4μg / mL.

Claims

1. A neutralizing monoclonal antibody or antigen-binding fragment thereof targeting the Gn glycoprotein of fever with thrombocytopenia syndrome virus, characterized in that: The monoclonal antibody or its antigen-binding fragment includes a heavy chain variable region and a light chain variable region; the heavy chain variable region includes three complementary determining regions CDR1, CDR2, and CDR3, and the amino acid sequences thereof are GFTFSDYY, IRNKAKGYTT, and ARDYRYDDEYYYAMDY, respectively; the light chain variable region includes three complementary determining regions CDR1, CDR2, and CDR3, and the amino acid sequences thereof are QGISNY, YTS, and QQYSKFPRT, respectively.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

9.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, wherein: The monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain having an amino acid sequence as shown in SEQ ID NO: 5, and a light chain having an amino acid sequence as shown in SEQ ID NO:

10.

4. The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein: The monoclonal antibody or antigen-binding fragment thereof further comprises any one of the following: Fab, Fab', Fab'-SH, scFv, F(ab')2 with the same antigen-binding fragment; The monoclonal antibody is humanized to obtain an antibody with the same or similar functions.

5. A polypeptide, characterized in that: It contains an amino acid sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 9, or an amino acid sequence selected from the group consisting of SEQ ID NO: 5 and SEQ ID NO:

10.

6. A polynucleotide, characterized in that: Encodes the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 or the polypeptide according to claim 5.

7. An expression vector, characterized in that: Comprising the polynucleotide according to claim 6.

8. A host cell, characterized in that: The host cell contains the expression vector according to claim 7.

9. Use of the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4 in the preparation of a product, characterized in that: The product comprises at least one of the following products: a drug or drug combination for treating or preventing fever with thrombocytopenia syndrome virus infection, a reagent or kit for detecting fever with thrombocytopenia syndrome virus or its Gn protein.

10. A product characterized by: comprising the monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 4; The product comprises at least one of the following products: a drug or drug combination for treating or preventing fever with thrombocytopenia syndrome virus infection, a reagent or kit for detecting fever with thrombocytopenia syndrome virus or its Gn protein.

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