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

Abstract

The present invention provides a protective monoclonal antibody S2A5 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 pseudovirus as an immunogen, and obtains a monoclonal antibody S2A5 that specifically targets SFTSV Gn and has high neutralizing ability and protective ability through flow sorting and single B cell PCR. The monoclonal antibody S2A5 has a very high in vitro neutralizing activity against SFTSV, and also has a neutralizing effect on the Gutul virus of the same genus. The monoclonal antibody S2A5 can effectively treat mice infected with SFTSV and can prevent SFTSV from infecting mice. It has extremely high application value in the clinical treatment and prevention of SFTSV and GTV infections, and can be used to prepare drugs for treating and preventing SFTSV and GTV infections, as well as SFTSV detection products.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology and relates to a highly neutralizing and protective monoclonal antibody S2A5 targeting the Gn protein of the fever with thrombocytopenia syndrome virus and its application. Background Art

[0002] Fever with thrombocytopenia syndrome virus (SFTSV) is a novel Bunyavirus first isolated and identified in my country in 2011. It belongs to the Bunyavirales order, Leucoviridae family, and Bunyavirus 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 multi-organ failure, with a mortality rate as high as 10%-30%. Since its initial report, cases have been reported in multiple provinces in my country, and the outbreak has gradually expanded to other parts of Asia, such as Japan and South Korea. Current prevention and control measures for SFTSV infection primarily focus on supportive care for symptoms, and there is still a lack of safe and effective vaccines and antiviral drugs. Meanwhile, Guertu virus (GTV) was isolated from tick samples in Xinjiang, my country. While no clinical cases have been reported, serological studies suggest that the virus may infect humans. Both SFTSV and GTV are tick-borne and closely related, belonging to the Bunyavirales order, Leucoviridae family, and Bundavirus genus. The development of effective vaccines and therapeutics for these viruses is crucial.

[0003] Antibody-mediated immune responses are one of the body's key defenses against viral infections and a key determinant of the preventive and protective effects of vaccines. Furthermore, highly effective neutralizing antibodies can be used for emergency treatment of viral outbreaks and to control the spread of epidemics, as well as to prevent viral infection in susceptible and high-risk populations. Antibody research and development also facilitates the rapid development of serological diagnostic kits, facilitating the rapid diagnosis of infectious diseases. Studies targeting various viruses have shown that isolated and purified monoclonal antibodies can effectively inhibit viral replication, while passive transfer of polyclonal sera and monoclonal antibodies to experimental animals can effectively prevent and protect against infection with the corresponding viruses. Antibody drugs are already commercially available for the treatment of viral infections (such as palivizumab for respiratory syncytial virus and ibalizumab for HIV), and numerous other antibody drugs targeting various viral infections are under development and clinical trials. Therefore, research on neutralizing antibodies against SFTSV and GTV is of great significance for the prevention and treatment of these two viruses.

[0004] The M segment of SFTSV encodes two envelope proteins, Gn and Gc. The heteromeric complex of Gn and Gc 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 of the Phlebovirus genus, 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. Furthermore, no effective neutralizing antibodies against GTV exist.

[0005] Therefore, there is an urgent need to develop protective neutralizing monoclonal antibodies against SFTSV and GTV. Summary of the Invention

[0006] The purpose of the present invention is to provide a protective monoclonal antibody targeting the Gn glycoprotein of fever with thrombocytopenia syndrome virus and its application. The present invention first immunizes BALB / c mice with SFTSV pseudovirus with a VSV backbone, takes the spleen and lymph nodes of the mice, grinds them into a single cell suspension, uses the expressed Gn protein as bait protein, and screens out 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 heavy chain variable region and light chain variable region nucleotide fragments of the antibody, which are cloned into an antibody expression vector containing a constant region. After expression and purification in Expi293 cells, the ability to bind to the antigen, the ability to neutralize the virus, and the ability to prevent or treat SFTSV-infected mice were verified, and a monoclonal antibody S2A5 with 100% protection against SFTSV infection was obtained, which also has a neutralizing effect on GTV.

[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 S2A5 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 (GYSFSDDN) with an amino acid sequence as shown in SEQ ID NO: 1, a CDR2 (IDPDNGGT) with an amino acid sequence as shown in SEQ ID NO: 2, and a CDR3 (AREDYYGSRAMDY) 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 (QSVDYAGDSY) with an amino acid sequence as shown in SEQ ID NO: 6, a CDR2 (AAS) with an amino acid sequence as shown in SEQ ID NO: 7, and a CDR3 (QQSYEDPRT) 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 S2A5 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 S2A5 or its antigen-binding fragment is shown in SEQ ID NO: 9.

[0010] In one embodiment, the heavy chain amino acid sequence of the monoclonal antibody S2A5 or its antigen-binding fragment is shown in SEQ ID NO: 5; the light chain amino acid sequence of the monoclonal antibody S2A5 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 or similar 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 thereof or polypeptides, including a sequence encoding the heavy chain variable region (e.g., SEQ ID NO: 12) and / or the light chain variable region (e.g., SEQ ID NO: 14) of the S2A5 antibody, and a sequence encoding the heavy chain (e.g., SEQ ID NO: 13) and / or the light chain (e.g., SEQ ID NO: 15) of the S2A5 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 S2A5 or its antigen-binding fragment or polypeptide in the preparation of a drug for treating or preventing infection with fever with thrombocytopenia syndrome virus and / or Gutul virus or a product for detecting SFTSV or its Gn protein.

[0019] A drug or drug combination for treating or preventing FET virus and / or Gutul virus infection, comprising the monoclonal antibody S2A5 or an antigen-binding fragment thereof.

[0020] A reagent or kit for detecting fever with thrombocytopenia syndrome virus or its Gn protein, comprising the monoclonal antibody S2A5 or its antigen-binding fragment.

[0021] The monoclonal antibody S2A5 provided by the present invention targets the key surface antigen Gn of SFTSV and has extremely high neutralizing activity. The half effective concentration (IC 50 ) is at the ng / mL level, and the IC 50 The IC value for WCH97 strain was 3 ng / mL. 50 At the same time, in vivo experiments have shown that the monoclonal antibody of the present invention can protect mice infected with SFTSV by 100% with a single dose and prevent SFTSV infection in mice. Moreover, the antibody also has a neutralizing effect on the GTV virus of the same genus, IC 50 The results of the present invention show that the monoclonal antibody S2A5 has a wide range of application prospects in the preparation of therapeutic drugs against SFTSV and / or GTV or SFTSV detection products. 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 purified S2A5 monoclonal antibody.

[0024] Figure 3 : ELISA curve of monoclonal antibody S2A5 binding to SFTSV Gn antigen.

[0025] Figure 4 : Neutralization of pseudoviruses of different SFTSV strains by monoclonal antibody S2A5 at the cellular level.

[0026] Figure 5 : Neutralization of SFTSV virulence by monoclonal antibody S2A5 at the cellular level.

[0027] Figure 6: Neutralization of GTV virulence by monoclonal antibody S2A5 at the cellular level.

[0028] Figure 7 : The protective effect of monoclonal antibody S2A5 in mice. 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] The Gn sequence (SEQ ID NO: 11) of the SFTSV strain WCH / 97 / HN / China / 2011 (abbreviated as WCH97) 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, and viral cDNA was obtained by reverse transcription PCR according to the manufacturer's instructions (Novozymes, R211-02). 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] 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 ion affinity chromatography (Ni-charged resin FF, GenScript). The highly pure SFTSV Gn extracellular domain protein was further purified 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. Packaging of replication-competent pseudoviruses of SFTSV and immunization of mice

[0038] Construction of pVSV-SFTSV-M vector: Using SFTSV-M (GenBank: QNR55510.1) as a template, PCR amplification was performed to obtain the M fragment of SFTSV (primers: upstream primer 5'-TAACAGAGATCGATCTG TTTACGCGTCACTATGATGAAAGTGATCTGGTT-3', downstream primers 5'-TCTGTTAGTTTT TTTCATACCTAGCAGGATTTGAGTTATCCGGCCAGCTTTGTCC-3' and 5'-CCTTGCT CACCATGGTGGCTAGCCGTGATATCTGTTAGTTTTTTTCATACCTAG-3', in two rounds of PCR, the upstream primers were the same, the first round used the first downstream primer to amplify the M fragment, and the second round used the second downstream primer to introduce the homology arm). At the same time, the pVSV-ΔG-eGFP (kerafast) vector was digested with MluI and NheI, and SFTSV was inserted into the vector by homologous recombination. The M fragment was inserted in front of eGFP to obtain the pVSV-SFTSV-M vector.

[0039] 293T cells were expressed at 1×10^ 6 / 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-SFTSV-M=3:5:8:1:5, VSV-N, P, G, L can all be purchased from Kerafast) for a total of 2.2 μg. After 6 hours of transfection, the medium was replaced with complete medium (DMEM, 4% FBS). The supernatant was collected 48-96 hours later and filtered with a 0.22 μm filter to remove VTF7-3 to obtain rVSV-SFTSV P0 virus.

[0040] Vero E6 cells were plated in 24-well plates and transfected with 0.75 μg of pCAGGS-VSV-G 24 hours in advance. Then, rVSV-SFTSV P0 (50 μL virus in 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 for another 24 hours. The supernatant was collected as the rVSV-SFTSV P1 progeny. Vero E6 cells were inoculated with rVSV-SFTSV P1 at a 1:50 ratio, and rVSV-SFTSV P2 and P3 progeny were amplified and used for mouse immunization. Viral titer was calculated by scanning for green fluorescent spots using ImmunoSpot.

[0041] BALB / c mice were intraperitoneally injected with the above-mentioned SFTSV replicable P3 pseudovirus (10 6 After three immunizations, 12.5 μg of Gn protein was injected intraperitoneally and intravenously for booster immunization three weeks later. The mice were killed 5 days later, and the spleen and lymph nodes were collected for flow cytometry sorting to obtain Gn-specific single B cells.

[0042] 2. Isolation of SFTSV Gn Protein-specific Single B Cells

[0043] 1) In a biosafety cabinet, place a grinding mesh on a plate containing 1640 (Mona, 2% FBS) medium. After blood collection, sacrifice and dissect the mouse. Remove the spleen (remove as much fat and other tissue as possible) and grind it in the grinding mesh to collect the suspension. After treating with red blood cell lysis buffer, wash twice with 1640 (2% FBS) and count the cells on a cell counter.

[0044] 2) Prepare cells according to the following requirements:

[0045] 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);

[0046] 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;

[0047] C. Cells to be sorted: 3 to 10 × 10 6 Resuspend cells in 100 μL staining buffer to prepare samples for sorting.

[0048] 3) Primary antibody staining:

[0049] 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.

[0050] B. Blank tube cells: not stained, but treated the same way as other samples.

[0051] 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.

[0052] 4) Wash cells twice with 100 μL staining buffer.

[0053] 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.

[0054] 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.

[0055] 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).

[0056] Example 3 Construction of Monoclonal Antibody Vectors from Single B Cell Clone

[0057] 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.

[0058] 2. Nested PCR:

[0059] 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.

[0060] Table 1. First round PCR primers

[0061]

[0062]

[0063] 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.

[0064] Nested PCR was performed using 1.5 μL of the first-round PCR product as a template. The amplification primers are shown in Table 2 below.

[0065] Table 2. Nested PCR primers

[0066]

[0067] 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.

[0068] 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 S2A5. Germline gene alignment of the antibody sequences was analyzed using IMGT online software.

[0069] The sequence alignment results of the S2A5 monoclonal antibody and the germline gene are shown in Tables 3 and 4 below:

[0070] Table 3. Germline genes of the S2A5 antibody heavy chain

[0071] VH alleles DH allele JH allele S2A5 IGHV1S135*01 IGHD1-1*02 IGHJ4*01

[0072] Table 4. Germline genes of the S2A5 antibody light chain

[0073] VK allele JK allele S2A5 IGKV3-4*01 IGKJ1*01

[0074] 3. Construction of Antibody Expression Vector

[0075] 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) are added before the heavy chain variable region and the light chain variable region, a restriction enzyme cleavage site SalI (5'-GTCGAC) is added to the tail of the heavy chain variable region, and a restriction enzyme cleavage site BsiWI (5'-CGTACG) is added to the tail of the light 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 S2A5 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 S2A5 antibody.

[0076] The amino acid sequence of the heavy chain variable region of the S2A5 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 amino acids set forth in SEQ ID NO:1, CDR2 with amino acids set forth in SEQ ID NO:2, and CDR3 with amino acids set forth in SEQ ID NO:3. The light chain variable region also includes three complementarity determining regions: CDR1 with amino acids set forth in SEQ ID NO:6, CDR2 with amino acids set forth in SEQ ID NO:7, and CDR3 with amino acids set forth in SEQ ID NO:8. The heavy chain amino acid sequence of the antibody S2A5 is shown in SEQ ID NO:5, and the light chain amino acid sequence is shown in SEQ ID NO:10.

[0077] Table 5. Cloning PCR primers

[0078]

[0079]

[0080]

[0081] Example 4 Expression and purification of S2A5 antibody

[0082] Using the Expi293 mammalian expression system, 200 mL of cells were passaged to 1.0 × 10 cells / mL 24 hours in advance. 6 / mL, when the density reaches 2.0×10 6 / mL, 150μg heavy chain and 180μg 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 0.45μm filter, it was purified using a Protein A affinity column (Tiandirenhe). The target antibody was eluted with 0.1M glycine at pH 2.7. The protein was concentrated by ultrafiltration tube (Millipore) and identified by SDS-PAGE. The results are as follows. Figure 2 As shown, a highly pure S2A5 antibody was obtained.

[0083] Example 5 Analysis of the Binding Activity of S2A5 Antibody to Gn

[0084] 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 S2A5 antibody purified in Example 4 was then diluted 10-fold from 10 μg / mL to 6 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 at a wavelength of 450 nm was detected by an enzyme-linked microplate reader. The results are as follows Figure 3 As shown, the S2A5 antibody bound to SFTSV Gn specifically and in a dose-dependent manner, indicating that S2A5 targeted SFTSV Gn.

[0085] Example 6S2A5 neutralizes SFTSV pseudovirus infection

[0086] Construction of the SFTSV M segment expression vector: Total RNA was extracted from authentic SFTSV strains QD02 and WCH97 using Trizol. Reverse transcription PCR was then 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 TTGTCCGGGACCGGAAGATCTGTTTGGTGCCCAGC-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 grown 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 The cells were infected with 10% TCID50 / mL of culture medium for 5 hours. After washing three times with PBS, the cells were supplemented with complete medium (DMEM, 4% FBS) containing VSV-G monoclonal antibody (purified from I1 hybridoma, 1 μg / mL). After 24 hours, the supernatants (SFTSV pseudoviruses) (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 S2A5 purified in Example 4 was diluted 4-fold in DMEM (2% FBS) starting at 8 μg / mL for 10 steps. The diluted antibody was mixed with 300 SFTSV QD02 strain or WCH97 strain VSV pseudoviruses packaged in this example, 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. The inhibition curve was plotted and the half inhibitory concentration (IC) was calculated. 50 , the results are as follows Figure 4 As shown. IC of S2A5 neutralizing QD02 pseudovirus 50 =0.001μg / mL, neutralizes the IC of WCH97 pseudovirus 50 =0.04μg / mL.

[0091] Example 7 S2A5 neutralizes SFTSV and GTV wild-type virus infection

[0092] The antibody S2A5 purified in Example 4 was serially diluted in DMEM (2% FBS) (the antibody of the QD02 strain was diluted as follows: starting from 4 μg / mL, 5-fold serial dilution for 8 steps; the antibody of the WCH97 strain was diluted as follows: starting from 167 μg / mL, 4-fold serial dilution for 10 steps; the antibody of the GTV DXM strain was diluted as follows: starting from 167 μg / mL, 4-fold serial dilution for 11 steps), and the antibody was diluted with 200 TCID 50 SFTSV WCH97 strain, QD02 strain or GTV DXM strain 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, washed 3 times with PBS, and then the cells were permeabilized with 0.5% Triton X-100 for 20 minutes, and blocked with PBST + 2% BSA for 2 hours. After incubation for 1 hour with a specific antibody against the N protein (polyclonal rabbit antibodies to the N protein of SFTSV and GTV were provided by the National Virus Resource Center), PBST was washed 3 times, and then FITC-conjugated 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-inhibitory concentration IC was calculated. 50 , the results are as follows Figure 5 and Figure 6 As shown. IC of S2A5 neutralizing SFTSV QD02 virulence 50 =0.003μg / mL, neutralizes the IC of SFTSV WCH97 virulence 50 =0.02μg / mL, neutralizing the IC of GTV virulence 50 =34μg / mL.

[0093] Example 8 Animal protection experiment of S2A5 antibody

[0094] In the antibody prevention virus infection experiment, 6-8 week old A129 mice (6 / group) were intraperitoneally injected with 400 μg of the purified S2A5 antibody described in Example 4. 24 hours later, 500 TCID 50 The survival status of mice was observed every day after the SFTSV HBMC5 virus was injected. The survival rate of mice in the S2A5 antibody group was 100%, while all mice in the control group died. Figure 7 shown.

[0095] In the antibody treatment of viral infection experiments, 600 TCID 50 SFTSV HBMC5 strain was injected intraperitoneally with 400 μg of the purified S2A5 antibody described in Example 4 24 hours later. The survival status of the mice was observed every day. The survival rate of the mice in the S2A5 antibody group was 100%, while all the mice in the control group died. Figure 7 shown.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof targeting the Gn protein 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, whose amino acid sequences are GYSFSDDN, IDPDNGGT, and AREDYYGSRAMDY, respectively; the light chain variable region includes three complementary determining regions CDR1, CDR2, and CDR3, whose amino acid sequences are QSVDYAGDSY, AAS, and QQSYEDPRT, 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 preparing a product, characterized in that: The product comprises at least one of the following products: a drug or drug combination for treating or preventing infection with fever with thrombocytopenia syndrome virus and / or Gurtu virus, and 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 infection with fever with thrombocytopenia syndrome virus and / or Gurtu virus, and a reagent or kit for detecting fever with thrombocytopenia syndrome virus or its Gn protein.

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