Protective monoclonal antibody targeting severe fever with thrombocytopenia syndrome virus gn glycoprotein and application thereof

Abstract

To provide a protective monoclonal antibody targeting severe fever with thrombocytopenia syndrome virus Gn glycoprotein and an application thereof.SOLUTION: A monoclonal antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable region and a light chain variable region, where the heavy chain variable region comprises three complementarity-determining regions having amino acid sequences of GYSFSDDN, IDPDNGGT, and AREDYYGSRAMDY, respectively, and the light chain variable region comprises three complementarity-determining regions having amino acid sequences of QSVDYAGDSY, AAS, and QQSYEDPRT, respectively. The monoclonal antibody has very high in-vitro neutralizing activity on the SFTSV, and also has a neutralizing effect on the Guertu virus of the same genus.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] The present invention belongs to the field of pharmaceutical technology and relates to a monoclonal antibody S2A5 that targets the Gn protein of severe fever with thrombocytopenia syndrome virus and has high neutralizing and protective capabilities, and its applications. [Background technology]

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel bunyavirus first isolated and identified in China in 2011. It is classified as a member of the genus Hantavirus, family Phenuiviridae, order Bunyavirales. This virus can infect wild and livestock animals as well as humans, causing acute fever, thrombocytopenia, leukopenia, vomiting, and diarrhea. Some severe cases can lead to death from multiple organ failure, with a case-fatality rate of 10% to 30%. Since its initial report, related cases have been discovered in multiple provinces across China, and the affected areas have gradually expanded, with outbreaks and epidemics occurring in other parts of Asia, including Japan and South Korea. Currently, prevention and treatment of SFTSV infection mainly relies on supportive care for symptoms, and safe and effective vaccines and antiviral drugs are not yet available in clinical practice. Meanwhile, Guertu virus (GTV) was isolated from tick samples in the Xinjiang Uyghur Autonomous Region of China. Although no clinical cases have been reported to date, serological association studies have indicated that this virus may be capable of infecting humans. SFTSV and GTV are both transmitted by ticks and are closely related. Both belong to the genus Hantavirus within the family Phenuiviridae of the order Bunyavirales. Therefore, research into effective vaccines and therapeutics against viruses in this group is crucial.

[0003] Antibody-mediated specific immune responses are one of the most important means by which the body fights viral infections and are one of the factors that determine the preventive and protective effects of vaccines. Highly effective neutralizing and protective antibodies can be used for emergency treatment of viral infection outbreaks and to control the spread of infectious diseases, as well as for preventing viral infection in susceptible and at-risk individuals. At the same time, antibody development has also contributed to the rapid development of serological diagnostic kits useful for the rapid diagnosis of infectious diseases. Research on various viruses has shown that isolated and purified monoclonal antibodies can effectively inhibit viral replication, while passive administration of polyclonal sera or monoclonal antibodies to experimental animals effectively prevents and protects the animals from infection with the corresponding viruses. Antibody drugs for the treatment of viral infections are already commercially available (e.g., palivizumab for respiratory syncytial virus infection and ibalizumab for the AIDS virus), and antibody drugs for various viral infections are currently under research and clinical trial. Therefore, research into neutralizing antibodies against SFTSV and GTV is extremely important for the prevention and treatment of these two viruses.

[0004] The M fragment of SFTSV encodes two envelope proteins, Gn and Gc, which form a heterodimer that covers the entire surface of the virus particle and are key proteins for virus adsorption and entry into host cells. Studies on Rift Valley fever virus (RVFV) and SFTSV have shown that Gn and Gc are important target antigens for eliciting specific immune protection, and that vaccine protection is positively correlated with the concentration of antibodies reactive to the viral surface glycoproteins. However, no monoclonal antibodies against SFTSV are currently in clinical trials or commercially available. At the same time, effective neutralizing antibodies against GTV have not yet been established. Therefore, there is an urgent need to develop protective neutralizing monoclonal antibodies against SFTSV and GTV. Summary of the Invention [Problem to be solved by the invention]

[0005] The objective of the present invention is to provide a protective monoclonal antibody targeting the Gn glycoprotein of severe fever with thrombocytopenia syndrome virus (SFTSSV) and its applications. In this study, BALB / c mice were immunized with a VSV-based SFTSV pseudovirus. The spleens and lymph nodes were harvested and disrupted to form single cell suspensions. Using the expressed Gn protein as a bait protein, single B cells were screened for those specifically binding to SFTSV Gn by flow sorting. Reverse transcription-PCR and nested PCR were then performed on the single B cells obtained from the screening. Nucleotide fragments of the antibody heavy and light chain variable regions were isolated and cloned into an antibody expression vector containing the constant region. The antibody was expressed in Expi293 cells, purified, and tested for antigen binding, virus neutralization, and prophylaxis or treatment of SFTSV-infected mice. The monoclonal antibody S2A5 was identified, which demonstrated 100% protection against SFTSV infection and neutralized GTV. [Means for solving the problem]

[0006] To achieve the above objectives, the technical solution of the present invention is configured as follows:

[0007] In one aspect, the present invention provides a monoclonal antibody S2A5 or an antigen-binding fragment thereof that targets the Gn protein of SFTSV, wherein the heavy chain variable region thereof comprises three complementarity determining regions: CDR1 (GYSFSDDN) having the amino acid sequence set forth in SEQ ID NO: 1, CDR2 (IDPDNGGT) having the amino acid sequence set forth in SEQ ID NO: 2, and CDR3 (AREDYYGSRAMDY) having the amino acid sequence set forth in SEQ ID NO: 3; and the light chain variable region thereof comprises three complementarity determining regions: CDR1 (QSVDYAGDSY) having the amino acid sequence set forth in SEQ ID NO: 6, CDR2 (AAS) having the amino acid sequence set forth in SEQ ID NO: 7, and CDR3 (QQSYEDPRT) having the amino acid sequence set forth in SEQ ID NO: 8.

[0008] In one embodiment, the amino acid sequence of the heavy chain variable region of the monoclonal antibody S2A5 or its antigen-binding fragment is set forth in SEQ ID NO: 4, and the amino acid sequence of the light chain variable region of the monoclonal antibody S2A5 or its antigen-binding fragment is set forth in SEQ ID NO: 9.

[0009] In one embodiment, the heavy chain amino acid sequence of the monoclonal antibody S2A5 or antigen-binding fragment thereof is set forth in SEQ ID NO:5, and the light chain amino acid sequence of the monoclonal antibody S2A5 or antigen-binding fragment thereof is set forth in SEQ ID NO:10.

[0010] In one embodiment, the monoclonal antibody further includes an antibody having the same or similar function obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody, or an antibody having the same or similar function obtained by humanizing a mouse-derived antibody variable region.

[0011] In one embodiment, the antigen-binding fragment is selected from Fab, Fab', Fab'-SH, scFv, and F(ab')2.

[0012] 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.

[0013] In another aspect, the present invention provides a polynucleotide encoding any of the above monoclonal antibodies or antigen-binding fragments thereof, wherein the polynucleotide comprises a sequence capable of encoding the heavy chain variable region (e.g., SEQ ID NO: 12) and / or light chain variable region (e.g., SEQ ID NO: 14) of the S2A5 antibody, and a sequence capable of encoding the heavy chain (e.g., SEQ ID NO: 13) and / or light chain (e.g., SEQ ID NO: 15) of the S2A5 antibody.

[0014] In another aspect, the present invention provides an expression vector comprising the polynucleotide, wherein the expression vector is capable of expressing the polynucleotide in a prokaryotic or eukaryotic host cell.

[0015] Specific examples of the expression vector include, but are not limited to, prokaryotic expression vectors, phage vectors, viral vectors, and mammalian expression vectors. In the present invention, mammalian expression vectors are particularly used.

[0016] In another aspect, the present invention provides a host cell comprising the expression vector, wherein the host cell comprises a prokaryotic or eukaryotic expression cell capable of expressing the expression vector.

[0017] 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 severe fever with thrombocytopenia syndrome virus and / or Glutuvirus infection, or in the preparation of a product for detecting SFTSV or its Gn protein.

[0018] In another aspect, the present invention provides a drug or drug combination for treating or preventing severe fever with thrombocytopenia syndrome virus and / or Glutuvirus infection, comprising the monoclonal antibody S2A5 or an antigen-binding fragment thereof.

[0019] In another embodiment, the present invention provides a reagent or reagent kit for detecting febrile thrombocytopenic syndrome virus or its Gn protein, comprising the monoclonal antibody S2A5 or an antigen-binding fragment thereof. [Effects of the Invention]

[0020] The monoclonal antibody S2A5 of the present invention targets Gn, an important surface antigen of SFTSV, and has extremely high neutralizing activity, with a median effective concentration (IC 50) at the ng / mL level, and IC against the QD02 virus strain 50 The IC against the WCH97 virus strain was 3 ng / mL. 50 The IC value is 20 ng / mL. In addition, in vivo experiments have demonstrated that the monoclonal antibody of the present invention can protect mice infected with SFTSV by a single dose, achieving a 100% protection rate, and can prevent SFTSV infection in mice. Furthermore, the antibody also exhibits a neutralizing effect against the same type of GTV virus, and its IC 50 The results of the present invention confirm that the monoclonal antibody S2A5 is expected to have a wide range of applications in the preparation of therapeutic drugs targeting SFTSV and / or GTV, or in the preparation of products for detecting SFTSV. [Brief explanation of the drawings]

[0021] [Figure 1] FIG. 1 shows the results of purification of the Gn extracellular domain protein of SFTSV by molecular sieving and identification by SDS-PAGE. [Figure 2] FIG. 1 shows the results of SDS-PAGE after purification of the S2A5 monoclonal antibody. [Figure 3] 1 is a graph showing the results of ELISA assays showing the binding of monoclonal antibody S2A5 to the Gn antigen of SFTSV. [Figure 4] FIG. 1 shows the neutralizing ability of monoclonal antibody S2A5 against pseudoviruses of different SFTSV strains at the cellular level. [Figure 5] FIG. 1 shows the neutralizing ability of monoclonal antibody S2A5 against SFTSV virus at the cellular level. [Figure 6] FIG. 1 shows the neutralizing ability of monoclonal antibody S2A5 against GTV virus at the cellular level. [Figure 7] FIG. 1 shows the protective effect of monoclonal antibody S2A5 on mice. DETAILED DESCRIPTION OF THE INVENTION

[0022] In order to make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail by way of specific examples below. It should be understood that these examples are merely illustrative of the present invention and are not intended to limit the scope of protection sought by the present invention.

[0023] Unless otherwise specified, all terms and techniques used herein have the same meaning as commonly understood by those skilled in the art to which this invention pertains. In the event of any discrepancy, the descriptions in this specification shall prevail. Furthermore, unless otherwise specified, all raw materials, reagents, instruments, consumables, etc. used in this invention can be purchased from commercial sources or prepared by existing methods.

[0024] The monoclonal antibody of the present invention, its preparation method, and application effects will be described in detail below based on specific examples and experimental data.

[0025] Example 1: Expression and purification of the extracellular domain of the Gn protein of SFTSV The Gn sequence (SEQ ID NO: 11) of the SFTSV WCH / 97 / HN / China / 2011 (hereafter referred to as "WCH97") virus strain was selected and an HRV3C protease cleavage site and a 6x histidine tag were added to its 3' end. Total RNA from the WCH97 virus strain was extracted with Trizol and then reverse transcription-PCR was performed according to the manufacturer's instructions (Novozymes, R211-02) to obtain viral cDNA. The reverse transcription-PCR reaction system contained 10 μL of 2x RT Mix, 2 μL of Histidine III Enzyme Mix, 1 μL of random hexamers, 3 μL of RNA, and 4 μL of water. The PCR procedure consisted of reverse transcription at 25°C for 5 minutes, 50°C for 45 minutes, followed by 85°C for 2 minutes. Using 2 μL of cDNA as template, PCR was performed with the forward primer 5'-GCGGAATTCGATACTGGACCGATCATTGC-3' and the reverse primer 5'-CCAAGGTCGACCCGCTTACCTCCAATGTTGC-3' to obtain the Gn fragment sequence of the WCH97 virus strain. The fragment was then introduced into the pFastBac1 vector using EcoRI and SalI endonuclease enzymes and transformed into competent DH10Bac strains. Correct clones were screened by blue-white spot assay, and Bacmids were extracted. SF9 cells were transformed to baculovirus, which was then amplified and transfected into Hi5 cells to express the Gn extracellular domain protein of SFTSV.

[0026] The supernatant from Hi5 cells was collected and centrifuged at 10,000 xg for 20 minutes at 4°C to remove cells and cell debris. The target protein was concentrated by nickel ion affinity chromatography (Ni-charged resin FF, Kingsley). It was then purified by gel filtration chromatography (Superdex200 Increase 10 / 300GL, Cytiva). The highly purified SFTSV Gn extracellular domain protein was obtained. The SDS-PAGE gel image showed the expected size of approximately 38 kDa. The results are shown in Figure 1.

[0027] Example 2: Isolation of single B cells specific for Gn of SFTSV 1) Construction of replication-competent SFTSV pseudovirus and immunization of mice The pVSV-SFTSV-M vector was constructed using SFTSV-M (GenBank: QNR55510.1) as a template and the following primers: forward primer 5'-TAACAGAGATCGATCTGTTTACGCGTCACTATGATGAAAGTGATCTGGTT-3'; and reverse primers 5'-TCTGTTAGTTTTTTTCATACCTAGCAGGATTTGAGTTATCCGGCCAGCTTTGTCC-3' and 5'-CCTGCTCACCATGGTGGCTAGCCGTGATATCTGTTAGTTTTTTCATACCTAG-3'. The SFTSV M fragment was amplified by PCR using two PCR reactions. The same forward primer was used in both PCR reactions, but the first PCR reaction used the first reverse primer to amplify the M fragment, and the second PCR reaction used the second reverse primer to introduce homologous flanking sequences. Separately, pVSV-ΔG-eGFP vector (kerafast) was digested with MluI and NheI, and the M fragment of SFTSV was inserted into the front end of eGFP by homologous recombination to obtain pVSV-SFTSV-M vector.

[0028] 293T cells were incubated at 1 × 10 6The cells were seeded at a density of 100 cells / mL in 12-well plates. On day 2, when the cells reached 90% confluence, the supernatant was discarded and 100 μL of vTF7-3 (a vaccinia virus expressing T7 RNA polymerase) and 100 μL of DMEM (without FBS or antibiotics) were added to infect the cells and cultured at 37°C for 1 hour. The infection medium was removed, and the cells were transduced using Genetwin (Vomid) with a total of 2.2 μg of plasmid (VSV-N:P:G:L:pVSV-SFTSV-M = 3:5:8:1:5, VSV-N, P, G, and L are all available from Kerafast). Six hours after transduction, the medium was replaced with complete medium (DMEM, 4% FBS). After 48–96 hours, the supernatant was collected and filtered through a 0.22 μm filter to remove VTF7-3, yielding the rVSV-SFTSV P0 generation virus.

[0029] Vero E6 cells were seeded in 24-well plates and transduced with 0.75 μg of pCAGGS-VSV-G 24 hours prior to transduction. rVSV-SFTSV P0 (50 μL virus + 150 μL LDMEM) was added at a 1:10 ratio. Approximately 8 hours later, 300 μL of complete medium was added and the cells were incubated at 34°C for 24 hours. The supernatant was collected and used as rVSV-SFTSV P1 generation virus. Vero E6 cells were inoculated with rVSV-SFTSV P1 virus at a 1:50 ratio, and the virus was amplified to rVSV-SFTSV P2 and P3 generation viruses, which were then used for immunization of mice. Green fluorescent spots were scanned using ImmunoSpot, and viral titers were calculated.

[0030] BALB / c mice were infected with the replication-competent P3 generation pseudovirus of the above SFTSV (10 6 After three immunizations, mice were given 12.5 μg of Gn protein intraperitoneally and intravenously at three-week intervals. Five days later, the mice were sacrificed, and the spleens and lymph nodes were removed and sorted by flow cytometry to obtain Gn-specific single B cells.

[0031] 2) Isolation of single B cells specific for the Gn protein of SFTSV 2-1) In a biosafety cabinet, a grinding mesh was placed on a plate containing 1640 (Monad Biotech, 2% FBS) medium. After blood collection, the mice were dissected and the spleens were collected (removing as much fat and other tissue as possible). The spleens were then ground using a grinding mesh to obtain a suspension. After treatment with red blood cell lysis solution, the spleens were washed twice with 1640 (2% FBS), and the cells were counted using a counting plate.

[0032] 2-2) Cells were prepared according to the following requirements: Single-stained tube cells A: To stain each fluorescent antibody independently for calibration, cells were divided into 8 equal parts, each containing approximately 5 × 10 5 The cells consisted of 50 μL of staining buffer (PBS, 2% FBS, 1 mM EDTA). Blank tube cell B: Prepare one cell of the same standard as the single stained tube as a separate unstained control, i.e., 5 x 10 5 The cells were suspended in 50 μL of staining buffer. Sorting cells C: 3-10 x 10 6 Each cell was taken and suspended in 100 μL of staining buffer to prepare a sample for sorting.

[0033] 2-3) Staining with primary antibodies was performed as follows. Single-stained tube cells A: FVS-780, CD3 / 4 / 8-BV510, CD19-PE-Cy7, IgD-PerCp-Cy5.5, CD138-BB515, CD95-PE, CD38-Pacific Blue, and highly expressed CD marker antibodies with APC fluorescent functional groups (all purchased from BD) were selected and added individually to the single-stained tube cells according to the dilution ratio recommended in the instruction manual, and then mixed uniformly. Blank tube cells B: Unstained and otherwise treated identically to the other samples. Sorting cells C: 0.5 μg / mL Biotin-SFTSV-Gn (EZ-Link NHS-PEG4-Biotin, ThermoScientific, Gn protein purified in Example 1 was biotinylated according to the instruction manual) was added, mixed well, and incubated at 4°C for 30 minutes.

[0034] 2-4) The cells were washed twice with 100 μL of staining buffer.

[0035] 2-5) Secondary antibody staining was performed as follows. Each antibody, CD3 / 4 / 8-BV510, CD19-PE-Cy7, IgD-PerCp-Cy5.5, CD138-BB515, CD95-PE, CD38-Pacific Blue, and Streptavidin-APC, was added to the sorting cells according to the ratios specified in the instruction manual and incubated at 4°C for 30 minutes in the dark.

[0036] 2-6) The cells were washed twice again, suspended in washing buffer, and transferred to flow cytometer tubes, which were then left to stand at 4°C in the dark in preparation for the next treatment.

[0037] Flow sorting was performed to collect CD19+, CD3 / 4 / 8-, IgD-, and APC+ target single B cells, which were then transferred to a 96-well plate containing a pre-prepared RNAase inhibitor (Promega).

[0038] Example 3: Construction of monoclonal antibody vectors from single B cell clones 1) Reverse transcription PCR was performed according to the manufacturer's instructions (Novozymes, R211-02) as follows. After sorting, plates containing 7 μL of single B cell suspension per well were transferred from -80°C to ice, incubated for 5 minutes, and centrifuged at 400 × g for 30 seconds at 4°C. The plates were incubated at 65°C for 5 minutes and then rapidly cooled on ice for 2 minutes. Meanwhile, the first-strand cDNA synthesis reaction mixture was prepared. The reverse transcription PCR reaction system consisted of 10 μL of 2x RT Mix, 2 μL of Hiscript III Enzyme Mix, and 1 μL of random hexamers. The PCR procedure consisted of reverse transcription at 25°C for 5 minutes, 50°C for 45 minutes, and then 85°C for 2 minutes.

[0039] 2) Nested PCR was performed as follows: 1 μL of the reverse transcription product was used as a template in the first PCR reaction to amplify the variable regions of antibodies H, κ, and λ. The amplification primers are shown in Table 1 below.

[0040] [Table 1]

[0041] A PCR reaction system was prepared according to the manufacturer's instructions (Novozymes, R211-02). Specifically, 10 μL of 2x Phanta Buffer, 0.4 μL of 10 mM dNTPs, 0.15 μL of forward primer mixture (5 μM each primer), 1 μL of reverse primer (10 μM), 0.5 μL of Phanta polymerase, and double-distilled water were added to a total volume of 20 μL. The PCR reaction program consisted of pre-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, followed by a final extension at 72°C for 10 minutes. Nested PCR was performed using 1.5 μL of the first PCR product as a template. The amplification primers are listed in Table 2.

[0042] [Table 2]

[0043] The PCR reaction system was prepared according to the manufacturer's instructions (CWBIO, EsTaq). Specifically, 10 μL of 2x EsTaq Mix, 1 μL of forward primer (10 μM), 1 μL of reverse primer (10 μM), and double-distilled water were added to a total volume of 20 μL. The PCR reaction program consisted of pre-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.

[0044] The PCR product was separated by 1.2% agarose gel electrophoresis, and the 400-500 bp band was sequenced. The heavy chain variable region sequence was determined to be as shown in SEQ ID NO: 12, and the light chain variable region sequence was determined to be as shown in SEQ ID NO: 14. The corresponding monoclonal antibody was named S2A5. The antibody sequences were also analyzed by comparing them with the germline gene using IMGT online software.

[0045] The sequence comparison results of the S2A5 monoclonal antibody and the germline gene are shown in Tables 3 and 4 below.

[0046] [Table 3]

[0047] [Table 4]

[0048] 3) The antibody expression vectors were constructed as follows. The first PCR product was used as a template, and the primers used were as shown in Table 5 (the 5'-end forward primers were combined to form a primer mixture, and a specific germline primer was used as the 3'-end reverse primer). A signal peptide sequence and the restriction enzyme cleavage site AgeI (5'-ACCGGT) were added to the beginning of the heavy and light chain variable regions using PCR. The restriction enzyme cleavage site SalI (5'-GTCGAC) was added to the end of the heavy chain variable region, and the restriction enzyme cleavage site BsiWI (5'-CGTACG) was added to the end of the light chain variable region. The AbVec2.0-IGG1 vector (Addgene), which contains the heavy chain constant region of human IgG1, was digested with the two restriction enzymes AgeI and SalI. The AbVec1.1-IgKC vector (Addgene), which contains the light kappa chain constant region, was digested with the two restriction enzymes AgeI and BsiWI. The heavy and light chain variable regions of the S2A5 antibody were introduced into expression vectors containing the constant regions using homologous recombination (Novozymes, C112-02), to construct heavy and light chain expression vectors for the S2A5 antibody.

[0049] The heavy chain variable region amino acid sequence of the S2A5 antibody was shown in SEQ ID NO: 4, and the light chain variable region amino acid sequence was as shown in SEQ ID NO: 9. The heavy chain variable region contained three complementarity determining regions: CDR1 having the amino acid sequence shown in SEQ ID NO: 1, CDR2 having the amino acid sequence shown in SEQ ID NO: 2, and CDR3 having the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region contained three complementarity determining regions: CDR1 having the amino acid sequence shown in SEQ ID NO: 6, CDR2 having the amino acid sequence shown in SEQ ID NO: 7, and CDR3 having the amino acid sequence shown in SEQ ID NO: 8. The heavy chain amino acid sequence of the antibody S2A5 was shown in SEQ ID NO: 5, and the light chain amino acid sequence was as shown in SEQ ID NO: 10.

[0050] [Table 5] JPEG2025118495000007.jpg158133

[0051] Example 4: Expression and purification of S2A5 antibody Using the Expi293 mammalian expression system, 200 mL of cells were cultured at 1.0 × 10 cells per well for 24 hours. 6 Subcultured to a density of 2.0 × 10 6 When the concentration reached 150 μg / mL, the cells were transfected with 150 μg of the heavy chain plasmid and 180 μg of the light chain plasmid using PEI MAX (Polysciences). After 6 days, the supernatant was collected, centrifuged at 10,000×g for 20 minutes at 4°C, filtered through a 0.45 μm membrane, and purified using a Protein A affinity column (Smart-Lifesciences). The target antibody was eluted with 0.1 M glycine, pH 2.7. The antibody was concentrated using an ultrafiltration tube (Millipore) and identified by SDS-PAGE. As shown in Figure 2, highly pure S2A5 antibody was obtained.

[0052] Example 5: Analysis of binding activity of S2A5 antibody and Gn The Gn protein of SFTSV purified in Example 1 was diluted to 3 μg / mL in coating buffer, and 50 μL per well was added to the labeling microplate. The plate was then coated overnight at 4°C. The labeling microplate was washed with PBST using a plate washer (BioTek) and then blocked with blocking solution (PBST + 1% BSA) for 2 hours. The S2A5 antibody purified in Example 4 was then diluted in six 10-fold gradients starting from 10 μg / mL in the blocking solution, added to the labeling microplate, and incubated at 37°C for 2 hours. After washing the plate again with PBST, an HRP-conjugated goat anti-human IgG (H+L, 1:20,000, ABclonal) secondary antibody was added, incubated at 37°C for 1 hour, and then washed. TMB (NCM Biotech) was added to develop color, and the reaction was stopped with 1 M hydrochloric acid. Absorbance was measured at 450 nm using a plate reader. As shown in Figure 3, the binding of the S2A5 antibody to Gn of SFTSV was dose-dependent, confirming that S2A5 targets Gn of SFTSV.

[0053] Example 6: Evaluation of S2A5 neutralizing ability against SFTSV pseudovirus infection The SFTSV M fragment expression vector was constructed as follows. Briefly, SFTSV QD02 and WCH97 virus strains were selected, and total RNA was extracted with Trizol. Then, reverse transcription-PCR was performed according to the manufacturer's instructions (Novozymes, R211-02) to obtain viral cDNA. The reverse transcription-PCR reaction system consisted of 10 μL of 2x 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 reaction procedure consisted of reverse transcription at 25°C for 5 minutes, 50°C for 45 minutes, and then 85°C for 2 minutes. PCR was performed using 2 μL of cDNA as a template to obtain the M fragment sequences of the SFTSV QD02 and WCH97 virus strains (for the QD02 virus strain, the forward primer 5'-CATTTTGGCAAAGAATTCACGCGTGCCACCATGATGAAAGTC-3' and the reverse primer 5'-CAGAGGGAAAAAGATCTTTATGCGGCCGCGAGCTCCTAAGCCAGCTTCGTCCTTG-3' were used; for the WCH97 virus strain, the forward primer 5'-CATTTTGGCAAAGAATTCACGCGTGCCACCATGATGAAAGTCGATCTGG-3' and the reverse primer 5'-TAGCTCGAGTTATCCGGCCAGCTTTGTCCGGGACCGGAAGATCTGTTTGGTGCCCAGC-3' were used). Then, they were digested with MluI and NotI, respectively, and introduced into the pCAGGS vector to obtain the pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97-M vectors, which can express the two full-length Gc and Gn envelope glycoproteins of SFTSV.

[0054] VSV-ΔG-eGFP was assembled as follows: 1 day before transfection, 5 × 10 BHK21 cells were transfected with 5 × 10 5The cells were seeded at a density of 1 / mL onto a 12-well plate. The next day, when the cells reached 90% confluence, the supernatant was discarded, and 100 μL of vTF7-3 (a vaccinia virus expressing T7 RNA polymerase) and 100 μL of DMEM (without FBS or antibiotics) were added to infect the cells, which were then incubated at 37°C for 1 hour. The infection solution was removed, and the cells were transduced using Genetwin (Vomid) with a total of 2.75 μg of plasmid (VSV-N:P:G:L:pVSV-ΔG-eGFP = 3:5:8:1:5, VSV-N, P, G, L, and pVSV-ΔG-eGFP are all available from Kerafast). Six hours after transduction, the liquid was replaced with complete medium (DMEM, 4% FBS). Approximately 48–54 hours later, the supernatant was collected and filtered through a 0.22 μm filter to remove vTF7-3, yielding the VSV-ΔG-eGFP P0 generation virus.

[0055] BHK21 cells were plated in 24-well plates and transduced with 0.75 μg of VSV-G 24 hours prior. VSV-ΔG-eGF PP0 was added at a 1:10 ratio (50 μL virus + 150 μL DMEM). Approximately 8 hours later, 300 μL of complete medium was added and the cells were incubated at 34°C. 24 hours post-infection, the supernatant was collected and used as VSV-ΔG-eGFP P1 generation virus. Green fluorescent spots were scanned using ImmunoSpot, and the virus titer was calculated.

[0056] Using Genetwin (Vomid), pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97-M plasmids were transduced into 293T cells. After 24 hours, VSV-ΔG-eGFP (1 × 10) diluted in DMEM (Moner) was transduced. 6The cells were infected with 100 TCID50 / mL for 5 hours, washed three times with PBS, and then supplemented with complete medium (DMEM, 4% FBS) containing a purified VSV-G monoclonal antibody (I1 hybridoma, 1 μg / mL). After 24 hours, the supernatants, i.e., 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 diluted to a concentration gradient and titrated on Vero E6 cells. The green fluorescent spots were scanned using ImmunoSpot to calculate the viral titer.

[0057] The S2A5 antibody purified in Example 4 was diluted 10 times in a 4-fold gradient starting from 8 μg / mL in DMEM (2% FBS), mixed with 300 SFTSV QD02 virus strains or VSV pseudovirus of WCH97 virus strains constructed in this example, and incubated at 37°C for 1 hour. It was then added to a 96-well plate seeded with Vero E6 cells. After 24 hours of incubation, the supernatant was discarded, and the fluorescent spots were scanned using ImmunoSpot. An infection inhibition graph was plotted, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The results are shown in Figure 4, and the IC of S2A5 neutralizing the QD02 pseudovirus was calculated. 50 IC50 of 0.001 μg / mL for neutralizing WCH97 pseudovirus 50 was 0.04 μg / mL.

[0058] Example 7: Evaluation of S2A5 neutralizing ability against SFTSV and GTV wild-type virus infection The S2A5 antibody purified in Example 4 was diluted with DMEM (2% FBS) to form a concentration gradient (the antibody against the QD02 virus strain was diluted 8 times with a 5-fold gradient starting from 4 μg / mL, the antibody against the WCH97 virus strain was diluted 10 times with a 4-fold gradient starting from 167 μg / mL, and the antibody against the GTV DXM virus strain was diluted 11 times with a 4-fold gradient starting from 167 μg / mL), and each antibody was diluted to 200 TCID 50The cells were mixed with SFTSV WCH97, QD02, or GTV DXM virus strains and incubated at 37°C for 1 hour. Then, they were added to a 96-well plate seeded with Vero E6 cells. After 36 hours of incubation, the culture medium was discarded and the cells were fixed with 4% paraformaldehyde. After washing three times with PBS, the cells were permeabilized with 0.5% Triton X-100 for 20 minutes and blocked with PBST + 2% BSA for 2 hours. After incubation with an antibody specific to the N protein (polyclonal rabbit antibodies against the N proteins of SFTSV and GTV were provided by the National Virus Resource Center) for 1 hour, the cells were washed three times with PBST. Then, a FITC-conjugated rabbit anti-human secondary antibody (Solarbio) was added and washed three times with PBST. After three washes with PBST, green fluorescence was scanned using ImmunoSpot, and an infection inhibition graph was plotted. The half-maximal inhibitory concentration (IC50) was calculated. 50 The results are shown in Figures 5 and 6, and the IC of S2A5 neutralizing the SFTSV QD02 virus was calculated. 50 The IC for neutralizing SFTSV WCH97 virus was 0.003 μg / mL. 50 The IC value for neutralizing the GTV virus was 0.02 μg / mL. 50 was 34 μg / mL.

[0059] Example: Animal protection experiment using 8S2A5 antibody In the experiment to prevent viral infection by antibody, 400 μg of the S2A5 antibody purified in Example 4 was intraperitoneally injected into 6- to 8-week-old A129 mice per group of 6 mice. 24 hours later, 500 TCID 50 After intraperitoneal injection of SFTSV HBMC5 virus, the survival status of the mice was monitored daily. The results are shown in Figure 7A. The survival rate of the mice in the S2A5 antibody-treated group was 100%, while all mice in the control group died.

[0060] On the other hand, in an antibody treatment experiment against viral infection, 500TCID 50The SFTSV HBMC5 virus strain was intraperitoneally injected into the mice, and 24 hours later, 400 μg of the S2A5 antibody purified in Example 4 was intraperitoneally injected, and the mice were monitored daily for survival. The results are shown in Figure 7B. The survival rate of the mice in the S2A5 antibody-treated group was 100%, while all mice in the control group died.

Claims

1. A monoclonal antibody or an antigen-binding fragment thereof that targets the Gn protein of severe fever with thrombocytopenia syndrome virus, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region; A monoclonal antibody or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises three complementarity determining regions whose amino acid sequences are GYSFSDDN, IDPDNGGT, and AREDYYGSRAMDY, respectively, and the light chain variable region comprises three complementarity determining regions 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 set forth in SEQ ID NO: 4 and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:

9.

3. The monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain having the amino acid sequence shown in SEQ ID NO: 5 and a light chain having the amino acid sequence shown in SEQ ID NO:

10.

4. The monoclonal antibody or antigen-binding fragment thereof Fab, Fab', Fab'-SH, scFv, F(ab') with the same antigen-binding fragment ２ , An antibody having the same function as the monoclonal antibody, which is obtained by substituting, deleting, and / or adding one or more amino acids to the amino acid sequence of the monoclonal antibody; and an antibody having the same or similar function obtained by humanizing the monoclonal antibody; The monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, further comprising any one of the following:

5. 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.

6. A polynucleotide encoding 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 comprising the polynucleotide of claim 6.

8. A host cell comprising the expression vector of claim 7.

9. 10. The application of the monoclonal antibody and its antigen-binding fragments according to any one of claims 1 to 4 to prepare a product, comprising: The product is A drug or drug combination for treating or preventing severe fever with thrombocytopenia syndrome virus and / or Glutuvirus infection, and A reagent or reagent kit for detecting severe fever with thrombocytopenia syndrome virus or its Gn protein; An application that includes one of the following:

10. A product comprising the monoclonal antibody and antigen-binding fragment thereof according to any one of claims 1 to 4, The product is A drug or drug combination for treating or preventing severe fever with thrombocytopenia syndrome virus and / or Glutuvirus infection, and A reagent or reagent kit for detecting severe fever with thrombocytopenia syndrome virus or its Gn protein; A product containing any one of the following:

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