Protective monoclonal antibody targeting the Gn glycoprotein of severe fever with thrombocytopenia syndrome virus and its application

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

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a novel bunyavirus first isolated and identified in Japan in 2011. It is classified under the order Bunyavirum, family Phenuiviridae, genus Hantavirus. This virus can infect wild animals, livestock, and humans, causing acute fever, thrombocytopenia, leukopenia, vomiting, and diarrhea. Some patients who develop severe symptoms may die from multiple organ failure, with a mortality rate of 10% to 30%. After the initial report of this pathogen, related cases were successively discovered in multiple provinces in Japan, and the affected area gradually expanded, with outbreaks and epidemics now being seen in other parts of Asia, including Japan and South Korea. Currently, the means of preventing and treating SFTSV infection mainly consist of supportive care for symptoms, and there are no safe and effective vaccines or antiviral drugs currently in clinical use. On the other hand, Guertu virus (GTV) is a virus isolated from tick specimens in the Xinjiang Uyghur Autonomous Region of China. Although no clinical cases have been reported to date, serological studies have indicated that this virus may be capable of infecting humans. Both SFTSV and GTV are transmitted by ticks, are closely related, and belong to the genus Hantavirus in the family Phenuiviridae, order Bunyaviraceae. Therefore, research into effective vaccines and treatments for viruses in this group is extremely important.

[0003] Antibody-mediated specific immune responses are one of the most important means by which organisms fight viral infections and are also one of the factors that determine the preventive and protective effects of vaccines. Furthermore, highly efficient neutralizing and protective antibodies can be used for emergency treatment during outbreaks of viral infections and for controlling the spread of infectious diseases, as well as for preventing viral infections in people who are susceptible to and at risk of viral infection. Simultaneously, antibody development contributes to the rapid development of serological diagnostic kits useful for the rapid diagnosis of infectious diseases. Studies on various viruses have shown that isolated and purified monoclonal antibodies can effectively inhibit viral replication, while passively administering polyclonal serum or monoclonal antibodies to experimental animals can effectively prevent and protect them from infection by the corresponding viruses. Antibody drugs for treating viral infections are already commercially available (for example, palivizumab for respiratory syncytial virus infection and ivalizumab for HIV), and various other antibody drugs for viral infections are in the research and clinical trial stages. 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. Gn and Gc form a heterodimer that covers the entire surface of the viral particle and are key proteins for viral adsorption and entry into host cells. Studies on Rift Valley fever virus (RVFV) and SFTSV have revealed that Gn and Gc are important target antigens for inducing specific immunoprotection, and that the protective effect of vaccines is positively correlated with the concentration of antibodies reactive to glycoproteins on the viral surface. However, there are currently no monoclonal antibodies against SFTSV that are in clinical trials or commercially available. At the same time, effective neutralizing antibodies against GTV have not yet been established. Therefore, the development of protective neutralizing monoclonal antibodies targeting SFTSV and GTV is urgently needed. [Overview of the project] [Problems that the invention aims to solve]

[0005] The object of the present invention is to provide a protective monoclonal antibody targeting the Gn glycoprotein of the Severe Fever with Thrombocytopenia Syndrome virus and its applications. In this invention, BALB / c mice were first immunized with SFTSV pseudovirus with a VSV backbone. After collecting the mouse spleen and lymph nodes, they were lysed to form a single-cell suspension, and the expressed Gn protein was used as a bait protein to screen for single B cells that specifically bind to SFTSV Gn by flow sorting. Reverse transcription PCR and nested PCR were then performed on the single B cells obtained from the screening to obtain nucleotide fragments of the heavy chain variable region and light chain variable region of the antibody, and these were cloned into an antibody expression vector containing the constant region. After expression and purification in Expi293 cells, the binding ability to the antigen, virus neutralizing ability, and prevention or treatment of SFTSV-infected mice were verified. As a result, a monoclonal antibody S2A5 was obtained that showed 100% protective effect against SFTSV infection and also had a neutralizing effect against GTV. [Means for solving the problem]

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

[0007] In one embodiment, 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 comprises three complementarity-determining regions: CDR1(GYSFSDDN) having the amino acid sequence shown in SEQ ID NO: 1, CDR2(IDPDNGGT) having the amino acid sequence shown in SEQ ID NO: 2, and CDR3(AREDYYGSRAMDY) having the amino acid sequence shown in SEQ ID NO: 3, and the light chain variable region comprises three complementarity-determining regions: CDR1(QSVDYAGDSY) having the amino acid sequence shown in SEQ ID NO: 6, CDR2(AAS) having the amino acid sequence shown in SEQ ID NO: 7, and CDR3(QQSYEDPRT) having the amino acid sequence shown 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 shown 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 shown in SEQ ID NO: 9.

[0009] 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, and the light chain amino acid sequence of the monoclonal antibody S2A5 or its antigen-binding fragment is shown 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 the antibody variable region derived from a mouse.

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

[0012] In another embodiment, 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 embodiment, the present invention provides a polynucleotide encoding any of the monoclonal antibodies or their antigen-binding fragments, the polynucleotide comprising a sequence capable of 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 capable of 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.

[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 said expression vector include, but are not limited to, prokaryotic expression vectors, phage vectors, viral vectors or mammalian expression vectors, and mammalian expression vectors are particularly used in the present invention.

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

[0017] In another aspect, the present invention provides use of said monoclonal antibody S2A5, or an antigen-binding fragment or polypeptide thereof, in the preparation of a medicament for treating or preventing severe fever with thrombocytopenia syndrome virus and / or Guertu virus infection, or in the preparation of a product for detecting SFTSV or the Gn protein thereof.

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

[0019] In another aspect, the present invention provides a reagent or a reagent kit comprising said monoclonal antibody S2A5 or an antigen-binding fragment thereof for detecting severe fever with thrombocytopenia syndrome virus or the Gn protein thereof. Effects of the Invention

[0020] The monoclonal antibody S2A5 according to the present invention targets Gn, which is an important surface antigen of SFTSV, has extremely high neutralizing activity, and has a half maximal effective concentration (IC 50is at the ng / mL level, and the IC against the QD02 virus strain 50 is 3 ng / mL, and the IC against the WCH97 virus strain 50 is 20 ng / mL. In addition, in vivo experiments have demonstrated that a single administration of the monoclonal antibody according to the present invention achieves a protection rate of 100% against SFTSV infection in mice, and can prevent SFTSV infection in mice. Furthermore, said antibody also exhibits a neutralizing effect against the homologous GTV virus, and its IC 50 is 34 μg / mL. The results of the present invention confirm that the monoclonal antibody S2A5 is expected to have broad applications in the preparation of therapeutic agents targeting SFTSV and / or GTV, or in the preparation of products for detecting SFTSV. [BRIEF DESCRIPTION OF THE DRAWINGS]

[0021] [Figure 1] It is a figure showing the results of purification by molecular sieve method and identification by SDS-PAGE of SFTSV Gn extracellular domain protein. [Figure 2] It is a figure showing the SDS-PAGE results after purification of S2A5 monoclonal antibody. [Figure 3] It is a graph showing the ELISA measurement results of binding of monoclonal antibody S2A5 to SFTSV Gn antigen. [Figure 4] It is a figure showing the neutralizing activity of monoclonal antibody S2A5 against pseudoviruses of different SFTSV strains at the cellular level. [Figure 5] It is a figure showing the neutralizing activity of monoclonal antibody S2A5 against SFTSV virus at the cellular level. [Figure 6] It is a figure showing the neutralizing activity of monoclonal antibody S2A5 against GTV virus at the cellular level. [Figure 7] It is a figure showing the protective effect of monoclonal antibody S2A5 on mice. [MODE FOR CARRYING OUT THE INVENTION]

[0022] The present invention will be described in more detail below with specific examples to further clarify its objectives, technical proposals, and advantages. It should be understood that these examples are merely illustrative and not intended to limit the scope of protection sought by the present invention.

[0023] All terms and techniques used herein have the same meaning as those generally understood by those skilled in the art to which the present invention pertains, unless otherwise specified. In case of any inconsistency, the provisions of this specification shall prevail. Furthermore, all raw materials, reagents, equipment, consumables, etc., used in the present invention are commercially available or can be prepared by existing methods, unless otherwise specified.

[0024] The monoclonal antibody of the present invention, its preparation method, and its 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 (hereinafter abbreviated as "WCH97") virus strain was selected, and an HRV3C protease cleavage site and a 6× histidine tag were added to its 3' end. After extracting the total RNA of the WCH97 virus strain with Trizol, reverse transcription PCR was performed according to the instructions for use (Novozymes, R211-02) to obtain the viral cDNA. The reverse transcription PCR reaction system consisted of 10 μL of 2× RT Mix, 2 μL of Hisscript III Enzyme Mix, 1 μL of Random hexamers, 3 μL of RNA, and 4 μL of water. The PCR procedure involved reverse transcription at 25°C for 5 minutes, followed by 45 minutes at 50°C, and then reaction at 85°C for 2 minutes. Using 2 μL of cDNA as a template, the Gn fragment sequence of the WCH97 virus strain was obtained by PCR using the forward primer 5'-GCGGAATTCGATACTGGACCGATCATTGC-3' and the reverse primer 5'-CCAAGGTCGACCCGCTTACCTCCAATGTTGC-3'. The fragment was introduced into the pFastBac1 vector using EcoRI and SalI nucleic acid endonucleases, transformed into competent DH10Bac cells, and after screening for accurate clones using the blue-white spot identification method, Bacmids were extracted. Baculoviruses were assembled by transforming SF9 cells, and after viral amplification, the SFTSV Gn extracellular domain protein was expressed by introducing Hi5 cells.

[0026] The supernatant of Hi5 cells was collected and centrifuged at 4°C and 10000g for 20 minutes to remove cells and cell debris. The target protein was concentrated by nickel-ion affinity chromatography (Ni-charged resin FF, Kingsley). Purification by gel filtration chromatography (Superdex200 Increase 10 / 300GL, Cytiva) yielded a high-purity SFTSV Gn extracellular domain protein. The SDS-PAGE gel plot showed a size of approximately 38kDa, as expected, and the results are shown in Figure 1.

[0027] Example 2: Isolation of Gn-specific single B cells of SFTSV 1) Assembly of a replicable pseudovirus of SFTSV and immunization of mice The pVSV-SFTSV-M vector was constructed using SFTSV-M (GenBank: QNR55510.1) as a template. The forward primer 5'-TAACAGAGATCGATCTGTTTACGCGTCACTATGATGAAAGTGATCTGGTT-3' and the reverse primers 5'-TCTGTTAGTTTTTTTCATACCTAGCAGGATTTGAGTTATCCGGCCAGCTTTGTCC-3' and 5'-CCTGCTCACCATGGTGGCTAGCCGTGATATCTGTTAGTTTTTTCATACCTAG-3' were used as primers, and the M fragment of SFTSV was obtained by PCR amplification. The PCR reaction was performed in two separate steps, using the same forward primer in both steps. However, in the first PCR reaction, the first reverse primer was used to amplify the M fragment, and in the second PCR reaction, the second reverse primer was used to introduce homology flanking. On the other hand, the pVSV-ΔG-eGFP vector (Kerafast) was digested using MluI and NheI, and the M fragment of SFTSV was inserted into the front of eGFP by homologous recombination to obtain the pVSV-SFTSV-M vector.

[0028] 293T cells 1 × 10⁶ days prior 6Cells were seeded at a density of 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 (vaccinia virus expressing T7 RNA polymerase) and 100 μL of DMEM (without FBS / antibiotics) were added to infect the cells. The cells were then cultured at 37°C for 1 hour. The infection fluid was removed, and 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) was transduced using Genetwin (Vomido). Six hours after transduction, the medium was changed to complete medium (DMEM, 4% FBS), and after 48-96 hours, the supernatant was collected and filtered through a 0.22 μm filter to remove VTF7-3, obtaining rVSV-SFTSV P0 generation viruses.

[0029] Vero E6 cells were seeded in 24-well plates and transduced with 0.75 μg of pCAGGS-VSV-G 24 hours prior to administration. rVSV-SFTSV P0 (50 μL virus + 150 μL LDMEM) was added in a 1:10 ratio, and approximately 8 hours later, 300 μL of complete medium was added. The cells were incubated at 34°C for 24 hours, and the supernatant was collected to obtain the rVSV-SFTSV P1 generation virus. Vero E6 cells were inoculated with the rVSV-SFTSV P1 virus in a 1:50 ratio to amplify to the rVSV-SFTSV P2 and P3 generations of virus, which were then used for mouse immunization. The viral titer was calculated by scanning the green fluorescent spot with ImmunoSpot.

[0030] In BALB / c mice, a replicable P3 generation pseudovirus of the above SFTSV (10 6 After administering FFU intraperitoneally every three weeks for three rounds of immunization, further enhanced immunization was performed by injecting 12.5 μg of Gn protein intraperitoneally and intravenously at three-week intervals. Five days later, the mice were killed, their spleens and lymph nodes were removed, and Gn-specific single B cells were obtained by sorting using flow cytometry.

[0031] 2) Isolation of single B cells specific to the Gn protein of SFTSV 2-1) In a biosafety cabinet, a grinding mesh was placed on a plate containing 1640 medium (manufactured by Monad Biotech, supplemented with 2% FBS). After blood collection from mice, the mice were dissected to collect spleens (from which fat and other tissues were removed as much as possible), the spleens were ground through the grinding mesh, and the suspension was recovered. After treatment with erythrocyte lysis solution, the cells were washed twice with 1640 (2% FBS), and counted using a counting plate.

[0032] 2-2) Cells were prepared according to the following requirements. Single-stained tube cells A: For individual staining of each fluorescent antibody for adjustment correction, equal amounts of cells were divided into 8 equal portions. Each equal portion was approximately 5×10 5 cells, and suspended in 50 µL of staining buffer (PBS, 2% FBS, 1 mM EDTA). Blank tube cells B: Separately, as an unstained control, cells equivalent to one portion of the single-stained tube with the same specification was prepared, that is, 5×10 5 cells were suspended in 50 µL of staining buffer. Sorting cells C: 3~10×10 6 cells were collected and suspended in 100 µL of staining buffer to obtain a sample for sorting.

[0033] 2-3) Staining with primary antibody 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 a highly expressed CD marker antibody with APC fluorescent functional group (all purchased from BD) were selected, each was individually added to single-stained tube cells according to the dilution ratio recommended in the instruction manual, and mixed uniformly. Blank tube cells B: No staining was performed, and other treatments were the same as those for other samples. Cell C for sorting: Add 0.5 μg / mL of Biotin-SFTSV-Gn (EZ-Link NHS-PEG4-Biotin, manufactured by ThermoScientific, which is the Gn protein purified in Example 1 and biotinylated according to the instructions for use) and mix well, then incubate at 4°C for 30 minutes.

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

[0035] 2-5) Staining with secondary antibodies 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 sorting cells according to the proportions specified in the instruction manual and incubated at 4°C in the dark for 30 minutes.

[0036] 2-6) After washing the cells twice again, they were suspended in washing buffer and transferred sequentially to flow cytometer tubes. They were then left to stand at 4°C under light-shielding conditions in preparation for the next treatment.

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

[0038] Example 3: Construction of a monoclonal antibody vector from a single B cell clone 1) Reverse transcription PCR was performed according to the instructions for use (Novozymes, R211-02). After sorting, plates containing 7 μL of single B cell suspension per well were transferred from -80°C to ice, allowed to stand for 5 minutes, and then centrifuged at 400×g for 30 seconds at 4°C. After standing at 65°C for 5 minutes, the plates were rapidly cooled on ice for 2 minutes, while the first-strand cDNA synthesis reaction mixture was prepared. The reverse transcription PCR reaction system consisted of 10 μL of 2×RT Mix, 2 μL of Hisscript III Enzyme Mix, and 1 μL of Random hexamers. The PCR procedure involved reverse transcription at 25°C for 5 minutes, followed by 45 minutes at 50°C, and then reaction at 85°C for 2 minutes.

[0039] 2) Nested PCR was performed as follows: Using 1 μL of reverse transcript as a template, the variable regions of antibody H, κ, and λ were amplified by the first PCR reaction, and the amplification primers are shown in Table 1 below.

[0040] [Table 1]

[0041] The PCR reaction system was prepared according to the instructions for use (Novozymes, R211-02). Specifically, 10 μL of 2× Phantasy Buffer, 0.4 μL of dNTPs (10 mM), 0.15 μL of forward primer mixture (5 μM each primer), 1 μL of reverse primer (10 μM), 0.5 μL of Phantasy polymerase, and double-distilled water were added to make a total volume of 20 μL. The PCR reaction program consisted of 50 cycles of preliminary denaturation at 95°C for 30 seconds, followed by denaturation at 95°C for 15 seconds, annealing at 46°C for 15 seconds, and extension at 72°C for 1 minute, followed by final extension at 72°C for 10 minutes. 1.5 μL of the first PCR product was used as a template for the nested PCR reaction, and nested PCR was performed. The amplification primers are shown in Table 2 below.

[0042] [Table 2]

[0043] The PCR reaction system was prepared according to the instructions for use (CWBIO, EsTaq). Specifically, 10 μL of 2×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 40 cycles of preliminary denaturation at 94°C for 2 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 57°C for 30 seconds, and extension at 72°C for 20 seconds, and finally final extension at 72°C for 10 minutes.

[0044] PCR products were separated by 1.2% agarose gel electrophoresis, and the base sequences were measured by taking bands of 400-500 bp. The sequence analysis results for the heavy chain variable region are shown in SEQ ID NO: 12, and the sequence analysis results for the light chain variable region are shown in SEQ ID NO: 14. The corresponding monoclonal antibody was named S2A5. Furthermore, the germline genes of the antibody sequences were compared and analyzed using IMGT online software.

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

[0046] [Table 3]

[0047] [Table 4]

[0048] 3) The antibody expression vector was constructed as follows: The first PCR product described above was used as a template, and the primers were specifically as shown in Table 5 (a primer mixture was formed by combining the 5'-end forward primer, and a specific germline primer was used as the 3'-end reverse primer). The signal peptide sequence and restriction enzyme cleavage site AgeI (5'-ACCGGT) were added to the front of the heavy chain variable region and light chain variable region using PCR, and the restriction enzyme cleavage site SalI (5'-GTCGAC) was added to the posterior of the heavy chain variable region, and the restriction enzyme cleavage site BsiWI (5'-CGTACG) was added to the posterior of the light chain variable region. The AbVec2.0-IGG1 vector (Addgene), containing the heavy chain constant region of human IgG1, was digested using the two restriction enzymes AgeI and SalI, and the AbVec1.1-IgKC vector (Addgene), containing the light chain κ chain constant region, was digested using the two restriction enzymes AgeI and BsiWI. S2A5 antibody heavy chain and light chain expression vectors were constructed by introducing the variable regions of the S2A5 antibody's heavy chain and light chain into expression vectors containing the aforementioned constant region, respectively, using homologous recombination (Novozymes, C112-02).

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

[0050] [Table 5] JPEG0007914963000006.jpg158133

[0051] Example 4: Expression and purification of S2A5 antibody Using the Expi293 mammalian expression system, 200 mL of cells were prepared 24 hours prior to the expression of 1.0 × 10⁶ cells. 6 Subculture until cells / mL reaches a density of 2.0 × 10⁶ 6 When the concentration reached 150 μg of heavy chain plasmid and 180 μg of light chain plasmid were transduced using PEI MAX (Polysciences). After 6 days, the supernatant was collected, centrifuged at 10000 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 using 0.1 M glycine at pH 2.7. Concentration was performed using an ultrafiltration tube (Millipore), and the protein was identified by SDS-PAGE. As shown in Figure 2, a highly pure S2A5 antibody was obtained.

[0052] Example 5: Analysis of the binding activity of S2A5 antibody and Gn The SFTSV Gn protein purified in Example 1 was diluted to 3 μg / mL in coating buffer, and 50 μL per well was added to the labeling microplate. Coating was performed by standing at 4°C overnight. 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 steps with the blocking solution from 10 μg / mL in a 10-fold gradient, added to the labeling microplate, and incubated at 37°C for 2 hours. After washing the plate again with PBST, HRP-conjugated goat anti-human IgG (H+L, 1:20000, 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 1M hydrochloric acid. Absorbance was measured at a wavelength of 450 nm using a plate reader. As shown in Figure 3, the binding of the S2A5 antibody to the Gn of SFTSV was dose-dependent, confirming that S2A5 targets the 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: SFTSV QD02 and WCH97 virus strains were selected, total RNA was extracted with Trizol, and then reverse transcription PCR was performed according to the instructions for use (Novozymes, R211-02) to obtain viral cDNA. The reverse transcription PCR reaction system consisted of 10 μL of 2×RT Mix, 2 μL of Hisscript III Enzyme Mix, 1 μL of Random hexamers, 3 μL of RNA, and 4 μL of water. The PCR reaction procedure involved reverse transcription at 25°C for 5 minutes, then at 50°C for 45 minutes, followed by reaction at 85°C for 2 minutes. Using 2 μL of cDNA as a template, PCR was performed to obtain the M fragment sequences of the SFTSV QD02 and WCH97 virus strains (for the QD02 virus strain, forward primer 5'-CATTTTGGCAAAGAATTCACGCGTGCCACCATGATGAAAGTC-3' and reverse primer 5'-CAGAGGGAAAAAGATCTTTATGCGGCCGCGAGCTCCTAAGCCAGCTTCGTCCTTG-3' were used, and for the WCH97 virus strain, forward primer 5'-CATTTTGGCAAAGAATTCACGCGTGCCACCATGATGAAAGTCGATCTGG-3' and reverse primer 5'-TAGCTCGAGTTATCCGGCCAGCTTTGTCCGGGACCGGAAGATCTGTTTGGTGCCCAGC-3' were used). Subsequently, by digesting them with MluI and NotI respectively and introducing them into pCAGGS vectors, we obtained the pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97-M vectors, which can express the two envelope glycoproteins of SFTSV, Gc and Gn, respectively.

[0054] The assembly of VSV-ΔG-eGFP was carried out as follows: BHK21 cells were assembled 1 day prior to the assembly of 5 × 10⁶ cells. 5Cells were plated in 12-well plates at a density of / mL. The supernatant was discarded the following day when the cells reached 90% confluence. 100 μL of vTF7-3 (vaccinia virus expressing T7 RNA polymerase) and 100 μL of DMEM (without FBS / antibiotics) were added to infect the cells, and the cells were incubated at 37°C for 1 hour. The infection solution was removed, and 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) were transduced using Genetwin (Vomido). Six hours after transduction, the liquid was replaced with a complete culture medium (DMEM, 4% FBS). After approximately 48-54 hours, the supernatant was collected and filtered through a 0.22 μm filter to remove vTF7-3, yielding VSV-ΔG-eGFP P0 generation viruses.

[0055] BHK21 cells were placed in a 24-well plate and transduced with 0.75 μg of VSV-G 24 hours prior to infection. VSV-ΔG-eGF PP0 was added in a 1:10 ratio (50 μL virus + 150 μL DMEM), and after approximately 8 hours, 300 μL of complete medium was added, and the cells were incubated at 34°C. The supernatant was collected 24 hours after infection and was used as the VSV-ΔG-eGFP P1 generation virus. The viral titer was calculated by scanning the green fluorescent spot with ImmunoSpot.

[0056] Using Genetwin (Vomid), pCAGGS-SFTSV_QD02-M and pCAGGS-SFTSV_WCH97--M plasmids were transduced into 293T cells, respectively. After 24 hours, VSV-ΔG-eGFP (1 × 10⁻¹⁰) diluted with DMEM (Mohner) was transduced. 6Cells were infected with TCID50 / mL over 5 hours, washed three times with PBS, and then supplemented with complete medium (DMEM, 4% FBS) containing VSV-G monoclonal antibody (I1 hybridoma, 1 μg / mL after purification). After 24 hours, the supernatant, i.e., SVTSV pseudoviruses (VSV-SFTSV-QD02 and VSV-SFTSV-WCH97), was collected, centrifuged at 3000 rpm for 10 minutes, aliquoted, and frozen at -80°C. The pseudoviruses were diluted to create a concentration gradient, titrated with Vero E6 cells, and viral titers were calculated by scanning the green fluorescence spot using ImmunoSpot.

[0057] The S2A5 antibody purified in Example 4 was diluted 10 times in DMEM (2% FBS) with a 4-fold gradient starting from 8 μg / mL. This was mixed with 300 SFTSV QD02 virus strains or WCH97 virus strain VSV pseudoviruses assembled in this example and incubated at 37°C for 1 hour. Subsequently, the mixture was added to a 96-well plate pre-seed with Vero E6 cells, incubated for 24 hours, discarded the supernatant, scanned for fluorescence spots using ImmunoSpot, plotted an infection inhibition graph, and the half-number inhibitory concentration IC50 was determined. 50 The IC was calculated. The results are shown in Figure 4, which is the IC when S2A5 neutralizes the QD02 pseudovirus. 50 The IC50 concentration is 0.001 μg / mL, and it is the IC50 concentration used to neutralize the WCH97 pseudovirus. 50 The concentration was 0.04 μg / mL.

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

[0059] Example: Animal protection experiment using 8S2A5 antibody In the antibody-based viral infection prevention experiment, 400 μg of the S2A5 antibody purified in Example 4 was intraperitoneally injected into 6 A129 mice (6-8 weeks old) per group, followed by 500 TCID 24 hours later. 50 The survival status of mice was observed daily after intraperitoneal injection of the SFTSV HBMC5 virus. The results are shown in Figure 7A, where the survival rate of the mice administered with S2A5 antibody was 100%, while all mice in the control group died.

[0060] On the other hand, in antibody therapy experiments against viral infection, 6 A129 mice aged 6-8 weeks were given 500 TCID per group. 50The SFTSV HBMC5 virus strain was injected intraperitoneally, and 24 hours later, 400 μg of the S2A5 antibody purified in Example 4 was injected intraperitoneally. The survival status of the mice was then observed daily. The results are shown in Figure 7B. The survival rate of the mice administered with S2A5 antibody was 100%, while all the mice in the control group died.

Claims

1. A monoclonal antibody or its antigen-binding fragment that targets the Gn protein of the severe fever with thrombocytopenia syndrome virus, The monoclonal antibody or its antigen-binding fragment comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region includes three complementarity-determining regions whose amino acid sequences are GYSFSDDN, IDPDNGGT, and AREDYYGSRAMDY, respectively, and the light chain variable region includes three complementarity-determining regions whose amino acid sequences are QSVDYAGDSY, AAS, and QQSYEDPRT, respectively. A monoclonal antibody or its antigen-binding fragment, 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.

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

10.

3. The antigen-binding fragments are Fab, Fab', Fab'-SH, scFv, and F(ab'). ２ A monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, selected from the above.

4. A polynucleotide encoding a monoclonal antibody or its antigen-binding fragment according to any one of claims 1 to 3.

5. An expression vector comprising the polynucleotide described in claim 4.

6. A host cell comprising the expression vector described in claim 5.

7. Use of a monoclonal antibody and its antigen-binding fragment for producing a product comprising the monoclonal antibody and its antigen-binding fragment according to any one of claims 1 to 3, The aforementioned product is Drugs or drug combinations for treating or preventing severe fever with thrombocytopenia syndrome virus and / or glutuvirus infection, and Reagents or reagent kits for detecting severe fever with thrombocytopenia syndrome virus or its Gn protein, The use of a monoclonal antibody and its antigen-binding fragment, which include any one of the following.

8. A product comprising a monoclonal antibody according to any one of claims 1 to 3 and the antigen-binding fragment thereof, The aforementioned product is Drugs or drug combinations for treating or preventing severe fever with thrombocytopenia syndrome virus and / or glutuvirus infection, and Reagents or reagent kits for detecting severe fever with thrombocytopenia syndrome virus or its Gn protein, A product that includes any one of the following.

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