Antibodies against severe fever with thrombocytopenia syndrome virus and their applications

Abstract

The present invention provides an antibody that binds to the SFTSV glycoprotein Gn, and the antibody has a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID No.1, 3, 5, 7, 9, 11, 13; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID No.2, 4, 6, 8, 10, 12, 14. The antibody of the present invention has very high in vitro neutralization activity (IC 50 can be as low as 3.1 ng / mL), and the in vivo detection results show that it can effectively prevent mice from being infected with SFTSV. In the therapeutic experiment, a single-dose treatment was received 5 days after infection with a lethal dose, and the survival efficiency could also reach 80%; the antibody of the present invention can effectively treat mice infected with SFTSV and prevent SFTSV from infecting mice, and has extremely high application value for clinical treatment and prevention of SFTSV infection.

Description

[0001] This application claims the priority of Chinese Patent Application No. 2024108142555, titled "Antibodies Against Severe Fever with Thrombocytopenia Syndrome Virus and Their Applications", filed on June 24, 2024. The entire content of the above application is incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biotechnology, and particularly to an antibody against severe fever with thrombocytopenia syndrome virus and its applications, and more particularly to a human monoclonal antibody against severe fever with thrombocytopenia syndrome virus and its applications. Background Art

[0003] Severe fever with thrombocytopenia syndrome (SFTS) is caused by the infection of a newly emerging tick-borne severe fever with thrombocytopenia syndrome virus (SFTSV). The harm of severe fever with thrombocytopenia syndrome is becoming increasingly serious. It is characterized by a sudden onset, and on average, it only takes 9 days for some patients to progress from the onset of symptoms to coma or death. In addition, if fungal infections occur during the treatment process, the severity of the disease is significantly increased. It is reported that the number of cases shows an overall increasing trend every year. Due to the complex transmission chain of SFTSV, the host and transmission vector are in a state of change, and combined with the characteristics of wide distribution of reported cases in provinces, fast spread, etc., and there are currently no effective vaccines and drugs against SFTSV, it is difficult to prevent and control. Therefore, there is an urgent need to develop drugs that can cure severe cases and reduce the fatality rate.

[0004] In this regard, antibody drugs have shown great potential. In 1998, the first neutralizing monoclonal antibody against respiratory syncytial virus - Palivizumab was approved for marketing, and it has been proven to effectively protect high-risk infants from the severe diseases caused by its infection. Currently, more and more neutralizing monoclonal antibody drugs are being applied to the treatment or prevention of infectious diseases, including influenza virus, human immunodeficiency virus, and novel coronavirus, etc. Therefore, the development of antibody drugs has become an important way to reduce the severity and fatality rate of infectious diseases.

[0005] The combined use of single-cell transcriptome and immune repertoire technologies can accurately determine the cell subtypes corresponding to specific B cell clones. By selecting B cell or plasma cell characteristic antibody sequences from recovered patients for verification, antibodies with potential clinical application value can be obtained. Since the results of screening monoclonal antibodies based on the BCR immune repertoire platform were reported in the journals *Science* in 2018 and *Cell* in 2019, international top academic journals have successively reported the acquisition of COVID-19 antibodies with strong neutralizing activity based on this platform in 2020 and 2021. The principle of this platform is to sort virus-reactive B cells after obtaining convalescent samples from several recovered patients, complete transcriptome and immune repertoire sequencing, and screen potential active antibodies and conduct functional verification based on information such as the patient's neutralizing antibody level, BCR clonality, and changes in cell subsets.

[0006] Monoclonal antibody drugs have been widely reported in antiviral therapy. Especially for members of the Bunyavirales order, such as Rift Valley fever virus, Hantavirus, Xinjiang hemorrhagic fever virus, etc., there have been continuous reports of human monoclonal antibodies, but the research and development of therapeutic antibodies for SFTS have been relatively slow. SFTS has typical viremia, and the virus mutation rate is relatively slow, and there is antigenic cross-reactivity between genotypes. Different scientific research institutions at home and abroad have carried out research and development of SFTSV antibody drugs and diagnostic reagents. Although there have been many reports of antibodies against SFTSV, there are still no effective antibody drugs available clinically. The M segment of SFTSV encodes the viral glycoprotein precursor, which is enzymatically cleaved into the N-terminal glycoprotein (Gn) and the C-terminal glycoprotein (Gc), which have been proven to be able to induce the production of neutralizing antibodies. Therefore, it is expected to obtain human antibodies with high antiviral activity after sequencing and functional verification of the antibody gene sequences in the B cell or plasma cell subsets with significant humoral immune changes in the acute and convalescent phases, provide a scientific basis for systematically elucidating the Gn protein epitope map and guide vaccine design, and provide new options for the clinical treatment of SFTSV infection. Summary of the Invention

[0007] To solve the above problems, the present invention discovers that neutralizing antibodies can be screened out with single Gn alone, which can be used to resist and eliminate SFTSV infection and become a drug for treating SFTSV infection. To obtain human neutralizing antibodies with neutralizing effects, the present invention first uses HEK 293T / F cells to express the Gn protein of SFTSV as a bait, and sorts B cells reactive to the SFTSV glycoprotein Gn from the PBMCs of SFTS recovered patients by flow cytometry. Then, the single-cell transcriptome and BCR repertoire of B cells are sequenced to obtain the transcriptome information and antibody heavy and light chain sequence information in B cells. According to information such as antibody sequence clone abundance, potential antibody sequence variable regions and constant regions are selected and ligated into an expression vector. After mammalian cell expression and purification, a series of biological property detections are carried out, including antigen-binding ability, virus-neutralizing activity, mouse experiments for treating SFTSV-infected mice, etc. Finally, 7 Gn human monoclonal antibodies (SD4, SD4-S, SD4-X, SD5, SD7, SD12, and SD22) with high efficiency in neutralizing SFTSV infection are identified.

[0008] On the one hand, the present invention provides an antibody that binds to the SFTSV glycoprotein Gn, and the antibody has a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as shown in any one of SEQ ID No.1, 3, 5, 7, 9, 11, 13; the amino acid sequence of the light chain variable region is as shown in any one of SEQ ID No.2, 4, 6, 8, 10, 12, 14.

[0009] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.1; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.2.

[0010] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.3; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.4.

[0011] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.5; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.6.

[0012] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.7; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.8.

[0013] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.9; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.10.

[0014] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 11; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 12.

[0015] In one embodiment, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No. 13; the amino acid sequence of the light chain variable region is as shown in SEQ ID No. 14.

[0016] In one embodiment, the antibody is a monoclonal antibody, a recombinant antibody or a polyclonal antibody, preferably, a monoclonal antibody.

[0017] In one embodiment, the antibody is a human antibody, a murine antibody or a chimeric antibody.

[0018] In a preferred embodiment, the antibody is a human antibody.

[0019] In one embodiment, the antibody further comprises a heavy chain constant region comprising IgG, for example, a heavy chain constant region of IgG selected from IgG1, IgG2, IgG3 or IgG4.

[0020] On the other hand, the present invention also provides a polynucleotide encoding the above antibody.

[0021] On the other hand, the present invention also provides a recombinant vector comprising the above polynucleotide.

[0022] On the other hand, the present invention also provides a host cell comprising the above recombinant vector.

[0023] On the other hand, the present invention also provides a pharmaceutical composition comprising the above antibody; optionally, further comprising a pharmaceutical carrier.

[0024] On the other hand, the present invention also provides the use of the above antibody in the preparation of a reagent or a drug for treating or preventing severe fever with thrombocytopenia syndrome.

[0025] On the other hand, the present invention also provides the use of the above antibody in the preparation of a reagent or a drug for neutralizing severe fever with thrombocytopenia syndrome virus.

[0026] On the other hand, the present invention also provides the use of the above antibody in the preparation of a reagent targeting the Gn protein of severe fever with thrombocytopenia syndrome virus.

[0027] The human monoclonal antibody of the present invention has very high in vitro neutralizing activity (IC 50It can be as low as 3.1 ng / mL), and in vivo detection results show that it can effectively prevent mice from being infected by SFTSV. In the therapeutic experiment, when treated 5 days after infection with a lethal dose, a survival efficiency of 20% can also be achieved. The antibody of the present invention can effectively treat mice infected with SFTSV and prevent SFTSV from infecting mice, and has extremely high application value for clinical treatment and prevention of SFTSV infection. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 . Purification results of SFTSV Gn and Gc proteins by molecular sieve purification; among them, (A) SDS-PAGE Coomassie brilliant blue staining identification results; (B) His-tag protein Western blot results; (C) Western blot results of sera from convalescent patients; among them, M. is protein Marker; 1. Normal cell control; 2. Purified Gc protein; 3. Purified Gn protein; (D) Molecular sieve diagram of SFTSV Gn protein.

[0029] Figure 2 . Molecular sieve chromatography results of SD4 purification.

[0030] Figure 3 . Molecular sieve chromatography results of SD4-S purification.

[0031] Figure 4 . Molecular sieve chromatography results of SD4-X purification.

[0032] Figure 5 . Molecular sieve chromatography results of SD5 purification.

[0033] Figure 6 . Molecular sieve chromatography results of SD7 purification.

[0034] Figure 7 . Molecular sieve chromatography results of SD12 purification.

[0035] Figure 8 . Molecular sieve chromatography results of SD22 purification.

[0036] Figure 9 . Neutralization curve of antibody against SFTSV.

[0037] Figure 10 . Kinetic curve of antibody binding to SFTSV Gn.

[0038] Figure 11 . Detection of competition spectra among different neutralizing antibodies; among them, A is the kinetic curve of different neutralizing antibodies competing for binding to the antigen, and B is the summary of competing epitopes among different neutralizing antibodies.

[0039] Figure 12. Effect of antibody in preventing mice from being infected with SFTSV.

[0040] Figure 13 . Effect of antibody in treating mice infected with SFTSV; wherein, A is the effect diagram of treating mice infected with SFTSV with single-dose antibody, and B is the effect diagram of treating mice infected with SFTSV with four-dose continuous antibody.

[0041] The sequence information involved in the present invention is as follows:

[0042] SEQ ID No.1: Heavy chain variable region of SD4

[0043] qvqlvesgggvvqpgrslrlscaasgfpfsgygmhwvrqapgkglewvavisydgseknyadsvkgrftisrdnskntlylqm nslrvedtavyycakdrdyygsgfydywgqgtlvtvss

[0044] SEQ ID No.2: Light chain variable region of SD4

[0045] diqmtqspsslsasvgdrvtitcqasqhisnhlnwyqqkpgtaptlliyeasnletgvpsrfsgsgsgtdfsftisslrpedfatyycq qydnfpltfgpgttmdik

[0046] SEQ ID No.3: Heavy chain variable region of SD4-S

[0047] qvqlvesgggvlqpgrslrlscaasgftfsgygmhwvrqapgkglewvavisydgseknygdsvkgrftisrdnskntlylqins lrtedravyycakdrdyfgsgffdywgqgtlvtvss

[0048] SEQ ID No.4: Light chain variable region of SD4-S

[0049] diqmtqspsslsasvgdrvtitcqasqditkylnwyqqkpgkapkllifeasnletgvpsrfsgsgsgtdftftisslqpedfatyycq qygdfpltfgggtkveik

[0050] SEQ ID No.5: Heavy chain variable region of SD4-X

[0051] qvqlvesgggvvqpgrslrlscaasgftfssygmhwvrqapgkglewvavisydgsnkyyadsvkgrftisrdnskntlylqm nslraedtavyycakdrdyygsgffdywgqgtlvtvss

[0052] SEQ ID No.6: SD4-X Light Chain Variable Region

[0053] diqmtqspsslsasvgdrvtitcqasqdisnhlnwyqekpgkvpklliyeasnvetgvpsrfsgsgsgtdftftisslqpediatyyc qqydnlpytfgqgtkleik

[0054] SEQ ID No.7: SD5 Heavy Chain Variable Region

[0055] evqlvesggglvrpggslritcsvsgftfsrcpihwvrqapgkgleyvsaissdggstyyadslkgrfiisrdnskntlhlqmsslrp edtavyycvkdrgcnghdseywgqgtlvtvss

[0056] SEQ ID No.: SD5 Light Chain Variable Region

[0057] dlqmtqspsslsasvgdrvtitcqashdisnyvnwyqqkpgkapilliydasfleagvpsrfsgsrsgtdftftirslqpediatyycq qyydvlsfgggtkveik

[0058] SEQ ID No.9: SD7 Heavy Chain Variable Region

[0059] qvqlqqwgagllkpsetlsltcavyggsfsgyfwswiwqppgkglewigeinhsgstnynpslksrvtisvdtsknqfslklrsvt aadtavyycargvrrfgfsythygmdvwgqgttvtvss

[0060] SEQ ID No.10: SD7 Light Chain Variable Region

[0061] aiqmtqspsslsasvgdrvtitcrasqgirndlgwyqqkpgqapklliytasslqsgvpsrfsgsgsgtdftltisslqpedfatyycl qdynypltfgggtkveik

[0062] SEQ ID No.11: Heavy chain variable region of SD12

[0063] evqllesggglvqpggslrlscaasgfifssyamswvrqapgkglewvsaisgsggstyyadsvkgrftisrdnskntlylqmnsl rdedtavyycakdrsyvssgffddwgqgtlvtvss

[0064] SEQ ID No.12: Light chain variable region of SD12

[0065] diqmtqspsslsasvgdrvtitcqasqdisnylnwyqqrpgkapkllifeasnletgvpsrfsgsgsgtdftftisslqpediatyycq qyenllytfgqgtkleik

[0066] SEQ ID No.13: Heavy chain variable region of SD22

[0067] qvqlvesgggvvqpgrslrlscaasgftfsgfgmhwvrqapgkglewvalisydgsdtyysdsvkgrftisrdnskntlylqlksl rpddtavyycvgdrdyfgsgffdhwgqgtlvtvss

[0068] SEQ ID No.14: Light chain variable region of SD22

[0069] diqmtqspsslsasigdrvtitcrasrhitnhlnwyqhkpgrapklliyeasnlqagvpsrfsgsgsgtdftftisslqpedfatyycq qydnlppafgggtkvdik

[0070] SEQ ID No.15: Amino acid sequence of Gn protein

[0071] gdsgpiicagpihsnksagiphllgysekicqidrlihvsswlrnhsqfqgyvgqrggrsqvsyypaensysrwsgllspcdadwlgmlvvkkaresdmivpgpsykgkvfferptfdgyvgwgcgsgksrtesgelcssdsgtssgllpsdrvlwigdvacqlmtpipeetflelksfsqsefpdickidgivfnqcegeslpqpfdvawmdvghshkiimrehktkwvqessskdfvcykegtgpcseseekacktsgscrgdmqfckvagcehgeetseakcrcslvhkpgevvvsyggmrvrpkcygfsrmmatlevnppeqrigqctgchlecinggvrlitltselrsatvcashfcssassgkksteihfhsgslvgktaihvkgalvdgteftfegscmfpdgcdavdctfcreflknpqcypakk

[0072] SEQ ID No.16: DNA sequence of Gn protein

[0073]

[0074] SEQ ID No.17: Amino acid sequence of Gc protein

[0075] cdemvhadsklvscrqgsgnmkecittgrallpavnpgqeaclhftapgspdskclkikvkrinlkckksssyfvpdarsrctsvrrcrwagdcqsgcpphftsnsfsddwagkmdraglgfsgcsdgcggaacgcfnaapscifwrkwvenphgiiwkvspcaawvpsavieltmpsgevrtfhpmsgiptqvfkgvsvtylgsdmevsgltdlceieelkskklalapcnqagmgvvgkvgeiqcsseesartikkdgciwnadlvgielrvddavcyskitsveavanysaipttigglrfershdsqgkisgsplditairgsfsvnyrglrlslseitatctgevtnvsgcyscmtgakvsiklhssknstahvrckgdetafsvlegvhsyivslsfdhavvdeqcqlncgghesqvtlkgnlifldvpkfvdgsymqtyhstvptganipsptdwln

[0076] SEQ ID No.18: DNA sequence of Gc protein

[0077] Detailed implementation manners

[0078] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention, and does not limit the present invention in other forms. Any person skilled in the relevant art may use the disclosed technical content to make equivalent embodiments with equivalent changes. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0079] To make the experimental content and technical solution of the present invention more clearly described, the present invention will be described in detail below in conjunction with specific embodiments and with reference to the accompanying drawings.

[0080] Example 1. Purification result of SFTSV Gn protein by molecular sieve purification

[0081] In this embodiment, the Gn and Gc proteins of severe fever with thrombocytopenia syndrome virus (SFTSV) are used for protein expression and purification; the specific operations are as follows:

[0082] The extracellular segment is designed as follows: protection base EcoRI - Kozak sequence - signal peptide - STFV - Gc / Gn - AVI - tag - His tag - stop codon - Hind III strategy.

[0083] The target gene sequences of the Gn protein and the Gc protein are shown in SEQ ID No.16 and SEQ ID No.18 respectively, and the amino acid sequences of the Gn protein and the Gc protein are shown in SEQ ID No.15 and SEQ ID No.17 respectively.

[0084] After the target gene is synthesized, it is constructed into the eukaryotic expression vector pcDNA3.4 (named: SFTFV - Gc / Gn_pcDNA3.4) for expression and purification. According to the SDS - PAGE results as Figure 1 shown, the Gc protein band in the purified cell supernatant is single and clear, indicating good purification effect; although there are trace impurity bands in the purified intracellular Gn protein, there is a clear target band at 52 kDa (as Figure 1 A). The antigenicity verification of the recombinant protein by Western blot shows that when detected with a 6×His antibody, the target band of the Gc recombinant protein exists near 70 kDa, and the target band of Gn exists near 52 kDa (as Figure 1B), indicating that Gn and Gc proteins were successfully extracted. When tested with recovered patient serum, Gn recombinant protein showed a specific band at around 52 kDa, and the size was consistent with expectations, proving that the extracted recombinant protein can react specifically with recovered patient serum, and that Gc cannot react specifically (such as Figure 1 C), this result shows that the preparation of Gn protein can be used as bait to catch virus-specific B cells. After the cell culture fluid containing the target protein is purified by ion affinity chromatography HisTrapTMHPGE and gel filtration chromatography Superose TM6Increase10 / 300GLGE, a relatively pure target protein (such as Figure 1 D). The purified Gn protein was used for subsequent experiments.

[0085] Example 2. Isolation of SFTSV Gn glycoprotein-specific B cells

[0086] From May 2020 to September 2022, the applicant cooperated with the Second Affiliated Hospital of Shandong First Medical University and Tai'an Center for Disease Control and Prevention to collect 10-20mL of blood and isolate peripheral blood mononuclear cells (PBMCs) with the informed consent of the patients. 7 The cells were incubated on ice for half an hour with a density of 100 nM SFTSV Gn protein at a final concentration of 100 nM, and then washed twice with PBS. CD3, CD16, CD56, CD45, CD19, CD27, and His tag antibodies were incubated on ice for half an hour, and then washed twice with PBS. - CD16 - CD56 - CD45 + CD19 + Gn + After collecting the cells by centrifugation at 1000g / 5min, the cell density reached more than 10,000 and was sent to the company for single-cell immune library and transcriptome library sequencing.

[0087] Example 3. SFTSV-Gn specific B cell immune repertoire sequencing and potential binding antibody selection and construction

[0088] We selected 23 antibody sequences from the total library for gene function verification based on cloning frequency, UMI number, and cell subtype. The human antibody construction strategy is as follows:

[0089] Heavy chain: CMV promoter-XbaI-Leader sequences-heavy chain variable region-CH-stop codon-EcoRV.

[0090] Light chain (K): CMV promoter - XbaI - Leader sequences - Light chain variable region - CL - Stop codon - EcoRV.

[0091] Light chain (L): CMV promoter - XbaI - Leader sequences - Light chain variable region - CL - Stop codon - EcoRV.

[0092] Twenty - three antibodies were screened, and the antibody information is shown in Table 1.

[0093] Table 1. Antibody sequence information

[0094]

[0095] The 23 purified antibodies obtained above were mixed with 100 TCID 50 and 10 TCID 50 SFTSV at a concentration of 100 μg / mL at 37°C for 1 hour, then added to a 48 - well plate pre - inoculated with Vero cells. After incubation at 37°C for 3 h, washed 3 times with PBS, added 400 μl of maintenance medium containing 2% penicillin - streptomycin, incubated for 4 days, and then the SFTSV nucleic acid was detected by fluorescence quantitative PCR method. At the same time, virus back - titration was set up, with 100 TCID 50 、10 TCID 50 、2.5 TCID 50 、0.625 TCID 50 、0.156 TCID 50 and three replicates for each negative control.

[0096] Table 2. Detection of potential neutralizing activity of antibodies

[0097]

[0098]

[0099] According to the results in Table 2, SD4, SD5, SD7, SD12, SD16 and SD22 have potential neutralizing activity. Since the expression effect of SD16 is not ideal, SD4, SD5, SD7, SD12 and SD22 were selected for further verification experiments.

[0100] To more accurately describe the IC 50 results, after incubating SFTSV with antibodies at different concentrations, the high - content immunofluorescence method was used to read the results. The results are as Figure 9 , and the IC 50They are 0.0035 μg / mL, 0.317 μg / mL, 0.111 μg / mL, 0.005 μg / mL, and 0.0045 μg / mL respectively. According to sequence similarity, two antibodies, SD4-S and SD4-X, were further verified. The IC 50 values of SD4-S and SD4-X are 0.0031 μg / mL and 0.01 μg / mL respectively.

[0101] Seven monoclonal antibodies with good verification effects: SD4, SD4-S, SD4-X, SD5, SD7, SD12, and SD22. Among them, the amino acid sequence of the heavy chain variable region of SD4 is shown in SEQ ID No.1, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.2; the amino acid sequence of the heavy chain variable region of SD4-S is shown in SEQ ID No.3, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.4; the amino acid sequence of the heavy chain variable region of SD4-X is shown in SEQ ID No.5, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.6; the amino acid sequence of the heavy chain variable region of SD5 is shown in SEQ ID No.7, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.8; the amino acid sequence of the heavy chain variable region of SD7 is shown in SEQ ID No.9, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.10; the amino acid sequence of the heavy chain variable region of SD12 is shown in SEQ ID No.11, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.12; the amino acid sequence of the heavy chain variable region of SD22 is shown in SEQ ID No.13, and the amino acid sequence of the light chain variable region is shown in SEQ ID No.14.

[0102] Example 4. Expression and purification of antibodies

[0103] HEK293F cells were co-transfected with the recombinant plasmids containing the light and heavy chain encoding genes of specific antibodies obtained in Example 3. The specific operation refers to the instruction manual of Sino Biological Inc. After culturing for 5 days, the supernatant was collected. The collected supernatant was centrifuged at 5,000 rpm for 1 h, filtered through a 0.22 μm filter membrane, and then bound to a pre-packed Protein A column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (pH 3.0), and the eluted protein was concentrated and subjected to size exclusion chromatography. The target peak was determined by SDS-PAGE. The size exclusion chromatography results of the typically purified antibodies SD4, SD4-S, SD4-X, SD5, SD7, SD12, and SD22 are shown respectively as Figures 2 - 8 shown.

[0104] Example 5. Performance detection of antibodies

[0105] The binding ability of the antibody to SFTSV Gn was detected by membrane diffraction technology. The binding force between the antigen and the antibody was detected using an Octect instrument. The specific steps were as follows: The Pro A sensor was used and pre-wetted in PBST buffer for 10 min. Then, the antibody was diluted to 15 μg / mL as the immobilized substance and immobilized on the ProA Biosensor type chip, and the threshold was set at 1 nm; the highest starting concentration of the antigen was 100 nM, and it was serially diluted 7 times at a 2-fold gradient with PBS + 0.05% Tween-20 buffer. The time for the baseline, immobilization, baseline, binding, dissociation, and regeneration experimental steps was set at 60 s, 60 s, 120 s, 120 s, 180 s, and 30 s, respectively. The antibody was immobilized on the Pro A sensor, and the immobilization height was set at the threshold of 1 nm, and the temperature was 30 °C. The kinetic curve was obtained, and the software globally fitted with a 1:1 model to calculate the affinity constant (KD), association rate constant (ka), and dissociation rate constant (kd). As Figure 10 shown, SD4, SD4-S, SD4-X, SD7, SD12, and SD22 showed fast binding and slow dissociation characteristics with the Gn protein. The binding constant of the SD4 antibody to Gn was 1.222E+05, and the dissociation constant was 1.177E-05. Therefore, the affinity of the SD4 antibody to SFTSV-Gn was 0.96 pM; also in the same order of magnitude were the SD4-S antibody (affinity: 19 nM) and the SD22 antibody (affinity: 20.5 nM), both of which were higher than the reported control antibodies. Therefore, the neutralizing active antibodies showed strong binding force to Gn.

[0106] Example 6. Competitive experiment between antibodies with confirmed binding targets detected by Octet

[0107] The BioLayer Interferometry Analyzer Octet RED 96 was used to detect whether there was competition between the binding of SD4 to the SFTSV-Gn glycoprotein and other antibodies. The HIS1K sensor chip was selected. After equilibration with 1×PBS + 0.05% Tween-20 buffer solution, the Gn glycoprotein with a His tag was immobilized on the chip at a concentration of 50 μg / mL. The antibody concentration was diluted to 100 - 500 nM (specifically according to the antibody binding force to ensure antibody binding saturation). After layout according to the experimental design, the machine parameters were set as follows: baseline: 30 s, immobilization: 180 s, baseline: 30 s, first antibody: 180 s, baseline: 30 s, second antibody: 180 s, regeneration: 30 s. After setting the temperature at 30 °C, the run was started. After completion, the two batches of data were merged and analyzed using the high-throughput analysis software - epitopebinning carried by the machine.

[0108] Layout detection was carried out according to the experimental design of epitope grouping (7×7). The epitope competition heatmap showed that in the absence of the primary antibody, the binding signal of the secondary antibody was about 0.2 nm. If the complete competition was defined as the intensity of the single secondary antibody binding signal being less than 30%, SD4, SD4-S, SD4-X, SD7, SD12, SD22, and Positive control (PC) were all below the threshold in the presence of the primary antibody ( Figure 11 A), indicating that the 7 antibody epitopes were basically the same; however, when the binding of SD4 was saturated, SD7 still had a certain binding signal ( Figure 11 B), suggesting that the binding epitopes of SD7 and other antibodies overlapped, showing an epitope cross phenomenon.

[0109] Example 7. Animal protection experiment

[0110] The preventive and therapeutic experimental methods were as previously described. The preventive effect evaluation showed that 24 h in advance, intraperitoneal injection (20 mg / kg) was administered, and then each A129 mouse was infected with 20 PFU of the SFTSV strain by subcutaneous injection in the hind limb. As a result, all the experimental mice in the SD4 antibody group survived completely (100%), and their body weights did not change significantly, while all the mice in the group only injected with the virus died ( Figure 12 ). At the same time, injecting the SD4 antibody only on the 1st or 2nd day after virus infection also achieved the same protective effect ( Figure 12 ).

[0111] To more accurately simulate the treatment status of patients when they come to the hospital, after A129 mice were infected with 20 PFU of SFTSV, a single dose of drug was administered on days 3 - 6. The body weights of the mice that started using the antibody on days 3 and 4 were significantly different from those of the virus-positive group on day 8, but there was no difference from the PBS control group; if the SD4 antibody was injected starting on day 5, the survival rate was 80%, and if the SD4 antibody was injected starting on day 6, the survival rate was 20%; the mice in the virus-positive group started to die 7 days after infection and all died on day 9, indicating that the SD4 antibody can effectively treat SFTSV infection ( Figure 13 A). To further explore whether multiple administrations of the antibody can improve the clinical treatment effect, that is, administration was started on days 3 - 6 and continued for 4 days. The results showed that the body weight changes of the experimental mice in the multiple administration group were basically the same as those in the single dose group, but when the SD4 antibody was injected starting on day 5, the survival rate increased to 100%, and when the SD4 antibody was injected starting on day 6, the survival rate was 40% ( Figure 13 B). It can be seen that continuous drug use can improve the clinical cure rate. The above experimental results at the animal level indicate that the SD4 antibody has good preventive and therapeutic effects on SFTSV infection.

[0112] Although the specific embodiments of the present invention have been described in detail, those skilled in the art will understand that various modifications and changes can be made to the details according to all the teachings that have been published, and such changes are within the scope of protection of the present invention. The entire scope of the present invention is given by the appended claims and any equivalents thereof.

Claims

1. An antibody that binds to the SFTSV glycoprotein Gn, said antibody having a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is as shown in SEQ ID No.1; the amino acid sequence of the light chain variable region is as shown in SEQ ID No.

2.

2. The antibody according to claim 1, wherein, The antibody is a monoclonal antibody or a recombinant antibody.

3. The antibody according to claim 1, wherein The antibody is a human antibody, a murine antibody or a chimeric antibody.

4. The antibody according to claim 1, wherein, The antibody further comprises the heavy chain constant region of IgG.

5. A polynucleotide encoding the antibody according to any one of claims 1-4.

6. A recombinant vector comprising the polynucleotide according to claim 5.

7. A host cell comprising the recombinant vector according to claim 6.

8. A pharmaceutical composition comprising the antibody according to any one of claims 1-4.

9. The pharmaceutical composition according to claim 8, characterized in that, The pharmaceutical composition further comprises a pharmaceutical carrier.

10. Use of the antibody according to any one of claims 1-4 in the preparation of a reagent or a drug for treating or preventing severe fever with thrombocytopenia syndrome.

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