Antibody of severe fever with thrombocytopenia syndrome virus and application thereof

Abstract

The invention provides an antibody combined with SFTSV glycoprotein Gn. The antibody is provided with a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown as any one of SEQ ID No.1, 3, 5, 7, 9, 11 and 13; the amino acid sequence of the light chain variable region is shown as any one of SEQ ID No.2, 4, 6, 8, 10, 12 and 14. The antibody provided by the invention has very high in-vitro neutralizing activity (IC50 can be as low as 3.1 ng / mL), and in-vivo detection results show that mice can be effectively prevented from being infected by SFTSV, and in a therapeutic experiment, 80% of survival efficiency can also be achieved after the mice are infected by a lethal dose for 5 days and are subjected to single-dose treatment; the antibody disclosed by the invention can be used for effectively treating mice infected by the SFTSV and preventing the infection of the mice by the SFTSV, and has extremely high application value in clinical treatment and prevention of the infection of the SFTSV.

Description

[0001] This application claims the priority of Chinese patent application No. 2024108142555, filed on June 24, 2024, entitled “Antibodies and Applications of Fever with Thrombocytopenia Syndrome Virus”, and the entire contents of the above application are incorporated herein by reference. Technical Field

[0002] The present invention relates to the field of biotechnology, and in particular to an antibody against a fever with thrombocytopenia syndrome virus and an application thereof, and in particular to a humanized monoclonal antibody against a fever with thrombocytopenia syndrome virus and an application thereof. Background Art

[0003] Severe fever with thrombocytopenia syndrome (SFTS) is caused by a new tick-borne fever with thrombocytopenia syndrome virus (SFTSV) infection. The harm of SFTSV is becoming more and more serious. Its characteristic is that it has an acute onset. Some patients only need an average of 9 days from the onset of symptoms to coma or death. In addition, if fungal infection is combined with the treatment process, the severity of the disease will be significantly increased. It is reported that the number of cases each year is increasing overall. Due to the complex transmission chain of SFTSV, its host and transmission medium are in a state of flux, and the reported cases are widely distributed in provinces and spread rapidly. There is currently no effective vaccine or drug for SFTSV, which makes it difficult to prevent and control. Therefore, it is urgent to develop drugs that can cure severe diseases and reduce mortality.

[0004] In this regard, antibody drugs show great potential. In 1998, the first neutralizing monoclonal antibody approved for respiratory syncytial virus, Palivizumab, was launched on the market and proved to be effective in protecting high-risk infants and young children from serious diseases caused by its infection. Currently, more and more neutralizing monoclonal antibodies are used to treat or prevent infectious diseases, including influenza virus, HIV and new coronavirus. Therefore, the development of antibody drugs has become an important way to reduce the severity and mortality of infectious diseases.

[0005] The combined use of single-cell transcriptome and immune repertoire technology can accurately determine the cell subtype corresponding to specific B cell clones, select characteristic antibody sequences of B cells or plasma cells from recovered patients for verification, and obtain antibodies with potential clinical application value. Since Science in 2018 and Cell in 2019 reported the results of screening monoclonal antibodies based on the BCR immune repertoire platform, top international academic journals in 2020 and 2021 have successively reported on the acquisition of strong neutralizing COVID-19 antibodies based on this platform. The principle of this platform is to sort virus-reactive B cells after obtaining samples from several recovered patients, complete transcriptome and immune repertoire sequencing, and screen potential active antibodies and perform 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 treatment. In particular, there are continuous reports of human monoclonal antibodies for members of the Bunyavirales order, such as Rift Valley fever virus, Hantavirus, Xinjiang hemorrhagic fever virus, etc., but the development of therapeutic antibodies for SFTS is relatively slow. SFTS has typical viremia, and the virus mutation rate is relatively slow. There is antigenic cross-talk between genotypes. Different scientific research institutions at home and abroad have carried out the development of SFTSV antibody drugs and diagnostic reagents. Although there have been many reports on 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), and has been proven to induce the production of neutralizing antibodies. Therefore, it is expected that human antibodies with high antiviral activity can be obtained after sequencing and functional verification of antibody gene sequences in B cells or plasma cell subsets with significant changes in humoral immunity during the acute and convalescent phases, providing a scientific basis for systematically clarifying the Gn protein antigen epitope map and guiding vaccine design, providing a new option for the clinical treatment of SFTSV infection. Summary of the invention

[0007] To solve the above problems, the present invention has found that neutralizing active antibodies can be screened out with a single Gn, which is used to resist and eliminate SFTSV infection and become a drug for treating SFTSV infection. In order to obtain human neutralizing antibodies with neutralizing effect, the present invention first uses HEK 293T / F cells expressing SFTSV's Gn protein as bait, and sorts B cells reactive to SFTSV glycoprotein Gn from PBMCs of SFTS recovered patients by flow cytometry, and then sequences the B cell single cell transcriptome and BCR repertoire to obtain transcriptome information and antibody light and heavy chain sequence information in B cells, and selects the variable region and constant region of the potential antibody sequence based on the antibody sequence clone abundance and other information and connects them to the expression vector. After expression and purification in mammalian cells, a series of biological property tests were carried out, including antigen binding ability, virus neutralization activity, and experiments on treating mice infected with SFTSV. Finally, 7 Gn human monoclonal antibodies (SD4, SD4-S, SD4-X, SD5, SD7, SD12 and SD22) that are highly effective in neutralizing SFTSV infection were identified.

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

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

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

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

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

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

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

[0015] In one embodiment, the amino acid sequence of the heavy chain variable region is shown as SEQ ID No.13; the amino acid sequence of the light chain variable region is shown as 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 of an IgG, for example, a heavy chain constant region of an 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, which comprises the above-mentioned antibody; optionally, further comprises a pharmaceutically acceptable carrier.

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

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

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

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

[0028] Figure 1 .SFTSV Gn and Gc protein purification molecular sieve purification results; wherein, (A) SDS-PAGE Coomassie Brilliant Blue staining identification results; (B) His-tagged protein Western blot protein blotting results; (C) recovered patient serum Western blot protein blotting results; wherein, M. is protein Marker; 1. normal cell control; 2. is purified Gc protein; 3. is purified Gn protein; (D) SFTSV Gn protein molecular sieve diagram.

[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 .The molecular sieve chromatography results of SD12 purification.

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

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

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

[0038] Fig.11 .Competition spectrum detection between different neutralizing antibodies; wherein A is the kinetic curve of competition between different neutralizing antibodies for antigen binding, and B is the summary of competition epitopes between different neutralizing antibodies.

[0039] Fig.12.The effect of antibodies in preventing mice from being infected with SFTSV.

[0040] Fig.13 .The effect of antibody treatment on SFTSV infection; wherein A is a graph showing the effect of a single dose of antibody treatment on mice infected with SFTSV, and B is a graph showing the effect of four consecutive doses of antibody treatment on mice infected with SFTSV.

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

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

[0043] qvqlvesgggvvqpgrslrlscaasgfpfsgygmhwvrqapgkglewvavisydgseknyadsvkgrftisrdnskntlylqm nslrvedtavyycakdrdyygsgfydywgqgtlvtvss

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

[0045] diqmtqspsslsasvgdrvtitcqasqhisnhlnwyqqkpgtaptlliyeasnletgvpsrfsgsgsgtdfsftisslrpedfatyycq qydnfpltfgpgttmdik

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

[0047] qvqlvesgggvlqpgrslrlscaasgftfsgygmhwvrqapgkglewvavisydgseknygdsvkgrftisrdnskntlylqins lrtedravyycakdrdyfgsgffdywgqgtlvtvss

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

[0049] diqmtqspsslsasvgdrvtitcqasqditkylnwyqqkpgkapkllifeasnletgvpsrfsgsgsgtdftftisslqpedfatyycq qygdfpltfgggtkveik

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

[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 DESCRIPTION

[0078] The present invention is further described below in conjunction with the embodiments. The following description is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change it into an equivalent embodiment 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 falls within the protection scope of the present invention.

[0079] In order to make the experimental contents and technical solutions of the present invention more clearly described, the present invention is described in detail below in combination with specific embodiments and with reference to the accompanying drawings.

[0080] Example 1. SFTSV Gn protein expression and purification results of molecular sieve purification

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

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

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

[0084] After the target gene was synthesized, it was constructed into the eukaryotic expression vector pcDNA3.4 (named: SFTFV-Gc / Gn_pcDNA3.4) for expression and purification. Figure 1 As shown in the figure, the Gc protein band in the purified cell supernatant is single and clear, indicating that the purification effect is good; although there are trace impurities in the intracellular Gn protein after purification, there is a clear target band at 52kDa (such as Figure 1 A). The antigenicity verification of the recombinant protein by Western blot showed that when detected with 6×His antibody, the Gc recombinant protein had a target band near 70kDa, and the Gn had a target band near 52kDa (such 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 their 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 10TCID 50 SFTSV was mixed and incubated at 37°C for 1 hour, then added to a 48-well plate pre-inoculated with Vero cells, incubated at 37°C for 3 hours, washed 3 times with PBS, and incubated for 4 days with 400 μl of maintenance solution containing 2% penicillin and streptomycin. SFTSV nucleic acid was detected by fluorescence quantitative PCR. Virus back titration was set at 100 TCID 50 、10TCID 50 , 2.5TCID 50 , 0.625TCID 50 , 0.156TCID 50 There were three replicate wells for each of the negative controls.

[0096] Table 2. Antibody potential neutralizing activity detection

[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 IC 50 Results: After SFTSV was incubated with antibodies at different concentrations, the results were read using high-content immunofluorescence. Fig. 9 , SD4, SD5, SD7, SD12 and SD22 ICs 50The values ​​were 0.0035μg / mL, 0.317μg / mL, 0.111μg / mL, 0.005μg / mL and 0.0045μg / mL respectively. Based on the sequence similarity, two antibodies, SD4-S and SD4-X, were verified. The IC values ​​of SD4-S and SD4-X were 50 0.0031μg / mL and 0.01μg / mL respectively.

[0101] The seven monoclonal antibodies with good validation effects were: SD4, SD4-S, SD4-X, SD5, SD7, SD12 and SD22, wherein 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 The amino acid sequence of the heavy chain variable region of SD22 is shown in SEQ ID No.12; the amino acid sequence of the light chain variable region 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 plasmid containing the light and heavy chain encoding genes of the specific antibody obtained in Example 3. The specific operation was referred to the instructions of Sino Biological, and the supernatant was collected after 5 days of culture. The collected supernatant was centrifuged at 5,000 rpm for 1 hour, filtered through a 0.22 μm filter membrane, and combined with a Protein A pre-packed column (5 mL, GE Healthcare). The bound protein was eluted with 10 mM glycine (Ph 3.0). The protein was collected and concentrated and then subjected to molecular sieve chromatography. The target peak was determined by SDS-PAGE. The molecular sieve chromatography results of the typical purified antibodies SD4, SD4-S, SD4-X, SD5, SD7, SD12 and SD22 were as follows: Figure 2-Figure 8 shown.

[0104] Example 5. Antibody performance testing

[0105] The binding ability of antibodies to SFTSV Gn was detected by membrane diffraction technology. The binding force between antigen and antibody was detected by Octect instrument. The specific steps are as follows: Use Pro A sensor and pre-wet it in PBST buffer for 10 minutes, then dilute the antibody to 15μg / mL as a solid, fix it to the ProA Biosensor type chip, and set the threshold to 1nm; the highest starting concentration of the antigen is 100nM, and use PBS+0.05% Tween-20 buffer to dilute 7 concentrations in a 2-fold gradient, set the baseline, solidification, baseline, binding, dissociation, regeneration experimental steps to 60s, 60s, 120s, 120s, 180s, 30s, respectively, the antibody is solidified on the Pro A sensor, the solidification height is set to a threshold of 1nm, the temperature is 30°C, and the kinetic curve is obtained. The software uses a 1:1 model global fitting to calculate the affinity constant (KD), binding rate constant (ka) and dissociation rate constant (kd). As Fig.10 As shown, SD4, SD4-S, SD4-X, SD7, SD12 and SD22 exhibit fast binding and slow dissociation characteristics with Gn protein. The binding constant of SD4 antibody with Gn is 1.222E+05, and the dissociation constant is 1.177E-05, so the affinity of SD4 antibody with SFTSV-Gn is 0.96pM; the same order of magnitude is also SD4-S antibody (affinity: 19nM) and SD22 antibody (affinity: 20.5nM), both higher than the reported control antibody. Therefore, the neutralizing active antibody shows strong binding with Gn.

[0106] Example 6. Competition experiment between confirmed target binding antibodies detected by Octet

[0107] Octet RED 96, a biomolecular interaction analyzer, was used to detect whether the binding of SD4 to SFTSV-Gn glycoprotein was competitive with other antibodies. HIS1K sensor chip was selected, and after equilibration with 1×PBS+0.05% Tween-20 buffer solution, Gn glycoprotein with His tag was fixed to the chip at a concentration of 50 μg / mL, and the antibody concentration was diluted to 100-500 nM (depending on the antibody binding force, the purpose is to saturate the antibody binding). After the layout was carried out according to the experimental design, the machine parameters were set to baseline: 30s, solidification: 180s, baseline: 30s, first antibody: 180s, baseline: 30s, second antibody: 180s, regeneration: 30s, and the temperature was set to 30°C before starting the operation. After completion, the high-throughput analysis software-epitopebinning provided by the machine was used to merge and analyze the two batches of data.

[0108] According to the epitope grouping experimental design, the layout (7×7) detection was carried out. The epitope competition heat map showed that in the absence of the first antibody, the second antibody binding signal was around 0.2nm. If the single second antibody binding signal intensity of less than 30% was used as the standard for complete competition, SD4, SD4-S, SD4-X, SD7, SD12, SD22 and Positive control (PC) were all below the threshold ( Fig.11 A), indicating that the epitopes of the seven antibodies are basically the same; however, when SD4 binds saturated, SD7 still has a certain binding signal ( Fig.11 B), it is speculated that the binding epitopes of SD7 and other antibodies overlap, and there is a phenomenon of epitope cross-talk.

[0109] Example 7. Animal protection experiment

[0110] The preventive and therapeutic experimental methods were as described above. The preventive effect evaluation showed that the mice in the SD4 antibody group survived completely (100%) without significant changes in body weight, and all mice in the virus injection group died ( Fig.12 ). At the same time, injecting SD4 antibody only on the first or second day after infection also had the same protective effect ( Fig.12 ).

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

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

Claims

1. An antibody that binds to SFTSV glycoprotein Gn, the antibody having a heavy chain variable region and a light chain variable region; the amino acid sequence of the heavy chain variable region is shown in any one of SEQ ID No.1, 3, 5, 7, 9, 11, and 13; the amino acid sequence of the light chain variable region is shown in any one of SEQ ID No.2, 4, 6, 8, 10, 12, and 14.

2. The antibody according to claim 1, characterized in that The antibody is a monoclonal antibody, a recombinant antibody or a polyclonal antibody.

3. The antibody according to claim 1, characterized in that The antibody is a human antibody, a murine antibody or a chimeric antibody.

4. The antibody according to claim 1, characterized in that The antibody also includes an IgG heavy chain constant region.

5. A polynucleotide encoding the antibody according to any one of claims 1 to 4. 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 to 4; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

9. Use of the antibody according to any one of claims 1 to 4 in the preparation of an agent or a medicine for treating or preventing fever with thrombocytopenia syndrome.

10. Use of the antibody according to any one of claims 1 to 4 in the preparation of an agent or a drug for neutralizing FEVER WITH THROMOCYTOPIA SYNDROME VIRUS.

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