Neutralizing antibody capable of potent neutralization of sftsv and use thereof

Abstract

A neutralizing antibody capable of potent neutralization of SFTSVs and the use thereof. The neutralizing antibody specifically recognizes amino acids at 26th, 39th, 42nd, 46th, 50th and 53rd sites of the sub-domain I and amino acids at 260th, 263rd, 268th, 271st and 301st sites of the sub-domain III of the extracellular domain of SFTSVGn. The neutralization activity of the neutralizing antibody ZS004-1C5 in inhibiting SFTSV(DBV) infection of half of cells is 67 thousands times that of MAb4-5 which is the only one fully-humanized neutralizing antibody known so far. Simply by means of one inoculation, the neutralizing antibody ZS004-1C5 can prevent STAT1- / - immunodeficient mice from a lethal attack caused by viruses with a 10-fold median lethal dose (LD50), and therefore has the potential to be put into clinical treatment, and may improve the survival rate for patients with a severe fever with thrombocytopenia syndrome (SFTS) and reduce the risk for intensive care.

Description

Neutralizing antibody strongly neutralizing SFTSV and application thereof TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and relates to a human monoclonal antibody with strong neutralizing activity against SFTSV and application thereof. BACKGROUND

[0002] Severe fever with thrombocytopenia syndrome (SFTS) is a zoonosis, and its main clinical manifestations include high fever, thrombocytopenia, leukopenia and multiple organ dysfunction, including lung, gastrointestinal tract, liver and kidney. The pathogen of SFTS is a novel virus in the family of Bunyaviridae, which is named as severe fever with thrombocytopenia syndrome virus (SFTSV, or DBV). It is of great significance to develop a therapeutic antibody against SFTSV for protecting the health of the people and maintaining the public health environment.

[0003] Antibody drugs play an important role in the field of infectious disease treatment. From the perspective of clinical first-line, SFTS patients have the following characteristics: 1, the age distribution is mainly elderly patients; 2, SFTS can lead to secondary infection of multiple pathogens after the onset, and new clinical symptoms or exacerbation of infection appear; 3, in addition to latent infection, most patients are severe when they are treated, and often need ICU monitoring and treatment for several days; 4, the patient's peripheral immune organs show severe inflammatory response. At present, only supportive therapy is adopted in the clinic, and there is no specific antibody drug for SFTSV (DBV) infection.

[0004] SUMMARY

[0005] The present application utilizes a single B cell antibody discovery platform to sort memory B cells that can bind to the surface glycoprotein of SFTSV from the peripheral blood mononuclear cells (PBMCs) of convalescents, and through transfection of linear DNA, high-throughput screening is carried out, and finally a specific antibody with super strong neutralizing activity is obtained, which is named as ZS004-1C5. At the same time, the application process successfully resolves the unique neutralizing epitope recognized by ZS004-1C5 by using cryo-EM technology, and the neutralizing mechanism of the antibody is elucidated from the perspective of structural immunology.

[0006] In one aspect, the present application provides a neutralizing antibody or a functional fragment thereof that strongly neutralizes SFTSV, which specifically recognizes the 26th, 39th, 42nd, 46th, 50th and 53rd amino acids of the I subdomain of the extracellular domain of SFTSV Gn, and the 260th, 263rd, 268th, 271st and 301st amino acids of the III subdomain.

[0007] The description of the I and III domains of SFTSV protein Gn and the amino acid sequences thereof can be found in (1) Du S, Peng R, Xu W, et al. Cryo-EM structure of severe fever with thrombocytopenia syndrome virus. Nat Commun. 2023; 14(1): 6333. Published 2023 Oct 10. doi: 10.1038 / s41467-023-41804-7; (2) Wu Y, Zhu Y, Gao F, et al. Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope. Proc Natl Acad Sci U S A. 2017; 114(36): E7564-E7573. doi: 10.1073 / pnas.1705176114; and RCSB Protein Data Bank (PDB) ID: 5Y10, which are hereby incorporated by reference herein.

[0008] In the specific embodiments, the neutralizing antibody is selected from the following two cases:

[0009] (1) a neutralizing antibody having a heavy chain and a light chain,

[0010] the variable region of the heavy chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 1, a CDR2 having an amino acid sequence of SEQ ID No.: 2, and a CDR3 having an amino acid sequence of SEQ ID No.: 3;

[0011] the variable region of the light chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 9, a CDR2 having an amino acid sequence of DVS, and a CDR3 having an amino acid sequence of SEQ ID No.: 10;

[0012] (2) a neutralizing antibody derived from (1) by substitution, deletion or addition of one or several amino acids to the amino acid sequences in (1) and having the activity of (1).

[0013] In the specific embodiments, the variable region of the heavy chain further comprises a FR1 having an amino acid sequence of SEQ ID No.: 4, a FR2 having an amino acid sequence of SEQ ID No.: 5, a FR3 having an amino acid sequence of SEQ ID No.: 6, and a FR4 having an amino acid sequence of SEQ ID No.: 7; and

[0014] The variable region of the light chain further comprises FR1 having the amino acid sequence of SEQ ID No.: 11, FR2 having the amino acid sequence of SEQ ID No.: 12, and FR3 having the amino acid sequence of SEQ ID No.: 13, and FR4 having the amino acid sequence of SEQ ID No.: 14.

[0015] In a specific embodiment, in case (2), the neutralizing antibody has a heavy chain and a light chain as follows:

[0016] The variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having the amino acid sequence of SEQ ID No.: 3 with one or several amino acids substituted, deleted or added in SEQ ID No.: 3; and

[0017] The variable region of the light chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 9, CDR2 having the amino acid sequence of DVS, and CDR3 having the amino acid sequence of SEQ ID No.: 10.

[0018] In a specific embodiment, in case (2), the neutralizing antibody has a heavy chain and a light chain as follows:

[0019] The variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having the amino acid sequence of SEQ ID No.: 3 with 1-3 amino acids substituted in SEQ ID No.: 3;

[0020] The variable region of the light chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 9, CDR2 having the amino acid sequence of DVS, and CDR3 having the amino acid sequence of SEQ ID No.: 10.

[0021] In a specific embodiment, in case (2), the neutralizing antibody has a heavy chain and a light chain as follows:

[0022] The variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having the amino acid sequence of SEQ ID No.: 18, SEQ ID No.: 19, SEQ ID No.: 20, SEQ ID No.: 21 or SEQ ID No.: 22;

[0023] The variable region of the light chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 9, a CDR2 having an amino acid sequence of SEQ ID No.: 10, and a CDR3 having an amino acid sequence of SEQ ID No.: 11.

[0024] In a specific embodiment, the neutralizing antibody has a light chain and a heavy chain selected from i to vi:

[0025] i. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 8,

[0026] ii. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 23;

[0027] iii. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 24;

[0028] iv. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 25;

[0029] v. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 26;

[0030] vi. the amino acid sequence of the variable region of the light chain is SEQ ID No.: 15; and the amino acid sequence of the variable region of the heavy chain is SEQ ID No.: 27.

[0031] In a specific embodiment, the neutralizing antibody has a heavy chain amino acid sequence of SEQ ID No.: 36 and a light chain amino acid sequence of SEQ ID No.: 37.

[0032] In another aspect, the present application provides a nucleic acid encoding the above-mentioned neutralizing antibody or antigen binding fragment thereof.

[0033] In still another aspect, the present application provides a vector comprising the above-mentioned nucleic acid.

[0034] In yet another aspect, the present application provides a host cell expressing the above-mentioned neutralizing antibody or antigen binding fragment thereof.

[0035] In yet another aspect, the present application provides a pharmaceutical composition comprising the above-mentioned neutralizing antibody or antigen binding fragment thereof, and a pharmaceutically acceptable excipient.

[0036] In yet another aspect, the present application provides the use of the neutralizing antibody or antigen-binding fragment thereof described above, or the pharmaceutical composition described above in the preparation of a medicament for inhibiting SFTSV virus and other Bunyavirus infection. Beneficial effects

[0037] Existing SFTSV (also known as DBV) research is mostly animal or humanized antibodies, and all are at the preclinical research level. The present application provides a human monoclonal antibody ZS004-1C5 with super strong neutralizing activity, which inhibits the neutralizing activity of half of the cells infected by SFTSV (DBV). It is 67,000 times that of the only known fully human neutralizing antibody MAb4-5. The ZS004-1C5 antibody only needs to be inoculated once, and it can make STAT1 - / - Immunodeficient mice are resistant to lethal attack by 10 times the median lethal dose (LD 50 ) virus, and have the potential to be used in clinical treatment, and are expected to improve the survival rate of SFTS severe patients and reduce the risk of intensive care.

[0038] In addition, SFTSV is a newly emerging tick-borne virus. According to the latest classification published by the International Committee on Virus Classification, SFTSV is officially named Dabie bunyavirus, also known as DBV, but scholars at home and abroad still use the original name SFTSV. Although the virus has been reclassified as a bunyavirus family and a bandavirus genus, it still belongs to the bunyavirus order. The surface glycoprotein substructure Gn of the virus members in the bunyavirus order has a domain with extremely similar spatial structure, so the ZS004-1C5 antibody of the present application can be used to identify other bunyaviruses in addition to SFTSV. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 shows the discovery and preparation process of the neutralizing antibody ZS004-1C5 of the present application.

[0040] Figure 2 shows the flow sorting scheme for isolating antigen-specific human memory B cells.

[0041] Figure 3 shows the in vitro ELISA binding activity of the neutralizing antibody ZS004-1C5 of the present application to SFTSV Gn protein.

[0042] Figure 4 shows the comparison of the neutralizing activity of the neutralizing antibody ZS004-1C5 of the present application and the MAb4-5 antibody to live virus.

[0043] Figure 5 shows the ZS004-1C5 antibody and SFTSV Gn protein complex Cryo-EM structure.

[0044] Figure 6 shows the SFTSV neutralizing epitope recognized by ZS004-1C5, in which the underlined amino acids are the neutralizing epitopes.

[0045] Figure 7 shows the preliminary validation of ZS004-1C5 therapy at 10 times the median lethal dose.

[0046] Figure 8 shows the in vitro ELISA binding activity (A) and virus neutralization activity (B) of the neutralizing antibody ZS004-1C5 of the present application and its variants to SFTSV Gn protein. DETAILED DESCRIPTION

[0047] The technical solutions of the present application are described in detail below through the specific embodiments, so that those skilled in the art can better understand the present application.

[0048] Materials and reagents

[0049] Cells:

[0050] Expi293F, source ThermoFisher Scientific, item number A14528

[0051] Instruments:

[0052] The PBS used in the examples is a commercial 0.01M phosphate buffer (brand: Sevibio; product number: G4202-500ML). The composition of PBS is as follows: 2.7mM KCl, 2.0mM KH2PO4, 137mM NaCl, 10mM Na2HPO4, pH 7.3-7.5, 0.1μm filtered to remove bacteria.

[0053] Example 1

[0054] Figure 1 shows the discovery and preparation process of the neutralizing antibody ZS004-1C5 of the present application, which first carries out the isolation of antigen-specific memory B cells, then carries out BCR amplification and antigen screening, and finally carries out antibody production and function identification, the detailed process is as follows.

[0055] I: Collection of blood samples from SFTSV convalescent patients and isolation of peripheral blood mononuclear cells (PMBC)

[0056] First, 50mL of blood was collected from each sample from SFTSV convalescent patients and healthy controls, respectively.

[0057] The density gradient centrifugation liquid Ficoll was pre-filled in the centrifuge tube, then the whole blood sample was mixed with phosphate buffer (PBS) at 1:1, and then slowly poured into the centrifuge tube pre-filled with gradient centrifugation liquid, ensuring that a clear interface was formed between the blood and the gradient liquid.

[0058] Subsequently, the PBMCs were obtained from the interface between the gradient liquid and the plasma after density gradient centrifugation.

[0059] The PBMCs in the white membrane layer were precisely pipetted and transferred into a new centrifuge tube. The PBMCs were washed twice with PBS containing 2% fetal bovine serum (FBS) to remove residual plasma and gradient liquid.

[0060] Finally, the obtained PBMCs were counted and subjected to subsequent experimental analysis.

[0061] Two: Flow cytometry sorting of Gn / Gc-specific B cells

[0062] The obtained PBMC samples from SFTSV-recovered patients and healthy controls were incubated with a variety of fluorescently labeled antibodies, including IgD FITC, IgM PerCP-Cy5.5, CD3 / CD235a PE-Cy5, CD27 PE-Cy7, CD38-AF700, CD19 APC-Cy7, CD14 BV605, and CD16 BV785, to accurately distinguish and identify the B cell population and its subgroups.

[0063] At the same time, in order to detect IgG capable of specifically recognizing Gn or Gc, the PBMC samples were incubated with strep-labeled Gn (amino acid sequence SEQ ID No.: 33) and Strep-PE, and with His-labeled Gc (amino acid sequence SEQ ID No.: 35) and APC anti-His.

[0064] The incubated mixture was incubated in the dark on ice. The incubated samples were analyzed using a flow cytometer, with the aim of sorting out Gn+ or Gn / Gc+ IgG-positive B cells.

[0065] The flow cytometry circle gate sorting logic was as follows:

[0066] 1. Adjust the PBMC machine voltage by forward and side scatter light signals (FSC and SSC) to obtain the cell population to be circled (A in FIG. 2);

[0067] 2. Remove the adherent cell bodies by forward scatter light signal area and intensity parameters (B in FIG. 2);

[0068] 3. Distinguish between dead and live cells by Zombie Aqua fluorescence channel (C in FIG. 2);

[0069] 4. Exclude NK cells by CD16 and CD14 marker fluorescence channels (D in FIG. 2);

[0070] 5. CD19 and CD3-CD235a marker fluorescence channel circle memory B cells (E in Figure 2);

[0071] 6. IgG BCR is obtained by excluding IgD and IgM BCR fluorescence channels (F in Figure 2);

[0072] 7. Specific memory B cells that can bind to Gn / Gc bait proteins are selected from the previous logic positive gate (G in Figure 2).

[0073] The sorted positive B cells are collected in a 96-well PCR plate preloaded with lysis solution and stored at -80°C for subsequent research or analysis.

[0074] Three: cloning of memory B cell surface receptors (BCR) and expression of recombinant antibodies

[0075] The extraction process refers to Gieselmann L, Kreer C, Ercanoglu MS, et al. Effective high-throughput isolation of fully human antibodies targeting infectious pathogens. Nat Protoc. 2021; 16(7): 3639-3671. doi: 10.1038 / s41596-021-00554-w, which is hereby incorporated by reference herein. The specific process is as follows.

[0076] 1. Obtain cell cDNA according to the manufacturer's method using a reverse transcription kit. All reagents used for reverse transcription are commercial reagents, reverse transcription kit (brand: Novozyme; product number: R211-02). The reverse transcription conditions are three-step temperature incubation: 25°C for 5 min; 50°C for 45 min; 85°C for 2 min.

[0077] 2. Use the nested PCR method to amplify the variable region sequences of IgH, IgK, and IgL using the cDNA obtained in step 1 as the template.

[0078] In the first round of PCR, 3 μL of cDNA was used as a template and mixed primers designed for IgH, IgK, and IgL were used to amplify in a 96-well plate.

[0079] The second round of PCR uses 5 μL of the product from the first round of PCR as a template. The primers used in this round contain homologous sequences to the expression vector, ensuring that the antibody variable sequences can be connected to the expression vector by homologous recombination in subsequent steps, thereby realizing the expression of the target antibody.

[0080] 3. After two rounds of PCR, the PCR products are loaded on a 1% gel for electrophoresis to verify whether the heavy chain and light chain variable region bands have been successfully amplified.

[0081] 4. Antibodies with both heavy and light chain bands are sent for sequencing analysis.

[0082] 5. The sequencing results of each antibody and its corresponding light and heavy chains are uploaded to the IMGT database for comparison, obtaining the antibody variable region information and annotation.

[0083] 6. The BCR two-round amplification products of the successfully annotated antibodies are enriched by DNA purification magnetic beads for the construction of linear expression frames.

[0084] Briefly, the construction of the linear expression frame is as follows: using the two-round amplification products as the main body, the upstream is added with a CMV promoter, a Kozak, and a secretory signal peptide dsDNA fragment, and the downstream is added with an antibody constant region, a stop codon, and a poly signal fragment by overlap extension PCR.

[0085] 7. The linear expression frame is transiently transfected into Expi293F cells, and the culture supernatant is collected after 48 h. The binding activity of each antibody to Gn or Gc protein is identified by ELISA. The BCR two-round amplification products of the antibodies with binding activity are used to construct recombinant antibody expression plasmids by homologous recombination.

[0086] 8. The constructed antibody plasmid is expressed by PEI transfection in 293F cells, and then purified by protein A chromatography column, finally obtaining the antibody named ZS004-1C5 for subsequent functional screening test.

[0087] The primer sequences, PCR amplification system, and conditions used above can be found in Chen Y, Zuiani A, Fischinger S, et al. Quick COVID-19 Healers Sustain Anti-SARS-CoV-2 Antibody Production. Cell. 2020; 183(6): 1496-1507.e16.

[0088] The nucleotide and amino acid sequence information related to the heavy chain and light chain of the obtained antibody ZS004-1C5 is as follows:

[0089] Heavy chain CDR1 : SEQ ID No.: 1

[0090] Heavy chain CDR2: SEQ ID No.: 2

[0091] Heavy chain CDR3: SEQ ID No.: 3

[0092] Heavy chain FR1 : SEQ ID No.: 4

[0093] Heavy chain FR2: SEQ ID No.: 5

[0094] Heavy chain FR3: SEQ ID No.: 6

[0095] Heavy chain FR4: SEQ ID No.: 7

[0096] Heavy chain variable region: SEQ ID No.: 8

[0097] Heavy chain variable region nucleotide sequence: SEQ ID No.: 16

[0098] Heavy chain: SEQ ID No.: 36

[0099] Nucleotide sequence of the heavy chain: SEQ ID No.: 38

[0100] Light chain CDR1 : SEQ ID No.: 9

[0101] Light chain CDR3: SEQ ID No.: 10

[0102] Light chain FR1 : SEQ ID No.: 11

[0103] Light chain FR2: SEQ ID No.: 12

[0104] Light chain FR3: SEQ ID No.: 13

[0105] Light chain FR4: SEQ ID No.: 14

[0106] Light chain variable region: SEQ ID No.: 15

[0107] Light chain variable region nucleotide sequence: SEQ ID No.: 17

[0108] Light chain: SEQ ID No.: 37

[0109] Nucleotide sequence of the heavy chain: SEQ ID No.: 39

[0110] Example 2: Screening of candidate antibodies for binding and neutralizing ability

[0111] 1. ELISA method for detecting the binding ability of antibodies to Gn / Gc for screening

[0112] Gn (sequence of SEQ ID No.: 34) and Gc (sequence of SEQ ID No.: 35) were coated in ELISA plates, respectively, using a coating concentration of 50 ng / well. The ELISA plates were stored at 4°C and maintained overnight to achieve complete adsorption.

[0113] Each well was supplemented with 200 μL of blocking solution, and then incubated at 37°C for 2 hours to reduce non-specific binding.

[0114] The harvested recombinant antibodies and positive control MAb4-5 (known SFTSV neutralizing antibody, see CN102942629A for details, which is incorporated herein by reference) were diluted to 10 μg / mL as the starting concentration using the blocking solution, and then serially diluted according to a three-fold gradient, forming a total of 12 concentration gradients.

[0115] 100 μL of the above gradient-diluted antibodies of 12 concentrations were added to each well of the coated ELISA plate. After incubation in a 37°C biochemical incubator for 2 hours, the unbound primary antibodies were removed by washing with a plate washer.

[0116] 100 μL of 1:5000 anti-human IgG-HRP was added to each well. After incubation in a 37°C biochemical incubator for 1 hour, the unbound secondary antibodies were removed by washing with a plate washer.

[0117] 100 μL of color developing solution was added to each well at room temperature for color development. After color development was complete, 50 μL of dilute sulfuric acid was quickly added to each well to terminate the color development reaction. The optical density (OD value) of each well was measured at a wavelength of 450 nm using an enzyme marker.

[0118] Subsequently, a curve was fitted according to the measured data to compare the EC50 values of each antibody to evaluate the binding ability of the antibodies to Gn / Gc, and the results are shown in Figure 3.

[0119] As can be seen from Figure 3, the half-binding concentration (IC 50 ) of ZS004-1C5 was 0.04598 μg / mL; the IC 50 of MAb4-5 was 0.05339 μg / mL; and the negative antibody did not bind to the Gn protein.

[0120] 2. SFTSV live virus neutralization experiment

[0121] The virus concentration in each well was 10 2 TCID 50After calculating the required virus amount, the virus solution was diluted with double-null (i.e., serum-free and antibiotic-free) DMEM. The antibody was also diluted with double-null DMEM to 100 μg / mL as the first hole concentration, and then diluted by two-fold gradient to a total of 12 concentrations.

[0122] In the 96-well cell culture plate, 100 μL of the antibody diluent was first added to each well, followed by 50 μL of the virus solution, and then incubated in a 37°C biochemical incubator for 1 hour to neutralize the virus by the antibody.

[0123] Then, 100 μL of the vero cell suspension was inoculated in the 96-well plate. At the same time, the cell control was set, and then incubated in a 37°C biochemical incubator for 48 hours.

[0124] The neutralization ability of the antibody was evaluated based on whether the cells were infected using immunofluorescence. The process was as follows: after the cells were fixed and the membranes were broken using 80% acetone, the cells were incubated and dyed with the rabbit primary antibody of Anti-SFTSV-NP and the Alexa fluor 488 Goat-anti-rabbit IgG secondary antibody. Under the fluorescence microscope, blue excitation light was used for observation. If the number of patchy green fluorescence observed under the microscope was less than that of the cell control well, it indicated that the corresponding antibody sample could neutralize the live SFTSV at this dilution, and was marked as a positive well. If the number of patchy green fluorescence observed under the microscope was consistent with that of the control group, it indicated that the corresponding antibody sample could not neutralize the live SFTSV at this dilution.

[0125] Finally, according to the measured data, the neutralization curve was fitted to compare the IC50 value of each antibody to evaluate the neutralization ability of the antibody to the live SFTSV, and the results are shown in FIG. 4.

[0126] As can be seen from FIG. 4, ZS004-1C5 has strong neutralization activity, and can protect 50% of the cells from virus infection at a concentration of 0.0003 μg / mL (half neutralization activity inhibition concentration). The half neutralization activity inhibition concentration of MAb4-5 is 20.15 μg / mL. It is calculated that the neutralizing antibody ZS004-1C5 of the present application has a neutralization activity of 67,000 times that of the human neutralizing antibody MAb4-5 to inhibit the infection of SFTSV (DBV) to half of the cells.

[0127] Example 3: Structure analysis of ZS004-1C5 and SFTSV-Gn complex

[0128] In order to analyze the epitope of the neutralizing antibody acting on Gn, the Cryo-EM technology was used to perform high-resolution structural analysis on the complex of the antibody and Gn. The specific operation is as follows.

[0129] Antibody ZS004-1C5 was co-incubated with Gn protein against the corresponding SFTSV GP substructure, and concentrated to prepare electron microscopy samples.

[0130] After the glow discharge hydrophilic treatment of the carbon grid, the complex was added dropwise and incubated for 3 seconds to form a sample liquid layer. Excess droplets were absorbed by filter paper, and the carbon grid was quickly frozen in liquid ethane.

[0131] The epitope of the antigen bound by the antibody was resolved using a Glacios cryogenic transmission electron microscope in an ultrahigh-resolution mode at a magnification of 81000x, and the Chimera software was used to analyze the antigen-antibody binding amino acid residue interaction surface and force.

[0132] The cryo-EM results showed that the ZS004-1C5 CDR3 region had 5 amino acid residues (E104, R116, Y109, Y107, and Y114) that interacted with Gn (Figure 5).

[0133] The SFTSV neutralizing epitope recognized by the antibody is shown in Figure 6, where the underlined amino acids are the neutralizing epitopes, including the 26th, 39th, 42nd, 46th, 50th, and 53rd amino acids of the I subdomain of the Gn extracellular domain, and the 260th, 263rd, 268th, 271st, and 301st amino acids of the III subdomain. These epitopes are novel antigen epitopes newly discovered in this paper.

[0134] Example 4: Establishment of SFTSV infection model and preliminary application of ZS004-1C5

[0135] To evaluate the availability of ZS004-1C5 for in vivo treatment, C57BL / 6-STATA1 - / - mice were used as an animal model to determine the median lethal dose of SFTSV. The specific operation is as follows.

[0136] The virus stock solution was diluted 10 times successively using medical water for injection to obtain dilutions with virus titers of 10 to 0.0001 TCID 50 , and the final volume of each dilution was 100 μL. C57BL / 6-STATA1 - / - mice were given 100 μL of each virus titer intraperitoneally using a 0.5 mL disposable insulin syringe, and each virus titer was set with 6 replicates.

[0137] The results showed that the dose that caused 50% of the animal models to die after viral infection was 0.15 TCID 50 . The mice were infected intraperitoneally at a dose of 10 TCID 50 (more than 66 times the LD 50 dose), and 500 μg of ZS004-1C5 was administered for treatment 24 hours later. The results showed that all the mice survived after administration (Figure 7).

[0138] Example 5: Affinity evolution of ZS004-1C5 based on yeast surface display system

[0139] In order to obtain antibody variants recognizing the neutralizing epitope described in the present application, and to discuss whether the affinity improved variants of ZS004-1C5 can still have strong neutralizing activity, a yeast surface display technology was used to construct a ZS004-1C5 CDR3 region amino acid mutation display library. The engineered displayed yeast library was subjected to directed evolution of the CDR3 region through multiple rounds of flow sorting. The final retained single yeast colonies were amplified by PCR method, and the corresponding CDR3 base information was obtained. The improved antibodies were expressed in vitro and subjected to binding verification, neutralizing activity verification and affinity detection. The specific operation is as follows:

[0140] According to the known amino acid residues of the antibody binding neutralizing epitope, the CDR3 amino acid sites of the antibody variable region that can bind the antigen epitope to play the main chemical bond function were analyzed, and a commercial random mutation kit (brand: Agilent; product number: G5903A) was used to construct the mutation library in the region according to the manufacturer's instructions. The mutation library was ligated to the pCTCON2 yeast display vector through Nhe I and BamH I enzyme digestion sites, and the ligated library was transformed into EBY100 Saccharomyces cerevisiae competent cells (brand: Shanghai Weidi Biology; product number: YC1110M) and cultured at 30°C on YPD solid plates overnight. The yeast colonies were scraped and transferred to SGCAA liquid medium for expansion culture, and the SGCAA medium was induced at 20°C for 24 h. After flow sorting was used to screen the yeast library, solid plate culture was performed, and the yeast genes were amplified by PCR and subjected to sequencing analysis. According to the sequencing results, the CDR3 amino acid sequence was analyzed. According to the analysis results, the antibody expression vector was reconstructed and expressed and purified, and 5 variants YD4, YB2, YF1, YD3 and YG6 were obtained, the nucleotide sequences of the heavy chain variable regions of which were SEQ ID No.: 28, SEQ ID No.: 29, SEQ ID No.: 30, SEQ ID No.: 31 and SEQ ID No.: 32, and the amino acid sequences were SEQ ID No.: 23, SEQ ID No.: 24, SEQ ID No.: 25, SEQ ID No.: 26 and SEQ ID No.: 27, respectively. The nucleotide sequence of the light chain variable region was SEQ ID No.: 17, and the amino acid sequence was SEQ ID No.: 15.

[0141] Culture medium description:

[0142] YPD solid plate medium: 10 g yeast extract, 20 g peptone, 20 g agar powder, 20 g glucose, volume 1 L;

[0143] SDCAA medium: 20 g glucose, 5 g casein, 6.7 g yeast nitrogen, 5.4 g Na2HPO4, 8.65 g NaH2PO4, volume 1 L;

[0144] SGCAA medium: 20 g galactose, 5 g casein, 6.7 g yeast nitrogen, 5.4 g Na2HPO4, 8.65 g NaH2PO4, volume 1 L.

[0145] The dissociation constant KD, ka, kdis, Full R^2 parameters of the antibodies before and after evolution were determined by BLI method, which were analyzed by a BLI tester (brand: SARTORIUS, machine name: Octet, machine model: R8) (machine self-analysis software name: Octet analysis studio, version: 12.2), and the results are shown in Table 1 below.

[0146] Table 1

[0147] At the same time, the ELISA binding ability and neutralization activity of the above 5 antibodies were characterized again using the same operation as in points 1 and 2 in Example 2, and the results are shown in Figure 8.

[0148] As can be seen from Figure 8, the 5 ZS004-1C5 variants obtained in this embodiment still have the ability to bind SFTSV Gn protein, and the neutralization activity of the 5 antibodies is still stronger than that of MAb4-5, but the data in Table 1 shows that the affinity of the 5 variants has not been improved.

Claims

1. A neutralizing antibody or a functional fragment thereof that strongly neutralizes SFTSV, which specifically recognizes amino acids at positions 26, 39, 42, 46, 50 and 53 of subdomain I and amino acids at positions 260, 263, 268, 271 and 301 of subdomain III of the Gn ectodomain of SFTSV.

2. The neutralizing antibody or functional fragment thereof of claim 1, wherein, The neutralizing antibody is selected from the following two cases: (1) a neutralizing antibody having a heavy chain and a light chain, the variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having the amino acid sequence of SEQ ID No.: 3; and the variable region of the light chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 9, CDR2 having the amino acid sequence of DVS, and CDR3 having the amino acid sequence of SEQ ID No.: 10; (2) a neutralizing antibody derived from (1) by substitution, deletion or addition of one or several amino acids to the amino acid sequence of (1) and having the activity of (1).

3. The neutralizing antibody or a functional fragment thereof according to claim 2, wherein, the variable region of the heavy chain further comprises FR1 having the amino acid sequence of SEQ ID No.: 4, FR2 having the amino acid sequence of SEQ ID No.: 5, FR3 having the amino acid sequence of SEQ ID No.: 6, and FR4 having the amino acid sequence of SEQ ID No.: 7; and the variable region of the light chain further comprises FR1 having the amino acid sequence of SEQ ID No.: 11, FR2 having the amino acid sequence of SEQ ID No.: 12, FR3 having the amino acid sequence of SEQ ID No.: 13, and FR4 having the amino acid sequence of SEQ ID No.:

14.

4. The neutralizing antibody or functional fragment thereof of claim 2, wherein, In case (2), the neutralizing antibody has a heavy chain and a light chain: the variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having an amino acid sequence obtained by substitution, deletion or addition of one or several amino acids to SEQ ID No.: 3; and the variable region of the light chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 9, CDR2 having the amino acid sequence of DVS, and CDR3 having the amino acid sequence of SEQ ID No.: 10, Preferably, the neutralizing antibody has a heavy chain and a light chain: the variable region of the heavy chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 1, CDR2 having the amino acid sequence of SEQ ID No.: 2, and CDR3 having an amino acid sequence obtained by substitution of 1-3 amino acids to SEQ ID No.: 3; and the variable region of the light chain comprises CDR1 having the amino acid sequence of SEQ ID No.: 9, CDR2 having the amino acid sequence of DVS, and CDR3 having the amino acid sequence of SEQ ID No.: 10, the variable region of the heavy chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 1, a CDR2 having an amino acid sequence of SEQ ID No.: 2, and a CDR3 having an amino acid sequence of SEQ ID No.: 18, SEQ ID No.: 19, SEQ ID No.: 20, SEQ ID No.: 21, or SEQ ID No.: 22; More preferably, the neutralizing antibody has a heavy chain and a light chain as follows: the variable region of the heavy chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 1, a CDR2 having an amino acid sequence of SEQ ID No.: 2, and a CDR3 having an amino acid sequence of SEQ ID No.: 18, SEQ ID No.: 19, SEQ ID No.: 20, SEQ ID No.: 21, or SEQ ID No.: 22; the variable region of the heavy chain comprises a CDR1 having an amino acid sequence of SEQ ID No.: 1, a CDR2 having an amino acid sequence of SEQ ID No.: 2, and a CDR3 having an amino acid sequence of SEQ ID No.: 18, SEQ ID No.: 19, SEQ ID No.: 20, SEQ ID No.: 21, or SEQ ID No.:

22.

5. The neutralizing antibody or a functional fragment thereof of claim 2, wherein, the neutralizing antibody has a light chain and a heavy chain selected from the following i to vi: i. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 8; ii. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 23; iii. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 24; iv. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 25; v. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 26; vi. the amino acid sequence of the light chain variable region is SEQ ID No.: 15, and the amino acid sequence of the heavy chain variable region is SEQ ID No.: 27; Preferably, the neutralizing antibody has a heavy chain amino acid sequence of SEQ ID No.: 36 and a light chain amino acid sequence of SEQ ID No.:

37.

6. A nucleic acid encoding the neutralizing antibody or an antigen-binding fragment thereof of any one of claims 1-5.

7. A vector comprising the nucleic acid of claim 6.

8. A host cell expressing the neutralizing antibody or an antigen-binding fragment thereof of any one of claims 1-5.

9. A pharmaceutical composition comprising the neutralizing antibody or an antigen-binding fragment thereof of any one of claims 1-5, and a pharmaceutically acceptable excipient.

10. Use of the neutralizing antibody or an antigen-binding fragment thereof of any one of claims 1-5, or the pharmaceutical composition of claim 9, in the manufacture of a medicament for inhibiting SFTSV virus and other Bunyavirus infection.

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