A single-domain antibody against Gc antigen of fever with thrombocytopenia syndrome virus

Abstract

This invention discloses a single-domain antibody and fusion protein that specifically binds to the Gc antigen of the virus in fever with thrombocytopenia syndrome, solving the problem of treating and preventing fever with thrombocytopenia syndrome and showing a highly efficient neutralization effect between pseudovirus and live virus.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a single-domain antibody against Gc antigen of fever with thrombocytopenia syndrome virus. Background Technology

[0002] Severe fever with thrombocytopenia syndrome virus (SFTSV) is a newly emerging tick-borne virus belonging to the genus Leukopivirus of the order Bunyavirales. Its primary vector is the Haemaphysalis longhorn tick, and it also carries the risk of human-to-human transmission through blood contact. It is widespread in several provinces in central and eastern my country, as well as in East Asian countries such as Japan, South Korea, and Vietnam, posing a serious threat to public health. Clinical manifestations of SFTSV infection initially include high fever, fatigue, and muscle aches, which rapidly progress to thrombocytopenia and leukopenia. Severe cases can lead to multiple organ failure, with a mortality rate as high as 12%–30%. It has been listed as a priority pathogen by the World Health Organization. Currently, prevention and control of SFTSV infection mainly involve symptomatic and supportive treatment. There is still a lack of safe and effective vaccines and specific antiviral drugs, and no monoclonal antibodies against SFTSV have entered clinical trials or are available for commercial use.

[0003] SFTSV's cell membrane proteins include two envelope proteins, Gn and Gc. The Gc protein is responsible for mediating the fusion of the virus with the host cell membrane. It is a key protein for the virus to adsorb and enter the host cell to complete replication and proliferation. It is also an important target antigen that induces the body to produce a specific immune protective response. The protective effect of the vaccine is positively correlated with the concentration of antibodies against this protein.

[0004] Antibody-mediated humoral immune responses are a crucial means for the body to combat viral infections. Highly effective neutralizing and protective antibodies can not only be used for emergency treatment of emerging viral infections and control of epidemic spread, but also help susceptible and high-risk populations prevent viral infection and facilitate the rapid development of serological diagnostic kits. Monoclonal antibodies can effectively inhibit viral replication; passive transfer of polyclonal serum and monoclonal antibodies to laboratory animals can effectively prevent and protect them from corresponding viral infections. Currently, antibody drugs targeting respiratory syncytial virus (RSV) and HIV are available on the market, and several other antibody drugs targeting different viral infections are in the research and clinical trial stages.

[0005] Single-domain antibodies, also known as nanobodies, are antibody fragments that contain only the heavy chain variable region (VHH). They have advantages such as small molecular weight, high affinity, strong specificity, good penetration, and ease of expression and modification. Compared with traditional monoclonal antibodies, they are more suitable for the prevention and treatment of tick-borne virus infections, especially in response to emerging infectious diseases, as they have the characteristics of short preparation cycle and rapid large-scale production.

[0006] Current treatments for SFTSV rely solely on symptomatic supportive measures such as antipyretics, plasma or platelet transfusions, lacking specific therapies and effective antibody treatments. Therefore, developing a single-domain antibody that specifically binds to the Gc protein of SFTSV and possesses highly efficient neutralizing activity is crucial for addressing this urgent clinical need and for the prevention and control of SFTSV. Summary of the Invention

[0007] To achieve the above objectives, this invention has developed a single-domain antibody capable of specifically binding to the Gc protein of SFTSV (Fever with Thrombocytopenia Syndrome Virus), resulting in superior clinical therapeutic effects. This specific antibody provides a new and effective means for the prevention and treatment of diseases caused by SFTSV infection.

[0008] Specifically, the present invention provides a single-domain antibody against Gc antigen of fever with thrombocytopenia syndrome virus. The single-domain antibody includes a frame region and three complementarity-determining regions CDR1, CDR2 and CDR3. The amino acid sequences of the complementarity-determining regions CDR1, CDR2 and CDR3 are shown in SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

[0009] In some embodiments, the single-domain antibody includes a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 4-6.

[0010] This invention provides a fusion protein comprising a single-domain antibody that specifically binds to the Gc antigen of fever with thrombocytopenia syndrome virus and a fusion protein Fc region.

[0011] In some embodiments, the Fc region of the fusion protein is selected from IgG or IgA.

[0012] In some embodiments, the Fc region of the fusion protein includes a CH2 domain and a CH3 domain.

[0013] In some embodiments, the Fc region of the fusion protein is selected from human IgG1, IgG2, IgG3 and / or IgG4, or an Fc region amino acid sequence that has one or more amino acid mutations with human IgG1, IgG2, IgG3, IgG4.

[0014] In some implementations, the amino acid mutation is a substitution or deletion of the amino acid.

[0015] In some embodiments, the fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 7-9.

[0016] The present invention provides a nucleic acid molecule that encodes the single-domain antibody or the fusion protein described herein.

[0017] The present invention also provides a recombinant vector comprising the nucleic acid molecule.

[0018] The present invention also provides a recombinant cell comprising the nucleic acid molecule and / or the recombinant vector, and capable of expressing the single-domain antibody or the fusion protein.

[0019] The present invention also provides a pharmaceutical composition comprising the single-domain antibody or the fusion protein and a pharmaceutically acceptable carrier.

[0020] The present invention also provides a detection kit comprising the single-domain antibody, the fusion protein, or the pharmaceutical composition.

[0021] The present invention also provides the use of the single-domain antibody, the fusion protein, the nucleic acid molecule, the recombinant vector, or the recombinant cell in the preparation of a medicament for treating or preventing diseases caused by viral infection with fever with thrombocytopenia syndrome.

[0022] Experimental verification has shown that the antibody provided by this invention can specifically bind to the Gc protein of fever with thrombocytopenia syndrome virus (FSH). This antibody not only exhibits highly efficient neutralizing ability in pseudovirus models but also shows significant inhibitory effects on live virus. Even after humanization, it maintains high purity. This achievement provides new insights for the development of therapeutic and preventative drugs against FSH infection, opening up new and effective avenues for the treatment and control of related diseases.

[0023] Abbreviations and Terminology Definitions

[0024] The following abbreviations are used in this article. CDR: Complementarity-determining region in the immunoglobulin variable region.

[0025] The term "single-domain antibody" refers to an antibody found in the peripheral blood of camels that is naturally absent from the light chain and contains only one heavy chain variable region (VHH). "Single-domain antibody" and "VHH" are used interchangeably, both referring to single-domain antibodies that specifically recognize and bind to Gc. A single-domain antibody is the variable region of a heavy chain antibody. Typically, a single-domain antibody contains three CDRs and four FRs.

[0026] The term "fusion protein" refers to a polypeptide or protein with single, dual, or multiple specificities, or with monovalent, divalent, or multivalent binding properties. It includes, but is not limited to, IgG, IgM, IgA, IgD, IgE, and all subclasses of fusion proteins, such as IgG subclasses IgG1, IgG2, IgG3, and IgG4 found or produced in animal cells, including human cells. "Heavy chain antibodies" formed by the fusion of a single-domain antibody (such as VHH) or its antigen-binding fragment with the Fc region of a fusion protein, as well as other antibodies or fragments with similar special structures, also fall within the scope of "fusion protein" as defined in this application.

[0027] The term "amino acid" refers to twenty common, naturally occurring amino acids. Naturally occurring amino acids include alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine ​​(Cys; C); glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G); histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Val; V). In some embodiments, the term "amino acid" also includes non-natural amino acids. Any suitable non-natural amino acid may be used. In some embodiments, the non-natural amino acid contains a reactive moiety for conjugating the agent with MIAC.

[0028] The term "complementarity-determining region" (CDR) refers to a discontinuous antigenic combination site found within the variable region of a heavy-chain polypeptide. CDRs have been described by Kabat et al., J. Biol. Chem. 252:6609-6616 (1977); Kabat et al., USDept. of Health and Human Services, "Sequences of proteins of immune interest" (1991); Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition includes overlaps or subsets of amino acid residues when compared with each other. Several methods exist for defining or describing CDRs, including Kabat, Chothia, IMGT, AbM, and Contact, with Kabat being the most commonly used.

[0029] The term "VHH" is used to distinguish the variable domain from the heavy chain variable domain and light chain variable domain present in conventional 4-chain antibodies. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (unlike the VH or VL domains in conventional 4-chain antibodies, where the epitope is recognized by both the VL and VH domains). The VHH domain is a small, stable, and efficient antigen-recognition unit formed by a single immunoglobulin domain. In some embodiments, the VHH domain comprises, or is composed of, the heavy chain variable region domain of a heavy chain antibody. In the context of this disclosure, the terms "single-domain antibody," "VHH," and "heavy chain variable region" are used interchangeably.

[0030] The term "specific" means that one of the molecules involved in specific binding does not exhibit significant binding to any molecules other than one or more of its binding partner molecules. Furthermore, the term is also used when a domain containing an antibody-variable region is specific to a particular epitope among multiple epitopes in an antigen. When the epitope bound by the domain containing the antibody-variable region is contained in several different antigens, an antigen-binding molecule containing the domain containing the antibody-variable region can bind to various antigens having said epitope.

[0031] The term "CH2 domain" refers to a heavy chain constant region in which a hinge connects to a CH3 domain within a heavy chain constant domain. In some embodiments, the term "CH2 domain" refers to a region of a fusion protein molecule spanning amino acids 238 to 340 (EU number). The term "CH2 domain" encompasses the wild-type CH2 domain and its variants (e.g., non-naturally occurring CH2 domains or modified CH2 domains). For example, the term "CH2 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH2 domains and variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH2 domains include CH2 domains with mutations that alter the biological activity of an antibody (such as ADCC, CDC, purification, dimerization, and half-life).

[0032] The term "CH3 domain" refers to the heavy chain constant region located at the C-terminus of the CH2 domain within the heavy chain constant domain. In some embodiments, the term "CH3 domain" refers to a region of a fusion protein molecule spanning amino acids 341 to 447 (EU number). The term "CH3 domain" encompasses the wild-type CH3 domain and its variants (e.g., non-naturally occurring CH3 domains or modified CH3 domains). For example, the term "CH3 domain" includes wild-type IgG1, IgG2, IgG3, and IgG4 CH3 domains and variants having 1, 2, 3, 4, 5, 1 to 3, 1 to 5, 3 to 5, and / or up to 5, 4, 3, 2, or 1 mutation (e.g., substitution, deletion, and / or addition). Exemplary CH3 domains include CH3 domains with mutations that alter the biological activity of the antibody (such as ADCC, CDC, purification, dimerization, and half-life).

[0033] The term "Fc" refers to a sequence of non-antigen-binding fragments (whether in monomeric or multimeric form) produced by antibody digestion or other means, and may contain a hinge region. The original fusion protein source of native Fc is preferably human, but can be any fusion protein. Fc molecules consist of monomeric polypeptides that can be covalently (i.e., disulfide bonds) and non-covalently associated to form dimers or multimers. Depending on the class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgA1, IgGA2, and IgG4), the number of intermolecular disulfide bonds between monomeric subunits of native Fc molecules ranges from 1 to 4.

[0034] The term "chimeric antibody" refers to an antibody in which a portion of the heavy chain originates from one source or species, while the remainder of the heavy chain originates from a different source or species. It refers to an antibody molecule in which a constant region or a portion thereof is altered, replaced, or exchanged, thereby linking the antigen-binding site to a different or altered constant region of a class and / or species, or to a completely different molecule (e.g., an enzyme, toxin, hormone, growth factor, drug), which imparts new properties to the chimeric antibody. For example, camel-derived antibodies can form chimeric antibodies by replacing their constant region with a constant region derived from human immunoglobulins. Due to the replacement with a human constant region, the chimeric antibody can retain its specificity in recognizing antigens while exhibiting reduced immunogenicity in humans, as compared to the original camel-derived antibody.

[0035] The term "humanized antibody" refers to a genetically engineered non-human antibody containing a human antibody constant domain and a non-human variable domain modified to contain a high level of sequence homology with the human variable domain. This can be achieved by transplanting the six non-human antibody complementarity-determining regions (CDRs) that together form the antigen-binding site onto the homologous human receptor frame region (FR). To fully reconstruct the binding affinity and specificity of the parent antibody, it may be necessary to replace frame residues of the parent antibody (i.e., the non-human antibody) into the human frame region (reversion mutation). Structural homology modeling can help identify amino acid residues in the frame region that are important for the antibody's binding properties. Therefore, a humanized antibody may contain a non-human CDR sequence, optionally a substantially human frame region containing one or more amino acid reversion mutations mutated into a non-human amino acid sequence, and a fully human constant region. Optionally, additional amino acid modifications, not necessarily reversion mutations, may be applied to obtain humanized antibodies with preferred characteristics, such as affinity and biochemical properties.

[0036] The term "frame region" or "FR" refers to the amino acid residues in the antibody variable region other than the CDR residues as defined above.

[0037] The term "amino acid mutation" refers to a mutation or change in amino acids in a variant protein or polypeptide compared to the original protein or polypeptide, including the insertion, deletion, or substitution of one or more amino acids based on the original protein or polypeptide.

[0038] The term "nucleic acid molecule" refers to both DNA and RNA molecules. Nucleic acid molecules can be single-stranded or double-stranded, but double-stranded DNA is preferred. Nucleic acids are "effectively linked" when placed in a functional relationship with another nucleic acid sequence.

[0039] The term "pharmaceutically acceptable carrier" refers to any inactive substance suitable for use in formulations for delivering bound molecules. Carriers can be anti-adhesives, adhesives, coating agents, disintegrants, fillers or diluents, preservatives (such as antioxidants, antibacterial agents, or antifungal agents), sweeteners, absorption delay agents, humectants, emulsifiers, buffers, etc. Examples of suitable pharmaceutically acceptable carriers include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, etc.), dextrose, vegetable oils (such as olive oil), saline, buffers, buffered saline, and isotonic agents such as sugars, polyols, sorbitol, and sodium chloride.

[0040] The term "neutralizing activity" refers to fusion proteins with antiviral activity that can specifically recognize viral antigens and effectively bind to and neutralize viral activity, preventing viral invasion of target cells and blocking viral replication in target cells, thus playing an important role in antiviral activity. Attached Figure Description

[0041] Figure 1 The results show the neutralizing activity of the antibody against the pseudovirus in this invention. Detailed Implementation

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. While methods and materials similar to or equivalent to those described and used herein may be used in the practice or testing of this application, suitable methods and materials are described below. In case of conflict, the patent specification shall prevail.

[0043] Example 1: Immunization of camels

[0044] Camels were immunized using SFTSV-Gc-his protein (SFTSV_AFB82725.1-2017 PNAS; Gc extracellular domain (Gc_ECD): 563-996) as the immunogen. Camel ear tags were recorded before immunization. The antigen and Freund's adjuvant were mixed at a 1:1 ratio, thoroughly emulsified to form a homogeneous mixture, and stored at 4°C for later use. During immunization, multiple injections were performed on both sides of the camel's neck near the lymph nodes, with two injection points on each side. 0.4 mL of the emulsified antigen was injected at each point. Camels were observed for 30 minutes after immunization to confirm they were in good condition and showed no signs of discomfort. The immunization schedule was once every two weeks, for a total of 7 immunizations.

[0045] Blood was collected for titer testing starting after the third immunization, and peripheral blood mononuclear cells (PBMCs) were isolated after the fourth immunization. Blood was collected 5-7 days after the sixth and seventh immunizations, with 25-30 mL collected each time and aliquoted into three blood collection tubes. Blood collection for immunization evaluation was scheduled before the fourth, fifth, and sixth immunizations, with 5 mL collected from the camel's jugular vein each time. On the day of blood collection, the blood was centrifuged at 400g for 30 minutes using a centrifuge pre-cooled to 25°C, and the supernatant serum was separated and stored.

[0046] Lymphocyte isolation was performed as follows: Add 3 mL of cell separation medium to a 15 mL centrifuge tube, then slowly add 3 mL of blood along the tube wall, taking care to maintain the liquid layer and avoid mixing the blood with the separation medium. Pre-cool the centrifuge to room temperature, centrifuge at 400g for 30 minutes, observe the layering after centrifugation, and carefully aspirate the middle cloud-like immune cell layer using a 200 μL pipette, transferring it to a new 15 mL centrifuge tube; transfer the upper serum layer to another new centrifuge tube and store at -80℃. Add 10 mL of room temperature PBS buffer to the collected immune cells, centrifuge at 25℃ and 400g for 20 minutes, and discard the supernatant; then add 5 mL of room temperature PBS buffer and centrifuge and wash once under the same conditions. After discarding the supernatant, count the cells using a hemocytometer. Depending on the cell count, resuspend the cells in an appropriate amount of RNAiso Plus lysis buffer to adjust the cell concentration to 102. 7 The lysis buffer was collected at a concentration of 100 μL / mL and transferred to cryovials for storage at -80°C.

[0047] Example 2: Gc database building and selection

[0048] Following the fifth immunization, peripheral blood mononuclear cells (PBMCs) were collected and isolated using lymphocyte separation fluid. Total RNA was extracted from the PBMCs, and cDNA was synthesized via reverse transcription using random primers. The VHH fragment was then amplified using specific primers, leading to the construction of two phage display libraries based on *E. coli*. To screen for specific antibodies, solid-phase panning was performed targeting the SFTSV-Gc-his recombinant protein. In each round of panning, phages binding to the solid-phase antigen were recovered by infecting *E. coli* TG1. The amplified phages were purified by PEG6000 / NaCl precipitation for the next round of screening. After three rounds of enrichment, phage ELISA was used to identify single clones, ultimately yielding positive single clones with good binding activity to the SFTSV-Gc-his protein.

[0049] Example 3 Construction and expression of human-camel chimeric antibody

[0050] Positive monoclonal antibodies obtained from phage screening were sequenced, and the corresponding VHH antibody fragments were synthesized based on the sequencing results and reconstructed into a human IgG antibody framework. Subsequently, the recombinant antibody gene was inserted into the pcDNA3.1 mammalian expression vector using molecular cloning technology. The plasmid was introduced into CHO cells using liposome transfection, and the fermentation supernatant was obtained through fed-batch culture. Finally, the supernatant was purified using a combination of affinity chromatography and ion exchange chromatography, successfully obtaining the target antibody.

[0051] Example 4: Binding activity of the human-camel chimeric antibody of the present invention to Gc protein on SFTSV

[0052] To detect the binding ability of the antibody to SFTSV-Gc, an ELISA method was used. After coating the Gc protein, serially diluted (0.1, 0.02, 0.004 μg / mL) human-camel chimeric antibody was added as the primary antibody, and detection was performed using HRP-labeled goat anti-human IgG-Fc. OD450 values ​​were read after TMB colorimetric development. The results are shown in Table 1, indicating that the selected human-camel chimeric antibody exhibits excellent binding activity to SFTSV-Gc protein.

[0053] Table 1. Binding activity of human-camel chimeric antibody to Gc protein

[0054]

[0055] Example 5: Neutralizing activity of single-concentration pseudoviruses of the human-camel chimeric antibody of the present invention.

[0056] Resuspend Vero cells in diluent (DMEM + 10% FBS) and adjust the cell density to 3 × 10⁶ cells / year. 4 Cells were seeded at a rate of 50 μL / well in a 96-well blank plate and incubated overnight. The antibody concentration was diluted to 1 μg / mL, and 30 μL / well was added to each 96-well plate, followed by 30 μL of HB29 pseudovirus (Pseudovirus-SFTS-HB29-M-Luciferase-GFP) diluted 1:25 with dilution buffer. The mixture was thoroughly mixed and incubated at 37°C for 1 h. After incubation, 50 μL of the antibody-pseudovirus mixture was added to each pre-seeded 96-well blank plate containing Vero cells. After incubation for another 22 h, 50 μL of luciferase assay reagent was added to each well, and the fluorescence intensity was measured using a microplate reader. The pseudovirus neutralization rate of the antibody was calculated, and the results are shown in Table 2. Human-camel chimeric antibodies with neutralizing activity higher than 40% were screened for further testing of pseudovirus neutralizing activity at gradient concentrations.

[0057] Table 2. Pseudovirus neutralization rate of human-camel chimeric antibodies.

[0058]

[0059] Example 6: Gradient concentration pseudovirus neutralizing activity of the human-camel chimeric antibody of the present invention

[0060] Resuspend Vero cells in diluent (DMEM + 10% FBS) and adjust the cell density to 3 × 10⁶ cells / year. 450 μL of antibody was seeded per well in a 96-well white plate and incubated overnight. The antibody was then serially diluted with the above diluent, and 30 μL per well was added to each well of the 96-well plate. Next, 30 μL of HB29 pseudovirus (Pseudovirus-SFTS-HB29-M-Luciferase-GFP) diluted 1:25 with the diluent was added, and the mixture was thoroughly mixed and incubated at 37°C for 1 h. After incubation, 50 μL of the antibody-pseudovirus mixture was added to a 96-well white plate pre-inoculated with Vero cells. After incubation for another 22 h, 50 μL of luciferase assay reagent was added to each well. The fluorescence value was measured using a microplate reader, and the pseudovirus neutralization rate of the antibody was calculated. The results are shown in Table 3. The experimental results indicate that the human-camel chimeric antibody of this invention possesses good neutralizing activity against HB29 pseudovirus.

[0061] Table 3. Pseudovirus neutralization rate of human-camel chimeric antibodies

[0062]

[0063] Example 7: Live virus neutralizing activity of the human-camel chimeric antibody of the present invention

[0064] Candidate antibodies with excellent neutralizing activity against SFTSV pseudovirus were selected for further testing of live virus neutralizing activity. 100 TCID50 units of SFTSV virus (subtype A) were thoroughly mixed with different concentrations of candidate antibodies and incubated at 37°C for 1 hour. After incubation, Vero cells were infected with this mixture and cultured for approximately 6 days. The cell state was observed under a microscope to determine the neutralizing activity of the antibody against the live virus. The criteria were as follows: complete cytopathic effect indicated ineffectiveness, 50% cytopathic effect indicated partial effectiveness, and no cytopathic effect indicated complete neutralization. P represents complete neutralization, M represents partial effectiveness, and N represents ineffectiveness. Specific test results are shown in Table 4. The experimental results show that the partial human-camel chimeric antibody of this invention has good live virus neutralizing activity against SFTSV virus (subtype A).

[0065] Table 4. Live virus neutralizing activity of the Gc human-camel chimeric antibody of the present invention

[0066]

[0067] Example 8: Human-centered engineering design

[0068] The variable region of the candidate antibody was humanized, and the modification process followed specific design principles: no protein modification sites such as glycosylation, deamidation, and isomerization were introduced, no integrin binding sites or cysteine ​​residues were introduced, and the reversion mutations of important amino acids in the framework region were required to maintain the original physicochemical and biochemical activities of the antibody. The specific modification methods are as follows.

[0069] Using the IgBLAST tool, the variable region of the human-camel chimeric antibody 26 was compared with the human Germline sequence. The antibody frame region (FR) was then replaced with the human Germline sequence with the highest sequence similarity. Based on this humanization modification, key amino acids affecting antibody affinity were reverse-mutated, i.e., mutated to the corresponding sites of the original camel-derived antibody frame region (FR). The humanization percentage was defined as the similarity ratio between the designed sequence frame region and the human Germline sequence frame region. The designed humanized sequences were compared and screened with human Germline sequences, and sequences with a humanization percentage of 85% or higher were finally selected. Detailed sequence information is shown in Table 5.

[0070] Table 5. Sequence numbers of some human-camel chimeric antibodies and humanized antibodies of the present invention.

[0071]

[0072] Example 9 Construction and expression of humanized antibodies

[0073] The designed antibody sequence was genetically synthesized and embedded within a human IgG antibody framework. Subsequently, molecular cloning techniques were used to insert the antibody fragment into a PVAC vector, thereby constructing a mammalian cell expression plasmid. The expression plasmid was introduced into a CHO host cell line using liposome transfection, and fermentation supernatant was obtained through fed-batch cell culture. This fermentation supernatant was then purified using affinity chromatography to obtain the target humanized antibody.

[0074] The expression levels of the fermentation supernatant before purification were compared and analyzed. Meanwhile, the purity of the antibody purified by one-step affinity chromatography was determined using size exclusion high-performance liquid chromatography (SEC-HPLC), and the specific results are shown in Table 6.

[0075] Table 6. SEC-HPLC Purity Detection Results

[0076]

[0077] Example 10: Neutralizing activity of humanized antibodies against pseudoviruses

[0078] The detection method is the same as in Example 6, and the results are shown in [example 6]. Figure 1 The isotype control 1 was Human IgG1, kappa IsotypeControl (supplier: Sino Biological; catalog number: HGEK). The results showed that the antibody of this invention had good neutralizing activity against the pseudovirus.

[0079] Example 11 Neutralizing activity of humanized antibody-live virus

[0080] Detection Method 1: The candidate antibody was serially diluted, starting with a final concentration of 50 µg / mL, and then serially diluted 3-fold. 50 µL of monoclonal antibody and 50 µL of a mixture containing 100 TCID50 viruses (SFTSV-JS2013-14, subtype E) were mixed together and incubated at 37°C for 1 h. The antibody-virus mixture was added to a 96-well cell plate and incubated at 37°C for 48 h. After washing once with PBS, 100 µL of 4% paraformaldehyde was added to each well for fixation at room temperature for 40 min. After washing once with PBS, 100 µL of 0.5% Triton was added to each well, and the plate was allowed to penetrate for 10 min at room temperature. After washing twice with PBS, 100 µL of 4% BSA was added to each well for blocking at room temperature for 1 h. The blocking solution was discarded, and the plate was washed three times with PBS. 40 µL of primary antibody (anti-NP rabbit antibody) was added to each well, and the plate was incubated at room temperature in the dark for 1 h. After washing three times with PBS, 40 µL of secondary antibody (anti-rabbit antibody Fc-Alex 488) was added to each well, and the plate was incubated at room temperature in the dark for 1 h. After washing three times with PBS, 40 µL of Hoechst was added to each well, and the plate was incubated at room temperature in the dark for 5 min. The cells were observed under a fluorescence microscope. ImageJ software was used to process the cells, calculate the number of infections, and compare the neutralization effect. The VC group served as a blank control without antibody. Inhibition rate = (1 - number of infected cells in sample group / number of infected cells in VC group) * 100%, and the results are shown in Table 7. The results indicate that the humanized antibody has good live virus neutralizing activity against SFTSV virus (SFTSV-JS2013-14, subtype E).

[0081] Table 7 Neutralizing activity of antibodies against live virus (E subtype)

[0082]

[0083] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. A single-domain antibody against Gc antigen of fever with thrombocytopenia syndrome virus, characterized in that, The single-domain antibody includes a framework region and three complementarity-determining regions (CDR1, CDR2, and CDR3), the amino acid sequences of which are shown in SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively.

2. The single-domain antibody against Gc antigen of fever with thrombocytopenia syndrome virus according to claim 1, characterized in that, The single-domain antibody includes a heavy chain variable region, the amino acid sequence of which is shown in any one of SEQ ID NO: 4-6.

3. A fusion protein, characterized in that, The single-domain antibody containing the anti-fever-thrombocytopenia syndrome virus Gc antigen as described in claim 1 or 2 and the Fc region of the fusion protein.

4. The fusion protein according to claim 3, characterized in that, The Fc region of the fusion protein is selected from IgG or IgA.

5. The fusion protein according to claim 3, characterized in that, The fusion protein's Fc region contains a CH2 domain and a CH3 domain.

6. The fusion protein according to claim 4, characterized in that, The Fc region of the fusion protein is selected from human IgG1, IgG2, IgG3 and / or IgG4, or the Fc region amino acid sequence that has one or more amino acid mutations with human IgG1, IgG2, IgG3, IgG4.

7. The fusion protein according to claim 6, characterized in that, The amino acid mutation is a substitution or deletion of an amino acid.

8. The fusion protein according to claim 3, characterized in that, The fusion protein comprises an amino acid sequence as shown in any one of SEQ ID NO: 7-9.

9. A nucleic acid molecule encoding a single-domain antibody as described in claim 1 or 2, or a fusion protein as described in any one of claims 3-8.

10. Use of a single-domain antibody according to claim 1 or 2, a fusion protein according to any one of claims 3-8, or a nucleic acid molecule according to claim 9 in the preparation of a medicament for the treatment or prevention of diseases caused by viral infection with fever accompanied by thrombocytopenia syndrome.

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