A human monoclonal antibody against Dabie Bandar virus and its application
The development of the monoclonal antibody DBV-G3 has solved the problem of the lack of effective drugs against Dabie Bandar virus in existing technologies, achieving the ability to efficiently bind to and neutralize the virus, and has significant preventive and therapeutic effects.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- NINGBO UNIV
- Filing Date
- 2026-02-13
- Publication Date
- 2026-05-26
Smart Images
Figure FT_1 
Figure FT_2 
Figure FT_3
Abstract
Description
Technical Field
[0001] This invention relates to the fields of immunology and molecular virology, and more specifically, to a monoclonal antibody against Dabie Bandar virus and its application. Background Technology
[0002] In 2010, Dabie Bandavirus (DBV) was first discovered to infect humans in the Dabie Mountains region of my country. Patients exhibit severe fever accompanied by thrombocytopenia, hence the disease was initially named Severe Fever with Thrombocytopenia Syndrome (SFTS), and the pathogen was initially named STSSV. DBV belongs to the Bunyaviridae family and is primarily transmitted through tick bites. It is a vector-borne virus that poses a serious threat to human health. Epidemics of this virus have been found in 27 provinces in my country, resulting in over 2,000 cases annually, with a case fatality rate as high as 30%. Besides China, the virus has also been found to be prevalent in countries such as South Korea and Japan. Furthermore, the longhorn tick, a vector for the virus, has been identified in the United States. Currently, there are no specific antiviral drugs or approved vaccines.
[0003] DBV is an enveloped, spherical virus with a genome consisting of three negative-strand RNA segments (L, M, and S segments). The M gene segment encodes two envelope proteins: Gn and Gc proteins. Both Gn and Gc proteins can mediate viral infection of cells and thus induce the production of neutralizing antibodies.
[0004] Currently, neutralizing antibodies are an effective treatment for viral diseases. Marketed drugs for treating and preventing viral infections include pallizumab (Synagis), nirsevimab, and clesrovimab for preventing respiratory syncytial virus (RSV) infection in children, and edalizumab (Trogarzo) and edalizumab for treating HIV infection, among others. Antibodies exert their therapeutic effects primarily through two mechanisms. First, neutralizing antibodies can block viral infection by binding to viral membrane proteins, thus preventing the virus from binding to cell receptors. Second, antibodies can clear free viruses or kill virus-infected cells through antibody-dependent cell-mediated cytotoxicity, antibody-mediated opsonization, and antigen-antibody complex activation of the complement system.
[0005] Given the increasing number of countries and regions where Dabie Bandar virus (DBV) is prevalent, it is urgent to screen for neutralizing monoclonal antibodies with higher affinity and preventive and therapeutic effects against DBV infection. Such antibodies can play an important role in protecting public life and health. Summary of the Invention
[0006] In this invention, unless otherwise stated, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, immunology, and virology experimental procedures used in this invention (if applicable) are all conventional procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.
[0007] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of four polypeptide chains: two identical "light" (L) chains and two identical "heavy" (H) chains. Antibody light chains can be classified into two types: κ and λ. Heavy chains can be classified into five types: μ, δ, γ, α, or ε, and antibodies composed of these heavy chains are correspondingly defined as IgM, IgD, IgG, IgA, and IgE. Within both light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. The constant regions of antibodies mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly degenerated regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of seven parts arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy / light chain pair form the antibody-binding sites. The allocation of amino acids to the regions or domains follows the definition in the Kabat Sequences of Proteins of Immunological Interest [National Institutes of Health, Bethesda, Md. (1987 and 1991)] or Chothia et al. (1989, Nature. 342:878-883). The term "antibody" is not limited to any particular method of producing antibodies. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.
[0008] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen bound by the full-length antibody and / or competes with the full-length antibody for specific binding to the antigen; it is also referred to as an "antigen-binding moiety." See also Fundamental Immunology, Ch. 7 [Paul, W., ed., 2nd ed., Raven Press, NY (1989)], which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA technology or by enzymatic or chemical cleavage of intact antibodies. In some cases, antigen-binding fragments include Fab, Fab', F(ab')2, Fd, Fv, dAb, and complementarity-determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabody (dAb), and polypeptides containing at least a portion of an antibody sufficient to confer specific antigen-binding ability to the polypeptide.
[0009] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), where the VL and VH domains pair to form a monovalent molecule by enabling them to generate linkers for a single polypeptide chain [see, for example, Bird et al., 1988, Science. 242:423-426. and Huston et al., 1988, Proc. Natl. Acad. Sci. USA. 85:5879-5883.]. Such scFv molecules may have a general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable prior art linkers consist of a repeating GGGGS amino acid sequence or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof can also be used (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 6444-6448.). Other connectors that can be used in this invention are described by Alfthan et al., 1995, Protein Eng. 8:725-731. Choi et al., 2001, Eur. J. Immunol. 31: 94-106.
[0010] In some cases, the antigen-binding fragment of an antibody is a biantibody, i.e., a bivalent antibody, in which the VH and VL domains are expressed on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thus forcing the domain to pair with a complementary domain on another chain and creating two antigen-binding sites [see, for example, Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6444 6448. and Poljak RJ et al., 1994, Structure. 2:1121-1123.]. Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibody DBV-G3 provided in this invention) can be obtained using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical fragmentation methods), and the antigen-binding fragments of the antibody can be specifically screened in the same manner as those used for intact antibodies.
[0011] In this invention, unless the context clearly indicates otherwise, when referring to the term "antibody," it includes not only the complete antibody but also the antigen-binding fragment of the antibody.
[0012] As used herein, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody derived from a group of highly homologous antibody molecules, i.e., a group of identical antibody molecules except for the possibility of spontaneous natural mutations. Monoclonal antibodies exhibit high specificity for a single epitope on an antigen. Polyclonal antibodies, as opposed to monoclonal antibodies, typically contain at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using the hybridoma technique first reported by Kohler et al. (1975, Nature. 256:495), but can also be obtained using recombinant DNA techniques [see Journal of virological methods, 2009. 158(1-2):171-179].
[0013] As used in this invention, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence (e.g., the ability to infect cells) of a target virus.
[0014] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.
[0015] As used in this invention, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells, animal cells such as CHO cells, or human cells such as HEK293 cells.
[0016] As used in this invention, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and the antigen it targets.
[0017] In this invention, amino acids are typically represented by single-letter or three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.
[0018] As used in this invention, the term "neutralizing activity" refers to the functional activity of an antibody or antibody fragment to bind to antigenic proteins on a virus, thereby preventing the virus from infecting cells and / or maturing and / or releasing viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.
[0019] As used in this invention, the term "severe fever with thrombocytopenia syndrome virus" refers to Dabie Bandavirus (DBV), officially classified and named by the International Committee on Taxonomy of Viruses (ICTV). The two terms have the same meaning and are used interchangeably. Similarly, as used in this invention, the term "severe fever with thrombocytopenia syndrome" refers to fever, rash, bleeding, thrombocytopenia, etc., caused by DBV infection; these two terms have the same meaning and are used interchangeably.
[0020] To address the problems of existing technologies, the present invention aims to provide a monoclonal antibody with high binding affinity to Dabie Bandar virus and its applications.
[0021] To achieve this objective, the present invention, after extensive experimental research, discovered an antibody that specifically recognizes and targets the Gn and Gc proteins of Dabie Bandar virus, particularly the extracellular regions of these two proteins, and can block the binding of the Gn and / or Gc proteins to cell surface receptors, demonstrating a highly efficient ability to neutralize the virus. Therefore, the antibody of the present invention is particularly suitable for the diagnosis, prevention, and treatment of Dabie Bandar virus and diseases associated with Dabie Bandar virus infection (e.g., severe fever with thrombocytopenia syndrome).
[0022] Specifically, the present invention provides the following technical solutions: A monoclonal antibody or its antigen-binding fragment, wherein the CDR1 of its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, the CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and the CDR3 has the amino acid sequence shown in SEQ ID NO: 3; and / or, Its light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO:4, CDR2 has the specific amino acid sequence AAS, and CDR3 has the amino acid sequence shown in SEQ ID NO:5.
[0023] Preferably, the monoclonal antibody or its antigen-binding fragment of the present invention has a heavy chain variable region having an amino acid sequence as shown in SEQ ID NO: 6 and a gene sequence as shown in SEQ ID NO: 12. And / or, its light chain variable region has an amino acid sequence as shown in SEQ ID NO: 7 and a gene sequence as shown in SEQ ID NO: 13.
[0024] In some preferred embodiments, the monoclonal antibody also has a signal peptide sequence at the N-terminus of the heavy chain variable region. In some preferred embodiments, the signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 10.
[0025] In some preferred embodiments, the monoclonal antibody also has a signal peptide sequence at the N-terminus of the light chain variable region. In some preferred embodiments, the signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 11.
[0026] Preferably, the signal peptide sequence of the present invention has the nucleotide sequence shown in SEQ ID NO: 16 and SEQ ID NO: 17.
[0027] The antigen-binding fragments of the present invention are selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragments, single-chain antibodies (e.g., scFv), human antibodies, chimeric antibodies, or bispecific or multispecific antibodies.
[0028] In some preferred embodiments, the monoclonal antibody further includes a heavy chain constant region. In some preferred embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 8. The gene sequence of the heavy chain constant region is shown in SEQ ID NO: 14.
[0029] In some preferred embodiments, the monoclonal antibody further includes a light chain constant region. In some preferred embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 9. The gene sequence of the light chain constant region is shown in SEQ ID NO: 15.
[0030] In some preferred embodiments, the light chain of the monoclonal antibody is of the kappa type.
[0031] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment can specifically bind to the envelope proteins (Gn and Gc proteins) of Dabie Bandar virus. In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment can target the extracellular regions of the Gn and Gc proteins of Dabie Bandar virus. In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment can inhibit receptor binding and / or membrane fusion processes mediated by the extracellular regions of the Gn and Gc proteins, thereby inhibiting viral infection of cells.
[0032] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment has neutralizing capability (e.g., capable of neutralizing Dabiebanda virus). In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of inhibiting Dabiebanda virus infection or entry into host cells. Thus, the monoclonal antibody or its antigen-binding fragment can neutralize Dabiebanda virus, thereby preventing and treating Dabiebanda virus infection.
[0033] The present invention also provides an isolated nucleic acid molecule encoding the monoclonal antibody or its antigen-binding fragment of the present invention. Such nucleic acid molecules are not limited by the method of their production and can be obtained using genetic engineering recombination techniques or chemical synthesis methods.
[0034] Specifically, the present invention also provides a nucleic acid molecule that encodes the above-mentioned monoclonal antibody or its antigen-binding fragment.
[0035] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 12 and / or SEQ ID NO: 13.
[0036] The nucleotide sequence shown in SEQ ID NO: 12 encodes the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention; the nucleotide sequence shown in SEQ ID NO: 13 encodes the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention.
[0037] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide sequence located at the 5' end of the heavy chain variable region of the nucleotide sequence capable of encoding the monoclonal antibody or its antigen-binding fragment of the present invention. In some preferred embodiments, the signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 10. In some preferred embodiments, the nucleotide sequence encoding the signal peptide sequence has the nucleotide sequence shown in SEQ ID NO: 16.
[0038] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a signal peptide sequence located at the 5' end of the nucleotide sequence encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention. In some preferred embodiments, the signal peptide sequence has the amino acid sequence shown in SEQ ID NO: 11. In some preferred embodiments, the nucleotide sequence encoding the signal peptide sequence has the nucleotide sequence shown in SEQ ID NO: 17.
[0039] In some preferred embodiments, the nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence and a heavy chain variable region capable of encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof; and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence and a light chain variable region capable of encoding the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
[0040] In some preferred embodiments, the nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO: 16 and a nucleotide sequence as shown in SEQ ID NO: 12; and a second polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO: 17 and a nucleotide sequence as shown in SEQ ID NO: 13.
[0041] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a heavy chain constant region capable of encoding the monoclonal antibody of the present invention or its antigen-binding fragment. In some preferred embodiments, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO:8. In some preferred embodiments, the nucleotide sequence encoding the heavy chain constant region capable of encoding the monoclonal antibody of the present invention or its antigen-binding fragment has a nucleotide sequence as shown in SEQ ID NO:14.
[0042] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding a light chain constant region capable of encoding the monoclonal antibody of the present invention or its antigen-binding fragment. In some preferred embodiments, the light chain constant region has an amino acid sequence as shown in SEQ ID NO:9. In some preferred embodiments, the nucleotide sequence encoding the light chain constant region capable of encoding the monoclonal antibody of the present invention or its antigen-binding fragment has a nucleotide sequence as shown in SEQ ID NO:15.
[0043] In some preferred embodiments, the nucleic acid molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence encoding a heavy chain variable region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof, and a nucleotide sequence encoding a heavy chain constant region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof; and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence encoding a light chain variable region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof, and a nucleotide sequence encoding a light chain constant region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof.
[0044] In some preferred embodiments, the nucleic acid molecule comprises a first polynucleotide comprising the nucleotide sequences shown in SEQ ID NO: 16, SEQ ID NO: 12 and SEQ ID NO: 14; and a second polynucleotide comprising the nucleotide sequences shown in SEQ ID NO: 17, SEQ ID NO: 13 and SEQ ID NO: 15.
[0045] The present invention also provides a vector comprising the aforementioned nucleic acid molecules. The vector of the present invention can be a cloning vector or an expression vector. In some preferred embodiments, the vector of the present invention is, for example, a plasmid, a granule, a bacteriophage, etc.
[0046] The present invention also provides a host cell comprising the aforementioned nucleic acid molecules or vectors. Such host cells include, but are not limited to, prokaryotic cells such as *Escherichia coli* cells, and eukaryotic cells such as yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The cells of the present invention can also be cell lines, such as 293T cells.
[0047] In another aspect, the present invention also provides a method for preparing the monoclonal antibody of the present invention or an antigen-binding fragment thereof, comprising culturing the host cell of the present invention under suitable conditions, and recovering the monoclonal antibody of the present invention or an antigen-binding fragment thereof from the cell culture.
[0048] The present invention also provides a composition comprising a monoclonal antibody or an antigen-binding fragment thereof as described above, a nucleic acid molecule, a vector or a host cell.
[0049] The composition may be a diagnostic agent or a therapeutic agent.
[0050] The present invention further provides a kit comprising the above-described monoclonal antibody or its antigen-binding fragment.
[0051] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment of the present invention further includes a detectable label. In some preferred embodiments, the kit further includes a second antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment of the present invention or an anti-idiotype antibody. Preferably, the second antibody further includes a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioisotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (e.g., horseradish peroxidase).
[0052] The present invention also provides a pharmaceutical composition comprising the above-described monoclonal antibody or its antigen-binding fragment, or further comprising a pharmaceutically acceptable carrier and / or excipient.
[0053] Preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as ribavirin.
[0054] In some preferred embodiments, the monoclonal antibody comprises: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO:1-3, and / or VL CDR1, 3 with amino acid sequences as shown in SEQ ID NO:4, 5, and VL CDR2 with a specific amino acid sequence of AAS; preferably, the monoclonal antibody comprises: VH as shown in SEQ ID NO:6 and / or VL as shown in SEQ ID NO:7.
[0055] The present invention also provides an application of the above-mentioned monoclonal antibody or its antigen-binding fragment in any of the following aspects: (1) Use in the preparation of products for the detection of the presence or level of Dabie Bandar virus or its Gn protein, Gc protein or extracellular region of Gn protein, Gc protein in a sample; (2) Application in the preparation of products for neutralizing the virulence of Dabie Bandar virus in samples; (3) Use in the preparation of a medicament, wherein the medicament is used to neutralize the virulence of Dabiebanda virus in a sample, or to prevent or treat Dabiebanda virus infection or disease associated with such infection in a subject.
[0056] In another aspect, the present invention provides a method for detecting the presence or level of Dabie Bandar virus or Gn protein, Gc protein, or the extracellular region of Gn protein or Gc protein in a sample, comprising using a monoclonal antibody or antigen-binding fragment of the present invention. In some preferred embodiments, the monoclonal antibody or antigen-binding fragment of the present invention further includes a detectable label. In another preferred embodiment, the method further includes using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment of the present invention. The method can be used for diagnostic purposes (e.g., the sample is from a patient) or for non-diagnostic purposes (e.g., the sample is a cell sample, not from a patient).
[0057] In another aspect, the present invention provides a method for diagnosing whether a subject is infected with Dabie Bandar virus, comprising: detecting the presence of Dabie Bandar virus or Gn protein, Gc protein, or the extracellular region of Gn protein or Gc protein in a sample from said subject using a monoclonal antibody or antigen-binding fragment thereof of the present invention. In some preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further includes a detectable marker. In another preferred embodiment, the method further includes using a second antibody carrying a detectable marker to detect the monoclonal antibody or antigen-binding fragment thereof of the present invention or an anti-idiotype antibody.
[0058] In another aspect, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof in the preparation of products, such as kits, for detecting the presence or level of Dabiebanda virus or the extracellular region of Gn protein, Gc protein or Gn protein, Gc protein in a sample, or for diagnosing whether a subject is infected with Dabiebanda virus.
[0059] In some preferred embodiments, the sample includes, but is not limited to, excrement, oral or nasal secretions, bronchoalveolar lavage fluid, blood, etc., from a subject (e.g., a mammal, preferably a human).
[0060] In some preferred embodiments, the monoclonal antibody is an antibody comprising: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3, and / or VLCDR1, 3 with amino acid sequences as shown in SEQ ID NO: 4, 5, and VL CDR2 with a specific amino acid sequence of AAS; preferably, it comprises: VH as shown in SEQ ID NO: 6 and / or VL as shown in SEQ ID NO: 7.
[0061] General methods for detecting the presence or level of a target virus or antigen (e.g., Dabie Bandar virus or Gn protein, Gc protein or the extracellular region of Gn protein, Gc protein) in a sample using monoclonal antibodies or their antigen-binding fragments are well known to those skilled in the art. In some preferred embodiments, the detection method may use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assays, and similar methods.
[0062] In another aspect, the present invention provides a method for neutralizing the virulence of Dabie Bandar virus in a sample, comprising contacting a sample containing Dabie Bandar virus with a monoclonal antibody or an antigen-binding fragment thereof of the present invention. Such methods can be used for therapeutic purposes or non-therapeutic purposes (e.g., the sample is a cell sample, rather than a patient or a sample from a patient).
[0063] In another aspect, the present invention provides the use of the monoclonal antibody of the present invention or its antigen-binding fragment for the preparation of a medicament for neutralizing the virulence of Dabie Bandar virus in a sample. In another aspect, the present invention provides the monoclonal antibody or its antigen-binding fragment as described above for neutralizing the virulence of Dabie Bandar virus in a sample.
[0064] In another aspect, the invention provides the use of the monoclonal antibody or antigen-binding fragment thereof in the preparation of a pharmaceutical composition for the prevention or treatment of Dabiebanda virus infection or related diseases (e.g., severe fever with thrombocytopenia syndrome) in a subject. In another aspect, the invention provides the monoclonal antibody or antigen-binding fragment thereof as described above for the prevention or treatment of Dabiebanda virus infection or related diseases (e.g., severe fever with thrombocytopenia syndrome) in a subject.
[0065] In another aspect, the present invention provides a method for preventing or treating a subject with Dabie Bandar virus infection or diseases associated with such virus infection (e.g., severe fever with thrombocytopenia syndrome), comprising administering to the subject in need a preventive or therapeutically effective amount of the monoclonal antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention.
[0066] In some preferred embodiments, the subject is a mammal, such as a human.
[0067] The monoclonal antibody or its antigen-binding fragment, or the pharmaceutical composition of the present invention, can be administered to a subject via any suitable route of administration. Such routes of administration include, but are not limited to, intramuscular injection, oral administration, oral administration, sublingual administration, topical administration, parenteral administration, rectal administration, or nasal administration.
[0068] In some preferred embodiments, the monoclonal antibody is an antibody comprising: VH CDR1-3 with amino acid sequences as shown in SEQ ID NO: 1-3, and / or VLCDR1, 3 with amino acid sequences as shown in SEQ ID NO: 4, 5, and VL CDR2 with a specific amino acid sequence of AAS; preferably, it comprises: VH as shown in SEQ ID NO: 6 and / or VL as shown in SEQ ID NO: 7.
[0069] The drugs or drug compositions provided by this invention can be used alone or in combination, or in combination with other pharmaceutically active agents (e.g., antiviral drugs such as ribavirin and interferon).
[0070] The beneficial effects of this invention are at least as follows: the monoclonal antibody of this invention (e.g., DBV-G3 monoclonal antibody) can bind to the extracellular regions of the Gn and Gc proteins of Dabie Bandar virus with high affinity, and exhibits strong neutralizing activity against Dabie Bandar virus. For example, the neutralizing titer (half-neutralizing concentration, NT) of the DBV-G3 monoclonal antibody against Dabie Bandar virus of this invention is shown in the data. 50 The monoclonal antibodies of this invention (e.g., DBV-G3 monoclonal antibody) are excellent. Therefore, the monoclonal antibodies of this invention have ideal clinical application value for the prevention and treatment of Dabie Bandar virus infection. Attached Figure Description
[0071] Figure 1 This is the SDS-PAGE electrophoresis result of the monoclonal antibody DBV-G3 expressed in Example 4 of this invention. In the gel image, "M" indicates a protein marker; "DTT" indicates that DTT (reducing SDS-PAGE) was added to the loading buffer.
[0072] Figure 2 This is a graph showing the binding curves of different concentrations of DBV-G3 monoclonal antibody to Gn and Gc proteins in Example 5 of the present invention. The horizontal axis represents antibody concentration (μg / ml), and the vertical axis represents OD450 value.
[0073] Figure 3 The graph shows the in vitro neutralizing activity of different concentrations of DBV-G3 monoclonal antibody against Dabie Bandar virus in Example 6 of this invention. The horizontal axis represents antibody concentration (μg / ml), and the vertical axis represents the neutralization percentage (%).
[0074] Figure 4 This invention, Example 7, describes an experiment to investigate the serum viral load of adult mice protected against lethal Dabie Bandar virus challenge with different doses of DBV-G3 monoclonal antibody. The horizontal axis represents treatment factors, and the vertical axis represents the serum viral load of mice 3 days after viral infection (3 dpi) [Log 10 (PFU / ml)]. Figure 4 a) and viral load in mouse brain tissue [Log 10 (PFU / g)] Figure 4 b). * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, **** indicates p < 0.0001.
[0075] Figure 5 This is Example 8 of the present invention, a survival experiment demonstrating the protection of adult mice against lethal Dabie Bandar virus challenge using the DBV-G3 monoclonal antibody. The horizontal axis represents the number of days post-infection, and the vertical axis represents the change in mouse body weight. Figure 5 a) Percentage survival rate ( Figure 5 (b) A red * indicates a significant difference between the 1 mg / kg DBV-G3 group and the control group, and a blue * indicates a significant difference between the 20 mg / kg DBV-G3 group and the control group. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001, and **** indicates p<0.0001. Detailed Implementation
[0076] The preferred embodiments of the present invention will now be described in detail with reference to specific examples. It should be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make various modifications and substitutions to the present invention without departing from its spirit and essence.
[0077] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in this invention are generally performed in accordance with the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989, and F.M. Usubel et al., A Concise Guide to Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the manufacturer's recommended conditions. Where specific conditions are not specified in the examples, they are performed under standard conditions or the manufacturer's recommendations. Reagents or instruments whose manufacturers are not specified are all commercially available products.
[0078] To obtain protective neutralizing antibodies, this invention first collects peripheral venous blood from recovered Dabie Bandar virus (DBV) patients, isolates B cells, extracts mRNA from the B cells, transcribes it into cDNA, amplifies the scFV gene sequence for monoclonal antibody production using PCR technology, and clones it into a phage vector, ultimately obtaining a phage library displaying the scFV antibody. The extracellular region of the Dabie Bandar virus Gc protein (DBV-Gc) expressed in *E. coli* is also described. 628-1024 As an antigen, DBV-Gc was obtained through multiple rounds of washing to specifically bind to it. 628-1024 Phages were selected, and then the selected single phages were sequenced to obtain the scFV gene sequence. Further, the sequence encoding the antibody variable region and the constant region gene were ligated into an expression vector, and expressed and purified in mammalian cells to obtain monoclonal antibody DBV-G3. A series of functional tests on monoclonal antibody DBV-G3 showed that it can specifically bind to the extracellular region of Gc protein (DBV-Gc). 628-1024 ) and the extracellular domain of Gn protein (DBV-Gn) 60-446 It inhibits the infection of Vero cells by Dabiebanda virus, protects mice against lethal Dabiebanda virus attack (specifically, it can significantly reduce viral load in mouse tissues and significantly reduce mouse mortality), and has neutralizing activity and preventive effect against Dabiebanda virus.
[0079] Information on some of the sequences involved in this invention is shown in Table 1 below.
[0080]
[0081] Example 1: Expression and purification of the extracellular regions of Gn and Gc proteins of Dabie Bandar virus 1. DBV-Gn, with its C-terminus tagged with a His tag (6 histidines) after codon optimization in E. coli, was used. 60-446 (Residues 60-446, virus strain HB154 / China / 2011) and DBV-Gc 628-1024 The coding sequence (residues 628-1024, viral strain HB154 / China / 2011) was cloned into the prokaryotic expression vector pET21a.
[0082] 2. The recombinant plasmid pET21a-DBV-Gn 60-446 pET21a-DBV-Gc 628-1024 The proteins were transformed into Escherichia coli BL21 strain and expressed using IPTG.
[0083] 3. Separation and purification of DBV-Gn using Ni-NTA affinity chromatography column. 60-446 DBV-Gc 628-1024 The purity of the two proteins was determined using SDS-PAGE assay.
[0084] Example 2: Specific recognition of DBV-Gc 628-1024 bacteriophage washing of proteins 1. Collect 50 ml of peripheral venous blood (EDTA anticoagulated) from recovered Dabie Bandar virus patients, and separate peripheral blood mononuclear cells (PBMCs) using human lymphocyte separation medium.
[0085] 2. The isolated PBMCs were mixed with a mixture of biotinylated antibodies from Miltenyi (containing biotinylated anti-human CD2 monoclonal antibody, anti-human CD3 monoclonal antibody, anti-human CD14 monoclonal antibody, anti-human CD43 monoclonal antibody, anti-human CD56 monoclonal antibody, and anti-human CD235a monoclonal antibody) in a flow cytometer and incubated at 4°C for 30 min to bind. Add pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA), add Anti-biotin microbeads, and incubate at 4°C for 30 min. Add pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA), centrifuge at 2000 rpm for 10 min, and discard the supernatant. Resuspend the cell pellet in pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA), transfer to a flow cytometry tube, insert a magnet and wait 3 min, collect unadsorbed cells, wash the adsorbed magnetic beads with pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA), insert a magnet and wait 3 min, collect unadsorbed cells. Transfer all unadsorbed cell solution to a 15 ml centrifuge tube; centrifuge at 2000 rpm for 10 min, discard the supernatant, and resuspend the cell pellet (rich in B cells) in pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA).
[0086] 3. Cells were lysed using Trizol, total RNA was purified using an RNA extraction kit, and RNA was reverse transcribed into cDNA using a reverse transcription kit.
[0087] 4. The variable regions (VH, VL, and Vk) of the antibody gene were amplified using primers from a fully human antibody library. The light and heavy chains were constructed into scFV through overlap extension PCR. The scFV was then molecularly cloned and recombined into the pCOMB3 series phage vector. The antibody was fused with a common tag and an amber stop codon was added after the tag. The recombinant vector was electroporated into the TG1 strain and fused with the phage pIII protein for expression. After infection with helper phages, it was displayed on the surface of the phage (this is the scFV phage).
[0088] 5. Washing and enrichment can be combined with DBV-GC. 628-1024 The scFV phage protein synthesis process is simplified as follows: (1) Washing: Use coating buffer to wash DBV-Gc 628-1024 Coat each well of a high-affinity ELISA plate (10 μg / ml, 100 μl / well) and incubate overnight at 4°C. Block with 5% skim milk / PBS. Add the above scFV phage library to the wells and incubate at room temperature for 1 h. Wash off the bound phages with elution buffer.
[0089] (2) Amplification of phages: Amplification of eluted phages was carried out using activated TG1 bacterial solution and helper phages.
[0090] (3) Repeat the above washing and amplification process 3 times to finally obtain the product with DBV-Gc. 628-1024 Bacteriophages with high affinity.
[0091] (4) The above phages were diluted to different gradients and then infected with TG1 bacteria. The phages were plated and incubated overnight at 37°C to obtain single colonies.
[0092] (5) Inoculate single colonies into 96-well deep-well plates, incubate at 37°C / 700 rpm for 4 h, add helper phage M13KO7, and incubate at 37°C / 180 rpm. Incubate for 48 hours.
[0093] (6) Single-phase ELISA: ELISA plate coated with DBV-Gc 628-1024 (1 μg / ml, 100 μl / well), coated overnight at 4°C. Blocked with 5% skim milk powder / PBS. Add the liquid from the above 96-well deep plate to the microplate and incubate at 37°C for 1 h. Wash the plate 5 times with PBST. Add HRP-labeled anti-M13 monoclonal antibody (1:5000) and incubate at 37°C for 1 h. Wash the plate 5 times with PBST. Add 40 μl TMB (20 μl of solution A + 20 μl of solution B prepared in our laboratory), react in the dark for 5-10 min, and stop the reaction by adding 20 μl of stop solution prepared in our laboratory. Read the OD450 value on the microplate reader. A phage with an OD450 value more than twice that of the control well is considered positive.
[0094] 6. The above-mentioned positive phages were sent to the company for sequencing to obtain the gene sequence encoding the monoclonal antibody scFV.
[0095] Example 3 Construction of recombinant expression vector for DBV monoclonal antibody Ten positive phages were obtained, and the gene sequences encoding the monoclonal antibody scFV had never been reported before. Through sequence analysis, we selected the variable regions (V regions) of the light and heavy chains of a monoclonal antibody named DBV-G3 for further study. The amino acid sequence of the heavy chain variable region of DBV-G3 is shown in SEQ ID NO: 6 (encoding gene shown in SEQ ID NO: 12). CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2, and CDR3 has the amino acid sequence shown in SEQ ID NO: 3. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 7 (encoding gene shown in SEQ ID NO: 13). CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, CDR2 has the specific amino acid sequence AAS, and CDR3 has the amino acid sequence shown in SEQ ID NO: 5. The V region genes of the heavy and light chains of DBV-G3 are shown in Tables 2-3.
[0096] Table 2 Genes in the V region of the heavy chain of DBV-G3 monoclonal antibody VH allele JH allele DBV-G3 IGHV1-18 IGHJ1 Table 3 Genes in the V region of the DBV-G3 monoclonal antibody light chain VL allele JL allele DBV-G3 IGKV1-16 IGKJ4 The nucleotide sequences encoding the variable regions of the DBV-G3 heavy and light chains were analyzed and then coupled into the expression vector pCAGGS (constructed in our laboratory) containing the corresponding nucleotide sequences encoding the constant regions of the heavy and light chains, respectively, to obtain recombinant expression vectors encoding the DBV-G3 monoclonal antibody heavy and light chains, respectively. The construction methods for the constructs expressing the heavy and light chains are as follows: Heavy chain coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence gene - VH gene - CH gene - Xho I restriction site; Light chain (k-type) coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence gene - VL gene - CL(k) gene - Xho I restriction site; The amino acid sequence of the signal peptide is shown in SEQ ID NO: 10 (encoding gene is shown in SEQ ID NO: 16), the amino acid sequence of CH is shown in SEQ ID NO: 8 (encoding gene is shown in SEQ ID NO: 14), and the amino acid sequence of CL is shown in SEQ ID NO: 9 (encoding gene is shown in SEQ ID NO: 15).
[0097] Example 4: Expression of DBV-G3 monoclonal antibody Heavy and light chain plasmids (recombinant expression vectors pCAGGS-DBV-G3H and pCAGGS-DBV-G3L, respectively encoding the heavy and light chains of the DBV-G3 monoclonal antibody obtained in Example 3) were co-transfected into 293T cells. The weight ratio of heavy chain plasmid pCAGGS-DBV-G3H to light chain plasmid pCAGGS-DBV-G3L was 1.07:0.93. Each 10 cm diameter plate of 293T cells was transfected with 2 μg of plasmid (heavy chain plasmid + light chain plasmid) and 4 μg of PEI. Cell supernatants were collected at 48 h and 96 h post-transfection and filtered through a 0.22 μm syringe filter. Purification was then performed using a Pierce protein A / G agarose column (Thermo Fisher), and the monoclonal antibody was concentrated using an ultrafiltration tube with a 30 kDa molecular weight cutoff. The purity of the expressed monoclonal antibody was subsequently assessed by SDS-PAGE (in the reducing state).
[0098] Result: As Figure 1 As shown, purified DBV-G3 monoclonal antibody was obtained, and light chain (around 25 kDa) and heavy chain (around 55 kDa) bands appeared in the reduced state (DTT added to the loading buffer).
[0099] Example 5: DBV-G3 monoclonal antibody and DBV-Gn 60-446 and DBV-Gc 628-1024 Assessment of protein binding ability In this embodiment, ELISA was used to evaluate the DBV-G3 monoclonal antibody and the DBV Gn protein (DBV-Gn). 60-446 ) and Gc protein (DBV-Gc) 628-1024 The binding ability of ) is determined by the following steps: 1. Coating antigen: DBV-Gn 60-446 or DBV-Gc 628-1024 Dissolve in 0.05 mol / L carbonate buffer (pH 9.6) to a final concentration of 20 μg / ml, add to a high affinity microplate (100 μl / well), and coat at 37°C for 1.5 h; 2. Wash the plate twice with phosphate-buffered saline (PBS); 3. Blocking plate: Add 100 μl of blocking buffer (5% skim milk powder / PBS) to each well and block at 37℃ for 1.5 h; 4. Wash the plate twice with PBS; 5. Add 50 μl of DBV-G3 monoclonal antibody diluted 3-fold (starting from 100 μg / ml, for a total of 12 dilutions) to each well and incubate at room temperature (25℃) for 1.5 h; 6. Wash the plate 5 times with PBST (PBS containing 0.5% Tween-20); 7. Add 50 μl of HRP-labeled mouse anti-human IgG monoclonal antibody (1:5000 dilution) to each well and incubate at room temperature for 1.5 h; 8. Wash the plate 5 times with PBST; 9. Add 40 μl of TMB solution (20 μl of solution A + 20 μl of solution B, freshly prepared) to each well and develop color at 37℃ for 15 min; 10. The OD450 value of each well was measured using an ELISA reader, and the data were analyzed and plotted using Prism 8 software.
[0100] Experimental results: such as Figure 2 As shown, the DBV-G3 monoclonal antibody can bind to the extracellular regions of Gn and Gc proteins (DBV-Gn, respectively) in a concentration-dependent manner. 60-446 and DBV-Gc 628-1024 DBV-G3 monoclonal antibody combined with DBV-Gn 60-446 Its capabilities are stronger than those combined with DBV-Gc 628-1024 The ability.
[0101] Example 6: Experimental neutralization of Dabie Bandar virus with DBV-G3 monoclonal antibody 1. Seed Vero cells in 24-well plates (1×10⁻⁶ cells / well). 5 (1 cell / well), cultured for 24 h.
[0102] 2. In a 96-well plate, serially dilute the DBV-G3 monoclonal antibody to be tested 3-fold (starting from 100 μg / ml), add the DBV virus to be tested [50 viral plaque forming units (PFU) / well] to each well, and incubate at 37°C for 1 h.
[0103] 3. Discard the Vero cell supernatant, add the virus / monoclonal antibody mixture to the cells, and infect at 37°C for 1.5 h.
[0104] 4. Discard the virus solution, add 1% methylcellulose culture medium to each well to cover the cells, and culture for 5 days.
[0105] 5. Add 4% paraformaldehyde to each well and fix at room temperature for 1 hour, then wash the plate.
[0106] 6. Add 0.5% crystal violet stain to each well and stain at room temperature for 10 min, then wash the plate.
[0107] 7. Count the number of viral plaques in each well and calculate the neutralizing titer (half-neutralizing concentration, NT) of the diluted monoclonal antibody against each virus. 50 value).
[0108] NT 50The analysis was performed using GraphPad Prism 8 software.
[0109] Experimental results: such as Figure 3 As shown, the DBV-G3 monoclonal antibody can inhibit DBV-infected cells with extremely high neutralizing activity, and its antiviral ability is directly proportional to the concentration of the monoclonal antibody, which neutralizes the NT of DBV. 50 The value was 1.6 ng / ml.
[0110] Example 7: Serum viral load experiment of DBV-G3 monoclonal antibody protecting adult mice against lethal Dabie Bandar virus challenge. 1. 100 PFU of DBV was injected into the peritoneal cavity of 6-week-old AB6 mice (with type I interferon receptor knocked out); 2. Two hours later, DBV-G3 monoclonal antibody at a dose of 1 mg / kg mouse body weight or 20 mg / kg mouse body weight was injected into the peritoneal cavity of DBV-infected mice, while control mice were injected with an equal volume of PBS into the peritoneal cavity.
[0111] 3. Three days later, the mice were sacrificed and their serum and brain tissue were collected. The viral load in the mouse serum and brain tissue was detected by viral plaque formation assay (PFA) based on Vero cells. The results are expressed as Log 10 (PFU / ml) and Log 10 (PFU / g), respectively.
[0112] 4. Use Prism 8 software to analyze, statistically analyze, and plot the data.
[0113] Experimental results: such as Figure 4 As shown, after DBV infection, the viral titers in the serum of mice administered DBV-G3 monoclonal antibody at 20 mg / kg body weight and 1 mg / kg body weight were significantly lower than those in the serum of the control group (administered PBS) (25 PFU / ml versus 1380384 PFU / ml, p<0.001, a 54953-fold decrease in viral titer; 13182 PFU / ml versus 1380384 PFU / ml, p<0.05, a 103-fold decrease in viral titer). Moreover, the viral titer in the serum of mice in the 20 mg / kg DBV-G3 group was also significantly lower than that in the serum of mice in the 1 mg / kg DBV-G3 group (25 PFU / ml versus 13182 PFU / ml, p<0.05). It is worth mentioning that the viral titer in the serum of mice in the 20 mg / kg DBV-G3 group was close to the detection limit, indicating that this dose of DBV-G3 can almost completely block DBV infection in mice.
[0114] Similarly, the viral load in the brain tissue of mice administered DBV-G3 monoclonal antibody at 20 mg / kg body weight and 1 mg / kg body weight was significantly lower than that in the brain tissue of the control group (administered with PBS) (7 PFU / g versus 4,677,354 PFU / g, p < 0.0001, a 64,564-fold decrease in viral titer; 12,882 PFU / g versus 467,735 PFU / g, p < 0.01, a 35-fold decrease in viral titer). Furthermore, the viral load in the brain tissue of mice in the 20 mg / kg DBV-G3 group was also significantly lower than that in the 1 mg / kg DBV-G3 group (7 PFU / g versus 12,882 PFU / g, p < 0.0001). The viral load in the brain tissue of mice in the 20 mg / kg DBV-G3 group was also close to the detection limit.
[0115] Example 8: Survival experiment of adult mice protected by DBV-G3 monoclonal antibody against lethal Dabiebanda virus challenge. 1. Inject 100 PFU DBV into the peritoneal cavity of 6-week-old AB6 mice.
[0116] 2. Two hours later, 1 mg / kg mouse body weight and 20 mg / kg mouse body weight of DBV-G3 were injected into the peritoneal cavity of the mice.
[0117] 3. Record the weight and mortality of the suckling mice daily (for a total of 2 weeks).
[0118] 4. Use Prism 8 to analyze, statistically analyze, and plot the data.
[0119] Experimental results: like Figure 5 As shown in a and 5b, 100% of the AB6 mice in the control group (n=6) who did not receive the monoclonal antibody died within 3 days of DBV infection. All AB6 mice injected with DBV-G3 monoclonal antibody at 1 mg / kg body weight also died within 5 days of DBV infection. However, all AB6 mice injected with DBV-G3 monoclonal antibody at 20 mg / kg body weight survived 21 days after DBV infection. The survival rate of the 20 mg / kg DBV-G3 group was significantly different from that of the control group (100% survival versus 0% survival, p<0.001), indicating that this dose of DBV-G3 could provide 100% protection against lethal dose DBV challenge in mice.
[0120] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.
Claims
1. A monoclonal antibody or its antigen-binding fragment, characterized in that, Its heavy chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 1, CDR2 has the amino acid sequence shown in SEQ ID NO: 2 and CDR3 has the amino acid sequence shown in SEQ ID NO: 3; and / or, its light chain variable region CDR1 has the amino acid sequence shown in SEQ ID NO: 4, the specific amino acid sequence of CDR2 is AAS and CDR3 has the amino acid sequence shown in SEQ ID NO:
5.
2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, Its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 6; and / or, its light chain variable region has an amino acid sequence as shown in SEQ ID NO:
7.
3. The monoclonal antibody or its antigen-binding fragment according to claim 1 or 2, characterized in that, The antigen-binding fragment is selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody, human antibody, chimeric antibody, or bispecific or multispecific antibody.
4. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.
5. The nucleic acid molecule according to claim 4, characterized in that, The nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 12 and / or SEQ ID NO:
13.
6. A carrier, characterized in that, It includes the nucleic acid molecule as described in claim 4 or 5.
7. A host cell, characterized in that, It comprises the nucleic acid molecule of claim 4 or 5 or the vector of claim 6.
8. A reagent kit, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3.
9. A pharmaceutical composition, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in any one of claims 1-3, or further comprises a pharmaceutically acceptable carrier and / or excipient.
10. The use of the monoclonal antibody or its antigen-binding fragment according to any one of claims 1-3 in any of the following aspects: (1) Use in the preparation of products for detecting the presence or level of Dabie Bandar virus or its Gn or Gc proteins in a sample; (2) Application in the preparation of products for neutralizing the virulence of Dabie Bandar virus in samples; (3) Use in the preparation of a medicament, wherein the medicament is used to neutralize the virulence of Dabiebanda virus in a sample, or to prevent or treat Dabiebanda virus infection or disease associated with such infection in a subject.