Monoclonal antibody of Japanese encephalitis virus and Zika virus and application thereof

Abstract

The invention discloses a monoclonal antibody of Japanese encephalitis virus and Zika virus and application of the monoclonal antibody, and belongs to the technical field of immunology and molecular virology. The monoclonal antibody or the antigen binding fragment thereof comprises VH CDR1, VH CDR2 and VH CDR3 of which the amino acid sequences are shown as SEQ ID NO: 1-3, and VL CDR1 and VL CDR3 of which the amino acid sequences are shown as SEQ ID NO: 4 and SEQ ID NO: 5, and the amino acid sequence is VL CDR2 of AAS. The monoclonal antibody LZY2086 provided by the invention can be combined with E protein extracellular regions of the encephalitis B virus and the Zika virus with high affinity, and has very strong neutralizing activity on the encephalitis B virus and the Zika virus. Therefore, as a broad-spectrum neutralizing antibody, the monoclonal antibody LZY2086 disclosed by the invention has ideal clinical application value in prevention and treatment of Japanese encephalitis virus and Zika virus infection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of immunology and molecular virology, and particularly relates to a monoclonal antibody for Japanese encephalitis virus and Zika virus and application thereof. BACKGROUND

[0002] Zika virus (ZIKV) and other mosquito-borne flaviviruses such as Japanese encephalitis virus (JEV) pose a serious threat to human health. The genome of such viruses is about 11 kb in length, encoding three structural proteins (C, prM / M and E proteins). Among them, the E protein mediates viral invasion by binding to the host cell surface receptor and is the key antigen that triggers neutralizing antibody response. ZIKV infection in adults can cause autoimmune neurological diseases such as Guillain-Barre syndrome, and infection in pregnant women can cause fetal microcephaly, but there is currently no vaccine against ZIKV approved for marketing. In addition, there is still a lack of specific antiviral drugs or antibody therapy for JEV and ZIKV infection in the clinic.

[0003] Neutralizing antibodies are one of the important means for the prevention and control of viral diseases. Some antibody drugs have been successfully applied in the clinic, such as Synagis for preventing respiratory syncytial virus (RSV) infection, and Trogarzo for HIV treatment, etc. The therapeutic mechanism of antibodies mainly includes two aspects: one is to block the interaction between virus surface protein and host receptor by binding to the virus surface protein, thereby neutralizing the virus infection ability; the other is to clear free viruses or infected cells by means of antibody-dependent cell-mediated cytotoxicity (ADCC), opsonophagocytosis and complement system activation, etc.

[0004] Therefore, it is particularly urgent to develop a broad-spectrum monoclonal antibody that can efficiently bind to viral antigens and has cross-neutralizing activity against JEV and ZIKV. Such antibodies have important application prospects in dealing with the spread of related viruses and protecting public health. SUMMARY

[0005] The present application aims to provide a monoclonal antibody for Japanese encephalitis virus and Zika virus and application thereof to solve the problems existing in the prior art. The monoclonal antibody provided by the present application can specifically recognize and target the E protein of Japanese encephalitis virus and Zika virus, especially the extracellular region of the E protein, and can block the binding of the E protein to the cell surface receptor, showing high efficiency in neutralizing viruses.

[0006] To achieve the above-mentioned purpose, the present application provides the following solutions.

[0007] The present application provides a monoclonal antibody or an antigen-binding fragment thereof, comprising a VH CDR1, a VH CDR2 and a VH CDR3 having an amino acid sequence as shown in SEQ ID NO: 1-3, and a VL CDR1 and a VL CDR3 having an amino acid sequence as shown in SEQ ID NO: 4 and SEQ ID NO: 5, and a VL CDR2 having an amino acid sequence of AAS.

[0008] Optionally, the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 6; and the light chain variable region has an amino acid sequence as shown in SEQ ID NO: 7.

[0009] The present application also provides a nucleic acid molecule encoding the monoclonal antibody or the antigen-binding fragment thereof.

[0010] Optionally, the nucleic acid molecule comprises a nucleotide sequence as shown in SEQ ID NO: 12 and / or SEQ ID NO: 13.

[0011] The present application also provides a recombinant vector comprising the nucleic acid molecule.

[0012] The present application also provides a recombinant host cell comprising the nucleic acid molecule or the recombinant vector.

[0013] The recombinant host cell is a non-plant cell.

[0014] The present application also provides a use of the nucleic acid molecule, the recombinant vector or the recombinant host cell in preparing a monoclonal antibody or an antigen-binding fragment thereof against Japanese encephalitis virus and Zika virus.

[0015] The present application also provides a use of the monoclonal antibody or the antigen-binding fragment thereof in any one of (1)-(3):

[0016] (1) preparing a product for detecting Zika virus, Japanese encephalitis virus, Japanese encephalitis virus E protein, Zika virus E protein, extracellular region of Japanese encephalitis virus E protein or extracellular region of Zika virus E protein;

[0017] (2) preparing a product for neutralizing the virulence of Zika virus or Japanese encephalitis virus;

[0018] (3) preparing a medicament for preventing or treating Zika virus and / or Japanese encephalitis virus infection.

[0019] The present application also provides a kit for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, extracellular region of Japanese encephalitis virus E protein or extracellular region of Zika virus E protein, comprising the monoclonal antibody or the antigen-binding fragment thereof.

[0020] The application also provides a pharmaceutical composition comprising the monoclonal antibody or the antigen-binding fragment thereof and a pharmaceutically acceptable adjuvant.

[0021] The application discloses the following technical effects:

[0022] The monoclonal antibody LZY2086 provided by the application can bind to the extracellular region of the E protein of the Japanese encephalitis virus and the Zika virus with high affinity, and has strong neutralizing activity on the Japanese encephalitis virus and the Zika virus. Therefore, as a broad-spectrum neutralizing antibody, the monoclonal antibody LZY2086 of the application has ideal clinical application value in preventing and treating Japanese encephalitis virus and Zika virus infection. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the application or the prior art, the following will briefly introduce the drawings needed in the embodiments. Obviously, the drawings in the following description are only some embodiments of the application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0024] Figure 1 WB detection results of the monoclonal antibody LZY2086 expressed in the embodiment 4 of the application; wherein, "M" on the gel map represents protein Marker; "DTT" represents that DTT (reducing SDS-PAGE) is added in the loading buffer; the detection antibody when doing WB is a single antibody of HRP-labeled anti-human IgG light chain and a single antibody of HRP-labeled anti-human IgG heavy chain;

[0025] Figure 2 Kinetic curve diagrams of the LZY2086 monoclonal antibody of different concentrations of the embodiment 5 of the application binding to the extracellular region of the E protein of the Japanese encephalitis virus (A) and the extracellular region of the E protein of the Zika virus (B);

[0026] Figure 3 Neutralizing activity of the LZY2086 monoclonal antibody of different concentrations of the embodiment 6 of the application on the Japanese encephalitis virus and the Zika virus in vitro;

[0027] Figure 4 Serum virus load experiment of the LZY2086 monoclonal antibody of different doses of the embodiment 7 of the application protecting mice against lethal Japanese encephalitis virus (A) and Zika virus (B) attack, the horizontal coordinate is the treatment factor, and the vertical coordinate is the serum virus load

Log 10 (PFU / ml)

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0032] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0033] The cell culture, molecular genetics, nucleic acid chemistry, immunology, and virology experimental procedures used in this invention (if applicable) are all standard procedures widely used in their respective fields. Furthermore, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0034] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair consisting of one "light" (L) chain and one "heavy" (H) chain. Antibody light chains can be classified as κ and λ light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, thereby defining antibodies as five classes: 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 denatured 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 types of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0035] 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.

[0036] 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 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.

[0037] 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 Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA .90:6444 6448. and Poljak RJ et al., 1994, Structure. 2:1121-1123.].

[0038] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibody LZY2086 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.

[0039] 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.

[0040] 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; that is, 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 comprise at least two or more different antibodies that typically recognize different epitopes on an antigen. Monoclonal antibodies are generally obtained using hybridoma techniques 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].

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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 its targeted antigen. In some embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific to an antigen) means that the antibody binds to less than about 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or smaller dissociation equilibrium constant (KD) binds to the antigen.

[0045] As used herein, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which describes the binding affinity between the antibody and the antigen. A smaller equilibrium dissociation constant indicates a stronger antibody-antigen binding and higher affinity between the antibody and the antigen. Typically, antibodies (e.g., the monoclonal antibody LZY2086 of this invention) have a KD value of less than approximately 10. -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10-9 M or 10 -10 The dissociation equilibrium constant (KD) of M or smaller binds to antigens (e.g., the E protein of Japanese encephalitis virus and Zika virus), and this value is determined using surface plasmon resonance (SPR) in a BIACORE 8K device.

[0046] 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.

[0047] 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.

[0048] As used in this invention, the term "Japanese encephalitis virus" refers to the Japanese encephalitis virus (JEV) as officially classified by the International Committee on Taxonomy of Viruses (ICTV). The two terms have the same meaning and can be used interchangeably.

[0049] As used in this invention, the terms "encephalitis B" and "Japanese encephalitis" refer to encephalitis caused by JEV infection; they have the same meaning and are used interchangeably. The term "Zika fever" refers to a fever-predominant disease caused by ZIKV infection; it has the same meaning and is used interchangeably.

[0050] This invention, based on extensive experimental research, has discovered an antibody that specifically recognizes and targets the E protein of Japanese encephalitis virus and Zika virus, particularly the extracellular region of the E protein, and blocks the binding of the E protein to cell surface receptors, demonstrating a highly efficient ability to neutralize the virus. Therefore, the antibody of this invention is particularly suitable for the diagnosis, prevention, and treatment of diseases related to Japanese encephalitis virus and Zika virus infection (e.g., Japanese encephalitis and Zika fever).

[0051] Specifically, the present invention provides a monoclonal antibody or its antigen-binding fragment thereof, wherein the CDR1 of the heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 1, the CDR2 has an amino acid sequence as shown in SEQ ID NO: 2, and the CDR3 has an amino acid sequence as shown in SEQ ID NO: 3;

[0052] And / or, the CDR1 of its light chain variable region has the amino acid sequence shown in SEQ ID NO: 4, the amino acid sequence of CDR2 is AAS and CDR3 has the amino acid sequence shown in SEQ ID NO: 5.

[0053] Optionally, its heavy chain variable region has an amino acid sequence as shown in SEQ ID NO: 6 and a gene sequence as shown in SEQ ID NO: 12;

[0054] And / or, its light chain variable region has an amino acid sequence as shown in SEQ ID NO: 7, or a gene sequence as shown in SEQ ID NO: 13.

[0055] 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.

[0056] 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.

[0057] Preferably, the signal peptide sequence of the present invention has the nucleotide sequence shown in SEQ ID NO: 16 and SEQ ID NO: 17.

[0058] 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.

[0059] In some preferred embodiments, the monoclonal antibody further includes a heavy chain constant region.

[0060] 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.

[0061] In some preferred embodiments, the monoclonal antibody further includes a light chain constant region.

[0062] 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.

[0063] In some preferred embodiments, the light chain of the monoclonal antibody is of the λ type.

[0064] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of specifically binding to the envelope protein (E protein) of Japanese encephalitis virus and Zika virus.

[0065] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of targeting the extracellular region of the E protein of Japanese encephalitis virus and Zika virus.

[0066] 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 region of the E protein, thereby inhibiting viral infection of cells.

[0067] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment has neutralizing ability (e.g., capable of neutralizing Japanese encephalitis virus and Zika virus).

[0068] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of inhibiting infection or entry into host cells by Japanese encephalitis virus and Zika virus. Thus, the monoclonal antibody or its antigen-binding fragment can neutralize Japanese encephalitis virus and Zika virus, thereby preventing and treating infection by these viruses.

[0069] 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.

[0070] Specifically, the present invention also provides a nucleic acid molecule that encodes the above-mentioned monoclonal antibody or its antigen-binding fragment.

[0071] Preferably, the nucleic acid molecule has a nucleotide sequence as shown in SEQ ID NO: 12 and / or SEQ ID NO: 13.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence capable of encoding a heavy chain constant region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof. 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 capable of encoding the heavy chain constant region of the monoclonal antibody of the present invention or an antigen-binding fragment thereof has a nucleotide sequence as shown in SEQ ID NO:14.

[0078] In some preferred embodiments, the nucleic acid molecule further comprises a nucleotide sequence of 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 of 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.

[0079] 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.

[0080] 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.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] The composition may be a diagnostic agent or a therapeutic agent.

[0086] The present invention further provides a kit comprising the above-described monoclonal antibody or its antigen-binding fragment.

[0087] 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).

[0088] 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.

[0089] Preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as ribavirin.

[0090] 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 and VL CDR3 with amino acid sequences as shown in SEQ ID NO:4-5, and VL CDR2 with an 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.

[0091] The present invention also provides an application of the above-mentioned monoclonal antibody or its antigen-binding fragment in any of the following aspects:

[0092] (1) Prepare products for detecting Japanese encephalitis virus, Zika virus or its E protein or extracellular region of E protein;

[0093] (2) To prepare products for neutralizing the virulence of Japanese encephalitis virus and Zika virus in samples;

[0094] (3) Prepare a drug for neutralizing the virulence of Japanese encephalitis virus and Zika virus in a sample, or for preventing or treating Japanese encephalitis virus, Zika virus infection or disease related to these viruses in a subject.

[0095] In another aspect, the present invention provides a method for detecting the presence or level of Japanese encephalitis virus, Zika virus, or their E protein or extracellular region in a sample, comprising using a monoclonal antibody or its antigen-binding fragment of the present invention. In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment of the present invention further includes a detectable label. In another preferred embodiment, the method further includes the step of using a second antibody carrying a detectable label to detect the monoclonal antibody or its 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).

[0096] In another aspect, the present invention provides a method for diagnosing whether a subject is infected with Japanese encephalitis virus or Zika virus, comprising the step of detecting the presence of Japanese encephalitis virus, Zika virus, or their E protein or extracellular region thereof in a sample from said subject using a monoclonal antibody of the present invention or an antigen-binding fragment thereof. In some preferred embodiments, the monoclonal antibody of the present invention or its antigen-binding fragment thereof 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 of the present invention or its antigen-binding fragment thereof, or an anti-idiotype antibody.

[0097] 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 Japanese encephalitis virus, Zika virus or its E protein or extracellular region in a sample, or for diagnosing whether a subject is infected with Japanese encephalitis virus or Zika virus.

[0098] In some preferred embodiments, the sample includes, but is not limited to, excrement, oral or nasal secretions, bronchoalveolar lavage fluid, etc., from a subject (e.g., a mammal, preferably a human).

[0099] 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 and VL CDR3 with amino acid sequences as shown in SEQ ID NO: 4-5, and VL CDR2 with an 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.

[0100] General methods for detecting the presence or level of a target virus or antigen (e.g., Japanese encephalitis virus, Zika virus, or its E protein or extracellular region) 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.

[0101] In another aspect, the present invention provides a method for neutralizing the virulence of Japanese encephalitis virus or Zika virus in a sample, comprising contacting a sample containing Japanese encephalitis virus or Zika virus with a monoclonal antibody or antigen-binding fragment 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).

[0102] 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 Japanese encephalitis virus and / or Zika virus in a sample. In another aspect, the present invention provides the monoclonal antibody or its antigen-binding fragment as described above, which is used to neutralize the virulence of Japanese encephalitis virus and / or Zika virus in a sample.

[0103] 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 Japanese encephalitis virus, Zika virus infection, or diseases associated with these viral infections (e.g., Japanese encephalitis, Zika fever) 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 Japanese encephalitis virus, Zika virus infection, or diseases associated with these viral infections (e.g., Japanese encephalitis, Zika fever) in a subject.

[0104] In another aspect, the present invention provides a method for preventing or treating Japanese encephalitis virus, Zika virus infection, or diseases associated with these viral infections (e.g., Japanese encephalitis, Zika fever) in a subject, comprising administering to the subject in need a preventive or therapeutically effective amount of a monoclonal antibody of the present invention or an antigen-binding fragment thereof, or a pharmaceutical composition of the present invention.

[0105] In some preferred embodiments, the subject is a mammal, such as a human.

[0106] 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, oral, oral, sublingual, topical, parenteral, rectal, or nasal routes.

[0107] 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 and VL CDR3 with amino acid sequences as shown in SEQ ID NO: 4-5, and VL CDR2 with an 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.

[0108] 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).

[0109] 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. Ausubel et al., A Concise Guide to Laboratory Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is in accordance with the conditions recommended by the product manufacturer. Where specific conditions are not specified in the examples, they are performed under standard conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0110] To obtain a protective neutralizing antibody, this invention first uses the extracellular region (E410) of the Zika virus E protein expressed in *E. coli* as an antigen. Memory B cells capable of specifically binding to ZIKV-E410 are screened from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteers (22 years old) who have been vaccinated with attenuated Japanese encephalitis virus using flow cytometry. Then, the selected single B cells are subjected to BCR 10Genomics sequencing to obtain the gene sequence encoding the antibody variable region in each B cell. Further, the sequence encoding the antibody variable region and the constant region gene are ligated into an expression vector, and expressed and purified in mammalian cells to obtain the monoclonal antibody LZY2086. A series of functional tests were performed on monoclonal antibody LZY2086. The results showed that monoclonal antibody LZY2086 can specifically bind to JEV-E406 and ZIKV-E410, inhibit the infection of Vero cells by Japanese encephalitis virus and Zika virus, protect mice against lethal Japanese encephalitis virus and Zika virus attack, and has broad-spectrum neutralizing activity against Japanese encephalitis virus and Zika virus infection.

[0111] Information on some of the sequences involved in this invention is shown in Tables 1 and 2 below.

[0112] Table 1. Amino acid and nucleotide sequences

[0113]

[0114] Table 2 Amino acid and nucleotide sequences

[0115]

[0116] The technical solution of the present invention will now be shown in detail with reference to specific embodiments:

[0117] Example 1: Expression and purification of the extracellular region of E protein of Japanese encephalitis virus and Zika virus

[0118] 1. The coding sequences of JEV-E406 (residues 1-406, GenBank accession no: MK558811) and ZIKV-E410 (residues 1-410, GenBank accession no: KX117076.1), which were optimized for E. coli codons and had 6 His tags at the C-terminus, were cloned into the prokaryotic expression vector pET21a to obtain recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410.

[0119] 2. The recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410 were transformed into Escherichia coli strain BL21 and protein expression was induced by IPTG.

[0120] 3. JEV-E406 and ZIKV-E410 proteins were separated and purified using a Ni-NTA affinity chromatography column. The purity of the two proteins was determined by SDS-PAGE and Western blotting.

[0121] Example 2: Isolation of memory B cells that specifically recognize ZIKV-E410 protein

[0122] 1. With informed consent from healthy volunteers who received the JEV live attenuated vaccine, 100 mL of peripheral venous blood was collected, and peripheral blood mononuclear cells (PBMCs) were separated using human lymphocyte separation fluid.

[0123] 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 (memory B cell) pellet in pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA).

[0124] 3. Add 5 mL of magnetic beads (1 μm in diameter) to a flow cytometry tube, insert a magnet and wait 3 min, then discard the supernatant; add 4 mL of PBS to suspend the magnetic beads, insert a magnet and wait 3 min, then discard the supernatant; add 4 mL of 5 μM imidazole solution, insert a magnet and wait 3 min, then discard the supernatant; add protein solution (2 mg ZIKV-E410) and an appropriate amount of binding buffer to 4 mL, tighten the cap and shake on a shaker for 40 min; insert a magnet and wait 3 min, then discard the supernatant; add 4 mL of PBS to suspend the magnetic beads, insert a magnet and wait 3 min, then discard the supernatant; add 4 mL of 5% BSA / PBS to suspend the magnetic beads, shake on a shaker for 10 min; insert a magnet and wait 3 min, then discard the supernatant; add 4 mL of PBS to suspend the magnetic beads, insert a magnet and wait 3 min, then discard the supernatant; suspend the above magnetic beads in 1 mL of memory B cell suspension (isolated from 100 mL of peripheral venous blood), shake on a shaker for 20 min; insert a magnet and wait 3 min, then discard the unbound solution; add 4... The magnetic beads were suspended in mL of pre-cooled buffer, the magnet was inserted and waited for 3 min. The unbound solution was discarded, and the adsorbed cells (i.e. target cells) were washed off with pre-cooled buffer.

[0125] 4. The above cells were sent to the company for 10× single-cell expression profiling sequencing + BCR library construction sequencing and analysis.

[0126] Example 3: Isolation and identification of LZY2086 monoclonal antibody and construction of recombinant expression vector

[0127] We sequenced the V region gene of the BCR of more than 6,000 memory B cells. Through sequence analysis, we selected the variable region (V region) sequence of the light and heavy chains of a monoclonal antibody named LZY2086 for further study.

[0128] The amino acid sequence of the heavy chain variable region of the LZY2086 monoclonal antibody 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 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 the LZY2086 monoclonal antibody are shown in Tables 3-4.

[0129] Table 3 Genes in the V region of the heavy chain of LZY2086 monoclonal antibody

[0130]

[0131] Table 4. Genes in the V region of the light chain of LZY2086 monoclonal antibody

[0132]

[0133] The nucleotide sequences encoding the variable regions of the LZY2086 heavy / light chains were analyzed and then coupled into the expression vector pCAGGS containing the corresponding nucleotide sequences encoding the constant regions of the heavy / laminar chains, respectively, to obtain recombinant expression vectors encoding the LZY2086 monoclonal antibody heavy and light chains, respectively. The construction methods for the heavy and light chain expression constructs are as follows:

[0134] Heavy chain coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence gene - VH gene - CH gene - Xho I restriction site;

[0135] Light chain (k) coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence gene - VL gene - CL(k) gene - Xho I restriction site;

[0136] The amino acid sequence of the heavy chain coding sequence signal peptide is shown in SED ID NO: 10 (the nucleotide sequence of the coding gene is shown in SEQ ID NO: 16), and the amino acid sequence of the light chain coding sequence signal peptide is shown in SED ID NO: 11 (the nucleotide sequence of the coding gene is shown in SEQ ID NO: 17).

[0137] The amino acid sequence of CH is shown in SED ID NO: 8 (encoding gene is shown in SEQ ID NO: 14), the amino acid sequence of CL is shown in SED ID NO: 9 (encoding gene is shown in SEQ ID NO: 15); the nucleotide sequence of VH gene is shown in SEQ ID NO: 12; and the nucleotide sequence of VL gene is shown in SEQ ID NO: 13.

[0138] Example 4 Expression of ZY2006 monoclonal antibody

[0139] Heavy and light chain plasmids (recombinant expression vectors pCAGGS-LZY2086H and pCAGGS-LZY2086L, respectively encoding the heavy and light chains of the LZY2086 monoclonal antibody obtained in Example 3) were co-transfected into 293T cells. The weight ratio of heavy chain plasmid pCAGGS-LZY2086H to light chain plasmid pCAGGS-LZY2086L was 1.07:0.93. Each 293T cell plate (10 cm in diameter) 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. The supernatants were then purified using a Pierce protein A / G agarose column (Thermo Fisher) and concentrated using ultrafiltration tubes with a molecular weight cutoff of 50 KD. Subsequently, SDS-PAGE (in both reducing and non-reducing states) was performed, followed by membrane transfer and Western blotting. The results are as follows: Figure 1 As shown, purified LZY2086 monoclonal antibody was obtained.

[0140] Example 5: Evaluation of the binding affinity of LZY2086 monoclonal antibody to JEV-E406 and ZIKV-E410

[0141] In this embodiment, surface plasmon resonance analysis was performed using the LifeDisc™ MetaSPR chip (Liangzhun [Hangzhou] Biotechnology Co., Ltd.). The specific steps are as follows:

[0142] First, JEV-E406 or ZIKV-E410 (20 μg / mL) was immobilized onto a LifeDisc™ MetaSPR chip (Liangzhun [Hangzhou] Biotechnology Co., Ltd.). Then, LZY2086, serially diluted with PBST solution at pH 7.4, was sequentially loaded through each channel (0.25, 0.5, 1, 2, and 3 mg / mL). The kinetic curves of LZY2086 binding to JEV-E406 or ZIKV-E410 proteins were recorded. Figure 2 As shown in Table 5), the kinetic constants were calculated using WeSPR One software (Liangzhun [Hangzhou] Biotechnology Co., Ltd.), and the fit was performed in "1:1 binding" mode. Figure 2 The results in Table 5 show that the LZY2086 monoclonal antibody can bind to the extracellular regions of the E proteins of JEV and ZIKV (JEV-E406 and ZIKV-E410) with high affinity. The amino acid sequences of JEV-406 and ZIKV-E410 are shown in Table 2.

[0143] Table 5. Affinity of LZY2086 monoclonal antibody with JEV-E406 and ZIKV-E410

[0144]

[0145] Example 6: In vitro neutralization of JEV and ZIKV by LZY2086 monoclonal antibody

[0146] 1. Seed Vero cells in 24-well plates (1 ∈ 10⁻⁶). 5 (each well), incubate for 24 h.

[0147] 2. In a 96-well plate, serially dilute the LZY2086 monoclonal antibody to be tested 3-fold (starting from 1 μg / mL), add the virus to be tested (JEV or ZIKV, 50 viral plaque forming units (PFU) / well) to each well, and incubate at 37°C for 1 h.

[0148] 3. Discard the Vero cell supernatant, add the virus / monoclonal antibody mixture to the cells, and infect at 37°C for 1.5 h.

[0149] 4. Discard the virus solution, add 1% methylcellulose culture medium to each well to cover the cells, and culture for 3-6 days.

[0150] 5. Add 4% paraformaldehyde to each well and fix at room temperature for 1 hour, then wash the plate.

[0151] 6. Add 0.5% crystal violet stain to each well and stain at room temperature for 10 min, then wash the plate.

[0152] 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).

[0153] NT 50 Analysis was performed using GraphPad Prism 6 software. The analysis results are shown below. Figure 3 See Table 6. It is evident that the LZY2086 monoclonal antibody can inhibit JEV and ZIKV with extremely high neutralizing activity.

[0154] Table 6 Neutralizing titers (half-neutralizing concentration, NT) of LZY2086 monoclonal antibody against JEV and ZIKV 50 )

[0155]

[0156] Example 7: Experiment on the protection of mice against lethal JEV or ZIKV challenge by LZY2086 monoclonal antibody.

[0157] 1. Inject 100 PFU of JEV or ZIKV subcutaneously into the back of 6-day-old C57BL / 6 mice or 1-day-old C57BL / 6 mice, respectively;

[0158] 2. Two hours later, 50 μg or 10 μg of LZY2086 monoclonal antibody was injected subcutaneously into the back of JEV-infected or ZIKV-infected mice, respectively. Three days later, the mice were sacrificed and their serum was collected. The viral load in the mouse serum was detected by viral plaque formation assay (PFA) based on Vero cells. The results are expressed as Log 10 (PFU / mL).

[0159] 3. Prism 6 was used to analyze, statistically analyze, and graph the data. The results are as follows: Figure 4 As shown, after ZIKV infection, the viral load in the serum of mice given 10 μg of LZY2086 monoclonal antibody was significantly lower than that in the control group (given PBS) (186 PFU / mL versus 5011 PFU / mL, P=0.0031), with a viral titer 26-fold lower; after JEV infection, the viral load in the serum of mice given 50 μg of LZY2086 monoclonal antibody was also significantly lower than that in the control group (363078 PFU / ml versus 1995262 PFU / ml, P=0.0029), with a viral titer more than 4-fold lower.

[0160] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment, characterized in that, It contains VH CDR1, VH CDR2 and VH CDR3 with amino acid sequences as shown in SEQ ID NO:1-3, and VL CDR1 and VL CDR3 with amino acid sequences as shown in SEQ ID NO:4 and SEQ ID NO:5, and VL CDR2 with amino acid sequence AAS.

2. The monoclonal antibody or its antigen-binding fragment according to claim 1, characterized in that, Its heavy chain variable region has the amino acid sequence shown in SEQ ID NO: 6; its light chain variable region has the amino acid sequence shown in SEQ ID NO:

7.

3. A nucleic acid molecule, characterized in that, It encodes the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleic acid molecule contains nucleotide sequences as shown in SEQ ID NO: 12 and / or SEQ ID NO:

13.

5. A recombinant vector, characterized in that, It includes the nucleic acid molecule as described in claim 3 or 4.

6. A recombinant host cell, characterized in that, Contains the nucleic acid molecule of claim 3 or 4 or the recombinant vector of claim 5; The recombinant host cell is a non-plant cell.

7. The use of a nucleic acid molecule as described in claim 3 or 4, a recombinant vector as described in claim 5, or a recombinant host cell as described in claim 6 in the preparation of monoclonal antibodies or antigen-binding fragments of Japanese encephalitis virus and Zika virus.

8. The use of a monoclonal antibody or antigen-binding fragment thereof as described in claim 1 or 2 in any one of the following (1)-(3): (1) Prepare products for detecting Zika virus, Japanese encephalitis virus, Japanese encephalitis virus E protein, Zika virus E protein, extracellular region of Japanese encephalitis virus E protein or extracellular region of Zika virus E protein; (2) To prepare products for neutralizing the virulence of Zika virus or Japanese encephalitis virus; (3) Application in the preparation of a medicine for the prevention or treatment of Zika virus and / or Japanese encephalitis virus infection.

9. A kit for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, the extracellular region of Japanese encephalitis virus E protein, or the extracellular region of Zika virus E protein, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2.

10. A pharmaceutical composition, characterized in that, It comprises the monoclonal antibody or its antigen-binding fragment as described in claim 1 or 2, and pharmaceutically acceptable excipients.

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