A human monoclonal antibody neutralizing zika virus and japanese encephalitis virus and application thereof

Abstract

This invention discloses a human monoclonal antibody capable of neutralizing Zika virus and Japanese encephalitis virus, and its applications, relating to the field of biotechnology. The human monoclonal antibody comprises VH CDR1 (amino acid sequence as shown in SEQ ID NO.1), VH CDR2 (amino acid sequence as shown in SEQ ID NO.2), VH CDR3 (amino acid sequence as shown in SEQ ID NO.3), VL CDR1 (amino acid sequence as shown in SEQ ID NO.4), VL CDR2 (amino acid sequence of AAS), and VL CDR3 (amino acid sequence as shown in SEQ ID NO.5). The human monoclonal antibody obtained by screening in this invention can simultaneously target and bind to the extracellular regions of the E protein of both Zika virus and Japanese encephalitis virus, and exhibits strong neutralizing activity against both.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a human monoclonal antibody that can neutralize Zika virus and Japanese encephalitis virus and its applications. Background Technology

[0002] Zika virus (ZIKV) and Japanese encephalitis virus (JEV), both mosquito-borne flaviviruses, pose a serious threat to human health. These flaviviruses have a genome of approximately 11 kb, encoding three structural proteins (C, prM / M, and E proteins). The E protein mediates viral infection by binding to receptors on the surface of sensitive cells and is therefore a key viral antigen inducing the production of neutralizing antibodies. ZIKV infection in adults can lead to neuroautoimmune diseases such as Guillain-Barré syndrome, and infection in pregnant women can cause microcephaly in newborns. Currently, there is no approved Zika vaccine. Furthermore, for JEV and ZIKV, there are no specific antiviral drugs or other therapies (such as antibody therapy) approved for clinical use.

[0003] Currently, neutralizing antibodies remain an effective treatment for viral diseases. Marketed drugs for treating and preventing viral infections include pallizumab (Synagis) for the prevention and treatment of respiratory syncytial virus (RSV) infection in children, and elbalizumab (Trogarzo) for the treatment of 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 (ADCC), antibody-mediated opsonization, and antigen-antibody complex activation of the complement system.

[0004] Therefore, it is urgent to screen for broad-spectrum neutralizing monoclonal antibodies with higher affinity that can simultaneously exhibit neutralizing activity against Zika virus and Japanese encephalitis virus. Summary of the Invention

[0005] The purpose of this invention is to provide a human monoclonal antibody capable of neutralizing Zika virus and Japanese encephalitis virus, and its application, to solve the problems existing in the prior art. The human monoclonal antibody provided by this invention can bind to the extracellular region of the E protein of Zika virus and Japanese encephalitis virus with high affinity, and has strong neutralizing activity against Zika virus and Japanese encephalitis virus.

[0006] To achieve the above objectives, the present invention provides the following solution: This invention provides a human monoclonal antibody or its antigen-binding fragment that can neutralize Zika virus and Japanese encephalitis virus, comprising VH CDR1 with the amino acid sequence shown in SEQ ID NO.1, VH CDR2 with the amino acid sequence shown in SEQ ID NO.2, VH CDR3 with the amino acid sequence shown in SEQ ID NO.3, VLCDR1 with the amino acid sequence shown in SEQ ID NO.4, VL CDR2 with the amino acid sequence AAS, and VL CDR3 with the amino acid sequence shown in SEQ ID NO.5.

[0007] Furthermore, the amino acid sequence of the heavy chain variable region of the human monoclonal antibody or its antigen-binding fragment capable of neutralizing Zika virus and Japanese encephalitis virus is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.7.

[0008] Furthermore, the human monoclonal antibody or its antigen-binding fragment capable of neutralizing Zika virus and Japanese encephalitis virus includes a heavy chain constant region and a light chain constant region.

[0009] Preferably, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO.8; and the amino acid sequence of the light chain constant region is shown in SEQ ID NO.9.

[0010] The present invention also provides a gene encoding a human monoclonal antibody or its antigen-binding fragment that can neutralize Zika virus and Japanese encephalitis virus.

[0011] Furthermore, the coding gene includes a light chain variable region coding gene and a heavy chain variable region coding gene; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.12; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.13.

[0012] The present invention also provides a recombinant expression vector comprising the above-described coding gene.

[0013] The present invention also provides a recombinant non-plant host cell, comprising the above-described recombinant expression vector.

[0014] Such non-plant host cells include, but are not limited to, prokaryotic cells and eukaryotic cells; prokaryotic cells include, for example, Escherichia coli cells; eukaryotic cells include, for example, yeast cells, insect cells, plant cells, and animal cells (such as mammalian cells, such as mouse cells, human cells, etc.). The recombinant non-plant host cells of the present invention can also be cell lines, such as HEK-293T cells.

[0015] The present invention also provides the use of the above-mentioned encoding gene, recombinant expression vector or recombinant non-plant host cell in the preparation of human monoclonal antibodies or antigen-binding fragments thereof capable of neutralizing Zika virus and Japanese encephalitis virus.

[0016] The present invention also provides the use of the above-mentioned human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus in (1) or (2): (1) Preparation of detection products for Zika virus and / or Japanese encephalitis virus; (2) Prepare drugs to inhibit Zika virus and / or Japanese encephalitis virus infection.

[0017] The present invention also provides a detection product for Zika virus and / or Japanese encephalitis virus, comprising the above-mentioned human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus.

[0018] The present invention also provides a drug for inhibiting Zika virus and / or Japanese encephalitis virus infection, wherein the active ingredient includes the above-mentioned human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus.

[0019] The present invention discloses the following technical effects: The human monoclonal antibody LZY2083 and its antigen-binding fragment obtained by screening in this invention can simultaneously target and bind to the extracellular regions of the E protein of Zika virus and Japanese encephalitis virus, and exhibit strong neutralizing activity against both Zika virus and Japanese encephalitis virus. ELISA experiments confirmed that the antibody can stably bind to both viral antigens in a concentration-dependent manner, with balanced binding affinity. In vitro neutralization experiments showed that its half-neutralizing concentrations (WMCs) for Japanese encephalitis virus and Zika virus were 331 ng / mL and 224 ng / mL, respectively, effectively blocking viral infection of host cells. Animal experiments further verified the in vivo protective effect: the antibody can significantly reduce the viral load in infected serum, decreasing the viral titer of Zika virus by 12-fold and the viral titer of Japanese encephalitis virus by more than 4-fold, effectively resisting lethal viral attacks.

[0020] The human monoclonal antibody LZY2083 provided by this invention has high safety and dual value for diagnostics and pharmaceuticals. On the one hand, it can be used to prepare detection reagents for two types of viruses, improving detection sensitivity; on the other hand, it can be developed into an antiviral drug, filling the market gap of lacking specific treatments for Zika and Japanese encephalitis viruses. It has good application prospects and industrialization potential in the fields of viral infection prevention and clinical treatment. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 The image shows the SDS-PAGE results of the monoclonal antibody LZY2083 expressed in Example 4; where "M" represents the protein marker; "non-reduced" means that DTT is not added to the loading buffer, and "reduced" means that DTT is added to the loading buffer; Figure 2 Figure 1 shows the ELISA results of different concentrations of monoclonal antibody LZY2083 binding to the extracellular region of the E protein of Japanese encephalitis virus and Zika virus. Figure 3 The figure shows the results of detecting the neutralizing activity of different concentrations of monoclonal antibody LZY2083 against Zika virus and Japanese encephalitis virus in vitro. Figure 4 Figure 1 shows the results of an experiment on serum viral load in mice protected against lethal Zika virus (A) and Japanese encephalitis virus (B) attacks by different doses of monoclonal antibody LZY2083. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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 experimental methods widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0029] 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 types. 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 the light and heavy chains, variable regions (V) and constant regions (C) 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) or four domains (CH1, CH2, CH3, and CH4). 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 variable regions [called hypervariable regions (HVRs) or complementarity-determining regions (CDRs)], interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of seven parts arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VHs and VLs) of each heavy / light chain pair form the antibody binding sites. The allocation of amino acids to various 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 antibody production. 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 subclasses), IgA (IgA1 or IgA2 subclasses), IgD, IgE, or IgM antibodies.

[0030] 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.

[0031] 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 produce linkers that are single polypeptide chains [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 an amino acid sequence (GGGGS) of 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.

[0032] 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.].

[0033] Antigen-binding fragments (e.g., the antibody fragments described above) of a given antibody (e.g., the monoclonal antibody LZY2083 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.

[0034] 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.

[0035] 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 monoclonal 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].

[0036] 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.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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 viral infection of cells and / or the maturation and / or release of progeny viruses. Antibodies or antibody fragments with neutralizing activity can prevent viral amplification, thereby inhibiting or eliminating viral infection.

[0042] 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.

[0043] 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 Laboratory 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.

[0044] To obtain a protective neutralizing antibody, this invention first uses the extracellular region of the Zika virus E protein (ZIKV-E) expressed in Escherichia coli. 410 Using ZIKV-E as an antigen, peripheral blood mononuclear cells (PBMCs) capable of specifically binding to ZIKV-E were screened from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteers (22 years old) who had been vaccinated with attenuated Japanese encephalitis virus vaccine using flow cytometry. 410 Memory B cells were selected, and then the selected single B cells were sequenced using BCR 10×Genomics to obtain the gene sequence encoding the antibody variable region in the single B cells. 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 LZY2083. A series of functional tests on monoclonal antibody LZY2083 showed that it can specifically bind to JEV-E. 406 and ZIKV-E 410 It inhibits the infection of Vero cells by Zika virus and Japanese encephalitis virus, protects mice against lethal Zika virus and Japanese encephalitis virus attacks, and has broad-spectrum neutralizing activity against Zika virus and Japanese encephalitis virus infection.

[0045] Information on some of the sequences involved in this invention is shown in Table 1 below.

[0046] Table 1. Sequence information involved in this invention Example 1: Expression and purification of the extracellular region of E protein of Japanese encephalitis virus and Zika virus 1. JEV-E, with its C-terminus optimized by E. coli and containing 6 His tags, was used. 406 (Residues 1-406, GenBank accession no: MK558811) and ZIKV-E 410The coding sequences (residues 1-410, GenBank accession no: KX117076.1) were cloned into the prokaryotic expression vector pET21a to obtain the recombinant plasmid pET21a-JEV-E. 406 and pET21a-ZIKV-E 410 .

[0047] 2. The recombinant plasmid pET21a-JEV-E 406 and pET21a-ZIKV-E 410 The proteins were transformed into Escherichia coli BL21 strain and expressed using IPTG.

[0048] 3. JEV-E was separated and purified using a Ni-NTA affinity chromatography column. 406 and ZIKV-E 410 Proteins. The purity of the two proteins was assessed using SDS-PAGE and WB experiments.

[0049] Example 2: Specific recognition of ZIKV-E 410 Isolation of protein-memory B cells 1. With informed consent from healthy volunteers who received the JEV live attenuated vaccine, 100 mL of peripheral venous blood was collected from them, and peripheral blood mononuclear cells (PBMCs) were separated using human lymphocyte separation fluid.

[0050] 2. The isolated PBMCs were mixed with a Miltenyi biotinylated antibody mixture (containing biotinylated anti-human CD2, anti-human CD3, anti-human CD14, anti-human CD43, anti-human CD56, and anti-human CD235a monoclonal antibodies) in a flow cytometry tube and incubated at 4°C for 30 min. Pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA) was added, followed by the addition of anti-biotin microbeads, and incubation at 4°C for 30 min. The mixture was then centrifuged at 2000 rpm for 10 min, and the supernatant was discarded. The cell pellet was resuspended in pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA), transferred to a flow cytometry tube, and a magnet was inserted for 3 min. Unbound cells were collected and resuspended in pre-chilled buffer (0.5% BSA / PBS, containing 20 mM EDTA). Wash the adsorbed magnetic beads with EDTA, insert the magnet and wait for 3 min, and collect the unadsorbed cells; transfer all the unadsorbed cell fluid into a 15 mL centrifuge tube; centrifuge at 2000 rpm for 10 min, discard the supernatant, and suspend the cells (memory B cells) in pre-cooled buffer (0.5% BSA / PBS, containing 20 mM EDTA) to precipitate the cells.

[0051] 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... Float the magnetic beads in mL of pre-cooled buffer, insert the magnet and wait for 3 min, discard the unbound solution, and add pre-cooled buffer to wash off the adsorbed cells (i.e. target cells). 4. The above cells were sent to the company for 10× single-cell expression profiling sequencing + BCR library construction sequencing and analysis.

[0052] Example 3: Isolation and identification of monoclonal antibody LZY2083 and construction of recombinant expression vector The genes of the V region of the BCR of more than 6,000 memory B cells were sequenced. Based on the sequence analysis, this invention selected a monoclonal antibody named LZY2083 for further research.

[0053] The sequence information of the monoclonal antibody LZY2083 is as follows: The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.6 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO.12), and the amino acid sequences of VH CDR1, VH CDR2 and VH CDR3 are shown in SEQ ID NO.1-3, respectively. The amino acid sequence of the light chain variable region is shown in SEQ ID NO.7 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO.13), the amino acid sequence of VL CDR1 is shown in SEQ ID NO.4, the amino acid sequence of VL CDR2 is AAS, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO.5.

[0054] The V region genes of the heavy and light chains of monoclonal antibody LZY2083 are shown in Tables 2-3.

[0055] Table 2 Genes of the heavy chain V region of monoclonal antibody LZY2083 Table 3. Genes of the V region of the light chain of monoclonal antibody LZY2083 The nucleotide sequences encoding the variable regions of the heavy and light chains of LZY2083, obtained from the analysis, were cloned along with the corresponding nucleotide sequences encoding the constant regions of the heavy and light chains into the expression vector pCAGGS, thereby obtaining the recombinant expression vector pCAGGS-LZY2083H for expressing the heavy chain and the recombinant expression vector pCAGGS-LZY2083L for expressing the light chain. The coding sequences for the heavy and light chains are as follows: Heavy chain coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence 1 encoding gene - VH gene - CH gene - Xho I restriction site; Light chain coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence 2 coding gene - VL gene - CL gene - Xho I restriction site; The amino acid sequence of signal peptide sequence 1 is shown in SEQ ID NO. 10 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 16); the amino acid sequence of signal peptide sequence 2 is shown in SEQ ID NO. 11 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 17); the amino acid sequence of CH is shown in SEQ ID NO. 8 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 14); and the amino acid sequence of CL is shown in SEQ ID NO. 9 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 15).

[0056] Example 4 Expression of monoclonal antibody LZY2083 The recombinant expression vectors pCAGGS-LZY2083H and pCAGGS-LZY2083L obtained in Example 3 were co-transfected into 293T cells. The weight ratio of pCAGGS-LZY2083H to pCAGGS-LZY2083L was 1.07:0.93. Each 10 cm diameter plate of 293T cells was transfected with 20 μg of plasmid (pCAGGS-LZY2083H + pCAGGS-LZY2083L) and 40 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 Pierceprotein A / G agarose column (Thermo Fisher) and concentrated using ultrafiltration tubes with a 50 kDa molecular weight cutoff. Subsequently, SDS-PAGE electrophoresis was performed.

[0057] Experimental results: such as Figure 1 As shown, this embodiment yielded purified monoclonal antibody LZY2083 with a light chain molecular weight of approximately 25 kDa and a heavy chain molecular weight of approximately 55 kDa.

[0058] Example 5: Monoclonal antibody LZY2083 and JEV-E 406 and ZIKV-E 410 Assessment of the ability to combine In this embodiment, ELISA was used to evaluate the monoclonal antibody LZY2083 and ZIKV-E. 410 and JEV-E 406 The binding ability. The specific steps are as follows: 1. Coating antigen: ZIKV-E 410 or JEV-E 406 Dissolve in 0.05 mol / L carbonate buffer (pH 9.6) to a final concentration of 1 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 3-fold serially diluted monoclonal antibody LZY2083 (starting from 100 μg / mL) 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. Measure the OD of each well using a microplate reader. 450 The values ​​were analyzed and plotted using Prism 8 software.

[0059] Experimental results: such as Figure 2 As shown, the monoclonal antibody LZY2083 can bind to the extracellular regions of the E proteins of ZIKV and JEV (ZIKV-E, respectively) in a concentration-dependent manner. 410 and JEV-E 406Monoclonal antibody LZY2083 binds to JEV-E. 406 Protein and ZIKV-E 410 The proteins have comparable capabilities.

[0060] Example 6: In vitro neutralization of JEV and ZIKV by monoclonal antibody LZY2083 1. Seed Vero cells in 24-well plates (1×10⁻⁶ cells / well). 5 (each well), incubate for 24 h.

[0061] 2. In a 96-well plate, serially dilute the monoclonal antibody LZY2083 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.

[0062] 3. Discard the Vero cell supernatant, add the virus / monoclonal antibody mixture to the cells, and infect at 37°C for 1.5 h.

[0063] 4. Discard the virus solution, add 1% methylcellulose culture medium to each well to cover the cells, and culture for 3-6 days.

[0064] 5. Add 4% paraformaldehyde to each well and fix at room temperature for 1 hour, then wash the plate.

[0065] 6. Add 0.5% crystal violet stain to each well and stain at room temperature for 10 min, then wash the plate.

[0066] 7. Count the number of viral plaques per well and analyze the experimental data using GraphPad Prism 6 soft analysis. Calculate the neutralizing titer (half-neutralizing concentration, NT) of the diluted monoclonal antibody against each virus. 50 value).

[0067] Experimental results: such as Figure 3 As shown in Table 4, the monoclonal antibody LZY2083 can inhibit JEV and ZIKV infection of cells in a concentration-dependent manner, with a lower NT ratio than JEV. 50 The value was 331 ng / mL, which is the NT value for neutralizing ZIKV. 50 The value was 224 ng / mL.

[0068] Table 4 Neutralizing titers (half-neutralizing concentration, NT) of monoclonal antibody LZY2083 against JEV and ZIKV 50 ) Example 7: Experiment on the protection of mice against lethal JEV or ZIKV challenge by monoclonal antibody LZY2083 1. 100 PFU of JEV or ZIKV were injected subcutaneously into the back of 6-day-old C57BL / 6 mice or 7-day-old A129 mice (with type 1 interferon receptor knocked out); 2.2 h later, 20 mg / kg or 4 mg / kg of the monoclonal antibody LZY2083 was injected subcutaneously into the back of JEV-infected or ZIKV-infected mice, respectively. Three days later, the mice were sacrificed, and serum was collected. Viral load in the mouse serum was detected using a plaque formation assay (PFA) based on Vero cells. The results are expressed as Log 10 (PFU / mL).

[0069] 3. Use Prism 6 to analyze, statistically analyze, and plot the data.

[0070] Experimental results: such as Figure 4 As shown, after ZIKV infection, the viral load in the serum of mice treated with 4 mg / kg monoclonal antibody LZY2083 was significantly lower than that in the control group (treated with PBS) (338,844 PFU / mL). versus The viral load was 4,466,836 PFU / mL (P=0.0079), a decrease of more than 12-fold. Following JEV infection, mice administered 20 mg / kg of the monoclonal antibody LZY2083 also had significantly lower serum viral loads than the control group (389,045 PFU / mL). versus The viral titer was 2041738 PFU / mL (P=0.01), a decrease of more than four times.

[0071] 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 human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus, characterized in that, Including VH CDR1 with amino acid sequence as shown in SEQ ID NO.1, VHCDR2 with amino acid sequence as shown in SEQ ID NO.2, VH CDR3 with amino acid sequence as shown in SEQ ID NO.3, VLCDR1 with amino acid sequence as shown in SEQ ID NO.4, VL CDR2 with amino acid sequence AAS and VL CDR3 with amino acid sequence as shown in SEQ ID NO.

5.

2. The human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus according to claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the human monoclonal antibody or its antigen-binding fragment capable of neutralizing Zika virus and Japanese encephalitis virus is shown in SEQ ID NO.6, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

7.

3. The encoding gene of a human monoclonal antibody or its antigen-binding fragment that can neutralize Zika virus and Japanese encephalitis virus as described in claim 1 or 2.

4. The encoding gene according to claim 3, characterized in that, The coding genes include light chain variable region coding genes and heavy chain variable region coding genes; The nucleotide sequence of the gene encoding the heavy chain variable region is shown in SEQ ID NO.12; The nucleotide sequence of the gene encoding the light chain variable region is shown in SEQ ID NO.

13.

5. A recombinant expression vector, characterized in that, Includes the coding gene as described in claim 3 or 4.

6. A recombinant non-plant host cell, characterized in that, Includes the recombinant expression vector as described in claim 5.

7. The use of the encoding gene as described in claim 3 or 4, the recombinant expression vector as described in claim 5, or the recombinant non-plant host cell as described in claim 6 in the preparation of the human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus as described in claim 1.

8. The use of a human monoclonal antibody or antigen-binding fragment thereof capable of neutralizing Zika virus and Japanese encephalitis virus as described in claim 1 or 2 in (1) or (2): (1) Preparation of detection products for Zika virus and / or Japanese encephalitis virus; (2) Prepare drugs to inhibit Zika virus and / or Japanese encephalitis virus infection.

9. A detection product for Zika virus and / or Japanese encephalitis virus, characterized in that, Includes the human monoclonal antibody or antigen-binding fragment thereof that can neutralize Zika virus and Japanese encephalitis virus as described in claim 1 or 2.

10. A drug for inhibiting Zika virus and / or Japanese encephalitis virus infection, characterized in that, The active ingredient includes the human monoclonal antibody or antigen-binding fragment thereof that can neutralize Zika virus and Japanese encephalitis virus as described in claim 1 or 2.

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