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 relates to the technical field of immunology and molecular virology. The monoclonal antibody or the antigen binding fragment thereof comprises a VH CDR1, a VH CDR2 and a VH CDR3 of which the amino acid sequences are as shown in SEQ ID NO: 1-3, and a VL CDR1, a VL CDR2 and a VL CDR3 of which the amino acid sequences are as shown in SEQ ID NO: 4-6. A large number of experimental studies show that the monoclonal antibody can specifically recognize and target extracellular regions of E proteins of the Japanese encephalitis virus and the Zika virus, can block combination of the E proteins and cell surface receptors, and shows efficient virus neutralizing capacity. Therefore, the antibody provided by the invention is particularly suitable for being used for diagnosing, preventing and / or treating diseases related to Japanese encephalitis virus and Zika virus infection.

Description

Technical Field

[0001] The present invention relates to the technical field of immunology and molecular virology, and in particular to a monoclonal antibody against Japanese encephalitis virus and Zika virus and application thereof. Background Art

[0002] Insect-borne flaviviruses such as Japanese encephalitis virus (JEV) and Zika virus (ZIKV) that are transmitted by mosquito bites pose a serious threat to human health. The genome of this type of flavivirus is approximately 11kb and encodes three structural proteins (C, prM / M and E proteins), of which the E protein mediates viral infection by binding to sensitive cell surface receptors and is therefore a key viral antigen for inducing the production of neutralizing antibodies. ZIKV infection in adults can cause neurological autoimmune diseases such as Guillain-Barré syndrome, and infection in pregnant women can cause microcephaly in newborns, but no Zika vaccine has been approved for use. In addition, for JEV and ZIKV, no specific antiviral drugs or other therapies (such as antibody therapy) have been approved for clinical use.

[0003] At present, neutralizing antibodies are still an effective method for treating viral diseases. Drugs that have been marketed for the treatment and prevention of viral infections include Synagis, which prevents respiratory syncytial virus (RSV) infection in children, and Trogarzo, which treats HIV infection. Antibodies mainly exert their therapeutic effects in two ways. First, neutralizing antibodies can block the binding of viruses to cell receptors by binding to viral membrane proteins, thereby blocking viral infection. Second, antibodies clear free viruses or kill virus-infected cells through antibody-dependent cell-mediated cytotoxicity, antibody-mediated opsonophagocytosis, and activation of the complement system by antigen-antibody complexes.

[0004] Therefore, it is urgent to screen out broadly neutralizing monoclonal antibodies with higher affinity and the ability to simultaneously exhibit neutralizing activity against Japanese encephalitis virus and Zika virus. Such antibodies can play an important role in protecting public life and health. Summary of the invention

[0005] The purpose of the present invention is to provide a monoclonal antibody against Japanese encephalitis virus and Zika virus and its application to solve the problems existing in the above-mentioned prior art. The monoclonal antibody 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 the ability to effectively neutralize the virus.

[0006] In the present invention, unless otherwise specified, the scientific and technical terms used have the meanings commonly understood by those skilled in the art. In addition, the cell culture, molecular genetics, nucleic acid chemistry, immunology, and virology experimental operation steps used in the present invention (if any) are conventional steps widely used in the corresponding fields. At the same time, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0007] As used in the present invention, the term "antibody" refers to an immunoglobulin molecule that is usually composed of two pairs of polypeptide chains [each pair has a "light" (L) chain and a "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, and antibodies are defined as IgM, IgD, IgG, IgA and IgE, a total of five categories. Within the light chain and heavy chain, the variable region and the constant region are connected by a "J" region of about 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 region of an antibody can 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 also be subdivided into regions with high variability [called complementary determining regions (CDRs)], interspersed with more conservative regions called framework regions (FRs). Each VH and VL has 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 (VH and VL) of each heavy chain / light chain pair form antibody binding sites, respectively. The allocation of amino acids to each region or domain follows the definition of 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 the antibody. For example, it includes recombinant antibodies, monoclonal antibodies and polyclonal antibodies. The antibody can be an antibody of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibody.

[0008] As used in the present invention, 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 possessed by the full-length antibody, and / or competes with the full-length antibody for specific binding to the antigen, which is also referred to as an "antigen-binding portion". See generally, Fundamental Immunology, Ch.7 [Paul, W., ed., 2nd edition, Raven Press, NY (1989)], which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be produced 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 complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv), chimeric antibodies, diabodies (diabody, dAb) and such polypeptides, which contain at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

[0009] In some cases, the antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired to form a monovalent molecule by a linker that enables them to be produced as a single polypeptide chain [see, e.g., 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 repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) 4 may be used, but variants thereof may also be used (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA. 90: 6444-6448.). Other linkers useful in the present invention are described by Alfthan et al., 1995, Protein Eng. 8:725-731. Choi et al., 2001, Eur. J. Immunol. 31:94-106.

[0010] In some cases, the antigen-binding fragment of an antibody is a diabody, i.e., a bivalent antibody in which the VH and VL domains are expressed on a single polypeptide chain, but with a linker that is too short to allow pairing between the two domains of the same chain, thereby forcing the domains to pair with the complementary domains of another chain and create two antigen-binding sites [see, e.g., Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA. 90:64446448. and Poljak RJ et al., 1994, Structure. 2:1121-1123.].

[0011] Antibody antigen-binding fragments (e.g., the above-mentioned antibody fragments) can be obtained from a given antibody (e.g., the monoclonal antibody LZY2006 provided by the present invention) using conventional techniques known to those skilled in the art (e.g., recombinant DNA technology or enzymatic or chemical cleavage methods), and the antibody antigen-binding fragments can be screened for specificity in the same manner as for intact antibodies.

[0012] In the present invention, unless the context clearly indicates otherwise, when the term "antibody" is mentioned, it includes not only intact antibodies but also antigen-binding fragments of antibodies.

[0013] As used in the present invention, the term "monoclonal antibody" refers to an antibody or a fragment of an antibody from a group of highly homologous antibody molecules, that is, a group of identical antibody molecules except for natural mutations that may occur spontaneously. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies are relative to monoclonal antibodies, and they usually contain at least 2 or more different antibodies, which usually recognize different epitopes on an antigen. Monoclonal antibodies can usually be obtained using the hybridoma technology first reported by Kohler et al. (1975, Nature. 256: 495), but can also be obtained using recombinant DNA technology [see Journal of virological methods, 2009. 158 (1-2): 171-179].

[0014] As used in the present invention, "neutralizing antibody" refers to an antibody or antibody fragment that can eliminate or significantly reduce the virulence of the target virus (eg, the ability to infect cells).

[0015] As used in the present invention, the term "vector" refers to a nucleic acid carrier into which a polynucleotide can be inserted. When a vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction or transfection, so that the genetic material elements it carries are expressed in the host cell. Vectors are well known to those skilled in the art, and include but are not limited to: plasmids; phagemids; 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 retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses (such as SV40). A vector can contain a variety of elements that control expression, including but not limited to promoter sequences, transcription initiation sequences, enhancer sequences, selection elements and reporter genes. In addition, the vector may also contain a replication initiation site.

[0016] As used in the present invention, the term "host cell" refers to cells that can be used to introduce vectors, 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.

[0017] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as a reaction between an antibody and an antigen to which it is directed. In certain embodiments, an antibody that specifically binds to an antigen (or has specificity for an antigen) refers to an antibody that binds to an antigen with a specificity of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 The dissociation equilibrium (KD) of M or less usually binds to the antigen.

[0018] As used in the present invention, the term "KD" refers to the dissociation equilibrium constant of a specific antibody-antigen interaction, which is used to describe the binding ability between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. Generally, antibodies (e.g., monoclonal antibody LZY2006 of the present invention) have a KD of less than about 10 -5 M, for example, less than about 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10-10 The PCR products bind to antigens (e.g., E protein of Japanese encephalitis virus and Zika virus) with a dissociation equilibrium constant (KD) of 2 M or less, as determined using surface plasmon resonance (SPR) in a BIACORE 8K instrument.

[0019] In the present invention, amino acids are generally represented by single-letter or three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

[0020] As used in the present invention, the term "neutralizing activity" refers to the functional activity of antibodies or antibody fragments that bind to antigenic proteins on viruses, thereby preventing virus-infected cells and / or the maturation of viral progeny and / or the release of viral progeny. Antibodies or antibody fragments with neutralizing activity can prevent the proliferation of viruses, thereby inhibiting or eliminating viral infection.

[0021] As used in the present invention, the term "Japanese encephalitis virus" refers to the Japanese encephalitis virus (JEV) officially classified by the International Committee on Taxonomy of Viruses (ICTV). The two have the same meaning and can be used interchangeably.

[0022] As used in the present invention, the terms "Japanese encephalitis" and "Japanese encephalitis" refer to encephalitis caused by JEV infection; the two have the same meaning and can be used interchangeably. The term "Zika fever" refers to a disease characterized by fever caused by ZIKV infection; the two have the same meaning and can be used interchangeably.

[0023] In view of the problems of the prior art, the object of the present invention is to provide a monoclonal antibody with high affinity to Japanese encephalitis virus and Zika virus and its application.

[0024] To achieve this purpose, the present invention has discovered an antibody after a large number of experimental studies, which 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 the ability to efficiently neutralize the virus. Therefore, the antibody of the present invention is particularly suitable for diagnosing, preventing and / or treating diseases related to Japanese encephalitis virus and Zika virus infection (such as Japanese encephalitis and Zika fever).

[0025] To achieve the above object, the present invention provides the following solutions:

[0026] The present invention provides a monoclonal antibody or an antigen-binding fragment thereof against Japanese encephalitis virus and Zika virus, comprising VH CDR1, VH CDR2 and VH CDR3 having amino acid sequences as shown in SEQ ID NOs: 1-3, and VL CDR1, VL CDR2 and VL CDR3 having amino acid sequences as shown in SEQ ID NOs: 4-6.

[0027] Furthermore, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:8.

[0028] In certain preferred embodiments, the monoclonal antibody further has a signal peptide at the N-terminus of the heavy chain variable region. In certain preferred embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO:11, and the nucleotide sequence of the gene encoding the signal peptide is shown in SEQ ID NO:17.

[0029] In certain preferred embodiments, the monoclonal antibody further has a signal peptide at the N-terminus of the light chain variable region. In certain preferred embodiments, the amino acid sequence of the signal peptide is shown in SEQ ID NO: 12, and the nucleotide sequence of the gene encoding the signal peptide is shown in SEQ ID NO: 18.

[0030] In certain preferred embodiments, the monoclonal antibody further comprises a heavy chain constant region. In certain preferred embodiments, the amino acid sequence of the heavy chain constant region is shown in SEQ ID NO: 9. The gene sequence of the heavy chain constant region is shown in SEQ ID NO: 15.

[0031] In certain preferred embodiments, the monoclonal antibody further comprises a light chain constant region. In certain preferred embodiments, the amino acid sequence of the light chain constant region is shown in SEQ ID NO: 10. The gene sequence of the light chain constant region is shown in SEQ ID NO: 16.

[0032] In certain preferred embodiments, the light chain of the monoclonal antibody is of the κ type.

[0033] In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment can specifically bind to the envelope protein (E protein) of Japanese encephalitis virus and Zika virus. In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment can target the extracellular region of the E protein of Japanese encephalitis virus and Zika virus. In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment can inhibit the receptor binding and / or membrane fusion process mediated by the extracellular region of the E protein, thereby inhibiting the infection of the virus to the cell.

[0034] In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment has neutralizing ability (e.g., can neutralize Japanese encephalitis virus and Zika virus). In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment can inhibit Japanese encephalitis virus and Zika virus from infecting or entering host cells. Thus, the monoclonal antibody or its antigen-binding fragment can neutralize Japanese encephalitis virus and Zika virus, and thus prevent and treat diseases related to Japanese encephalitis virus and Zika virus infection.

[0035] The present invention also provides a gene encoding the above-mentioned monoclonal antibody or its antigen-binding fragment.

[0036] Preferably, the coding gene comprises a DNA molecule with nucleotide sequences as shown in SEQ ID NO: 13 and SEQ ID NO: 14. The DNA molecule shown in SEQ ID NO: 13 can encode the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention; the DNA molecule shown in SEQ ID NO: 14 can encode the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0037] In certain preferred embodiments, the coding gene further comprises a DNA molecule encoding a signal peptide sequence, which is located at the 5' end of the DNA molecule capable of encoding the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 11. In certain preferred embodiments, the DNA molecule encoding the signal peptide has a nucleotide sequence as shown in SEQ ID NO: 17.

[0038] In certain preferred embodiments, the DNA molecule further comprises a nucleotide sequence encoding a signal peptide, which is located at the 5' end of the DNA molecule capable of encoding the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the signal peptide has an amino acid sequence as shown in SEQ ID NO: 12. In certain preferred embodiments, the DNA molecule encoding the signal peptide sequence has a nucleotide sequence as shown in SEQ ID NO: 18.

[0039] In certain preferred embodiments, the DNA molecule comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding a heavy chain variable region of a monoclonal antibody or an antigen-binding fragment thereof of the present invention; and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding a light chain variable region of a monoclonal antibody or an antigen-binding fragment thereof of the present invention.

[0040] In certain preferred embodiments, the encoding gene comprises a first polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:17 and a nucleotide sequence as shown in SEQ ID NO:13; and, a second polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:18 and a nucleotide sequence as shown in SEQ ID NO:14.

[0041] In certain preferred embodiments, the coding gene further comprises a nucleotide sequence capable of encoding a heavy chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the heavy chain constant region has an amino acid sequence as shown in SEQ ID NO: 9. In certain preferred embodiments, the nucleotide sequence capable of encoding a heavy chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the present invention has a nucleotide sequence as shown in SEQ ID NO: 15.

[0042] In certain preferred embodiments, the coding gene further comprises a nucleotide sequence capable of encoding a light chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the light chain constant region has an amino acid sequence as shown in SEQ ID NO: 10. In certain preferred embodiments, the nucleotide sequence capable of encoding a light chain constant region of a monoclonal antibody or antigen-binding fragment thereof of the present invention has a nucleotide sequence as shown in SEQ ID NO: 16.

[0043] In certain preferred embodiments, the encoding gene comprises a first polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence capable of encoding a heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a nucleotide sequence capable of encoding a heavy chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention; and a second polynucleotide comprising a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence capable of encoding a light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a nucleotide sequence capable of encoding a light chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention.

[0044] In certain preferred embodiments, the encoding gene comprises a first polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:17, SEQ ID NO:13 and SEQ ID NO:15; and a second polynucleotide comprising a nucleotide sequence as shown in SEQ ID NO:18, SEQ ID NO:14 and SEQ ID NO:16.

[0045] The present invention also provides a recombinant vector comprising the above-mentioned coding gene. The vector of the present invention can be a cloning vector or an expression vector. In certain preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, etc.

[0046] The present invention also provides a recombinant host cell, comprising the above-mentioned encoding gene or recombinant vector. 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 cell of the present invention can also be a cell line, such as 293T cells.

[0047] The present invention also provides the use of the above-mentioned encoding gene, recombinant vector or recombinant host cell in preparing monoclonal antibodies or antigen-binding fragments thereof against Japanese encephalitis virus and Zika virus.

[0048] The present invention also provides a method for preparing the monoclonal antibody or antigen-binding fragment thereof of the present invention, comprising the steps of culturing the recombinant host cell of the present invention under suitable conditions, and recovering the monoclonal antibody or antigen-binding fragment thereof of the present invention from the cell culture.

[0049] The present invention also provides the use of the above-mentioned monoclonal antibody or its antigen-binding fragment in any one of the following (1)-(3):

[0050] (1) Preparation of products for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region;

[0051] (2) Preparation of products for neutralizing the virulence of Japanese encephalitis virus or Zika virus;

[0052] (3) Preparing a drug; the drug is used to prevent and / or treat diseases related to Japanese encephalitis virus and / or Zika virus infection.

[0053] The present invention also provides a product for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region, including the above-mentioned monoclonal antibody or its antigen-binding fragment.

[0054] In certain preferred embodiments, the product further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof or anti-idiotypic antibody of the present invention. Preferably, the second antibody further comprises 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) and the like.

[0055] The present invention also provides a pharmaceutical composition, comprising a pharmaceutically acceptable adjuvant and the above-mentioned monoclonal antibody or an antigen-binding fragment thereof.

[0056] In certain preferred embodiments, the auxiliary material is a carrier and / or an excipient.

[0057] Preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as ribavirin and the like.

[0058] The present invention discloses the following technical effects:

[0059] The monoclonal antibody LZY2006 of the present invention can bind to the extracellular region of the E protein of Japanese encephalitis virus and Zika virus with high affinity, and has strong neutralizing activity against Japanese encephalitis virus and Zika virus, and therefore, serves as a broad-spectrum neutralizing antibody. The monoclonal antibody LZY2006 of the present invention has ideal clinical application value for preventing and treating Japanese encephalitis virus and Zika virus infection, and is expected to play an important role in protecting public life and health. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0061] Figure 1 This is the SDS-PAGE electrophoresis diagram of the monoclonal antibody LZY2006 expressed in Example 4 of the present invention; wherein, "no DTT" on the gel diagram indicates that DTT was not added (non-reducing SDS-PAGE), and "DTT" indicates that DTT was added (reducing SDS-PAGE);

[0062] Figure 2 The kinetic curves of different concentrations of LZY2006 monoclonal antibody binding to the extracellular region of E protein of Japanese encephalitis virus (A) and the extracellular region of E protein of Zika virus (B) of Example 5 of the present invention; in A and B, the abscissa is time (seconds) and the ordinate is response value (RU);

[0063] Figure 3 The figure is a graph showing the results of the in vitro neutralization activity test of the LZY2006 monoclonal antibody of different concentrations against Japanese encephalitis virus and Zika virus in Example 6 of the present invention; the ordinate Neutralization (%) represents the neutralization percentage, and the abscissa represents the concentration;

[0064] Figure 4The results of the survival experiment of different doses of LZY2006 monoclonal antibody in Example 7 of the present invention to protect mice against lethal Japanese encephalitis virus and Zika virus attack; wherein, A is the percentage of the body weight of mice given 1 μg of LZY2006 monoclonal antibody to the original body weight under JEV (10 PFU) infection; B is the percentage survival rate of mice given 1 μg of LZY2006 monoclonal antibody under JEV (10 PFU) infection; C is the percentage of the body weight of mice given 25 μg of LZY2006 monoclonal antibody to the original body weight under JEV (10 PFU) infection; D is the percentage survival rate of mice given 25 μg of LZY2006 monoclonal antibody under JEV (10 PFU) infection; E is the percentage of the body weight of mice given 1 μg of LZY2006 monoclonal antibody to the original body weight under ZIKV (1000 PFU) infection; F is the percentage of the body weight of mice given 1 μg of LZY2006 monoclonal antibody to the original body weight under ZIKV (1000 PFU) infection. The percentage survival rate of mice infected with LZY2006 monoclonal antibody; G is the percentage of body weight of mice infected with ZIKV (1000 PFU) and given 25 μg LZY2006 monoclonal antibody to the original body weight; H is the percentage survival rate of mice infected with ZIKV (1000 PFU) and given 25 μg LZY2006 monoclonal antibody; in A, C, E and G, the horizontal axis is the number of days after virus infection, and the vertical axis is the percentage of mouse body weight to the original body weight; in B, D, F and H, the horizontal axis is the number of days after virus infection, and the vertical axis is the percentage survival rate of mice. DETAILED DESCRIPTION

[0065] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0066] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0067] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0068] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0069] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0070] Unless otherwise specified, the molecular biology experimental methods and immunoassays used in the present invention are basically carried out with reference to the methods described in J. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, 1989, and FM Ausubel et al., A Compiled Guide to 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. If the specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used without indicating the manufacturer are all conventional products that can be obtained commercially.

[0071] The information of the partial sequences involved in the present invention is shown in Table 1 below.

[0072] Table 1 Amino acid and nucleotide sequences

[0073]

[0074]

[0075]

[0076] In order to obtain neutralizing antibodies with protective effects, the present invention first uses the extracellular region (E410) of Zika virus E protein expressed by Escherichia coli as an antigen, and screens memory B cells that can specifically bind to ZIKV-E410 from peripheral blood mononuclear cells (PBMCs) of healthy adult volunteers (22 years old) vaccinated with Japanese encephalitis virus attenuated vaccine by flow sorting, and then performs BCR 10×Genomics sequencing on the single B cells obtained by screening to obtain the gene sequence encoding the variable region of the antibody in the single B cell. Further, the sequence encoding the variable region of the antibody is connected to the constant region gene into an expression vector, and expressed and purified in mammalian cells to obtain the monoclonal antibody LZY2006. A series of functional tests were performed on the monoclonal antibody LZY2006, and the results showed that the monoclonal antibody LZY2006 could specifically bind to JEV-E406 and ZIKV-E410, inhibit the infection of Japanese encephalitis virus and Zika virus on Vero cells, protect mice against lethal Japanese encephalitis virus and Zika virus attacks, and had broad-spectrum neutralizing activity against Japanese encephalitis virus and Zika virus infection. The details are as follows:

[0077] Example 1 Expression and purification of the extracellular region of E protein of Japanese encephalitis virus and Zika virus

[0078] 1. The codon-optimized JEV-E406 (residues 1-406, GenBank accession no: MK558811) and ZIKV-E410 (residues 1-410, GenBank accession no: KX117076.1) coding gene sequences with six 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.

[0079] 2. The recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410 were transformed into Escherichia coli BL21 strain and IPTG was used to induce protein expression.

[0080] 3. The JEV-E406 and ZIKV-E410 proteins were separated and purified using Ni-NTA affinity chromatography columns. The purity of the two proteins was detected by SDS-PAGE and WB experiments.

[0081] Example 2 Isolation of memory B cells that specifically recognize ZIKV-E410 protein

[0082] 1. With the informed consent of healthy volunteers who received JEV live attenuated vaccine, 100 mL of peripheral venous blood was collected and peripheral blood mononuclear cells (PBMCs) were separated using human lymphocyte separation medium.

[0083] 2. The separated PBMCs were mixed with a biotinylated antibody mixture 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 cytometry tube and incubated at 4°C for 30 minutes. Add pre-cooled buffer (0.5% BSA / PBS, containing 20mM EDTA), add Anti-biotin microbeads, and incubate at 4°C for 30min; add pre-cooled buffer (0.5% BSA / PBS, containing 20mM EDTA), centrifuge at 2000rpm for 10min, and discard the supernatant; add pre-cooled buffer (0.5% BSA / PBS, containing 20mM EDTA) to suspend the cell pellet, transfer it to a flow tube, insert a magnet and wait for 3min, collect the unadsorbed cells, use pre-cooled buffer (0.5% BSA / PBS, containing 20mM EDTA) to wash the adsorbed magnetic beads, insert a magnet and wait for 3min, and collect the unadsorbed cells; transfer all unadsorbed cell fluids to a 15mL centrifuge tube; centrifuge at 2000rpm for 10min, discard the supernatant, and use pre-cooled buffer (0.5% BSA / PBS, containing 20mM EDTA) to suspend the cell (memory B cell) pellet.

[0084] 3. Take 5mL magnetic beads (1μm diameter) and add them to the flow tube, insert the magnet and wait for 3min, and discard the supernatant; add 4mL PBS to suspend the magnetic beads, insert the magnet and wait for 3min, and discard the supernatant; add 4mL 5μM imidazole solution, insert the magnet and wait for 3min, and discard the supernatant; add protein solution (2mg ZIKV-E410) and an appropriate amount of binding buffer to 4 mL, tighten the lid and shake on a shaker for 40 minutes; insert a magnet and wait for 3 minutes, discard the supernatant, add 4 mL PBS to suspend the magnetic beads, insert a magnet and wait for 3 minutes, discard the supernatant; add 4 mL 5% BSA / PBS to suspend the magnetic beads, and shake on a shaker for 10 minutes; insert a magnet and wait for 3 minutes, discard the supernatant, add 4 mL PBS to suspend the magnetic beads, insert a magnet and wait for 3 minutes, discard the supernatant; suspend the above magnetic beads with 1 mL memory B cell suspension (separated from 100 mL peripheral venous blood), and shake on a shaker for 20 minutes; insert a magnet and wait for 3 minutes, discard the unbound liquid; add 4 mL pre-cooled buffer to suspend the magnetic beads, insert a magnet and wait for 3 minutes, discard the unbound liquid, and add pre-cooled buffer to wash the adsorbed cells (i.e., target cells).

[0085] 4. Send the above cells to the company for 10× single-cell expression profiling sequencing + BCR library construction sequencing and analysis.

[0086] Example 3 Isolation and identification of LZY2006 monoclonal antibody and construction of recombinant expression vector

[0087] A total of more than 6,000 genes of the BCR V region of memory B cells were detected. Through sequence analysis, the present invention selected the variable region (V region) sequences of the light chain and heavy chain of a monoclonal antibody named LZY2006 for subsequent research.

[0088] The amino acid sequence of the heavy chain variable region of LZY2006 monoclonal antibody is shown in SEQ ID NO: 7 (the encoding gene is shown in SEQ ID NO: 13), the amino acid sequence of VH CDR1 of the heavy chain variable region is shown in SEQ ID NO: 1, the amino acid sequence of VH CDR2 is shown in SEQ ID NO: 2, and the amino acid sequence of VH CDR3 is shown in SEQ ID NO: 3. The amino acid sequence of the light chain variable region is shown in SEQ ID NO: 8 (the encoding gene is shown in SEQ ID NO: 14), the amino acid sequence of VL CDR1 of the light chain variable region is shown in SEQ ID NO: 4, the amino acid sequence of VL CDR2 is shown in SEQ ID NO: 5, and the amino acid sequence of VL CDR3 is shown in SEQ ID NO: 6. The V region genes of the heavy chain and light chain of LZY2006 monoclonal antibody are shown in Tables 2-3.

[0089] Table 2 LZY2006 monoclonal antibody heavy chain V region gene

[0090] Monoclonal antibody VH alleles JH allele LZY2006 IGHV4-59 IGHJ6

[0091] Table 3 LZY2006 monoclonal antibody light chain V region gene

[0092] Monoclonal antibody VL allele JL allele LZY2006 IGLV3-25 IGLJ2

[0093] The nucleotide sequences encoding the variable regions of the heavy and light chains of LZY2006 were respectively inserted into the corresponding expression vector pCAGGS (constructed in our laboratory) containing the nucleotide sequences encoding the constant regions of the heavy and κ chains, thereby obtaining the recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the heavy and light chains of the LZY2006 monoclonal antibody, respectively.

[0094] Heavy chain coding sequence (5'-3'): CMV promoter-EcoR I restriction site-signal peptide sequence gene-VH gene-CH gene-Xho I restriction site;

[0095] Light chain (κ) coding sequence (5'-3'): CMV promoter-EcoR I restriction site-signal peptide sequence gene-VL gene-CL (κ) gene-Xho I restriction site;

[0096] Wherein, the amino acid sequence of the signal peptide sequence of the heavy chain coding sequence is shown in SED ID NO: 11 (the nucleotide sequence of the coding gene is shown in SEQ ID NO: 17), and the amino acid sequence of the signal peptide sequence of the light chain coding sequence is shown in SED ID NO: 12 (the nucleotide sequence of the coding gene is shown in SEQ ID NO: 18);

[0097] The nucleotide sequence of the VH gene is shown in SEQ ID NO: 13; the nucleotide sequence of the VL gene is shown in SEQ ID NO: 14;

[0098] The amino acid sequence of CH is shown in SED ID NO:9 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO:15), and the amino acid sequence of CL is shown in SED ID NO:10 (the nucleotide sequence of the encoding gene is shown in SEQ ID NO:16).

[0099] Example 4 Expression of LZY2006 monoclonal antibody

[0100] Heavy chain and light chain plasmids (recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the heavy chain and light chain of LZY2006 monoclonal antibody respectively obtained in Example 3) were co-transfected into 293T cells. The weight ratio of heavy chain plasmid pCAGGS-LZY2006H to light chain plasmid pCAGGS-LZY2006L was 1.07:0.93, and 293T cells per cell dish (10 cm in diameter) were transfected with 2 μg of plasmid (heavy chain plasmid + light chain plasmid) and 4 μg of PEI. Cell supernatants were collected 48 h and 96 h after transfection and filtered with a 0.22 μm syringe filter, then purified using a Pierce protein A / G agarose column (Thermo Fisher), and the monoclonal antibody was concentrated using an ultrafiltration tube with a molecular weight cutoff of 50 KDa. Then, the samples were electrophoresed by SDS-PAGE (under reducing and non-reducing conditions). Figure 1 As shown, purified LZY2006 monoclonal antibody was obtained.

[0101] Example 5 Evaluation of the binding ability of LZY2006 monoclonal antibody to JEV-E406 and ZIKV-E410

[0102] In this example, the surface plasmon resonance analysis was performed using LifeDiscTM MetaSPR chip (Quantum Precision [Hangzhou] Biotechnology Co., Ltd.). The specific steps are as follows:

[0103] First, JEV-E406 or ZIKV-E410 (20 μg / mL) was fixed to the LifeDiscTM MetaSPR chip (Quantum Precision [Hangzhou] Biotechnology Co., Ltd.), and then LZY2006 serially diluted with pH 7.4 PBST solution was sequentially passed through each channel (40, 80, 160, 320 μg / mL [i.e., 0.267 μM, 0.534 μM, 1.068 μM, 2.136 μM] were loaded one by one). The kinetic curve of LZY3412 binding to JEV-E406 or ZIKV-E410 protein was recorded ( Figure 2 The kinetic constants (as shown in Table 4) were calculated using WeSPR One software (Quantum Precision Biotechnology Co., Ltd., Hangzhou) and fitted in the "1:1 binding" mode. Figure 2 The results in Table 4 show that LZY2006 monoclonal antibody can bind to the extracellular region of the E protein of JEV and ZIKV (JEV-E406 and ZIKV-E410) with high affinity. The amino acid sequence of JEV-406 and the amino acid sequence of ZIKV-E410 are shown in Table 1.

[0104] Table 4 Affinity of LZY2006 mAb to JEV-E406 and ZIKV-E410

[0105]

[0106] Example 6 Experiment of neutralization of JEV and ZIKV by LZY2006 monoclonal antibody in vitro

[0107] 1. Plate Vero cells in 24-well plates (1×10 5 / well) and cultured for 24 h.

[0108] 2. The LZY2006 monoclonal antibody to be tested was diluted 3-fold in a 96-well plate (starting from 3.67 μg / mL), and the virus to be tested (JEV or ZIKV, 50 viral plaque-forming units (PFU) / well) was added to each well and incubated at 37°C for 1 h.

[0109] 3. Discard the Vero cell supernatant, add the virus / monoclonal antibody mixture to the cells, and infect at 37°C for 1.5 hours.

[0110] 4. Discard the virus solution, add 1% methylcellulose culture medium to each well to cover the cells, and culture for 3-6 days.

[0111] 5. Add 4% paraformaldehyde to each well and fix at room temperature for 1 hour, then wash the plate.

[0112] 6. Add 0.5% crystal violet staining solution to each well and stain for 10 minutes at room temperature, then wash the plate.

[0113] 7. Count the number of virus plaques in each well and calculate the neutralization titer (half inhibitory concentration, IC) of the diluted monoclonal antibody against each virus. 50 value).

[0114] IC 50 GraphPad Prism 6 software was used for analysis. The analysis results are shown in Figure 3 and Table 5. It can be seen that LZY2006 mAb can inhibit JEV and ZIKV with extremely high neutralizing activity.

[0115] Table 5 Neutralization titers (half inhibitory concentration, IC) of LZY2006 monoclonal antibody against JEV and ZIKV 50 )

[0116]

[0117] Example 7 Experiment on the protection of mice by LZY2006 monoclonal antibody against lethal JEV or ZIKV attack

[0118] 1. Inject JEV (10 PFU) or ZIKV (1000 PFU) subcutaneously into the back of 1-day-old mice;

[0119] 2.2 hours later, 1 μg or 25 μg of LZY2006 monoclonal antibody was injected subcutaneously into the back of mice for a mouse survival experiment (mouse body weight and mortality were recorded daily).

[0120] 3. Use Prism 6 to analyze, count and plot the data. The results are as follows: Figure 4 shown.

[0121] The results showed that after JEV infection, the percentage survival rate of mice given 1 μg or 25 μg of LZY2006 monoclonal antibody was significantly higher than that of mice not given LZY2006 monoclonal antibody (16.7% versus 0, P<0.0001; 42.9% versus 0, P=0.0001, respectively); after ZIKV infection, the percentage survival rate of mice given 25 μg of LZY2006 monoclonal antibody was significantly higher than that of mice not given LZY2006 monoclonal antibody (50% versus 0, P=0.0011).

[0122] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoclonal antibody or antigen-binding fragment thereof against Japanese encephalitis virus and Zika virus, It is characterized in that It comprises VH CDR1, VH CDR2 and VH CDR3 whose amino acid sequences are shown in SEQ ID NOs: 1-3, and VL CDR1, VL CDR2 and VL CDR3 whose amino acid sequences are shown in SEQ ID NOs: 4-6.

2. The monoclonal antibody or antigen-binding fragment thereof according to claim 1, It is characterized in that The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO:7; the amino acid sequence of the light chain variable region is shown in SEQ ID NO:

8.

3. A gene encoding the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2.

4. The coding gene according to claim 3, It is characterized in that A DNA molecule comprising the nucleotide sequence shown in SEQ ID NO: 13 and / or SEQ ID NO:

14.

5. A recombinant vector, It is characterized in that Comprising the coding gene according to claim 3 or 4.

6. A recombinant host cell, It is characterized in that Comprising the coding gene according to claim 3 or 4 or the recombinant vector according to claim 5.

7. Use of the encoding gene according to claim 3 or 4, the recombinant vector according to claim 5 or the recombinant host cell according to claim 6 in preparing monoclonal antibodies or antigen-binding fragments thereof against Japanese encephalitis virus and Zika virus.

8. Use of the monoclonal antibody or antigen-binding fragment thereof as claimed in claim 1 or 2 in any one of the following (1) to (3): (1) Preparation of products for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region; (2) Preparation of products for neutralizing the virulence of Japanese encephalitis virus or Zika virus; (3) Preparing a drug; the drug is used to prevent and / or treat diseases related to Japanese encephalitis virus and / or Zika virus infection.

9. A product for detecting Japanese encephalitis virus, Zika virus, Japanese encephalitis virus E protein, Zika virus E protein, Japanese encephalitis virus E protein extracellular region or Zika virus E protein extracellular region, It is characterized in that The invention comprises the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

10. A pharmaceutical composition, It is characterized in that The invention comprises pharmaceutically acceptable excipients and the monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2.

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