A monoclonal antibody against Japanese encephalitis virus and Zika virus and its application

Abstract

The present invention discloses a monoclonal antibody against Japanese encephalitis virus and Zika virus and its application, which relates to the technical fields of immunology and molecular virology. The monoclonal antibody or its antigen-binding fragment comprises VH CDR1, VH CDR2 and VH CDR3 with amino acid sequences shown in SEQ ID NO: 1-3, and VL CDR1, VL CDR2 and VL CDR3 with amino acid sequences shown in SEQ ID NO: 4-6. After a large number of experimental studies, the present invention discovers a monoclonal antibody which can specifically recognize and target the extracellular region of the E protein of Japanese encephalitis virus and Zika virus, and can block the binding of the E protein to the cell surface receptor, showing a high ability to 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.

Description

Technical Field

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

[0002] Arboviruses such as Japanese encephalitis virus (i.e., Japanese encephalitis virus, JEV) and Zika virus (ZIKV) transmitted by mosquito bites pose a serious threat to human health. The genomes of these flaviviruses are approximately 11 kb in length and encode three structural proteins (C, prM / M, and E proteins). Among them, the E protein mediates virus infection by binding to the surface receptor of sensitive cells, and thus is the key viral antigen that induces the production of neutralizing antibodies. Zika virus infection in adults can lead to neurological autoimmune diseases such as Guillain-Barré syndrome, and infection in pregnant women can lead to microcephaly in newborns. However, there is currently no Zika vaccine approved for use. In addition, 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 method for treating viral diseases. Commercially available drugs for treating and preventing virus infections include palivizumab (Synagis) for preventing respiratory syncytial virus (RSV) infection in children, ibalizumab (Trogarzo) for treating HIV infection, and so on. Antibodies play a therapeutic role mainly in two aspects. First, neutralizing antibodies can bind to viral membrane proteins and block the binding of the virus to cell receptors, thereby blocking virus infection. Second, antibodies can 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 has become extremely urgent to screen broad-spectrum neutralizing monoclonal antibodies with higher affinity and capable of showing neutralizing activity against both 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 object 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 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 cell surface receptors, showing a highly efficient ability to neutralize the virus.

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

[0007] As used in the present invention, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains, each pair having 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, based on which antibodies are defined into five classes: IgM, IgD, IgG, IgA, and IgE. Within the light and heavy chains, the variable and constant regions are joined 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 the immunoglobulin 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 further divided into regions with high variability [called complementarity-determining regions (CDRs)], interspersed with more conserved regions called framework regions (FRs). Each VH and VL are arranged in the following order of seven parts 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 the antibody binding site. The assignment of amino acids to each region or domain follows the definitions 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 by any specific method of antibody production. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be antibodies of different isotypes, e.g., IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0008] As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind the same antigen as 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 ed., Raven Press, N.Y. (1989)], which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of antibodies can be generated by recombinant DNA techniques 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, diabodies (dAb), and such polypeptides that comprise at least a portion of an antibody sufficient to confer upon the polypeptide the ability to specifically bind an antigen.

[0009] In some cases, an antigen-binding fragment of an antibody is a single-chain antibody (e.g., scFv), in which the VL and VH domains are paired by a linker that enables them to be produced as a single polypeptide chain to form a monovalent molecule [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 can have the 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 can be used, but variants thereof can also be used (Holliger et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6444-6448.). Other linkers that can be used 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 on the same chain, thus forcing the domains to pair with the complementary domains on the other chain and generating two antigen-binding sites [see, e.g., Holliger P. et al., 1993, Proc. Natl. Acad. Sci. USA. 90:6444-6448. and Poljak R.J. et al., 1994, Structure. 2:1121-1123.].

[0011] Antigen-binding fragments of an antibody (e.g., the above 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 techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of the antibody 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 referring to the term "antibody", 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, i.e., a group of identical antibody molecules except for possible spontaneous natural mutations. Monoclonal antibodies have high specificity for a single epitope on an antigen. Polyclonal antibodies, in contrast to monoclonal antibodies, generally contain at least two or more different antibodies that usually recognize different epitopes on an antigen. Monoclonal antibodies can generally be 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, e.g., Journal of virological methods, 2009. 158(1-2):171-179].

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

[0015] As used in the present invention, the term "vector" refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, the vector is called an expression vector. A vector can be introduced into a host cell by transformation, transduction, or transfection, enabling the genetic elements it carries to be 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 (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages such as lambda 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, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, polyomaviruses (such as SV40). A vector can contain various elements for controlling expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector can also contain an origin of replication.

[0016] As used in the present invention, the term "host cell" refers to a cell into which a vector can be introduced, 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 in the present 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. In certain embodiments, an antibody that specifically binds to an antigen (or an antibody that is specific for an antigen) refers to an antibody that binds the antigen with a dissociation equilibrium constant (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 M or less.

[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 an antibody and an 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, an antibody (e.g., the monoclonal antibody LZY2006 of the present invention) binds with 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 An antibody with a dissociation equilibrium constant (KD) of M or less binds to an antigen (e.g., the E protein of Japanese encephalitis virus and Zika virus), and this value is determined using surface plasmon resonance (SPR) on a BIACORE 8K instrument.

[0019] In the present invention, amino acids are generally represented by single-letter or three-letter abbreviations well-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 an antibody or antibody fragment that binds to the antigenic protein on a virus, thereby preventing the virus from infecting cells and / or the maturation of viral progeny and / or the release of viral progeny. An antibody or antibody fragment with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating viral infection.

[0021] As used in the present invention, the term "Japanese encephalitis virus" refers to Japanese encephalitis virus (JEV), which has the same meaning as the official classification name of the International Committee on Taxonomy of Viruses (ICTV), and the two can be used interchangeably.

[0022] As used in the present invention, the terms "Japanese encephalitis" and "encephalitis B" 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 mainly characterized by fever caused by ZIKV infection, and the two have the same meaning and can be used interchangeably.

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

[0024] To achieve this object, after a large number of experimental studies, the present invention has discovered an antibody that can specifically recognize and target the E protein of Japanese encephalitis virus and Zika virus, particularly the extracellular region of the E protein, and can block the binding of the E protein to cell surface receptors, showing a high ability to 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 with amino acid sequences as shown in SEQ ID NO: 1-3, and VL CDR1, VL CDR2 and VL CDR3 with amino acid sequences as shown in SEQ ID NO: 4-6.

[0027] Furthermore, the amino acid sequence of the heavy chain variable region is as shown in SEQ ID NO: 7; the amino acid sequence of the light chain variable region is as 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 as shown in SEQ ID NO: 11, and the nucleotide sequence of its encoding gene is as 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 as shown in SEQ ID NO: 12, and the nucleotide sequence of its encoding gene is as shown in SEQ ID NO: 18.

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

[0031] In certain preferred embodiments, the monoclonal antibody further includes a light chain constant region. In certain preferred embodiments, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO: 10. The gene sequence of the light chain constant region is as 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, and inhibit the infection of cells by the virus.

[0034] In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment has neutralizing ability (e.g., capable of neutralizing Japanese encephalitis virus and Zika virus). In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of inhibiting the infection or entry of Japanese encephalitis virus and Zika virus into host cells. Thus, the monoclonal antibody or its antigen-binding fragment can neutralize Japanese encephalitis virus and Zika virus, and thereby prevent and treat diseases associated with Japanese encephalitis virus and Zika virus infections.

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

[0036] Preferably, the coding gene comprises a DNA molecule having a nucleotide sequence 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 its antigen-binding fragment 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 its antigen-binding fragment 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 its antigen-binding fragment 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 its antigen-binding fragment 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, which comprises a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding the heavy chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention; and a second polynucleotide, which comprises a nucleotide sequence encoding a signal peptide and a nucleotide sequence capable of encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention.

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

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

[0042] In certain preferred embodiments, the encoding gene further comprises a nucleotide sequence capable of encoding the light chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the light chain constant region has the amino acid sequence shown in SEQ ID NO: 10. In certain preferred embodiments, the nucleotide sequence capable of encoding the light chain constant region of the monoclonal antibody or antigen-binding fragment thereof of the present invention has the nucleotide sequence 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 the heavy chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a nucleotide sequence capable of encoding the 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 the light chain variable region of the monoclonal antibody or antigen-binding fragment thereof of the present invention, and a nucleotide sequence capable of encoding the 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 the nucleotide sequences shown in SEQ ID NO: 17, SEQ ID NO: 13, and SEQ ID NO: 15; and a second polynucleotide comprising the nucleotide sequences 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, and the like.

[0046] The present invention also provides a recombinant host cell comprising the above-mentioned coding 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, for example, mouse cells, human cells, etc.). The cells of the present invention can also be cell lines, such as 293T cells.

[0047] The present invention also provides the use of the above-mentioned coding gene, recombinant vector, or recombinant host cell in the preparation of 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 antigen-binding fragment thereof in any one of the following (1)-(3):

[0050] (1) Preparing a product 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;

[0051] (2) Preparing a product for neutralizing the virulence of Japanese encephalitis virus or Zika virus;

[0052] (3) Preparing a drug; the drug is used for preventing and / or treating 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, extracellular region of Japanese encephalitis virus E protein, or extracellular region of Zika virus E protein, comprising the above-mentioned monoclonal antibody or antigen-binding fragment thereof.

[0054] In certain preferred embodiments, the product further comprises a second antibody that specifically recognizes the monoclonal antibody or antigen-binding fragment thereof of the present invention or an anti-idiotypic antibody. 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, radioactive isotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (such as horseradish peroxidase), etc.

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

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

[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. Therefore, 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 infections, 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 technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0061] Figure 1 It is the SDS-PAGE electrophoresis diagram of the monoclonal antibody LZY2006 expressed in Example 4 of the present invention; wherein, "without DTT" on the gel diagram indicates that DTT is not added (non-reducing SDS-PAGE), and "DTT" indicates that DTT is added (reducing SDS-PAGE);

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

[0063] Figure 3 It is the detection result diagram of the neutralizing activity of different concentrations of the LZY2006 monoclonal antibody in vitro against Japanese encephalitis virus and Zika virus in Example 6 of the present invention; the ordinate Neutralization (%) represents the neutralization percentage, and the abscissa is the concentration;

[0064] Figure 4Survival experiment results 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 attacks; wherein, A is the percentage of the body weight of mice administered with 1 μg of LZY2006 monoclonal antibody to the original body weight under JEV (10 PFU) infection; B is the percentage survival rate of mice administered with 1 μg of LZY2006 monoclonal antibody under JEV (10 PFU) infection; C is the percentage of the body weight of mice administered with 25 μg of LZY2006 monoclonal antibody to the original body weight under JEV (10 PFU) infection; D is the percentage survival rate of mice administered with 25 μg of LZY2006 monoclonal antibody under JEV (10 PFU) infection; E is the percentage of the body weight of mice administered with 1 μg of LZY2006 monoclonal antibody to the original body weight under ZIKV (1000 PFU) infection; F is the percentage survival rate of mice administered with 1 μg of LZY2006 monoclonal antibody under ZIKV (1000 PFU) infection; G is the percentage of the body weight of mice administered with 25 μg of LZY2006 monoclonal antibody to the original body weight under ZIKV (1000 PFU) infection; H is the percentage survival rate of mice administered with 25 μg of LZY2006 monoclonal antibody under ZIKV (1000 PFU) infection; in A, C, E and G, the abscissa is the number of days after virus infection, and the ordinate is the percentage of the body weight of mice to the original body weight; in B, D, F and H, the abscissa is the number of days after virus infection, and the ordinate is the percentage survival rate of mice. Detailed implementation manners

[0065] The various exemplary implementation manners 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, characteristics and implementation schemes of the present invention.

[0066] It should be understood that the terms used in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0067] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0068] Without departing from the scope or spirit of the present invention, various modifications and variations to the specific embodiments of the description of the present invention will be apparent to those skilled in the art. Other embodiments obtained from the description of the present invention will be apparent to those skilled in the art. The description and examples of the present invention are merely exemplary.

[0069] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0070] Unless otherwise specified, the molecular biology experimental methods and immunoassay methods used in the present invention are basically carried out according to 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., Current Protocols in Molecular Biology, 3rd Edition, John Wiley & Sons, Inc., 1995; the use of restriction endonucleases is carried out according to the conditions recommended by the product manufacturer. For those not specified in the examples, they are carried out according to the conventional conditions or the conditions recommended by the manufacturer. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained commercially.

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

[0072] Table 1 Amino acid and nucleotide sequences

[0073]

[0074]

[0075]

[0076] To obtain neutralizing antibodies with protective effects, the present invention first uses the extracellular region of Zika virus E protein (E410) expressed by Escherichia coli as an antigen, and through flow cytometry sorting, memory B cells that can specifically bind to ZIKV-E410 are screened from the peripheral blood mononuclear cells (PBMCs) of adult healthy volunteers (22 years old) who have been vaccinated with a live attenuated Japanese encephalitis virus vaccine. Then, the single B cells obtained by screening are subjected to BCR 10×Genomics sequencing to obtain the gene sequences encoding the antibody variable regions in individual B cells. Further, the sequences encoding the antibody variable regions are ligated to the constant region genes into an expression vector and expressed and purified in mammalian cells, thereby obtaining the monoclonal antibody LZY2006. A series of functional tests are carried out on the monoclonal antibody LZY2006, and the results show that the monoclonal antibody LZY2006 can specifically bind to JEV-E406 and ZIKV-E410, inhibit the infection of Japanese encephalitis virus and Zika virus to Vero cells, protect mice from lethal Japanese encephalitis virus and Zika virus attacks, and has broad-spectrum neutralizing activity against Japanese encephalitis virus and Zika virus infections. The specific details are as follows:

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

[0078] 1. The gene sequences encoding JEV-E406 (residues 1-406, GenBank accession no: MK558811) and ZIKV-E410 (residues 1-410, GenBank accession no: KX117076.1) with 6 His tags at the C-terminus optimized for Escherichia coli codons are respectively cloned into the prokaryotic expression vector pET21a to obtain the recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410.

[0079] 2. The recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410 are respectively transformed into Escherichia coli BL21 strain and the proteins are induced to express using IPTG.

[0080] 3. The Ni-NTA affinity chromatography column is used to separate and purify JEV-E406 and ZIKV-E410 proteins. SDS-PAGE and WB experiments are used to detect the purity of the two proteins.

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

[0082] 1. With the informed consent of healthy volunteers vaccinated with the live attenuated JEV vaccine, 100 mL of peripheral venous blood is collected from them, and peripheral blood mononuclear cells (PBMC) are separated using human lymphocyte separation medium.

[0083] 2. Mix the isolated PBMC with the 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, anti-human CD235a monoclonal antibody) in a flow tube and incubate at 4 °C for 30 min for binding. Add pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA), then add Anti-biotin microbeads and incubate at 4 °C for 30 min; add pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA), centrifuge at 2000 rpm for 10 min, and discard the supernatant; add pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA) to resuspend the cell pellet, transfer it to a flow tube, insert a magnet and wait for 3 min, collect the unadsorbed cells, wash the adsorbed magnetic beads with pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA), insert a magnet and wait for 3 min, collect the unadsorbed cells; transfer all the 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 with pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA).

[0084] 3. Take 5 mL of magnetic beads (1 μm in diameter) and add them to a flow tube, insert a magnet and wait for 3 min, discard the supernatant; add 4 mL of PBS to resuspend the magnetic beads, insert a magnet and wait for 3 min, discard the supernatant; add 4 mL of 5 μM imidazole solution, insert a magnet and wait for 3 min, discard the supernatant; add protein solution (2 mg of ZIKV-E410) and an appropriate amount of binding buffer to make up to 4 mL, tighten the lid and shake on a shaker for 40 min; insert a magnet and wait for 3 min, discard the supernatant, add 4 mL of PBS to resuspend the magnetic beads, insert a magnet and wait for 3 min, discard the supernatant; add 4 mL of 5% BSA / PBS to resuspend the magnetic beads and shake on a shaker for 10 min; insert a magnet and wait for 3 min, discard the supernatant, add 4 mL of PBS to resuspend the magnetic beads, insert a magnet and wait for 3 min, discard the supernatant; resuspend the above magnetic beads with 1 mL of memory B cell suspension (isolated from 100 mL of peripheral venous blood) and shake on a shaker for 20 min; insert a magnet and wait for 3 min, discard the unbound solution; add 4 mL of pre-cooled buffer to resuspend the magnetic beads, insert a magnet and wait for 3 min, discard the unbound solution, and add pre-cooled buffer to wash down the adsorbed cells (i.e., the 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 Monoclonal Antibody LZY2006 and Construction of Recombinant Expression Vector

[0087] More than 6,000 genes in the V region of the BCR 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 the 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 the LZY2006 monoclonal antibody are shown in Table 2 - Table 3.

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

[0090] Monoclonal antibody V-H allele J-H allele LZY2006 IGHV4-59 IGHJ6

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

[0092] Monoclonal antibody V-L allele J-L allele LZY2006 IGLV3-25 IGLJ2

[0093] The nucleotide sequences encoding the heavy chain and light chain variable regions of LZY2006 obtained by analysis were respectively inserted into the expression vector pCAGGS (constructed in our laboratory) containing the nucleotide sequence encoding the constant region of the heavy chain / κ chain, thereby obtaining the recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the heavy chain and light chain 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] Among them, 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 coding 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 coding gene is shown in SEQ ID NO: 16).

[0099] Example 4 Expression of LZY2006 Monoclonal Antibody

[0100] The heavy chain and light chain plasmids (the recombinant expression vectors pCAGGS-LZY2006H and pCAGGS-LZY2006L encoding the heavy chain and light chain of LZY2006 monoclonal antibody obtained in Example 3) were co-transfected into 293T cells. The weight ratio of the heavy chain plasmid pCAGGS-LZY2006H to the light chain plasmid pCAGGS-LZY2006L was 1.07:0.93. 2 μg of plasmid (heavy chain plasmid + light chain plasmid) and 4 μg of PEI were transfected into 293T cells in each cell culture dish (10 cm in diameter). The cell supernatant was collected at 48 h and 96 h after transfection and filtered through 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 50KDa cut-off molecular weight. Subsequently, electrophoresis was performed by SDS-PAGE (in reducing and non-reducing states). The results are as Figure 1 shown, and 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, surface plasmon resonance analysis was performed using a LifeDiscTM MetaSPR chip (Quantassay [Hangzhou] Biotechnology Co., Ltd.). The specific steps are as follows:

[0103] First, fix JEV-E406 or ZIKV-E410 (20 μg / mL) to the LifeDiscTM MetaSPR chip (Quantum [Hangzhou] Biotechnology Co., Ltd.). Then, successively load LZY2006 serially diluted with PBST solution at pH 7.4 through each channel (40, 80, 160, 320 μg / mL [i.e., 0.267 μM, 0.534 μM, 1.068 μM, 2.136 μM] one by one). Record the kinetic curves of LZY3412 binding to JEV-E406 or ZIKV-E410 protein ( Figure 2 as shown), and calculate the kinetic constants using WeSPR One software (Quantum [Hangzhou] Biotechnology Co., Ltd.) (as shown in Table 4), and fit in the "1:1 binding" mode. Figure 2 The results in and Table 4 show that the monoclonal antibody LZY2006 can bind to the extracellular regions of the E proteins of JEV and ZIKV (JEV-E406 and ZIKV-E410) with high affinity. Among them, the amino acid sequences of JEV-406 and ZIKV-E410 are shown in Table 1.

[0104] Table 4 Affinity of monoclonal antibody LZY2006 for JEV-E406 and ZIKV-E410

[0105]

[0106] Example 6 In vitro neutralization experiments of LZY2006 monoclonal antibody against JEV and ZIKV

[0107] 1. Seed Vero cells in a 24-well plate (1×10 5 cells / well) and culture for 24 h.

[0108] 2. In a 96-well plate, serially dilute the LZY2006 monoclonal antibody to be tested (starting from 3.67 μg / mL), add the virus to be tested (JEV or ZIKV, 50 plaque-forming units (PFU) / well) to each well, and incubate at 37 °C for 1 h.

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

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

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

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

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

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

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

[0116]

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

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

[0119] 2. After 2 h, inject 1 μg or 25 μg of the LZY2006 monoclonal antibody subcutaneously into the back of the mice for the mouse survival experiment (record the body weight and death of the mice daily).

[0120] 3. Analyze, statistically process, and plot the data using Prism 6, and the results are as shown in Figure 4 .

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

[0122] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A monoclonal antibody or its antigen-binding fragment against Japanese encephalitis virus and Zika virus, characterized in that, VH CDR1, VH CDR2 and VH CDR3 comprising the amino acid sequences as shown in SEQ ID NO: 1-3, and VL CDR1, VL CDR2 and VL CDR3 comprising the amino acid sequences as shown in SEQ ID NO: 4-6.

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

8.

3. A coding gene for the monoclonal antibody or its antigen-binding fragment as claimed in claim 1 or 2.

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

14.

5. A recombinant vector, characterized in that, Comprising the coding gene as claimed in claim 3 or 4.

6. A recombinant host cell, characterized in that, Comprising the coding gene as claimed in claim 3 or 4 or the recombinant vector as claimed in claim 5; The recombinant host cell is a non-plant cell.

7. Use of the coding gene as claimed in claim 3 or 4, the recombinant vector as claimed in claim 5 or the recombinant host cell as claimed in claim 6 in the preparation of a monoclonal antibody or its antigen-binding fragment against Japanese encephalitis virus and Zika virus.

8. Use of the monoclonal antibody or its antigen-binding fragment as claimed in claim 1 or 2 in any one of the following (1)-(3): (1) Preparation of a product 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; (2) Preparation of a product for neutralizing the virulence of Japanese encephalitis virus or Zika virus; (3) Preparation of a drug; the drug is used for treating Japanese encephalitis virus infection and / or Zika virus infection.

9. A product 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, characterized in that, Comprising the monoclonal antibody or its antigen-binding fragment as claimed in claim 1 or 2.

10. A pharmaceutical composition, characterized in that, Comprising a pharmaceutically acceptable excipient and the monoclonal antibody or its antigen-binding fragment as claimed in claim 1 or 2.

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