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

Abstract

The present invention provides a human monoclonal antibody against Japanese encephalitis virus and Zika virus and its application, belonging to the cross-field of immunology and molecular virology. The monoclonal antibody provided by the present invention includes: CDR1 of the heavy chain variable region shown in SEQ ID No.1, CDR2 of the heavy chain variable region shown in SEQ ID No.2, CDR3 of the heavy chain variable region shown in SEQ ID No.3, CDR1 of the light chain variable region shown in SEQ ID No.4, CDR2 of the light chain variable region shown in SEQ ID No.5, and CDR3 of the light chain variable region shown in SEQ ID No.6. The human monoclonal antibody provided by 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 has ideal clinical application value for preventing and treating Japanese encephalitis virus and Zika virus infections.

Description

Technical Field

[0001] The present invention relates to the cross - field of immunology and molecular virology. Specifically, it relates to a human monoclonal antibody against Japanese encephalitis virus and Zika virus and its applications. Background Art

[0002] Arboviruses such as Japanese encephalitis virus (i.e., Japanese B encephalitis virus, JEV) and Zika virus (ZIKV), which are transmitted by mosquito bites, seriously threaten human health. The genomes of these flaviviruses are approximately 11 kb and encode three structural proteins (C, prM / M, and E protein). Among them, the E protein mediates virus infection by binding to the surface receptors of sensitive cells, and thus is the key viral antigen that induces the production of neutralizing antibodies.

[0003] Currently, Japanese encephalitis virus is often detected by reverse transcriptase polymerase chain reaction (RT - PCR) testing and IgM detection in serum / cerebrospinal fluid. Since the viremia period of Japanese encephalitis virus infection is short, and the detection results are affected by other febrile diseases (such as dengue fever, malaria, leptospirosis, and other arbovirus infections), the detection rate is relatively low. At the same time, there is currently no specific antiviral drug or other therapy for the treatment of Japanese encephalitis virus infection.

[0004] Currently, the diagnosis of Zika virus is often confirmed by reverse transcriptase polymerase chain reaction (RT - PCR) testing. However, the clinical manifestations of Zika virus infection are similar to those of many febrile diseases (such as dengue fever, malaria, leptospirosis, and other arbovirus infections), and its geographical distribution is similar to that of other arboviruses. Therefore, the diagnosis of Zika virus infection requires laboratory confirmation by one of the following: 1) NAAT to detect viral RNA in serum or urine; 2) serological tests (enzyme - linked immunosorbent assay [ELISA] to detect IgM, plaque reduction neutralization test [PRNT] to detect Zika virus antibodies). This makes the existing detection techniques relatively cumbersome. At the same time, the window period after Zika virus infection is extremely short, and the false - negative rate of RT - PCR is relatively high. At the same time, there is currently no specific antiviral drug or other therapy for the treatment of Zika virus infection.

[0005] Therefore, it is extremely urgent to screen monoclonal antibodies with high affinity and showing activity against Japanese encephalitis virus and Zika virus. Such antibodies are of great significance for protecting public life and health. Summary of the Invention

[0006] The technical problem to be solved by the present invention is to provide a monoclonal antibody with high affinity and showing neutralizing activity against both Japanese encephalitis and Zika viruses simultaneously.

[0007] To solve the above problems, a first aspect of the present invention provides a monoclonal antibody against Japanese encephalitis virus and Zika virus, and the monoclonal antibody comprises: CDR1 of the heavy chain variable region shown in SEQ ID No.1, CDR2 of the heavy chain variable region shown in SEQ ID No.2, CDR3 of the heavy chain variable region shown in SEQ ID No.3, CDR1 of the light chain variable region shown in SEQ ID No.4, CDR2 of the light chain variable region shown in SEQ ID No.5, and CDR3 of the light chain variable region shown in SEQ ID No.6.

[0008] The human monoclonal antibody provided by the present invention is a neutralizing antibody, 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 cell surface receptors, showing a highly efficient ability to neutralize the virus. Therefore, the antibody of the present invention is particularly suitable for diagnosing, preventing and treating diseases related to Japanese encephalitis virus and Zika virus infection (such as Japanese encephalitis and Zika fever).

[0009] Preferably, the monoclonal antibody further comprises FR1 of the heavy chain variable region framework region shown in SEQ ID No.7, FR2 of the heavy chain variable region framework region shown in SEQ ID No.8, FR3 of the heavy chain variable region framework region shown in SEQ ID No.9, FR4 of the heavy chain variable region framework region shown in SEQ ID No.10, FR1 of the light chain variable region framework region shown in SEQ ID No.11, FR2 of the light chain variable region framework region shown in SEQ ID No.12, FR3 of the light chain variable region framework region shown in SEQ ID No.13, and FR4 of the light chain variable region framework region shown in SEQ ID No.14.

[0010] In some preferred embodiments, the amino acid sequence of the heavy chain variable region of the monoclonal antibody provided by the present invention is as shown in SEQ ID No.15, and the amino acid sequence of the light chain variable region is as shown in SEQ ID No.16.

[0011] Preferably, the monoclonal antibody comprises:

[0012] (a) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.15;

[0013] (b) a light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.16;

[0014] (c) the heavy chain variable region sequence as in (a) and the light chain variable region sequence as in (b).

[0015] In some preferred embodiments, the monoclonal antibody comprises:

[0016] 1) A heavy chain variable region sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID No. 15;

[0017] 2) A light chain variable region sequence having at least 95% sequence identity with the amino acid sequence shown in SEQ ID No. 16;

[0018] 3) The heavy chain variable region sequence as in 1) and the light chain variable region sequence as in 2).

[0019] Preferably, the monoclonal antibody further comprises a signal peptide sequence at the N-terminus of the heavy chain variable region and / or a signal peptide sequence at the N-terminus of the light chain variable region.

[0020] In some preferred embodiments, the signal peptide sequence at the N-terminus of the heavy chain variable region is as shown in SEQ ID No. 19.

[0021] In some preferred embodiments, the signal peptide sequence at the N-terminus of the light chain variable region is as shown in SEQ ID No. 20.

[0022] In some preferred embodiments, the antigen-binding fragment of the monoclonal antibody provided by the present invention is selected from any one or more of Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity-determining region fragment, single-chain antibody (e.g., scFv), human antibody, chimeric antibody, or bispecific or multispecific antibody.

[0023] In some preferred embodiments, the monoclonal antibody provided by the present invention further comprises a heavy chain constant region and a light chain constant region, wherein the amino acid sequence of the heavy chain constant region is as shown in SEQ ID No. 17, and the amino acid sequence of the light chain constant region is as shown in SEQ ID No. 18.

[0024] In some preferred embodiments, the light chain of the monoclonal antibody provided by the present invention is of the κ type or λ type, preferably the κ type.

[0025] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment can specifically bind to the envelope proteins (E proteins) of Japanese encephalitis virus and Zika virus.

[0026] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment can target the extracellular region of the E proteins of Japanese encephalitis virus and Zika virus.

[0027] In some 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.

[0028] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment has neutralizing ability.

[0029] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment is capable of inhibiting 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 thereby achieve the purpose of preventing and treating infections of Japanese encephalitis virus and Zika virus.

[0030] Furthermore, a second aspect of the present invention provides a nucleic acid molecule for encoding the monoclonal antibody or its antigen-binding fragment described in the first aspect. The nucleic acid molecule includes nucleotide sequences shown in SEQ ID No. 21 and SEQ ID No. 22. Such nucleic acid molecules are not limited by the methods for their production and can be obtained by genetic engineering recombinant techniques or chemical synthesis methods.

[0031] In some preferred embodiments, the nucleic acid molecule includes a first polynucleotide, which contains a nucleotide sequence encoding a signal peptide sequence, 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 nucleotide sequence capable of encoding the heavy chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention; and a second polynucleotide, which contains a nucleotide sequence encoding a signal peptide sequence, a nucleotide sequence capable of encoding the light chain variable region of the monoclonal antibody or its antigen-binding fragment of the present invention, and a nucleotide sequence capable of encoding the light chain constant region of the monoclonal antibody or its antigen-binding fragment of the present invention.

[0032] In some preferred embodiments, the nucleic acid molecule further includes a nucleotide sequence encoding a heavy chain constant region sequence. The heavy chain constant region of the monoclonal antibody provided by the present invention can be obtained by transcription and translation of the nucleotide sequence shown in SEQ ID No. 23.

[0033] In some preferred embodiments, the nucleic acid molecule further includes a nucleotide sequence encoding a light chain constant region sequence. The light chain constant region of the monoclonal antibody provided by the present invention can be obtained by transcription and translation of the nucleotide sequence shown in SEQ ID No. 24.

[0034] In some preferred embodiments, the nucleic acid molecule further includes a nucleotide sequence encoding a signal peptide sequence, which is located at the 5' end of the nucleotide sequence capable of encoding the heavy chain variable region of the monoclonal antibody of the present invention.

[0035] In some preferred embodiments, the signal peptide sequence located at the N-terminus of the heavy chain variable region can be obtained by transcription and translation of the nucleotide sequence shown in SEQ ID No. 25.

[0036] In some preferred embodiments, the signal peptide sequence located at the N-terminus of the light chain variable region can be obtained by transcription and translation of the nucleotide sequence shown in SEQ ID No. 26.

[0037] Furthermore, a third aspect of the present invention provides a vector, which comprises the nucleotide sequences shown in SEQ ID No. 21 and SEQ ID No. 22.

[0038] In some preferred embodiments, the vector provided by the present invention further comprises any one or more nucleic acid molecules selected from SEQ ID No. 23 to SEQ ID No. 26. The vector provided by the present invention can be a cloning vector or an expression vector.

[0039] In some preferred embodiments, the vector of the present invention is, for example, a plasmid, a cosmid, a phage, and the like.

[0040] Furthermore, a fourth aspect of the present invention provides a host cell, which contains the nucleic acid molecule described in the second aspect or the vector described in the third aspect, and the host cell is a prokaryotic cell or a eukaryotic cell.

[0041] In some preferred embodiments, the host cell is a prokaryotic cell or a eukaryotic cell.

[0042] More preferably, the host cell is selected from Escherichia coli cells, yeast cells, insect cells, plant cells, mammalian cells, mouse cells, and human cells. The cells of the present invention can also be a cell line, such as 293T cells.

[0043] In some preferred embodiments, the present invention also provides a method for preparing the monoclonal antibody or its antigen-binding fragment of the present invention, which comprises culturing the host cell of the present invention under suitable conditions and recovering the monoclonal antibody or its antigen-binding fragment from the cell culture.

[0044] Furthermore, a fifth aspect of the present invention provides a pharmaceutical composition, which comprises:

[0045] (i) any one of the monoclonal antibodies described in claims 1 to 4, the nucleic acid molecule described in claim 5, the vector described in claim 6, and the host cell described in claim 7; and

[0046] (ii) a pharmaceutically acceptable carrier and / or excipient.

[0047] More preferably, the pharmaceutical composition further comprises other pharmaceutically active agents, such as ribavirin.

[0048] In some preferred embodiments, the pharmaceutical composition may be a diagnostic agent or a therapeutic agent.

[0049] Furthermore, a sixth aspect of the present invention provides an application, specifically including:

[0050] 1) The application of the monoclonal antibody described in the first aspect in the preparation of diagnostic products for Japanese encephalitis virus and / or Zika virus infection;

[0051] 2) The application of the monoclonal antibody described in the first aspect in the preparation of drugs for the treatment of Japanese encephalitis virus and / or Zika virus infection;

[0052] 3) The application of the nucleic acid molecule described in the second aspect in the preparation of drugs for the treatment of Japanese encephalitis virus and / or Zika virus infection;

[0053] 5) The application of the vector described in the third aspect in the preparation of drugs for the treatment of Japanese encephalitis virus and / or Zika virus infection;

[0054] 6) The application of the host cell described in the fourth aspect in the preparation of drugs for the treatment of Japanese encephalitis virus and / or Zika virus infection;

[0055] 7) The application of the pharmaceutical composition described in the fifth aspect in the preparation of drugs for the treatment of Japanese encephalitis virus and / or Zika virus infection.

[0056] In some preferred embodiments, the monoclonal antibody or its antigen-binding fragment provided by the present invention further includes a detectable label.

[0057] In some preferred embodiments, the aforementioned kit further includes a second antibody that specifically recognizes the monoclonal antibody or its antigen-binding fragment of the present invention or an anti-idiotypic antibody. Preferably, the second antibody further includes a detectable label. Such detectable labels are well known to those skilled in the art and include, but are not limited to, radioactive isotopes, fluorescent substances, luminescent substances, colored substances, and enzymes (such as horseradish peroxidase), etc.

[0058] More preferably, the present invention provides a method for detecting the presence or level of Japanese encephalitis virus, Zika virus, or their E protein or the extracellular region of the E protein in a sample, which includes using the monoclonal antibody or its antigen-binding fragment of the present invention. In certain preferred embodiments, the monoclonal antibody or its antigen-binding fragment of the present invention further includes a detectable label. In another preferred embodiment, the method further includes using a second antibody carrying a detectable label to detect the monoclonal antibody or its antigen-binding fragment of the present invention. The method can be used for diagnostic purposes (for example, the sample is a sample from a patient), or non-diagnostic purposes (for example, the sample is a cell sample, rather than a sample from a patient).

[0059] More preferably, the present invention provides a method for diagnosing whether a subject is infected with Japanese encephalitis virus or Zika virus, which comprises: detecting the presence of Japanese encephalitis virus, Zika virus or their E protein or the extracellular region of the E protein in a sample from the subject using the monoclonal antibody or antigen-binding fragment thereof of the present invention. In certain preferred embodiments, the monoclonal antibody or antigen-binding fragment thereof of the present invention further comprises a detectable label. In another preferred embodiment, the method further comprises using a second antibody carrying a detectable label to detect the monoclonal antibody or antigen-binding fragment thereof or the anti-idiotypic antibody of the present invention.

[0060] More preferably, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof of the first aspect in the preparation of a product, such as a kit, for detecting the presence or level of Japanese encephalitis virus, Zika virus or their E protein or the extracellular region of the E protein in a sample, or for diagnosing whether a subject is infected with Japanese encephalitis virus or Zika virus.

[0061] In some preferred embodiments, the sample includes but is not limited to excreta, oral or nasal secretions, bronchoalveolar lavage fluid, etc. from a subject (such as a mammal, preferably a human). The general methods of using a monoclonal antibody or antigen-binding fragment thereof to detect the presence or level of a target virus or antigen (such as Japanese encephalitis virus, Zika virus or their E protein or the extracellular region of the E protein) in a sample are well known to those skilled in the art. In certain preferred embodiments, the detection method can use enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay, chemiluminescent immunoassay, radioimmunoassay, fluorescence immunoassay, immunochromatography, competitive assay and similar detection methods.

[0062] More preferably, the present invention provides a method for neutralizing the virulence of Japanese encephalitis virus and Zika virus in a sample, which comprises contacting the sample containing Japanese encephalitis virus and Zika virus with the monoclonal antibody or antigen-binding fragment thereof of the present invention. Such methods can be used for therapeutic purposes or non-therapeutic purposes (for example, the sample is a cell sample, rather than a patient or a sample from a patient).

[0063] More preferably, the present invention provides the use of the monoclonal antibody or antigen-binding fragment thereof provided in the first aspect in the preparation of a drug for neutralizing the virulence of Japanese encephalitis virus and Zika virus in a sample. In another aspect, the present invention provides the monoclonal antibody or antigen-binding fragment thereof as described above for neutralizing the virulence of Japanese encephalitis virus and Zika virus in a sample.

[0064] More preferably, the present invention provides the use of the monoclonal antibody or its antigen-binding fragment provided in the first aspect in the preparation of a pharmaceutical composition for preventing or treating Japanese encephalitis virus, Zika virus infection in a subject or diseases associated with these viral infections (such as Japanese encephalitis, Zika fever).

[0065] More preferably, the present invention provides the monoclonal antibody or its antigen-binding fragment as described above for preventing or treating Japanese encephalitis virus, Zika virus infection in a subject or diseases associated with these viral infections (such as Japanese encephalitis, Zika fever).

[0066] Compared with the prior art, the beneficial effects of the present invention are as follows: The monoclonal antibody provided by 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. Specifically, the monoclonal antibody provided by the present invention has a very high affinity for the extracellular region of the E protein of Japanese encephalitis virus and Zika virus, and the neutralization titer data (half-inhibitory concentration, IC 50 ) against Japanese encephalitis virus and Zika virus are very excellent. Therefore, as a broad-spectrum neutralizing antibody, the monoclonal antibody of the present invention has ideal clinical application value for preventing and treating Japanese encephalitis virus and Zika virus infections. BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 It is the WB detection result of the monoclonal antibody LZY3412 expressed in Example 4 of the specific embodiment of the present invention;

[0068] Figure 2 It is the kinetic curve result of different concentrations of the monoclonal antibody LZY3412 binding to the extracellular region of the E protein of Japanese encephalitis virus (JEV-E406) and the extracellular region of the E protein of Zika virus (ZIKV-E410) in Example 5 of the specific embodiment of the present invention;

[0069] Figure 3 It is the graph of the neutralizing activity test results of different concentrations of the monoclonal antibody LZY3412 against Japanese encephalitis virus and Zika virus in vitro in Example 6 of the specific embodiment of the present invention;

[0070] Figure 4 It is the graph of the survival experiment results of different doses of the monoclonal antibody LZY3412 protecting mice against lethal Japanese encephalitis virus and Zika virus attacks in Example 7 of the specific embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0071] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description of specific embodiments of the present invention. It should be noted that the following embodiments are only used to illustrate the implementation methods and typical parameters of the present invention, and are not used to limit the parameter ranges described in the present invention. Reasonable variations derived therefrom are still within the scope of protection of the claims of the present invention.

[0072] It should be noted that the endpoints and any values within the ranges disclosed in this document are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the values between the endpoints of each range, between the endpoints of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed in this document.

[0073] Unless otherwise defined, all terms, symbols, and other scientific terms used in this document are intended to have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In some cases, for the purpose of clarification or convenient reference, terms with conventional meanings are defined in this document. Such definitions in this document should not be construed as indicating a significant difference from the conventional understanding in the art. The technical methods described or cited in this document are generally well understood by those skilled in the art and are adopted by conventional methods. Unless otherwise specified, the use of commercially available kits, reagents, and instruments follows the protocols and parameters provided by the manufacturers.

[0074] Term Explanation:

[0075] Antibody: An immunoglobulin molecule 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, and based on this, antibodies are defined as 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 the 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 of the classical complement system (C1q). The VH and VL regions can also be further divided into regions of high variability, called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is 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)

[0076] Antigen-binding fragment: A polypeptide that comprises a fragment of a full-length antibody and that retains the ability to specifically bind the same antigen to which the full-length antibody binds, and / or competes with the full-length antibody for specific binding to the antigen, also referred to as an “antigen-binding portion”. See generally, Fundamental Immunology, Ch. 7

Paul, W., ed., 2nd ed., Raven Press, N.Y. (1989)

see, e.g., Bird et al., 1988, Science. 242:423-426. and Huston et al., 1988, Proc. Natl. Acad. Sci. USA. 85:5879-5883.

[0077] Monoclonal antibody: 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, typically 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]. 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).

[0078] Vector: A nucleic acid vehicle into which a polynucleotide can be inserted. When the vector enables the expression of 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, enabling the genetic material 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, etc. 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 multiple elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector can also contain an origin of replication.

[0079] Host cell: A cell that can be used to introduce a vector, including, but not limited to, prokaryotic cells such as Escherichia coli, fungal cells such as yeast cells, insect cells such as S2 Drosophila cells, animal cells such as CHO cells, or human cells such as HEK293 cells, etc.

[0080] Specific binding: 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) means that the antibody binds to the antigen with a dissociation equilibrium constant (KD) of less than approximately 10 -5 M, for example less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10 M or less.

[0081] 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 (for example, the monoclonal antibody LZY3412 of the present invention) binds to the antigen with a KD of less than approximately 10 -5 M, for example less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, or 10 -10A 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 measured using surface plasmon resonance (SPR) in a BIACORE 8K device.

[0082] Neutralizing activity: refers to the functional activity of an antibody or antibody fragment that can bind to the antigen protein on the virus, thereby preventing the virus from infecting cells and / or the maturation of virus progeny and / or the release of virus progeny. Antibodies or antibody fragments with neutralizing activity can prevent the amplification of the virus, thereby inhibiting or eliminating virus infection.

[0083] Japanese encephalitis virus: refers to Japanese encephalitis virus (JEV), which is the official classification name of the International Committee on Taxonomy of Viruses (ICTV). The two have the same meaning and can be used interchangeably.

[0084] Japanese encephalitis and Japanese B encephalitis: refer to encephalitis caused by JEV infection; the two have the same meaning and can be used interchangeably.

[0085] Zika fever: refers to a disease mainly characterized by fever caused by ZIKV infection. The two have the same meaning and can be used interchangeably.

[0086] As described in the background art, currently, for Japanese encephalitis virus infection and Zika virus infection, there are technical defects such as a high missed detection rate in detection and no specific drugs for these viruses. To solve the above problems, the specific embodiments of the present invention provide a monoclonal antibody against Japanese encephalitis virus and Zika virus, 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 a high ability to neutralize the virus. Therefore, the antibody of the present invention is particularly suitable for diagnosing, preventing, and treating diseases related to Japanese encephalitis virus and Zika virus infection (such as Japanese encephalitis and Zika fever).

[0087] Specifically, the monoclonal antibody provided by the specific embodiments of the present invention includes: the heavy chain variable region CDR1 (VH-CDR1) shown in SEQ ID No.1, the heavy chain variable region CDR2 (VH-CDR2) shown in SEQ ID No.2, the heavy chain variable region CDR3 (VH-CDR3) shown in SEQ ID No.3, the light chain variable region CDR1 (VL-CDR1) shown in SEQ ID No.4, the light chain variable region CDR2 (VL-CDR2) shown in SEQ ID No.5, and the light chain variable region CDR3 (VL-CDR3) shown in SEQ ID No.6.

[0088] In the monoclonal antibody provided in the above embodiment, it further includes the heavy chain variable region framework region FR1 (VH-FR1) shown in SEQ ID No.7, the heavy chain variable region framework region FR2 (VH-FR2) shown in SEQ ID No.8, the heavy chain variable region framework region FR3 (VH-FR3) shown in SEQ ID No.9, the heavy chain variable region framework region FR4 (VH-FR4) shown in SEQ ID No.10, the light chain variable region framework region FR1 (VL-FR1) shown in SEQ ID No.11, the light chain variable region framework region FR2 (VL-FR2) shown in SEQ ID No.12, the light chain variable region framework region FR3 (VL-FR3) shown in SEQ ID No.13, and the light chain variable region framework region FR4 (VL-FR4) shown in SEQ ID No.14.

[0089] Specifically, in the above embodiment, the amino acid sequence of the heavy chain variable region (VH) of the monoclonal antibody provided by the present invention is as shown in SEQ ID No.15, and the amino acid sequence of the light chain variable region (VL) is as shown in SEQ ID No.16.

[0090] More specifically, the amino acid sequence of the heavy chain constant region (CH) of the monoclonal antibody provided by the present invention is as shown in SEQ ID No.17; the amino acid sequence of the light chain constant region (CL) is as shown in SEQ ID No.18.

[0091] More specifically, in the above embodiment, the monoclonal antibody provided by the present invention should include:

[0092] (a) A heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.15;

[0093] (b) A light chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.16;

[0094] (c) The heavy chain variable region sequence as in (a) and the light chain variable region sequence as in (b).

[0095] In the above embodiment, the monoclonal antibody further includes a signal peptide sequence at the N-terminus of the heavy chain variable region and / or a signal peptide sequence at the N-terminus of the light chain variable region.

[0096] More specifically, the signal peptide sequence at the N-terminus of the heavy chain variable region is preferably as shown in SEQ ID No.19; the signal peptide sequence at the N-terminus of the light chain variable region is preferably as shown in SEQ ID No.20.

[0097] The specific embodiments of the present invention further provide a nucleic acid molecule, which is used to encode the aforementioned monoclonal antibody or its antigen-binding fragment, and should preferably include the nucleotide sequences shown in SEQ ID No.21 and SEQ ID No.22.

[0098] More specifically, the nucleic acid molecule further includes the nucleotide sequence shown in SEQ ID No.23 for encoding the heavy chain constant region and the nucleotide sequence shown in SEQ ID No.24 for encoding the light chain constant region.

[0099] More specifically, the nucleic acid molecule further includes the nucleotide sequence shown in SEQ ID No.25 for encoding the signal peptide at the N-terminus of the heavy chain variable region and the nucleotide sequence shown in SEQ ID No.26 for encoding the signal peptide at the N-terminus of the light chain variable region.

[0100] The specific embodiments of the present invention further provide a vector, which includes the nucleotide sequences shown in SEQ ID No.21 and SEQ ID No.22.

[0101] More specifically, the vector further includes any one or more nucleic acid molecules among SEQ ID No.23 to SEQ ID No.26.

[0102] The specific embodiments of the present invention further provide a host cell, which is a prokaryotic cell or a eukaryotic cell.

[0103] The technical solutions of the present invention will be further described below through specific examples.

[0104] In the specific embodiments of the present invention, first, the extracellular region of Zika virus E protein (E410) expressed by Escherichia coli was used as an antigen. Through flow cytometry sorting, memory B cells that can specifically bind to ZIKV-E410 were screened from the peripheral blood mononuclear cells (PBMCs) of adult healthy volunteers (22 years old) who had been vaccinated with the attenuated Japanese encephalitis virus vaccine. Then, the single B cells obtained by screening were subjected to BCR 10Genomics sequencing to obtain the gene sequences encoding the antibody variable regions in the single B cells. Further, the sequences encoding the antibody variable regions were ligated to the expression vector and expressed and purified in mammalian cells to obtain the monoclonal antibody LZY3412. A series of functional tests were carried out on the monoclonal antibody LZY3412. The results showed that the monoclonal antibody LZY3412 could specifically bind to JEV-E406 and ZIKV-E410, inhibit the infection of Japanese encephalitis virus and Zika virus on Vero cells, protect mice from lethal Japanese encephalitis virus and Zika virus attacks, and had broad-spectrum neutralizing activity against Japanese encephalitis virus and Zika virus infections.

[0105] Example 1

[0106] Expression and Purification of the Extracellular Region of the E Protein of Japanese Encephalitis Virus and Zika Virus

[0107] The coding sequences of JEV-E406 (residues 1-406, GenBank accession no: MK558811) and ZIKV-E410 (residues 1-410, GenBank accession no: KX117076.1) with a 6-His tag at the C-terminus optimized for E. coli codons were cloned into the prokaryotic expression vector pET21a. Among them, the amino acid sequence of JEV-406 is shown in SEQ ID No. 27, and the amino acid sequence of ZIKV-E410 is shown in SEQ ID No. 28.

[0108] The recombinant plasmids pET21a-JEV-E406 and pET21a-ZIKV-E410 were respectively transformed into E. coli BL21 strain and induced to express proteins with IPTG.

[0109] The JEV-E406 and ZIKV-E410 proteins were separated and purified using a Ni-NTA affinity chromatography column. The purity of the two proteins was detected by SDS-PAGE and WB experiments.

[0110] Example 2

[0111] Isolation of Memory B Cells Specifically Recognizing the ZIKV-E410 Protein

[0112] With the informed consent of healthy volunteers vaccinated with the live attenuated JEV vaccine, 100 ml of peripheral venous blood was collected from them, and peripheral blood mononuclear cells (PBMCs) were isolated using human lymphocyte separation medium.

[0113] Mix the isolated PBMC with a biotinylated antibody mixture (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), 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 suspend the cell pellet, transfer 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, and collect the unadsorbed cells; transfer all the unadsorbed cell suspension to a 15 ml centrifuge tube; centrifuge at 2000 rpm for 10 min, discard the supernatant, and suspend the cell (memory B cell) pellet with pre-cooled buffer (0.5% BSA / PBS containing 20 mM EDTA).

[0114] 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, and discard the supernatant; add 4 ml of PBS to suspend the magnetic beads, insert a magnet and wait for 3 min, and discard the supernatant; add 4 ml of 5 μM imidazole solution, insert a magnet and wait for 3 min, and discard the supernatant; add protein solution (2 mg of ZIKV-E410) and an appropriate amount of binding buffer to 4 ml, tighten the cap and shake on a shaker for 40 min; insert a magnet and wait for 3 min, discard the supernatant, add 4 ml of PBS to suspend the magnetic beads, insert a magnet and wait for 3 min, and discard the supernatant; add 4 ml of 5% BSA / PBS to suspend 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 suspend the magnetic beads, insert a magnet and wait for 3 min, and discard the supernatant; suspend 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, and discard the unbound solution; add 4 ml of pre-cooled buffer to suspend the magnetic beads, insert a magnet and wait for 3 min, and discard the unbound solution, and add pre-cooled buffer to wash down the adsorbed cells (i.e., the target cells).

[0115] Perform 10× single-cell expression profiling sequencing + BCR library construction sequencing and analysis on the above cells.

[0116] Example 3

[0117] Isolation and Identification of LZY3412 Monoclonal Antibody and Construction of Recombinant Expression Vector

[0118] More than 6,000 memory B cell BCR V-region genes were measured. Through sequence analysis, the variable region (V-region) sequences of the light and heavy chains of a monoclonal antibody named LZY3412 were selected for subsequent research. The amino acid sequence of the heavy chain variable region of LZY3412 monoclonal antibody is shown in SEQ ID No.15 (the encoding gene is shown in SEQ ID No.21). The CDR1 of the heavy chain variable region has the amino acid sequence shown in SEQ ID No.1, CDR2 has the amino acid sequence shown in SEQ ID No.2, and CDR3 has the amino acid sequence shown in SEQ ID No.3. The amino acid sequence of the light chain variable region is shown in SEQ ID No.16 (the encoding gene is shown in SEQ ID No.22). The CDR1 of the light chain variable region has the amino acid sequence shown in SEQ ID No.4, CDR2 has the amino acid sequence shown in SEQ ID No.5, and CDR3 has the amino acid sequence shown in SEQ ID No.6. The V-region genes of the heavy and light chains of LZY3412 monoclonal antibody are shown in Table 1 and Table 2.

[0119] Table 1 Heavy Chain V-Region Gene of LZY3412 Monoclonal Antibody

[0120] V-H allele J-H allele LZY3412 IGHV4-39 IGHJ6

[0121] Table 2 Light Chain V-Region Gene of LZY3412 Monoclonal Antibody

[0122] V-L allele J-L allele LZY3412 IGKV1D-39 IGKJ1

[0123] The nucleotide sequences encoding the heavy / light chain variable regions of LZY3412 obtained by analysis were synthesized and then cloned into the expression vector pCAGGS containing the nucleotide sequences encoding the constant regions of the heavy / κ chains respectively, so as to obtain the recombinant expression vectors encoding the heavy and light chains of LZY3412 monoclonal antibody. The construction methods of the constructs expressing the heavy and light chains are as follows:

[0124] Heavy chain coding sequence (5'-3'): CMV promoter - EcoR I restriction site - signal peptide sequence gene - VH gene - CH gene - Xho I restriction site;

[0125] 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;

[0126] Among them, the amino acid sequence of the signal peptide sequence is shown in SED ID No. 19 (the encoding gene is shown in SEQ ID No. 25), the amino acid sequence of CH is shown in SED ID No. 17 (the encoding gene is shown in SEQ ID No. 23), and the amino acid sequence of CL is shown in SED ID No. 18 (the encoding gene is shown in SEQ ID No. 24).

[0127] Example 4

[0128] The heavy chain and light chain plasmids (the recombinant expression vectors pCAGGS-LZY3412H and pCAGGS-LZY3412L encoding the heavy chain and light chain of the LZY3412 monoclonal antibody obtained in Example 3) were co-transfected into 293T cells. The weight ratio of the heavy chain plasmid pCAGGS-LZY3412H to the light chain plasmid pCAGGS-LZY34IP was 1.07:0.93. 20 μg of plasmid (heavy chain plasmid + light chain plasmid) and 40 μg of PEI were transfected into 293T cells in each cell culture dish (diameter 10 cm). The cell supernatant was collected at 48 h and 96 h after transfection and filtered through a 0.22 μm syringe filter, and then purified using a Pierce protein A / G agarose column (Thermo Fisher), and concentrated using a 50 kDa cut-off molecular weight ultrafiltration tube (Millipore). Subsequently, SDS-PAGE (in reducing and non-reducing states) electrophoresis and membrane transfer were performed for WB experiments. The results are as Figure 1 shown. Among them, "M" on the gel diagram represents the protein Marker; "without DTT" means no DTT was added (non-reducing SDS-PAGE), and "DTT" means DTT was added (reducing SDS-PAGE); "Anti-human IgG L chain" means the detection antibody for WB was a monoclonal antibody against human IgG light chain labeled with HRP, and "Anti-human IgG H chain" means the detection antibody for WB was a monoclonal antibody against human IgG heavy chain labeled with HRP. The results showed that the purified LZY3412 monoclonal antibody was obtained.

[0129] Example 5

[0130] Evaluation of the binding ability of LZY3412 monoclonal antibody to JEV-E406 and ZIKV-E410

[0131] In this example, surface plasmon resonance analysis was performed using a LifeDiscTM MetaSPR chip (Quanzhun [Hangzhou] Biotechnology Co., Ltd.). The specific steps are as follows:

[0132] First, fix JEV-E406 or ZIKV-E410 (20 μg / ml) to the LifeDiscTM MetaSPR chip (Quantassay [Hangzhou] Biotechnology Co., Ltd.). Then, sequentially load LZY3412 serially diluted with PBST solution at pH 7.4 through each channel (loading one by one at 40, 80, 160, 320 μg / ml [i.e., 0.267 μM, 0.534 μM, 1.068 μM, 2.136 μM]). 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 (Quantassay [Hangzhou] Biotechnology Co., Ltd.) (as shown in Table 3), and fit in the "1:1 binding" mode. Figure 2 The results in and Table 3 show that the LZY3412 monoclonal antibody 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 sequence of JEV-406 is as shown in SEQ ID No.27, and the amino acid sequence of ZIKV-E410 is as shown in SEQ ID No.28.

[0133] Table 3 Affinity of LZY3412 Monoclonal Antibody with JEV-E406 and ZIKV-E410

[0134] Antigen Affinity KD (nM) JEV-E406 440 ± 16 ZIKV-E410 482.5 ± 91.5

[0135] Example 6

[0136] In vitro neutralization of JEV and ZIKV by LZY3412 monoclonal antibody

[0137] Seed Vero cells in a 24-well plate (1×10 5 cells / well) and culture for 24 h.

[0138] In a 96-well plate, serially dilute the LZY3412 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.

[0139] Discard the supernatant of Vero cells, add the virus / monoclonal antibody mixture to the cells, and infect at 37°C for 1.5 h.

[0140] Discard the virus solution, cover the cells with 1% methylcellulose culture medium, and culture for 3 - 6 days.

[0141] Add 4% paraformaldehyde to each well and fix at room temperature for 1 h, then wash the plate.

[0142] Add 0.5% crystal violet staining solution to each well and stain at room temperature for 10 min, then wash the plate.

[0143] Count the number of viral plaques in each well and calculate the neutralization titer (half-inhibitory concentration, IC 50 value) of the diluted monoclonal antibody against each virus.

[0144] IC 50 Analyze using GraphPad Prism 6 software. The analysis results are shown in Table 4 and Figure 3 . It can be seen that the LZY3412 monoclonal antibody can inhibit JEV and ZIKV with extremely high neutralizing activity.

[0145] Table 4 Neutralization titers (half-inhibitory concentration, IC 50 ) of LZY3412 monoclonal antibody against JEV and ZIKV

[0146] Virus <![CDATA[Half inhibitory concentration, IC 50 (ng / ml)]]> JEV 35.7 ± 4.1 ZIKV 2211 ± 951.5

[0147] Example 7

[0148] Experiment on the protection of mice by LZY3412 monoclonal antibody against lethal JEV or ZIKV challenge

[0149] Inject JEV (10 PFU) or ZIKV (1000 PFU) subcutaneously into the back of 1-day-old C57BL / 6 strain mice;

[0150] Two hours later, inject 1 or 10 μg of LZY3412 monoclonal antibody subcutaneously into the back of the mice for the mouse survival experiment (record the body weight and death of the mice daily).

[0151] Use Prism 6 to analyze, statistically process, and plot the data. The results are as Figure 4 shown. After JEV infection, the percentage survival rates of mice given 1 μg or 10 μg of LZY3412 monoclonal antibody were significantly higher than those of mice not given LZY3412 monoclonal antibody (33.3% versus 7.7%, P = 0.0196; 50% versus 8.3%, P = 0.0444); after ZIKV infection, the percentage survival rates of mice given 1 μg or 10 μg of LZY3412 monoclonal antibody were significantly higher than those of mice not given LZY3412 monoclonal antibody (28.6% versus 0, P = 0.0329; 50% versus 0, P = 0.0011).

[0152] Although the present disclosure is disclosed as above, the scope of protection of the present disclosure is not limited thereto. Those skilled in the art can make various changes and modifications without departing from the spirit and scope of the present disclosure, and these changes and modifications will all fall within the scope of protection of the present invention.

Claims

1. A human monoclonal antibody against Japanese encephalitis virus and Zika virus, characterized in that, The monoclonal antibody comprises: CDR1 of the heavy chain variable region shown in SEQ ID No.1, CDR2 of the heavy chain variable region shown in SEQ ID No.2, CDR3 of the heavy chain variable region shown in SEQ ID No.3, CDR1 of the light chain variable region shown in SEQ ID No.4, CDR2 of the light chain variable region shown in SEQ ID No.5, and CDR3 of the light chain variable region shown in SEQ ID No.

6.

2. The monoclonal antibody according to claim 1, wherein The monoclonal antibody further comprises FR1 of the heavy chain variable region framework region shown in SEQ ID No.7, FR2 of the heavy chain variable region framework region shown in SEQ ID No.8, FR3 of the heavy chain variable region framework region shown in SEQ ID No.9, FR4 of the heavy chain variable region framework region shown in SEQ ID No.10, FR1 of the light chain variable region framework region shown in SEQ ID No.11, FR2 of the light chain variable region framework region shown in SEQ ID No.12, FR3 of the light chain variable region framework region shown in SEQ ID No.13, and FR4 of the light chain variable region framework region shown in SEQ ID No.

14.

3. The monoclonal antibody according to claim 1, wherein The monoclonal antibody comprises: (a) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.15; (b) a heavy chain variable region sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID No.16; (c) the heavy chain variable region sequence in (a) and the light chain variable region sequence in (b).

4. The monoclonal antibody according to claim 1, characterized in that, The monoclonal antibody further comprises a signal peptide sequence at the N-terminus of the heavy chain variable region and / or a signal peptide sequence at the N-terminus of the light chain variable region.

5. A nucleic acid molecule, characterized in that, The nucleic acid molecule is used for encoding any one of the monoclonal antibodies or antigen-binding fragments thereof as claimed in claims 1 to 4, and the nucleic acid molecule comprises the nucleotide sequences shown in SEQ ID No.21 and SEQ ID No.

22.

6. A carrier, characterized in that, The vector comprises the nucleotide sequences shown in SEQ ID No.21 and SEQ ID No.

22.

7. A host cell, characterized in that, The host cell contains the nucleic acid molecule as claimed in claim 5 or the vector as claimed in claim 6, and the host cell is selected from any one or more of Escherichia coli cells, yeast cells, insect cells, and mammalian cells.

8. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises: (i) any one of the monoclonal antibodies as claimed in claims 1 to 4, the nucleic acid molecule as claimed in claim 5, the vector as claimed in claim 6, and the host cell as claimed in claim 7; and (ii) a pharmaceutically acceptable carrier and / or excipient.

9. An application, characterized in that, Comprises: 1) The use of any one of the monoclonal antibodies as claimed in claims 1 to 4 in the preparation of a diagnostic product for Japanese encephalitis virus and / or Zika virus infection; 2) The use of any one of the monoclonal antibodies as claimed in claims 1 to 4 in the preparation of a drug for treating Japanese encephalitis virus and / or Zika virus infection; 3) The use of the nucleic acid molecule as claimed in claim 5 in the preparation of a drug for treating Japanese encephalitis virus and / or Zika virus infection; 5) Use of the vector according to claim 6 in the preparation of a medicament for treating Japanese encephalitis virus and / or Zika virus infection; 6) Use of the host cell according to claim 7 in the preparation of a medicament for treating Japanese encephalitis virus and / or Zika virus infection; 7) Use of the pharmaceutical composition according to claim 8 in the preparation of a medicament for treating Japanese encephalitis virus and / or Zika virus infection.

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