Antibody for Detection Specific for Non-structural protein 1 of ZIKA virus and Use thereof

Abstract

The present invention relates to an antibody specific to the non-structural protein 1 (NS1) of the Zika virus. More specifically, the antibody of the present invention has the ability to specifically bind to NS1 of the Zika virus and is not specific to other flaviviruses, so it is very useful for the diagnosis of the Zika virus.

Description

Technology Field

[0001] The present invention relates to a diagnostic antibody capable of specifically binding to non-structural protein 1 (NS1) of the Zika virus and its use. Background Technology

[0003] The Zika virus is one of the representative mosquito-borne viruses. The Zika virus is a single positive-stranded RNA virus and belongs to the Flavivirus genus, a sub-genus of the Flaviviridae family. In addition to the Zika virus, flaviviruses include the Dengue virus, Yellow Fever virus, Japanese Encephalitis virus, St. Louis Encephalitis virus, and West Nile virus.

[0004] All flaviviruses are spherical and have a common structure consisting of approximately 10,000 to 11,000 bases. The proteins that make up flaviviruses can be broadly classified into structural proteins and non-structural proteins. Although the functions or roles of all proteins have not yet been elucidated, it is understood that precursor membrane proteins, envelope proteins, non-structural protein 1, parts of non-structural proteins 2A and 2B, and parts of non-structural proteins 4A and 4B are exposed on the outside of the virus. Therefore, when attempting to detect the Zika virus—which is a flavivirus—using antibodies, the envelope protein, non-structural protein 1, non-structural proteins 2A and 2B, and non-structural proteins 4A and 4B, which are exposed on the outside of the Zika virus and facilitate access and binding (or reaction) with antibodies, can be considered useful biomarkers (targets, target substances).

[0005] The recent increase in pediatric infections and potential microcephaly and other neurological disorders associated with Zika virus infection has led to a growing demand for laboratory tests to detect Zika virus infection. In this context, high specificity of antibodies is required to distinguish Zika virus infection from infections of other flaviviruses. However, known anti-Zika antibodies are typically cross-reactive with other flaviviruses and are therefore not useful for distinguishing Zika virus infection from infections of other flaviviruses.

[0006] Therefore, there is a need to develop antibodies that are specific only to the Zika virus but not specific to other flaviviruses, as well as diagnostic methods using such antibodies. The problem to be solved

[0008] To solve the above problem, the inventors developed an antibody having a specific binding ability to the non-structural protein 1 (NS1) of the Zika virus, and completed the present invention by confirming that this antibody has excellent binding ability to the Zika virus.

[0009] One aspect is to provide a Zika virus-specific antibody or a functional fragment thereof that specifically binds to non-structural protein 1 of the Zika virus (ZIKV) and includes a heavy chain variable region and a light chain variable region.

[0010] Another aspect is to provide a polynucleotide encoding the above antibody or its binding fragment.

[0011] Another aspect is to provide an expression vector into which the above nucleic acid molecule is inserted.

[0012] Another aspect is to provide a hybridoma cell that produces the aforementioned antibody or its binding fragment.

[0013] Another aspect is to provide a Zika virus diagnostic kit containing a reagent for diagnosing Zika virus (ZIKV) antigens.

[0014] Another aspect is to provide a composition for diagnosing Zika virus comprising the above antibody or a binding fragment thereof. means of solving the problem

[0016] 1. It specifically binds to Non-structural Protein 1 of the Zika virus (ZIKV), and

[0017] A Zika virus-specific antibody or a binding fragment thereof comprising a heavy chain variable region and a light chain variable region.

[0018] 2. In the above 1, the heavy chain variable region comprises CDR1-VH containing the amino acid sequence of SEQ ID NO. 1, CDR2-VH containing the amino acid sequence of SEQ ID NO. 2, and CDR3-VH containing the amino acid sequence of SEQ ID NO. 3, and

[0019] A Zika virus-specific antibody or a binding fragment thereof, characterized in that the light chain variable region comprises CDR1-VK containing the amino acid sequence of SEQ ID NO. 4, CDR2-VK containing the amino acid sequence of SEQ ID NO. 5, and CDR3-VK containing the amino acid sequence of SEQ ID NO. 6.

[0020] 3. In the above 1, the heavy chain variable region comprises CDR1-VH containing the amino acid sequence of SEQ ID NO. 17, CDR2-VH containing the amino acid sequence of SEQ ID NO. 18, and CDR3-VH containing the amino acid sequence of SEQ ID NO. 19, and

[0021] A Zika virus-specific antibody or a binding fragment thereof, characterized in that the light chain variable region comprises CDR1-VK containing the amino acid sequence of SEQ ID NO. 20, CDR2-VK containing the amino acid sequence of SEQ ID NO. 21, and CDR3-VK containing the amino acid sequence of SEQ ID NO. 22.

[0022] 4. In any one of claims 1 to 3 above, the antibody or its binding fragment is an antibody or its binding fragment which is a scFv fragment, a scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody.

[0023] 5. In the above 4, the antibody or its binding fragment is an antibody or its binding fragment that is a Fab fragment.

[0024] 6. A polynucleotide encoding the antibody of any one of claims 1 to 3 above or a binding fragment thereof.

[0025] 7. The polynucleotide of 6 above, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 7 and the base sequence of SEQ ID NO. 8.

[0026] 8. The polynucleotide of 6 above, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 23 and the base sequence of SEQ ID NO. 24.

[0027] 9. An expression vector into which the polynucleotide of 6 above is inserted.

[0028] 10. An expression vector according to 9, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO. 7 and the nucleotide sequence of SEQ ID NO. 8.

[0029] 11. An expression vector according to 9, wherein the polynucleotide comprises the nucleotide sequence of SEQ ID NO. 23 and the nucleotide sequence of SEQ ID NO. 24.

[0030] 12. A hybridoma cell that produces the antibody or a binding fragment thereof of any one of claims 1 to 3 above.

[0031] 13. A Zika virus diagnostic kit comprising a preparation for diagnosing Zika virus (ZIKV) antigens,

[0032] The above viral antigen is non-structural protein 1 of the Zika virus, and

[0033] The above-mentioned formulation is a diagnostic kit that is an antibody of any one of claims 1 to 3 or a binding fragment thereof.

[0034] 14. A composition for diagnosing Zika virus comprising an antibody or a binding fragment thereof according to any one of claims 1 to 3 above. Effects of the invention

[0036] The antibody of the present invention has a specific binding ability to the non-structural protein 1 (NS1) of the Zika virus and is not specific to other flaviviruses, so that Zika virus antibodies present in biological samples derived from patients infected with the Zika virus can be effectively bound. Effective binding of antibodies that recognize Zika virus-specific antigens can dramatically increase sensitivity and accuracy during diagnosis. Brief explanation of the drawing

[0038] Figure 1 is a figure showing a structure for the production of Zika virus NS1 protein (ZIKV NS1 protein). Figure 2 shows the isolation and purification of the non-structural protein 1 (NS1) antigen protein of the Zika virus. Figure 3 shows the Fusion screening results for Zika NS1_CTD. Figure 4 shows the ELISA test results for Zika NS1 and Dengue NS1 of the Zika NS1_CTD fusion clone. Figure 5 shows the results of SDS-PAGE analysis of the purified protein of the Zika NS1_CTD monoclonal antibody. Figure 6 is a figure confirming the degree of binding of Zika virus NS1-specific single-chain antibodies to Zika virus NS1 antigens and dengue virus NS1 antigens. Figure 7 shows a rapid diagnostic kit for point-of-care testing containing a pair of antibodies for diagnosing the Zika virus. Figure 7a shows the Zika virus-specific diagnostic results according to the concentration of the Zika virus-specific antibody. Figure 7b shows that the Zika virus-specific antibody did not bind to the dengue virus even when the concentration of the antibody changed. Figure 8 shows the nucleotide sequences of VH and VL of the 4B1 antibody among Zika virus-specific antibodies. Figure 9 shows the nucleotide sequences of VH and VL of the 5D12 antibody, which is a Zika virus-specific antibody. Specific details for implementing the invention

[0039] The inventors have developed an antibody or a binding fragment thereof that has a specific binding ability to the non-structural protein 1 (NS1) of the Zika virus and does not specifically bind to other viruses, and have confirmed that said antibody or its binding fragment can be very useful for the diagnosis, prevention, and treatment of the Zika virus.

[0040] The present invention will be described in detail below.

[0042] The present invention provides an antibody that specifically binds to non-structural protein 1 (NS1) of the Zika virus (ZIKV).

[0043] The antibody of the present invention has excellent binding ability to non-structural protein 1 of the Zika virus (ZIKV). In one embodiment, the antibody of the present invention can exhibit excellent diagnostic, preventive, and therapeutic effects against the Zika virus by specifically binding only to NS1 of the Zika virus.

[0044] The term “binding molecule” as used in the present invention comprises an intact immunoglobulin comprising a monoclonal antibody, such as a chimeric, humanized, or human monoclonal antibody, or an antigen-binding fragment that is an immunoglobulin binding to an antigen.

[0045] In one embodiment of the present invention, the binding molecule that specifically binds to NS1 of the Zika virus may be any one selected from the group consisting of antibodies, peptides, peptide analogs, aptamers, and compounds that specifically bind to NS1 of the Zika virus. Specifically, the binding molecule may be an antibody or a fragment thereof.

[0046] In one embodiment of the present invention, the antibody may be a Zika virus-specific antibody or a binding fragment thereof that specifically binds to non-structural protein 1 of the Zika virus and includes a heavy chain variable region and a light chain variable region.

[0047] In the present invention, the Zika virus refers to one of the representative mosquito-borne viruses, a single positive-stranded RNA virus, and a virus belonging to the Flavivirus genus, a sub-genus of the Flaviviridae family.

[0048] The above Zika virus has a spherical structure with a size of 40 to 65 nm and consists of about 10,000 to 11,000 bases. The envelope protein and the icosahedral-like nucleocapsid protein may be symmetrical. The proteins that make up the Zika virus can be broadly classified into structural proteins and non-structural proteins. Structural proteins include capsid proteins, precursor membrane proteins, membrane proteins, and envelope proteins, while non-structural proteins include non-structural protein 1 (NS1), non-structural protein 2A (NS2A), non-structural protein 2B (NS2B), non-structural protein 3 (NS3), non-structural protein 4A (NS4A), non-structural protein 4B (NS4B), and non-structural protein 5 (NS5).

[0049] In the present invention, the non-structural protein 1 (NS1) is an intracellular dimer, and three of these dimers are assembled to form a hexamer lipoprotein particle which is secreted into the extracellular space. NS1 is required for viral replication and is a major antigenic marker of viral infection involved in immune evasion and disease development.

[0050] In the present invention, we intended to produce an antibody that recognizes the Zika virus-specific antigen, for example, non-structural protein 1 (NS1). Accordingly, using the antigen, two monoclonal antibodies, 4B1 and 5D12, which exhibit excellent binding affinity for Zika virus non-structural protein 1 (NS1), were selected.

[0051] In this specification, the term "antibody" is used in the broadest possible sense and specifically includes intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies formed from two or more intact antibodies (e.g., bispecific antibodies), and antibody fragments exhibiting desired biological activity. An antibody is a protein produced by the immune system capable of recognizing and binding to a specific antigen. Structurally, an antibody typically has a Y-shaped protein composed of four amino acid chains (two heavy chains and two light chains). Each antibody primarily has two regions: a variable region and a constant region. The variable region, located at the distal end of the Y-arm, binds to and interacts with the target antigen. The variable region includes a complementation determining region (CDR) that recognizes and binds to specific binding sites on the specific antigen. The constant region, located at the tail end of the Y, is recognized and interacts with by the immune system. The target antigen generally has multiple binding sites, called epitopes, which are recognized by the CDRs on the antibody. Each antibody that specifically binds to a different epitope has a different structure. Therefore, an antigen can have one or more corresponding antibodies.

[0052] In one embodiment of the present invention, the Zika virus-specific antibody or its binding fragment may comprise a heavy chain variable region comprising CDR1-VH having the amino acid sequence of SEQ ID NO. 1, CDR2-VH having the amino acid sequence of SEQ ID NO. 2, and CDR3-VH having the amino acid sequence of SEQ ID NO. 3, and a light chain variable region comprising CDR1-VK having the amino acid sequence of SEQ ID NO. 4, CDR2-VK having the amino acid sequence of SEQ ID NO. 5, and CDR3-VK having the amino acid sequence of SEQ ID NO. 6.

[0053] In one embodiment of the present invention, the Zika virus-specific antibody or its binding fragment may comprise a heavy chain variable region comprising CDR1-VH having the amino acid sequence of SEQ ID NO. 17, CDR2-VH having the amino acid sequence of SEQ ID NO. 18, and CDR3-VH having the amino acid sequence of SEQ ID NO. 19, and a light chain variable region comprising CDR1-VK having the amino acid sequence of SEQ ID NO. 20, CDR2-VK having the amino acid sequence of SEQ ID NO. 21, and CDR3-VK having the amino acid sequence of SEQ ID NO. 22.

[0054] In one embodiment of the present invention, the antibody or its binding fragment may be, but is not limited to, a scFv fragment, a scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody. One embodiment of the present invention provides a Fab fragment that binds to a Zika virus non-structural protein 1 (NS1) region.

[0055] In addition, the present invention includes functional variants of the binding molecule. Binding molecules are considered functional variants of the binding molecule of the present invention if they can compete with the binding molecule of the present invention to specifically bind to the Zika virus non-structural protein 1 (NS1) region and possess the ability to bind to the Zika virus non-structural protein 1 (NS1) region. Functional variants include, but are not limited to, derivatives having substantially similar primary structural sequences, and include, for example, in vitro or in vivo modifications, chemical agents and / or biochemical agents, which are not found in the parent monoclonal antibody of the present invention. Such modifications include, for example, acetylation, acylation, covalent bonding of nucleotides or nucleotide derivatives, covalent bonding of lipids or lipid derivatives, crosslinking, disulfide bond formation, glycosylation, hydroxylation, methylation, oxidation, pegylation, proteolysis, and phosphorylation. The functional variant may optionally be an antibody comprising an amino acid sequence containing one or more substitutions, insertions, deletions, or combinations thereof compared to the amino acid sequence of the parent antibody. Furthermore, the functional variant may comprise a truncated form of the amino acid sequence at either or both the amino terminus or the carboxy terminus. The functional variant of the present invention may have the same or different, higher or lower binding affinity compared to the parent antibody of the present invention, but may still bind to NS1 of the Zika virus. For example, the amino acid sequence of a variable region, including but not limited to the backbone structure, hypervariable region, and particularly the complementarity-determining region (CDR) of the light chain or heavy chain, may be modified. Generally, the light chain or heavy chain region comprises three hypervariable regions, including three CDR regions, and a more conserved region, namely the backbone region (FR).The hypervariable region includes amino acid residues from the CDR and amino acid residues from the hypervariable loop. Functional variants within the scope of the present invention may have about 50% to 99%, about 60% to 99%, about 80% to 99%, about 90% to 99%, about 95% to 99%, or about 97% to 99% amino acid sequence homogeneity with the parent antibody of this specification. Among computer algorithms known to those skilled in the art, Gap or Bestfit may be used to optimally arrange the amino acid sequences to be compared and to define similar or identical amino acid residues. Functional variants may be obtained by modifying the parent antibody or a part thereof by known general molecular biological methods, including PCR methods, mutagenesis using oligomeric nucleotides, and partial mutagenesis, or by organic synthesis methods, but are not limited thereto.

[0056] In one embodiment of the present invention, the antibody or its binding fragment may be produced by a hybridoma cell.

[0057] In one embodiment of the present invention, it was confirmed that the Zika virus diagnostic effect is excellent when the above-mentioned Zika virus-specific antibodies are used in combination. Specifically, when two types of Zika virus-specific antibodies (4B1 and 5D12) are used as a pair, an excellent Zika virus diagnostic effect can be achieved.

[0059] In addition, the present invention provides a nucleic acid molecule encoding the antibody. Specifically, the nucleic acid molecule may be a polynucleotide encoding the antibody or a binding fragment thereof.

[0060] The nucleic acid molecules of the present invention include all nucleic acid molecules in which the amino acid sequence of the antibody provided in the present invention is translated into a polynucleotide sequence as is known to those skilled in the art. Therefore, various polynucleotide sequences can be prepared by an open reading frame (ORF), and all of these are also included in the nucleic acid molecules of the present invention.

[0061] In one embodiment of the present invention, the polynucleotide may include the base sequence of SEQ ID NO. 7 and the base sequence of SEQ ID NO. 8.

[0062] In one embodiment of the present invention, the polynucleotide may include the base sequence of SEQ ID NO. 23 and the base sequence of SEQ ID NO. 24.

[0064] In addition, the present invention provides an expression vector into which the nucleic acid molecule is inserted. In one embodiment, the expression vector may be an expression vector into which the polynucleotide is inserted.

[0065] In one embodiment of the present invention, the expression vector may include the nucleotide sequence of SEQ ID NO. 7 and the nucleotide sequence of SEQ ID NO. 8.

[0066] In one embodiment of the present invention, the expression vector may include the nucleotide sequence of SEQ ID NO. 23 and the nucleotide sequence of SEQ ID NO. 24.

[0067] The above expression vector may be selected from the group consisting of Celltrion’s proprietary expression vector MarEx vector (refer to Korean Patent Registration No. 10-1076602), commercially widely used pCDNA vectors, F, R1, RP1, Col, pBR322, ToL, Ti vectors; cosmids; phages such as lambda, lambdoid, M13, Mu, p1 P22, Qμ, T-even, T2, T3, T7, etc.; and plant viruses, but is not limited thereto. Any expression vector known to those skilled in the art as an expression vector may be used in the present invention, and the selection of the expression vector depends on the properties of the target host cell. When introducing the vector into the host cell, it may be performed by calcium phosphate transfection, viral infection, DEAE-dextran regulated transfection, lipofectamine transfection, or electroporation, but is not limited thereto. Those skilled in the art may select and use an introduction method suitable for the expression vector and the host cell to be used. For example, the vector contains one or more screening markers, but is not limited thereto, and screening can also be performed using a vector that does not contain a screening marker, depending on whether a product is produced. The selection of the screening marker is made by the target host cell, and since this utilizes methods already known to those skilled in the art, the present invention is not limited thereto.

[0068] To facilitate the purification of the antibody of the present invention, a tag sequence can be inserted into an expression vector and fused.

[0069] The above tags include, but are not limited to, hexahistidine tags, hemagglutinin tags, myc tags, or flag tags, and any tag known to those skilled in the art that facilitates purification can be used in the present invention.

[0071] In addition, the present invention provides a hybridoma cell that produces an antibody or a binding fragment thereof. In one embodiment, the hybridoma cell may be a cell into which the expression vector has been introduced.

[0072] The term "hybridoma cell" in the present invention refers to a cell created by artificially fusing two different types of cells, and means a cell or cell line in which two or more homologous or heterologous cells are fused using a substance that induces cell fusion, such as polyethylene glycol (PEG), or a certain type of virus, thereby integrating different functions of the different cells into one cell.

[0073] The above hybridoma cell line can be prepared through the steps of: immunizing a mouse using 'Zika virus non-structural protein 1'; fusing the splenocytes of the immunized mouse with myeloma cells and culturing them; and selecting a hybridoma cell line that produces an antibody specific to the 'Zika virus non-structural protein 1' protein or a binding fragment thereof from the fused hybridoma cell line.

[0074] Additionally, the antibody or its binding fragment specific to 'Zika virus non-structural protein 1' of the present invention can be prepared by a fusion method well known in the art to which the present invention belongs (see Khler G. & Milstein C., Nature (1975) Vol. 256, pp. 495-497., Galfre G. & Milstein C., Methods Enzymol. (1981) Vol. 73, pp. 3-46.). To create a 'hybridoma cell line' secreting said antibody or its binding fragment, one of the two cell populations to be fused may use cells from an immunologically suitable host animal, such as a mouse injected with 'Zika virus non-structural protein 1', and the other may use a cancer or myeloma cell line. These two cell populations are fused by a method known in the art to which the present invention belongs, such as polyethylene glycol, and then the antibody-producing cells are proliferated by a standard tissue culture method. After obtaining a uniform cell population by subcloning using the limited dilution technique, hybridoma cell lines capable of producing antibodies specific to 'Zika virus non-structural protein 1' are selected using an enzyme-linked immunosorbent assay (ELISA) or Western blot analysis, and can be cultured in large quantities in vitro or in vivo according to standard techniques. The above-mentioned large-scale in vivo culture refers to injecting the hybridoma cell line into the peritoneal cavity of a mouse to induce high-concentration antibody production, and then isolating it from ascites.

[0075] The monoclonal antibody produced by the above hybridoma cell line may be used without purification, or it may be used after being purified to a high purity (e.g., 90% or more) according to methods well known in the art to which the present invention belongs. Such purification techniques may include separating it from a culture medium or ascites using methods such as gel electrophoresis, dialysis, salt precipitation, ion exchange chromatography, and affinity antigen column chromatography.

[0076] In one embodiment of the present invention, the hybridoma cell may be a cell into which an expression vector comprising the nucleotide sequence of SEQ ID NO. 7 and the nucleotide sequence of SEQ ID NO. 8 has been introduced.

[0077] In one embodiment of the present invention, the hybridoma cell may be a cell into which an expression vector comprising the nucleotide sequence of SEQ ID NO. 23 and the nucleotide sequence of SEQ ID NO. 24 has been introduced.

[0079] In addition, the present invention provides a Zika virus diagnostic kit comprising a preparation for diagnosing a Zika virus (ZIKV) antigen. In one specific example, the virus antigen is non-structural protein 1 of the Zika virus, and the preparation may be a diagnostic kit that is the antibody or a binding fragment thereof.

[0080] In one embodiment of the present invention, the antibody of the present invention used in a diagnostic kit may be labeled in a diagnostic manner. Various methods available for labeling biomolecules are well known to those skilled in the art and are considered within the scope of the present invention. Examples of types of labels that may be used in the present invention include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds, and bioluminescent compounds. Commonly used labels include fluorescent substances (e.g., fluorescein, rhodamine, Texas Red, etc.), enzymes (e.g., horseradish peroxidase, β-galactosidase, alkaline phosphatase), radioisotopes (e.g., 32P or 125I), biotin, digoxigenin, colloidal metals, and chemiluminescent or bioluminescent compounds (e.g., dioxetane, luminol, or acridinium). Labeling methods such as the covalent bonding method of enzymes or biotinyl groups, iodination, phosphorylation, and biotinylation are well known in the art. Diagnostic methods include, but are not limited to, autoradiography, fluorescence microscopy, and direct and indirect enzymatic reactions. Commonly used diagnostic analysis methods include radioisotope or non-radioisotope methods. These include Western blotting, overlay analysis, RIA (Radioimmuno Assay), IRMA (ImmuneRadioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (Enzyme Linked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay), and CLIA (Chemioluminescent Immune Assay).

[0081] The diagnostic kit of the present invention can be used to diagnose the presence of the Zika virus by contacting a sample with the antibody and confirming the reaction. The sample may be any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus from lung tissue, respiratory tissue, and saliva of a subject, but is not limited thereto, and the sample can be prepared by conventional methods known to those skilled in the art.

[0082] One embodiment of the present invention may be a kit comprising a) the antibody; and b) a container. Specifically, the antibody of a) may be an antibody or a binding fragment thereof that specifically binds to non-structural protein 1 of the Zika virus.

[0083] In addition, the present invention provides a diagnostic kit for Zika virus-related diseases comprising an antibody or a binding fragment thereof that binds to a region of a Zika virus non-structural protein (NS1).

[0084] In the diagnostic kit of the present invention, the kit container may include a solid carrier. The antibody of the present invention may be attached to the solid carrier, and such a solid carrier may be porous or non-porous, planar or non-planar.

[0086] In addition, the present invention provides a composition for diagnosing Zika virus comprising the antibody or a binding fragment thereof.

[0088] In addition, in another embodiment of the present invention, the present invention provides a method for diagnosing the Zika virus using the diagnostic kit.

[0089] In addition, in another embodiment of the present invention, the present invention provides a method for diagnosing a Zika virus-related disease using the diagnostic kit.

[0091] Each of the above features described herein may be used in combination, and the fact that each of the above features is described in different dependent claims of the patent claims does not indicate that they cannot be used in combination.

[0093] The present invention will be explained in more detail below through examples and experimental examples. However, these examples are intended to illustrate the present invention, and the scope of the present invention is not limited to these examples and experimental examples.

[0095] Examples and Experimental Examples

[0096] Experimental Example 1. Isolation and Purification of Zika Virus NS1 Recombinant Antigen Protein

[0097] Genes were cloned for the amino acid sequences of the Zika virus NS1 protein at regions 1-352 (full-length) and 172-352 (see Fig. 1).

[0098] Using an E. coli expression system, a refolding isolation and purification technique for the viral antigen recombinant 172-352 site protein (Zika NS1_CTD) was established to produce more than 10 mg of antigen protein with high purity, which was used for the development of mouse immune antibodies (see Fig. 2).

[0100] Experimental Example 2. Cloning of Zika virus NS1-specific antibody

[0101] Using the isolated antigen protein, Zika virus-specific single-chain antibodies capable of binding to the antigen protein were identified (see Figures 3a to 3d). Forty-one clones that reacted with the antigen protein (OD > 0.5) were identified by ELISA (ELISA conditions: antigen coating: 100 ng / well, 2nd Ab dilution: 1:10,000, substrate: TMB) (indicated in red; H12 well is a positive control).

[0102] After transferring the above 41 positive clones to a 24-well plate, they were confirmed by ELISA (ELISA conditions: antigen coating: 100 ng / well, 2nd Ab dilution: 1:10,000, substrate: TMB). As a result, 22 clones were identified that showed no cross-reactivity with dengue virus NS1 and reacted specifically with the Zika NS1 antigen (see Fig. 4).

[0103] Based on the above ELISA results, clones #4B1, #5D12, #5G3, #8D11, #9E3, #4D2, #7H8, #8H5, #9C10, and #9E5 were selected, and a total of 10 types of monoclonal antibodies were cloned and confirmed by ELISA, and then stored in liquid nitrogen.

[0104] As a result, a total of eight monoclonal antibodies were identified, excluding those with low purity or those showing cross-reactivity with dengue NS1 (see Tables 1 and 2).

[0105]

[0107]

[0109] Experimental Example 3. Purification of Zika virus NS1-specific antibody and confirmation of binding affinity

[0110] 10 mL of mouse ascites from the cloned Zika NS1_CTD monoclonal antibodies #9E3, #9C10, #8H5, #9E5, #4D2, #4B1, #5D12, and #8D11 were each purified using a Protein A column, and IgG was quantified by the BCA method (using BGG standard). The purity of the purified antibodies was confirmed by SDS-PAGE followed by staining with Coomassie blue (see Fig. 5).

[0111] Western blot (WB) and Dot blot (DB) experiments confirmed that the eight purified Zika-specific monoclonal antibodies mentioned above have binding affinity to HEK293 cell expression antigens.

[0112] As a result, four of the eight Zika virus-specific single-chain antibodies had binding affinities similar to commercially available antibodies. In addition, it was confirmed that none of the four antibodies showed a specific reaction to dengue virus NS1, but they specifically bound to Zika virus NS1 (see Fig. 6).

[0113] These results mean that the aforementioned antibodies can be used for Zika virus-specific diagnosis.

[0115] Experimental Example 3. Analysis of Zika virus-specific antibody sequences

[0116] The sequences of two of the four Zika virus-specific single-chain antibodies mentioned above were analyzed.

[0117] Table 3 is the sequence for 4B1 among the above single-chain antibodies.

[0118] Sequence number order designation 1 GYTFTDYY CDR 1_VH 2 IYPGSGNI CDR 2_VH 3 ARPTVYFDY CDR 3_VH 4 QSIGTS CDR 1_VK 5 YAS CDR 2_VK 6 QQSNSWPLT CDR 3_VK 7 CAGATCCAGCTGCAGCAGTCTGGACCTGGGGCCAAGGCACCACTCTCTGAGCTGGTGAAGCCTGGGGCTTCACTGAGGATATCCTGCAAGCCTTCTGGCTACACCTTCACTGACTACTATATAAACTGGGTGAAGCAGAAGCCTGGACAGGGACTTGAGTTGGATTGGATGGATT TATCCTGGAAGCGGTAATATTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCTGACATCTGAGGACACTGCTGTCTATTTCTGTGCAAGACCTACGGTCTATTTTGACTATACAGTCTCCTCA VH nucleotide sequence 8 GACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAGCATTGGCACAAGCATACACTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGAGT CTATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAGCTGGCCACTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA VL nucleotide sequence 9 QIQLQQSGPELVKPGASLRISCKPS FR1_VH 10 INWVKQKPGQGLEWIGW FR2_VH 11 KYNEKFKGKATLTVDTSSSTAYMQLSSLTSEDTAVYFC FR3_VH 12 WGQGTTLTVSS FR4_VH 13 DILLTQSPAILSVSPGERVSFSCRAS FR1_VK 14 IHWYQQRTNGSPRLLIK FR2_VK 15 ESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYC FR3_VK 16 FGAGTKLELK FR4_VK

[0120] Table 4 is the sequence for 5D12 among the above single-chain antibodies.

[0121] 서열번호 서열 명칭 17 GHTFTRYW CDR 1_VH 18 IFPSDSYT CDR 2_VH 19 TSSSNYYYGSSWFAY CDR 3_VH 20 QSLLYSSNQKNY CDR 1_VK 21 WAS CDR 2_VK 22 QQYYSYPRT CDR 3_VK 23 CAGGTCCAACTGCAGCAGCCTGGGGCTGAGGTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCCACACCTTCACCAGGTACTGGATAAACTGGGTGAAGCAGAGGCCTGGACAAGGCCTTGAGTGGATCGGAAATATTTTTCCTTCTGATAGTTATACTAACTACAATCAAAAGTTCAAGGACAAGGCCACATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAGCTCAGCAGCCCGACATCTGAGGACTCTGCGGTCTATTACTGTACAAGCTCCTCCAATTATTACTACGGTAGTAGCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCA VH 염기서열 24 GACATTGTGATGTCACAGTCTCCATCCTCCCTAGCTGTGTCAGTTGGAGAGAAGGTTACTATGAGCTGCAAGTCCAGTCAGAGCCTTTTATATAGTAGCAATCAAAAGAACTACTTGGCCTGGTACCAGCAGAAACCAGGGCAGTCTCCTAAACTGCTGATTTACTGGGCATCCACTAGGGAATCTGGGGTCCCTGATCGCTTCACAGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTGTGAAGGCTGAAGACCTGGCAGTTTATTACTGTCAGCAATATTATAGCTATCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAA VL nucleotide sequence 25 QVQLQQPGAEVVRPGASVKLSCKAS FR1_VH 26 INWVKQRPGQGLEWIGN FR2_VH 27 NYNQKFKDKATLTVDKSSSTAYMQLSSPTSEDSAVYYC FR3_VH 28 WGQGTLVTVSA FR4_VH 29 DIVMSQSPSSLAVSVGEKVTMSCKSS FR1_VK 30 LAWYQQKPGQSPKLLIY FR2_VK 31 TRESGVPDRFTGSGSGTDFTLTISSVKAEDLAVYYC FR3_VK 32 FGGGTKLEIK FR4_VK

[0123] Experimental Example 4. Preparation of a Zika virus diagnostic kit

[0124] A Zika virus diagnostic kit containing the two types of antibodies (4B1 and 5D12) analyzed above was manufactured.

[0125] Zika virus samples and dengue virus samples were treated with the above-manufactured diagnostic kit at different concentrations to check whether a specific diagnostic reaction occurred.

[0126] As a result, it was confirmed that a diagnostic reaction occurred for the Zika virus sample (see Fig. 7a), but no reaction occurred for the dengue virus sample (see Fig. 7b).

[0127] These results mean that the Zika virus can be diagnosed quickly and accurately using a Zika virus diagnostic kit containing the two types of antibodies mentioned above.

Claims

Claim 1 A Zika virus-specific antibody or a binding fragment thereof that specifically binds to non-structural protein 1 of the Zika virus (ZIKV) and comprises a heavy chain variable region and a light chain variable region, wherein (i) the heavy chain variable region comprises CDR1-VH composed of the amino acid sequence of SEQ ID NO. 1, CDR2-VH composed of the amino acid sequence of SEQ ID NO. 2, and CDR3-VH composed of the amino acid sequence of SEQ ID NO. 3, and the light chain variable region comprises CDR1-VK composed of the amino acid sequence of SEQ ID NO. 4, CDR2-VK composed of the amino acid sequence of SEQ ID NO. 5, and CDR3-VK composed of the amino acid sequence of SEQ ID NO. 6; or (ii) a Zika virus-specific antibody or a binding fragment thereof, wherein the heavy chain variable region comprises CDR1-VH composed of the amino acid sequence of SEQ ID NO. 17, CDR2-VH composed of the amino acid sequence of SEQ ID NO. 18, and CDR3-VH composed of the amino acid sequence of SEQ ID NO. 19, and the light chain variable region comprises CDR1-VK composed of the amino acid sequence of SEQ ID NO. 20, CDR2-VK composed of the amino acid sequence of SEQ ID NO. 21, and CDR3-VK composed of the amino acid sequence of SEQ ID NO.

22. Claim 2 delete Claim 3 delete Claim 4 The antibody or its binding fragment according to claim 1, wherein the antibody or its binding fragment is an antibody or its binding fragment that is a scFv fragment, a scFv-Fc fragment, a Fab fragment, an Fv fragment, a diabody, a chimeric antibody, a humanized antibody, or a human antibody. Claim 5 In claim 4, the antibody or its binding fragment is an antibody or its binding fragment that is a Fab fragment. Claim 6 A polynucleotide encoding the antibody of claim 1 or a binding fragment thereof. Claim 7 The polynucleotide of claim 6, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 7 and the base sequence of SEQ ID NO.

8. Claim 8 The polynucleotide of claim 6, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 23 and the base sequence of SEQ ID NO.

24. Claim 9 An expression vector having the polynucleotide of claim 6 inserted therein. Claim 10 An expression vector according to claim 9, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 7 and the base sequence of SEQ ID NO.

8. Claim 11 An expression vector according to claim 9, wherein the polynucleotide comprises the base sequence of SEQ ID NO. 23 and the base sequence of SEQ ID NO.

24. Claim 12 A hybridoma cell that produces the antibody of claim 1 or a binding fragment thereof. Claim 13 A diagnostic kit for Zika virus comprising a preparation for diagnosing a Zika virus (ZIKV) antigen, wherein the virus antigen is non-structural protein 1 of the Zika virus, and the preparation is the antibody of claim 1 or a binding fragment thereof. Claim 14 A composition for diagnosing Zika virus comprising the antibody of claim 1 or a binding fragment thereof.

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