How to determine if you have a virus infection

Abstract

Provided is a method for determining whether a subject is infected with a virus, the method involving (1) testing for whether a protein that includes at least a portion of the amino acid sequence of the helicase region of the MDA5 protein and has a molecular weight of approximately 60 kDa is detected in a sample taken from the subject and (2) determining that the subject is infected with a virus when the protein has been detected. Also provided is a monoclonal antibody that bonds to the protein.

Description

[Technical Field]

[0001] This patent application claims priority to Japanese Patent Application No. 2020-141874, the entire contents of which are incorporated herein by reference. The present disclosure relates to methods for determining whether a subject is infected with a virus, as well as proteins and antibodies that may be used in the methods. [Background technology]

[0002] Viral RNA is known to be sensed by the RIG-I-like receptor (RLR) family. The RLR family includes three members: RIG-I (DDX58), LGP2 (DHX58), and MDA5 (IFIH1). These three RLRs are expressed in the cytoplasm of cells. MDA5 detects infection by picornaviruses such as polio and encephalomyocarditis virus (EMCV) and recognizes long double-stranded RNA (dsRNA). Like other RLRs, MDA5 activates NF-κB and interferon regulatory factors (IRFs) via its N-terminal region containing a caspase recruitment domain (CARD). MDA5 can also recognize single-stranded RNA (ssRNA) viruses, such as mouse hepatitis virus (MHV), calicivirus, and flavivirus families, as well as RNA derived from DNA viruses. A pediatric patient with a homozygous deletion missense mutation (K365E) in MDA5 has been previously reported (Non-Patent Document 2). This patient had a history of recurrent respiratory infections caused by ssRNA viruses (such as human rhinovirus (HRV), influenza virus, respiratory syncytial virus (RSV), and coronavirus), DNA viruses (adenovirus), and bacteria (such as Haemophilus influenza, Mycoplasma pneumoniae, and Staphylococcus aureus). Non-Patent Document 3 also discloses that MDA5 is essential for defense against coronavirus infection, a positive single-stranded RNA virus. Specifically, MDA5 plays an important role in defense against dsRNA, ssRNA, DNA, and bacteria. However, the mechanism of MDA5-mediated defense against infection has not been elucidated. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Yueh-Ming Loo, et al., Journal of Virology, 82, 335-345, 2008 [Non-patent document 2] Ian T. Lamborn et al., The Journal of Experimental Medicine, 214, 1949-1972, 2017 [Non-patent document 3] Zachary B. Zalinger et al., Journal of Virology, 89, 12330-12340, 2015 Summary of the Invention [Problem to be solved by the invention]

[0004] One object of the present disclosure is to provide a method for determining whether a subject is infected with a virus. [Means for solving the problem]

[0005] We found that a protein with a molecular weight of approximately 60 kDa, which contains at least a portion of the amino acid sequence of the helicase domain of the MDA5 protein, is detected in the serum of healthy individuals, and that this protein is rapidly released from the cytoplasm of peripheral blood mononuclear cells (PBMCs) upon stimulation with virus-mimicking double-stranded RNA. Therefore, this protein can be used as a marker for viral infection.

[0006] In one aspect, the present disclosure provides a method for determining whether a subject is infected with a virus, comprising: (1) Testing whether a protein having at least a partial amino acid sequence of the helicase region of the MDA5 protein and a molecular weight of approximately 60 kDa is detected in a sample collected from the subject; and (2) determining that the subject is infected with the virus when the protein is detected; The present invention provides a method comprising:

[0007] In one aspect, the present disclosure provides a method for producing a pharmaceutical composition comprising: (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or a monoclonal antibody that competes with said monoclonal antibody for binding to MDA5.

[0008] In one aspect, the present disclosure provides a kit for determining whether a subject is infected with a virus, the kit comprising an antibody that binds to a protein comprising the amino acid sequence of at least a portion of the helicase region of the MDA5 protein and having a molecular weight of approximately 60 kDa. In one aspect, the disclosure provides a protein comprising the amino acid sequence of at least a portion of the helicase region of the MDA5 protein, and having a molecular weight of approximately 60 kDa. In one aspect, the present disclosure provides for the use of the above proteins as diagnostic markers for viral infection. [Effects of the Invention]

[0009] The present disclosure provides methods for determining whether a subject is infected with a virus, as well as proteins and antibodies that can be used in the methods. [Brief explanation of the drawings]

[0010] [Figure 1]The amino acid sequences of the heavy and light chain variable regions of the mouse anti-human MDA5 monoclonal antibody (H27) are shown. Each CDR is indicated by a box. [Figure 2] The amino acid sequences of the heavy chain variable region and light chain variable region of the mouse anti-human MDA5 monoclonal antibody (H46) are shown. Each CDR is indicated by a box. [Figure 3] 1 shows MDA5 mRNA levels in human normal tissues (lung, spleen, pancreas, muscle, and placenta) measured by real-time quantitative PCR (RT-qPCR). [Figure 4] Immunohistochemical analysis of normal tonsils and pancreas with mouse anti-human MDA5 monoclonal antibody (H27) is shown (bar: 200 μm). [Figure 5] Western blot analysis of normal lung tissue and lung cancer using mouse anti-human MDA5 monoclonal antibody (H27). Lanes 1 and 2: normal lung tissue (50 mg / lane), Lanes 3 and 4: lung cancer (50 mg / lane). [Figure 6] Figure 1 shows the levels of soluble MDA5 protein in serum. Serum levels of soluble MDA5 protein were analyzed in 32 healthy donors using a highly sensitive sandwich immunoassay system. The detection limit of this assay was 80 pg / mL. [Figure 7] Characterization of soluble human MDA5 protein. Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood. Cells (2 x 106 cells / mL) were stimulated with 250 μg / mL of polyinosinic acid-polycytidylic acid sodium salt (poly I:C) at 37°C. The levels of soluble MDA5 in the supernatant were analyzed. Solid line: poly I:C stimulation; dashed line: control. [Figure 8] Western blot analysis of MDA5 protein is shown. Lane 1: total PBMC. Lane 2: supernatant (0 min). Lane 3: supernatant, no stimulation (15 min). Lane 4: supernatant, poly I:C stimulation (15 min). Lane 5: supernatant, no stimulation (1 h). Lane 6: supernatant, poly I:C stimulation (1 h). Lane 7: supernatant, no stimulation (2 h). Lane 8: supernatant, poly I:C stimulation (2 h). DETAILED DESCRIPTION OF THE INVENTION

[0011] Unless otherwise specified, terms used herein have the meanings commonly understood by those skilled in the art of organic chemistry, medicine, pharmacology, molecular biology, microbiology, etc. Definitions of some terms used herein are provided below, but these definitions take precedence over common understandings in this specification.

[0012] As used herein, when a numerical value is accompanied by the term "about," it is intended to encompass a range of ±10% of that value. For example, "about 20" includes "18 to 22." A range of numerical values ​​includes all values ​​between and including the endpoints. When "about" refers to a range, it applies to both endpoints of the range. Thus, for example, "about 20 to 30" includes "18 to 33."

[0013] The MDA5 protein is a member of the RIG-I-like receptor (RLR) family and is a cytoplasmic protein with a molecular weight of approximately 120 kDa. The MDA5 protein is known to sense intracellular RNA derived from double-stranded RNA (dsRNA), single-stranded RNA (ssRNA), or DNA viruses, and to activate NF-κB and interferon regulatory factors (IRFs), thereby playing a role in defense against viral infection.

[0014] In the present disclosure, the MDA5 protein may be from any species, typically mammalian (e.g., human, mouse, rat, hamster, rabbit, cat, dog, cow, sheep, monkey, etc.), and preferably human. The amino acid sequences of MDA5 proteins derived from various species can be easily obtained using publicly known databases. A representative amino acid sequence of human MDA5 protein is registered in GenBank under accession number AF095844.1 (SEQ ID NO: 1). In the present disclosure, MDA5 protein includes the products of its naturally occurring alleles.

[0015] In the present disclosure, the MDA5 protein may contain a sequence in which one or several amino acids have been deleted, substituted, or added to the original amino acid sequence (e.g., the amino acid sequence of SEQ ID NO: 1), so long as its functions (i.e., RNA recognition ability and NF-κB / IRF activation ability) are maintained. Note that "several" preferably means 2 to 7, more preferably 2 to 5, and most preferably 2 to 3 amino acids. Conservative substitutions between similar amino acid residues are preferred.

[0016] Furthermore, as long as its function is maintained, the MDA5 protein may contain an amino acid sequence that has at least about 70% or more, preferably about 80% or more, more preferably about 90% or more, particularly preferably about 95% or more, and most preferably about 97%, about 98%, or about 99% or more identity to the original amino acid sequence (e.g., the amino acid sequence of SEQ ID NO: 1) when calculated using BLAST or the like (e.g., when using the initial condition parameters of BLAST).

[0017] "Sequence identity" is determined by comparing two sequences that are optimally aligned across the entire region of the sequences to be compared. The sequences to be compared may have additions or deletions (e.g., gaps) in the optimal alignment of the two sequences. Sequence identity can be calculated using programs such as FASTA, BLAST, and CLUSTAL W provided in public databases (e.g., DDBJ (http: / / www.ddbj.nig.ac.jp)). Alternatively, commercially available sequence analysis software (e.g., Vector NTI (登録商標) Software, GENETYX (登録商標) ver. 12).

[0018] As demonstrated in the Examples below, when an animal is infected with a virus, a protein containing a partial amino acid sequence of the MDA5 protein and having a molecular weight of approximately 60 kDa is secreted into the blood. Here, the molecular weight was measured by SDS-PAGE under reducing conditions. This protein contains at least a portion of the helicase domain of the MDA5 protein. The helicase domain of the MDA5 protein corresponds to positions 306 to 873 of SEQ ID NO: 1. In the present disclosure, this protein is referred to as a "secreted MDA5 protein."

[0019] Thus, for example, whether a subject is infected with a virus can be determined by a method comprising the following steps: (1) testing whether secreted MDA5 protein is detected in a sample collected from a subject; and (2) If secreted MDA5 protein is detected, it is determined that the subject is infected with the virus. This method can also be used as a method to assist in determining whether or not a person is infected with a virus.

[0020] The subject may be of any species, typically including mammals (e.g., humans, mice, rats, hamsters, rabbits, cats, dogs, cows, pigs, sheep, monkeys, etc.) and birds (chickens, quail, turkeys, ducks, geese, etc.), preferably humans.

[0021] The sample may be blood, plasma, or serum collected from a subject. Blood samples may be collected by conventional methods, for example, from a vein or artery. Plasma or serum samples may be prepared by appropriately treating the blood using methods well known to those skilled in the art. This treatment is not particularly limited and may be any clinically acceptable treatment. For example, addition of an anticoagulant, centrifugation, etc. may be performed. The sample may also be other body fluids collected from a subject, such as saliva, nasal discharge, sputum, pleural effusion, or ascites. The collected sample may be stored at a low temperature during or after its preparation prior to use, for example, frozen. The collected sample may also be appropriately concentrated or diluted as needed before use.

[0022] The secreted MDA5 protein can be detected by immunological techniques using antibodies that bind to the secreted MDA5 protein. Examples of immunological techniques include enzyme-linked immunosorbent assays (ELISA, e.g., direct, indirect, sandwich, or competitive), immunochromatography, Western blotting, flow cytometry, and radioisotope immunoassay (RIA), with sandwich ELISA being preferred.

[0023] In the context of this disclosure, antibody refers to an affinity ligand based on an immunoglobulin framework, including monoclonal and polyclonal antibodies of any origin, including murine, rat, rabbit, goat, human, and other antibodies, as well as chimeric antibodies containing sequences from multiple species, e.g., partially humanized antibodies, e.g., partially humanized mouse antibodies. The variable region of an antibody typically consists of three complementarity-determining regions (also referred to as CDRs) flanked by four framework regions. In this disclosure, the amino acid positions assigned to the CDRs and frameworks of an antibody variable region are defined according to Kabat (see Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, Md., (1987) and (1991)).

[0024] Antibodies can be produced by existing general production methods using the secreted MDA5 protein or an antigenic partial peptide of the protein as an immunogen. For example, polyclonal antibodies can be produced by immunizing animals with an antigen, and monoclonal antibodies can be produced using hybridoma technology. Alternatively, antibodies can be produced using the full-length MDA5 protein as an immunogen, and antibodies that bind to the secreted MDA5 protein can be selected. Alternatively, commercially available antibodies can be used.

[0025] The secreted MDA5 protein can be obtained by known methods, such as genetic engineering techniques, by constructing an expression vector containing a polynucleotide encoding it and expressing it in cells. Specifically, an expression vector is constructed so that a polynucleotide encoding the secreted MDA5 protein is expressed under an expression control region such as an enhancer or promoter, and host cells are transformed with this expression vector to express the secreted MDA5 protein. Thus, the present disclosure also provides a polynucleotide encoding the secreted MDA5 protein, an expression vector containing the polynucleotide, and a transformed cell containing the polynucleotide or expression vector.

[0026] Alternatively, cells expressing the full-length MDA5 protein (e.g., peripheral blood mononuclear cells) or cultured cells expressing the full-length MDA5 protein can be stimulated with polyinosinic acid-polycytidylic acid sodium salt (poly I:C) to secrete the secreted MDA5 protein into the culture supernatant, and the secreted MDA5 protein can be recovered from the culture supernatant using, for example, an existing anti-MDA5 antibody.

[0027] The antibody may be a fragment or derivative thereof, so long as it can selectively interact with the secreted MDA5 protein. Antibody fragments and derivatives include, for example, a Fab fragment consisting of the first heavy chain constant domain (CH1), light chain constant domain (CL), heavy chain variable domain (VH), and light chain variable domain (VL) of an intact immunoglobulin protein; an Fv fragment consisting of two variable antibody domains, VH and VL; a single-chain Fv fragment (scFv) consisting of two VH and VL domains linked by a flexible peptide linker; and a minibody based on the variable heavy chain domain.

[0028] The antibody may be labeled. The label may be appropriately selected by one skilled in the art. Non-limiting examples of labels include fluorescent dyes (e.g., fluorescein, rhodamine, phycoerythrin, fluorescamine), chromophoric dyes (e.g., rhodopsin), chemiluminescent compounds (e.g., luminal, imidazole), and bioluminescent proteins (e.g., luciferin, luciferase), haptens (e.g., biotin), enzymes (e.g., peroxidase, alkaline phosphatase, beta-lactamase), radioisotopes (e.g., 3 H, 14 C. 32 P, 35 S or 125 I), particles (e.g., metal particles such as gold), and fluorescent semiconductor nanocrystals (quantum dots). Various labels can be bound to the desired antibody using various chemical reactions well known to those skilled in the art, such as amine or thiol reactions. Reactive groups other than amine and thiol, such as aldehyde, carboxylic acid, and glutamine, can also be used. Alternatively, the antibody may be unlabeled. In this case, a labeled secondary antibody that recognizes the antibody for the secreted MDA5 protein may be used.

[0029] The antibody may be bound to a suitable support. The support is not particularly limited as long as it can immobilize the antibody, and may be of any shape or material. Examples of supports include membranes such as nylon membranes, beads, glass, plastic, and metal.

[0030] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising a sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 2, and / or a light chain variable region comprising a sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 3. Antibodies that have no alterations in the CDRs of the heavy chain variable region and / or light chain variable region are preferred.

[0031] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising a sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 10, and / or a light chain variable region comprising a sequence that has 80% or more, preferably 85% or more, more preferably 90% or more, and even more preferably 95% or more sequence identity with the amino acid sequence of SEQ ID NO: 11. Antibodies that have no alterations in the CDRs of the heavy chain variable region and / or light chain variable region are preferred.

[0032] In one embodiment, a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:2 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO:3 may be used. In one embodiment, a monoclonal antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11 may be used.

[0033] In one embodiment, a monoclonal antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and / or a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3 may be used. In one embodiment, a monoclonal antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10 and / or a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11 may be used.

[0034] In one embodiment, a heavy chain variable region comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 2; and / or a light chain variable region comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 3; Monoclonal antibodies including:

[0035] In one embodiment, a heavy chain variable region comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 10; and / or a light chain variable region comprising the amino acid sequences of CDR1, CDR2, and CDR3 in the amino acid sequence of SEQ ID NO: 11; Monoclonal antibodies including:

[0036] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 4, CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and CDR3 comprising the amino acid sequence of SEQ ID NO: 6, and / or a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 7, CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and CDR3 comprising the amino acid sequence of SEQ ID NO: 9.

[0037] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 12, CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and CDR3 comprising the amino acid sequence of SEQ ID NO: 14, and / or a light chain variable region comprising CDR1 comprising the amino acid sequence of SEQ ID NO: 15, CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and CDR3 comprising the amino acid sequence of SEQ ID NO: 17.

[0038] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 4, CDR2 consisting of the amino acid sequence of SEQ ID NO: 5, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 6, and / or a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 7, CDR2 consisting of the amino acid sequence of SEQ ID NO: 8, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 9.

[0039] In one embodiment, a monoclonal antibody may be used that comprises a heavy chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 12, CDR2 consisting of the amino acid sequence of SEQ ID NO: 13, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 14, and / or a light chain variable region comprising CDR1 consisting of the amino acid sequence of SEQ ID NO: 15, CDR2 consisting of the amino acid sequence of SEQ ID NO: 16, and CDR3 consisting of the amino acid sequence of SEQ ID NO: 17.

[0040] In one embodiment, a monoclonal antibody that competes with any of the above monoclonal antibodies for binding to MDA5 can be used. Such antibodies can be identified by competitive assays well known to those skilled in the art, such as cross-blocking assays, preferably competitive ELISA assays. For example, such antibodies inhibit the binding of any of the above monoclonal antibodies to MDA5 by at least 20%, 30%, 40%, or 50% in a competitive assay.

[0041] Alternatively, a monoclonal antibody that competes with any of the above monoclonal antibodies for binding to MDA5 may be produced by existing general production methods using a peptide containing the epitope of any of the above monoclonal antibodies as an immunogen.

[0042] As shown in the Examples described below, a monoclonal antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 2 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 3, and a monoclonal antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11, recognize a secreted MDA5 protein containing a portion of the MDA5 protein of SEQ ID NO: 1. Therefore, the secreted MDA5 protein contains a region corresponding to the epitope of these monoclonal antibodies.

[0043] A monoclonal antibody comprising a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 10 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11 recognizes the amino acid sequence QILENSLLNL (SEQ ID NO: 18) at positions 415 to 424 of SEQ ID NO: 1 as an epitope. Thus, the secreted MDA5 protein comprises a region corresponding to positions 415 to 424 of SEQ ID NO: 1. Positions 415 to 424 of SEQ ID NO: 1 are included in the helicase region (positions 306 to 873 of SEQ ID NO: 1) responsible for the helicase activity of the MDA5 protein, and therefore the secreted MDA5 protein comprises at least a portion of the helicase region of the MDA5 protein (the region corresponding to positions 306 to 873 of SEQ ID NO: 1). The term "a region corresponding to positions 415 to 424 of SEQ ID NO: 1" refers to a region in an MDA5 protein that matches the region from positions 415 to 424 of SEQ ID NO: 1 when the amino acid sequence of that MDA5 protein is aligned with the amino acid sequence of SEQ ID NO: 1 in an optimal state (maximum amino acid identity). The same applies to "a region corresponding to positions 306 to 873 of SEQ ID NO: 1."

[0044] In step (2), if the secreted MDA5 protein is detected in step (1), the subject is determined to be infected with a virus. Viruses include picornaviruses, coronaviruses (such as SARS-related coronaviruses (SARS-CoV, SARS-CoV-2), MERS coronavirus, and mouse hepatitis virus), positive single-stranded RNA viruses such as encephalomyocarditis virus, rhinovirus, Japanese encephalitis virus, hepatitis C virus, West Nile virus, and dengue virus, negative single-stranded RNA viruses such as influenza virus, Sendai virus, and vesicular stomatitis virus, double-stranded RNA viruses such as reovirus, and DNA viruses such as poxviruses and adenoviruses.

[0045] In another embodiment, a kit for determining whether a subject is infected with a virus is provided, comprising an antibody that binds to secreted MDA5 protein. The antibody may be dissolved in water or a suitable buffer, such as phosphate-buffered saline (PBS), or lyophilized and provided in a suitable container. Suitable containers include bottles, vials, syringes, test tubes, plates, membranes, etc. The container may be made of a variety of materials, such as glass or plastic. The kit may further include other components or reagents necessary for detecting secreted MDA5 protein. For example, the kit may further include a labeled secondary antibody, a chromogenic substrate, a blocking solution, a washing buffer, an ELISA plate, a blotting membrane, etc. The kit may further include other materials desirable from a commercial and user standpoint, such as a package insert containing instructions for use.

[0046] In one aspect, there is provided a method for determining whether a subject is infected with a virus, comprising: (1) collecting a sample from a patient; (2) testing whether secreted MDA5 protein is detected in the sample; and (3) determining that the subject is infected with a virus when the secreted MDA5 protein is detected; A method is provided that includes:

[0047] In one embodiment, an antibody that binds to the secreted MDA5 protein is provided for determining whether a subject is infected with a virus. In one embodiment, there is provided the use of an antibody that binds to secreted MDA5 protein to manufacture a kit for determining whether a subject is infected with a virus.

[0048] For example, the present disclosure provides the following embodiments. [1] A method for determining whether a subject is infected with a virus, comprising: (1) Testing whether a protein containing a partial amino acid sequence of the MDA5 protein and having a molecular weight of approximately 60 kDa is detected in a sample collected from the subject; and (2) determining that the subject is infected with the virus when the protein is detected; A method comprising: [2] A method for determining whether a subject is infected with a virus, comprising: (1) Testing whether a protein having at least a partial amino acid sequence of the helicase region of the MDA5 protein and a molecular weight of approximately 60 kDa is detected in a sample collected from the subject; and (2) determining that the subject is infected with the virus when the protein is detected; A method comprising: [3] The method according to item 1 or 2, wherein the protein comprises a region corresponding to positions 415 to 424 of SEQ ID NO: 1. [4] The method according to any one of items 1 to 3, wherein the virus is an RNA virus. [5] The method according to any one of items 1 to 4, wherein in step (1), the protein is detected by sandwich ELISA. [6] The method according to any one of items 1 to 5, wherein in step (1), the protein is detected using a monoclonal antibody comprising (i) or (ii) or a monoclonal antibody that competes with the monoclonal antibody for binding to MDA5; (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9 (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17. [7] The method according to claim 6, wherein in step (1), the protein is detected using a monoclonal antibody comprising (i) or (ii). [8] Monoclonal antibodies, (i) comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, or (ii), wherein the heavy chain variable region comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and the light chain variable region comprises an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 11. [9] (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or a monoclonal antibody that competes with said monoclonal antibody for binding to MDA5.

[10] The monoclonal antibody according to item 9, which is a monoclonal antibody comprising (i) or (ii).

[11] (i) and comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, or (ii) A monoclonal antibody according to claim 10, comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 11.

[12] A kit for determining whether a subject is infected with a virus, comprising an antibody that binds to a protein that contains a partial amino acid sequence of the MDA5 protein and has a molecular weight of approximately 60 kDa.

[13] A kit for determining whether a subject is infected with a virus, comprising an antibody that binds to a protein having an amino acid sequence of at least a portion of the helicase region of the MDA5 protein and a molecular weight of approximately 60 kDa.

[14] The kit according to item 12 or 13, wherein the protein comprises a region corresponding to positions 415 to 424 of SEQ ID NO: 1.

[15] The kit according to any one of items 12 to 14, comprising the monoclonal antibody according to any one of items 9 to 11.

[16] A protein containing a partial amino acid sequence of the MDA5 protein and having a molecular weight of approximately 60 kDa.

[17] A protein containing at least a portion of the amino acid sequence of the helicase region of the MDA5 protein and having a molecular weight of approximately 60 kDa.

[18] The protein according to item 16 or 17, which comprises a region corresponding to the epitope of the monoclonal antibody according to any one of items 9 to 11.

[19] The protein according to any one of items 16 to 18, comprising a region corresponding to positions 415 to 424 of SEQ ID NO: 1.

[20] Use of the protein according to any one of items 16 to 19 as a marker for determining viral infection.

[0049] All documents cited herein are hereby incorporated by reference. All of the above descriptions are non-limiting and can be modified without departing from the scope of the invention as defined in the appended claims. Furthermore, all of the following examples are non-limiting and are provided solely to illustrate the invention. [Example]

[0050] Real-time quantitative PCR Complementary DNA (cDNA) isolated from human normal tissues (lung, spleen, pancreas, muscle, and placenta) was purchased from BioChain (Newark, CA). Real-time quantitative PCR (RT-qPCR) was performed in duplicate using SYBR Green Mastermix (Qiagen) and primers for human β-actin and MDA5 (Qiagen, Tokyo, Japan) on an Mx3000p PCR machine (Stratagene, La Jolla, CA). Average relative expression was calculated using the previously reported threshold cycle method (S. Takenaka, et al., Biochem Biophys Res Commun 445 (2014) 597-601; M. Tominaga, et al., Respir Investig 55 (2017) 293-299).

[0051] Generation of anti-human MDA5 monoclonal antibody Full-length human MDA5 cDNA (GenBank accession no. AF095844) containing a 6X His tag, GST, and turbo3C protease cleavage site at the N-terminus was subcloned into the pPSC8 expression vector (Protein Sciences Corporation, Meriden, CT) to form pPSC8 / human MDA5. Recombinant human MDA5 protein was isolated from SF9 cells cotransfected with the baculovirus AcNPV and pPSC8 / human MDA5. Anti-human MDA5 monoclonal antibodies (mAbs) were obtained by fusing the mouse myeloma cell line X-63·Ag8 / 653 with spleen cells isolated from BALB / c mice immunized with recombinant human MDA5 protein. Anti-human MDA5 mAbs (clones H27 [mouse IgG1], H46 [mouse IgG2b], H77 [mouse IgG2b], and H85 [mouse IgG1]) were established. As previously reported, purified mouse anti-human MDA5 mAb was also produced in our laboratory (Y. Kitasato, et al., Am J Respir Cell Mol Biol 31 (2004) 619-625; S. I. Takenaka, et al., Biochem Biophys Rep 4 (2015) 386-391). The amino acid sequences of the heavy and light chain variable regions of clones H27 and H46 are shown in Figures 1 and 2, respectively. The epitope of clone H46, as determined by hydrogen-deuterium exchange mass spectrometry (HDX-MS), was identified as QILENSLLNL (SEQ ID NO: 18), located at amino acid positions 415 to 424 of SEQ ID NO: 1.

[0052] Immunohistochemical staining Immunohistochemical staining was performed as previously reported (Y. Kitasato, et al., supra; T. Nouno, et al., J Thorac Dis 11 (2019) 4005-4017). Briefly, lung tissue was fixed in 10% buffered formalin and embedded in paraffin wax. Serial sections (4 μm thick) were cut from the paraffin-embedded tissue and placed on poly-l-lysine-coated slides. Deparaffinized sections were autoclaved in 10 mM citrate buffer (pH 6.0) for 3 minutes. To inhibit endogenous peroxidase activity, sections were incubated in 0.3% H2O2 for 10 minutes and stained with our own mouse anti-human MDA5 mAb (H27 [mouse IgG1], 1 μg / mL). Mouse IgG1 (BioLegend, Tokyo, Japan) was used as a control antibody. These antibodies were applied to sections for 18 hours at 4°C or 1–2 hours at room temperature. Positive reactions were identified using goat anti-mouse and anti-rabbit immunoglobulins (Ig) conjugated with peroxidase-labeled polymers (En Vision Dual Link system-HRP, Agilent Dako, Tokyo, Japan) and a liquid DAB substrate / color developer system (Agilent Dako).

[0053] subject Lung tissue and lung cancer samples were obtained from two patients with lung squamous cell carcinoma (a 67-year-old male and a 71-year-old male) who underwent surgical resection at Kurume University Hospital. Serum and / or urine were obtained from 32 healthy donors (23 males and 9 females, ages 28–54 years). Formalin-fixed, paraffin-embedded (FFPE) tissues of tonsils (a 4-year-old female) and pancreas (a 51-year-old female) were purchased from Bio-options, Inc. (Brea, CA). The study was approved by the Kurume University Hospital Institutional Review Board (approval date: July 31, 2019; approval number: 19090) in accordance with the Declaration of Helsinki (2013). Informed consent to participate in this study was obtained from all patients.

[0054] PBMC isolation and in vitro stimulation Peripheral blood mononuclear cells (PBMCs) were isolated from peripheral blood using Lymphoprep™ (STEMCELL TECHNOLOGIES, Vancouver, Canada). Cells were then washed twice with cold PBS and incubated at 2 x 10 cells. 6 The cells were suspended in cold PBS at 250 μg / mL. The cells were stimulated with 250 μg / mL of polyinosinic acid-polycytidylic acid sodium salt (poly I:C) (catalog no. P1530, Merck, Tokyo, Japan) at 37°C. The supernatants were collected at various time points.

[0055] Western blot analysis NuPAGE (登録商標) Western blot analysis was performed using 7% Tris-Acetate Midi Gel (Thermo Fisher Scientific, Tokyo, Japan) according to the manufacturer's protocol. Anti-human MDA5 mAb (clone H27 or clone H46, 1 μg / mL) was used for immunoblotting as previously reported (T. Hoshino, et al., Am J Respir Crit Care Med 176 (2007) 49-62).

[0056] Establishment of a human MDA5 sandwich immunoassay system MESOSCALE DISCOVERY (登録商標)A human MDA5 sandwich immunoassay system was established using electrochemiluminescence in an assay system (Meso Scale Japan, Tokyo, Japan). Briefly, mouse anti-human MDA5 monoclonal antibody (clone H46) was dissolved at 2 μg / mL in phosphate-buffered saline (PBS) as the primary mAb and dispensed into the plate at 25 μL / well aliquots and incubated overnight at 4°C. The plate was washed three times with 150 μL of Quantikine Wash Buffer 1 (R&D Systems, Minneapolis, MN, USA), and 100 μL / well of Block Ace blocking solution (Nakarai Tesque, Kyoto, Japan) was added. The plate was then incubated at room temperature for at least 1 hour to prevent nonspecific adsorption of the secondary antibody to the plate. The plate was then washed three times. Samples were dispensed at 25 μL / well. Recombinant human MDA5 protein diluted to 50 ng / mL, 10 ng / mL, 2 ng / mL, 400 pg / mL, 80 pg / mL, 16 pg / mL, and 3.2 pg / mL was used as a standard. After incubation at room temperature for 1 hour, each well was washed three times. Next, 2 μg / mL of biotin-labeled mouse anti-human MDA5 secondary mAb (clone H27) was dispensed at 25 μL / well, incubated at room temperature for 1 hour, and each well was washed three times. This was followed by 50 μL of 0.5 μg / mL MSDSULFO-TAG (商標) Labeled streptavidin was added to each well, and the plate was incubated at room temperature for 30 minutes. Each well was washed three times. The amount of human MDA5 protein was measured using a MESO QuickPlex SQ 120 according to the manufacturer's protocol.

[0057] result Characterization of MDA5 expression in tissues To examine MDA5 mRNA levels in human normal tissues, real-time quantitative PCR was performed. Repeated analysis revealed that MDA5 mRNA was constitutively expressed in the lung, spleen, pancreas, and placenta, and weakly expressed in muscle (Figure 3). Normal tonsils and pancreas were obtained. Immunohistochemical analysis was performed to analyze MDA5 protein expression using the in-house anti-MDA5 mAb clone H27. MDA5 protein was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and pancreatic cells in the pancreas (Figure 4). Normal lung tissue and lung cancer tissue were obtained from two patients with squamous cell lung carcinoma. Both the normal lung tissue and lung cancer tissue strongly and constitutively expressed MDA5 protein. As previously reported (DC Kang, et al., Proc Natl Acad Sci USA 99 (2002) 637-642), the molecular weight of human MDA5 was approximately 120 kDa. Furthermore, a weak band of MDA5 protein was observed around 60 kDa in both normal lung tissue and lung cancer (Fig. 5). The same results were obtained with two anti-human MDA5 mAbs (clone H27 or clone H46).

[0058] Soluble MDA5 protein in serum We predicted that a soluble form of human MDA5 protein exists in human serum. First, we established an in-house enzyme-linked immunosorbent assay (ELISA) using two human MDA5 mAbs (clones H27 and H46), similar to a previous report (S.I. Takenaka, et al., supra). The detection range was typically 400 pg / mL to 25 ng / mL. Using this in-house MDA5-ELISA, we analyzed MDA5 protein in the serum of seven healthy donors. MDA5 protein was detectable in the serum of one of the seven donors (395 pg / mL). However, 395 pg / mL was close to the limit of the detectable level of this ELISA assay. Therefore, we performed MESOSCALE DISCOVERY. (登録商標)We constructed a highly sensitive sandwich immunoassay system using electrochemiluminescence in an assay system (Meso Scale Japan, Tokyo, Japan) and analyzed MDA5 protein in the serum of 32 healthy donors (Figure 6). The detection range of this system was typically 80 pg / mL to 50 ng / mL. Soluble MDA5 protein was detectable in the serum of 14 donors (3314 ± 7215 pg / mL, n = 32). The soluble MDA5 protein level in the serum of three donors exceeded 10 ng / mL. In contrast, soluble MDA5 was undetectable in the serum of 18 donors. Interestingly, soluble MDA5 protein was undetectable in the urine of six healthy donors. This suggests that normal kidneys do not excrete soluble MDA5, approximately 60 kDa, through the nephron.

[0059] Characteristics of soluble MDA5 protein Immunohistochemical analysis of tonsils showed that lymphoid cells strongly expressed MDA5. We also found a soluble form of human MDA5 protein in serum from healthy donors. Therefore, we expected that PBMCs might produce soluble MDA5 protein. PBMCs were isolated from the peripheral blood of four healthy donors. RPMI 1640 containing 10% FCS stimulated PBMCs, and 2x10 PBMCs were cultured in RPMI 1640 containing 10% FCS. 6The cells were suspended in cold PBS at 1000 cells / mL. PBMCs were stimulated with 250 μg / mL polyinosinic acid-polycytidylic acid sodium salt (poly I:C) at 37°C. MDA5 detects poly I:C, a synthetic dsRNA analog (H. Kato, et al., Nature 441 (2006) 101-105). Supernatants were collected at various time points. A highly sensitive sandwich immunoassay system revealed that MDA5 protein levels increased in PMBC supernatants within 15 minutes after poly I:C stimulation. MDA5 protein levels in PMBC supernatants decreased after 30 minutes and were almost undetectable after 2 hours. Representative data are shown in Figure 7. Western blotting also showed the same results; MDA5 protein increased in PMBC supernatants within 15 minutes after poly I:C stimulation and was almost undetectable after 2 hours. The molecular weight of soluble MDA5 was approximately 60 kDa. Interestingly, PBMCs expressed MDA5 proteins with molecular weights of approximately 60 and 120 kDa (Figure 8). In this study, a weak band of MDA5 protein around 60 kDa was also observed in normal lung tissue and lung cancer (Figure 5). The full-length MDA5 (IFIH1) cDNA encodes 1,025 amino acids (DC Kang, et al., supra). The predicted overall molecular weight is 116,686.83. These results suggest that soluble MDA5 protein may be generated in some tissues by alternative splicing of MDA5 mRNA or protease-mediated cleavage of the MDA5 protein. [Industrial Applicability]

[0060] The method of the present disclosure can determine whether a subject is infected with a virus, and the results obtained can be used for diagnosis.

Claims

1. 1. A method for determining whether a subject is infected with a virus, comprising: (1) testing a sample collected from a subject to determine whether a protein having an amino acid sequence of at least a portion of the helicase domain of the MDA5 protein and a molecular weight of approximately 60 kDa is detected; (2) when the protein is detected, it indicates that the subject is infected with the virus; method.

2. The method of claim 1, wherein the protein comprises a region corresponding to positions 415 to 424 of SEQ ID NO:

1.

3. The method according to claim 2, wherein the region corresponding to positions 415 to 424 of SEQ ID NO: 1 consists of the amino acid sequence QILENSLLNL (SEQ ID NO: 18).

4. The method of claim 1 , wherein the virus is an RNA virus.

5. The method according to claim 1, wherein in step (1), the protein is detected by sandwich ELISA.

6. The method according to any one of claims 1 to 5, wherein in step (1), the protein is detected using a monoclonal antibody comprising (i) or (ii) or a monoclonal antibody that competes with the monoclonal antibody for binding to MDA5; (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO:7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO:8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:9 (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and A light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO:

17.

7. The method according to claim 6, wherein in step (1), the protein is detected using a monoclonal antibody comprising (i) or (ii).

8. The monoclonal antibody (i) comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, or (ii) and comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:

11.

9. (i) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 4, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 5, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 6; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 7, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 8, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 9; or (ii) a heavy chain variable region comprising a heavy chain CDR1 comprising the amino acid sequence of SEQ ID NO: 12, a heavy chain CDR2 comprising the amino acid sequence of SEQ ID NO: 13, and a heavy chain CDR3 comprising the amino acid sequence of SEQ ID NO: 14; and a light chain variable region comprising a light chain CDR1 comprising the amino acid sequence of SEQ ID NO: 15, a light chain CDR2 comprising the amino acid sequence of SEQ ID NO: 16, and a light chain CDR3 comprising the amino acid sequence of SEQ ID NO: 17; or a monoclonal antibody which competes with said monoclonal antibody for binding to MDA5.

10. The monoclonal antibody of claim 9, which is a monoclonal antibody comprising (i) or (ii).

11. (i) comprising a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 3, or The antibody of claim 10, comprising (ii) a heavy chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising an amino acid sequence having 90% or more identity to the amino acid sequence of SEQ ID NO:

11.

12. A kit for determining whether a subject is infected with a virus, comprising an antibody that binds to a protein that comprises at least a partial amino acid sequence of the helicase region of the MDA5 protein and has a molecular weight of approximately 60 kDa.

13. A protein consisting of a portion of the amino acid sequence of the MDA5 protein, containing at least a portion of the amino acid sequence of the helicase region of the MDA5 protein, containing a region corresponding to positions 415 to 424 of SEQ ID NO: 1, and having a molecular weight of approximately 60 kDa.

14. Use of a protein having a molecular weight of approximately 60 kDa and comprising at least a portion of the amino acid sequence of the helicase region of the MDA5 protein as a marker for determining viral infection.

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