JP2007099724A - Antiviral agent targeting sulfatide - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an antiviral agent effective for various viral diseases to begin with that of influenza virus. <P>SOLUTION: This antiviral agent targeting sulfatides is provided by using the antiviral agent targeting the sulfatides and containing an antisulfatide antibody as an active ingredient, the antiviral agent targeting the sulfatides and containing an arylsulfatase A (ASA) as an active ingredient, the antiviral agent targeting the sulfatides and containing an arylsulfatase A (ASA) gene as an active ingredient, the antiviral agent targeting the sulfatides and containing a sulfatide synthase inhibitor as an active ingredient, the antiviral agent targeting the sulfatides and containing an antisense strand of the sulfatide synthase gene as an active ingredient, and the antiviral agent targeting the sulfatides and containing an siRNA targeting the mRNA of the sulfatide synthase as an active ingredient. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an antiviral agent effective against influenza virus targeting sulfatide.

  As a therapeutic agent for influenza virus, a proton channel inhibitor such as amantadine that binds to the M2 protein of influenza A virus and exhibits antiviral activity and one of surface glycoproteins of influenza A and B viruses have been used so far. Sialic acid derivative 2,3-didehydro-2,4-dideoxy-4-guanidino-N-acetylneuraminic acid (trade name zanamivir) which shows antiviral action by binding to neuraminidase and inhibiting sialidase activity Has been developed. In addition, as an influenza virus therapeutic agent, an anti-influenza virus agent containing a bayberry solvent extract as an active ingredient (for example, refer to Patent Document 1), an Izumo native soba extract is used as an active ingredient Anti-influenza virus agent (for example, see Patent Document 2), anti-influenza virus agent (for example, see Patent Document 3) characterized by containing cromoglycic acid or a salt thereof as an effective ingredient, and effective caffeic acid phenethyl ester Proliferation inhibitors of influenza viruses contained as components (for example, see Patent Document 4) have been proposed.

  In addition, a recognized site of a virus receptor selected from the group comprising ganglioside GM4, ganglioside GM3, ganglioside GD3, sialylparaglobond, sulfatide, which is effective against viruses such as influenza A virus and HIV-1 virus. An antiviral agent containing a glycosphingolipid having a sugar chain containing an active ingredient (see, for example, Patent Document 5), or administered orally or parenterally to prevent the influenza virus from adsorbing to the tissue, Influenza virus adsorption inhibitor containing sulfatide as an active ingredient capable of protecting against infection (see, for example, Patent Document 6), and inhibitor of neutrophil surface adhesion molecule selectin consisting of biologically derived sulfated glycolipid such as sulfatide A, B, C, non-ABC hepatitis c Sulfatides (sulfated glycolipids) such as mitigation of liver damage due to lupus infection, immunodeficiency due to infection with various retroviruses, suppression of cell degeneration effective for suppression of cranial nerve damage and organ toxicity mitigation agent (for example, see Patent Document 7) Antiviral agents as active ingredients have also been proposed.

  On the other hand, sulfatide is one of the major sulfated glycolipids abundantly expressed in organs including intestines and lungs of various mammals (see, for example, Non-Patent Documents 1 to 7). Sulfatide is also detected in cultured cell lines such as canine, bovine, and monkey kidneys where influenza virus is primarily isolated and proliferated. It is known that sulfatide interacts with extracellular matrix, adhesion molecule, HIV virus (for example, see Non-Patent Documents 8 and 9), bacteria, and malaria parasite (for example, see Non-Patent Documents 10 to 12). . The biosynthesis of sulfatide is Golgi, and is performed by transferase, ceramide galactosyltransferase (CGT), cerebroside (galactosylceramide) sulfotransferase (CST) (see, for example, Non-Patent Documents 2 and 13). CGT converts ceramide to galactosylceramide, a precursor of sulfatide. As shown in FIG. 1, 3′-O sulfation by CST occurs while sulfatide degradation is performed by an enzyme that desulfates galactose residues in the sulfatide molecule, lysosomal allylsulfatase A (ASA). Subsequently, sulfatide is synthesized (for example, see Non-Patent Document 14).

  The present inventors have previously shown that sulfatide binds to influenza A virus particles and inhibits viral infection and sialidase activity at low pH (see, for example, Non-Patent Documents 15 and 16). However, the role of sulfatide for influenza A virus infection remained unclear.

JP 2004-331608 A Japanese Patent Application Laid-Open No. 2004-2361 JP 2004-352712 A JP 2004-91446 A JP 2001-233773 A JP-A-10-72355 JP-A-8-109134 J. Biochem. (Tokyo) 100, 825-835 (1986) J. Biol. Chem. 272, 4864-4868 (1997) Eur. J. Biochem. 267, 1909-1917 (2000) Vet. Microbiol. 36, 307-316 (1993) J. Natl. Cancer. Inst. 63, 1153-1160 (1979) FEMS Microbiol. Lett. 209, 183-188 (2002) J. Biochem. (Tokyo) 130, 279-283 (2001) Virology 246, 211-220 (1998) AIDS 6, 1213-1214 (1992) Infect. Immun. 64, 624-628 (1996) Exp. Parasitol. 84, 42-55 (1996) J. Biol. Chem. 268, 10356-10363 (1993) Biochem. Biophys. Res. Commun. 32, 449-453 (1997) J. Biol. Chem. 264, 1252-1259 (1989) Biochem. J. 318, 389-393 (1996) FEBS Lett. 553, 355-359 (2003)

  Proton channel inhibitors such as amantadine, which have been developed as a therapeutic agent for influenza virus, are not effective against influenza B and C viruses, and side effects and the emergence of resistant viruses are problematic. In addition, neuraminidase inhibitors have not been established as safe for children and the elderly, and are not effective against influenza C viruses. On the other hand, no effective therapeutic agent has been developed for human parainfluenza virus infection that is known to cause severe symptoms such as bronchitis and pneumonia in infants. An object of the present invention is to provide an antiviral agent effective for various viral diseases including influenza virus.

  The present inventors have found that influenza A virus exhibits strong binding properties not only to sialic acid-containing sugar chains but also to glycolipids such as sulfatides that do not contain sialic acid at all. Sulfatide is widely distributed in animal tissues, and high expression is observed in tissues such as kidney, bronchus and lung. Furthermore, it is known to exist in kidney-derived epithelial cell lines such as dogs, cows, monkeys and the like that are widely used for isolation and culture of influenza viruses and parainfluenza viruses. Therefore, as a result of producing an anti-sulfatide monoclonal antibody and examining the influenza virus infection inhibitory effect, it was found that the monoclonal antibody against sulfatide strongly inhibits the proliferation of influenza virus infection. On the other hand, the monoclonal antibody against other neutral glycolipid (Gb3) did not show influenza virus infection inhibitory activity. Infection with influenza virus also occurs when the action of an enzyme involved in the biosynthesis of sulfatide is suppressed by the antisense nucleic acid method or when the gene of allylsulfatase A, which is known to degrade sulfatide, is highly expressed. It became clear that growth was inhibited. Furthermore, it has been found that monoclonal antibodies against sulfatide also strongly inhibit human parainfluenza virus and Sendai virus infection. The present invention has been completed based on the above findings.

  That is, the present invention relates to (1) an antiviral agent targeting sulfatide, (2) an antiviral agent targeting sulfatide containing an anti-sulfatide antibody as an active ingredient, and (3) an anti-sulfatide antibody. The antiviral agent targeting sulfatide as described in (2) above, which is an antibody, or (4) the anti-sulfatide monoclonal antibody is a mouse anti-sulfatide monoclonal antibody (GS-5, IgM), (3) Antiviral agent targeting sulfatide, (5) Antiviral agent targeting sulfatide having allylsulfatase A (ASA) as an active ingredient, (6) Allylsulfatase A (ASA) gene An antiviral agent targeting sulfatide containing an expression vector as an active ingredient, or (7) An antiviral agent targeting sulfatide, which comprises a fatide synthase inhibitor as an active ingredient, (8) an antiviral agent targeting sulfatide, which comprises an antisense strand of a sulfatide synthase gene as an active ingredient, and (9) sulfatide synthesis Antiviral agent targeting sulfatide with siRNA (small interfering RNA) targeting enzyme mRNA as active ingredient and (10) siRNA expression vector targeting sulfatide synthase mRNA as active ingredient (7) to (7) above, wherein the antiviral agent targeting sulfatide and (11) sulfatide synthase are cerebroside sulfotransferase (CST) and / or ceramide galactosyltransferase (CGT). 10) Select one of the sulfatides And antiviral agents and that, (12) viruses, relates to an anti-viral agent targeting any of sulfatide above, wherein the is an influenza virus (1) to (11).

  The present invention shows that antibodies and gene function inhibitors targeting sulfated glycolipids (sulfatides) are effective not only as influenza viruses but also as antiviral agents such as parainfluenza viruses. And antiviral agents effective against various viral diseases including influenza virus, and the results of the present invention are expected to contribute to avoiding social damage caused by viruses such as influenza. .

  The antiviral agent of the present invention includes an antiviral agent targeting sulfatide, that is, an antiviral agent targeting sulfatide having an anti-sulfatide antibody as an active ingredient, and sulfatide having allyl sulfatase A (ASA) as an active ingredient. Antiviral agent targeted, sulfatide targeting allylsulfatase A (ASA) gene expression vector as active ingredient, antiviral agent targeting sulfatide containing sulfatide synthase inhibitor as active ingredient, sulfatide Antiviral agents targeting sulfatide with the antisense strand of the synthase gene as an active ingredient, and antiviral agents targeting sulfatide with siRNA targeting the sulfatide synthase mRNA as an active ingredient are particularly limited. This is not something The virus agent "refers to a composition having an infection control action, control the action of virus growth after viral infection of the virus.

  Viruses that can be expected to have antiviral effects include influenza virus, Newcastle disease virus, parainfluenza virus, Sendai virus, measles virus belonging to the same Paramyxoviridae family as human parainfluenza virus and Sendai virus ), Mumps virus (a virus that causes mumps), and rubella virus, among which influenza virus can be preferably exemplified.

  Specific examples of the anti-sulfatide antibody include monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single chain antibodies, humanized antibodies, and immunospecific antibodies such as bifunctional antibodies capable of simultaneously recognizing two epitopes. These can be prepared by conventional methods using sulfatide as an antigen, among which monoclonal antibodies are more preferable in terms of their specificity. Specific examples of anti-sulfatide monoclonal antibodies include mouse anti-sulfatide monoclonal antibodies (GS-5, IgM).

  Antibodies against sulfatides are produced by administering to animals (preferably non-human) sulfatide or a fragment containing an epitope, or cells expressing the sulfatide on the membrane surface using conventional protocols, for example, preparation of monoclonal antibodies Include the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV-, which give rise to antibodies produced by continuous cell line cultures. Any method such as a hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used. Hereinafter, as a sulfatide, a method for producing a mouse monoclonal antibody that specifically binds to a human-derived sulfatide, that is, a mouse anti-h sulfatide monoclonal antibody will be described by taking human-derived sulfatide as an example.

  A mouse anti-h sulfatide monoclonal antibody-producing hybridoma is prepared by, for example, immunizing a BALB / c mouse with a sulfatide obtained from human, and spleen cells of the immunized mouse and mouse NS-1 cells (ATCC TIB-18). Anti-h sulfatide monoclonal antibody-producing hybridomas can be produced by cell fusion using conventional methods and screening using immunofluorescent staining patterns. In addition, as a method for separating and purifying the monoclonal antibody, any method can be used as long as it is a method generally used for protein purification, and liquid chromatography such as affinity chromatography can be specifically exemplified. .

  In order to produce a single-chain antibody against the sulfatide of the present invention, a single-chain antibody preparation method (US Pat. No. 4,946,778) can be applied. In addition, in order to express humanized antibodies, transgenic mice or other mammals can be used, clones expressing sulfatides can be isolated and identified using the above antibodies, and sulfatides can be identified by affinity chromatography. It can also be purified.

Moreover, as an anti-sulfatide antibody, the Fab fragment of the said antibody couple | bonded specifically with a sulfatide, F (ab ') ₂ fragment | piece, etc. can be used. For example, the Fab fragment can be prepared by treating an antibody with papain or the like, and the F (ab ′) ₂ fragment can be prepared with pepsin or the like.

Examples of the anti-sulfatide monoclonal antibody include fluorescent substances such as FITC (fluorescein isocyanate) or tetramethylrhodamine isocyanate, radioisotopes such as ¹²⁵ I, ³² P, ¹⁴ C, ³⁵ S or ³ H, Perform functional analysis of the sulfatide by using fusion proteins fused with fluorescent proteins such as alkaline phosphatase, peroxidase, β-galactosidase or phycoerythrin, and fluorescent proteins such as green fluorescent protein (GFP) Can do. Examples of the immunological measurement method include RIA method, ELISA method, fluorescent antibody method, plaque method, spot method, hemagglutination method, and octalony method.

  The anti-sulfatide monoclonal antibody can be produced by culturing an anti-sulfatide monoclonal antibody-producing hybridoma in vivo or in vitro by a conventional method. For example, in an in vivo system, it can be obtained by culturing in the abdominal cavity of a rodent, preferably a mouse or a rat. In an in vitro system, it can be obtained by culturing in an animal cell culture medium. As a medium for culturing a hybridoma in an in vitro system, a cell culture medium such as RPMI 1640 or MEM containing an antibiotic such as streptomycin or penicillin can be exemplified.

  Allyl sulfatase A (cerebroside sulfatase) is an enzyme that uses sulfatide as a substrate and decomposes its sulfate group, has all the species examined, and is known as an enzyme localized in lysosomes. The allylsulfatase A may be extracted and purified from animal or plant cells or microorganisms, but can also be prepared by gene recombination technology using the allylsulfatase A gene. The enzyme can be purified by affinity chromatography, ion exchange chromatography, or hydrophobic chromatography. As a method for measuring the activity of allylsulfatase A, generally, [35S] sulfatide and p-nitrocatechol sulfate can be used for enzymes derived from animal tissues, and p-nitrocatechol sulfate can be used for enzymes derived from microorganisms. Alternatively, sulfatide can be used as a substrate, and the activity of sulfuric acid produced by an enzymatic reaction can be measured by ion chromatography. Moreover, as a method for introducing allylsulfatase A into cells, a macromolecule and a non-covalent conjugate are formed, the structure of the macromolecule such as a protein is changed, and the macromolecule such as a protein is delivered into the cell. A non-cytotoxic drug such as Chariot (manufactured by Active Motif) can be used.

  Moreover, as an allylsulfatase A gene expression vector capable of expressing allylsulfatase A, for example, an adenovirus vector (Science, 252) used for transient expression in all cells (other than blood cells) including non-sorted cells. , 431-434, 1991), retroviral vectors used for long-term expression in dividing cells (Microbiology and Immunology, 158, 1-23, 1992) and non-pathogenic, non-dividing cells, Specific examples include adeno-associated virus vectors (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992) and liposomes used for expression, but are not limited thereto. Among these, an adenovirus vector that can express a gene directly in a living body without establishing a cell line orally and can express a gene in a cell system or a living organ with high efficiency is particularly preferable. In addition, introduction of allylsulfatase A gene into these expression vectors can be performed by a conventional method. For example, an expression vector can be obtained by inserting an allylsulfatase A gene (such as NC_000022) downstream of an appropriate promoter in these expression vectors. Can be built. Expression vectors in which the allylsulfatase A gene is incorporated into these viruses can be expressed locally by infecting target cells.

  Examples of the antiviral agent of the present invention include those that inhibit the expression and action of sulfatide synthase such as cerebroside sulfotransferase (CST) and ceramide galactosyltransferase (CGT), which inhibit the action of sulfatide synthase. Illustrate sulfatide synthase inhibitors such as anti-CST monoclonal antibody and anti-CGT monoclonal antibody, the antisense strand of sulfatide synthase gene that inhibits the expression of sulfatide synthase, and siRNA targeting sulfatide synthase mRNA. Can do.

  The antisense strand of the sulfatide synthase gene that inhibits the expression of sulfatide synthase is an antisense polynucleotide having a base sequence complementary to or substantially complementary to DNA encoding sulfatide synthase, preferably an anti-sense polynucleotide. Sense DNA can be mentioned. The base sequence substantially complementary to the DNA encoding sulfatide synthase is, for example, about 70% or more, preferably about 70% or more of the total base sequence or partial base sequence of the base sequence complementary to the sense strand of sulfatide synthase gene DNA. May include base sequences having a homology of about 80% or more, more preferably about 90% or more, and most preferably about 95% or more. Antisense polynucleotides are usually composed of about 10 to 40 bases, preferably about 15 to 30 bases, and in order to prevent degradation by a hydrolase such as nuclease, the phosphate residues of each nucleotide constituting the antisense DNA. The group (phosphate) may be substituted with a chemically modified phosphate residue such as phosphorothioate, methylphosphonate, phosphorodithionate, and the like. These antisense polynucleotides can be produced using a known DNA synthesizer or the like based on the base sequence information of sulfatide synthase gene DNA.

  The antisense strand of the sulfatide synthase gene of the present invention may contain modified / modified sugars, bases, and bonds, and is provided in a special form such as liposome or microsphere, or applied by gene therapy. Or can be administered in an added form. Examples of such additional forms include polycationic substances such as polylysine that act to neutralize the charge of the phosphate group skeleton, lipids that enhance interaction with cell membranes and increase nucleic acid uptake ( For example, hydrophobic substances such as phospholipids and cholesterols). Preferred lipids for addition include cholesterol and derivatives thereof (eg, cholesteryl chloroformate, cholic acid, etc.), which can be attached to the 3 ′ end or 5 ′ end of nucleic acids, It can also be attached via a sugar or intramolecular nucleoside bond. Further, in order to prevent degradation by nucleases such as exonuclease and RNase, modification can be performed with a cap group specifically arranged at the 3 'end or 5' end of the nucleic acid. Examples of the capping group include hydroxyl protecting groups known in the art, including glycols such as polyethylene glycol and tetraethylene glycol. The inhibitory activity of the antisense strand can be examined using a transformant, a gene expression system in vivo or in vitro, or a translation system of sulfatide synthase in vivo or in vitro.

  The siRNA targeting the sulfatide synthase mRNA can suppress the expression of the sulfatide synthase gene and is composed of a sense strand RNA and an antisense strand RNA that are homologous to a specific sequence that is the target of the sulfatide synthase mRNA. It is not particularly limited as long as it is a single-stranded RNA, and the origin of the sulfatide synthase gene is not particularly limited, but a human-derived sulfatide synthase gene is preferred. The specific sequence to be a target of the sulfatide synthase mRNA is a partial sequence of a specific region of the sulfatide synthase mRNA, preferably a partial sequence having a base length of 19 to 21 bp. As a target sequence of the sulfatide synthase mRNA, A sequence specific for sulfatide synthase mRNA is preferred.

  The base sequence of such siRNA can be designed based on the sequence information of the sulfatide synthase gene. SiRNA design guidelines have already been published in many literatures and books, and sulfatide synthase-specific siRNAs can be designed according to these guidelines. For example, siRNA can be prepared by appropriately using a known method such as a synthetic method and a method using a gene recombination technique.

  In the synthesis method, double-stranded RNA can be synthesized by a conventional method based on sequence information. That is, in order to synthesize siRNA, which is a double-stranded oligoribonucleotide, first, a sense strand oligoribonucleotide and an antisense strand oligoribonucleotide are respectively synthesized. In general, synthesis is performed by organic synthesis using a synthesizer using four types of ribonucleotides with protecting groups. RNA is a polymer molecule in which four types of nucleosides (adenosine, guanosine, cytidine, uridine) are linked by a phosphodiester bond. The synthesis is amidite, and 2'-bis (acetoxyethoxy) is added to the 2 'end of the sugar that forms the backbone. -A methyl ether (2'-ACE) protecting group is attached and the bases are phosphate-coupled (coupled) one by one. The generated single-stranded RNA is deprotected by reacting with 100 mM TEMED-Acetate (pH 3.8) buffer at 60 ° C. for 30 minutes, and quality check is performed using HPLC. When high-purity RNA is required, RNA is further purified from the gel or subjected to HPLC after denaturing polyacrylamide gel electrophoresis. Next, both strands are annealed. An aqueous solution of both strands having a concentration of about 50 μM (50 pmol / μL) was prepared, and a solution obtained by mixing each aqueous solution and 5 × annealing buffer (500 mM potassium acetate, 150 mM HEPES-KOH pH 7.4, 10 mM magnesium acetate) was mixed with 90 solutions. Heat at 1 ° C. for 1 minute, then hold at 37 ° C. for 60 minutes. As a result, the sense oligonucleotide and the antisense oligonucleotide are annealed with each other to form a double strand.

  In the method using gene recombination technology, an expression vector incorporating a sense strand DNA or an antisense strand DNA is constructed, and the sense strand RNA or antisense strand RNA generated by transcription after introducing the vector into a host cell. Can be prepared by obtaining a double-stranded RNA expression cassette comprising a sense strand DNA-linker-antisense strand DNA of a specific sequence of a sulfatide synthase gene, and the double-stranded RNA expression cassette Is preferably ligated downstream of the promoter of the expression vector, and the desired double-stranded RNA is produced by in vivo expression and construction.

  siRNA can be directly introduced into cells by a lipofection method or the like. Although a liposome composed of phosphatidylserine (PS) having a negative charge can be used, N- [1- (2,3-dioleyloxy) propyl]-, which is a cationic lipid that forms a more stable liposome. N, N, N-triethylammonium chloride (DOTMA, trade name: Transfectam, Lipofectamine) can also be used. When a complex of these cationic lipids and negatively charged siRNA is formed, positively charged liposomes are adsorbed on the surface of negatively charged cells and fused with the cell membrane so that siRNA can be Can be introduced in. In actual treatment, a complex of siRNA and liposome is directly injected into the affected area or the surrounding tissue.

  In addition, siRNA expression vectors targeting sulfatide synthase mRNA have the advantage that siRNA can be present in the cell longer than when siRNA is directly introduced into the cell, and the efficiency of gene expression suppression is increased. Have. The siRNA expression vector targeting the mRNA of the sulfatide synthase inserts a double-stranded RNA expression cassette consisting of sense strand DNA-linker-antisense strand DNA of a specific sequence of the sulfatide synthase gene downstream of the promoter. Can be produced more advantageously. Moreover, although the vector for siRNA expression can use a well-known thing including a commercial item, when introducing into a mammal, it is preferable that it is a viral vector. Examples of viral vectors include mouse leukemia retrovirus vectors (Microbiology and Immunology, 158, 1-23, 1992), adeno-associated virus vectors (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992), Specific examples include adenoviral vectors (Science, 252, 431-434, 1991) and liposomes, but HIV lentiviral vectors are capable of efficient long-term expression in non-dividing cells. It is preferable at having a point. Moreover, these expression systems may contain not only the expression but also a control sequence that regulates the expression. Introduction of double-stranded RNA expression cassettes into these expression vectors can be performed by conventional methods. For example, by inserting a double-stranded RNA expression cassette downstream of an appropriate promoter in these expression vectors, siRNA expression vectors can be obtained. Can be built.

  The antiviral agent of the present invention can be used as a prophylactic / therapeutic agent for viral diseases. When administering or introducing it into a living body, tissue, cell, etc. of a mammal as a therapeutic agent, a pharmaceutically acceptable carrier, binder, stabilizer, excipient, diluent, pH buffer ordinarily used in this field It can be used together with various compounding ingredients for preparations such as agents, disintegrants, solubilizers, solubilizers, isotonic agents. The pharmaceutical composition used together with the pharmaceutically acceptable carrier is used in the pharmaceutical field according to its administration form, for example, oral (including buccal or sublingual) administration or parenteral administration (injection etc.). Then, it can be formulated in a formulation form known per se. Specific examples of the dosage form include tablets, capsules, fine granules, syrups, suppositories, ointments, injections, and the like. Specific examples of the carrier for the preparation include crystalline cellulose, gelatin, lactose, starch, magnesium stearate, talc, vegetable and animal fats, oil, gum, and polyalkylene glycol. The antiviral agent of the present invention can be contained in the preparation in an amount of 0.001 to 100%. For example, anti-sulfatide antibody (GS-5) can be used at 0.3% and plasmid vector at 0.001%.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

[Inhibition of influenza A virus growth by anti-sulfatide antibody]
1. Anti-sulfatide antibody (GS-5) acts on cells infected with influenza A virus and suppresses the growth of the virus.
Influenza A virus strain: A / WSN / 33 (H1N1) was used. Cells at 15 hours after infection in the presence or absence of acetyltrypsin (AT), which cleaves virus HA and promotes virus growth, were fixed with methanol at room temperature for 30 seconds, and mouse anti-influenza A virus nucleoprotein (NP) The monoclonal antibody (4E6) was reacted at room temperature for 30 minutes. Reaction was carried out with horseradish peroxidase (HRP) -labeled anti-mouse IgG + M at room temperature for 30 minutes. The results are shown in FIG.
Before: Canine kidney-derived (MDCK) cells not infected with virus and anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) were incubated at 37 ° C. for 30 minutes, and then infected at 34 ° C. for 1 hour.
During: Anti-sulfatide monoclonal antibody GS-5 (300 μg / ml)
Infect MDCK cells at 34 ° C for 1 hour.
After: After MDCK cells were infected with MDCK cells at 34 ° C. for 1 hour, anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) was continuously incubated.

  FIG. 2 (a) shows that 10 ml of 100 mM citrate buffer (pH 6.0) is 1 μl of 30% hydrogen peroxide, 200 μl of 60 mM N, N-diethyl-p-phenylenediamine (DEPDA), 100 mM 4-chloro. -Naphtol (4-CN) After mixing 200 μl, the filtered coloring solution was added, and coloring was performed for 10 to 30 minutes at room temperature (DEPDA coloring, virus-infected cells stained blue). FIG. 2 (b) shows the unit The number of virus-infected cells per area is shown. From these results, it can be seen that the anti-sulfatide antibody (GS-5) acts on cells infected with influenza A virus and suppresses the growth of the virus.

2. Anti-sulfatide antibody (GS-5) is effective on infected cells after 4 hours after influenza A virus infection, and reduces the amount of infectious progeny virus produced from infected cells.
Influenza A virus strain: A / WSN / 33 (H1N1) was infected to MDCK cells at 34 ° C. for 1 hour, and then treated with anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) at the times shown in FIG. . Those not treated with GS-5 were used as controls (Con). The virus infectivity titer in the supernatant 24 hours after infection was measured. FIG. 3 shows the results in terms of% relative infectivity when Con is 100%. From FIG. 3, it was found that the anti-sulfatide antibody (GS-5) showed an effect on infected cells after 4 hours after infection with influenza A virus and reduced the amount of infectious progeny virus produced from the infected cells.

3. Anti-sulfatide antibody (GS-5) inhibits influenza A virus growth.
Influenza A virus strain: A / WSN / 33 (H1N1) was infected to MDCK cells at 34 ° C. for 1 hour, and anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) or mouse anti-glycolipid Gb3Cer monoclonal antibody (TU-1) Cultivated in serum-free medium (containing 2.5 μg / ml acetyltrypsin) containing subclass IgM) (500 μg / ml). A medium containing no antibody was used as a control (Con). Cells 26 hours after infection were fixed with methanol at room temperature for 30 seconds, and a mouse anti-influenza A virus NP monoclonal antibody (4E6) was reacted at room temperature for 30 minutes. The mixture was reacted with HRP-labeled anti-mouse IgG + M for 30 minutes at room temperature. FIG. 4 (a) shows the result of color development of infected cells by DEPDA, and FIG. 4 (B) shows the result of measuring the virus infectivity (plaque forming unit; pfu / ml) in the supernatant. From these results, it was found that the anti-sulfatide antibody (GS-5) suppressed the growth of influenza A virus, but the anti-glycolipid Gb3Cer antibody (TU-1) did not suppress the growth of influenza A virus.

4). Anti-sulfatide antibody (GS-5) also suppresses the growth of influenza A viruses other than WSN strains.
Influenza A virus strain: A / Memphis / 1/71 (H3N2) or A / duck / 313/4/78 (H5N3) was infected to MDCK cells at 34 ° C. for 1 hour, and anti-sulfatide monoclonal antibody GS-5 (300 μg / Ml) or a serum-free medium (containing 2.5 μg / ml acetyltrypsin) containing mouse anti-glycolipid Gb3Cer monoclonal antibody (TU-1; subclass IgM) (500 μg / ml). A medium containing no antibody was used as a control (Con). Cells 24 hours after infection were fixed with methanol at room temperature for 30 seconds, and mouse anti-influenza A virus NP monoclonal antibody (4E6) was reacted at room temperature for 30 minutes. The infected cells were reacted with HRP-labeled anti-mouse IgG + M at room temperature for 30 minutes to develop DEPDA color. The results are shown in FIG. FIG. 5 shows that anti-sulfatide antibody (GS-5) suppresses the growth of influenza A virus strains: A / Memphis / 1/71 (H3N2) and A / duck / 313/4/78 (H5N3), It was found that the glycolipid Gb3Cer antibody (TU-1) does not suppress the growth of these influenza A viruses.

5. Anti-sulfatide antibody (GS-5) inhibits influenza A virus growth.
MDCK cells were infected with influenza A virus strain: A / Memphis / 1/71 (H3N2) at 34 ° C. for 1 hour, and serum-free medium (acetyltrypsin (anti-trypsin (300 μg / ml) containing anti-sulfatide monoclonal antibody GS-5) AT) (GS-5 / AT; ●) or no (GS-5; ▪)) was cultured for 0, 2, 6, 18, 30, and 45 hours. Serum-free medium (containing AT (Con / AT; ◯) or not containing (Con; □)) was used as a control. The cells were fixed with methanol for 30 seconds at room temperature, and mouse anti-influenza A virus NP monoclonal antibody (4E6) was reacted at room temperature for 30 minutes, and then reacted with HRP-labeled anti-mouse IgG + M for 30 minutes at room temperature. FIG. 6 (A) shows the result of measuring DEPDA (N, N-Diethyl-p-phenylenediamine) color and measuring the hemagglutination unit (HAU) by the virus in the culture supernatant for 45 hours. Moreover, the result of counting the number of infected cells per unit area at each time is shown in FIG. 6B, and a photograph of infected cells after 30 hours of culture (100 magnification) is shown in FIG. 6C. From these results, it was found that the anti-sulfatide antibody (GS-5) suppresses the growth of influenza A virus even in the presence of acetyltrypsin (AT) that promotes virus growth.

[Inhibition of growth of parainfluenza A virus by anti-sulfatide antibody]
1. Anti-sulfatide antibody (GS-5) suppresses the growth of parainfluenza virus (hPIV1, hPIV3, SV, NDV).
hPIV1: human parainfluenza type 1, hPIV3: human parainfluenza type 3, SV: Sendai virus and NDV: Newcastle disease virus were tested. Monkey kidney-derived LLCCMK2 cells were infected with each virus at 34 ° C. for 1 hour, and serum-free medium containing anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) or anti-glycolipid Gb3Cer monoclonal antibody TU-1 (500 μg / ml) ( 1 μg / ml AT) was cultured at 37 ° C. for 48 hours. A medium containing no antibody was used as a control (Con). The cells were fixed with methanol at room temperature for 30 seconds, and a rabbit polyclonal antibody against each virus was reacted at room temperature for 30 minutes. Reaction was carried out with horseradish peroxidase (HRP) -labeled Protein A at room temperature for 30 minutes. To 10 ml of 100 mM citrate buffer (pH 6.0), 1 μl of 30% hydrogen peroxide, 200 μl of 60 mM DEPDA, and 200 μl of 100 mM 4-CN (4-chloro-naphtol) are added, and then the filtered coloring solution is added. For 10 to 30 minutes. The results are shown in FIG. 7 (DEPDA coloration, virus-infected cells stained blue). From these results, it was found that the anti-sulfatide antibody (GS-5) suppressed the growth of parainfluenza virus, but the anti-glycolipid Gb3Cer antibody (TU-1) did not suppress the growth of parainfluenza virus.

  In addition, monkey kidney-derived LLCCMK2 cells were infected with each virus at 34 ° C. for 1 hour, and cultured in a serum-free medium (containing 1 μg / ml AT) containing GS-5 or TU-1 at 37 ° C. for 48 hours. A medium containing no antibody was used as a control (Con). The infectious progeny virus titer of the culture supernatant after 48 hours was measured. The results are shown in FIG. In the measurement of the virus titer of the culture supernatant, anti-sulfatide antibody (GS-5) inhibits the growth of parainfluenza virus, but anti-glycolipid Gb3Cer antibody (TU-1) does not inhibit the growth of parainfluenza virus. all right.

2. Anti-sulfatide antibody (GS-5) suppresses the growth of parainfluenza virus (hPIV1, hPIV3, SV, NDV) in a concentration-dependent manner.
Monkey kidney-derived LLCCMK2 cells were infected with each virus of hPIV1, hPIV3, SV and NDV at 34 ° C. for 1 hour, and contained serum-free anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) diluted 1 to 8 times. The cells were cultured in a medium (containing 1 μg / ml AT) at 37 ° C. for 48 hours. A medium containing no antibody was used as a control (No GS-5). The cells were fixed with methanol at room temperature for 30 seconds, and a rabbit polyclonal antibody against each virus was reacted at room temperature for 30 minutes. Reaction was carried out with horseradish peroxidase (HRP) -labeled Protein A at room temperature for 30 minutes. To 10 ml of 100 mM citrate buffer (pH 6.0), 1 μl of 30% hydrogen peroxide, 200 μl of 60 mM DEPDA, and 200 μl of 100 mM 4-CN (4-chloro-naphtol) are added, and then the filtered coloring solution is added. For 10 to 30 minutes. The results are shown in FIG. 9 (DEPDA coloration, virus-infected cells stained blue). From these results, it was found that the anti-sulfatide antibody (GS-5) suppresses the growth of parainfluenza virus in a concentration-dependent manner.

3. Anti-sulfatide antibody (GS-5) does not inhibit the initial infection of parainfluenza virus, but reduces the amount of infectious progeny virus produced from infected cells.
Anti-sulfatide monoclonal antibody GS-5 (300 μg / ml) or anti-glycolipid Gb3Cer monoclonal antibody TU-1 (30 minutes before virus infection of hPIV1, hPIV3, SV and NDV and during virus infection (34 ° C., 1 hour) Monkey kidney-derived LLCCMK2 cells were treated with 500 μg / ml). The untreated antibody was used as a control (Con). Cells were cultured for 16 hours in serum-free medium. The cells were fixed with methanol at room temperature for 30 seconds, and a rabbit polyclonal antibody against each virus was reacted at room temperature for 30 minutes. The mixture was reacted with HRP-labeled Protein A at room temperature for 30 minutes to develop DEPDA color. FIG. 10 (A) shows the results shown as the number of relative infected cells when Con is 100%. In addition, each virus was infected at 34 ° C. for 1 hour under the condition where all LLCCMK2 cells were infected. FIG. 10 (B) shows the results of culturing in a serum-free medium containing GS-5 or TU-1 for 24 hours and measuring the infectious virus infectivity titer (TCID ₅₀ /0.1 ml) in the supernatant. From these results, it was found that the anti-sulfatide antibody (GS-5) does not inhibit the initial infection of parainfluenza virus, but reduces the amount of infectious progeny virus produced from infected cells.

[Inhibition of virus growth by sulfatide-degrading enzyme]
1. Sulfatide-degrading enzyme (allyl sulfatase A; ASA) expression in canine kidney-derived MDCK cells reduces the amount of sulfatide and also reduces influenza A virus growth.
A pTriEx3-neo vector (ASA) into which a human ASA gene (NC — 000022) was incorporated or a vector alone (Vector) was introduced into MDCK cells and cultured under neomycin G418 (1 mg / ml) for 2 weeks or more. After fixing the cells with methanol, mouse anti-sulfatide monoclonal antibody (GS-5) was reacted on ice for 1 hour. FIG. 11 (a) shows the result of comparing the amount of sulfatide in each cell by reacting FITC-labeled anti-mouse IgM-PBS 100-fold diluted solution on ice for 1 hour, measuring the amount of sulfatide in each cell with a flow cytometer. Further, the cells were fixed with methanol, and the same procedure as in (a) above was performed. The result of observation with a confocal laser microscope and comparing the amount of sulfatide is shown in FIG. 11B (sulfatide; green, nucleus; DAPI staining, blue).

  Influenza A virus strain: A / WSN / 33 (H1N1) was infected at a low infectivity at 34 ° C. for 1 hour, and cultured in a serum-free medium containing 2.5 μg / ml acetyltrypsin (AT) for 26 hours. The virus infectivity (plaque forming unit; pfu / ml) of the supernatant was measured, and the results of comparison of virus growth are shown in FIG. Infected cells were cultured in a serum-free medium containing influenza virus sialidase inhibitor zanamivir (1 μM) for 16 hours. The cells were fixed with methanol at room temperature for 30 seconds, and reacted with a mouse anti-influenza A virus nucleoprotein (NP) monoclonal antibody (4E6) at room temperature for 30 minutes. It was made to react with a horseradish HRP labeled anti-mouse IgG + M 3000 times dilute solution at room temperature for 30 minutes. Mixing 10 μl of 100 mM citrate buffer (pH 6.0) with 1 μl of 30% hydrogen peroxide, 200 μl of 60 mM DEPDA (N, N-Diethyl-p-phenylenediamine), 200 μl of 100 mM 4-CN (4-chloro-naphtol) Thereafter, a filtered color developing solution was added, and color was allowed to develop for 10 to 30 minutes at room temperature (DEPDA color development, virus-infected cells stained blue). FIG. 11 (d) shows the result of counting the number of infected cells per unit area and comparing the number of initial infected cells. FIG. 11 (e) shows the result of the above (c), in which infected cells were stained in the same manner as in (d), observed in a 40 × microscope field of view, and virus growth was compared.

[Inhibition of virus growth by RNAi]
1. RNA interference (RNAi) of sulfatide synthase (CST) in MDCK cells reduces the amount of sulfatide and reduces influenza A virus growth.
The 401st-419th, 337-357th and 29th-47th GFP genes of CST gene were incorporated into RNAi vector pSilencer3.1-H1 neo, and the gene was introduced into MDCK cells. G418 drug selection and cloning were performed for over 2 weeks. The amount of CST mRNA in the cells was quantified by real-time PCR based on the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. The primer pair used in real-time PCR was shown as a relative value (%) of the amount of mRNA of the parent MDCK strain, and the RNAi effect on CST mRNA was compared. The results are shown in FIG. Further, after fixing the cells with methanol, GS-5 was reacted on ice for 1 hour. FITC-labeled anti-mouse IgM-PBS 100-fold diluted solution was allowed to react on ice for 1 hour, and the amount of sulfatide in each cell was measured and compared with a flow cytometer. The results are shown in FIG. In addition, the virus was infected at a low infectivity titer at 34 ° C. for 1 hour and cultured in a serum-free medium containing 2.5 μg / ml AT for 26 hours. The virus infectivity (pfu / ml) of the supernatant was measured, and the virus growth was compared. The results are shown in FIG. The cells of (c) were fixed with methanol at room temperature for 30 seconds, and 4E6 was reacted at room temperature for 30 minutes. The mixture was reacted with an HRP-labeled anti-mouse IgG + M 3000-fold dilute solution at room temperature for 30 minutes. DEPDA color was allowed to develop for 10-30 minutes at room temperature. Observation was performed with a 40-power microscope field, and virus growth was compared. The results are shown in FIG.

[Sulphatide highly expressing COS7 cells]
1. Monkey kidney-derived COS7 cells into which two canine-derived sulfatide synthases have been introduced have greatly increased influenza A virus proliferative ability as compared to parent COS7 cells.
A pIRES-neo vector incorporating sulfatide synthases CST and CGT was introduced into COS7 cells with very little sulfatide expression, G418 drug selection and cloning were performed, and two sulfatide-expressing COS7 cells were cloned, that is, SulCOS1: sulfatide high An expressed COS7 cell clone 1 (a 3-base CAG was inserted at position 132 from the start codon of CST) and SulCOS2: a sulfatide highly expressing COS7 cell clone 2 were obtained. After fixing the cells with methanol, anti-sulfatide antibody (GS-5) was reacted on ice for 1 hour. FITC-labeled anti-mouse IgM-PBS 100-fold diluted solution was reacted on ice for 1 hour, and the amount of sulfatide in each cell was measured with a flow cytometer and compared. The results are shown in FIG. Further, the cells were fixed with methanol, and the same procedure as in (a) above was performed. Observed with a confocal laser microscope, the amount of sulfatide was compared. The results are shown in FIG. 13 (b) (sulfatide; green, nucleus; DAPI staining, blue). In addition, the virus was infected at a low infectious titer at 34 ° C. for 1 hour and cultured in a serum-free medium containing 2.5 μg / ml acetyltrypsin (AT) for 24 hours. The virus infectivity (pfu / ml) of the supernatant was measured, and the virus growth was compared. The results are shown in FIG. Infected cells were cultured in a serum-free medium containing influenza virus sialidase inhibitor zanamivir (1 μM) for 15 hours. The cells were fixed with methanol at room temperature for 30 seconds, and 4E6 was reacted at room temperature for 30 minutes. The mixture was reacted with an HRP-labeled anti-mouse IgG + M 3000-fold dilute solution at room temperature for 30 minutes. The number of infected cells per unit area developed with DEPDA for 10 to 30 minutes at room temperature was counted, and the initial number of infected cells was compared. The results are shown in FIG. In addition, the results of (c) above were stained for infected cells in the same manner as (d), and observed under a 40-power microscope field to compare virus growth. The results are shown in FIG.

2. Anti-sulfatide antibody (GS-5) suppresses influenza A virus growth in COS7 cells that highly express sulfatide.
A serum-free medium (AT 2.5 μg / ml) containing anti-sulfatide monoclonal antibody GS-5 or anti-glycolipid Gb3Cer monoclonal antibody TU-1 by infecting sulfatide-highly expressing COS7 cells (SulCO1, SulCOS2) with virus at 34 ° C. for 1 hour. Containing) for 20 hours. As a control, only a serum-free medium (containing AT 2.5 μg / ml) was used (Con). The virus infection titer (pfu / ml) of the supernatant was measured, and the result of comparison of the virus growth inhibitory effect of the antibody is shown in FIG. 14 (a). The cells were fixed with methanol at room temperature for 30 seconds, and NP monoclonal antibody (4E6) was reacted at room temperature for 30 minutes. By reacting with HRP-labeled anti-mouse IgG + M at room temperature for 30 minutes, infected cells were developed with DEPDA color, and the virus growth inhibitory effect of the antibody was compared. The results are shown in FIG.

3. Influenza A viruses other than WSN strains also grow in COS7 cells that highly express sulfatide.
Influenza A virus strain: A / Memphis / 1/71 (H3N2) or A / duck / 313/4/78 (H5N3) was infected to SulCOS1 cells at 34 ° C. for 1 hour, and serum-free medium (2.5 μg / ml Cultured with AT). Cells 24 hours after infection were fixed with methanol at room temperature for 30 seconds, and NP monoclonal antibody (4E6) was reacted at room temperature for 30 minutes. The infected cells were reacted with HRP-labeled anti-mouse IgG + M for 30 minutes at room temperature to develop DEPDA color. The results are shown in FIG.

[Promotion of apoptosis induction by sulfatide]
1. Sulfatide promotes apoptosis induction of influenza A virus.
Cells were infected with highly infectious virus for 1 hour at 34 ° C., 2% FBS-containing medium containing anti-sulfatide monoclonal antibody GS-5 or anti-glycolipid Gb3Cer monoclonal antibody TU-1, COS7 cells were 16 hours, MDCK cells were Cultured for 24 hours. Peel off cells with 0.125% trypsin and stain FITC-labeled annexin V (an indicator of apoptosis that recognizes and binds translocation of lipid phosphatidylserine inside the cell membrane to the outside of the membrane that occurs early in apoptosis) and dead cells Stained with propidium iodide (PI) for 15 minutes at room temperature. A 2% FBS-containing medium containing no antibody was used as a control (Con). Stained cells were analyzed with a flow cytometer. There was almost no difference in PI staining in any cell. FIG. 16 (a) shows the results of virus-induced apoptosis in sulfatide highly expressing SulCOS1 cells and the anti-apoptotic effect of anti-sulfatide antibody (GS-5). In addition, FIG. 16B shows the results of virus-induced apoptosis in COS7 cells and the effect of GS-5. Moreover, the virus-induced apoptosis comparison result of ASA expression MDCK cell and MDCK cell is shown in FIG.16 (c).

[Inhibition of NP transport by anti-sulfatide antibody (GS-5), etc.]
1. Although the cell membrane transport of the nascent viral membrane proteins hemagglutinin (HA) and neuraminidase (NA) in sulfatide-expressing SulCOS1 cells is normal, the transport of the nascent viral nucleoprotein (NP) from the nucleus to the cytoplasm is compared to the parent COS7 cells. Promoted. Furthermore, anti-sulfatide antibody (GS-5) suppresses NP transport.
Highly infectious influenza A virus strain: A / Memphis / 1/71 (H3N2) was infected to sulfatide highly expressing SulCOS1 cells and parent COS7 cells at 34 ° C. for 1 hour. The cells were cultured in a medium containing 1% FBS at 37 ° C. for 7 hours and fixed with methanol for 30 seconds. Anti-influenza A virus H3HA monoclonal antibody (2E10), N2NA monoclonal antibody (SI-4) or NP monoclonal antibody (4E6) was reacted overnight at 4 ° C. TRITC-labeled goat anti-mouse IgG 100-fold diluted solution was reacted at 4 ° C. for 2 hours, and an anti-fading agent was added. Nuclei were DAPI stained. The nascent virus protein was observed under a confocal laser microscope. The results are shown in FIG. In FIG. 17, (a)-(c) and (g)-(h) are SulCOS1 cells (SulCOS1), (d)-(f) and (i)-(j) are COS7 cells (Parent), (A), (d), (g), (h) are HA staining, (b), (e) are NA staining, (c), (f), (i), (j) are NP staining. (G)-(h) represent the results of culturing at 37 ° C. for 7 hours in a medium containing 1% FBS containing GS-5 or TU-1 after virus infection, and (g), (i) are GS- (H), (j) shows TU-1 processing. In addition, the white line of (a) shows 50 micrometers.

2. Although the transport of nascent viral membrane protein HA in MDCK cells is normal, transport of nascent NP from the nucleus to the cytoplasm is suppressed in ASA-expressing MDCK cells. Furthermore, the anti-sulfatide antibody (GS-5) does not affect the cell membrane transport of the nascent virus HA and NA in MDCK cells, but suppresses the transport of the nascent virus NP to the cytoplasm.
MDCK cells were infected with a highly infectious influenza A virus strain: A / Memphis / 1/71 (H3N2) at 34 ° C. for 1 hour. The cells were cultured in a medium containing 1% FBS at 37 ° C. for 7 hours and fixed with methanol for 30 seconds. The reaction was carried out overnight at 4 ° C. with H3HA monoclonal antibody (2E10), N2NA monoclonal antibody (SI-4) or NP monoclonal antibody (4E6). TRITC-labeled goat anti-mouse IgG 100-fold diluted solution was reacted at 4 ° C. for 2 hours, and an anti-fading agent was added. Nuclei were DAPI stained. The nascent virus protein was observed under a confocal laser microscope. The results are shown in FIG. In FIG. 18, (a), (d), (g)-(o) are MDCK cells (Parent), (b), (e) are MDCK cells (Vector) into which only Vector has been introduced, (c). , (F) are ASA-expressing MDCK cells (ASA), (a)-(c) and (g)-(i) are HA staining, (j)-(l) are NA staining, (d) -(F) and (m)-(o) represent NP staining, (g)-(o) is a medium containing 1% FBS containing GS-5 or TU-1 at 37 ° C for 7 hours after virus infection. The culture results are shown. (G) and (i) represent GS-5 processing, and (h) and (j) represent TU-1 processing. In addition, the white line of (a) shows 50 micrometers.

[Comparison of progeny virus production under each virus-infected cell or GS-5 treatment]
Each cell was infected with a highly infectious influenza A virus strain: A / WSN / 33 (H1N1) at 34 ° C. for 1 hour. In order to prevent multistage infection of the virus, the cells were cultured in a serum-free medium in the absence of AT. The collected supernatant was treated with AT (10 μl / ml) at 37 ° C. for 30 minutes to infect the progeny virus, and the infectivity titer (pfu / ml) was measured. FIG. 19 (a) shows the measurement results of the progeny virus infectivity titer in the supernatant 20 hours after infection in COS7 cells (SulCOS1, SulCOS2) and parent COS7 cells (Parent) that highly express sulfatide. Moreover, the measurement result of the progeny virus infectious titer in the serum-free medium supernatant containing GS-5 or TU-1 16 hours after infection in the sulfatide highly expressing SulCOS1 cells is shown in FIG. A serum-free medium containing no antibody was used as a control (Con). In addition, FIG. 19 (c) shows the measurement results of the progeny virus infectivity titer in the supernatant 16 hours after infection in ASACK-expressed MDCK cells (ASA), MDCK cells into which only vector was introduced (Vector), and parent MDCK cells (Parent). Show. And the measurement result of the progeny virus infectivity titer in the serum-free medium supernatant containing GS-5 or TU-1 16 hours after infection in MDCK cells is shown in FIG. 19 (d). A serum-free medium containing no antibody was used as a control (Con).

[Method]
1. Cells and Virus MDCK (Madin-Dearby canine kidney) cells and MDCK cells introduced with plasmids were cultured in EMEM (Eagle's minimum essential medium) medium supplemented with 5% FBS (Fetal bovine serum). COS-7 cells and COS-7 cells introduced with the plasmid were cultured in DMEM (Dulbecco's modified Eagle's medium) supplemented with 10% FBS. Several influenza A viruses [A / WSN / 33 (H1N1), A / Menphis / 1/71 (H3N2), A / duck / 313/4/78 (H5N3)] The eggs were grown at 34 ° C. for 2 days, and sucrose density gradient centrifugation according to the method described in Reference 7 (Takahashi, T. et al. J. Biochem. (Tokyo) 130, 279-283 (2001)). separated.

2. Antibodies Mouse anti-sulfatide monoclonal antibodies (GS-5, IgM) and mouse anti-sphingoglycolipid (Gb ₃ Cer) antibodies (TU-1, IgM) are described in the literature [Konno, A et al. Int. Immunol. 8, 1905- 1913 (1996), Ding, Z. et al. FEBS Lett. 418, 310-314 (1997), Shikata, K. et al. J. Pathol. 188, 93-99 (1999), Miyamoto, D. et al. Glycoconj. J. 14, 379-388 (1997)] was used. In experiments on viral infection and replication, GS-5 (300 μg / ml) and TU- in the supernatant of mouse hybridoma cells cultured in serum-free medium hybridoma-SFM (Invitrogen Corp., Carlsbad, Calif., USA). 1 (500 μg / ml) was used. Mouse anti-NP (4E6), HA (2E10), NA (SI-4) monoclonal antibody (IgG) uses A / Menphis / 1/71 (H3N2) which is influenza virus, and the literature (Miyamoto, D. et al Glycoconj. J. 14, 379-388 (1997)).

3. Total RNA cloning and plasmid transfer cells TRIzol reagent extracts (Invitrogen Corp., Carlsbad, CA, USA) ^{at, Takara RNA PCR TM Kit (AMV} ) ver.3.0 (Takara Bio Inc., Shiga. Japan) with And converted to cDNA. MDCK cell-derived CGT gene and CST gene are determined using 3'-full RACE Core set (Takara Bio Inc., Shiga. Japan) and 5'-full RACE Core set (Takara Bio Inc., Shiga. Japan) did. Two ORF (Open reading frame) sequences were obtained from CST mRNA. One was identical to the ORF of human CST, while the other had 3 bases inserted between the 131st and 132nd bases counted from the start codon. The CST gene is pfu Ultra high fidelity DNA polymerase using a primer having a sequence containing EcoRI enzyme cleavage site of 5′-CGGAATTCCGATGCCGCTGCCGCCAGAAGAAAGC-3 ′ [SEQ ID NO: 1] and 5′-CCGGAATTCCGGTCACCACCCTCAGAAAGCCCGGATGA-3 ′ [SEQ ID NO: 2]. , CA, USA) and was amplified by PCR. By treating the PCT fragment of the CST gene inserted with 3 bases and the non-inserted CST gene with EcoRI and incorporating each using the EcoRI enzyme cleavage site of the pGEM-T easy vector (Promega, Madison, WI, USA), Two pGEM-CST vectors were made. The ORF partial fragment of the CST gene excised from the pGEM-CST vector using EcoRI was incorporated into a pIRES-neo vector (BD Biosciences, CA, USA).

  CGT gene is 5'-CGGCCGCGGCCGTCTCGCCATGAAGTCTCTACACTCCGTTATTCATGC-3 '[SEQ ID NO: 3] and 5'-CGGCGGCGTCTCGAATTTT is used as primer und Amplified. The pTRAGET-CGT vector was prepared by incorporating this amplified gene fragment into the pTRAGET vector (Promega, Madison, WI, USA) by TA cloning. Using pTRAGET-CGT vector as a template, 5′-ATAAGAATGCGCCCGCTAAAACTATAGAGATCTCTACACTCCGTATTTTCATGCTCTCTG-3 ′ [SEQ ID NO: 5] and 5′-ATAAGAAATCGCGCCGCCTAAACTATTCATTCTTCTTTTATCTT A fragment with a cleavage site was obtained. The Not I-treated CGT ORF PCR fragment was incorporated using the Not I cleavage site of the pIRES-neo vector in which the CST ORF had already been incorporated. Simultaneous expression of CST and CGT protein from the same mRNA, two bicistronic pIRES-CST-CGT expression vectors (one with no insertion in the CST gene or one with 3 base insertions) A vector was prepared.

  As for the ASA gene, a primer having an EcoRI cleavage site of 5'-GGAATTCCATGGGGGCACCCGCGGT-3 '[SEQ ID NO: 7] and 5'-CGGAATTCCGTCAGGCATGGGGATCTGGGC-3' [SEQ ID NO: 8] is used as a pfu ultra high fidelity DNA polymerase stratagene, CA, USA) and was amplified by PCR. The pGEM-ASA vector was incorporated into the pGEM-T easy vector using the EcoRI site. The ORF of ASA utilizes pGEM-ASA, and 5′-CATGCCATGGGGGCACCCGGGTC-3 ′ [SEQ ID NO: 9] (Nco I restriction enzyme site) and 5′-CGGAATTCCGGGGCATGGGGATCTGGGCA-3 ′ [SEQ ID NO: 10] (EcoRI restriction enzyme site) And the stop codon for tagging the histidine tag) were amplified and inserted between the Nco I and EcoRI cleavage sites of the pTriEx3-neo vector (Novagen, Darmstad, Germany). Thus, a bicistronic pTriEx3-ASA vector capable of expressing ASA and neomycin phosphotransferase protein from the same mRNA was constructed. The entire sequence of the gene fragment inserted into the plasmid was confirmed by ABI PRISM 310NT Genetic Analyzer (Applied Biosystems, CA, USA).

  COS-7 cells were transfected with pIRES-CST-CGT vector (both CST gene with or without 3-base insertion) using Trans IT-293 (Panvera, Madison, WI, USA) reagent. . After cloning, the transfected cells were selected by culturing for 3 weeks or longer in the presence of 1 mg / ml G418 (Promega, Madison, WI, USA). Selection of cells rich in sulfur lipids using GS-5 antibody and Fluorescein isothiocyanate (FITC) -conjugated anti-mouse IgG goat antibody (Sigma-Aldrich Corp., Missouri, USA) as secondary antibodies This was confirmed by observation with a fluorescence microscope (OLYMPUS IX70, Olympus, Tokyo, Japan). Is it possible to obtain two sulfatide-rich cell clones (one with a CST gene with a 3-base insertion) and whether the CST gene mRNA is constantly expressed from pIRES-CST-CGT in each cell? Using PCR primers with sequences such as 5′-AGTACTTAATACGACTCACTATAGG-3 ′ [SEQ ID NO: 11] (T7 promoter primer) and 5′-ACGGTGATGAAGGTGGC-3 ′ [SEQ ID NO: 12] (CST antisense primer) Confirmed by law. At that time, the expression of mRNA of GAPDH (glyceraldehyde 3-phosphate dehydrogenase) which is a housekeeping gene was used as a control.

  In MDCK cells, pTriEx3-ASA was introduced using Trans IT-293 reagent. After cloning, the transfected cells were selected by culturing for 3 weeks or longer in the presence of 1 mg / ml G418. Transfected cells were screened by confirming ASA activity and sulfur sulfate depletion. The sulfatase activity in 120-150 μg of the transfected cells was determined using literature (Yaghootfam, A. et al. J. Biol. Chem. 278, 32653-32661 (2003) using 4-methylumbelliferyl sulfate (Reserch Organics, Ohio, USA). )).

  Confirmation of sulfur lipid depletion in the transfected cells is achieved by using GS-5 antibody and FITC (Fluorescein isothiocyanate) -conjugated anti-mouse IgG goat antibody (Sigma-Aldrich Corp., Missouri, USA) as the primary antibody. As for whether or not the ASA gene mRNA is constantly expressed from the pTriEx3-ASA vector obtained by observing with a fluorescence microscope (OLYMPUS IX70, Olympus, Tokyo, Japan), 5′-AGTACTTAATACGACTCACTATAGG-3 ′ PCR was carried out using primers having sequences such as [SEQ ID NO: 11] (T7 promoter primer) and 5′-AGCCAGGAACTTCGGC-3 ′ [SEQ ID NO: 13] (ASA antisense primer). As a control at that time, GAPDH mRNA expression was used.

4). Virus Infection and Replication Influenza A virus strain: Cells infected with A / WSN / 33 (H1N1) are left in a medium without serum at 34 ° C. for 1 hour. In order to confirm the initial infection with influenza A virus, cells were cultured in a serum-free medium supplemented with zanamivir (1 μM), a sialidase inhibitor specific to influenza A virus, to prevent multiple replication. In order to confirm viral multiple replication, the infected cells were activated at 34 ° C. without the GS-5 antibody, TU-1 antibody, or antibody to activate the virus fusion activity required for multiple replication. The cells were cultured in a serum-free medium supplemented with acetyltrypsin (2.5 μg / ml) that dissociates the HA, HA1, and HA2 subunits. Infected cells were fixed with cold methanol for 30 seconds, reacted with anti-NP monoclonal antibody (4E6) for 30 minutes, and then HRP (Hourse radish peroxidase) -conjugated goat anti-mouse IgG + M antibody (Jackson Immuno research, West Grove, PA) ) For 30 minutes at room temperature. The NP of the virus in the infected cell is determined according to the method described in the literature (Suzuki, T. et al. Biochem. J.318,389-393 (1996)), hydrogen peroxide, N, N-diethyl-p-phenylendiamin dihydrocholoride. And matured by adding 4-chloro-1-naphtol. The progeny virus titer in the supernatant of infected cells was determined by plaque assay.

5. Plaque assay and virus titration To measure virus titer, MDCK cells confluent in a single layer were reacted with a log dilution of virus in liquid medium without serum for 1 hour at 34 ° C. Infected monolayer cells were further supplemented with serum-free liquid medium containing acetyltrypsin (2.5 μg / ml) and 0.8% agarose. Monolayer cells were cultured at 34 ° C. for 2-3 days until plaques were visible.

6). Fluorescence activated cell sorter (FACS) analysis of virus-induced apoptosis COS-7 cells and MDCK cells are influenza A virus strains: A / WSN / 33 (H1N1), MOI is 1 or 2 pfu / cell, 34 Each was infected for 1 hour at 0 ° C. COS-7 cells and MDCK cells are cultured at 34 ° C. in a liquid medium containing GS-5 or TU-1 and supplemented with FBS, and removed from the culture dish with 0.125% trypsin 16 and 24 hours after infection. It was collected. As a control, a liquid medium containing no antibody and containing 2% FBS was used. Expression of phosphatidylserine on the cell surface as a result of virus-induced apoptosis was examined by two-color analysis of FITC-conjugated annexin V conjugated with propidium iodide according to the manufacturer's manual. Fluorescence to the cells was excited with an 488 nm wavelength argon laser from an EPICS XL flow cytometer (Beckman Coulter Inc., Fullerton, Calif.). A minimum of 10,000 cells were examined for each sample.

7). Fluorescence microscopy The cells were cultured on coverslips (Teflon printed glass slides, Erie Scientific Company, Portsmouth, NH) and visualized in cells using cold methanol for 30 seconds in order to visualize the expression of sulfated lipids in the cells. Permeabilized and reacted with GS-5 antibody and then reacted with FITC-conjugated goat anti-mouse IgM secondary antibody. In order to visualize viral NP, HA, and NA in virus-infected cells, the cells were seeded with the influenza A virus species A / Memphis / 1/71 (H3N2), MOI of 5 pfu / cell at 34 ° C. for 1 hour. Infected and cultured at 37 ° C. in liquid medium supplemented with 1% FBS. Seven hours after infection, the infected cells were fixed and permeabilized with cold methanol for 30 seconds. Infected cells were reacted with mouse anti-NP (4E6), HA (2E10), or NA (SI-4) monoclonal antibody, followed by tetramethyl rhodamin (TRITC) -conjugated goat anti-mouse IgG secondary antibody (Sigma-Aldrich Corp. , Missouri, USA). Nuclei were stained with DAPI (4'6-diamino-2-phenylindole dihydrochloride) (Dojindo Labratories, Kumamoto, Japan). The cells were observed under an LSM510 confocal microscope (Carl Zeiss Inc., Thornwood, NY).

8). RNAi
As shown in FIG. 20, 401st to 419th CST gene (number from the start codon) 5′-GCTTCAACATCATCTGCA-3 ′ [SEQ ID NO: 14], 337 to 357 (start) to RNAi vector pSilencer3.1-H1 neo The number from the codon) 5′-CGACTTCGAACTACCCCGGCC-3 ′ [SEQ ID NO: 15], 29th to 47th (number from the start codon) 5′-CTGGAGTGTCCCAATTCT-3 ′ [SEQ ID NO: 16] of GFP (Green fluorescence protein), respectively Integration and gene transfer into MDCK cells. The gene-introduced MDCK cells were subjected to drug selection by G418 for a period of 2 weeks or longer.

  Using the selected MDCK cells, the amount of intracellular CST mRNA was quantified by real-time PCR based on the amount of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA. As a primer pair for real-time PCR, CST: forward, 5′-AGCACGGGCTCAAGTTC-3 ′ [SEQ ID NO: 17] (CST302-318) / reverse, 5′-ACGGTGATGAAGGTGGGC-3 ′ [SEQ ID NO: 12] (CST469-485), GFP: forward, 5′-TCAACGGGATTGGCCGTATTGG-3 ′ [SEQ ID NO: 18] (GAPDH17-38) / reverse, 5′-TGAAGGGGTCATTGATGGCG-3 ′ [SEQ ID NO: 19] (GAPDH97-106) was used.

It is a figure which shows the synthetic | combination and decomposition | disassembly metabolic pathway of sulfated glycolipid (sulfatide). It is a figure which shows the result by which the anti-sulfatide antibody (GS-5) of this invention acts on influenza A virus infection cell, and suppresses the proliferation of a virus. It is a figure which shows the result by which the anti-sulfatide antibody (GS-5) of this invention shows an effect on the infected cell after 4 hours after influenza A virus infection, and reduces the amount of infectious progeny virus produced from an infected cell. . It is a figure which shows that the anti- sulfatide antibody (GS-5) of this invention suppresses the proliferation of influenza A virus. It is a figure which shows that the anti- sulfatide antibody (GS-5) of this invention also suppresses the proliferation of influenza A viruses other than a WSN strain. It is a figure which shows that the anti-sulfatide antibody (GS-5) of this invention suppresses the proliferation of influenza A virus also in presence of acetyltrypsin (AT) which accelerates | stimulates viral growth. It is a figure which shows that the anti- sulfatide antibody (GS-5) of this invention suppresses the proliferation of parainfluenza virus. It is a figure which shows that the anti-sulfatide antibody (GS-5) of this invention of this invention suppresses the proliferation of parainfluenza virus (culture supernatant virus titer measurement). It is a figure which shows that the anti- sulfatide antibody (GS-5) of this invention suppresses the proliferation of parainfluenza virus depending on density | concentration. It is a figure which shows that the anti-sulfatide antibody (GS-5) of this invention does not inhibit the initial infection of parainfluenza virus, but reduces the amount of infectious progeny virus produced from an infected cell. It is a figure which shows that the sulfatide-degrading enzyme (allyl sulfatase A; ASA) expression in a canine kidney origin MDCK cell reduces the amount of sulfatides, and also reduces the proliferation of influenza A virus. FIG. 2 shows that RNA interference (RNAi) of sulfatide synthase (CST) in MDCK cells decreases the amount of sulfatide and decreases the proliferation of influenza A virus. It is a figure which shows that the proliferative ability of influenza A virus increases greatly in the monkey kidney origin COS7 cell which introduce | transduced two canine origin sulfatide synthetase gene compared with a parent COS7 cell. It is a figure which shows that the anti- sulfatide antibody (GS-5) of this invention suppresses the proliferation of influenza A virus in a sulfatide high expression COS7 cell. It is a figure which shows that influenza A viruses other than a WSN strain | stump | stock also proliferate in a sulfatide high expression COS7 cell. It is a figure which shows that sulfatide accelerates | stimulates the apoptosis induction of influenza A virus. Although the cell membrane transport of the nascent viral membrane proteins hemagglutinin (HA) and neuraminidase (NA) in sulfatide-expressing SulCOS1 cells is normal, the transport of the nascent viral nucleoprotein (NP) from the nucleus to the cytoplasm is compared to the parent COS7 cells It is a figure which is further accelerated | stimulated and an anti-sulfatide antibody (GS-5) suppresses transport of NP. The transport of nascent viral membrane protein HA in MDCK cells is normal, but transport of nascent NP from the nucleus to the cytoplasm is suppressed in ASA-expressing MDCK cells, and anti-sulfatide antibody (GS-5) It is a figure which does not affect the cell membrane transport of newborn virus HA and NA, but suppresses transport of newborn virus NP to the cytoplasm. It is a figure which shows the comparison result of the progeny virus production amount under each virus infection cell or GS-5 process. It is a schematic diagram which shows the outline | summary of siRNA preparation using RNAi vector pSilencer3.1-H1 neo.

Claims (12)

Antiviral agent targeting sulfatide. An antiviral agent targeting sulfatide, which comprises an anti-sulfatide antibody as an active ingredient. The antiviral agent targeting sulfatide according to claim 2, wherein the anti-sulfatide antibody is an anti-sulfatide monoclonal antibody. The antiviral agent targeting sulfatide according to claim 3, wherein the anti-sulfatide monoclonal antibody is a mouse anti-sulfatide monoclonal antibody (GS-5, IgM). An antiviral agent targeting sulfatide, comprising allylsulfatase A (ASA) as an active ingredient. An antiviral agent targeting sulfatide, comprising an allylsulfatase A (ASA) gene expression vector as an active ingredient. An antiviral agent targeting sulfatide, comprising a sulfatide synthase inhibitor as an active ingredient. An antiviral agent targeting sulfatide, characterized by comprising an antisense strand of a sulfatide synthase gene as an active ingredient. An antiviral agent targeting sulfatide, wherein siRNA targeting sulfatide synthase mRNA is an active ingredient. An antiviral agent targeting sulfatide, characterized by comprising an siRNA expression vector targeting sulfatide synthase mRNA as an active ingredient. The sulfatide-targeting antiviral agent according to any one of claims 7 to 10, wherein the sulfatide synthase is cerebroside sulfotransferase (CST) and / or ceramide galactosyltransferase (CGT). The antiviral agent targeting sulfatide according to any one of claims 1 to 11, wherein the virus is an influenza virus.