JP2019214555A - Composition for prevention of virus infection - Google Patents

Abstract

To provide a composition for prevention of virus infection such as influenza.SOLUTION: The present invention provides a composition for prevention of virus infection of a human or a nonhuman animal that is administered to an epithelial cell of the respiratory tract, gastrointestinal tract or eye tissue of the human or nonhuman animal, and comprises an antagonist bound to a virus receptor of the epithelial cell as an active ingredient.SELECTED DRAWING: Figure 2

Description

  The present invention relates to a composition for preventing a viral infection such as influenza. More specifically, by administering to epithelial cells such as the respiratory tract and gastrointestinal tract and treating in advance, there is no need for injection, and infection can be suppressed simply and inexpensively, for human or non-human animals. The present invention relates to a composition for preventing a viral infection and a method for preventing the same.

  Even in the modern age of medical treatment, influenza A and influenza B are prevalent worldwide every winter, and there is concern about pandemix due to the occurrence of highly pathogenic viruses. Avian influenza, a zoonotic disease, is also feared to be transmitted to humans, and if it is found in poultry farms, large-scale killings have been carried out.

  Pathogenic viruses, such as influenza, that have invaded the body from the outside, invade the cells and grow by binding to specific receptors expressed on the surface of human or animal host cells. Vaccination is at the heart of modern viral infection control, which involves pre-injecting attenuated or inactivated viruses and their components to induce immunity against specific viruses and increase the body's defenses. It is a way to put.

  Here, lectin, which is a sugar-binding protein, has a binding specificity derived from its structure, and is used for searching for glycoconjugates on the cell surface and for purifying glycoproteins and glycolipids. In recent years, attention has been paid to the antiviral effect of lectins on influenza virus, and application to therapeutic and prophylactic drugs has been studied.

  For example, Patent Document 1 discloses a method for treating and preventing a microbial infectious disease containing mannose-binding lectin (MBL) and the like, which is adapted to be directly inhaled into the respiratory tract or directly transmitted to other epidermal cell infection sites. And the like are disclosed. By administering this spray-dried powder composition, it is said that MBL can activate the immune system to treat the site of infection, and can block the invasion of viruses and bacteria to prevent infection.

  Patent Document 2 discloses a therapeutic agent for influenza and the like containing a plant-derived lectin as an active ingredient. It is said that by administering this therapeutic agent, a situation in which a patient becomes serious in the early stage of influenza infection can be effectively prevented.

  In this document, the action point of lectin is analyzed, and hairy vetch lectin and Fuji lectin have been shown to have an infection-suppressing effect in the process after virus adsorption, indicating the potential for therapeutic use. In addition, Hilochawantake lectin and Aspergillus oryzae lectin have been shown to have an infection-suppressing effect in the process prior to virus adsorption, indicating their potential for prophylactic use.

JP 2008-526735 A JP 2014-201587 A

  However, in the spray-dried powder composition for treatment and prevention disclosed in Patent Document 1, the lectin whose effect has been confirmed is genetically modified human MBL, and it is difficult to stably prepare it in a large amount, and the cost increases. I will. The mechanism of action of prevention is said to be that MBL binds to the surface of a virus or bacterium, thereby physically blocking entry into host cells and suppressing infection, but the binding has been confirmed. It is not a virus but only some bacteria such as Staphylococcus aureus.

  In addition, although the therapeutic agent for influenza disclosed in Patent Document 2 has been suggested to have therapeutic and prophylactic uses, the antiviral activity of various lectins is only confirmed by screening, and the mechanism of action is clear. Has not been. Furthermore, it is described that amaryllis lectin and concanavalin A (ConA) did not show significant antiviral activity.

  The present invention has been made in view of the above problems, and has been made to clarify the mechanism of action of the antiviral effect by focusing on the binding between a lectin and a virus receptor of a host cell, which has not been conventionally considered. The first purpose. And, by applying its mechanism of action, it is possible to adapt to various viral infectious diseases, and to suppress infection easily and inexpensively, a composition for preventing viral infectious diseases for human or non-human animals. And a preventive method using the same.

  Means for Solving the Problems As a result of intensive studies to solve the above-mentioned problems, the present inventors have found that lectins have high affinity for virus receptors on host cells and can physically inhibit the binding of viruses to host cells. By administering an antagonist that specifically binds to a viral receptor on epithelial cells such as the respiratory tract and gastrointestinal tract prior to the onset of a viral infection, the viral receptor is embedded in advance, and the host cells of the virus The inventor has found that the intrusion into the material can be suppressed, and as a result of further research, the present invention has been completed.

  That is, the present invention is applied to human or non-human animal airway, gastrointestinal tract or epithelial cells of the eye tissue, and contains an antagonist that binds to a virus receptor of the epithelial cells as an active ingredient. A composition for preventing viral infection. According to the present invention, virus infection can be prevented simply and inexpensively without injection.

  In the composition for preventing viral infection of the present invention, the antagonist may be mannose-binding lectin (MBL), and the concentration thereof may be 30 μg / mL or more. MBL has high affinity for the virus receptor and can be expected to have an excellent effect, and low-cost and highly safe plant-derived MBL can be used.

  In the composition for preventing viral infection of the present invention, the antagonist may be an inactivated virus, and the concentration of the inactivated virus may be 6 μg / mL or more as the HA content. The inactivated virus has a high affinity for the virus receptor and can be expected to have an excellent effect, and an inactivated vaccine that has already been manufactured and approved as a pharmaceutical can be used.

  Further, the composition for preventing viral infection of the present invention, wherein the viral infection is selected from the group consisting of influenza virus, measles virus, mumps virus, herpes simplex virus types 1 and 2, avian influenza virus, and Newcastle disease virus. The infection may be caused by one of the above. These infectious diseases are highly infectious and have been recurring in various places, and their preventive agents are expected to make a significant social contribution in the medical field.

  Further, the composition for preventing viral infection of the present invention is a drug or a quasi-drug, and may be in a form to be administered via the nasal cavity, oral cavity, or eyes, and may be a spray, a gargle, a troche, orally. It may be one selected from the group consisting of a liquid preparation, an oral solid preparation, and an eye drop. By using these dosage forms, they can be simply and efficiently administered to epithelial cells of the respiratory tract, digestive tract or eye tissue.

  Further, the composition for preventing a viral infection of the present invention is a food or feed, and may be in a form to be administered via the oral cavity, and may be one selected from the group consisting of gum, candy, beverage, and powdered beverage. It may be. By adopting these forms, it can be inexpensively and easily administered to epithelial cells of the digestive tract.

  In addition, the present invention provides a method for administering a virus infection to a non-human animal by administering the above-described composition for preventing a virus infection and treating a virus receptor with an antagonist before the non-human animal becomes infected with the virus. How to prevent. According to the present invention, the health of pets and livestock can be maintained simply and inexpensively, and zoonotic diseases can be prevented.

  The composition for preventing a viral infection of the present invention is administered to epithelial cells of the respiratory tract, gastrointestinal tract, or ocular tissue before afflicted with the viral infection, and the virus receptor is pretreated with an antagonist, thereby injecting the composition. There is no need, and infection can be suppressed simply and inexpensively.

It is explanatory drawing which shows the binding property between an influenza virus and concanavalin A (ConA), and a virus receptor. It is an explanatory view showing the mechanism of action of the antiviral effect of the composition for preventing virus infection of the present invention. 4 is a graph showing the antiviral effect of ConA on avian influenza virus in Example 1. 4 is a graph showing the antiviral effect of ConA on Newcastle disease virus in Example 2. It is a graph which shows the relationship between the light absorbency in the quantification test of ConA concentration, and the added ConA concentration. It is a graph which shows the standard curve showing the relationship between the light absorbency calculated in the quantification test of ConA concentration, and ConA concentration. It is a graph which shows the relationship between the ConA density | concentration of the cell surface layer quantified using the standard curve, and the added ConA density | concentration. 5 is a graph showing the antiviral effect of an inactivated whole particle virus against influenza virus in Example 3. 7 is a graph showing the antiviral effect of ConA on influenza virus in Example 3. It is a schematic diagram showing the evaluation result of the antiviral test of ConA against rubella virus in Example 4. It is a schematic diagram showing the evaluation result of the antiviral test of ConA against measles virus in Example 4. It is a schematic diagram showing the evaluation result of the antiviral test of ConA against mumps virus in Example 4. FIG. 11 is a schematic diagram showing the evaluation results of an antiviral test of ConA against herpes simplex virus type 1 in Example 4. FIG. 14 is a schematic diagram showing the evaluation results of an antiviral test of ConA against herpes simplex virus type 2 in Example 4. It is a schematic diagram showing the evaluation result of ConA antiviral test against RS virus in Example 4.

  Hereinafter, the virus infectious disease preventive composition of the present invention and a preventive method using the same will be described in detail. The components, compositions, manufacturing methods, and the like, whose description is omitted, can be the same or substantially the same as those known to those skilled in the art.

  The composition for preventing viral infection of the present invention is for human or non-human animals. Non-human animals are not particularly limited as long as they are mammals or birds that infect the epithelial cells of the respiratory tract, gastrointestinal tract or eye tissues with the virus. Specifically, mammals such as cattle, pigs, horses, sheep, and goats that become livestock, birds such as chickens, duck, quail, and turkey, mammals such as dogs, cats, and rabbits that become pets, parakeets, and beetle pine And birds.

  The composition for preventing a viral infection of the present invention is administered to epithelial cells of the respiratory tract, gastrointestinal tract or eye tissue. The airway is the passage of air from the nose to the lungs and is divided into the upper and lower airways. The upper airway is from the nose to the nasal cavity, nasopharynx, pharynx, and larynx, and the lower airway is the trachea, bronchi, bronchioles, and lungs on the lung side of the larynx.

  The gastrointestinal tract is a passage from the mouth to the anus for food and its digestive substances, and is divided into an upper gastrointestinal tract and a lower gastrointestinal tract. The upper gastrointestinal tract is from the mouth to the oral cavity, pharynx, esophagus, stomach, and small intestine (duodenum); the lower gastrointestinal tract is the small intestine (jejunum, ileum) and the large intestine (cecum, appendix, colon, rectum) that are anal to the duodenum. is there.

  The eye is a visual organ located in the orbit, and is divided into an eyeball and its appendages. The eye tissue to which the present invention is administered is a part in contact with or close to the outside world, and includes eyelids, horns, conjunctiva, lacrimal organ, cornea, sclera, orbital tissue, and the like.

  The target tissue to which the composition for preventing a viral infection of the present invention is administered is an epithelium of the respiratory tract, gastrointestinal tract, or ocular tissue in which a receptor that specifically binds to a virus that causes a viral infection to be prevented is expressed in a large amount. Cells. The main target tissue differs depending on the type of virus. For example, in the case of an influenza virus, epithelial cells of the upper respiratory tract such as the human nasopharynx, pharynx, and larynx. In the case of avian influenza virus, it is epithelial cells of the lower gastrointestinal tract such as the small intestine and large intestine of birds.

  The composition for preventing a viral infection of the present invention comprises a composition containing, as an active ingredient, an antagonist (antagonist) that binds to a virus receptor present on the surface of epithelial cells of the respiratory tract, gastrointestinal tract, or eye tissue of a human or non-human animal. (Prophylactic agent). By administering the composition for preventing viral infection of the present invention before suffering from viral infection and pre-embedding the viral receptor with an antagonist, the binding of the virus is physically inhibited. Is prevented.

  The main use of the composition for preventing viral infections of the present invention is for prevention, but in cases where infection is suspected or at an early stage of infection, the treatment is administered to suppress the onset of symptoms and the deterioration of symptoms. Uses shall be included.

  Here, FIG. 1 is an explanatory diagram showing the binding between influenza virus and concanavalin A (ConA) and a virus receptor, and FIG. 2 shows the mechanism of action of the antiviral effect of the composition for preventing virus infection of the present invention. FIG. When viruses adsorb to host cells and enter inside, many viruses recognize sugar chains expressed on the cell membrane of host cells as specific receptors and bind.

  As shown in FIG. 1, the virus receptor to which the influenza virus specifically binds is such that monosaccharides such as mannose, glucosamine, and galactose present on the surface of epithelial cells such as the upper respiratory tract are condensed and sialic acid is condensed to the terminal. It is a receptor consisting of a sugar chain structure.

  Proteins such as hemagglutinin (HA) and neuraminidase (NA) protrude in a spike form from the outer envelope of the virus particles. Since this hemagglutinin (HA) has a high affinity for a sialic acid residue at the terminal of the receptor, influenza virus can specifically bind to the receptor.

  On the other hand, as shown in FIG. 1, concanavalin A (ConA), which is an antagonist, has high affinity for mannose constituting the receptor. Therefore, as shown in FIG. 2, by administering ConA before the virus binds to the receptor to establish infection and pre-embedding the virus receptor with ConA, the virus binding is physically inhibited. Therefore, infection can be prevented.

  The antagonist contained as an effective substance in the composition for preventing viral infection of the present invention is a substance that specifically binds to a virus receptor and acts as an antagonist as described above. Specifically, a mannose binding lectin (Mannose binding lectin, MBL) having a high affinity for a mannose site of a sugar chain is exemplified. A plant-derived MBL that can be manufactured at low cost, has high safety, and has little effect on aroma and taste even when added to pharmaceuticals and foods is preferable.

  As plant-derived MBLs, there are classified into Class 3 in the lectin classification based on recognition of monosaccharides of Makela, from bean (ConA), pea (PSA), lentil (LCA), and broad bean (VFA). MBL and derivatives thereof are preferred, and concanavalin A (ConA) is more preferred.

  The concentration (content) of mannose-binding lectin (MBL) contained in the composition for preventing virus infection of the present invention can exert a predetermined antiviral effect, provide a suitable aroma and taste, and reduce production costs. If it is, there is no particular limitation. It is preferably at least 30 μg / mL (0.003% by weight), more preferably at least 60 μg / mL (0.006% by weight), still more preferably at least 125 μg / mL (0.0125% by weight), and still more preferably 250 μg / mL. mL (0.025% by weight) or more.

  Alternatively, an inactivated virus may be used as an antagonist. Hemagglutinin (HA), which is a virus envelope protein, has a high affinity for sialic acid residues at the terminal of the virus receptor, and thus can act as an antagonist similarly to mannose-binding lectin (MBL). Specific examples include an inactivated whole particle virus inactivated while retaining the structure of the virus particle, and an inactivated split virus inactivated by destroying the virus particle. It is preferable to use formalin-inactivated whole particle vaccine (Whole virus vaccine) and SP vaccine (HA vaccine) that have been approved for production as pharmaceuticals.

  The concentration (content) of the inactivated virus contained in the virus infectious disease prevention composition of the present invention can exert a predetermined antiviral effect, has stability under certain conditions, and has a low production cost. There is no particular limitation as long as it exists. The HA content is preferably at least 6 μg / mL (0.0006% by weight), more preferably at least 12 μg / mL (0.0012% by weight), still more preferably at least 23 μg / mL (0.0023% by weight), and even more. It is preferably at least 47 μg / mL (0.0047% by weight).

  The commercial form of the composition for preventing virus infection of the present invention is not particularly limited as long as it can exhibit a predetermined antiviral effect and can be administered simply, efficiently and inexpensively. Pharmaceuticals for humans or animals and quasi-drugs specified by the Act on Pharmaceuticals may be used. Further, it may be a food, a health food, or a food for specified health use prescribed by the law on foods. Furthermore, feed for livestock and pet food for pets may be used.

  Pharmaceuticals or quasi-drugs take the form of administration via the nasal cavity, oral cavity or eye. Dosage forms administered to epithelial cells of the respiratory tract include sprays sprayed into the nasal cavity or oral cavity, aerosol formulations (MDI) or dry powder formulations (DPI) inhaled through the oral cavity, lozenges contained in the oral cavity, and dilutions at the time of use Or a mouthwash and a mouthwash which are dissolved and used. Dosage forms to be administered to epithelial cells of the digestive tract include solid oral preparations such as tablets, capsules, granules and powders, internal liquids such as syrups, elixirs, decoctions, suppositories, enemas and the like. . Dosage forms administered to epithelial cells of eye tissues include eye drops, eye ointments and the like.

  In the formulation design of the above various formulations, known means known to those skilled in the pharmaceutical field can be used. For example, in the case of a spray, the viscosity and properties may be adjusted according to the type and content of the thickener in order to improve the adhesion to the tissue. For inhalants, the particle size and size distribution of the drug may be optimized to improve its transmissibility to tissues. In a solid oral preparation, an enteric capsule or an enteric coating may be used to prevent inactivation of the active ingredient by digestive juice.

  For example, as a spray agent, 95 to 99% by weight of pure water, 0.01 to 0.5% by weight of a surfactant (such as polysorbate 80), and 0.01 to 0.5% of a preservative (such as benzalkonium chloride). % By weight, 0.01 to 0.5% by weight of antioxidant (sodium edetate hydrate), appropriate amount of tonicity agent (such as sodium chloride), appropriate amount of buffer (sodium citrate hydrate), pH An appropriate amount of a regulator (such as dilute hydrochloric acid), an appropriate amount of a thickening agent (such as carmellose sodium), etc., and 0.25% by weight of concanavalin A (ConA) as an active ingredient are mixed by a conventional method to prepare a drug solution. A fixed amount spraying type spray agent filled in is exemplified. This is sprayed into the nasal cavity or oral cavity once to several times a day to administer ConA to epithelial cells of the upper respiratory tract.

  A spray containing an inactivated virus as an active ingredient may contain an appropriate amount of vaccine-derived components such as formalin and thimerosal. In addition, it is known that when an inactivated virus is administered to a living body through a nasal or oral route, a local immune system is stimulated to induce a local antibody. However, an adjuvant that enhances such a local immune effect is known. May be added.

  As the gargle, 90 to 97% by weight of pure water, 0.01 to 0.5% by weight of a surfactant (such as polysorbate 80), 0.5 to 5% by weight of a preservative (such as ethanol), and a humectant (such as glycerin) ) 0.5-5% by weight, fragrance (natural vegetable essential oil etc.) 0.05-5% by weight, antioxidant (sodium edetate hydrate etc.) 0.01-0.5% by weight, buffer (phosphorus) An appropriate amount of sodium hydrogen oxyhydrate, etc., and 2.5% by weight of concanavalin A (ConA) as an active ingredient are mixed by a conventional method to prepare a stock solution, which is then diluted with a gargle for use at the time of use. Is exemplified. This is diluted 10 times with water, gargled once to several times a day, and ConA is administered to epithelial cells of the upper respiratory tract.

  In the case of food or feed, it takes a form to be administered via the oral cavity. Examples of the food include solid foods, semi-solid foods, liquid foods, beverages, powdered drinks, sweets such as candy and gum, and the like. Examples of feed or pet food include feed and pet food having various properties such as solid, semi-solid, fluid, and liquid.

  For example, as gum, gum base (vegetable resin, vinyl acetate resin, ester gum, etc.) 20 to 40% by weight, sweetener (sugar, glucose, starch syrup, etc.) 50 to 80% by weight, flavor (natural vegetable essential oil, etc.) 0 0.05-1% by weight, acidulant (citric acid, malic acid, etc.) 0.05-1% by weight, softener (water, glycerin, etc.) suitable amount, antioxidant (ascorbic acid, etc.) proper amount, etc., and as an active ingredient Granular gum is prepared by mixing 0.25% by weight of concanavalin A (ConA) by a conventional method to prepare a gum material, molding a predetermined amount thereof, and coating a sugar coating. This is chewed orally several times a day and ConA is administered to epithelial cells of the upper respiratory tract or gastrointestinal tract.

  As candy, sugar raw materials (sugar, glucose, starch syrup, etc.) 90 to 95% by weight, acidulants (citric acid, malic acid, etc.) 0.05 to 3% by weight, flavors (natural vegetable essential oils, etc.) 0.05 to 1 A candy dough is prepared by mixing an appropriate amount of a colorant (such as a natural pigment), an appropriate amount of an antioxidant (such as ascorbic acid), and 0.25% by weight of concanavalin A (ConA) as an active ingredient in a conventional manner. A hard candy molded in a predetermined amount is exemplified. This is dissolved in the oral cavity several times a day, and ConA is administered to the epithelial cells of the upper respiratory tract or digestive tract.

  Further, the present invention provides a method for preventing viral infection of a non-human animal by administering the above-mentioned composition for preventing viral infection and treating a virus receptor with an antagonist before the non-human animal becomes infected with the virus. How to prevent. The non-human animal is as described above. The prophylactic composition to be administered includes both a case where a prophylactic composition distributed as a commercial product is administered and a case where a self-prepared prophylactic composition is administered.

  In the above-mentioned medicines and quasi-drugs, foods and feeds of the present invention, the raw materials, various additives, composition ratios, storage containers, production methods, etc., of the active ingredients such as mannose-binding lectin and inactivated virus, etc. A general substance which is appropriately set within a range that does not impair the action and is widely used in the field of pharmaceuticals, foods and the like can be adopted.

  In addition, the number of daily administrations and the dosage of the virus infectious disease preventive composition of the present invention may be appropriately increased or decreased according to the type, sex, body weight, age, symptoms, etc., in order to maximize the effects of the present invention. Is done.

  Hereinafter, the composition for preventing a virus infection of the present invention will be specifically described with reference to examples and the like. It should be noted that the present invention is not limited to these embodiments, and various changes can be made without departing from the technical idea of the present invention.

[Example 1]
<Concanavalin A anti-avian influenza virus test>
Concanavalin A (ConA) (for biochemistry, manufactured by Wako Pure Chemical Industries, Ltd.) was used as a test material, and low pathogenic avian influenza virus (A / duck / Aomori / 395/04 [H7N1]) was used as a test virus. AIV), dog kidney passage cells (MDCK cells) were used as sensitive cells.

  ConA was dissolved in PBS (10 mM, pH 7.2) and adjusted to a concentration of 25 mg / mL. This ConA solution was further diluted with a minimum essential medium (MEM) to adjust to six concentrations of 1000, 500, 250, 125, 62.5, and 31.25 μg / mL.

MDCK cells were cultured on a 60 mm Petri dish to form a monolayer sheet. This single layer sheet was washed twice with MEM. Next, 6 mL of ConA solution was added at 4 mL per petri dish, and sensitization was performed at 37 ° C. in a 5% CO ₂ incubator for 30 minutes. Thereafter, the Petri dish was washed three times with 2 mL of PBS to remove ConA.

Subsequently, 200 μL of the challenge virus (AIV) diluted and adjusted for the infectious titer was inoculated per 200 μL of petri dish, infected, spread on a monolayer sheet, and cultured for 1 hour to adsorb the virus. After washing with MEM to remove the inoculum, a primary agar medium supplemented with trypsin was overlaid and cultured at 34 ° C. in a 5% CO ₂ incubator for 48 hours. Further, a secondary agar medium was overlaid under the same conditions and cultured for 24 hours. After formalin fixation and methylene blue staining, the number of concentrically formed plaques was counted. The evaluation results are shown in Tables 1 and 2 below and in FIG. 3 in cases where the amount of infectious virus (the number of plaques) is large and small.

[Example 2]
<Concanavalin A anti-Newcastle disease virus test>
Newcastle disease virus Sato strain (NDV) was used as a test virus, and chicken embryo fibroblasts (CEF cells) were used as susceptible cells. The test was performed under the same conditions as in Example 1 except that trypsin was not added to the overlaid primary and secondary agar media, and the primary agar medium was overlaid and cultured for 72 hours. Similarly, the evaluation results are shown in Tables 3 and 4 below and in FIG. 4 for cases where the amount of infectious virus (the number of plaques) is large and small.

  For AIV, from the results in Table 1 and FIG. 3, even when the amount of infectious virus (the number of plaques) was relatively large, a plaque reduction of about 90% was observed when the concentration of ConA was 125 μg / mL, indicating a high anti-virus. It turns out that it shows an effect. Further, from the results in Table 2 and FIG. 3, when the amount of infectious virus (the number of plaques) was relatively small, a 100% reduction in plaque was observed even when the ConA concentration was as low as 62.5 μg / mL. It can be seen that the virus was inactivated.

  Regarding NDV, the results of Tables 3 and 4 and FIG. 4 show that a plaque reduction of about 85 to 98% was observed regardless of the amount of infectious virus (the number of plaques), indicating a high antiviral effect.

  From the above results, it was shown that by adding Concanavalin A (ConA) to the cells in advance, the subsequent virus infection of the cells was suppressed. This inhibitory effect was observed even after ConA was washed out of the cells using PBS, so that ConA did not act directly on the virus, but specifically on MDCK and CEF cell surface receptors or glycoproteins. Binding to inhibit the binding of the virus to the specific receptor.

Further, since similar results were obtained in MDCK (canine-derived) and CEF (chicken-derived) cells, ConA binds not only to sugar chains of mammals such as humans and dogs but also to sugar chains of birds, It is thought to suppress the adsorption of the virus to host cells.
Even if ConA was sensitized to the cells at a concentration of 1000 μg / mL, the cells did not die, and their cytotoxicity is considered to be extremely low.

Next, in order to demonstrate the mechanism of action of the antiviral effect of Concanavalin A (ConA), a quantitative test of ConA bound to the cell surface was performed.
[Test Example 1]
<Quantitative test of concanavalin A bound to cell surface>
To the MDCK cells adsorbed on the 96-well microplate, a ConA solution adjusted to 11 steps from 4 μg / mL to 0.003906 μg / mL by 2-fold serial dilution was added at 100 μL per well to add ConA to the cell surface. Combined.

  Unbound ConA was removed by washing with PBS-T, and the cells were immobilized on a plate using a cell fixing solution. After blocking the surface of the 96-well microplate, a 1000-fold diluted HRP-labeled anti-ConA secondary antibody solution was added. The absorbance at 450 nm was measured to determine the amount of ConA on the surface of the MDCK cells. The measurement results are shown in the middle column of Table 5 below and in FIG.

  In addition, by the same method as described above, a ConA solution adjusted from 500 μg / mL to 11 steps by two-fold serial dilution was added to a 96-well microplate and immobilized. An HRP-labeled anti-ConA secondary antibody solution was added, the absorbance at 450 nm was measured, and a ConA standard curve (calibration curve) was calculated. The graph of the standard curve is shown in FIG. The ConA concentration of the surface layer of MDCK cells quantified from this standard curve and the above measurement results is shown in the right column of Table 5 below. FIG. 7 is a graph showing the correlation between the concentration of the added ConA solution and the concentration of ConA in the cell surface layer.

As shown in Table 5 and FIG. 5, it can be seen that the amount of ConA in the cell surface increases depending on the concentration of the added ConA solution. In addition, as shown in FIG. 7, the correlation coefficient between the ^two was R ² = 0.9435, indicating a high correlation. From the above results, it is considered that the added ConA is bound to a receptor or glycoprotein on the surface of MDCK cells.

  According to the mechanism of action of ConA's antiviral effect discussed from the above results, it is presumed that a substance having a high affinity for the virus receptor acts as an antagonist (antagonist) and exhibits the same antiviral effect. To demonstrate this, an antiviral test was performed using an inactivated whole particle virus inactivated while retaining hemagglutinin (HA), which has a high affinity for the sialic acid residue at the end of the virus receptor.

[Example 3]
<Anti-influenza virus test for inactivated whole particle virus>
As a test material, an inactivated whole particle virus solution (A / Singapore / GP190 / 2015 (IVR-190) (H1N1) pdm09) was used. This inactivated virus solution contained 150 μg / mL as the HA content, and the amount of protein was measured with a photoelectric colorimeter to find that it was 4640 μg / mL (in terms of BSA).

  In order to remove preservatives such as thimerosal and formalin contained in the inactivated virus solution, 5 ml of the inactivated virus solution was put into Amicon Ultra-15 (50K), centrifuged and concentrated to 100 μl. When this concentrated solution was reconstituted to 5 mL with PBS and the amount of protein was measured, it was found to be 1450 μg / mL (in terms of BSA), indicating that the amount of protein was reduced to about 1/3. The inactivated virus solution from which the preservative was removed by concentration and reconstitution was diluted two-fold with PBS to prepare a six-stage solution from the stock solution to 32-fold.

As a test virus, an influenza virus (A / California / 07/09 (H1N1) pdm09) (IV) which was passaged 15 times with MDCK cells and highly proliferative on MDCK cells was used. The infectious titer of this challenge virus is 6.6 Log PFU / 0.1 mL, but since the infectious titer used in this test is 100 PFU / 0.1 mL, it was diluted to 1 × 10 ⁻⁵ and 2 × 10 ^−5. Used for the test. The virus type is pdm09 virus, which is the same as the inactivated virus solution.

  MDCK cells were used as sensitive cells. In addition, ConA was used as a comparison material for the antiviral effect. ConA was serially diluted two-fold with PBS to prepare six-step solutions from 500 μg / mL to 15.6 μg / mL. In addition, MEM medium was used as a control.

After culturing MDCK cells in a 6-well plate for 3 days, the culture solution for cell growth was removed and washed once with MEM. Next, 200 μL of each of the cell treatment solutions of the inactivated whole particle virus solution, ConA solution, and control MEM was added per well. The plate was repeatedly rocked so that the cell treatment solution was in extensive contact with the cell surface, and sensitized at 37 ° C. in a 5% CO ₂ incubator for 60 minutes. Thereafter, each cell treatment solution was removed by washing three times with MEM.

Subsequently, the infectious virus (HV) adjusted for the infectious titer was inoculated by inoculating 100 μL per well. After culturing for 1 hour, virus adsorption was performed. After washing with MEM to remove the inoculum, MEM agar medium containing 2 μg / mL trypsin and 0.01% DEAE dextran was overlaid on the cells. After the agar medium was solidified, the plate was inverted, and culturing was performed for 48 hours in a 5% CO ₂ incubator at 37 ° C. After formalin fixation and methylene blue staining, the number of concentrically formed plaques was counted. The evaluation results of the inactivated whole particle virus are shown in Table 6 and FIG. 8 below, and the evaluation results of ConA are shown in Table 7 and FIG. 9 below.

  From the results shown in Table 6 and FIG. 8, it can be seen that a plaque reduction of about 90% was observed in the 4-fold diluted solution of the inactivated whole particle virus, indicating a high antiviral effect. Here, when the HA content of the stock solution to the 32-fold dilution is estimated, the HA content of the first inactivated whole particle virus solution is 150 μg / mL, and the protein amount is reduced to about ３ after concentration and reconstitution. Therefore, 46.9 (stock solution), 23.4 (2 times), 11.7 (4 times), 5.86 (8 times), 2.93 (16 times), 1.46 (32 times), respectively. ) Estimated as μg / mL. Even if cells were sensitized with a stock solution of inactivated whole particle virus, the cells were not killed, and their cytotoxicity is considered to be extremely low.

  Also, from the results in Table 7 and FIG. 9, when the ConA concentration was 62.5 μg / mL, a plaque reduction of about 90% was observed, indicating that the antiviral effect against IV as well as AIV and NDV was high. I understand. These antiviral effects did not show a large difference even when the amount of inoculated challenge virus (infection titer) was changed.

  The above-mentioned virus suppression effect depends on the concentration of the cell treatment solution, and was observed even after washing the cell treatment solution with PBS. Therefore, inactivated whole particle virus having high affinity for sialic acid residues, and mannose It is believed that ConA, which has a high affinity for the site, embeds the viral receptor on the MDCK cell surface and inhibits the binding of the attacking virus.

  In addition, it is known that when an inactivated virus is administered to a living body through a nasal or oral route, the local immune system is stimulated to induce a local antibody (such as IgA). When the prophylactic agent of the present invention is contained in a living body, two effects of local infection protection by virus receptor embedding and local antibody induction by local immunostimulation can be expected.

[Example 4]
<Antiviral test of concanavalin A against human infectious virus>
Concanavalin A (ConA) (manufactured by Wako Pure Chemical Industries, Ltd.) was used as a test material. ConA was dissolved in PBS (10 mM, pH 7.2), adjusted to a concentration of 25 mg / mL, and sterile filtered. This ConA solution was further serially diluted two-fold with a 199M-Md medium to prepare six-stage solutions of 1000, 500, 250, 125, 62.5, and 31.25 μg / mL.

The following six types of test viruses, rubella virus (Baylor strain), measles virus (Edmonston strain), mumps virus (Enders strain), herpes simplex virus type 1 (HF strain), and herpes simplex virus type 2 (UW-238 strain) ), RS virus (Long strain) was used. Each virus strain was diluted to 10 ⁻³ to 10 ⁻⁷ using a 199 M-Md medium to adjust the infectious titer.

Vero560 was used as test cells. Vero cells were diluted with E-MEM (5% FBS) medium to a concentration of 1 × 10 ⁵ cells / mL. This cell solution was added to a 24-well plate at 1 mL per well, and cultured at 37 ° C. in a CO ₂ incubator for 3 days to form a sheet. This sheet was washed once with PBS.

Next, 0.1 mL of ConA solution having six concentrations was added per well, and sensitization was performed at room temperature of 25 ° C. for 30 minutes. Thereafter, ConA was removed by washing once with PBS.
Subsequently, 0.1 mL per well of each diluted virus solution was inoculated into a 24-well plate to infect the cells, and the cells were cultured in a CO ₂ incubator at 37 ° C. for 60 minutes to carry out virus adsorption. As a control, 0.1 mL of 199M-Md medium was added per well. The addition of the ConA solution to the 24-well plate and the inoculation of each virus diluent were performed according to the 4 × 6 grid shown in FIGS.

Further, 1 mL of 199M-Md medium was added per well, and the cells were cultured at 37 ° C. in a CO ₂ incubator. The state of the cytopathic effect (CPE) was confirmed, and judgment was performed 4 to 7 days later. The results are shown in Table 8 below and FIGS.

  From the results in Table 8 and FIGS. 10 to 15, it can be seen that ConA exhibits a growth inhibitory effect on measles, mumps and herpes simplex virus type 1 virus from a low concentration region. In addition, it can be seen that the herpes simplex virus type 2 has a growth inhibitory effect in a high concentration region. On the other hand, for rubella and RS virus, no growth inhibitory effect was observed at 1000 μg / mL or less.

  These inhibitory effects depend on the ConA concentration and are observed even after the ConA has been washed away with PBS. Therefore, as in Examples 1 to 3, ConA embeds the virus receptor on the Vero cell surface. It is considered that the binding of each attacking virus was inhibited. Since the inhibitory effect on various viruses was observed, the mechanism of action of the present invention is not limited to a specific virus, and epithelial cells in contact with the outside world such as the respiratory tract, gastrointestinal tract, ocular tissues, etc. It has been suggested that the cells can adapt to various viruses that invade the living body as cells and can suppress the infection.

The composition for preventing viral infections of the present invention is injected by administering it to epithelial cells of the respiratory tract, gastrointestinal tract or eye tissue before treating the virus with a viral infection such as influenza. There is no need, and simple and inexpensive infection protection can be realized. Therefore, the present invention is particularly useful not only in the medical field of humans or animals, but also in industrial fields such as pharmaceuticals, quasi-drugs, foods, health foods, foods for specified health use, pet foods, feeds, etc. for humans or animals.

Claims (11)

  For the prevention of viral infections for human or non-human animals, which is administered to epithelial cells of the respiratory tract, gastrointestinal tract or ocular tissue of human or non-human animals and contains as an active ingredient an antagonist that binds to a viral receptor of the epithelial cells. Composition.   The composition according to claim 1, wherein the antagonist is mannose-binding lectin (MBL).   The composition for preventing a viral infection according to claim 2, wherein the concentration of mannose-binding lectin (MBL) is 30 µg / mL or more.   The composition for preventing viral infection according to claim 1, wherein the antagonist is an inactivated virus.   The composition for preventing a virus infection according to claim 4, wherein the concentration of the inactivated virus is 6 µg / mL or more as an HA content.   The viral infection is an infection caused by one selected from the group consisting of influenza virus, measles virus, mumps virus, herpes simplex virus types 1 and 2, avian influenza virus, and Newcastle disease virus. A composition for preventing a viral infection according to any one of the above.   The composition according to any one of claims 1 to 6, which is a drug or a quasi-drug, and is administered via the nasal cavity, the oral cavity, or the eye.   The composition for preventing a viral infection according to claim 7, wherein the drug or the quasi-drug is one selected from the group consisting of a spray, a mouthwash, a troche, an oral liquid, an oral solid, and an eye drop. object.   The composition according to any one of claims 1 to 3, which is a food or feed and is administered via the oral cavity.   The composition for preventing a virus infection according to claim 9, wherein the food is one kind selected from the group consisting of gum, candy, beverage, and powdered beverage. Before administering a virus infection to a non-human animal, administering the composition for preventing a virus infection according to any one of claims 1 to 10 and pre-treating the virus receptor with an antagonist, whereby a non-human animal can be obtained. How to prevent viral infections in animals.