Pharmaceutical composition for topical administration

Abstract

To provide a novel pharmaceutical composition for topical administration and a novel immunopotentiating composition for topical administration.SOLUTION: According to the present invention, a pharmaceutical composition for topical administration and an immunopotentiating composition for topical administration, each comprising a lactic acid bacterium as an active ingredient, are provided. The lactic acid bacterium as the active ingredient in the present invention is preferably Lactococcus lactis subsp. lactis. According to the present invention, topical administration (particularly nasal administration) is advantageous in that it can further enhance the infection-preventative effect and the immunopotentiating effect of the lactic acid bacterium as the active ingredient.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application claims the benefit of priority of Japanese Patent Application No. 2022-206490 (filing date: December 23, 2022), and the entire disclosure thereof is incorporated herein by reference.

[0002] The present invention relates to a pharmaceutical composition for topical administration.

Background Art

[0003] The worldwide spread of infectious diseases caused by viruses or bacteria such as the novel coronavirus (SARS-CoV-2) has become a social problem. As a therapeutic agent for coronavirus disease 2019 (COVID-19) caused by SARS-CoV-2, for example, the development of antibody pharmaceuticals using neutralizing monoclonal antibodies has been underway (Non-Patent Documents 1 to 3). On the other hand, there is no prophylactic agent for COVID-19 that has been recognized as effective as a pharmaceutical, and the development thereof is demanded. In particular, since viruses are likely to produce mutant strains, the development of therapeutic and prophylactic agents that can be expected to be effective against mutant strains is demanded.

[0004] Lactic acid bacteria are abundantly contained in fermented milk such as yogurt and have been used as food and drink for a long time. In addition, lactic acid bacteria are known to bring about various physiological activities in the intestine and the like by oral ingestion, but the effects of topical administration for pharmaceutical use are not known.

Prior Art Documents

Non-Patent Documents

[0005]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0006] The present invention aims to provide a novel pharmaceutical composition for topical administration.

Means for Solving the Problems

[0007] The present inventors have found that the prophylactic effect of infectious diseases can be enhanced by topical administration of lactic acid bacteria. The present inventors have also found that an immunostimulatory effect is exhibited by topical administration of lactic acid bacteria. The present invention is based on these findings.

[0008] According to the present invention, the following inventions are provided. [1] A pharmaceutical composition for topical administration containing lactic acid bacteria as an active ingredient. [2] The pharmaceutical composition according to [1] above, wherein the topical administration is nasal administration, sublingual administration or inhalation administration. [3] The pharmaceutical composition according to [1] or [2] above, wherein the topical administration is administration to the upper respiratory tract and / or lower respiratory tract. [4] The pharmaceutical composition according to [3] above, wherein the upper respiratory tract is the nasal cavity. [5] The pharmaceutical composition according to any one of [1] to [4] above, for inducing the production of interferon by plasmacytoid dendritic cells in the upper respiratory tract and / or lower respiratory tract. [6] The pharmaceutical composition according to [5] above, wherein after the administration target of the pharmaceutical composition has contracted an infectious disease, the production of IFN is induced in the target. [7] The pharmaceutical composition according to any one of [1] to [6] above, for the prevention or treatment of infectious diseases, or for use in reducing the risk of the administration target contracting an infectious disease. [8] The pharmaceutical composition according to [7] above, wherein the infectious disease is a viral infectious disease. [9] The pharmaceutical composition according to [8] above, wherein the prevention of the viral infectious disease is prevention of the onset of the viral infectious disease or prevention of the aggravation of the viral infectious disease.

[10] The pharmaceutical composition according to [8] or [9] above, wherein the viral infectious disease is COVID-19 or an influenza virus infectious disease.

[11] The pharmaceutical composition according to any one of [8] to

[10] above, wherein the viral infectious disease is caused by infection with a causative virus of a respiratory infectious disease.

[12] The pharmaceutical composition according to

[11] above, wherein the causative virus of the respiratory viral infectious disease is SARS-CoV-2 or an influenza virus.

[13] The pharmaceutical composition according to any one of [1] to

[12] above, which is administered to a mammal.

[14] The pharmaceutical composition according to

[13] above, wherein the mammal is a human.

[15] The pharmaceutical composition according to any one of [1] to

[14] above, wherein the number of administrations is 2 or more.

[16] The pharmaceutical composition according to any one of [1] to

[15] above, wherein the administration interval is 1 day or more.

[17] The pharmaceutical composition according to any one of [8] to

[16] above, wherein the effective period of prevention of the viral infectious disease is 56 days from the last day of local administration.

[18] The pharmaceutical composition according to any one of [1] to

[17] above, wherein the daily dose of lactic acid bacteria (based on an adult body weight of 50 kg) is 1 mg or more and 1000 mg or less.

[19] The pharmaceutical composition according to any one of [1] to

[18] above, wherein the lactic acid bacteria is Lactococcus lactis subsp. lactis JCM5805 strain.

[20] A composition for local administration containing Lactococcus lactis subsp. lactis as an active ingredient.

[21] The composition for local administration according to

[20] above, which is a pharmaceutical composition.

[22] The pharmaceutical composition according to any one of [8] to

[19] above, which is locally administered to the viral infection site.

[23] An immunopotentiating composition for topical administration, containing lactic acid bacteria as an active ingredient.

[24] The immunopotentiating composition according to

[22] or

[23] above, for enhancing the expression of IFN-inducible antiviral genes (ISGs) in submandibular lymph nodes and / or the spleen.

[25] The immunopotentiating composition according to

[24] above, wherein the ISG is one or more selected from the group consisting of Viperin, Isg15, and Mx1.

[26] The immunopotentiating composition according to any one of

[22] to

[25] above, for increasing the ratio of pDCs to lymphocytes in the spleen and / or for increasing the ratio of pDCs to lymphocytes in the nasal mucosa.

[27] CD11b + Siglec-H + The immunopotentiating composition according to any one of

[22] to

[26] above, for increasing the ratio of cells.

[28] A method for preventing or treating an infectious disease, a method for reducing the risk of contracting an infectious disease, or an immunopotentiating method, including the step of topically administering an effective amount of lactic acid bacteria or a composition containing the same to a subject in need thereof.

[29] For the manufacture of a prophylactic or therapeutic agent for an infectious disease for topical administration, for the manufacture of an agent for reducing the risk of contracting an infectious disease for topical administration, or for the manufacture of an immunopotentiating agent for topical administration, or as a prophylactic or therapeutic agent for an infectious disease for topical administration, as an agent for reducing the risk of contracting an infectious disease for topical administration, or as an immunopotentiating agent for topical administration, or in a method for preventing or treating an infectious disease by topical administration, a method for reducing the risk of contracting an infectious disease by topical administration, or a method for immunopotentiating by topical administration, the use of lactic acid bacteria.

[30] Lactic acid bacteria for use in preventing or treating an infectious disease by topical administration, for reducing the risk of contracting an infectious disease by topical administration, or for immunopotentiating by topical administration.

Advantages of the Invention

[0009] According to the present invention, it is advantageous in that the preventive effect and immunostimulatory effect of lactic acid bacteria as an active ingredient against infectious diseases can be further enhanced by local administration (particularly nasal administration).

Brief Description of the Drawings

[0010]

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Mode for Carrying Out the Invention

[0011] The present invention is a pharmaceutical composition for topical administration containing lactic acid bacteria as an active ingredient.

[0012] The lactic acid bacteria, which is the active ingredient of the present invention, is not particularly limited, but is preferably a lactic acid bacterium having the property of inducing interferon (IFN: Interferon) production, and more preferably a lactic acid bacterium that activates plasmacytoid dendritic cells (pDC) and has the property of inducing at least IFN production. For example, a lactic acid bacterium having the property of inducing IFN-α production of 50 pg / mL or more, preferably 100 pg / ml or more, when added to a culture solution of plasmacytoid dendritic cells (pDC) derived from human peripheral blood mononuclear cells at 10 μg / ml can be used.

[0013] The lactic acid bacteria, which is the active ingredient of the present invention, can also be a lactic acid bacterium having the property of inducing IFN production in a subject after the subject suffering from an infectious disease.

[0014] The lactic acid bacteria having the property of inducing IFN production of the present invention can preferably induce the production of any one or more of Type I IFN (Type I interferon), Type II IFN (Type II interferon), and Type III IFN (Type III interferon). Type I IFN is a cytokine effective against viral infection and includes IFN-α (1, 2, 4, 5, 6, 7, 8, 10, 13, 14, 16, 17, 21), IFN-β, and the like. Type II IFN includes IFN-γ, and Type III IFN includes IFN-λ. Among them, the lactic acid bacteria having the property of inducing IFN production of the present invention are preferably those having the activity of inducing the production of Type I IFN in particular. When pDC is activated by the lactic acid bacteria having the property of inducing IFN production of the present invention, cell protrusions, which are characteristics of activated pDC, appear, the production of Type I IFN and Type III IFN is induced, and furthermore, the production of Type II IFN such as IFN-γ from NK cells or Th1 cells can also be induced.

[0015] The IFN that the lactic acid bacteria having the property of inducing IFN production, which is an active ingredient of the present invention, can induce production of is not particularly limited, but is preferably one or more selected from the group consisting of IFN-α, IFN-β, and IFN-λ, more preferably two or more selected from the group consisting of IFN-α, IFN-β, and IFN-λ, even more preferably two or more selected from the group consisting of IFN-α, IFN-β, and IFN-λ, and at least one of the two or more IFNs is IFN-α, even more preferably at least two of the two or more IFNs are IFN-α and IFN-β, and particularly preferably three types of IFN-α, IFN-β, and IFN-λ.

[0016] Whether the lactic acid bacteria having the property of inducing IFN production, which is the active ingredient of the present invention, can activate pDC and at least induce IFN production can be confirmed, for example, by measuring whether pDC is activated and IFN production is induced when candidate lactic acid bacteria are cultured in the presence of mammalian bone marrow cells such as mice. For example, the production of IFN-α can be measured by measuring the concentration of IFN-α in the culture supernatant by ELISA or the like. Specifically, mouse bone marrow cells from which erythrocytes have been removed are suspended in RPMI medium (SIGMA) containing 10% fetal bovine serum (FCS) and 2 μM β-mercaptoethanol to a concentration of 5×10 5 cells / mL, Flt-3L is added to the resulting cell suspension as a pDC-inducing cytokine at a final concentration of 100 ng / ml, and the cells are cultured at 37°C and 5% CO2 in a CO2 incubator. After 7 days, a lactic acid bacterial strain is added at 10 μg / ml, and after 48 hours, the culture supernatant is collected, and the concentration of IFN-α in the culture supernatant can be measured by ELISA using an IFN-α measurement kit (PBL).

[0017] The lactic acid bacteria that are the active ingredient of the present invention are not particularly limited, and examples include lactic acid cocci. For example, lactic acid bacteria belonging to the genus Lactococcus, Leuconostoc, Pediococcus, Streptococcus, Enterococcus, Lactobacillus, and Oenococcus, Bifidobacterium, Weissella, and Tetragenococcus can be mentioned. The lactic acid bacteria that are the active ingredient of the present invention are preferably lactic acid bacteria belonging to the genus Lactococcus or Pediococcus. In addition, as the lactic acid bacteria that are the active ingredient of the present invention, one bacterial species or strain can be used, or two or more bacterial species or strains can be used in combination.

[0018] Examples of bacteria belonging to the genus Lactococcus include, for example, Lactococcus lactis, Lactococcus lactis subsp. lactis, Lactococcus garvieae, Lactococcus lactis subsp. cremoris, Lactococcus lactis subsp. hordniae, etc. From the viewpoint of activating pDC and inducing at least IFN production, Lactococcus lactis subsp. lactis is preferred.

[0019] Specific examples of bacteria belonging to the genus Lactococcus include, for example, Lactococcus lactis subsp. lactis JCM5805, Lactococcus lactis subsp. lactis NBRC12007, Lactococcus lactis subsp. lactis NRIC1150, Lactococcus lactis subsp. lactis JCM20101, Lactococcus lactis subsp. lactis JCM7638, Lactococcus lactis subsp. lactis ATCC11454, Lactococcus garvieae NBRC100934, Lactococcus lactis subsp. cremoris JCM16167, Lactococcus lactis subsp. cremoris NBRC100676, Lactococcus lactis subsp. hordniae JCM1180, Lactococcus lactis subsp. hordniae JCM11040, and Lactococcus plantarum JCM11056, etc. From the viewpoint of activating pDC and inducing at least IFN production, Lactococcus lactis subsp. lactis JCM5805 is preferred.

[0020] Examples of Leuconostoc bacteria include, for example, Leuconostoc lactis. Specific examples of Leuconostoc bacteria include, for example, Leuconostoc lactis NBRC12455.

[0021] Examples of Pediococcus bacteria include, for example, Pediococcus acidilactici, Pediococcus pentosaceus, Pediococcus cellicola, Pediococcus claussenii, Pediococcus damnosus, Pediococcus ethanolidurans, Pediococcus inopinatus, Pediococcus parvulus, Pediococcus stilesii, etc. Specific examples of Pediococcus bacteria include, for example, Pediococcus acidilactici JCM8797, Pediococcus acidilactici K15, and Pediococcus damnosus JCM5886.

[0022] Examples of Streptococcus bacteria include, for example, Streptococcus thermophilus. Specific examples of Streptococcus bacteria include, for example, Streptococcus thermophilus SBC8781.

[0023] Examples of Enterococcus bacteria include, for example, Enterococcus alcedinis.

[0024] Examples of bacteria belonging to the genus Lactobacillus include, for example, Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, Lactobacillus rhamnosus, Lactobacillus plantarum, Lactobacillus gasseri, Lactobacillus acidophilus, and Lactobacillus bulgaricus. Specific examples of bacteria belonging to the genus Lactobacillus include, for example, Lactobacillus paracasei KW3110, Lactobacillus paracasei MCC1849, Lactobacillus paracasei K71, Lactobacillus rhamnosus GG, Lactobacillus rhamnosus CRL1505, Lactobacillus gasseri SBT2055, Lactobacillus parakefir (Lentilactobacillus parakefir in the new classification) JCM8573, Lactobacillus pentosus (Lactiplantibacillus pentosus in the new classification) ONRICb0240, Lactobacillus plantarum (Lactiplantibacillus plantarum in the new classification) L-137, and Lactobacillus acidophilus L-92 or Lactobacillus bulgaricus OLL1073R-1 (including the strain having exopolysaccharide) and the like. In the present invention, from the viewpoint of excellent water dispersibility in beverages, the bacteria belonging to the genus Lactobacillus can be, for example, one or more selected from the group consisting of Lactobacillus paracasei, Lactobacillus delbrueckii, Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus fructivorans, Lactobacillus hilgardii, and Lactobacillus rhamnosus.

[0025] Examples of bacteria belonging to the genus Oenococcus include, for example, Oenococcus oeni. Specific examples of bacteria belonging to the genus Oenococcus include Oenococcus oeni JCM6125.

[0026] Examples of bacteria belonging to the genus Bifidobacterium include, for example, Bifidobacterium animalis subsp. lactis and Bifidobacterium longum subsp. infantis. Specific examples of bacteria belonging to the genus Bifidobacterium include Bifidobacterium animalis subsp. lactis JCM10602 and Bifidobacterium longum subsp. infantis JCM1222.

[0027] Examples of bacteria belonging to the genus Weissella include, for example, Weissella paramesenteroides and Weissella viridescens. Specific examples of bacteria belonging to the genus Weissella include Weissella paramesenteroides JCM9890 and Weissella viridescens JCM1174.

[0028] Examples of bacteria belonging to the genus Tetragenococcus include, for example, Tetragenococcus halophilus. Specific examples of bacteria belonging to the genus Tetragenococcus include Tetragenococcus halophilus NRIC0098.

[0029] In the present invention, lactic acid bacteria can be obtained from known depository institutions or the like. For example, among the above-mentioned Lactococcus strains, the JCM strain can be obtained from the RIKEN BioResource Center, Microbial Material Development Laboratory (1-1 Higashi, 3-chome, Tsukuba, Ibaraki), the NBRC strain can be obtained from the Biological Genetic Resources Department of the National Institute of Technology and Evaluation (8-5 Kazusa Kamashima 2-chome, Kisarazu, Chiba), the NRIC strain can be obtained from the Strain Preservation Laboratory of the Tokyo University of Agriculture (1-1 Sakuragaoka 1-chome, Setagaya, Tokyo), and the ATCC strain can be obtained from the American type culture collection (USA), respectively.

[0030] In the present invention, the lactic acid bacteria used as an active ingredient may contain a culture of lactic acid bacteria. The culture includes viable cells, dead cells, disrupted products of viable cells or dead cells, freeze-dried products of viable cells or dead cells, disrupted products of the freeze-dried products, enzyme-treated products of viable cells or dead cells, culture solutions, culture solution extracts, etc., and also includes a part of lactic acid bacteria and processed products of lactic acid bacteria. Dead cells can be obtained by treating viable cells. For example, they can be obtained by any one or a combination of two or more of heat treatment, treatment with drugs such as antibiotics, treatment with chemical substances such as formalin, treatment with ultraviolet rays, and treatment with radiation such as γ-rays. Processed products include, for example, heat-treated cells (dead cells), their freeze-dried products, cultures containing these, and further include disrupted solutions of cells by ultrasonic waves or the like and enzyme-treated solutions of cells. The processed products also include processed products obtained by removing the cell wall by enzymatic or mechanical means. Furthermore, it also includes a nucleic acid-containing fraction obtained by dissolving the cells with a surfactant or the like and then precipitating with ethanol or the like. Furthermore, the lactic acid bacteria used as an active ingredient in the present invention can also contain dead cells. Furthermore, the above-mentioned culture of lactic acid bacteria contains DNA and RNA derived from lactic acid bacteria, and the DNA and RNA derived from lactic acid bacteria preferably activate pDC and can at least induce IFN production.

[0031] The cultivation of lactic acid bacteria can be carried out by known methods using known media. As the medium, for example, MRS medium, GAM medium, LM17 medium, etc. can be used, and inorganic salts, vitamins, amino acids, antibiotics, serum, etc. can be appropriately added and used. The cultivation can be carried out at 25 to 40 °C for several hours to several days.

[0032] After cultivation, the lactic acid bacteria cells can be obtained by collecting the lactic acid bacteria cells by centrifugation or filtration. When used as dead cells, they may be sterilized and inactivated by an autoclave or the like before use.

[0033] In the present invention, local administration means local administration to the site where the lactic acid bacteria, which are the active ingredient, directly act. For example, nasal administration, sublingual administration, inhalation administration, buccal administration, transdermal administration, rectal administration, vaginal administration, pulmonary administration, airway administration (intratracheal administration), and eye drop administration can be mentioned. As local administration, in terms of enhancing the infection prevention effect or infection suppression effect and reducing the administration burden, nasal administration, sublingual administration, and inhalation administration are preferably used. In local administration, it is assumed that the lactic acid bacteria, which are the active ingredient, are absorbed through the mucosa at local sites such as the nasal cavity, sublingual region, pharynx, larynx, airway, bronchus, lung, vagina, skin, eye, and intestine. Also, when administering lactic acid bacteria from the nasal cavity, preferably, the administration site is the Waldeyer's pharyngeal ring (for example, pharyngeal tonsil, superior pharynx (nasopharynx), palatine tonsil, middle pharynx, or lingual tonsil). Local administration is advantageous in terms of enhancing the infection prevention effect or infection suppression effect because it can directly act on pDCs and conventional dendritic cells (cDCs) etc. present in the local periphery with lactic acid bacteria having the property of activating pDCs, which are the active ingredient, and inducing at least IFN production, compared to oral administration as an oral preparation etc.

[0034] Dosage forms (formulations) suitable for topical administration in the present invention are well known to those skilled in the art and can be appropriately selected according to the purpose. Examples of dosage forms include, for example, nasal drops (e.g., nasal powder, nasal solution), inhalants (e.g., inhalation aerosol, inhalation powder, inhalation solution), sublingual tablets, buccal administration agents, injections (including intradermal injection, subcutaneous injection, intramuscular injection, intravenous injection), ointments, creams, gels, suppositories, patches, and poultices. These dosage forms can be formulated (manufactured) using pharmaceutically acceptable carriers (including solvents) by methods commonly practiced in the art (e.g., known methods described in the General Rules for Preparations of the Japanese Pharmacopoeia, 18th Edition, etc.).

[0035] In the present invention, the nasal drops are not particularly limited. For example, they can be prepared by dispersing lactic acid bacteria in a liquid such as purified water or physiological saline or in a powder composed of a carrier. Also, they can be provided as nasal drops by filling a known nebulizer or the like for nasal drops.

[0036] In the present invention, the inhalants are not particularly limited. For example, they can be prepared by dispersing lactic acid bacteria in a liquid such as purified water or physiological saline or in a powder composed of a carrier, and then provided as inhalants by filling a known inhaler or the like for inhalants. Also, they can be provided by means of inhaling water particles or water vapor together with lactic acid bacteria orally or nasally or the like. Specific means include a method of inhaling by an inhaler capable of inhaling water particles such as a nebulizer, a method of inhaling by a steam generator capable of generating heat by reacting with oxygen in the air and generating water vapor and supplying the warmed water vapor to the subject, etc.

[0037] The nebulizer is preferably an ultrasonic nebulizer, a mesh nebulizer, or a compressor nebulizer.

[0038] The particle size of the inhaled water particles is preferably less than 10 μm, more preferably 1 to 8 μm. Here, the particle size of the water particles can be measured by the light scattering method.

[0039] In formulation, pharmaceutically acceptable carriers include, but are not limited to, for example, excipients, thickeners, lubricants, binders, disintegrants, solvents, solubilizers, suspending agents, emulsifiers, isotonic agents, buffers, soothing agents, and stabilizers. Further, additives such as preservatives, pH adjusters, cooling agents, antioxidants, wetting agents, adhesives, and odor correctives can be included as necessary.

[0040] Examples of excipients include lactose, sucrose, D-mannitol, starch, corn starch, crystalline cellulose, and light anhydrous silicic acid.

[0041] Examples of thickeners include polyhydric alcohols such as glycerin, celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose, hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, carboxyvinyl polymer, and sodium carboxymethylcellulose, sodium alginate, chondroitin sulfate, and polyethylene glycol.

[0042] Examples of lubricants include magnesium stearate, calcium stearate, talc, and colloidal silica.

[0043] Examples of binders include crystalline cellulose, sucrose, dextrin, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, starch, sucrose, gelatin, methylcellulose, and sodium carboxymethylcellulose.

[0044] Examples of disintegrants include starch, carboxymethylcellulose, calcium carboxymethylcellulose, and L-hydroxypropylcellulose.

[0045] Examples of solvents include water (including purified water, pure water, etc.), ethanol, isopropyl alcohol, and propylene glycol.

[0046] Examples of solubilizing agents include celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose; polyethylene glycol, propylene glycol, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate, and polyvinylpyrrolidone.

[0047] Examples of suspending agents include surfactants such as sodium lauryl sulfate, lauryl aminopropionic acid, lecithin, benzalkonium chloride, glyceryl monostearate, polyoxyethylene hydrogenated castor oil, and polysorbate; polyhydric alcohols such as glycerin; saccharides such as sorbitol, mannitol, and sucrose; celluloses such as methylcellulose, carboxymethylcellulose, and hydroxypropylmethylcellulose; hydrophilic polymers such as polyvinyl alcohol, polyvinylpyrrolidone, and carboxyvinyl polymer; and chondroitin sulfate.

[0048] Examples of isotonic agents include glucose, D-sorbitol, sodium chloride, glycerin, potassium chloride, propylene glycol, and sucrose.

[0049] Examples of buffering agents include phosphates (such as sodium hydrogen phosphate and sodium dihydrogen phosphate), boric acid, borax, acetates (such as sodium acetate), carbonates (such as sodium carbonate, calcium carbonate, and potassium carbonate), citric acid, and sodium L-glutamate.

[0050] Examples of soothing agents include benzyl alcohol, chlorobutanol, propylene glycol, ethyl aminobenzoate, and lidocaine.

[0051] Examples of stabilizers include sulfur compounds such as sodium sulfite, sodium bisulfite, sodium metabisulfite, sodium thiosulfate, Rongalit, thioglycerol, thioglycolic acid, thiolactic acid, cysteine, glutathione, thioacetic acid, methionine, thiosorbitol, thioglucose, thiourea, etc.; inorganic acids and their salts such as boric acid, borax, phosphoric acid, metaphosphoric acid, sodium carbonate, sodium bicarbonate, etc.; organic acids and their salts (such as sodium edetate) such as formic acid, oxalic acid, tartaric acid, citric acid, edetic acid, etc.; acid amides such as acetamide, diethylacetamide, nicotinamide, urea, barbital, etc., urea derivatives, polyhydric alcohols such as glycol, propylene glycol, glycerin, polyethylene glycol, glucose, ascorbic acid, etc., saccharides, phenols such as phenol, quinone, coumarone, isocoumarone, etc., amino acids and proteins such as dibutylhydroxytoluene, glycine, glutamic acid, lysine, phenylalanine, casein, edestin, etc.

[0052] Examples of emulsifiers include glycerin esters (glycerol monooleate), sucrose fatty acid esters, lecithin (such as vegetable lecithin, egg yolk lecithin, soybean lecithin, etc.), various surfactants (alkylbenzene sulfonate type emulsifiers, benzalkonium chloride, sorbitan sesquioleate, dodecylbenzene sulfonic acid, etc.), and triethanolamine, etc.

[0053] Examples of preservatives include paraoxybenzoic acid esters such as propyl paraoxybenzoate, butyl paraoxybenzoate, etc., parabens such as methylparaben, ethylparaben, propylparaben, butylparaben, etc., reverse soaps such as benzalkonium chloride, benzethonium chloride, chlorhexidine gluconate, cetylpyridinium chloride, etc., alcohol derivatives such as benzyl alcohol, phenethyl alcohol, etc., organic acids and their salts such as sodium dehydroacetate, sorbic acid, sodium sorbate, etc., and phenols such as parachloromethoxyphenol and parachlorometacresol, etc.

[0054] Examples of pH adjusters include sodium hydroxide, potassium hydroxide, trisodium phosphate, disodium hydrogen phosphate, hydrochloric acid, nitric acid, citric acid, boric acid, acetic acid, and the like.

[0055] Examples of cooling agents include 1-menthol, camphor, peppermint water, and the like. Examples of antioxidants include sulfites, ascorbic acid, citric acid, sodium edetate, and the like.

[0056] Examples of wetting agents include propylene glycol, polysorbate, polyethylene glycol, glycerin, and the like.

[0057] Examples of adhesives include hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxyvinyl polymer, propylene glycol, polysorbate 80, and the like.

[0058] Examples of odor correctors include trehalose, malic acid, maltose, anise essential oil, vanilla essential oil, cardamom essential oil, and the like.

[0059] The pharmaceutical composition of the present invention can be used for the prevention or treatment of infectious diseases.

[0060] The pharmaceutical composition of the present invention can be used to reduce the risk of contracting an infectious disease in the administration subject or to reduce the risk of infection by a pathogen in the administration subject. The administration subject is a subject to whom the pharmaceutical composition of the present invention is administered, and can be a subject at risk of contracting an infectious disease or a subject at risk of infection by a pathogen. Here, in the present invention, "reduction of the risk of contracting an infectious disease" means that the probability of contracting an infectious disease is reduced, and "reduction of the risk of infection by a pathogen" means that the probability of infection by a pathogen is reduced.

[0061] Also, as one aspect of the present invention, a composition for topical administration containing Lactococcus lactis subsp. lactis as an active ingredient can be used, and preferably the composition is a pharmaceutical composition.

[0062] In the present invention, the infectious disease is not particularly limited, but is a disease caused by the infection of a pathogen. Examples thereof include viral infectious diseases, bacterial infectious diseases, rickettsia-chlamydia infectious diseases, fungal infectious diseases, parasitic infectious diseases, and prion infectious diseases, etc., and preferably viral infectious diseases and bacterial infectious diseases.

[0063] In the present invention, examples of the viral infectious disease include COVID-19, influenza virus infection, rabies, Japanese encephalitis, West Nile fever, dengue fever, chikungunya fever, Zika virus infection, tick-borne encephalitis, hepatitis E, severe fever with thrombocytopenia syndrome (SFTS), Ebola hemorrhagic fever, severe acute respiratory syndrome (SARS), and Middle East respiratory syndrome (MERS), etc.

[0064] In the present invention, examples of the bacterial infectious disease include Q fever, plague, salmonellosis, leptospirosis, cat scratch disease, brucellosis, Capnocytophaga canimorsus infection, Corynebacterium ulcerans infection, campylobacteriosis, and anthrax, etc.

[0065] In the present invention, examples of the rickettsia-chlamydia infectious disease include Japanese spotted fever, scrub typhus, and psittacosis, etc.

[0066] In the present invention, examples of the fungal infectious disease include dermatophytosis and cryptococcosis, etc.

[0067] In the present invention, examples of the parasitic infectious disease include toxoplasmosis, ascariasis, echinococcosis, cryptosporidiosis, and anisakiasis, etc.

[0068] In the present invention, examples of the prion infectious disease include variant Creutzfeldt-Jakob disease (vCJD), etc.

[0069] In the present invention, prevention of viral infectious diseases is used in the sense of including prevention of onset of viral infectious diseases and prevention of aggravation of viral infectious diseases. Here, the presence or absence of onset of viral infectious diseases can be determined by known methods such as PCR tests, antigen tests, and antibody tests. The presence or absence of the severity of viral infectious diseases can be determined, for example, using the presence or absence of respiratory failure, oxygen saturation concentration, and pneumonia findings as indicators. When the viral infectious disease is COVID-19, the severity classification described in the COVID-19 treatment guidelines by the National Institutes of Health (NIH) of the United States (https: / / www.covid19treatmentguidelines.nih.gov / tables / management-of-hospitalized-adults-summary / ) and the like can be referred to.

[0070] The causative virus of the viral infectious disease to be prevented or treated by the pharmaceutical composition of the present invention is not particularly limited. For example, SARS-CoV-2, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome coronavirus, common human coronaviruses (229E, NL63, OC43, and HKU1), influenza virus, parainfluenza virus, adenovirus, RSV, human metapneumovirus, rhinovirus, dengue virus, varicella-zoster virus, herpes simplex virus, measles virus, parainfluenza virus, enterovirus, rhinovirus, human metapneumovirus, alphavirus, cytomegalovirus, Epstein-Barr virus, yellow fever virus, echovirus, coxsackievirus, herpes simplex virus, measles virus, rubella virus, varicella virus, arbovirus, arenavirus, filovirus, and human papillomavirus, etc. can be mentioned. In the present invention, the causative virus of the viral infectious disease is preferably the causative virus of respiratory infectious diseases, and more preferably SARS-CoV-2.

[0071] In the present invention, SARS-CoV-2 includes not only the virus strain first discovered but also its mutant strains (e.g., B.1.1.7 line (alpha strain), B.1.351 line (beta strain), P.1 line (gamma strain), B.1.617.2 line (delta strain), and B.1.1.529 line (omicron strain), etc.). Note that SARS-CoV-2 is synonymous with severe acute respiratory syndrome coronavirus-2, and the disease and symptoms caused by SARS-CoV-2 infection are called COVID-19 (so-called novel coronavirus infection).

[0072] The lactic acid bacterium having the property of inducing IFN production, which is the active ingredient of the present invention, can induce IFN production in vivo and does not directly administer IFN to the living body. When directly administering IFN to the living body, there can be oral administration and parenteral administration. Here, when directly administering IFN to the living body orally, IFN has weak resistance to gastric juice and intestinal juice, decomposition is caused, and the possibility of being absorbed by the living body as IFN is low. Furthermore, when directly administering IFN to the living body parenterally, for example, subcutaneous administration, etc. can be possible, but the intake burden is high, and the possibility of side effects (fever, headache, depression, etc.) has also been reported. Therefore, the lactic acid bacterium having the property of inducing IFN production, which is the active ingredient of the present invention, can be said to be superior to the case of directly administering IFN to the living body in terms of the point that the administration burden can be reduced, the point that the possibility of side effects can be reduced, and the preventive and therapeutic effects against infectious diseases.

[0073] Regarding the mechanism of virus infection, for example, in the case of SARS-CoV-2, it is known that the virus binds to and adsorbs to the ACE2 (angiotensin converting enzyme 2) receptor on the cell surface, and using this as a foothold, the virus enters the cell, leading to infection. Therefore, by locally administering the lactic acid bacteria, which are the active ingredient of the present invention, to tissues with high expression of the ACE2 receptor, a higher preventive effect and therapeutic effect against infectious diseases can be exerted. Tissues with high expression of the ACE2 receptor include the respiratory organs (e.g., nasal cavity, pharynx, larynx, trachea, bronchi, lungs), preferably the upper respiratory tract (more preferably the nasal cavity) and the lower respiratory tract.

[0074] As shown in the examples described later, the lactic acid bacteria, which are the active ingredient of the present invention, may have an immunostimulatory effect when administered locally. Therefore, according to another aspect of the present invention, there is provided an immunostimulatory composition for local administration containing lactic acid bacteria as an active ingredient. The immunostimulatory composition of the present invention can be a pharmaceutical composition. Here, in the present invention, immunostimulation means maintaining or enhancing the immune function, maintaining or enhancing immunity, maintaining or enhancing the immune action, or maintaining or enhancing the immune response. The effect of immunostimulation can be confirmed, for example, by an increase in the ratio of pDC to lymphocytes, as shown in the examples described later.

[0075] As shown in the following Examples, the lactic acid bacteria, which are the active ingredient of the present invention, may have the property of enhancing the expression of IFN-inducible antiviral genes (ISGs: IFN-stimulated genes) in the submandibular lymph nodes and / or spleen. That is, the pharmaceutical composition of the present invention can be used to enhance the expression of ISGs in the submandibular lymph nodes and / or spleen. Examples of ISGs whose expression can be enhanced by the lactic acid bacteria, which are the active ingredient of the present invention, include, but are not limited to, Viperin, Isg15, Mx1, Oasl2, IFITM, ISG20, and RyDEN. In the pharmaceutical composition of the present invention, the ISG is one or more selected from the group consisting of Viperin, Isg15, and Mx1, preferably one or more selected from the group consisting of Viperin, Isg15, and Mx1, and more preferably Viperin or ISG15. Here, the Viperin protein encoded by Viperin is a radical SAM domain-containing molecule having various antiviral activities, the ISG15 encoded by Isg15 is a ubiquitin-like small molecule having antiviral properties, and the MX1 encoded by Mx1 is a dynamin-like GTPase, which targets the viral nucleocapsid and is known to suppress the virus before replication is established (Schoggins JW., Curr Opin Virol., 2014: 40-46.).

[0076] As shown in the following examples, the lactic acid bacteria, which are the active ingredient of the present invention, can increase the ratio of pDCs to lymphocytes in the spleen and can increase the ratio of pDCs to lymphocytes in the nasal mucosa. That is, the pharmaceutical composition of the present invention can be used to increase the ratio of pDCs to lymphocytes in the spleen and / or to increase the ratio of pDCs to lymphocytes in the nasal mucosa. Here, in the present invention, the ratio of pDCs to lymphocytes in the spleen means the proportion of pDCs in all lymphocytes contained in the spleen, and the ratio of pDCs to lymphocytes in the nasal mucosa means the proportion of pDCs in all lymphocytes contained in the nasal mucosa. The ratio of pDCs to lymphocytes in the spleen and / or the ratio of pDCs to lymphocytes in the nasal mucosa is not limited, but for example, as shown in the following examples, in the analysis using FACS (Fluorescence-activated cell sorter), it can be calculated as the ratio (proportion) of pDCs to all lymphocytes in the lymphocyte fraction.

[0077] In the present invention, "increase in the ratio of pDCs to lymphocytes in the spleen" means that the ratio of pDCs to lymphocytes in the spleen when lactic acid bacteria are administered is increased compared to the ratio when lactic acid bacteria are not administered. Specifically, when the ratio of pDCs to lymphocytes in the spleen of the subject after administration of lactic acid bacteria is, for example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times or 3.0 times or more compared to the ratio of the subject before administration, it can be considered that the ratio of pDCs to lymphocytes in the spleen has increased.

[0078] In the present invention, the "increase in the ratio of pDCs to lymphocytes in the nasal mucosa" means that the ratio of pDCs to lymphocytes in the nasal mucosa when lactic acid bacteria are administered increases compared to the ratio when lactic acid bacteria are not administered. Specifically, when the ratio of pDCs to lymphocytes in the nasal mucosa of a subject after administration of lactic acid bacteria is, for example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times or 5.0 times or more compared to the ratio of the subject before administration, it can be considered that the ratio of pDCs to lymphocytes in the nasal mucosa has increased.

[0079] As shown in the following examples, the lactic acid bacteria, which are the active ingredient of the present invention, can increase the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa. That is, the immunopotentiating composition of the present invention can be used to increase the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa. Here, in the present invention, the ratio of CD11b + Siglec-H + cells to lymphocytes means the proportion of CD11b + Siglec-H + cells in all lymphocytes contained in the nasal mucosa. The ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa is not limited, but for example, as shown in the following examples, in the analysis using FACS, it can be calculated as the ratio (proportion) of CD11b + Siglec-H + cells to all lymphocytes in the lymphocyte fraction.

[0080] In the present invention, "increase in the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa" means that when lactic acid bacteria are administered, the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa increases as compared to the ratio when lactic acid bacteria are not administered. Specifically, the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa of the subject after administration of lactic acid bacteria is, for example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times, 3.5 times, 3.6 times, 3.7 times, 3.8 times, 3.9 times, 4.0 times, 4.1 times, 4.2 times, 4.3 times, 4.4 times, 4.5 times, 4.6 times, 4.7 times, 4.8 times, 4.9 times or 5.0 times or more as compared to the ratio of the subject before administration. In this case, it can be said that the ratio of CD11b + Siglec-H + cells to lymphocytes in the nasal mucosa has increased.

[0081] The pharmaceutical composition of the present invention can be administered to mammals. There is no particular limitation on the mammals, and examples include primates, rodents, carnivores, etc., and preferably primates. Examples of primates include humans, chimpanzees, rhesus monkeys, marmosets, dogs, cats, cows, horses, pigs, and sheep, and preferably humans.

[0082] The pharmaceutical composition of the present invention can be administered either before or after the onset of an infectious disease (or before or after infection with a pathogen). When used for the prevention of an infectious disease, it can be said that it is desirable to administer it before infection. However, even after infection, it may be administered in the early stage after infection (for example, within 5 days after infection) to prevent the disease from worsening. The administration time can also be determined in consideration of the prevalence of viral infectious diseases and the periods when they are likely to spread.

[0083] The number of administrations of the pharmaceutical composition of the present invention can be arbitrarily set according to the purpose of administration, the constitution, physical condition, symptoms, etc. of the administration subject, but it can be once or multiple times (for example, 2 or more times, 3 or more times, 4 or more times, 5 or more times). The administration interval in the case of multiple administrations can be 1 to 24 hours or 1 to 10 days, and for example, it can be once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days. When used for the prevention or treatment of an infectious disease, from the viewpoints of maintaining the preventive or therapeutic effect and saving medical costs, it is preferable to administer it 2 or more times at an administration interval of once a day.

[0084] When the pharmaceutical composition of the present invention is used for the prevention of a viral infectious disease, from the viewpoint of immediate efficacy, the start date of the effective period during which the active ingredient exhibits a preventive effect is the 0th day, 1st day, 2nd day, 3rd day, 4th day, 5th day, 6th day or 7th day from the last day of administration.

[0085] When the pharmaceutical composition of the present invention is used for the prevention of a viral infectious disease, from the viewpoint of maintaining the effect, the end date of the effective period during which the active ingredient exhibits a preventive effect is the 7th day, 14th day, 28th day or 56th day from the last day of administration.

[0086] In the pharmaceutical composition of the present invention, the above start period and the above end period can be arbitrarily combined. For example, it can be from day 0 to day 56, from day 0 to day 28, from day 0 to day 14, from day 0 to day 7, from day 3 to day 56, from day 3 to day 28, from day 3 to day 14, from day 3 to day 7, from day 5 to day 56, from day 5 to day 28, from day 5 to day 14, from day 5 to day 7, from day 7 to day 56, from day 7 to day 28, from day 7 to day 14.

[0087] When the pharmaceutical composition of the present invention is used for the prevention of viral infectious diseases, the effective period during which the active ingredient exhibits a preventive effect is within 56 days, within 28 days, within 14 days, within 7 days, within 6 days, within 5 days, within 4 days or within 3 days from the last day of administration.

[0088] The daily dosage of the active ingredient of the present invention can be appropriately adjusted and determined according to the weight of the subject to which it is administered. In the case of an adult (for example, an adult human), based on a body weight of 50 kg, for example, it can be specified by the dry cell mass. From the viewpoint of the infection prevention effect or the infection suppression effect, the lower limit value (equal to or exceeding) thereof can be 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg, 36 mg, 37 mg, 38 mg, 39 mg, 40 mg, 41 mg, 42 mg, 43 mg, 44 mg, 45 mg or 50 mg, and the upper limit value (equal to or less than) thereof can be 1000 mg, 950 mg, 900 mg, 850 mg, 800 mg, 750 mg, 700 mg, 650 mg, 600 mg, 550 mg, 500 mg, 475 mg, 450 mg, 425 mg, 400 mg, 375 mg, 350 mg, 325 mg, 300 mg, 275 mg, 250 mg, 225 mg, 200 mg, 190 mg, 180 mg, 170 mg, 160 mg, 150 mg, 140 mg, 130 mg, 120 mg, 110 mg or 100 mg, 90 mg, 80 mg, 70 mg, 60 mg or 50 mg. These upper and lower limit values can be arbitrarily combined with each other, and the above dosage range can be, for example, 1 mg or more and 1000 mg or less, 10 mg or more and 500 mg or less, 25 mg or more and 450 mg or less, 30 mg or more and 400 mg or less, 35 mg or more and 375 mg or less, 40 mg or more and 350 mg or less, 40 mg or more and 325 mg or less, 45 mg or more and 300 mg or less, 45 mg or more and 250 mg or less, 45 mg or more and 200 mg or less, 45 mg or more and 150 mg or less, 45 mg or more and 100 mg or less, 45 mg or more and 90 mg or less, 45 mg or more and 80 mg or less, 45 mg or more and 70 mg or less or 45 mg or more and 60 mg or less.

[0089] The daily dosage of the active ingredient of the present invention can also be specified by the number of bacteria. In the case of an adult (for example, an adult human), based on a body weight of 50 kg, from the viewpoint of the infection prevention effect or the infection suppression effect, the lower limit value (equal to or exceeding) is 1×10 8 cells, 1×10 9 cells, 1×10 10 cells or 1×10 11 cells, and the upper limit value (less than or equal to) can be 1×10 14 cells, 1×10 13 cells, 1×10 12 cells, 5×10 11 cells or 1×10 11 cells. These upper and lower limit values can be arbitrarily combined respectively. The range of the above dosage can be, for example, 1×10 8 ~1×10 14 cells, 1×10 9 ~1×10 13 cells, 1×10 10 ~1×10 12 cells, 5×10 10 ~5×10 11 cells or 1×10 11 cells. The number of lactic acid bacteria can be measured, for example, by a fluorescence staining method, flow cytometry, or a culture method. From the viewpoint of accuracy, it is preferably measured by flow cytometry.

[0090] It is preferable to continue the administration of the active ingredient of the present invention within the period expecting the infection prevention effect or the infection suppression effect. From the viewpoint of better exerting the infection prevention effect or the infection suppression effect, the administration period of the active ingredient of the present invention can be, for example, the above daily dosage for 1 week or more, 2 weeks or more, 3 weeks or more, 1 month or more (4 weeks or more). The administration interval of the active ingredient of the present invention can be once every 3 days, once every 2 days, or once a day with the above daily dosage, and preferably once a day.

[0091] The active ingredient of the present invention may also be administered before an event or a time period when infection prevention is expected, such as an increase in the likelihood or probability of contracting an infectious disease (susceptibility to infection by a pathogen). Examples of events for which an infection prevention effect is expected include actions that increase the likelihood or probability of contracting an infectious disease (susceptibility to infection by a pathogen) (e.g., participation in an event with a high risk of infection, travel to an epidemic area, infection of cohabitants such as family members), etc. Examples of time periods for which an infection prevention effect is expected include the epidemic period of an infectious disease. Examples of the administration timing (administration time point) before an event for which an infection prevention effect is expected include, for example, 1 day or more before, 3 days or more before, 1 week or more before, 2 weeks or more before, 3 weeks or more before, 1 month or more before (4 weeks or more before), or 2 months or more before (8 weeks or more before). Also, although not particularly limited, depending on the case, it is also possible to continue ingestion at intervals after starting the administration until an event for which an infection prevention effect is expected. In the present invention, as a preferable administration timing before an event for which an infection prevention effect is expected, for example, it can be 2 times or more including 1 day before and 3 days before, 2 times or more including 4 days before and 6 days before, 2 times or more including 7 days before and 9 days before, 2 times or more including 10 days before and 12 days before, or 2 times or more including 14 days before and 16 days before.

[0092] The active ingredient of the present invention may also be administered after an event or a time period when an infection prevention effect is expected. Examples of the administration timing after an event for which an infection prevention effect is expected include, for example, 1 day or more after, 3 days or more after, 1 week or more after, 2 weeks or more after. Also, although not particularly limited, when administering after an event for which an infection prevention effect is expected, depending on the case, it is also possible to continue administration at intervals. In the present invention, particularly preferably, the administration of the active ingredient of the present invention is started before an event for which an infection prevention effect is expected, and it is also possible to administer until after the event.

[0093] The administration of the active ingredient of the present invention may be combined with known methods for preventing or suppressing infection. Examples of known methods for preventing or suppressing infection include, for example, the administration of immunostimulants (e.g., polysaccharides such as fucoidan and β-glucan, lactoferrin, propolis, and bifidobacteria), the administration of antiviral agents (e.g., M2 ion-channel inhibitors (amantadine or rimantadine), neuraminidase inhibitors (oseltamivir or zanamivir) and their salts, etc.), the administration of neutralizing antibody drugs (tixagevimab and cilgavimab), and the administration of vaccines (e.g., live vaccines, inactivated vaccines, recombinant protein vaccines, mRNA (messenger RNA) vaccines, DNA vaccines, and viral vector vaccines).

[0094] According to another aspect of the present invention, there is provided a method for preventing or treating a viral infectious disease, a method for reducing the risk of contracting an infectious disease or reducing the risk of infection by a pathogen, or an immunostimulating method, which comprises the step of topically administering an effective amount of lactic acid bacteria or a composition comprising the same to a subject in need thereof. The method of the present invention can be carried out in accordance with the description of the pharmaceutical composition of the present invention.

[0095] According to another aspect of the present invention, there is also provided the use of lactic acid bacteria for the manufacture of a prophylactic or therapeutic agent for viral infectious diseases for topical administration, for the manufacture of an agent for reducing the risk of contracting an infectious disease or reducing the risk of infection by a pathogen for topical administration, or for the manufacture of an immunostimulant for topical administration. According to the present invention, there is also provided the use of lactic acid bacteria as a prophylactic or therapeutic agent for viral infectious diseases for topical administration, as an agent for reducing the risk of contracting an infectious disease or reducing the risk of infection by a pathogen for topical administration, or as an immunostimulant for topical administration. According to the present invention, there is also provided the use of lactic acid bacteria in a method for preventing or treating an infectious disease by topical administration, a method for reducing the risk of contracting an infectious disease or reducing the risk of infection by a pathogen by topical administration, or an immunostimulating method by topical administration. The use of the present invention can be carried out in accordance with the description of the pharmaceutical composition and the method of the present invention.

[0096] According to another aspect of the present invention, there is also provided a lactic acid bacterium for use in preventing or treating viral infections by topical administration, reducing the risk of contracting an infection by topical administration or reducing the risk of infection by a pathogen by topical administration, or for use in immunostimulation by topical administration. The lactic acid bacterium of the present invention can be implemented according to the descriptions regarding the pharmaceutical composition and the method of the present invention.

Examples

[0097] The present invention will be described more specifically based on the following examples and the like, but the present invention is not limited to these examples.

[0098] Example 1: Examination of the preventive effect against SARS-CoV-2 infection by nasal administration (1) In Example 1, the preventive effect of SARS-CoV-2 infection by nasal administration of lactic acid bacteria was examined.

[0099] (1) Method A Administration of lactic acid bacteria, infection with SARS-CoV-2 BALB / c mice (17 weeks old, female, body weight of about 20 g, Japan SLC Inc.) were divided into a total of 6 groups: a control group (non-lactic acid bacteria-administered group) and 5 lactic acid bacteria-administered groups, and heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) were administered under the conditions shown in Table 1 (the number of mice in each group was 2 or 3). Mice in each group were infected with a mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. (2022) 8(1). eabh3827) established at the National Institute of Infectious Diseases under anesthesia on day 0 of infection at 0.5×LD 50 (30 μl). Also, body weight was measured before infection and 3 days after infection. Then, the mice were sacrificed 3 days after infection, and the lungs and bronchoalveolar lavage fluid (BALF) were collected.

[0100]

Table 1

[0101] Extraction and Preparation of RNA The collected lungs were immersed in TRIzol (Thermo Fisher Scientific) and dispersed in a gentleMACS M tube (Miltenyi Biotec). After centrifugation, the supernatant was collected, chloroform (Nacalai Tesque) was added, and after centrifugation, the upper aqueous layer was collected into a tube containing 2-propanol (Nacalai Tesque). After centrifugation, the supernatant was removed, the pellet was suspended in 75% ethanol and centrifuged, the supernatant was completely removed, and after drying, it was suspended in RNase-Free Water (GIBCO). In addition, RNA from the collected BALF was prepared using the QIAmp Viral RNA Mini Kit (QIAGEN).

[0102] Quantification of RNA The extracted RNA was quantified by real-time PCR. Real-time PCR was performed using the QuantiTect Probe RT-PCR Kit (QIAGEN) and gene-specific SARS-CoV-2 primers and probes, according to the general reaction composition, with a reaction at 50°C for 30 minutes, 95°C for 15 minutes, followed by 45 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The primers shown in Table 2 were used (Shirato K, et al., Jpn. J. Infect. Dis. 2020; 73(4): 304-307).

[0103] [Table 2]

[0104] Quantification of the Virus For BALF, the infectious SARS-CoV-2 virus was quantified. Specifically, a 10-fold serial dilution series from 10 to 10^6 was prepared with DMEM (GIBCO) supplemented with 10% (w / v) FCS. 100 μl of each BALF dilution was inoculated in quadruplicate into a 96-well plate (CORNING) seeded with Vero cells (African green monkey kidney cell line (JCRB1819 VeroE6 / TMPRSS2, JCRB cell bank)), and viral infection was evaluated by the presence or absence of cytopathic effect 4 days after inoculation. The viral titer was calculated as TCID50 / ml using the Reed-Muench formula.

[0105] (2) Results The results were as shown in Figs. 1 to 3. From the ratio of body weight on day 3 of infection (about 72 hours after infection) to the body weight before SARS-CoV-2 infection shown in Fig. 1, no weight loss was observed in any of the lactic acid bacteria-administered groups, whereas in the control group (non-lactic acid bacteria-administered group), a weight loss of about 90% of the pre-infection weight was observed. Since the inhibitory effect on weight loss was also observed in the Day(-2 / -7) group and the Day(-3 / -8) group, the inhibitory effect on infection by nasal administration of lactic acid bacteria was maintained for several days (at least 3 days). Also, in the control group, compared with the lactic acid bacteria-administered groups, the hair condition became worse, and a decrease in activity level and a decrease in the number of diet intakes were observed.

[0106] In Fig. 2, the amount of SARS-CoV-2 RNA (copy number) in the lung and BALF 3 days after SARS-CoV-2 infection (about 72 hours after infection) was significantly decreased in the Day(-1 / -3) group, the Day(-2 / -4) group, and the Day(-3 / -5) group compared with the control group, and it was also confirmed to be decreased in the Day(-1 / -3)(low) group, the Day(-2 / -7) group, and the Day(-3 / -8) group. Since a decrease in the amount of RNA was also observed in the Day(-2 / -7) group and the Day(-3 / -8) group, the inhibitory effect on infection by nasal administration of lactic acid bacteria was maintained for several days (at least 3 days).

[0107] In Figure 3, the viral load of infectious SARS-CoV-2 in BALF 3 days after SARS-CoV-2 infection (about 72 hours after infection) was significantly decreased in the Day(-1 / -3) group, Day(-2 / -4) group, and Day(-3 / -5) group compared with the control group, and it was also confirmed to be decreased in the Day(-1 / -3)(low) group, Day(-2 / -7) group, and Day(-3 / -8) group. Since a decrease in the viral load was also observed in the Day(-2 / -7) group and Day(-3 / -8) group, the infection inhibitory effect by the nasal administration of lactic acid bacteria was maintained for several days (at least 3 days).

[0108] These results indicated that administration of lactic acid bacteria not only suppressed infection but also had an effect of suppressing disease progression.

[0109] Example 2: Examination of the preventive effect against SARS-CoV-2 infection by nasal administration (2) In Example 2, the effects of nasal administration of lactic acid bacteria on IFN-α production in blood and BALF after SARS-CoV-2 infection were examined.

[0110] (1) Method BALB / c mice (17-week-old, female, body weight about 20 g, Japan SLC Inc.) were divided into a total of 6 groups: a control group (non-lactic acid bacteria-administered group) and 5 lactic acid bacteria-administered groups, and heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) were administered under the conditions shown in Table 3 (n number of each group was 2 or 3). Mice in each group were infected with the mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. 2022; 8(1): eabh3827) established at the National Institute of Infectious Diseases under anesthesia on day 0 of infection at 0.5×LD 50 (30 μl). Then, they were dissected 3 days after infection (about 72 hours later), and the interferon-α (IFN-α) concentration in blood and bronchoalveolar lavage fluid (BALF) was measured using an interferonα All Subtype ELISA Kit (PBL BIOMEDICAL LABORATORIES).

[0111]

Table 3

[0112] (2) Results The results were as shown in Fig. 4. In the control group, it was confirmed that the average value of the IFN-α concentration in the blood 3 days after SARS-CoV-2 infection (about 72 hours after infection) exceeded 100 pg / ml, and the average value of the IFN-α concentration in the BALF exceeded 300 pg / ml. In contrast, in the lactic acid bacteria administration group, it was confirmed that the average value of the IFN-α concentration in the blood 3 days after SARS-CoV-2 infection (about 72 hours after infection) was 50 pg / ml or less, and the average value of the IFN-α concentration in the BALF was 200 pg / ml or less.

[0113] Example 3: Examination of the preventive effect against SARS-CoV-2 infection by nasal administration (3) In Example 3, the suppression of weight loss after SARS-CoV-2 infection by intranasal administration of lactic acid bacteria was examined.

[0114] (1) Method BALB / c mice (16 weeks old, female, body weight about 20 g, Japan SLC Inc.) were divided into two groups: a control group (non-lactic acid bacteria administration group) and a lactic acid bacteria administration group, and heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) were administered under the conditions shown in Table 4 (n = 6 for each group). Mice in each group were infected with the mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. 2022; 8(1): eabh3827) established at the National Institute of Infectious Diseases under anesthesia on day 0 of infection at 0.5×LD 50 (30 μl). Then, the body weight was measured daily until 7 days after infection, and the ratio of the body weight to the body weight before infection was calculated.

[0115]

Table 4

[0116] (2) Results The results were as shown in Fig. 5. In the control group, significant weight loss was observed, with the peak occurring 3 to 4 days after infection. In contrast, in the lactic acid bacteria administration group, no obvious weight loss was observed after infection, and the weight loss was significantly suppressed compared to the control group.

[0117] Example 4: Examination of the preventive effect against SARS-CoV-2 infection by nasal administration (4) In Example 4, the persistence of the preventive effect of intranasal administration of lactic acid bacteria against SARS-CoV-2 infection was examined.

[0118] (1) Method A Administration of lactic acid bacteria, infection with SARS-CoV-2 BALB / c mice (15-week-old females, body weight approximately 20 g, purchased from Japan SLC, Inc.) were divided into a total of 6 groups: a control group (non-lactic acid bacteria administration group) and 5 lactic acid bacteria administration groups. Heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) were administered under the conditions shown in Table 5 (n = 4 or 5 for each group). On day 0 of infection, the mice in each group were anesthetized and infected with the mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. 2022; 8(1): eabh3827) established at the National Institute of Infectious Diseases at a dose of 5 × LD 50 (5 μl). Then, on day 2 after infection, the mice were dissected, and nasal-associated lymphoid tissues (NALT) were collected.

[0119]

Table 5

[0120] B Extraction and preparation of RNA RNA was extracted and prepared from the collected NALT in the same manner as described in Example 1(1)B.

[0121] C Quantification of RNA RNA quantification was performed in the same manner as described in Example 1(1)C.

[0122] (2) Results The results were as shown in Fig. 6. In the Day(-1 / -3) group and the Day(-4 / -6) group, the amounts of viral RNA and subgenomic RNA of SARS-CoV-2 were significantly lower compared to the control group. Also, in the Day(-7 / -9) group, the Day(-10 / -12) group, and the Day(-14 / -16) group, the amounts of viral RNA and subgenomic RNA tended to be about 10-fold lower compared to the control group.

[0123] Example 5: Examination of the preventive effect against influenza A virus infection by nasal administration In Example 5, the preventive effect of intranasal administration of lactic acid bacteria against influenza A virus (H1N1) infection was examined.

[0124] (1) Method a Administration of lactic acid bacteria, infection with influenza A virus BALB / c mice (12-week-old, female, body weight about 20 g, from Japan SLC, Inc.) were divided into two groups: a control group (non-lactic acid bacteria-administered group) and a lactic acid bacteria-administered group, and heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) were administered under the conditions shown in Tables 6 and 7 (the number of mice in each group was 5 or 7). The mice in each group of Tables 6 and 7 were infected with A / PR8 / 34 (PR8) or A / Narita / 1 / 2009 (Narita) of influenza A virus (obtained from the National Institute of Infectious Diseases, Ikeda K, Ainai A, et al., Vaccine. 2015; 33(45): 6066-9. or Adachi Y, et al., J Exp Med. 2015; 212(10): 1709-23.) under anesthesia on the day of infection 0 at 40×LD 50 (5 μl) or 5×LD 50 (5 μl), respectively. Then, they were dissected 2 days after infection, and nasal lavage fluid was collected.

[0125]

Table 6

[0126]

Table 7

[0127] Extraction and preparation of influenza virus RNA RNA was prepared from the collected nasal lavage fluid using the QIAmp ViralRNA Mini Kit (QIAGEN).

[0128] Quantification of influenza virus RNA The extracted RNA was quantified by real-time PCR. Real-time PCR was performed using the QuantiTect Probe RT-PCR Kit (QIAGEN) and gene-specific primers and probes for influenza A virus, and according to the general reaction composition, reacting at 50°C for 30 minutes, 95°C for 15 minutes, and then at 94°C for 15 seconds and 56°C for 75 seconds for 40 cycles. The primers shown in Table 8 were used (Nakauchi M, et al., Journal of Virological Methods. 2011; 7171(1): 156-162).

[0129]

Table 8

[0130] (2) Results The results were as shown in Figure 7. In the lactic acid bacteria administration group, the amount of influenza A virus RNA in the nasal lavage fluid was significantly lower in both PR8 infection and Narita infection compared to the control group.

[0131] Example 6: Examination of the immunostimulatory effect by nasal administration (1) In Example 6, the immunostimulatory effect by intranasal administration of lactic acid bacteria was examined.

[0132] (1) Method Administration of lactic acid bacteria BALB / c mice (16 weeks old, female, body weight of approximately 20 g, purchased from Japan SLC, Inc.) were divided into four groups as shown in Table 9, and a nasal administration test of lactic acid bacteria was conducted. The control group was administered PBS (GIBCO), and the administration groups were administered heat-killed cells of lactic acid bacteria (Lactococcus lactis subsp. lactis JCM5805 strain) under the conditions shown in Table 9 (n = 3 for each group). Note that the control group was administered only PBS corresponding to the administration dose of the administration group. Then, three days after the administration of lactic acid bacteria, the mice were euthanized by whole blood collection from the heart under anesthesia and dissected.

[0133]

Table 9

[0134] Extraction of RNA RNA extraction from each tissue specimen was performed according to the following procedures (i) to (vi). (i) Each tissue specimen was collected into an M tube (Miltenyi Biotec) containing 1 ml of TRIzol (Thermo Fisher Scientific) and stored frozen at -80°C. (ii) The frozen tissue specimen was thawed, the tissue was disrupted using GentleMACS (Miltenyi Biotec), allowed to stand at room temperature for 2 to 3 minutes, and then centrifuged at 3,000×g for 5 minutes at 4°C. The supernatant was collected into an Eppendorf tube and further centrifuged at 12,000×g for 10 minutes at 4°C. (iii) The supernatant was collected into an Eppendorf tube, 180 μl of chloroform (FUJIFILM Wako Pure Chemical Corporation) was added, allowed to stand at room temperature for 2 to 3 minutes, and then centrifuged at 12,000×g for 15 minutes at 4°C. (iv) The upper aqueous layer of the supernatant was collected into an Eppendorf tube containing 450 μl of 2-propanol (FUJIFILM Wako Pure Chemical Corporation), allowed to stand at room temperature for 10 minutes, and then centrifuged at 12,000×g for 10 minutes at 4°C. (v) The supernatant was removed, 1 ml of 75% ethanol (FUJIFILM Wako Pure Chemical Corporation) was added, vortexed, and then centrifuged at 7,500×g for 5 minutes at 4°C. (vi) The supernatant was removed, and the pellet was air-dried for 15 minutes. Subsequently, it was suspended in RNase Free Water (QIAGEN), heat-treated at 56 °C for 10 minutes, and then stored at -80 °C.

[0135] Quantification of viral RNA Quantification of the mRNA expression levels of Viperin, Isg15, and Mx1, which are IFN-inducible antiviral genes (ISGs), was performed on the extracted RNA. Specifically, it was carried out according to the following procedures (i) to (v). (i) After measuring the RNA concentration of each sample using a nanodrop, it was diluted with Nuclease Free Water of the iScript cDNA synthesis kit (BIORAD) to a concentration of 1000 ng / 15 μl. (ii) Master Mix (4 μl of 5× iScript reaction mix / sample, 1 μl of iScript reverse transcript / sample) was prepared and 5 μl was added to each RNA solution. (iii) Subsequently, cDNA synthesis was performed using a thermal cycler and stored at -20 °C. (iv) Each cDNA sample was diluted 5-fold with MilliQ water, and a standard solution prepared by mixing all samples was adjusted to concentrations of ×1, ×2, ×4, ×8, ×16, ×32, ×64, and ×128. (v) The reaction solution shown in Table 10 was applied to a 96-well plate, and real-time PCR was performed using a LightCycler 480 with a reaction of 45 cycles of a set of 10 seconds at 95 °C, 10 seconds at 60 °C, and 10 seconds at 72 °C. The primers shown in Table 11 were used.

[0136]

Table 10

[0137]

Table 11

[0138] (2) Results The results were as shown in Figures 8 to 11. In Figure 8, from the ratios of the body weights on Day (-2) (2 days before dissection), Day (-1) (1 day before dissection (the second intranasal administration day of lactic acid bacteria)), and Day (0) (the day of dissection) to the body weight on Day (-3) (3 days before dissection (the first intranasal administration day of lactic acid bacteria)), when comparing the Low volume (-) group and Low volume (+) on Day (0) (the day of dissection), no significant difference was observed. Also, when comparing the High Volume (-) group and High Volume (+) group on Day (0) (the day of dissection), no significant difference was observed. Moreover, in any of the groups, no effects on the hair condition and activity level were observed.

[0139] In Figure 9, the Low volume (+) group showed a significant increase in the mRNA expression levels of Viperin and Isg15 in the submandibular lymph nodes compared to the Low volume (-) group, and the mRNA expression level of Mx1 also tended to increase. In contrast, the High Volume (+) group showed no significant increase compared to the High Volume (-) group. Low volume (+) and High Volume (+) were respectively made to act on the nasal cavity and the lungs due to the difference in the administered volume of the lactic acid bacteria solution. This result suggested that the nasal cavity was preferable as the local administration site.

[0140] In Figure 10, the Low volume (+) group showed a significant increase in the expression levels of Viperin and Isg15 in the spleen compared to the Low volume (-) group, and the expression level of Mx1 also tended to increase. In contrast, the High Volume (+) group showed no significant increase compared to the High Volume (-) group.

[0141] In Figure 11, the Low volume (+) group showed a tendency for an increase in the expression level of Isg15 in the lungs compared to the Low volume (-) group.

[0142] Example 7: Examination of the immunostimulatory effect by nasal administration (2) In Example 7, using the spleen, submandibular lymph nodes, and nasal mucosa excised by dissection in Example 6, the effect of transnasal administration of lactic acid bacteria on pDCs was examined.

[0143] (1) Method The number of pDCs in each of the spleen, submandibular lymph nodes, and nasal mucosa excised by dissection in Example 6 was quantified by the following procedure. Each tissue was stained with the fluorescently labeled antibodies shown in Table 12, and after staining, the cells were washed once with FACS buffer, resuspended in FACS buffer, and subjected to FACS analysis. Then, data were acquired using FACS Cant II (BD Biosciences) and analyzed using FlowJosoftware (Tree Star). Here, cells expressing CD11c and Siglec-H (CD11c + SIglec-H + cells) or cells not expressing CD11b but expressing Siglec-H (CD11b - Siglec-H + cells) were defined as pDCs, and the ratio of pDCs to lymphocytes in each tissue was quantified from the ratio of pDCs to all lymphocytes included in the lymphocyte fraction of FACS analysis. Also, the expression levels of MHC class II (I-A / I-E) and CD86, which are cell surface activation markers of pDCs, were measured. In addition, from the ratio of cells expressing CD11b and Siglec-H (CD11b + Siglec-H + cells) included in the lymphocyte fraction of FACS analysis, the ratio of CD11b + Siglec-H + cells to lymphocytes in each tissue was quantified.

[0144]

Table 12

[0145] (2) Results The results were as shown in Figs. 12 to 17. In Fig. 12, in the Low volume administration group (Low volume(+) group), the ratio of pDCs to lymphocytes in the spleen tended to increase compared to the Low volume control group (Low volume(-) group). On the other hand, in Fig. 13, no significant difference was observed in the expression levels of MHC class II (I-A / I-E) (Fig. 13A) and CD86 (Fig. 13B) in the pDCs of the spleen.

[0146] In Fig. 14, in the Low volume administration group (Low volume(+) group), the ratio of pDCs to lymphocytes in the submandibular lymph nodes significantly decreased (p < 0.01) compared to the Low volume control group (Low volume(-) group). On the other hand, in Fig. 15, no significant difference was observed in the expression levels of MHC class II (I-A / I-E) (Fig. 15A) and CD86 (Fig. 15B) in the pDCs of the submandibular lymph nodes.

[0147] In Fig. 16, in the Low volume administration group (Low volume(+) group), the ratio of pDCs to lymphocytes in the nasal mucosa significantly increased (p < 0.05) compared to the Low volume control group (Low volume(-) group) (Fig. 16A (solid line enclosure) and Fig. 16B (left)). On the other hand, in Fig. 17A, no significant difference was observed in the expression levels of MHC class II (I-A / I-E) and CD86 in the pDCs of the nasal mucosa.

[0148] In Fig. 16, also, in the Low volume administration group (Low volume(+) group), CD11b + Siglec-H + The ratio of cells to lymphocytes in the nasal mucosa significantly increased (p < 0.01) compared to the Low volume control group (Low volume(-) group) (Fig. 16A (dotted line enclosure) and Fig. 16B (right)). On the other hand, in Fig. 17B, no significant difference was observed in the expression levels of MHC class II (I-A / I-E) and CD86 in the CD11b + Siglec-H + cells of the nasal mucosa.

[0149] Example 8: Examination of the immunostimulatory effect by nasal administration (3) In Example 8, using nasal mucosa cells collected from the nasal mucosa excised by dissection in Example 6, the immunostimulatory effect by administration of lactic acid bacteria was further examined.

[0150] (1) Method The nasal mucosa excised by dissection in Example 6 was temporarily stored in RPMI+ solution, and then the nasal mucosa was squeezed using forceps in HBSS buffer solution. After passing the solution through a 70 μm cell strainer, after centrifugation (1,500 rpm, 4°C, 5 minutes), the supernatant was removed and RPMI+ was added to suspend the cell pellet. Next, the cell count was performed, and it was adjusted to 6×10 5 cells / ml, and seeded in a 48-well plate at 500 μl / well. Then, for the medium, a non-added one, one with ODN1585 (Invivogen) added at 1 μM as CpG-A, and one with inactivated influenza virus H1N1 (HyTest) (H1N1) added at 5 μg / ml were added to each well, and cultured in a CO2 incubator for 24 hours. After the culture was completed, the cell solution was collected and centrifuged (5,000 rpm, 4°C, 2 minutes), and the culture supernatant was collected. The IFN-α concentration in the culture supernatant was measured using VeriKine Interferonα ELISA Kit, Mouse (PBL Assay Science), the IFN-β concentration was measured using VeriKine Interferonβ ELISA Kit (PBL Assay Science), and the IFN-λ concentration was measured using Mouse IL-28B / IFN-lambda3 DuoSet ELISA (R&D systems), respectively.

[0151] (2) Results The results were as shown in Figs. 18 and 19. In Fig. 18, when CpG-A was added (CpG-A 1 μM), the amount of IFN-α production in nasal mucosa cells collected from the Low volume administration group (Low volume(+)) was significantly increased (p<0.01) compared to the nasal mucosa cells collected from the Low volume control group (Low volume(-)). On the other hand, when CpG-A was not added (CpG-A 0 μM), the amount of IFN-α production was not detected. From these results, it was shown that administration of lactic acid bacteria induced IFN-α production after infection with pathogens such as bacteria and viruses, and exhibited a preventive effect against infectious diseases.

[0152] In Fig. 19, when inactivated H1N1 was added (H1N1 5 μg / ml), the amounts of production of IFN-α, IFN-β, and IFN-γ in nasal mucosa cells collected from the Low volume administration group (Low volume(+)) all tended to increase compared to the nasal mucosa cells collected from the Low volume control group (Low volume(-)). On the other hand, when inactivated H1N1 was not added (H1N1 0 μg / ml), the amount of IFN-α production was not detected. From these results, it was shown that administration of lactic acid bacteria induced production of IFN-α, IFN-β, and INF-λ after infection with influenza virus, and exhibited a preventive effect against infectious diseases.

[0153] Reference Example: Examination of the preventive effect against SARS-CoV-2 infection by oral administration In the reference example, the preventive effect of oral administration of lactic acid bacteria, a known administration method, against SARS-CoV-2 infection was examined.

[0154] (1) Method a Viral upper respiratory tract inoculation test group BALB / c mice (20 weeks old, female, body weight approximately 20 g) were divided into two groups, a control group (standard diet intake group) and a test diet intake group (n = 8 for each group), and were fed ad libitum with standard diet or test diet (containing 0.029% by mass of heat-killed cells of Lactococcus lactis subsp. lactis JCM5805 strain as lactic acid bacteria) from 14 days before SARS-CoV-2 infection until 2 days or 3 days after infection. Mice in each group were infected on day 0 of infection with 5 × LD 50 (1.8×10 4 TCID50) (5 μl) of the mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. 2022; 8(1): eabh3827) established at the National Institute of Infectious Diseases under anesthesia. Then, they were dissected 2 days or 3 days after infection, and nasal-associated lymphoid tissues (NALT) were collected.

[0155] Group for virus lower respiratory tract inoculation test BALB / c mice (21 weeks old, female, body weight approximately 20 g) were divided into two groups, a control group (standard diet intake group) and a test diet intake group (n = 8 for each group), and were fed ad libitum (orally administered) with standard diet or test diet (containing 0.05% by mass of heat-killed cells of Lactococcus lactis subsp. lactis JCM5805 strain as lactic acid bacteria) from 14 days before SARS-CoV-2 infection until 3 days after infection. Mice in each group were infected on day 0 of infection with 10 × LD 50 (3.5×10 4 TCID50) (30 μl) of the mouse-adapted SARS-CoV-2 QHmusX strain (Iwata-Yoshikawa N et al., Science Advances. 2022; 8(1): eabh3827) established at the National Institute of Infectious Diseases under anesthesia. Then, they were dissected 3 days after infection, and the lungs were collected.

[0156] Extraction and preparation of viral RNA In the same manner as described in Example 1(1), RNA was extracted and prepared from the collected NALT and lungs.

[0157] Quantification of RNA In the same manner as described in Example 1(1), the RNA was quantified.

[0158] (2) Results The results regarding the viral upper respiratory tract inoculation test group ((1) Method A) are as shown in Fig. 20, and the results regarding the viral lower respiratory tract inoculation test group ((1) Method I) are as shown in Fig. 21. In the viral upper respiratory tract inoculation test group, as a result of allowing free intake (oral administration) of the standard diet or the test diet from 14 days before SARS-CoV-2 infection to 2 days after or 3 days after infection (for a total of 16 days or 17 days), it was confirmed that each mouse ingested an average of 2.38 g per day (1.19 mg of lactic acid bacteria as the test diet). On the other hand, in the viral lower respiratory tract inoculation test group, as a result of allowing free intake (oral administration) of the standard diet or the test diet from 14 days before SARS-CoV-2 infection to 3 days after infection (for a total of 17 days), it was confirmed that each mouse ingested an average of 2.2 g per day (0.64 mg of lactic acid bacteria as the test diet).

[0159] In Fig. 20, it was confirmed that the amount of SARS-CoV-2 RNA in the NALT 2 days or 3 days after SARS-CoV-2 infection (about 48 hours or about 72 hours after infection) decreased in the lactic acid bacteria administration group compared to the control group.

[0160] In Fig. 21, it was confirmed that the amount of SARS-CoV-2 RNA in the NALT 3 days after SARS-CoV-2 infection (about 72 hours after infection) decreased in the lactic acid bacteria administration group compared to the control group.

Claims

1. A pharmaceutical composition for topical administration, comprising lactic acid bacteria as an active ingredient.

2. 2. The pharmaceutical composition of claim 1, wherein the topical administration is nasal, sublingual or inhalation administration.

3. The pharmaceutical composition according to claim 1 , wherein the local administration is to the upper and / or lower respiratory tract.

4. The pharmaceutical composition of claim 3 , wherein the upper respiratory tract is the nasal cavity.

5. 3. The pharmaceutical composition according to claim 1 or 2, for inducing interferon (IFN) production by plasmacytoid dendritic cells in the upper and / or lower respiratory tract.

6. The pharmaceutical composition according to claim 5, wherein the production of IFN is induced in a subject to which the pharmaceutical composition is administered after the subject is infected with an infectious disease.

7. 3. The pharmaceutical composition according to claim 1 or 2, for use in preventing or treating an infectious disease, or for reducing the risk of contracting an infectious disease in a subject to which the composition is administered.

8. The pharmaceutical composition according to claim 7 , wherein the infectious disease is a viral infection.

9. The pharmaceutical composition according to claim 8 , wherein the prevention of a viral infection is prevention of the onset of a viral infection or prevention of the aggravation of a viral infection.

10. The pharmaceutical composition of claim 8, wherein the viral infection is COVID-19 or an influenza virus infection.

11. The pharmaceutical composition according to claim 8, wherein the viral infection is caused by a virus that causes a respiratory tract infection.

12. The pharmaceutical composition according to claim 11, wherein the causative virus of the respiratory viral infection is SARS-CoV-2 or influenza virus.

13. The pharmaceutical composition according to claim 1 or 2, which is administered to a mammal.

14. The pharmaceutical composition of claim 13, wherein the mammal is a human.

15. The pharmaceutical composition according to claim 1 or 2, which is administered two or more times.

16. The pharmaceutical composition according to claim 1 or 2, wherein the administration interval is one day or more.

17. The pharmaceutical composition according to claim 8, wherein the effective period of prevention of viral infection is 56 days from the last day of topical administration.

18. 3. The pharmaceutical composition according to claim 1, wherein the daily dosage of the lactic acid bacteria (based on an adult body weight of 50 kg) is 1 mg to 1000 mg.

19. 3. The pharmaceutical composition according to claim 1 or 2, wherein the lactic acid bacterium is Lactococcus lactis subsp. lactis JCM5805 strain.

20. A composition for topical administration comprising Lactococcus lactis subsp. lactis as an active ingredient.

21. 21. The composition for topical administration according to claim 20, which is a pharmaceutical composition.

22. The pharmaceutical composition of claim 8 , which is administered locally to the site of viral infection.

23. An immunostimulatory composition for topical administration, comprising lactic acid bacteria as an active ingredient.

24. The immunostimulatory composition according to claim 22 or 23, for enhancing expression of IFN-inducible antiviral genes (ISGs) in submandibular lymph nodes and / or the spleen.

25. The immunostimulatory composition according to claim 24, wherein the ISG is one or more selected from the group consisting of Viperin, Isg15 and Mx1.

26. The immunostimulatory composition according to claim 22 or 23, for increasing the ratio of pDC to lymphocytes in the spleen and / or for increasing the ratio of pDC to lymphocytes in the nasal mucosa.

27. CD11b on lymphocytes in the nasal mucosa ＋ Siglec-H ＋ The immunostimulatory composition according to claim 22 or 23 for increasing the proportion of cells.

28. A method for preventing or treating an infectious disease, a method for reducing the risk of contracting an infectious disease, or a method for immunostimulating the immune system, comprising the step of locally administering an effective amount of lactic acid bacteria or a composition comprising the same to a subject in need thereof.

29. Use of lactic acid bacteria for the manufacture of an agent for preventing or treating infectious diseases for local administration, for the manufacture of an agent for reducing the risk of contracting infectious diseases for local administration, or for the manufacture of an immunostimulant for local administration, or as an agent for preventing or treating infectious diseases for local administration, as an agent for reducing the risk of contracting infectious diseases for local administration, or as an immunostimulant for local administration, or in a method for preventing or treating infectious diseases by local administration, a method for reducing the risk of contracting infectious diseases by local administration, or a method for stimulating the immune system by local administration.

30. A lactic acid bacterium for use in preventing or treating an infectious disease by local administration, for use in reducing the risk of contracting an infectious disease by local administration, or for use in immunostimulation by local administration.

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