JP7720054B2 - Composition for promoting interferon-λ production and method for producing the same - Google Patents

Description

NPMD NITE P-03535

Article 30, paragraph 2 of the Patent Act applies [Publication by email] Sent on: October 5, 2020 To: National Center for Global Health and Medicine, Hepatitis and Immunology Research Center

The present invention relates to a composition containing lactic acid bacteria as an active ingredient that promotes interferon-λ production, and a method for producing the same.

Immunity is the body's self-defense system that constantly monitors and repels cancer cells that develop within the body, as well as bacteria and viruses that invade from outside. Research into compositions and foods that boost immunity has been ongoing for a long time.

Interferon-λ (IFN-λ) is a physiologically active substance (cytokine) belonging to the type III interferon family. In humans, the IFN-λ family is composed of four proteins: IFN-λ1 (IL-29), IFN-λ2 (IL-28A), IFN-λ3 (IL-28B), and IFN-λ4. Its production has been confirmed in dendritic cells, hepatocytes, intestinal epithelial cells, and lung epithelial cells. IFN-λ activates natural immunity, including antiviral activity, and may exhibit antiviral activity, immunostimulatory activity, inhibitory activity against viral infections (e.g., hepatitis B virus, hepatitis C virus), antitumor growth activity, antitumor activity, and anticancer activity. Therefore, it is expected to be used in foods and beverages, therapeutic agents, preventative agents, ameliorative agents, and palliatives that possess these activities (Non-Patent Document 1).

In addition to directly administering IFN-λ into the body, increasing the expression and production levels of IFN-λ in the body -- that is, administering a substance that enhances IFN-λ production -- is expected to induce the physiological activity of IFN-λ in the body. Patent Document 1 reports that Lactococcus lactis JCM20101 and JCM5805 strains enhance IFN-λ production in plasmacytoid dendritic cells. Patent Document 2 reports that Tetragenococcus halophilus KK221 strain enhances IFN-λ production in BDCA3DC dendritic cells. Patent Document 3 reports that in intestinal epithelial cells (HT-29 cells) mock-infected with synthetic double-stranded RNA poly:IC, Bifidobacterium breve MCC1274 strain, M-16V strain, Bifidobacterium longum subsp. longum BB536 strain, and Bifidobacterium longum subsp. infantis M-63 strain enhance IFN-λ production.

International Publication No. 2012/091081 International Publication No. 2017/175774 JP 2019-167327 A

Lazear H.M. et al., Shared and distinct functions of type I and type III interferons. Immunity 50 (4): 907-923 (2019)

The lactic acid bacteria described in Patent Documents 1 and 2 have the effect of further enhancing the IFN-producing ability of dendritic cells, which already have a high IFN-producing ability. On the other hand, the lactic acid bacteria described in Patent Document 3 enhance the IFN-λ producing ability of intestinal epithelial cells, but this effect was confirmed in cells that had already been immunologically activated. Therefore, there is a need for lactic acid bacteria that significantly promote IFN-λ production in cells other than dendritic cells, without the need for other immunostimulatory treatments.

The present invention aims to provide a composition containing lactic acid bacteria that exhibits a high IFN-λ production-promoting effect even in cells other than dendritic cells. The present invention also aims to provide a method for producing a composition containing lactic acid bacteria that exhibits a high IFN-λ production-promoting effect even in cells other than dendritic cells.

To achieve the above-mentioned objective, the inventors conducted extensive research and discovered that Enterococcus bacteria increase the amount of IFN-λ produced by colonic epithelial cells without the need for other immunostimulatory treatments, leading to the completion of the present invention.

The present invention encompasses the following.
(1) A composition for enhancing interferon-λ production in a subject, comprising a culture or a processed product of Enterococcus as an active ingredient.
(2) The composition according to (1), wherein the Enterococcus bacterium is Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535).
(3) The composition according to (1) or (2), wherein the culture or treated product of the Enterococcus bacterium has been sterilized.
(4) The composition according to any one of (1) to (3), which increases the expression level of a target antiviral protein gene.
(5) The composition described in (4), wherein the antiviral protein gene is at least one gene selected from the group consisting of the Mx dynamin-like GTPase 1 (Mx1) gene, the 2'-5'-oligoadenylate synthetase 1 (OAS1) gene, and the interferon-stimulated gene 15 (ISG15) gene.
(6) A pharmaceutical composition comprising the composition according to any one of (1) to (5).
(7) A food composition comprising the composition according to any one of (1) to (5).
(8) A composition for external use, comprising the composition according to any one of (1) to (5).
(9) A method for producing a composition for enhancing interferon-λ production in a subject, the method comprising the steps of culturing Enterococcus bacteria and obtaining a culture or treated product of the Enterococcus bacteria.
(10) The method according to (9), wherein the Enterococcus bacterium is Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535).

The present invention provides a composition containing lactic acid bacteria that exhibits a high IFN-λ production-promoting effect even in cells other than dendritic cells. The present invention also provides a method for producing a composition containing lactic acid bacteria that exhibits a high IFN-λ production-promoting effect even in cells other than dendritic cells.

1 is a graph showing the relationship between the amount of Enterococcus cathelliflavus KB1733 strain culture added and the amount of IFN-λ1 produced by WiDr cells. Error bars in the graph indicate standard error. An "*" in the graph indicates that a significant difference was observed from the control (p<0.05) by Steel's test (n=6). 1 is a graph showing the relationship between the amount of Enterococcus cathelliflavus KB1733 strain culture added and the amount of IFN-λ3 produced by WiDr cells. Error bars in the graph indicate standard error. An "*" in the graph indicates that a significant difference was observed from the control (p<0.05) by Steel's test (n=6). 1 is a graph showing the relationship between the treatment time with a culture of Enterococcus cathelliflavus KB1733 strain and the expression level of the Mx1 gene in WiDr cells. Error bars in the graph indicate standard error. An * in the graph indicates that a significant difference was observed compared to time 0 (p<0.05) by Steel's test (n=6). 1 is a graph showing the relationship between the treatment time with a culture of Enterococcus cathelliflavus KB1733 strain and the OAS1 gene expression level in WiDr cells. Error bars in the figure indicate standard error. An * in the figure indicates a significant difference (p<0.05) from time 0 by Steel's test (n=6). 1 is a graph showing the relationship between the treatment time with a culture of Enterococcus cathelliflavus KB1733 strain and the expression level of the ISG15 gene in WiDr cells. Error bars in the figure indicate standard error. An * in the figure indicates a significant difference (p<0.05) from time 0 by Steel's test (n=6).

1. Composition The composition of the present invention is a composition for enhancing interferon-λ (IFN-λ) production in a subject, comprising a culture or processed product of Enterococcus as an active ingredient. The composition of the present invention is characterized in that, when administered to a subject by oral ingestion or other means, it can promote IFN-λ production in the subject.

The inventors have discovered that Enterococcus bacteria, a type of lactic acid bacteria, promote IFN-λ production in WiDr cells, a colon cancer cell line. WiDr cells are derived from the colon epithelium and are not cells that naturally produce high amounts of IFN like dendritic cells, but rather are closer to normal somatic cells. Colon epithelial cells often come into contact with pharmaceutical and food ingredients when orally administered, and promotion of IFN-λ production in these cells is believed to contribute significantly to improving the body's immune function.

The inventors further found that Enterococcus bacteria promote the expression of various IFN-inducible antiviral protein genes in WiDr cells. This indicates that Enterococcus bacteria not only promote IFN-λ production in colon epithelial cells, but also actually induce immune defense functions in these cells.

As used herein, the term "subject" refers to a human or non-human animal, or cells derived from a human or non-human animal. Non-human animals are not particularly limited, but include, for example, mammals, birds, reptiles, amphibians, and fish, and preferably chickens, cows, horses, pigs, dogs, and cats. The composition of the present invention is characterized in that, when administered to a subject, it can increase the amount of IFN-λ produced by the subject.

The IFN-λ whose production is increased by the composition of the present invention may be any of IFN-λ1, IFN-λ2, IFN-λ3, and IFN-λ4, but IFN-λ1 and/or IFN-λ3 are particularly preferred.

As used herein, "IFN-λ1" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 1. The amino acid sequence of SEQ ID NO: 1 is the sequence of Accession No. AAN86125.

As used herein, "IFN-λ2" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 2. The amino acid sequence of SEQ ID NO: 2 is the sequence of Accession No. AAN86126.

As used herein, "IFN-λ3" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 3. The amino acid sequence of SEQ ID NO: 3 is the sequence of Accession No. AAN86127.

As used herein, "IFN-λ4" refers to a protein having an amino acid sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the amino acid sequence of SEQ ID NO: 4. The amino acid sequence of SEQ ID NO: 4 is the sequence of Accession No. NP_001263183.

As used herein, "sequence identity" refers to the percentage of identical amino acids or bases in the entire overlapping amino acid sequence (including the amino acid that serves as the translation start point) or base sequence (including the start codon) in the optimal alignment when two amino acid sequences or base sequences are aligned with or without gaps, and is calculated using formula (1). Sequence identity can be easily determined using BLAST (Basic Local Alignment Search Tool), an algorithm commonly used in this field. For example, BLAST is available to anyone from websites such as NCBI (National Center for Biotechnology Information) and KEGG (Kyoto Encyclopedia of Genes and Genomes), and sequence identity can be easily determined using default parameters.
Sequence identity (%) = number of matches (gap ignoring) / length of shorter sequence (length excluding gaps) × 100 Formula (1)

The composition of the present invention comprises a culture or processed product of Enterococcus bacteria. As used herein, a "culture" of bacteria refers to live cells, dead cells, disrupted live or dead cells, freeze-dried live or dead cells, disrupted freeze-dried products, culture fluid, culture fluid extract, etc. The culture may contain parts of the cells or processed products of the cells, and may also contain DNA, RNA, etc. extracted from the cells. A "processed product" of bacteria refers to cells that have been subjected to, for example, enzyme treatment, heat treatment, extraction treatment, salting-out treatment, ethanol precipitation treatment, drying treatment, etc.

As used herein, "Enterococcus bacteria" refers to lactic acid bacteria belonging to the phylum Firmicutes, also known as Enterococcus bacteria. They are Gram-positive cocci that primarily exist in the intestinal tract of animals as diplococci or short streptococci.

The Enterococcus bacterium contained in the composition of the present invention is preferably Enterococcus casselliflavus. Enterococcus casselliflavus strain KB1733 is particularly preferred. Enterococcus casselliflavus strain KB1733 was received for international deposit under the Budapest Treaty at the Patent Microorganisms Depositary of the National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture) on September 10, 2021, under the accession number NITE AP-03535. Enterococcus casselliflavus strain KB1733 was isolated from pickles, a traditional fermented food, and has been used as a food. It can be said to be a highly safe bacterium, especially when taken orally.

In the composition of the present invention, the culture or processed product of Enterococcus bacteria may contain viable bacteria, but is preferably sterilized, i.e., the bacteria are contained in the composition in a state in which they are unable to grow or multiply. It has been confirmed that the culture or processed product of Enterococcus bacteria, particularly the Enterococcus cathelliflavus KB1733 strain, which is the active ingredient in the composition of the present invention, is effective even without containing viable bacteria. Sterilizing the composition of the present invention has the advantage that, compared to when viable bacteria are contained, it is easier to manage the temperature, time, etc. during transportation and storage when distributing it on the market as a pharmaceutical, food, etc.

Interferons (IFNs) are classified into three types: type I, type II, and type III, and each type induces a specific immune response. IFN-mediated signaling promotes the upregulation of MHC class I and II molecules, activating numerous downstream signaling cascades and resulting in antiviral mechanisms, particularly the production of various antiviral proteins. More than 120 types of IFN-inducible antiviral proteins are known, including Mx dynamin-like GTPase 1 (Mx1), 2'-5'-oligoadenylate synthetase 1 (OAS1), interferon-stimulated gene 15 (ISG15), viperin, protein kinase R (PKR), and interferon regulatory factor 7 (IRF7).

As used herein, "Mx dynamin-like GTPase 1 (Mx1) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 5. Mx1 is known as a protein that exhibits intracellular antiviral activity against a wide range of viruses, including influenza, parainfluenza, measles, coxsackievirus, and hepatitis B. The nucleotide sequence set forth in SEQ ID NO: 5 is the sequence of Accession No. NG_027788.

As used herein, "2'-5'-oligoadenylate synthetase 1 (OAS1) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 6. OAS1 binds to and activates inactive RNase L, degrading mRNA and rRNA and suppressing the growth of viruses and bacteria. The nucleotide sequence of SEQ ID NO: 6 is the sequence of Accession No. NG_011530.

As used herein, "interferon-stimulated gene 15 (ISG15) gene" refers to a gene having a nucleotide sequence that shares 60% or more, 65% or more, 70% or more, 80% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100% sequence identity with the nucleotide sequence set forth in SEQ ID NO: 7. The nucleotide sequence of SEQ ID NO: 7 is the sequence of Accession No. NG_033033.

The composition of the present invention preferably has the effect of increasing the expression level of an antiviral protein gene in a subject. The antiviral protein gene is not particularly limited as long as it is a gene for a protein derived from the subject that has antiviral activity, but it is preferably at least one gene selected from the group consisting of the Mx1 gene, the OAS1 gene, and the ISG15 gene, which are particularly useful indicators of IFN activity.

The form of the composition of the present invention is not particularly limited and may be any of an aqueous solution, dry powder, freeze-dried product, frozen product, etc., but the form can be appropriately selected depending on the intended use of the composition, as described below.

The composition of the present invention is a composition for administration to a subject. The administration form is not particularly limited as long as it allows the active ingredient to be taken up by the subject, and may be oral or parenteral administration, although the administration form may be selected appropriately depending on the intended use of the composition, as described below. The composition of the present invention is highly safe when administered to a subject by oral ingestion, etc. Regardless of the administration form, it has the effect of promoting IFN-λ production in the subject, and, in relation to IFN, may have antiviral, immunostimulatory, anti-infectious, anti-hepatitis B, anti-hepatitis C, anti-tumor growth, anti-tumor, and anti-cancer effects in the subject.

2. Uses of the Composition The uses of the composition of the present invention are not particularly limited, and may be incorporated into, for example, pharmaceuticals, quasi-drugs, cosmetics, food and beverages, feed, etc. The pharmaceuticals, quasi-drugs, and cosmetics referred to here are those defined in the "Act on Ensuring Quality, Efficacy, and Safety of Pharmaceuticals, Medical Devices, etc. (PMD Act)." Below, the compositions of the present invention will be described separately as "pharmaceutical compositions,""ediblecompositions," and "external compositions" according to their uses, but these are not completely separate and may overlap with each other.

2-1 Pharmaceutical Composition The pharmaceutical composition of the present invention is characterized by containing the composition of the present invention described in Section "1. Composition." As used herein, the term "pharmaceutical composition" refers to a composition for treating and/or preventing a disease in humans or non-human animals, and primarily includes pharmaceuticals under the Pharmaceutical and Medical Device Act and some quasi-drugs.

The pharmaceutical composition of the present invention contains a culture or processed product of Enterococcus bacteria as an active ingredient. In addition to the active ingredient, the pharmaceutical composition of the present invention may also contain a pharmaceutically acceptable carrier, if necessary. "Pharmaceutically acceptable carrier" here refers to additives commonly used in the pharmaceutical formulation field. Examples include excipients, binders, disintegrants, fillers, emulsifiers, flow additives, lubricants, etc.

Examples of excipients include sugars such as monosaccharides, disaccharides, cyclodextrins, and polysaccharides (more specifically, but not limited to, glucose, sucrose, lactose, raffinose, mannitol, sorbitol, inositol, dextrin, maltodextrin, starch, and cellulose), metal salts (e.g., sodium chloride, sodium or calcium phosphate, calcium sulfate, magnesium sulfate, calcium carbonate), citric acid, tartaric acid, glycine, low, medium, and high molecular weight polyethylene glycols (PEG), pluronics, kaolin, silicic acid, or combinations thereof.

Examples of binders include starch paste made from corn, wheat, rice, or potato starch, simple syrup, glucose solution, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, shellac, and/or polyvinylpyrrolidone.

Disintegrants include the above-mentioned starches, lactose, carboxymethyl starch, cross-linked polyvinylpyrrolidone, agar, laminaran powder, sodium bicarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, stearic acid monoglyceride, or salts thereof.

Examples of fillers include the aforementioned sugars and/or calcium phosphate (e.g., tricalcium phosphate or calcium hydrogen phosphate).

Examples of emulsifiers include sorbitan fatty acid esters, glycerin fatty acid esters, sucrose fatty acid esters, and propylene glycol fatty acid esters.

Examples of flow regulators and lubricants include silicates, talc, stearates, or polyethylene glycol.

Such carriers are primarily used to facilitate the formation of the dosage form and to maintain the dosage form and pharmacological effect, and may be used appropriately as needed. In addition to the above-mentioned additives, flavoring agents, solubilizers, suspending agents, diluents, surfactants, stabilizers, absorption enhancers, bulking agents, wetting agents, humectants, adsorbents, disintegration inhibitors, coating agents, colorants, preservatives, antioxidants, fragrances, flavorings, sweeteners, buffers, etc. may also be included as needed.

The pharmaceutical composition of the present invention may also contain other drugs to the extent that the effect of promoting IFN-λ production is not lost. For example, it may contain a predetermined amount of another antibiotic.

The dosage form of the pharmaceutical composition of the present invention is not particularly limited, as long as it does not inactivate the active ingredient, a culture or processed product of Enterococcus bacteria, or other additional active ingredients. For example, it may be liquid, solid, or semisolid. Specific dosage forms include, for oral administration, tablets such as sugar-coated tablets, buccal tablets, coated tablets, and chewable tablets, lozenges, pills, powders, capsules including soft capsules, granules, suspensions, emulsions, syrups including dry syrups, and liquids such as elixirs. For parenteral administration, injectables such as intravenous injection, subcutaneous injection, intraperitoneal injection, and intramuscular injection are available, as well as transdermal absorption tapes, aerosols, and suppositories for transdermal, nasal, pulmonary, enteral, buccal, and transmucosal administration. From the standpoint of convenience and versatility, oral administration forms are preferred.

The pharmaceutical composition of the present invention preferably contains a culture or processed product of Enterococcus bacteria in an amount that is effective in promoting IFN production and is highly unlikely to cause serious side effects. The dose of the culture or processed product of Enterococcus bacteria is not particularly limited, but can be adjusted to, for example, 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, particularly 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, calculated in terms of bacterial cell count.

The number of times the pharmaceutical composition of the present invention is administered is not particularly limited, as long as the IFN production-promoting effect is sufficient and no serious side effects occur. For example, once every three days to five times a day, and particularly once to three times a day, is preferred. The administration period of the pharmaceutical composition of this embodiment is not particularly limited, but can be, for example, 1 to 24 weeks, and particularly 8 to 12 weeks.

2-2 Edible Composition The edible composition of the present invention is characterized by containing the composition of the present invention described in Section "1. Composition." As used herein, "edible composition" refers to a composition that is to be orally ingested as nutrition by humans or non-human animals. An edible composition suitable for human consumption is also referred to as a food or drink, and an edible composition suitable for non-human animal consumption is also referred to as a feed.

The form of the edible composition (food, drink, feed) of the present invention is not particularly limited, and examples include processed foods, health foods (nutritional supplements, functional foods, foods for the sick, foods for specified health uses, foods with functional claims, etc.), supplements, foods for the sick (hospital food, sick food, nursing care food, etc.), confectionery, oils and fats, dairy products, retort foods, microwave foods, frozen foods, seasonings, health supplements, beverages, energy drinks, etc.

The shape and properties of the edible composition (food, drink, feed) of the present invention are not particularly limited, and examples include solid, semi-solid, gel, liquid, and powder. In particular, when used as a health food or supplement, it is preferable to make it in the form of granules, capsules, tablets, chewable tablets, powdered drinks, energy drinks, smoothies, jellies, gummies, candies, gum, etc., to allow for continuous and convenient intake.

The amount of cultured or processed Enterococcus bacteria contained in the edible composition of the present invention is not particularly limited as long as it is an amount that is unlikely to cause health damage, but can be adjusted, for example, so that the amount is 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, particularly 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, converted into bacterial cell count.

2-3 Composition for External Use The composition for external use of the present invention is characterized by containing the composition of the present invention described in Section "1. Composition." As used herein, "composition for external use" refers to a composition used to treat and/or cure diseases of the body surface, such as the skin, hair, or nails of humans or non-human animals, or to improve and/or maintain the health or beauty of the body surface, and is intended to be applied to the body surface by sticking, rubbing, spraying, or a similar method. This primarily includes some pharmaceuticals and quasi-drugs under the Pharmaceutical and Medical Device Act, as well as cosmetics.

The form of the topical composition of the present invention is not particularly limited as long as it can be applied to the body surface, and may be in any form, such as a transdermal absorption tape, aerosol, patch, lotion, gel, cream, spray, or pack.

The topical composition of the present invention may contain other components as needed in addition to a culture or processed product of Enterococcus bacteria. Examples of other components include excipients, binders, disintegrants, fillers, emulsifiers, flow regulators, lubricants, etc. Details of each component are as described in Section "2-1 Pharmaceutical Compositions."

The amount of cultured or processed Enterococcus bacteria contained in the topical composition of the present invention is not particularly limited as long as it is an amount that is unlikely to cause health damage, but can be adjusted, for example, to 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, particularly 1 x ¹⁰ to 1 x ¹⁰ cells/individual/day, converted into bacterial cell count.

3. Method for Producing a Composition The production method of the present invention is a method for producing a composition for enhancing IFN-λ production in a subject, and is characterized by comprising the steps of culturing bacteria of the genus Enterococcus, and obtaining a culture or a treated product of the bacteria of the genus Enterococcus.

The production method of the present invention is not particularly limited in terms of the culture conditions for Enterococcus bacteria, collection method of the culture or processed product as the active ingredient, or processing method, as long as it produces a culture or processed product of Enterococcus bacteria that exhibits the effect of enhancing IFN-λ production and antiviral protein gene expression.

In the production method of the present invention, the Enterococcus bacterium is preferably Enterococcus casseliflavus. Enterococcus casseliflavus strain KB1733 is particularly preferred.

The production method of the present invention includes a step of culturing Enterococcus bacteria. One example of the culturing step is to inoculate Enterococcus bacteria into an MRS medium and culture them for 20 to 24 hours at 30 to 37°C, which is the optimum temperature for the bacteria, to obtain a culture solution.

3-2 Processing Step The production method of the present invention includes a step of obtaining a culture or a treated product of Enterococcus bacteria. More specifically, it includes a processing step of processing the culture solution obtained in the culturing step to obtain a culture or a treated product that can be used as a composition.

As an example of the processing step, the following method can be used. Bacteria are collected from the culture medium obtained in the culturing step and washed with sterilized water or physiological saline. The obtained bacteria are then dried, either after heat sterilization or without treatment, to obtain a dried sterilized powder or a dried powder as the active ingredient. Note that the active ingredient is not limited to a powder, and may also be in the form of an aqueous solution or frozen product.

The drying method used in the processing step is not particularly limited, but methods such as natural drying, air drying, and freeze drying can be used. The sterilization method is also not particularly limited, but methods such as heat sterilization, pressure sterilization, and osmotic sterilization can be used.

The composition obtained by the manufacturing method of the present invention can promote IFN-λ production in a subject by administering it to the subject via oral ingestion or other means. Furthermore, in relation to IFN, the composition may have antiviral, immunostimulatory, anti-infectious, anti-hepatitis B, anti-hepatitis C, anti-tumor growth, anti-tumor, and anti-cancer effects in the subject.

The present invention will be explained in more detail below using examples, but the technical scope of the present invention is not limited to the following examples.

Example 1. Evaluation test of promotion of IFN-λ production in WiDr cells by Enterococcus lactic acid bacteria
[1-1] Preparation of Test Samples A cryopreserved bacterial solution of Enterococcus bacteria (Enterococcus casselliflavus KB1733 strain) was added to MRS liquid medium (Thermo Fisher Scientific) and then cultured at 30°C for 24 hours.

After 24 hours of incubation, the culture medium was added back to MRS liquid medium and further incubated at 30°C for 24 hours. It was then inoculated into 40 mL of MRS liquid medium in a 50 mL centrifuge tube and cultured statically at 30°C for 20-24 hours. The mixture was then centrifuged at 2,590 x g and 4°C for 10 minutes, and the supernatant was removed. Physiological saline was added to the bacterial cells, and the cells were suspended by vortexing. The mixture was then centrifuged under the same conditions, and the supernatant was removed. 3 mL of ultrapure water was added, and the bacterial cells were suspended by vortexing. The entire suspension was transferred to a 15 mL centrifuge tube, sterilized by heating at 100°C for 30 minutes, cooled under running water, and frozen overnight in a -80°C deep freezer. The lid of the 15 mL centrifuge tube containing the frozen suspension was removed, and the opening of the tube was covered with a Bemcot filter sterilized at 121 °C for 15 minutes. The tube was then dried for 6 days using a vacuum freeze dryer (AGC TECHNO GLASS, FREEZE-DRYER, SRG-40M). After drying, the Bemcot filter was removed from the 15 mL centrifuge tube containing the sterilized cells. The dried sample was finely pulverized using a microspatula, and 10 mg of bacterial powder was transferred to a 1.5 mL tube. 1 mL of DMEM (high glucose) (Thermo Fisher Scientific) medium supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL penicillin-streptomycin (hereinafter also referred to as "complete medium") was added and suspended by vortexing to obtain a sterilized cell suspension at a concentration of 10 mg/mL. The fungicide suspension was serially diluted with complete medium to prepare fungicide suspensions with concentrations of 1, 3, 10, and 30 μg/mL, which were used as test samples.

[1-2] Cell Culture Test WiDr cells (JCRB Cell Bank, National Institutes of Biomedical Innovation, Health and Nutrition, No. JCRB0224) were maintained in a 100 mm diameter dish (AGC TECHNO GLASS) at 37°C and 5% _CO2 . The medium was changed every 2-3 days. Subculture was performed twice a week. After removing the medium and washing the cells with 10 mL of PBS, 2 mL of 0.25% (w/v) trypsin-1 mM EDTA solution was added and incubated at 37°C and 5% _CO2 for 3 minutes to detach the cells from the bottom of the dish. 8 mL of complete medium was added to stop the reaction, and the detached cells were collected. After counting the cell number using trypan blue staining, complete medium was prepared so that the viable cell count was 1.1 x ¹⁰⁶ cells/dish, and the cells were passaged in a 100 mm dish. After confirming that the passaged WiDr cells had reached 80-90% confluence, WiDr cells were seeded at 1 x ¹⁰ cells/well in a 6-well plate (AGC TECHNO GLASS) and cultured at 37°C in a 5% _CO2 environment. After 48 hours of culture, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added and the cells were washed again. After removing the PBS, 1,000 μL of each concentration of the sterilized cell suspension was added to each well and incubated for 48 hours. As a control, complete medium without the sterilized cell suspension was added and incubated in the same manner. After incubation, the cell supernatant was collected in a 1.5 mL tube and centrifuged (1,500 x g, 4°C, 5 minutes). After centrifugation, the supernatant was collected in a 1.5 mL tube and stored at -80°C. The IFN-λ1 and IFN-λ3 concentrations in the cell supernatant were measured. IFN-λ1 concentration was measured using an IL-29 Human ELISA Kit (Thermo Fisher Scientific) according to the protocol provided with the kit. IFN-λ3 concentration was measured by chemiluminescence enzyme immunoassay (CLEIA) according to the method described in M. Sugiyama, et al., Hepatology Research, 42(11) pp. 1089-1099 (2012).

[1-3] Results The concentrations of IFN-λ1 and IFN-λ3 in the WiDr cell culture medium when each of the sterile cell suspensions was added are shown in Figures 1 and 2, respectively. It was confirmed that the addition of sterile cell suspensions significantly increased the production of IFN-λ1 and IFN-λ3 by WiDr cells.

Example 2: Evaluation test of the amount of antiviral protein gene expression increased by Enterococcus lactic acid bacteria
[2-1] Preparation of test sample A suspension of the fungicide at a concentration of 10 μg/mL was prepared under the same conditions as in Example 1 and used as a test sample.

[2-2] Cell Culture Test WiDr cells were maintained in a 100 mm diameter dish (AGC TECHNO GLASS) at 37°C and 5% _CO2 . The medium was changed every 2-3 days, and subculture was performed twice a week. After removing the medium and washing the cells with 10 mL of PBS, 2 mL of 0.25% (w/v) trypsin-1 mM EDTA solution was added and reacted for 3 minutes at 37°C and 5% _CO2 to detach the cells from the bottom of the dish. 8 mL of complete medium was added to stop the reaction, and the detached cells were collected. After counting the cell number using trypan blue staining, the cells were passaged in a 100 mm dish in complete medium so that the viable cell count was 1.1 x ¹⁰⁶ cells/dish. After confirming that the passaged WiDr cells reached 80-90% confluence, WiDr cells were seeded onto 35 mm dishes (AGC TECHNO GLASS) at 1 x ¹⁰ cells/dish and cultured at 37°C in a 5% _CO2 environment. After 48 hours of culture, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added again, and the cells were washed again. After removing the PBS, 1,000 μL of a 10 μg/mL sterilized cell suspension was added to each well and incubated for 0, 4, 8, 24, and 48 hours. After each incubation, the medium was removed, and 0.5 mL of PBS was added to wash the cells. After removing the PBS, 0.5 mL of PBS was added again, and the cells were washed again. The 35 mm dishes from which the supernatant had been removed were stored at -80°C.

[2-3] Total RNA Extraction Cells from each well were detached using 900 μL of TRIzol™ (Thermo Fisher Scientific) and pipetted, and the cell extract was collected in a 1.5 mL ringlock tube. The sample was placed on ice, 180 μL of chloroform was added, and the mixture was stirred with a vortex mixer and then left to stand at room temperature for at least 3 minutes. Centrifuged at 12,000 × g, 4 ° C, and 15 minutes, 250 μL of the supernatant was transferred to a 1.5 mL tube, 200 μL of isopropanol was added, and the mixture was stirred with a vortex mixer. After standing at room temperature for 10 minutes, the mixture was centrifuged at 12,000 × g, 4 ° C, and 10 minutes. The supernatant was removed, and the tube was air-dried for approximately 10 minutes. 75% ethanol was added to the precipitate, and the mixture was centrifuged at 12,000 × g, 4 ° C, and 5 minutes, and the supernatant was removed. The same procedure was repeated twice. After drying the RNA at room temperature, it was dissolved in 30-50 μL of RNase-free distilled water to prepare an RNA extraction solution. 0.1 volumes of 3 M sodium acetate aqueous solution and 2.5 volumes of ethanol were added to the RNA extraction solution, mixed gently, and then allowed to stand overnight at -80°C. Centrifugation was performed at 12,000 × g, 4°C, and 10 minutes. The supernatant was removed, and the tube was air-dried for approximately 10 minutes. 75% ethanol was added to the precipitate, and centrifuged at 12,000 × g, 4°C, and 5 minutes. The supernatant was removed, and the RNA was dried at room temperature, then dissolved in 30 μL of RNase-free distilled water to prepare an RNA solution.

[2-4] cDNA synthesis. Reverse transcription from RNA to cDNA was performed using the Prime Script RT reagent Kit (Takara Bio) according to the manufacturer's instructions. Specifically, the enzyme mix, extracted RNA solution, and water from the kit were placed in a microtube, and the reverse transcription reaction was carried out by heating at 37°C for 15 minutes and then at 85°C for 5 seconds using a thermal cycler. The prepared cDNA solution was stored at -80°C.

[2-5] Quantitative PCR
Using SYBR Premix ExTaq II (Takara Bio), real-time PCR was performed on a 7500 Fast Real-time PCR system (Applied Biosystems) to measure the gene abundance (equivalent to mRNA abundance) of Mx1, OAS1, ISG15, and GAPDH in each cDNA solution. SYBR Premix Ex Taq II, PCR primers (forward primer (F) and reverse primer (R)), each cDNA sample, and RNase-free distilled water were added to a 96-well PCR plate, which was then placed in a PCR machine and subjected to PCR. The PCR temperature conditions were an initial denaturation (95°C for 30 seconds), followed by 40 cycles of temperature changes from 95°C for 5 seconds to 60°C for 30 seconds. The sequences of the primers used are shown in Table 1 as SEQ ID NOs: 8 to 15. The expression level of each gene was corrected with the expression level of glyceraldehyde 3-phosphate dehydrogenase (GAPDH), an internal standard gene, and then analyzed as a relative value, with the gene expression level at 0 hours of reaction under the same conditions set at 1.

[2-6] Results The gene expression levels of Mx1, OAS1, and ISG15 in WiDr cells upon addition of the killer cell suspension are shown in Figures 3, 4, and 5, respectively. It was confirmed that addition of the killer cell suspension promoted the expression of various IFN-inducible antiviral protein genes.

Claims (9)

A composition for enhancing interferon-λ production in a subject, containing a killed form of Enterococcus cathelliflavus as an active ingredient. The composition described in claim 1, wherein the Enterococcus casselliflavus is Enterococcus casselliflavus strain KB1733 (Accession No. NITE AP-03535). The composition described in claim 1 or 2, which increases the expression level of an antiviral protein gene in a subject. The composition of claim 3, wherein the antiviral protein gene is at least one gene selected from the group consisting of the Mx dynamin-like GTPase 1 (Mx1) gene, the 2'-5'-oligoadenylate synthetase 1 (OAS1) gene, and the interferon-stimulated gene 15 (ISG15) gene. A pharmaceutical composition for enhancing interferon-λ production in a subject, comprising the composition of any one of claims 1 to 4. An edible composition for enhancing interferon-λ production in a subject, comprising the composition of any one of claims 1 to 4. A topical composition for enhancing interferon-λ production in a subject, comprising the composition of any one of claims 1 to 4. A method for producing a composition for enhancing interferon-λ production in a subject, comprising the steps of culturing Enterococcus cathelliflavus and obtaining a killed form of Enterococcus cathelliflavus. The production method described in claim 8, wherein the Enterococcus casselliflavus is the Enterococcus casselliflavus KB1733 strain (accession number NITE AP-03535).