WO2020071463A1

WO2020071463A1 - Method for inducing production of iga targeting ilc2

Info

Publication number: WO2020071463A1
Application number: PCT/JP2019/039036
Authority: WO
Inventors: 尚子佐藤; 大野　博司
Original assignee: 国立研究開発法人理化学研究所
Priority date: 2018-10-04
Filing date: 2019-10-03
Publication date: 2020-04-09
Also published as: JP7444459B2; JPWO2020071463A1

Abstract

Provided is an agent for inducing ILC2 in the stomach, said agent comprising at least one kind of bacterium having the following characteristics: (1) sensitivity to vancomycin; and (2) tolerance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin and metronidazole.

Description

Method for inducing IgA production targeting ILC2

The present invention relates to an IgA production inducer targeting ILC2 present in the stomach.

The stomach is maintained strongly acidic, and until now it was thought that its role as an organ was only to digest and kill food. Helicobacter pylori (also referred to as "H. pylori", "H. pylori", etc.) has begun to focus on the relationship between the stomach and H. pylori, starting with reports of engraftment in the stomach. Most of the research is about the "stomach as tissue."

研究 In fact, research on stomach and bacteria reported in various scientific journals is mainly concerned with elucidation of the mechanism of gastric cancer onset by H. pylori infection and the elucidation of the infection mechanism from the structure of H. pylori itself. As far as the immune response is concerned, there are a few groups that are studying T cell responses and cancer, but there are few reports of the relationship between commensal bacteria present in the stomach and immunocompetent cells so far.

近年 In recent years, natural lymphocytes (Innate Lymphoid cells) (ILC) have been found to play an important role in maintaining homeostasis in various tissues in the body. The ILCs are classified into three subsets, ILC1, ILC2, and ILC3, depending on the properties of cells and differences in expression of transcription factors (Non-Patent Documents 2 to 6). These ILC studies have elucidated, for the first time, the pathogenesis of diseases that could not be explained by acquired immunity alone, and are now a group of cells that have attracted attention worldwide (Non-Patent Document 1).

However, our studies also reveal that the stomach also has an important immunological role (Non-Patent Document 7), and that this response is mainly due to bacterial-induced ILC2. Have been. The stomach is not just an organ for decomposing and disinfecting food, but it is important to screen flora before it reaches the intestinal tract by chemically and immunologically controlling the risk of infection that rises with food intake It is suggested that it is a healthy organ. However, at present, it is not clear which bacteria induces ILC2.

An object of the present invention is to elucidate the role of immunocompetent cells (especially ILC2) present in the stomach and its relation to the symbiotic bacterial flora of the stomach, thereby leading to the development of a novel anti-infective therapeutic agent and the like. I do.

The present inventors have conducted intensive studies on the above problems and found that (1) the presence of commensal bacteria in the stomach can affect the abundance of ILC2 in the stomach; (3) Bacteria that cause the induction of ILC2 in the stomach are sensitive to vancomycin and are affected by ampicillin, colistin, neomycin, and metronidazole. (4) that the bacterium that mainly induces the induction of ILC2 in the stomach is a bacterium belonging to the family S24-7; (5) that the bacterium that belongs to the family of the S24-7 family that mainly produces the induction of ILC2 in the stomach is And a bacterium having a nucleotide sequence represented by SEQ ID NO: 28 (eg, Muribaculum intestinale) and the like. This has led to the completion of the present invention by further research Te. That is, the present invention is as follows.

[A1] An ILC2 inducer in the stomach comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[A2] The inducer according to [A1], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[A3] The inducer according to [A1] or [A2], wherein the bacterium is a bacterium belonging to the family S24-7.
[A4] The inducing agent according to any one of [A1] to [A3], wherein the bacterium contains the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[A5] An agent for inducing stomach IgA production, comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[A6] The production inducer according to [A5], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[A7] The production inducer according to [A5] or [A6], wherein the bacterium belongs to the family S24-7.
[A8] The production inducer according to any one of [A5] to [A7], wherein the bacterium contains the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[A9] A therapeutic or prophylactic agent for oral infection, comprising a bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[A10] The therapeutic or prophylactic agent according to [A9], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[A11] The therapeutic or prophylactic agent according to [A9] or [A10], wherein the bacterium is a bacterium belonging to the family S24-7.
[A12] The therapeutic or prophylactic agent according to any one of [A9] to [A11], wherein the bacterium contains the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[A13] The therapeutic or prophylactic agent according to any one of [A9] to [A12], wherein the oral infection is at least one of infections selected from the following groups:
H. pylori infections, O: 157 infections, and Salmonella infections.
[B1] A method for inducing ILC2 in the stomach, comprising orally administering to a subject at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[B2] The induction method according to [B1], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[B3] The induction method according to [B1] or [B2], wherein the bacterium is a bacterium belonging to the family S24-7.
[B4] The induction method according to any one of [B1] to [B3], wherein the bacterium contains the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[B5] A method for inducing IgA production in the stomach, comprising orally administering to a subject at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and (2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[B6] The production induction method according to [B5], wherein the bacterium further has the following characteristics:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[B7] The production induction method according to [B5] or [B6], wherein the bacterium is a bacterium belonging to the family S24-7.
[B8] The production induction method according to any one of [B5] to [B7], wherein the bacterium contains the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[B9] A method for treating or preventing an oral infection, comprising orally administering a bacterium having the following properties to a subject:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[B10] The treatment or prevention method according to [B9], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[B11] The method according to [B9] or [B10], wherein the bacterium is a bacterium belonging to the family S24-7.
[B12] The treatment or prevention method according to any of [B9] to [B11], wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[B13] The treatment or prevention method according to any of [B9] to [B12], wherein the oral infection is at least one of infections selected from the following groups:
H. pylori infections, O: 157 infections, and Salmonella infections.
[C1] At least one bacterium having the following properties for use in a method for inducing ILC2 in the stomach:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[C2] The bacterium according to [C1], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[C3] The bacterium according to [C1] or [C2], wherein the bacterium is a bacterium belonging to the family S24-7.
[C4] The bacterium according to any one of [C1] to [C3], wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[C5] At least one bacterium having the following properties for use in a method for inducing the production of IgA in the stomach:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[C6] The bacterium according to [C5], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[C7] The bacterium according to [C5] or [C6], wherein the bacterium is a bacterium belonging to the family S24-7.
[C8] The bacterium according to any one of [C5] to [C7], wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[C9] A bacterium having the following properties for use in a method for treating or preventing an oral infection:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[C10] The bacterium according to [C9], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[C11] The bacterium according to [C9] or [10], wherein the bacterium is a bacterium belonging to the family S24-7.
[C12] The bacterium according to any one of [C9] to [C11], wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[C13] The bacterium according to any one of [C9] to [C12], wherein the oral infection is at least one of infections selected from the following group:
H. pylori infections, O: 157 infections, and Salmonella infections.
[D1] Use of at least one bacterium having the following properties for producing a medicament for inducing ILC2 in the stomach:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[D2] Use according to [D1], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[D3] The use according to [D1] or [D2], wherein the bacterium is a bacterium belonging to the family S24-7.
[D4] Use according to any one of [D1] to [D3], wherein the bacterium comprises the following nucleotides (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[D5] Use of at least one bacterium having the following properties for producing a medicament for inducing IgA production in the stomach:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[D6] Use according to [D5], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[D7] The use according to [D5] or [D6], wherein the bacterium is a bacterium belonging to the family S24-7.
[D8] Use according to any one of [D5] to [D7], wherein the bacterium comprises the following nucleotides (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[D9] Use of oral infections, including bacteria having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
[D10] Use according to [D9], wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
[D11] The use according to [D9] or [D10], wherein the bacterium is a bacterium belonging to the family S24-7.
[D12] Use according to any one of [D9] to [D11], wherein the bacterium comprises the following nucleotides (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
[D13] The use according to any one of [D9] to [D12], wherein the oral infection is at least one of infections selected from the following groups:
H. pylori infections, O: 157 infections, and Salmonella infections.

According to the present invention, ILC2 can be induced in the stomach of a mammal. In addition, IgA production in the stomach can be enhanced as a result of induction of ILC2. This makes it possible to treat and / or prevent diseases and the like caused by infection with microorganisms that can be removed by IgA, such as H. pylori.

FIG. 1 is a diagram showing the results of studies on the proportions of various ILCs in the stomach of mice. (A) Outline of the present embodiment. (B), (c) and (d) Cell numbers or percentages of various ILCs in the small intestine and stomach. FIG. 2 shows the expression of IL-33Ra in mouse gastric ILC2. (A) Number of KLRG1 and Sca1 expressing cells in small intestine and stomach. (B) Percentage of cells expressing IL-33Rα in the small intestine and stomach. FIG. 3 is a diagram showing the effect of commensal bacteria on gastric ILC2. (A) Outline of the present embodiment. (B) and (c) ILC2 cell numbers in SPF and GF mice. (D) Percentage of cells expressing IL-33Rα in SPF and GF mice. FIG. 4 shows the effect of commensal bacteria on the function of gastric ILC2. (A) and (b) Cell numbers of IL-5 or IL-13 expressing cells in the stomach in SPF and GF mice. (C) and (d) Cell numbers of CD3 and CD4 positive cells (ie, T cells) and CD19 and B220 positive cells (ie, B cells) in SPF and GF mice. Under normal conditions, there was no change in the number of T cells and B cells in the stomach. FIG. 5 is a diagram showing induction of gastric ILC2 in GF mice by oral ingestion of gastric microbiota from SPF mice. (A) Outline of the present embodiment. (B), (c) and (d) Time course of the number of IL-5 expressing cells or IL-13 expressing cells in the stomach of GF mice orally ingesting the stomach-derived microflora of SPF mice. FIG. 6 is a graph showing the time course of the bacterial flora in the stomach or feces of a GF mouse due to oral ingestion of the gastric flora of the SPF mouse. (A) Changes over time in the percentage of bacterial species present in the stomach or feces of GF mice. (B) Time-dependent change in the proportion of Bacteroidetes present. (C) Changes over time in the proportion of S24-7 families. FIG. 7 is a view showing characteristics of a symbiotic bacterium group affecting the induction of gastric ILC2 concerning drug resistance. (A) Outline of the present embodiment. (B) and (c) Changes in ILC2 cell number after antibiotic treatment. FIG. 8 is a diagram showing characteristics of various bacterial groups related to drug resistance. (A) Changes in the numbers of various bacterial groups after antibiotic treatment. (B) Changes in abundance ratio of Actinobacteria and Bacteroidetes after antibiotic treatment. FIG. 9 is a diagram showing the localization of S24-7 in the stomach. FIG. 10 is a diagram showing the relationship between the number of ILC2 in the stomach and the number of bacteria. (A) Heatmap profile of the relative population of each bacterium at the family level in gastric contents from mice treated with each antibiotic. (B) Scatter profile of each bacterial population and ILC2 count at the family level in gastric contents from mice treated with antibiotics (ampicillin, neomycin, metronidazole, and colistin) or SPF mice (control). . FIG. 11-A shows various data on the immune response to Helicobacter pylori infection. (A) Detection of CagA protein in H. pylori-infected mice. (B) Time course of the number of ILC2 cells after infection with H. pylori. (C) H. pylori CFU present in mouse stomach tissue after H. pylori infection. (D) to (f) Changes over time in the numbers of B cells and T cells after infection with H. pylori. FIG. 11-B shows various data on the immune response to Helicobacter pylori infection. (A) and (b) Changes over time in the number of ILC2 cells in the stomach after H. pylori infection. (C) Time course of B and T cell numbers in the stomach after H. pylori infection. (D) Changes over time in the amount of IgA due to anti-IL-5 antibody treatment. (E) Temporal change in IgA level after H. pylori infection. FIG. 11-C is a diagram showing various data on an immune response to infection with Helicobacter pylori. (A) and (b) Time course of the number of IL-5 or IL-13 producing ILC2 cells after H. pylori infection. (C) Time course of the amount of IL-33 in the stomach after H. pylori infection. (D) and (e) Reduction of IgA producing cells by anti-IL-5 antibody treatment. (F) Changes over time in the expression level of pLgR after infection with H. pylori. FIG. 12 is a diagram showing the amount of bacterial-bound IgA in the stomach of vancomycin-treated mice. (A) and (b) Amount of IgA antibodies binding to bacteria in gastric contents and gastric mucus in SPF and vancomycin treated mice. FIG. 13 is a diagram showing various data relating to an immune response to infection with Muribaculum intestinale (YL27). (A) Outline of the present embodiment. (B) and (c) Time course of the number of ILC2 cells in the stomach after YL27 infection. (D) Time course of the number of cells of ILC2 producing IL-5 or IL-13. FIG. 14 is a diagram showing changes in the expression level of pIgR in response to YL27 infection and IgA production in the stomach contents or feces. FIG. 15 is a photograph showing that YL27 infected to GF mice adheres to the stomach of GF mice. FIG. 16 is a diagram illustrating an outline of the twelfth embodiment. FIG. 17 is a diagram showing IgA production in stomach contents or feces in mice infected with H. pylori after being infected with YL27. FIG. 18 is a diagram showing changes in the expression level of pIgR in the stomach of mice infected with H. pylori after being infected with YL27. FIG. 19 is a diagram showing suppression of H. pylori infection by YL27 based on measurement of the amount of H. pylori present in the stomach.

1. The present invention provides an ILC2 inducer in the stomach (hereinafter sometimes referred to as "the ILC2 inducer of the present invention") comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. The ILC2 inducer of the present invention can induce the expression of ILC2, which is a kind of immunocompetent cell, in the stomach of an application subject by the function of the specific bacteria to be added.

The bacterium to be combined with the ILC2 inducer of the present invention is characterized as a bacterium having sensitivity to vancomycin and resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. Attached. In one embodiment, the bacterium formulated in the ILC2 inducer of the present invention is susceptible to vancomycin and resistant to at least two drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. A bacterium, preferably a bacterium that is sensitive to vancomycin and resistant to at least three drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole, more preferably, The bacterium may be a bacterium that is sensitive to vancomycin and resistant to at least three drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. It has that sensitivity, and can be a bacterium having ampicillin, colistin, neomycin, and resistance to all metronidazole. In one embodiment, the bacterium formulated in the ILC2 inducer of the present invention has a nucleotide sequence represented by SEQ ID NO: 1. In another aspect, the bacterium contained in the ILC2 inducer of the present invention is a bacterium belonging to the family S24-7.

細菌 A bacterium belonging to the family S24-7 refers to a gram-negative bacterium belonging to the order Bacteroidetes Bacteroidales, S24-7 family. It has been reported that bacteria belonging to the S24-7 family widely coexist with homeothermic animals (Ormerod {KL} et al., Microbiome. 2016 {Jul} 7; 4 (1): 36.). As used herein, the term “thermophilic animal” refers to mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, humans, and the like. First, there is no particular limitation as long as it is a constant-temperature animal in which bacteria having the above-mentioned drug characteristics can coexist. The bacteria belonging to the family S24-7 have sensitivity to vancomycin and resistance to all of ampicillin, colistin, neomycin, and metronidazole, as shown in the following Examples. Can be confirmed on the basis of. Alternatively, as shown in the examples below, bacteria belonging to the family S24-7 may have the nucleic acid sequence shown in SEQ ID NO: 1. Therefore, in some cases, its presence can be confirmed by using the nucleic acid sequence shown in SEQ ID NO: 1 as a probe.

において In one embodiment of the present invention, the bacterium may be, but is not limited to, Muribulculum intestinale (also referred to as “YL27” or the like). In addition, YL27 has 16sΔrRNA consisting of the sequence represented by SEQ ID NO: 28. Therefore, in one embodiment, the bacterium used in the present invention is (1) a bacterium containing the nucleotide shown in SEQ ID NO: 28, or (2) at least 90% or more (more preferably 91% or more) the nucleotide shown in SEQ ID NO: 28 %, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more).

細菌 Bacteria to be incorporated in the ILC2 inducer of the present invention are bacteria that coexist with homeostatic animals and are present in the stomach, small intestine, large intestine, feces, and the like. Therefore, bacteria mixed with the ILC2 inducer of the present invention can be easily obtained by collecting bacteria from such a bacterial source. In one embodiment, after a bacterium collected from such a bacterium source is suspended in pure water or a buffer to form a bacterial suspension, a method known per se (for example, JP-A-2015-188407, JP-A-2012-175973, 2010-041967, International Publication WO2007 / 023711, JP-A-2005-229837, etc.). In addition, during the culture, unnecessary microorganism contamination may be removed by adding an appropriate antibiotic or the like. Specifically, the bacterium contained in the ILC2 inducer of the present invention has resistance to ampicillin, colistin, neomycin, and metronidazole. Therefore, at least one of them, preferably at least two, more preferably By adding at least three, particularly preferably these four types of drugs during the culture, the purity of the bacteria to be added to the inducer of the present invention can be increased.

As a specific example of a method for preparing a bacterium to be mixed with the ILC2 inducer of the present invention, a method of laparotomy of a constant temperature animal (for example, a mouse) and collecting contents and / or mucus contained in the gastrointestinal tract is exemplified. You. The collected contents and / or mucus include bacteria that are sensitive to vancomycin and resistant to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. . Therefore, the content and / or the mucus can be used as an active ingredient as it is, and among the bacteria contained therein, the bacterium is sensitive to vancomycin and is selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. Bacteria having resistance to at least one selected agent may be isolated and / or cultured using the above-described method, and then blended as an active ingredient. In this specification, the term "isolated" means that an operation for removing a factor other than the target bacterium has been performed and the state of the bacterium other than the target bacterium has been removed. In addition to the embodiment in which the number of target bacteria occupies the majority, other embodiments in which a small amount of other bacteria are present may be included in addition to the embodiment in which the bacteria are completely removed.

The amount of the bacterium to be added to the ILC2 inducer of the present invention is not particularly limited as long as a desired effect can be obtained, and is usually 0.001 to 100% by weight based on the total weight of the ILC2 inducer of the present invention. It can be preferably 0.01 to 100% by weight, more preferably 1 to 100% by weight, particularly preferably 10 to 100% by weight, but is not particularly limited thereto.

The subject to which the ILC2 inducer of the present invention can be suitably applied is not particularly limited as long as it is a constant temperature animal having ILC2 as an immunocompetent cell in the stomach, but preferably is mouse, rat, hamster, guinea pig, rabbit, cat, dog, Cattle, horses, sheep, monkeys, and humans, most preferably humans.

The dosage form of the ILC2 inducer of the present invention is not particularly limited as long as a desired effect can be obtained, and can be made into an oral administration preparation together with excipients and the like usually used in medicine. The dosage form of the oral administration preparation is not particularly limited as long as the desired effect can be obtained, but it is semi-solid or liquid so that the bacteria mixed with the ILC2 inducer of the present invention can remain alive in the preparation. Preferably, there is. The preparation for oral administration may contain excipients and / or additives commonly used in medicine.

Alternatively, the ILC2 inducer of the present invention may be provided in the form of a food composition. When provided in a food composition, the food composition may also be semi-solid or liquid so that the bacteria incorporated in the ILC2 inducer of the present invention can maintain a viable state in the food composition. preferable. Examples of such food compositions include, but are not limited to, yogurt, jelly, beverages, and the like.

In one aspect, the present invention provides a method for inducing ILC2 in the stomach of a subject, comprising orally administering the ILC2 inducer of the present invention to the subject. The method can be performed by orally administering to the subject an ILC2 inducer described above.

2. IgA production inducer The present invention also provides a gastric IgA production inducer (hereinafter sometimes referred to as "the IgA production inducer of the present invention"), comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. The IgA production inducer of the present invention can induce the expression of ILC2, which is a type of immunocompetent cell, in the stomach of an application subject by the function of the specific bacteria to be incorporated. The induced ILC2 produces / secretes cytokines such as IL-5 and IL-13 and stimulates B cells present in the stomach. The stimulated B cells differentiate into antibody-producing cells, and as a result, can induce and enhance IgA production in the stomach of the application target.

Regarding bacteria to be blended with the IgA production inducer of the present invention, its drug characteristics, its preparation method, its blending amount, and the application target and dosage form of the IgA production inducer of the present invention, "1. ILC2 inducer" Can be the same as described above.

In one aspect, the present invention provides a method for inducing IgA production in the stomach of a subject, comprising orally administering the IgA production-inducing agent of the present invention to the subject. This method can be carried out by orally administering the above-mentioned IgA production inducer to a subject.

In one embodiment, the IgA production inducer of the present invention can be provided in the form of a pharmaceutical or a food. When provided in the form of a food composition, the food composition must be semi-solid or liquid so that the bacteria contained in the IgA production-inducing agent of the present invention can remain alive in the food composition. Is preferred. Examples of such food compositions include, but are not limited to, yogurt, jelly, beverages, and the like.

3. Therapeutic and / or prophylactic agent for oral infection The present invention also relates to a therapeutic or prophylactic agent for oral infection (hereinafter referred to as "the therapeutic or prophylactic agent of the present invention") comprising at least one bacterium having the following characteristics. May provide):
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. The therapeutic or prophylactic agent of the present invention can induce the expression of ILC2, which is a type of immunocompetent cell, in the stomach of a subject by the function of the specific bacterium to be added. The induced ILC2 produces / secretes cytokines such as IL-5 and IL-13 and stimulates B cells present in the stomach. The stimulated B cells differentiate into antibody-producing cells, and as a result, IgA production in the stomach of the target is induced / enhanced, and the treatment and / or prevention of oral infections including H. pylori is achieved.

Regarding the bacteria to be incorporated into the therapeutic or prophylactic agent of the present invention, the drug characteristics thereof, the preparation method thereof, the amount of the bacterium, the application target and the dosage form of the IgA production inducer of the present invention, etc., refer to “1. Can be the same as described above.

治療 The therapeutic or prophylactic agent of the present invention promotes the elimination of pathogenic microorganisms present in the stomach by inducing and enhancing the production of secretory IgA antibodies in the gastric mucosa. In view of such viewpoints, the therapeutic or prophylactic agent of the present invention can be suitably used for oral infections, especially for H. pylori infections. Since S24-7 is also detected as a major bacterium in the intestinal tract, it is possible that the intestinal tract may be protected from infectious enteritis caused by infectious bacterial species such as O: 157 and Salmonella infection. Therefore, in one aspect, the therapeutic or prophylactic agent of the present invention can be used for treating or preventing H. pylori infection, O: 157 infection, and Salmonella infection.

In one aspect, the therapeutic or prophylactic agent of the present invention can be provided in the form of a medicament or food. When provided in a food composition, the food composition must be semi-solid or liquid so that the bacteria to be added to the therapeutic or prophylactic agent of the present invention can maintain a viable state in the food composition. Is preferred. Examples of such food compositions include, but are not limited to, yogurt, jelly, beverages, and the like.

対象 The therapeutic agent of the present invention is applied to the above-mentioned oral infection patients. The subject to which the prophylactic agent of the present invention is applied is a healthy subject or a subject who has cured the above-mentioned oral infection.

The therapeutic agent of the present invention is administered in such an amount that the subject can ingest a therapeutically effective amount of bacteria of the family S24-7. The therapeutically effective amount can vary depending on the height, weight, sex, administration route, administration schedule, and the like of the subject to which the therapeutic agent is administered, but can be appropriately determined by those skilled in the art.

The prophylactic agent of the present invention is administered in such an amount that a subject can take a prophylactically effective amount of bacteria of the S24-7 family. The prophylactically effective amount can vary depending on the height, weight, sex, administration route, administration schedule, and the like of the subject to which the prophylactic agent is administered, but can be appropriately determined by those skilled in the art.

In one aspect, the present invention provides a method for treating or preventing an oral infection, comprising orally administering a therapeutic or prophylactic agent of the present invention to a subject. The present method can be carried out by orally administering the above-mentioned agent for treating or preventing oral infection to a subject.

In addition, “treatment” in the present specification may include not only cure of the disease but also remission of the disease and improvement of the degree of the disease.

As used herein, the term "prevention" includes, in addition to preventing the onset of the disease, delaying the onset of the disease, and comparing the state of the subject at the time of the onset of the disease with the normal state of onset of the disease. And mild. In addition, the term "prevention" as used herein includes preventing the recurrence of the disease after treatment, delaying the recurrence of the disease after treatment, and usually controlling the condition of the subject at the time of recurrence of the disease after treatment. Of the disease as compared to the state at the time of recurrence of the disease.

The present invention will be described more specifically in the following examples, but the present invention is not limited to these examples.

[Materials and methods]
1. Mouse C57BL / 6N mice were purchased from CLEA (CLEA JAPAN, INC) and bred at RIKEN under specific pathogen-free (SPF) conditions for at least 4 weeks. Sterile (GF) mice with a C57BL / 6N background were purchased from CLEA and maintained on a sterile vinyl isolator at RIKEN or Yokohama City University. Rag2 ^{- / -} mice (.. N.Satoh-Takayama et al , Microbial flora drives interleukin 22 production in intestinal NKp46 + cells that provide innate mucosal immune defense Immunity 29, 958-970 (2008)), Il-33 gfp / gfp mice (.. K.Oboki et al, IL -33 is a crucial amplifier of innate rather than acquired immunity Proc Natl Acad Sci U S A 107, 18581-18586 (2010)), CD3ε - / - mice (M.Malissen et al . , Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene. EMBO J 14, 4641-4653 (1995), RORγt ^gfp / t mice. RORgamma (t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 5, 64-73 (2004)) was provided by Dr. Di Santo, Dr. Nakae, and Dr. Marissen, respectively. All mice (8-18 weeks of age) were used for experiments that adhered to guidelines provided by the Animal Research Committee of the RIKEN Yokohama Branch or the Research Committee of Yokohama City University, and were in accordance with Japanese law. It was conducted.

2. Cell preparation, flow cytometry open cytometry analysis and intracellular staining stomach tissue quickly after picked up from the body of the mouse, then washed gently gastric contents with cold PBS buffer or RPMI medium. Stomach tissue was cut into small pieces and digested with 1.0 mg / ml collagenase (Wako Pure Chemical Industries Ltd). The resulting supernatants were pooled and finally passed through a 40 μm mesh filter. Isolation and flow cytometric analysis of small intestinal lamina propria lymphocytes (LPL) is described in N.W. Satoh-Takayama et al. , Lymphotoxin-beta receptor-independent development of intestinal IL-22-producing NKp46 (+) innate lymphoid cells. Eur J Immunol 41, 780-786 (2011) and Y. Sasagawa et al. , Quartz-Seq: a high reproducible and sensitive single-cell RNA sequencing method, reviews non-genetic gene-expression heterogeneity. This was performed using the method described in Genome Biol 14, R31 (2013). For flow cytometry analysis, after blocking the Fc receptor with 2.4G2 mAb (BioLegend), all cells were stained at 4 ° C. using each surface antibody, and dead cells were LIVE / DEAD fixable Aqua Dead Cell Stain (Thermo). Fisher). Surface or intracellular staining was performed using specific fluorescent dye-linked antibodies (BioLegend, eBioScience and Southern Biotech, see Table 1 below). The anti-mouse DCLK antibody was labeled with alexa488 according to the manufacturer's instructions (Thermo Fisher scientific). For intracellular cytokine staining, isolated cells were incubated with PMA (50 ng / ml) and ionomycin (2 μg / ml) or without PMA and ionomycin for 2 hours at 37 ° C. in the presence of GolgiPlug (BD biosciences). did. After staining with the surface antibody, the incubated cells were fixed with PBS containing 4% PFA for 15 minutes at room temperature. The fixed cells were permeabilized with 1 × permeabilization buffer (included in Foxp3 / TF buffer set (Thermo Fisher Scientific)) at 4 ° C. for 60 minutes. For transcription factor detection in cells, isolated cells were stained with intracellular antibodies using Foxp3 / TF staining buffer set according to the manufacturer's instructions (Thermo Fisher Scientific). For cell analysis and sorting, cells were analyzed with FACSAria III (BD Biosciences). For all lymphocyte analysis by flow cytometry, lymphocytes were first defined by FSC and SSC intensity, and then carefully gated based on CD45 expression. Data was analyzed with FlowJosoftware (TreeStar).

3. RNA-seq and quantitative PCR
For RNA-seq, 1000 cells extracted from stomach or small intestine with FACS Aria III (BD Biosciences) at 98% or better purity were directly sorted into 50 μl lysis buffer (QIAGEN). All steps after sorting followed the Quartz-seq method (library; KAPA library preparation kit, illumina. Adapter; Next Multiplex Oligo for illumina, NEB) with some modifications (see Y. Sasagawa. , Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reviews non-genetic gene-exposure gene. All samples were sequenced in HiSeq 1500, rapid mode. For quantitative PCR analysis, RNA total RNA was extracted from purified gastric or intestinal cell preparations using RNeasy micro kit (QIAGEN). For total tissue RNA expression analysis, the stomach or intestine was frozen in liquid nitrogen. Frozen tissue was triturated in liquid nitrogen and RNA was extracted from powdered tissue using lysis buffer (QIAGEN). The cDNA is generated using reverse transcriptase (Revatra Ace, TOYOBO) according to the manufacturer's instructions, followed by primers (Eurofins genomics; see Table 2) or TaqMan probes for Il25 and Il33 (Thermo Fisher Scientific; see Table 3). ) Was used to perform quantitative PCR analysis using RT SYBR Green qPCR Master Mix (TAKARA SYBR Premix ExTaq II).

4. RNA-seq Read Alignment and Gene Qualification
Differential gene expression was analyzed using the DEseq package in the R software, and in each analysis these genes were identified by log2 fold change Value, p-Value and FDR correction value. P-Value and FDR correction value for multiple tests were calculated using the Benjamini-Hochberg method. Biological replicates include each cell type or condition.

5.16S rRNA Sample Preparation and Data Processing and Analysis Gastric contents and mucus samples were stocked at -80 ° C immediately after centrifugation and removal of supernatant. DNA extraction was performed by T.I. Jinnohara et al. , IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake. J Exp Med, (2017) and T.M. Kato et al. , Multiple omics uncovers host-gut microbiological mutulims dur ing prebiotic fructooligosaccharide supplementation. DNA Res 21, 469-480 (2014) was performed using a slightly modified method. For analysis, the V4 variable region (515F-806R) was sequenced with the Illumina Miseq library prepared according to the manufacturer's (Takara Bio, Inc) protocol. The sample library with 20% denatured PhiX (spike-in) was sequenced using the Miseq 500 cycle kit to obtain 2 × 250 bp paired-end reads.

分類 Taxonomic assignments of sequenced data and estimation of relative abundance were performed using the analysis pipeline of the QIIME software package. The chimera check was performed using UCHIME. Operational taxonomic units (OTUs) were defined with 97% similarity. OTU is based on T.A. Kato et al. , Multiple omics ｃｏuncovers host-gut microbial mutualism during prebiotics fructooligosaccharide supplementation. Taxonomies were assigned based on comparisons in the Greengenes database using the RDP classifier described in {DNA Res 21,469-480 (2014).

6. Antibiotic treatment, stomach contents and mucus preparation, and transplantation experiments Mice in SPF condition were added with ampicillin (0.1 g / L), colistin (1 g / L) or vancomycin (0.5 g / L) to drinking water. Orally for 3 weeks. Stomach contents and mucus were obtained from C57BL / 6N mice or antibiotic-treated mice under SPF conditions as controls. The open stomach was gently washed in sterile PBS. The collected contents were homogenized and vortexed sufficiently. The tube was allowed to stand for 30 seconds to remove large particles. For analysis of the mucus area, the gastric tissue after washing the gastric contents was scraped with a curved needle. After spin down to remove epithelium and large particles, the supernatant was used for gastric mucus analysis. For oral transplantation experiments, 300 μl of a mixture of gastric contents and mucus was orally ingested once or once a week for 4 weeks in GF mice. To reduce intestinal microbiota, litter CD3ε ^{+ / +} and CD3ε ^{− / −} pregnant female mice were treated with ampicillin (0.1 g / L), vancomycin (1 g / L), neomycin (1 g / L), Antibiotics were added to the drinking water by adding an antibiotic cocktail containing and metalonidazole (1 g / L) to the pups and orally ingested until weaning (21 days after delivery).

7. Evaluation of IgA-Coated Bacteria by Flow Cytometry Analysis Stomach contents were obtained from control GF mice or H. pylori infected mice. The open stomach was gently washed with 1 ml of sterile PBS. The contents were centrifuged at 700 g for 5 minutes, and the supernatant was further centrifuged at 12,000 g for 5 minutes to remove unbound IgA. The bacteria are Kawamoto et al. , Foxp3 (+) T cells regulatory immunoglobulin a selection and facilitate diversification of bacterial species responsible for immune. Stained with PE-conjugated anti-mouse IgA antibody and DAPI as described in Immunity 41, 152-165 (2014). After washing with PBS, the bacteria were suspended in FACS buffer and analyzed using FACSAria III.

8. H. pylori culture, infection and infection check H. pylori strain used herein; PMSS1 was grown on a sheep blood agar plate and Satoh-Takayama et al. , Lymphotoxin-beta-receptor-independent development of intestinal IL-22-producing NKp46 + internal lymphoid cells. The cells were cultured in a liquid medium described in Eur J Immunol 41, 780-786 (2011). Mice were infected orally with 1 × 10 ⁹ CFU / mouse and mice were sacrificed for analysis at 2 or 9 weeks post-infection. In all the gastrointestinal tracts of these mice, infection with H. pylori was confirmed by expression of the CagA gene of the PMSS strain.

9. Immunohistology Immunohistological analysis of 8 μm frozen sections of the stomach was performed as described by N.W. Satoh-Takayama et al. , Microflora drives interleukin 22 production in intestinal NKp46 + cells that provide innate mucosal immune defense. Immunity 29, 958-970 (2008) and N.I. Satoh-Takayama et al. , Lymphotoxin-beta-receptor-independent development of intestinal IL-22-producing NKp46 + internal lymphoid cells. Eur J Immunol 41, 780-786 (2011). As the primary antibody, a rat anti-B220 antibody (RA3-62B; BioLegend) was used. Sections were stained using DAPI. Slides were viewed using a Leica AF6000.

10. Statistical analysis experimental results are means ± s. e. m. Indicated by Statistical differences between groups were determined using the unpaired Student's t-test. Statistical comparisons between two or more groups used one-way analysis of variance (one-way ANOVA). P values <0.05 were considered statistically significant. All data was analyzed using Graph-pad Prism software.

[Reference Example 1] Examination of the ratio of various ILCs present in the stomach The small intestine of SPF mice used Collagenase (Wako) adjusted to a concentration of 1 mg / L with 2% FBS / RPMI-1640 medium, and the large intestine was similarly used at 1 mg / L. The mixture was stirred at 37 ° C for 15 minutes in Collagenase (SIGMA) adjusted to L concentration. Fifteen minutes later, only the supernatant was recovered, and then Collagenase was added to the remaining intestinal fragments, and the same procedure was repeated twice to recover the supernatant. The collected supernatant was filtered using a 100 μm Cell strainer (BD Bioscience), and used for lymphocyte isolation after centrifugation. After centrifugation of the cell suspension, the cells were suspended in 4 ml of 40% Percoll and transferred to a 15 ml Falcon tube. Thereafter, an equal volume of 70% Percoll was slowly added using a Pasteur pipette, and the mixture was layered downward at 2000 rpm at 20 ° C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer generated by the density gradient was collected and suspended in a 2% FBS / RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cell strainer to remove mucus and the like.

The lymphocytes collected by the above method were suspended in an appropriate amount of 2% FBS / RPMI-1640 medium, and seeded at 1 × 10 ⁶ cells / well on a Tissue Culture Plate (96 Well, Flat Butt with Low Evaporation Lid, FALCON). . After centrifugation, Purified Rat Anti-mouse CD16 adjusted to a final concentration of 0.1 mg / ml with a 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS / D-PBS (-)). / CD32 (BD Biosciences) was added, and the mixture was allowed to stand at 4 ° C for 15 minutes to perform blocking and staining of dead cells. Thereafter, the cells were washed with Facs buffer, and after centrifugation, each antibody diluent (CD3, CD45, TCRb, CD19, CD127, CD90.2) was added to the cells, and the cells were allowed to stand at 4 ° C. for 20 minutes, and the cell surface was removed. Was expressed with a fluorescently labeled antibody.

抗原 Antigen expressed in cells was stained by intracellular staining. After staining with a cell surface antibody, the cells were washed with D-PBS (-). After centrifugation, Fixation / Permeabilization solution (Foxp3 / Transscription Factor Staining Buffer Set, ThermoFisher) was added, and the mixture was allowed to stand at 4 ° C. for 30 minutes in a light-shielded state to perform cell fixing and cell membrane permeation. After the cells were fixed, the cells were washed with D-PBS (-), and the supernatant was removed by centrifugation. Next, the cells were washed again with Permeabilization buffer (Foxp3 / Transscription Factor Factor Staining Buffer Set, ThermoFisher). After centrifugation, each of the nuclear staining antibodies (GATA3 and RORgt) diluted with Permeabilization @ buffer was added to each well, and the cells were allowed to stand at 4 ° C. for 30 minutes in the light-shielded state to perform staining. After staining, the cells were washed with Permeabilization buffer and Facs buffer, then suspended in an appropriate amount in FACS buffer, and used for flow cytometry analysis. FACSAria III (BD @ Biosciences) was used for the stained cells, and flow cytometry analysis was performed. Analysis of the obtained data was performed using FlowJo (Tree @ Star, Inc.). The obtained flow cytometry results show only a cell population that does not express CD3, TCRb, and CD19 but expresses CD45.

通り As shown in FIG. 1, it was shown that ILC2 was extremely predominant in the stomach (ILC 1: 5%, ILC2: 94%, ILC3: 0.6%) as compared to the small intestine.

[Reference Example 2] Confirmation of expression of IL-33Ra in gastric ILC2

The small intestine of the SPF mouse used Collagenase (Wako) adjusted to a concentration of 1 mg / L in a 2% FBS / RPMI-1640 medium, and the large intestine was similarly treated in Collagenase (SIGMA) adjusted to a concentration of 1 mg / L at 37 ° C. And stirred for 15 minutes. Fifteen minutes later, only the supernatant was recovered, and then Collagenase was added to the remaining intestinal fragments, and the same procedure was repeated twice to recover the supernatant. The collected supernatant was filtered using a 100 μm CellStrainer (BD Bioscience), centrifuged, and used for lymphocyte isolation. After centrifugation of the cell suspension, the cell suspension was suspended in 4 ml of 40% Percoll and transferred to a 15 ml Falcon tube. Then, an equivalent volume of 70% percoll was slowly added using a Pasteur pipette, and the mixture was layered downward at 2000 rpm at 20 ° C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer generated by the density gradient was collected and suspended in a 2% FBS / RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cell strainer to remove mucus and the like.

The lymphocytes collected by the above method were suspended in an appropriate amount of a 2% FBS / RPMI-1640 medium, and seeded at 1 × 10 ⁶ cells / well on a Tissue Culture Plate (96 Well, Flat Butt with Low Evaporation Lid, FALCON). After centrifugation, Purified Rat Anti-mouse CD16 adjusted to a final concentration of 0.1 mg / ml with a 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS / D-PBS (-)). / CD32 (BD Biosciences) was added, and the mixture was allowed to stand at 4 ° C. for 15 minutes to perform blocking and staining of dead cells. Thereafter, the cells were washed with Facs buffer, and after centrifugation, the respective antibody diluents (CD3, CD45, TCRb, CD19, CD127, CD90.2, Sca1, KLRG1, and IL33Ra) were added to the cells according to Table 3 or 4. Then, the mixture was allowed to stand at 4 ° C. for 20 minutes, and the antigen expressed on the cell surface was stained with a fluorescently labeled antibody. The obtained flow cytometry results show only a cell population that does not express CD3, TCRb, and CD19 but expresses CD45. IL33Ra expression is further shown for cells that express both Sca1 and KLRG1.

ＩＬAs shown in FIG. 2, it was shown that IL-33Ra was highly expressed in gastric ILC2 as compared with small intestine ILC2 (SI PLL: 5.6, Stomach: 24).

[Example 1] Investigation of the effect of commensal bacteria on gastric ILC2 In the small intestine of SPF mice and germ-free (GF) mice, Collagenase (Wako) adjusted to a concentration of 1 mg / L with 2% FBS / RPMI-1640 medium was used. The large intestine was also stirred at 37 ° C. for 15 minutes in Collagenase (SIGMA) adjusted to a concentration of 1 mg / L. Fifteen minutes later, only the supernatant was recovered, and then Collagenase was added to the remaining intestinal fragments, and the same procedure was repeated twice to recover the supernatant. The collected supernatant was filtered using a 100 μm Cell strainer (BD Bioscience), and used for lymphocyte isolation after centrifugation. After centrifugation of the cell suspension, the cells were suspended in 4 ml of 40% Percoll, transferred to a 15 ml Falcon tube, and then slowly added with an equal volume of 70% Percoll using a Pasteur pipette, and overlaid at 2000 rpm at 20 ° C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer generated by the density gradient was collected and suspended in a 2% FBS / RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cell strainer to remove mucus and the like.

The lymphocytes collected by the above method were suspended in an appropriate amount of 2% FBS / RPMI-1640 medium, and seeded at 1 × 10 ⁶ cells / well on a Tissue Culture Plate (96 Well, Flat Butt with Low Evaporation Lid, FALCON). . After centrifugation, Purified Rat Anti-mouse CD16 adjusted to a final concentration of 0.1 mg / ml with a 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS / D-PBS (-)). / CD32 (BD Biosciences) was added, and the mixture was allowed to stand at 4 ° C for 15 minutes to perform blocking and staining of dead cells. Thereafter, the cells were washed with Facs buffer, and after centrifugation, the respective antibody diluents (CD3, CD45, TCRb, CD19, CD127, CD90.2, Sca1, KLRG1, and IL33Ra) were added to the cells according to Table 3 or 4. Then, the mixture was allowed to stand at 4 ° C. for 20 minutes, and the antigen expressed on the cell surface was stained with a fluorescently labeled antibody. The obtained flow cytometry results show only a cell population that does not express CD3, TCRb, and CD19 but expresses CD45. IL33Ra expression is further shown for cells that express both Sca1 and KLRG1 (ie, ILC2).

通り As shown in FIG. 3, the abundance of gastric ILC2 was significantly different between the SPF mouse and the GF mouse, indicating that symbiotic bacteria affect the abundance of gastric ILC2.

[Example 2] Investigation of the effect of commensal bacteria on the function of gastric ILC2 Lymphocytes collected by the above-described method were suspended in a complete buffer containing 10% FBS / RPMI-1640 medium supplemented with L-glutamine, penicillin, and streptomycin. Was used to confirm some cytokine production. Lymphocytes were seeded on a Tissue Culture Plate (96 Well, Flat Butt with Low Evaporation Lid) at 1 × 10 ⁶ cells / well. Golgi Plug (Biosciences), which is an inhibitor of intracellular protein transport, was added without stimulation or together with PMA (50 ng / ml) and Ionomycin (1 ig / ml), and the mixture was allowed to stand at 37 ° C. in a 25% CO ₂ incubator for 2 hours. . After standing for 2 hours, centrifugation was performed, and cell surface molecules were stained as described above. The stained cells were washed twice with D-PBS (-), centrifuged, added with 4% PFA / D-PBS (-), and allowed to stand at room temperature for 15 minutes to fix the cells. After centrifugation, the cells were washed with Permeabilization Buffer. After the permeation treatment, each of the IL-5 and IL-13 antibodies was diluted with Permeabilization Buffer, the diluted solution was added to each well, and the plate was allowed to stand still at 4 ° C. for 60 minutes in a light-shielded state to perform staining. FACSAria III (BD Biosciences) was used for the stained cells, and flow cytometry analysis was performed. Analysis of the obtained data was performed using FlowJo (Tree Star, Inc). FIG. 4 compares the production of IL-5 and IL-13 for cells that do not express CD3, TCRb, and CD19 but that express CD45, Scal, and KLRG1 (ie, ILC2).

通り As shown in FIG. 4, it was shown that there was a large difference in the amount of IL-5 and IL-13 expressing cells between the SPF mouse and the GF mouse. In addition, it was confirmed that there was no difference in the number of T cells and B cells, which are cells that mainly produce IL-5 and / or IL-13 other than ILC2 in the stomach (FIG. 4 (c) and FIG. (D)). That is, it was shown that the presence or absence of symbiotic bacteria did not affect T cells or B cells, but only affected the number and function of ILC2 in the stomach.

Example 3 Induction of Gastric ILC2 by Oral Intake of Stomach-Derived Microbiota Stomach contents and gastric mucosal tissue of SPF mice were scraped, suspended in PBS, and filtered using 100 μm Cell strainer (BD Bioscience) for a total of 3 ml. , And 300 μl thereof was orally administered. Mice that received a single dose and mice that were repeated once a week four times were used for analysis one week after the last oral dose. The stomach was removed from the orally administered mouse and a germ-free mouse as a control, and an experiment was performed using an intracellular cytokine production detection method. The lymphocytes collected by the above-described method were suspended in a complete buffer obtained by adding L-glutamine, penicillin, and streptomycin to 10% FBS / RPMI-1640 medium, and partially used for confirming cytokine production. Lymphocytes were seeded on a Tissue Culture Plate (96 Well, Flat Butt with Low Evaporation Lid) at 1 × 10 ⁶ cells / well. The collected lymphocytes were added with Golgi Plug (Biosciences), an intracellular protein transport inhibitor, and allowed to stand at 37 ° C. in a 25% CO ₂ incubator for 2 hours. After standing for 2 hours, centrifugation was performed, and cell surface molecules were stained as described above. The stained cells were washed twice with D-PBS (-), centrifuged, added with 4% PFA / D-PBS (-), and allowed to stand at room temperature for 15 minutes to fix the cells. After centrifugation, the cells were washed with Permeabilization Buffer. After the permeation treatment, each of the IL-5 and IL-13 antibodies was diluted with Permeabilization Buffer, the diluted solution was added to each well, and the plate was allowed to stand still at 4 ° C. for 60 minutes in a light-shielded state to perform staining. FACSAria III (BD Biosciences) was used for the stained cells, and flow cytometry analysis was performed. Analysis of the obtained data was performed using FlowJo (Tree Star, Inc). FIG. 5A is a diagram illustrating an outline of the present embodiment. FIGS. 5 (b) and (d) show that for cells expressing CD45, Scal, and KLRG1 that do not express CD3, TCRb, or CD19 (ie, ILC2), IL-5 or IL-13 production was determined using FACS. FIG. 5 (b)) and further quantified (FIG. 5 (d)). FIG. 5C is a diagram showing the relationship between the number of times of oral ingestion and the number of cells.

As shown in FIG. 5, when the contents and / or mucus from the stomach of the SPF mouse were orally ingested into the GF mouse, the bacterial abundance in the stomach of the GF mouse increased in proportion to the number of ingestions, and the IL content increased. The number of cells secreting -5 or IL-13 (ie, ILC2) increased. This indicates that ILC2 in the stomach of the GF mouse was induced by the symbiotic bacterium contained in the contents and / or mucus from the stomach of the SPF mouse engrafted in the stomach of the GF mouse. In other words, the results in FIG. 5 indicate that gastric commensal bacteria affect the number and function of ILC2.

[Example 4] Analysis of bacterial flora by oral ingestion of gastric microbial flora Microflora of mice to which stool, stomach contents and gastric mucosal layer were orally administered were analyzed. Feces and stomach contents were weighed at 10 mg and used for DNA extraction. After adding 450 μl of Tris-10 × EDTA (10 mmol / l Tris-HCl, 10 mmol / l EDTA, pH 8.0) to a 10 mg stool sample, suspending with vortex, adding 25 μl of Lysozyme stock (wako) 300 mg / ml, 37 Incubate for 1 hour at 1000C with shaking at 1000 rpm. Thereafter, 55 μl of 20,000 U / ml of Achromopeptidase stock (wako) was added, and the mixture was incubated at 37 ° C. for 0.5 hour with shaking at 1000 rpm. After 61.5 μl of 10% SDS was added, and 25.7 μl of Proteinase K stock (Merck) 25 mg / ml was added, the mixture was incubated at 55 ° C. for 1 hour with shaking at 1000 rpm. 650 μl of phenol / CHCl ₃ / IAA (Nacalai) was added and shaken on a rotator for 5 minutes. Thereafter, the mixture was centrifuged at 13000 rpm for 5 minutes, and the supernatant was recovered. 650 μl of phenol / CHCl ₃ / IAA was added to the collected supernatant and shaken with a rotator for 5 minutes. Thereafter, the mixture was centrifuged at 13000 rpm for 5 minutes, and 300 µl of the supernatant was recovered. To the collected supernatant, 50 μl of 3M-CH ₃ COONa (Nakarai) and 1000 μl of 100% EtOH were added and suspended. After standing at −20 ° C., the mixture was centrifuged at 15000 rpm and 4 ° C. for 10 minutes, and the supernatant was aspirated, and 1000 μl of 70% EtOH was added to Pellet. Thereafter, the pellet was centrifuged again at 15000 rpm at 4 ° C. for 10 minutes, and the pellet was dried at 40 ° C. for 10 minutes and suspended in 250 μl of TE. To 200 μl of 250 μl, 2 μl of RNase (10 mg / ml) was added and incubated at 37 ° C. for 1 hour. 200 μl of 10% PEG6000-2.5M NaCl was added and incubated for 30 minutes. After centrifuging at 15000 rpm at 4 ° C. for 20 minutes to remove the supernatant, washing with 1000 μl of 70% EtOH, centrifuging again at 15000 rpm at 4 ° C. for 20 minutes, removing the supernatant, and drying the pellet at 40 ° C. for 10 minutes I let it. Thereafter, the cells were suspended in 50 μl of TE, and the DNA concentration was measured with Nanodrop (Thermo). The extracted DNA was diluted to 5 ng / μl, and the V4 region of 16S rRNA was amplified using primers of 515F (GTGCCAGCMGCCGCGGTAA: SEQ ID NO: 26) and 806R (GGACTACHVGGGGTWTCTAAT: SEQ ID NO: 27). The PCR product was purified using AMPure beads, and a barcode sequence was provided by a PCR reaction using Nextra XT Index Kit v2 (Illumina). After the PCR, the PCR product was purified again using AMPure beads, and the DNA concentration was measured using PicoGreen (Invitrogen). From the results of the concentration measurement, all the samples were diluted to 1 ng / μl, and after confirming the size and concentration of the DNA by TapeStation (Agilent), KAPA qPCR was performed. Gene sequences are sequenced by Miseq (Illumina), and the gene sequences obtained using the package software QIIME (http://qime.org/) are clustered with a sequence similarity of 97% to create an Operational taxonomic unit (OTU). After that, the obtained OTU was searched for a database using Ribosomal Database Project (https://rdp.cme.msu.edu) to identify the bacterial species.

As shown in FIG. 6, as the content and / or mucus from the stomach of the SPF mouse was orally ingested, the amount of bacteria belonging to the S24-7 family increased in the stomach and feces of the GF mouse. Indicated. That is, it was shown that an increase in the amount of bacteria belonging to the family S24-7 in the stomach may have a great effect on the induction of ILC2 in the stomach.

[Example 5] Identification of symbiotic bacteria affecting the induction of gastric ILC2 1
To mice in SPF condition, drinking water was added with ampicillin (0.1 g / L), colistin (1 g / L), neomycin (1 g / L), metronidazole (1 g / L) or vancomycin (0.5 g / L). Orally for 3 weeks. Lymphocytes were collected from the stomach of C57BL / 6N mice or antibiotic-treated mice under SPF conditions as a control, and subjected to flow cytometry analysis according to the above-described procedure.

As shown in FIG. 7, in mice fed ampicillin (Amp), colistin (Colistin), neomycin (Neo), and metronidazole (MNZ), the cell number of gastric ILC2 was significantly changed as compared with the control (SPF). Was not seen. On the other hand, in the mice to which vancomycin was taken, the number of cells of gastric ILC2 was decreased. Therefore, it was shown that commensal bacteria that affect the induction of gastric ILC2 are resistant to ampicillin, colistin, neomycin, and metronidazole but sensitive to vancomycin.

[Example 6] Identification of symbiotic bacteria affecting the induction of gastric ILC2 2
Gene sequences are sequenced by Miseq (Illumina), and the gene sequences obtained using the package software QIIME (http://qime.org/) are clustered with a sequence similarity of 97% to create an Operational taxonomic unit (OTU). After that, the obtained OTU was searched for a database using Ribosomal Database Project (https://rdp.cme.msu.edu) to identify the bacterial species. The flora information was appropriately normalized by the above-described method, and the ratio of various flora to each antibiotic administration group was calculated. FIG. 8 shows the results.

As shown in FIG. 8, the symbiotic bacteria that are sensitive to vancomycin and resistant to ampicillin, colistin, neomycin, and metronidazole include bacteria belonging to the family S14-7. Was.

[Example 7] Examination of localization of S24-7 To a sample excised from a mouse, 1 ml of Carnoy's fixative (60% methanol, 30% chloroform, 10% acetic anhydride) was added and fixed for 6 hours. After the fixation, the solution was replaced with 1 ml of methanol, and left at room temperature for 30 minutes. After 30 minutes, methanol was added again, and the mixture was left at room temperature for 30 minutes. After 30 minutes, the mixture was replaced with dehydrated ethanol and allowed to stand at room temperature for 20 minutes. The sample was placed in a cassette, and a program for ethanol (1 hour), xylene (2 hours, 3 times) and paraffin (3 hours, 3 times) was performed using a Laica tissue processor, and embedded in paraffin after completion of the program. The block was sliced to 5 μm and attached to a slide glass. After stretching at 37 ° C., it was dried in an oven at 60 ° C. for 10 minutes. The slide glass was placed in xylene and dipped for 10 minutes, and then the slide glass was placed in 99.5% ethanol for 5 minutes. Then it was air-dried. The probe was diluted with a pre-warmed hybridization buffer (0.9 M NaCl, 20 mM pH 7.4 Tris-HCl, 0.1% SDS) to 10 nM. The sample was overlaid and reacted at 48 ° C. for 2 hours. After completion of the reaction, 100 μl of a hybridization buffer (0.9 M NaCl, 20 mM pH 7.4 Tris-HCl, 10% formamide) was added thereto, and the mixture was reacted at 48 ° C. for 5 minutes. After completion of the reaction, the plate was washed three times with PBS, and detection was performed with Laica SP8.

プローブ The probe used above was prepared as follows. Attached to the stomach contents of control (SPF mice), ampicillin-treated mice, colistin-treated mice, neomycin-treated mice, and metronidazole-treated mice, but not decreased, but vancomycin-treated mice had decreased. The amount of the nucleic acid sequence of the bacteria to be treated was determined using Miseq (Illumina). As a result of the analysis, it was confirmed that the sequence of bacteria belonging to the family S24-7 satisfies this condition (11 types of sequences were determined). The nucleic acid sequence (SEQ ID NO: 1) common to these 11 types of sequences was determined. A probe was prepared by labeling the 3 'end of SEQ ID NO: 1 with a fluorescent protein (A555). FIG. 9 shows the results.

ＳAs shown in FIG. 9, S24-7 was found to be present in the epithelium of the stomach. These results also strongly suggested that S24-7 engrafted in the stomach and constituted a bacterial flora, and had a great effect on the induction of ILC2 in the stomach.

[Example 8] Investigation on correlation between the number of ILC2 cells in the stomach and the number of S24-7 individuals In mice under SPF condition, ampicillin (0.1 g / L), colistin (1 g / L), neomycin were added to drinking water. (1 g / L), metronidazole (1 g / L) or vancomycin (0.5 g / L), and orally taken for 3 weeks. Thereafter, the stomach contents and feces of the mice were collected and analyzed as described in Examples 4 and 6. At the same time, lymphocytes collected from the stomach were analyzed by flow cytometry according to the above-described procedure. With the upper limit of the value of 1% or more for each bacterial count obtained by the microflora analysis, a correlation diagram between the bacterial species (family) and the flow cytometry analysis results was created as a heat map and a scatter plot.

通り As shown in FIG. 10, a strong positive correlation was shown between the bacterial population of S24-7 and the number of ILC2 cells (r = 0.8, p = 0.03). On the other hand, such a relationship was not found between the number of bacteria other than S24-7 family and the number of ILC2 cells. The results showed that the S24-7 bacteria induced an increase in ILC2 in the stomach.

Example 9 Detailed Examination of Immune Response to Helicobacter pylori Infection Sterile mice were orally administered at 1 × 10 ⁹ CFU / 300 μl PBS. Two weeks after the infection, when the number of Helicobacter pylori was highest, and nine weeks after the stomach exhibited inflammatory symptoms were examined. Bacterial binding IgA was detected by the method described above. Next, the stomach 2 weeks and 9 weeks after the infection was removed, the stomach was opened, and the contents were lightly washed with PBS. The powdered stomach was suspended in Lysis buffer attached to QIAGEN RNAeasy kit, and RNA was extracted according to the attached protocol. The extracted RNA was reverse-transcribed using a SuperScript III enzyme to obtain cDNA. The cDNA was examined by performing quantitative PCR using pIgR-specific primers. The stomach two weeks after infection was compared with a germ-free mouse according to the above-described intracellular cytokine production protocol. The results are shown in FIGS. 11A to 11C.

In order to confirm the infection of Helicobacter pylori, the CagA protein secreted by the bacterium was detected, and it was confirmed that the protein was clearly present at 2 weeks after the infection, whereas the protein was confirmed at 9 weeks after the infection. No CagA protein was detected in the small intestine (SI) at any time point (FIG. 11-A (a)), and the number of ILC2 cells in the stomach of Helicobacter pylori-infected mice was Maximized at 2 weeks post-infection (FIGS. 11-A (b), 11-B (a) and (b)), while PMSS1 in the stomach as shown in FIG. 11-A (c). CFU peaked at 1 week post-infection, B cells increased significantly at 2 weeks post-infection, and cell numbers remained elevated at 9 weeks post-infection. Was On the other hand, T cells did not increase until 3 weeks after infection (FIGS. 11-A (d), (e), (f) and FIG. 11-B (c)). At the time point, ILC2 expressing IL-5 or IL-13 was significantly increased (Fig. 11-C (a) and (b)). At two weeks after infection, IL- The production amount of Helicobacter pylori was significantly increased (FIG. 11-C (c)), and it was strongly suggested that cytokine production by ILC2 is important as a response immunity to Helicobacter pylori. When mice were treated with anti-IL-5 antibody, CD19 ⁻ B220 ⁻ IgA ⁺ plasma B cells and CD19 ⁺ B220 ⁻ IgA ⁺ plasmablast B cells in the stomach were significantly reduced at 2 weeks post infection ( Figures 11-C (d) and (e)) have been shown to result in a concomitant reduction in IgA secretion in gastric tissue (Figure 11-B (d)). As shown, IgA binding to Helicobacter pylori was highest at 2 weeks post infection and then decreased, with a significant decrease in Helicobacter pylori at 9 weeks post infection due to clearance of the bacteria by IgA. In addition, as shown in Fig. 11-C (f), the expression level of pIgR, which is important for making IgA secretory, is enhanced in the stomach as compared with the expression level in the intestinal tract. This is also considered to be indirect evidence that IgA is involved in eliminating Helicobacter pylori.

The above suggests that the following protective responses to Helicobacter pylori infection occur: (1) production of IL-33 by gastric tissue, (2) activation of ILC2 by IL-33, ( 3) production of IL-5 and / or IL-13 by ILC2, (4) activation of B cells by IL-5 and / or IL-13 produced by ILC2, (5) production of IgA by B cells, And (6) binding and clearance of Helicobacter pylori by IgA.

[Example 10] Investigation of the amount of bacterial-bound IgA in the stomach of vancomycin-treated mice The stomach contents and the gastric mucus layer of SPF mice treated with vancomycin-administered mice for 3 weeks and SPF mice were collected as described above. The collected sample was suspended by adding 1 ml of sterile PBS to 1 g, and then centrifuged at 8000 g for 5 minutes. The supernatant was collected as a bacterial layer and centrifuged again at 15,000 g for 15 minutes. The precipitate was collected as IgA-bound bacteria, washed with 1% FBS / PBS, and blocked with 2.4G2 antibody for 15 minutes. After washing with 1% FBS / PBS, the cells were stained four times with an anti-IgA antibody conjugated with PE as a fluorescent dye for 30 minutes. After staining, the cells were washed three times with PBS, stained with DAPI for 5 minutes, and finally washed once again with PBS, and subjected to flow cytometry analysis.

As shown in FIG. 12, the amount of IgA bound to bacteria in the stomach contents and gastric mucus (biofilm) of vancomycin-treated SPF mice was lower than that of control (vancomycin-untreated SPF mice). It has been shown. This means that vancomycin treatment reduces the number of S24-7 individuals in the stomach and concomitantly reduces the number of ILC2 cells. The decrease in ILC2 reduces the production of IL-5 and / or IL-13 and reduces B cell activation. As a result, it is considered that the amount of IgA secreted in the stomach was reduced. Thus, this example demonstrates that commensal bacteria of the family S24-7 can directly affect secretory IgA levels in the stomach.

[Example 11] Induction of gastric ILC2 and IgA by Muribabaculum intestinale A type strain, Muribaculum intestinale (also referred to as “YL27”), was orally administered once to a GF mouse once at 1 × 10 ⁸ CFU / mouse, and two weeks after the infection, Mice were dissected and stomach cells were analyzed by flow cytometry. FIG. 13A shows an outline of this experiment, and FIGS. 13B to 13D show the experimental results.

される As shown in FIG. 13, in GF mice infected with YL27, ILC2 present in the stomach was significantly increased. Type 2 cytokines (IL-5 and IL-13) were also increased. Therefore, YL27 was shown to be a bacterium capable of inducing ILC2 in the stomach.

Furthermore, the expression of plgR in the stomach tissue of the infected mouse was confirmed by qPCR. At the same time, the amount of IgA contained in the stomach contents and feces was confirmed by ELISA. FIG. 14 shows the results.

ＰAs shown in FIG. 14, in the stomach tissue of the mouse infected with Y27, the expression level of plgR was significantly increased (FIG. 14 (a)). In addition, IgA levels were increased in both the stomach contents and feces of the mice infected with Y27.

[Example 12] Confirmation of presence of YL27 in stomach It was confirmed that YL27 was present in the stomach of a mouse infected with YL27. A fluorescent dye (Alexa488) was bound to a probe (EUB338 (5′-GCTGCCTCCCGTAGGAGT-3 ′: SEQ ID NO: 29)) for detecting 338-355 of the 16s rRNA of Y27, and YL27 was obtained by the FISH method (see above for details of the protocol). Was confirmed in the stomach, and the results are shown in FIG.

通り As shown in FIG. 15, it was shown that YL27 was present in the stomach of YL27-infected mice.

Example 13 Examination of YL27 Infection Prevention Effect on H. pylori Infection was performed by orally administering YL27 (1 × 10 ⁸ / mouse) to sterile mice. One week after the administration, YL27 (1 × 10 ⁸ / mouse) was administered again. The day after the second administration of YL27, H. pylori was orally infected. Two weeks after H. pylori infection, the infected mice were dissected, and (1) IgA production in the stomach contents or feces of the infected mice, (2) mRNA expression of pIgR in the stomach of the infected mice, and (3) The amount of H. pylori in the stomach tissue of infected mice was analyzed. The analysis method used in (1) and (2) is as described above. The outline of the analysis method (3) is as follows: Two weeks after the infection with H. pylori, the mice were sacrificed and the stomach was collected. The contents of the collected stomach were removed, and the weight of the stomach tissue was measured, cut into small pieces with scissors, and suspended in a H. pylori (PMSS1) culture liquid medium. The suspension was allowed to stand for a while, and after removing large tissues, the suspension was cultured for 2 days on an agar medium for culturing H. pylori (BD BBLPlate).

実験 The outline of the experiment schedule of this example is shown in FIG. 16, and the results of (1) to (3) are shown in FIGS. 17 to 19, respectively.

される As shown in FIG. 17, the IgA production was not significantly different in the stomach contents, but the IgA amount was increased, and the IgA amount in feces was significantly increased. In addition, as shown in FIG. 18, the expression of pIgR in gastric tissue was also significantly induced by administration of YL27 before infection.

Furthermore, as shown in FIG. 19, the number of H. pylori present in the stomach of the mice to which YL27 had been administered in advance was significantly reduced as compared with the control.

The above results indicate that YL27 can suppress H. pylori infection. In other words, it was shown that ingestion of YL27 could prevent H. pylori infection.

According to the present invention, it is possible to induce the expression of ILC2, a type of immunocompetent cell, in the stomach of a subject. The induced ILC2 produces / secretes cytokines such as IL-5 and IL-13 and stimulates B cells present in the stomach. The stimulated B cells are differentiated into antibody-producing cells, and as a result, IgA production in the stomach of the subject is induced and enhanced, so that oral infections including H. pylori can be treated and / or prevented. Therefore, the present invention is very useful in the field of medicine and the like.

This application is based on Japanese Patent Application No. 2018-189489 filed in Japan (filing date: October 4, 2018), the contents of which are incorporated in full herein.

Claims

An ILC2 inducer in the stomach comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
2. The inducer of claim 1, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
誘導 The inducer according to claim 1 or 2, wherein the bacterium is a bacterium belonging to the family S24-7.
The inducer according to any one of claims 1 to 3, wherein the bacterium comprises the following nucleotides (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
An inducer of IgA production in the stomach comprising at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
The production inducer according to claim 5, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
7. The production inducer according to claim 5, wherein the bacterium is a bacterium belonging to the family S24-7.
The production inducer according to any one of claims 5 to 7, wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
An agent for treating or preventing an oral infection, comprising a bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
The therapeutic or prophylactic agent according to claim 9, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
治療 The therapeutic or preventive agent according to claim 9 or 10, wherein the bacterium is a bacterium belonging to the family S24-7.
The therapeutic or prophylactic agent according to any one of claims 9 to 11, wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
The therapeutic or prophylactic agent according to any one of claims 9 to 12, wherein the oral infection is at least one of infections selected from the following group:
H. pylori infections, O: 157 infections, and Salmonella infections.
A method for inducing ILC2 in the stomach, comprising orally administering to a subject at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
15. The method according to claim 14, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
誘導 The induction method according to claim 14 or 15, wherein the bacterium is a bacterium belonging to the family S24-7.
The induction method according to any one of claims 14 to 16, wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
A method for inducing IgA production in the stomach, comprising orally administering to a subject at least one bacterium having the following properties:
(1) sensitivity to vancomycin, and (2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
19. The method of inducing production according to claim 18, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
The production induction method according to claim 18 or 19, wherein the bacterium is a bacterium belonging to the family S24-7.
The production induction method according to any one of claims 18 to 20, wherein the bacterium comprises the following nucleotides (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
A method for treating or preventing an oral infection comprising orally administering to a subject a bacterium having the following properties:
(1) sensitivity to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole.
23. The method of treatment or prevention according to claim 22, wherein the bacterium further has the following properties:
(3) It has the nucleotide sequence shown in SEQ ID NO: 1.
24. The method according to claim 22 or 23, wherein the bacterium is a bacterium belonging to the family S24-7.
The treatment or prevention method according to any one of claims 22 to 24, wherein the bacterium comprises the following nucleotide (1) or (2):
(1) a nucleotide represented by SEQ ID NO: 28, or
(2) a nucleotide having at least 90% or more identity to the nucleotide shown in SEQ ID NO: 28;
The treatment or prevention method according to any one of claims 22 to 25, wherein the oral infection is at least one of infections selected from the following group:
H. pylori infections, O: 157 infections, and Salmonella infections.