JP7444459B2 - Method for inducing IgA production targeting ILC2 - Google Patents

Description

Application of Article 30, Paragraph 2 of the Patent Act ▲1▼December 14, 2017 Presented at the 46th Academic Meeting of the Japanese Society of Immunology at the Sendai International Center ▲2▼May 22, 2018 https://confit. atlas. jp/guide/event/jscbjsdb2018/subject/P3-1F-126/tables? cryptoId=https://confit. atlas. Presented through jp/guide/event/jscbjsdb2018/proceedings/list ▲3▼June 8, 2018 Presented at the 70th Japanese Society of Cell Biology, Tower Hall Funabori

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

The stomach remains highly acidic, and until now it was thought that its role as an organ was only to digest and sterilize food. Starting with reports that Helicobacter pylori (also referred to as ``H. pylori'' or ``H. pylori'') colonized the stomach, attention began to be focused on the relationship between the stomach and Helicobacter pylori. Most of the research is related to the ``stomach as a tissue.''

In fact, the research on the stomach and bacteria reported in various scientific journals mainly focuses on the mechanism of gastric cancer development due to Helicobacter pylori infection, and the elucidation of the infection mechanism from the structure of Helicobacter pylori itself. As far as immune responses are concerned, there are a few groups conducting research on T cell responses and cancer, but so far there have been few reports on the relationship between commensal bacteria present in the stomach and immunocompetent cells.

Furthermore, in recent years, it has been found that innate lymphoid cells (ILCs) play an important role in maintaining homeostasis in various tissues within the body. These ILCs are classified into three subsets, ILC1, ILC2, and ILC3, depending on the characteristics of the cells and the expression of transcription factors (Non-Patent Documents 2 to 6). Research on these ILCs has revealed for the first time the mechanism of disease onset that could not be explained by acquired immunity alone, and these cell groups are currently attracting attention around the world (Non-Patent Document 1).

However, research by the present inventors has revealed that the stomach also plays an important immunological role (Non-Patent Document 7), and that this response is mainly due to ILC2 induced by bacteria. It's here. The stomach is not only an organ for breaking down and sterilizing food, but it is also important to chemically and immunologically control the risk of infection, which increases with food intake, and to screen the bacterial flora before it reaches the intestinal tract. It was suggested that this is a similar organ. However, at present it is not clear what kind of bacteria induces ILC2.

SpitsH. et al. , Nat Rev Immunol. 2013 Feb; 13(2):145-9. Satoh-Takayama N. et al. , Immunity. 2008 Dec 19;29(6):958-70. Satoh-Takayama N. et al. , Immunity. 2014 Nov 20;41(5):776-88. Maloy KJ. et al. , Immunity. 2013 Apr 18;38(4):630-2. Moro K. et al. , Nature. 2010 Jan 28;463(7280):540-4. Neill DR. et al. , Nature. 2010 Apr 29;464(7293):1367-70. Muhammad JS. et al. , J Pak Med Assoc. 2012 Sep;62(9):955-9.

The purpose of the present invention is to clarify the role of immunocompetent cells (particularly ILC2) present in the stomach and their relationship with the commensal bacterial flora of the stomach, thereby leading to the development of new anti-infective drugs. do.

As a result of intensive investigation into the above issues, the present inventors found that (1) the presence of commensal bacteria in the stomach can affect the amount of ILC2 in the stomach; (2) the presence of commensal bacteria in the stomach can affect the amount of ILC2 in the stomach (3) the bacteria leading to the induction of ILC2 in the stomach are sensitive to vancomycin and sensitive to ampicillin, colistin, neomycin, and metronidazole; (4) The bacteria that mainly induce ILC2 in the stomach belong to the S24-7 family; (5) The bacteria that mainly induce the induction of ILC2 in the stomach belong to the S24-7 family. , a bacterium (eg, Muribaculum intestinale) having the nucleotide sequence represented by SEQ ID NO: 28, and further research based on this knowledge led to the completion of the present invention. That is, the present invention is as follows.

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

According to the present invention, ILC2 can be induced in the stomach of mammals. Furthermore, as a result of inducing ILC2, IgA production in the stomach can be enhanced. This makes it possible to treat and/or prevent diseases caused by infection with microorganisms that can be removed by IgA, including Helicobacter pylori.

FIG. 1 is a diagram showing the results of an examination of the abundance ratio of various ILCs in the mouse stomach. (a) Outline of this example. (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 the small intestine and stomach. (b) Percentage of IL-33Rα expressing cells in the small intestine and stomach. FIG. 3 is a diagram showing the influence of commensal bacteria on gastric ILC2. (a) Outline of this example. (b) and (c) ILC2 cell counts in SPF and GF mice. (d) Percentage of IL-33Rα expressing cells in SPF and GF mice. FIG. 4 shows the influence of commensal bacteria on the function of gastric ILC2. (a) and (b) Cell numbers of IL-5-expressing cells or IL-13-expressing cells in the stomach in SPF mice and GF mice. (c) and (d) Cell counts of CD3 and CD4 positive cells (i.e. T cells) and CD19 and B220 positive cells (i.e. B cells) in the stomach in SPF and GF mice. Under normal conditions, no changes were observed 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 this example. (b), (c) and (d) Changes over time in the number of IL-5-expressing cells or IL-13-expressing cells in the stomachs of GF mice that orally ingested the stomach-derived microbiota of SPF mice. FIG. 6 is a diagram showing changes over time in the bacterial flora in the stomach or feces of GF mice due to oral ingestion of stomach-derived microflora from SPF mice. (a) Changes over time in the proportion of bacterial species in the stomach or feces of GF mice. (b) Changes in the abundance ratio of Bacteroidetes over time. (c) Changes in the abundance ratio of the S24-7 family over time. FIG. 7 is a diagram showing the drug resistance characteristics of commensal bacterial groups that affect the induction of gastric ILC2. (a) Outline of this example. (b) and (c) Changes in ILC2 cell numbers after antibiotic treatment. FIG. 8 is a diagram showing characteristics related to drug resistance possessed by various bacterial groups. (a) Changes in the abundance of various bacterial groups after antibiotic treatment. (b) Changes in the 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 population of each bacteria. (a) Heat map profile of the relative abundance of each bacterium at the family level in the stomach contents from mice treated with each antibiotic. (b) Scatter profile of each bacterial population and ILC2 count at the family level in stomach contents from mice treated with antibiotics (ampicillin, neomycin, metronidazole, and colistin) or SPF mice (control). . FIG. 11-A is a diagram showing various data regarding the immune response to Helicobacter pylori infection. (a) Detection of CagA protein in H. pylori infected mice. (b) Time course of ILC2 cell number after H. pylori infection. (c) H. pylori CFU present in mouse gastric tissue after H. pylori infection. (d) to (f) Time course of B cell and T cell numbers after H. pylori infection. FIG. 11-B is a diagram showing various data regarding the immune response to Helicobacter pylori infection. (a) and (b) Time course of ILC2 cell number in the stomach after H. pylori infection. (c) Time course of cell numbers of B cells and T cells in the stomach after H. pylori infection. (d) Changes in IgA amount over time due to anti-IL-5 antibody treatment. (e) Changes in IgA amount over time after infection with Helicobacter pylori. FIG. 11-C is a diagram showing various data regarding the immune response to Helicobacter pylori infection. (a) and (b) Time course of the number of IL-5 or IL-13 producing ILC2 cells after H. pylori infection. (c) Changes in the amount of IL-33 in the stomach after H. pylori infection over time. (d) and (e) Reduction of IgA-producing cells by anti-IL-5 antibody treatment. (f) Changes in the expression level of pLgR over time after infection with Helicobacter pylori. FIG. 12 is a diagram showing the amount of bacteria-bound IgA in the stomach of vancomycin-treated mice. (a) and (b) Amount of IgA antibodies binding to bacteria in gastric contents and mucus in SPF and vancomycin-treated mice. FIG. 13 is a diagram showing various data regarding the immune response to infection with Muribaculum intestinalale (YL27). (a) Outline of this example. (b) and (c) Time course of ILC2 cell number in the stomach after YL27 infection. (d) Changes over time in the number of ILC2 cells producing IL-5 or IL-13. FIG. 14 is a diagram showing changes in the expression level of pIgR and the amount of IgA produced in stomach contents or feces in response to YL27 infection. FIG. 15 is a photographic diagram showing that YL27 infected with GF mice adheres to the stomachs of GF mice. FIG. 16 is a diagram showing an overview of Example 12. FIG. 17 is a diagram showing the amount of IgA produced in the stomach contents or feces of mice infected with YL27 and then infected with Helicobacter pylori. FIG. 18 is a diagram showing changes in the expression level of pIgR in the stomachs of mice infected with YL27 and then infected with Helicobacter pylori. FIG. 19 is a diagram showing the suppression of Helicobacter pylori infection by YL27 based on the measurement of the amount of Helicobacter pylori present in the stomach.

1. ILC2 Inducer The present invention provides an ILC2 inducer in the stomach (hereinafter sometimes referred to as "ILC2 inducer of the present invention") comprising at least one type of bacteria having the following properties:
(1) Susceptibility 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 type of immunocompetent cell, in the stomach of the subject to which it is applied, depending on the function of the specific bacteria added.

The bacteria included in the ILC2 inducer of the present invention are characterized as bacteria that are sensitive to vancomycin and resistant to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. can be attached. In one embodiment, the bacteria included in the ILC2 inducer of the present invention are susceptible to vancomycin and resistant to at least two drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. Preferably, the bacteria may be susceptible to vancomycin and resistant to at least three drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole, and more preferably, The bacterium may be susceptible to vancomycin and resistant to at least three drugs selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole, particularly preferably susceptible to vancomycin and , ampicillin, colistin, neomycin, and metronidazole, all of which can be resistant to bacteria. In one embodiment, the bacterium included in the ILC2 inducer of the present invention is characterized by having the nucleotide sequence shown in SEQ ID NO: 1. In another embodiment, the bacteria included in the ILC2 inducer of the present invention are bacteria belonging to the S24-7 family.

Bacteria belonging to the S24-7 family means Gram-negative bacteria belonging to the S24-7 family, order Bacteroidales, phylum Bacteroidetes. Bacteria belonging to the S24-7 family have been reported to coexist widely with warm-blooded animals (Ormerod KL et al., Microbiome. 2016 Jul 7;4(1):36.). Note that in this specification, warm-blooded animals include mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, and humans, but bacteria belonging to the S24-7 family are exemplified. The animal is not particularly limited as long as it is a warm-blooded animal with which bacteria having the drug properties described above can coexist. In addition, as shown in the following example, bacteria belonging to the S24-7 family are sensitive to vancomycin and resistant to all of ampicillin, colistin, neomycin, and metronidazole. The existence can be confirmed based on . Alternatively, as shown in the Examples below, a bacterium belonging to the S24-7 family may have the nucleic acid sequence shown in SEQ ID NO: 1. Therefore, its existence may also be confirmed by using the nucleic acid sequence shown in SEQ ID NO: 1 as a probe.

In one aspect of the invention, the bacterium can be, but is not limited to, Muribaculum intestinale (also referred to as "YL27", etc.). Note that YL27 has 16s rRNA consisting of the sequence represented by SEQ ID NO: 28. Therefore, in one aspect, the bacteria used in the present invention are (1) bacteria containing the nucleotide shown in SEQ ID NO: 28, or (2) at least 90% (more preferably 91%) of the nucleotide shown in SEQ ID NO: 28. % or more, 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).

The bacteria contained in the ILC2 inducer of the present invention are bacteria that coexist with warm-blooded animals and exist in their stomachs, small intestines, large intestines, feces, and the like. Therefore, the bacteria to be incorporated into the ILC2 inducer of the present invention can be easily obtained by collecting bacteria from such bacterial sources. In one embodiment, bacteria collected from such a bacterial source are suspended in pure water or a buffer solution to form a bacterial suspension, and then the bacteria are suspended using a method known per se (for example, JP 2015-188407, JP 2012-175973, JP 2012-175973, JP 2010-041967, International Publication WO 2007/023711, JP 2005-229837, etc.). Further, during these cultures, unnecessary microbial contamination may be removed by adding an appropriate antibiotic or the like. Specifically, since the bacteria included in the ILC2 inducer of the present invention are resistant to ampicillin, colistin, neomycin, and metronidazole, at least one of these, preferably at least two, and more preferably By adding at least three, particularly preferably these four types of drugs during culture, it is possible to increase the purity of the bacteria contained in the inducer of the present invention.

A specific example of a method for preparing bacteria to be incorporated into the ILC2 inducer of the present invention is a method in which the abdomen of a warm-blooded animal (for example, a mouse) is opened and the contents and/or mucus contained in the gastrointestinal tract are collected. Ru. The collected contents and/or mucus contain bacteria that are susceptible to vancomycin and resistant to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. . Therefore, the contents and/or mucus can be used as an active ingredient as is, or among the bacteria contained therein, those bacteria are susceptible to vancomycin and are selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. Bacteria that are resistant to at least one selected drug can also be isolated and/or cultured using the methods described above and then formulated as an active ingredient. In this specification, "isolation" means that an operation has been performed to remove factors other than the target bacteria, and the state that exists in nature has been removed. In addition to the embodiment in which the target bacteria are completely removed, it may also include an embodiment in which the number of target bacteria is the majority, and other bacteria are present in trace amounts.

The amount of bacteria added to the ILC2 inducer of the present invention is not particularly limited as long as the 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. The content is 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.

Suitable subjects for application of the ILC2 inducer of the present invention are not particularly limited as long as they are warm-blooded animals that have ILC2 as immunocompetent cells in the stomach, but are preferably mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, Bovine, equine, ovine, simian, and human, most preferably human.

The dosage form of the ILC2 inducer of the present invention is not particularly limited as long as the desired effect can be obtained, and it can be made into an oral preparation together with excipients commonly used in pharmaceuticals. The dosage form of the oral preparation is not particularly limited as long as the desired effect can be obtained, but it may be semi-solid or liquid so that the bacteria contained in the ILC2 inducer of the present invention can remain viable in the preparation. It is preferable that there be. Orally administered preparations may contain excipients and/or additives commonly used in pharmaceuticals.

Alternatively, the ILC2 inducer of the present invention may be provided in the form of a food composition. When provided as a food composition, the food composition may be in a semi-solid or liquid form so that the bacteria incorporated in the ILC2 inducer of the present invention can remain viable in the food composition. preferable. Examples of such food compositions include, but are not limited to, yogurt, jelly, drinks, 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. This method can be carried out by orally administering the above-described ILC2 inducer to a subject.

2. IgA Production Inducer The present invention also provides an IgA production inducer in the stomach (hereinafter sometimes referred to as "IgA production inducer of the present invention") comprising at least one type of bacteria having the following characteristics:
(1) Susceptibility 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 the subject to which it is applied, depending on the function of the specific bacteria 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, it is possible to induce and enhance IgA production in the stomach of the subject.

Regarding the bacteria to be incorporated into the IgA production inducer of the present invention, its drug characteristics, its preparation method, its blending amount, as well as the applicable targets and dosage form of the IgA production inducer of the present invention, please refer to "1. ILC2 inducer". It can be similar to that explained in .

In one aspect, the present invention provides a method for inducing IgA production in the stomach of a subject, which comprises orally administering to the subject the IgA production inducing agent of the present invention. 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 food product. When provided as a food composition, the food composition must be semi-solid or liquid so that the bacteria incorporated in the IgA production inducer of the present invention can remain viable in the food composition. is preferred. Examples of such food compositions include, but are not limited to, yogurt, jelly, drinks, and the like.

3. Treatment and/or prevention agent for oral infections The present invention also provides a treatment or prevention agent for oral infections (hereinafter referred to as "the treatment or prevention agent of the present invention"), which contains at least one type of bacteria having the following characteristics. ):
(1) Susceptibility to vancomycin, and
(2) Resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole. The therapeutic or preventive agent of the present invention can induce the expression of ILC2, which is a type of immunocompetent cell, in the stomach of the subject to which it is applied, depending on the function of the specific bacteria 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 subject is induced and enhanced, thereby achieving treatment and/or prevention of oral infections including Helicobacter pylori.

Regarding the bacteria to be incorporated into the therapeutic or prophylactic agent of the present invention, its drug properties, its preparation method, its blending amount, and the applicable targets and dosage forms of the IgA production inducer of the present invention, please refer to "1. ILC2 inducer". It can be similar to that explained in .

The therapeutic or preventive 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. Considering this point of view, the therapeutic or preventive agent of the present invention can be suitably used for Helicobacter pylori infection among oral infections. Furthermore, since S24-7 is also detected as a major bacterium in the intestinal tract, it is possible that it may protect the intestinal tract from infectious enteritis caused by infectious bacterial species such as O:157 and Salmonella infection. Therefore, in one embodiment, the therapeutic or preventive agent of the present invention can be used to treat or prevent Helicobacter pylori infection, O:157 infection, and Salmonella infection.

In one embodiment, the therapeutic or preventive agent of the present invention can be provided in the form of a pharmaceutical or food product. When provided as a food composition, the food composition must be semi-solid or liquid so that the bacteria incorporated in the therapeutic or preventive agent of the present invention can remain viable in the food composition. is preferred. Examples of such food compositions include, but are not limited to, yogurt, jelly, drinks, and the like.

The therapeutic agent of the present invention is applied to patients suffering from the above-mentioned oral infections. Furthermore, the preventive agent of the present invention is applied to healthy subjects or subjects who have been cured of the above-mentioned oral infections.

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

Furthermore, the prophylactic agent of the present invention is administered in an amount that allows the subject to ingest a prophylactically effective amount of bacteria of the S24-7 family. The prophylactically effective amount may vary depending on the height, weight, sex, administration route, administration schedule, etc. of the subject to whom the prophylactic agent is administered, and can be appropriately determined by those skilled in the art.

In one aspect, the present invention provides a method for treating or preventing oral infections, which comprises orally administering the therapeutic or preventive agent of the present invention to a subject. This method can be carried out by orally administering the above-mentioned oral infectious disease treatment or prevention agent to a subject.

Note that "treatment" as used herein may include not only cure of a disease but also remission of the disease and improvement of the degree of the disease.

In addition to preventing the onset of the disease, "prevention" as used herein includes delaying the onset of the disease, and comparing the condition of the subject at the onset of the disease with the normal state of onset of the disease. This includes mild symptoms. In addition, "prevention" as used herein includes preventing the recurrence of the disease after treatment, delaying the recurrence of the disease after treatment, and the condition of the subject at the time of recurrence of the disease after treatment. It also includes being mild compared to the condition at the time of recurrence of the disease.

The present invention will be explained in more detail in the following examples, but the present invention is not limited to these examples in any way.

[Materials and methods]
1. mouse
C57BL/6N mice were purchased from CLEA (CLEA JAPAN, INC.) and kept at RIKEN under specific pathogen-free (SPF) conditions for over 4 weeks. Germ-free (GF) mice with a C57BL/6N background were purchased from CLEA and maintained in sterile vinyl isolators at RIKEN or Yokohama City University. Rag2 ^−/− mice (N. Satoh-Takayama et al., Microbial flora drives interleukin 22 production in international NKp46+ cells that provide Innate mucosal immune defense. Immunity 29, 958-970 (2008)), Il-33 ^gfp/gfp mouse (K. Oboki et al., IL-33 is a crucial amplifier of innate rather than acquired immunity. Proc Natl Acad Sci USA 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 ^{gf p} /t mouse (G. Eberl et al., An essential function for the nuclear receptor RORgamma(t) in the generation of fetal lymphoid tissue inducer cells. Nat Immunol 5, 64-73 (2004)) by Dr. Di Santo, Nakae , Dr. Malissen, and Dr. Littman, respectively. All mice (8-18 weeks old) were used in experiments that adhered to the guidelines provided by the RIKEN Yokohama Branch Animal Care and Use Committee or the Yokohama City University Research Committee, and in accordance with Japanese law. It was conducted.

2. Cell Preparation, Flow Cytometry Analysis, and Intracellular Staining The gastric tissue was opened immediately after being picked up from the mouse body, and then the gastric contents were gently washed 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 cytometry analysis of small intestinal lamina propria lymphocytes (LPL) was performed by N. Satoh-Takayama et al. , Lymphotoxin-beta receptor-independent development of experimental IL-22-producing NKp46(+) innate lymphoid cells. Eur J Immunol 41, 780-786 (2011) and Y. Sasagawa et al. , Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expressio n heterogeneity. This was performed using the method described in Genome Biol 14, R31 (2013). For flow cytometry analysis, after blocking Fc receptors with 2.4G2 mAb (BioLegend), all cells were stained with each surface antibody at 4°C, and dead cells were stained with LIVE/DEAD fixable Aqua Dead Cell Stain (Thermo Fisher). Surface or intracellular staining was performed using specific fluorescent dye-conjugated antibodies (BioLegend, eBioScience and SouthernBiotech, see Table 1 below). 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 or without PMA (50 ng/ml) and ionomycin (2 μg/ml) in the presence of GolgiPlug (BD biosciences) for 2 h at 37 °C. did. After staining with surface antibodies, the incubated cells were fixed with PBS containing 4% PFA for 15 minutes at room temperature. Fixed cells were permeabilized with 1× permeabilization buffer (included in the Foxp3/TF buffer set (Thermo Fisher Scientific)) for 60 min at 4°C. For transcription factor detection in cells, isolated cells were stained with intracellular antibodies using the Foxp3/TF staining buffer set according to the manufacturer's instructions (Thermo Fisher Scientific). For cell analysis and sorting, cells were analyzed on a FACSAriaIII (BD Biosciences). For all lymphocyte analyzes by flow cytometry, lymphocytes were first defined by FSC and SSC intensity and then carefully gated based on CD45 expression. Data were analyzed with FlowJo software (TreeStar).

3. RNA-seq and quantitative PCR
For RNA-seq, 1000 cells extracted from stomach or small intestine were sorted directly into 50 μl of lysis buffer (QIAGEN) using a FACS AriaIII (BD Biosciences) at 98% or higher purity. All steps after sorting followed the Quartz-seq method (library; KAPA library preparation kit, illumina; adapter; Next Multiplex Oligo for illumina, NEB) with slight modifications (details in Y. Sas Agawa et al. , Quartz-Seq: a highly reproducible and sensitive single-cell RNA sequencing method, reveals non-genetic gene-expressio n heterogeneity. Genome Biol 14, R31 (2013)). All samples were sequenced on HiSeq1500, rapid mode. For quantitative PCR analysis, total RNA was extracted from purified gastric or intestinal cell preparations using the RNeasy micro kit (QIAGEN). For whole tissue RNA expression analysis, stomach or intestine was frozen in liquid nitrogen. Frozen tissue was triturated in liquid nitrogen and RNA was extracted from the powdered tissue using lysis buffer (QIAGEN). cDNA was generated using reverse transcriptase (Revatra Ace, TOYOBO) according to the manufacturer's instructions and then using primers (Eurofins genomics; see Table 2) or TaqMan probes for Il25 and Il33 (Thermo Fisher Scientific; see Table 3). ), quantitative PCR analysis was performed using RT SYBR Green qPCR MasterMix (TAKARA SYBR Premix ExTaq II).

4. RNA-seq read alignment and Gene Qualification
Differential gene expression was analyzed using the DEseq package in R software, and in each analysis these genes were identified by log2 fold change Value, p-Value and FDR correction value. The 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 the supernatant. DNA extraction was performed using T. Jinnohara et al. , IL-22BP dictates characteristics of Peyer's patch follicle-associated epithelium for antigen uptake. J Exp Med, (2017) and T. Kato et al. , Multiple omics uncovers host-gut microbial mutualism during prebiotic fructooligosaccharide supplementation. This was carried out using the method described in DNA Res 21, 469-480 (2014) with some modifications. For analysis, the V4 variable region (515F-806R) was sequenced with an Illumina Miseq library prepared according to the manufacturer's protocol (Takara Bio, Inc). A sample library with 20% denatured PhiX (spike-in) was sequenced using the Miseq 500 cycle kit, yielding 2 x 250 bp paired-end reads.

Taxonomic assignment and relative abundance estimation of sequenced data was performed using the analysis pipeline of the QIIME software package. Chimera check was performed using UCHIME. Operational taxonomic units (OTUs) were defined at 97% similarity. OTU is T. Kato et al. , Multiple omics uncovers host-gut microbial mutualism during prebiotic fructooligosaccharide supplementation. Taxonomy was assigned based on comparison in the Greengenes database using the RDP classifier described in DNA Res 21, 469-480 (2014).

6. Antibiotic treatment, gastric contents and mucus preparation, and transplantation experiments Mice in SPF conditions were supplemented with ampicillin (0.1 g/L), colistin (1 g/L), or vancomycin (0.5 g/L) in their drinking water. , orally for 3 weeks. Stomach contents and mucus were obtained from C57BL/6N mice under SPF conditions or antibiotic-treated mice as controls. The opened stomach was gently washed in sterile PBS. The collected contents were homogenized and vortexed thoroughly. The tube was left undisturbed for 30 seconds to remove large particles. For analysis of the mucus area, the gastric tissue was scraped with a curved needle after washing the gastric contents. After spinning down to remove epithelium and large particles, the supernatant was used for gastric mucus analysis. For oral implantation experiments, GF mice received 300 μl of a mixture of gastric contents and mucus orally once or weekly for 4 weeks. To reduce the gut microbiota, CD3ε ^+/+ and CD3ε ^−/− pregnant female mice from the same litter were treated with ampicillin (0.1 g/L), vancomycin (1 g/L), neomycin (1 g/L), Antibiotic treatment was performed by adding an antibiotic cocktail containing 1 g/L and metallonidazole to the drinking water and ingesting the mouse orally until the pups were weaned (21 days after birth).

7. Evaluation of IgA-coated bacteria by flow cytometric analysis Gastric contents were obtained from control GF mice or H. pylori infected mice. The opened 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. Bacteria, S. Kawamoto et al. , Foxp3(+) T cells regulate immunoglobulin a selection and facilitate diversification of bacterial species responsible for i mmune homeostasis. Stained with PE-conjugated anti-mouse IgA antibody and DAPI as described in Immunity 41, 152-165 (2014). After washing with PBS, bacteria were suspended in FACS buffer and analyzed using FACSAriaIII.

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

9. Immunohistology Immunohistological analysis of 8 μm frozen sections of the stomach was performed using 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) and N. Satoh-Takayama et al. , Lymphotoxin-beta receptor-independent development of intestinal IL-22-producing NKp46+ innate lymphoid cells. The method described in Eur J Immunol 41, 780-786 (2011) was used. As the primary antibody, 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 mean ± s. e. m. Indicated by Statistical differences between groups were determined using an unpaired Student's t-test. One-way ANOVA was used for statistical comparisons between two or more groups. A P value <0.05 was considered statistically significant. All data were analyzed using Graph-pad Prism software.

[Reference Example 1] Examination of the abundance ratio of various ILCs in the stomach The small intestine of SPF mice was treated with Collagenase (Wako) adjusted to a concentration of 1 mg/L in 2% FBS/RPMI-1640 medium, and the large intestine was also treated with 1 mg/L. The mixture was stirred at 37° C. for 15 minutes in Collagenase (SIGMA) adjusted to a L concentration. After 15 minutes, only the supernatant was collected, and then Collagenase was added to the remaining intestinal fragments, and the same operation was repeated twice to collect 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, suspend in 4 ml of 40% Percoll and transfer to a 15 ml Falcon tube. Using a Pasteur pipette, slowly add an equal amount of 70% Percoll to layer the cells downward, and incubate at 2000 rpm at 20°C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer produced by the density gradient was collected and suspended in 2% FBS/RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cellstrainer 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 plated at 1×10 ⁶ cells/well on a Tissue Culture Plate (96 Well, Flat Buttom with Low Evaporation Lid, FALCON). Sowed 1 each . After centrifugation, Purified Rat Anti-mou was adjusted to a final concentration of 0.1 mg/ml using 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS/D-PBS (-)). se CD16 /CD32 (BD Biosciences) was added and allowed to stand at 4°C for 15 minutes to perform blocking and staining of dead cells. After that, the cells were washed with Facs buffer, and after centrifugation, each antibody dilution solution (CD3, CD45, TCRb, CD19, CD127, CD90.2) was added to the cells and left to stand at 4°C for 20 minutes to remove the cell surface. The antigen expressed in the cells was stained with a fluorescently labeled antibody.

For antigens expressed within cells, staining was performed using an intracellular staining method. After staining with cell surface antibodies, the cells were washed with D-PBS(-). After centrifugation, Fixation/Permeabilization solution (Foxp3/Transcription Factor Staining Buffer Set, ThermoFisher) was added, and the cells were left to stand at 4°C for 30 minutes in the dark to perform cell fixation and cell membrane permeation. It was. After fixing the cells, the cells were washed with D-PBS(-), and the supernatant was removed by centrifugation. Next, the cells were washed again with Permeabilization Buffer (Foxp3/Transcription Factor Staining Buffer Set, ThermoFisher). After centrifugation, each intranuclear staining antibody (GATA3 and RORgt) diluted with Permeabilization buffer was added to each well, and the wells were allowed to stand at 4°C for 30 minutes while shielded from light for staining. After staining, the cells were washed with Permeabilization buffer and FACS buffer, and then suspended in an appropriate amount in FACS buffer and used for flow cytometry analysis. The stained cells were analyzed by flow cytometry using FACSAriaIII (BD Biosciences). 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, or CD19, but expresses CD45.

As shown in FIG. 1, ILC2 was shown to be extremely predominant in the stomach compared to the small intestine (ILC1: 5%, ILC2: 94%, ILC3: 0.6%).

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

The small intestine of SPF mice was prepared using Collagenase (Wako) adjusted to a concentration of 1 mg/L in 2% FBS/RPMI-1640 medium, and the large intestine was prepared in Collagenase (SIGMA) adjusted to a concentration of 1 mg/L in the same manner at 37°C. , and stirred for 15 minutes. After 15 minutes, only the supernatant was collected, and then Collagenase was added to the remaining intestinal fragments, and the same operation was repeated twice to collect 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, suspend in 4 ml of 40% Percoll and transfer to a 15 ml Falcon tube. Using a Pasteur pipette, slowly add an equal amount of 70% Percoll to layer the cells downward, and incubate at 2000 rpm at 20°C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer produced by the density gradient was collected and suspended in 2% FBS/RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cellstrainer 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 plated at 1×10 ⁶ cells/well on a Tissue Culture Plate (96 Well, Flat Buttom with Low Evaporation Lid, FALCON). The seeds were sown in liters. After centrifugation, Purified Rat Anti-mou was adjusted to a final concentration of 0.1 mg/ml using 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS/D-PBS (-)). se CD16 /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. After that, wash the cells with Facs buffer, and after centrifugation, add each antibody dilution solution (CD3, CD45, TCRb, CD19, CD127, CD90.2, Sca1, KLRG1, and IL33Ra) to the cells according to Table 3 or 4. The cells were 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, or CD19, but expresses CD45. Expression of IL33Ra is also shown for cells co-expressing Sca1 and KLRG1.

As shown in FIG. 2, IL-33Ra was shown to be highly expressed in stomach ILC2 compared to small intestine ILC2 (SI LPL: 5.6, Stomach: 24).

[Example 1] Examination of the influence of commensal bacteria on gastric ILC2 The small intestines of SPF mice and germ-free (GF) mice were treated with Collagenase (Wako) adjusted to a concentration of 1 mg/L in 2% FBS/RPMI-1640 medium. The large intestine was similarly stirred at 37° C. for 15 minutes in Collagenase (SIGMA) adjusted to a concentration of 1 mg/L. After 15 minutes, only the supernatant was collected, and then Collagenase was added to the remaining intestinal fragments, and the same operation was repeated twice to collect 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, suspend in 4 ml of 40% Percoll and transfer to a 15 ml Falcon tube. Using a Pasteur pipette, slowly add an equal amount of 70% Percoll to layer the cells downward, and incubate at 2000 rpm at 20°C. Centrifugation was performed for 20 minutes. After centrifugation, the intermediate layer produced by the density gradient was collected and suspended in 2% FBS/RPMI-1640 medium to isolate lymphocytes. On the other hand, cells collected from the stomach were filtered using a 40 μm Cellstrainer 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 plated at 1×10 ⁶ cells/we on a Tissue Culture Plate (96 Well, Flat Buttom with Low Evaporation Lid, FALCON). Seed in 1 liter . After centrifugation, Purified Rat Anti-mou was adjusted to a final concentration of 0.1 mg/ml using 1/500 volume of Zombie Aqua Fixable Viability Kit (Biolegend) and FACS buffer (2% FBS/D-PBS (-)). se CD16 /CD32 (BD Biosciences) was added and allowed to stand at 4°C for 15 minutes to perform blocking and staining of dead cells. After that, wash the cells with Facs buffer, and after centrifugation, add each antibody dilution solution (CD3, CD45, TCRb, CD19, CD127, CD90.2, Sca1, KLRG1, and IL33Ra) to the cells according to Table 3 or 4. The cells were 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, or CD19, but expresses CD45. Expression of IL33Ra is also shown for cells that co-express Sca1, KLRG1 (ie, ILC2).

As shown in Figure 3, the abundance of gastric ILC2 was significantly different between SPF mice and GF mice, indicating that commensal bacteria influence the abundance of gastric ILC2.

[Example 2] Examination of the influence of commensal bacteria on the function of gastric ILC2 Lymphocytes collected by the method described above were suspended in Complete Buffer containing 10% FBS/RPMI-1640 medium with L-glutamine, penicillin, and streptomycin added. , were partially used to confirm cytokine production. Lymphocytes were seeded at 1×10 ⁶ cells/well on a Tissue Culture Plate (96 Well, Flat Buttom with Low Evaporation Lid). Golgi Plug (Biosciences), an intracellular protein transport inhibitor, was added without stimulation or together with PMA (50 ng/ml) and Ionomycin (1 ig/ml), and the cells were left standing in a 37°C, 25% CO ₂ incubator for 2 hours. . After standing for 2 hours, centrifugation was performed, and cell surface molecules were stained in the same manner as described above. The stained cells were washed twice with D-PBS (-), and after centrifugation, 4% PFA/D-PBS (-) was added and left at room temperature for 15 minutes to fix the cells. After centrifugation, the cells were washed with Permeabilization Buffer. After permeabilization, each IL-5 and IL-13 antibody was diluted with Permeabilization Buffer, the diluted solution was added to each well, and the wells were allowed to stand at 4° C. for 60 minutes while shielded from light for staining. The stained cells were analyzed by flow cytometry using FACSAriaIII (BD Biosciences). Analysis of the obtained data was performed using FlowJo (Tree Star, Inc.). FIG. 4 shows a comparison of IL-5 and IL-13 production in cells (ie, ILC2) that do not express CD3, TCRb, or CD19 but express CD45, Sca1, and KLRG1.

As shown in FIG. 4, there was a large difference in the abundance of IL-5 and IL-13 expressing cells between SPF mice and GF mice. In addition, it was confirmed that in the stomach, 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 (Figures 4(c) and 4(c)). (d)). In other words, it was shown that the presence or absence of commensal bacteria does not affect T cells or B cells, but only the number of ILC2s in the stomach and their functions.

[Example 3] Induction of gastric ILC2 by oral ingestion of gastric microflora The gastric contents and gastric mucosal tissue of SPF mice were scraped, suspended in PBS, and filtered using a 100 μm Cellstrainer (BD Bioscience) to a total of 3 ml. of which 300 μl was orally administered. Mice treated once and mice treated once a week four times were simultaneously used for analysis one week after the last oral administration. Stomachs were removed from mice to which oral administration was administered and germ-free mice as controls, and experiments were conducted using a method for detecting intracellular cytokine production. Lymphocytes collected by the above-described method were suspended in Complete Buffer containing 10% FBS/RPMI-1640 medium to which L-glutamine, penicillin, and streptomycin were added, and a portion was used to confirm cytokine production. Lymphocytes were seeded at 1×10 ⁶ cells/well on a Tissue Culture Plate (96 Well, Flat Buttom with Low Evaporation Lid). Golgi Plug (Biosciences), an intracellular protein transport inhibitor, was added to the collected lymphocytes, and the cells were left standing in a 25% CO ₂ incubator at 37° C. for 2 hours. After standing for 2 hours, centrifugation was performed, and cell surface molecules were stained in the same manner as described above. The stained cells were washed twice with D-PBS (-), and after centrifugation, 4% PFA/D-PBS (-) was added and left at room temperature for 15 minutes to fix the cells. After centrifugation, the cells were washed with Permeabilization Buffer. After permeabilization, each IL-5 and IL-13 antibody was diluted with Permeabilization Buffer, the diluted solution was added to each well, and the wells were allowed to stand at 4° C. for 60 minutes while shielded from light for staining. The stained cells were analyzed by flow cytometry using FACSAriaIII (BD Biosciences). Analysis of the obtained data was performed using FlowJo (Tree Star, Inc.). FIG. 5(a) is a diagram showing an overview of this embodiment. Figures 5(b) and (d) show IL-5 or IL-13 production using FACS for cells that do not express CD3, TCRb, CD19 but express CD45, Sca1, KLRG1 (i.e., ILC2). Fig. 5(d) shows the results of comparison (Fig. 5(b)) and further quantification (Fig. 5(d)). FIG. 5(c) is a diagram showing the relationship between the number of oral intakes and the number of cells.

As shown in Figure 5, when GF mice orally ingested the contents and/or mucus derived from the stomachs of SPF mice, the amount of bacteria in the stomachs of GF mice increased in proportion to the number of ingestions, and IL The number of cells secreting -5 or IL-13 (ie, ILC2) increased. This indicates that commensal bacteria contained in the stomach-derived contents and/or mucus of SPF mice colonized the stomachs of GF mice, thereby inducing ILC2 in the stomachs of GF mice. In other words, the results in FIG. 5 can be said to indicate that commensal bacteria in the stomach influence the number of ILC2s and their functions.

[Example 4] Analysis of bacterial flora by oral ingestion of stomach-derived microflora Bacterial flora was analyzed in mice to which feces, stomach contents, and gastric mucosal layer were orally administered. 10 mg of feces and stomach contents were measured and used for DNA extraction. Add 450 μl of Tris-10×EDTA (10 mmol/l Tris-HCl, 10 mmol/l EDTA, pH 8.0) to 10 mg of fecal sample, suspend with vortex, add 25 μl of Lysozyme stock (wako) 300 mg/ml, 37 It was incubated for 1 hour at 1000 rpm with shaking at 1000 rpm. Thereafter, 55 μl of Achromopeptidase stock (WAKO) 20,000 U/ml was added and incubated at 37° C. for 0.5 hour while shaking at 1000 rpm. After adding 61.5 μl of 10% SDS and 25.7 μl of Proteinase K stock (Merck) 25 mg/ml, 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 the mixture was shaken on a rotator for 5 minutes. Thereafter, the mixture was centrifuged at 13,000 rpm for 5 minutes, and the supernatant was collected. 650 μl of phenol/CHCl ₃ /IAA was added to the collected supernatant, and the mixture was shaken on a rotator for 5 minutes. Thereafter, the mixture was centrifuged at 13,000 rpm for 5 minutes, and 300 μl of supernatant was collected. 50 μl of 3M-CH ₃ COONa (Nacalai) and 1000 μl of 100% EtOH were added to the collected supernatant and suspended. After standing at -20°C, centrifugation was performed at 15,000 rpm at 4°C for 10 minutes, the supernatant was aspirated, and 1,000 μl of 70% EtOH was added to the pellet. Thereafter, the pellet was centrifuged again at 15,000 rpm for 10 minutes at 4°C, then dried at 40°C for 10 minutes, and suspended in 250 μl of TE. 2 μl of RNase (10 mg/ml) was added to 200 μl of the 250 μl and incubated at 37° C. for 1 hour. 200 μl of 10% PEG6000-2.5M NaCl was added and incubated for 30 minutes. Centrifuge at 15,000 rpm, 4°C for 20 minutes, remove the supernatant, wash with 1,000 μl of 70% EtOH, centrifuge again at 15,000 rpm, 4°C for 20 minutes, remove the supernatant, and dry the pellet at 40°C for 10 minutes. I let it happen. Thereafter, it was suspended in 50 μl of TE, and the DNA concentration was measured using Nanodrop (Thermo). The extracted DNA was diluted to 5 ng/μl, and the V4 region of 16S rRNA was amplified using primers 515F (GTGCCAGCMGCCGCGGTAA: SEQ ID NO: 26) and 806R (GGACTACHVGGGTWTCTAAT: SEQ ID NO: 27). The PCR product was purified using AMPure beads, and a barcode sequence was added by PCR reaction using Nextra XT Index Kit v2 (Illumina). After PCR, the PCR product was purified again using AMPure beads, and the DNA concentration was measured using PicoGreen (Invitrogen). Based on the concentration measurement results, all samples were diluted to 1 ng/μl, and after confirming the DNA size and concentration using TapeStation (Agilent), KAPA qPCR was performed. Gene sequences were sequenced using Miseq (Illumina), and the obtained gene sequences were clustered with a sequence similarity of 97% using the package software QIIME (http://qiime.org/) to create operational taxonomic units (OTUs). After that, the obtained OTU was searched in a database using Ribosomal Database Project (https://rdp.cme.msu.edu) to identify the bacterial species.

As shown in Figure 6, as the contents and/or mucus derived from the stomachs of SPF mice were orally ingested, the abundance of bacteria belonging to the S24-7 family increased in the stomachs and feces of GF mice. Shown. That is, it was shown that an increase in the amount of bacteria belonging to the S24-7 family in the stomach may have a large effect on the induction of ILC2 in the stomach.

[Example 5] Identification of commensal bacterial groups that affect the induction of gastric ILC2 1
Ampicillin (0.1 g/L), colistin (1 g/L), neomycin (1 g/L), metronidazole (1 g/L), or vancomycin (0.5 g/L) was added to the drinking water of mice in SPF condition. , orally for 3 weeks. Lymphocytes were collected from the stomachs of C57BL/6N mice under SPF conditions or antibiotic-treated mice as controls, and subjected to flow cytometry analysis according to the procedure described above.

As shown in Figure 7, there was a significant change in the number of gastric ILC2 cells in mice ingested with ampicillin (Amp), colistin (Colistin), neomycin (Neo), and metronidazole (MNZ) compared to the control (SPF). was not seen. On the other hand, in mice ingested with vancomycin, the number of gastric ILC2 cells decreased. Therefore, commensal bacteria that affect the induction of gastric ILC2 were shown to be resistant to ampicillin, colistin, neomycin, and metronidazole, but sensitive to vancomycin.

[Example 6] Identification of commensal bacterial groups that affect the induction of gastric ILC2 2
Gene sequences were sequenced using Miseq (Illumina), and the obtained gene sequences were clustered with a sequence similarity of 97% using the package software QIIME (http://qiime.org/) to create operational taxonomic units (OTUs). After that, the obtained OTU was searched in a database using Ribosomal Database Project (https://rdp.cme.msu.edu) to identify the bacterial species. The bacterial flora information was appropriately normalized using the method described above, and the proportion occupied by each type of bacterial flora was calculated for each antibiotic administration group. The results are shown in FIG.

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

[Example 7] Study of localization of S24-7 1 ml of Carnoy's fixative (60% methanol, 30% chloroform, 10% acetic anhydride) was added to a sample extracted from a mouse and fixed for 6 hours. After fixation, the mixture 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 standing at room temperature for 30 minutes. After 30 minutes, the mixture was replaced with dehydrated ethanol and left at room temperature for 20 minutes. The sample was placed in a cassette and programmed with ethanol (1 hour), xylene (2 hours, 3 times), and paraffin (3 hours, 3 times) using a Laica tissue processor, and after the program was completed, it was embedded in paraffin. The block was sliced into 5 um slices and attached to a slide glass. After stretching at 37°C, it was placed in an oven at 60°C for 10 minutes to dry. A glass slide was placed in xylene and soaked for 10 minutes, and then placed in 99.5% ethanol for 5 minutes. It was then air-dried. The probe was diluted to 10 nM in pre-warmed hybridization buffer (0.9M NaCl, 20mM pH 7.4 Tris-HCl, 0.1% SDS). The sample was layered and reacted at 48°C for 2 hours. After the reaction was completed, 100 µl of hybridization buffer (0.9M NaCl, 20mM pH 7.4 Tris-HCl, 10% formamide) was added, and the reaction was carried out at 48°C for 5 minutes. After the reaction was completed, it was washed three times with PBS and detected using Laica SP8.

Note that the probe used above was created as follows. To determine the bacteria that were reduced in vancomycin-treated mice but not in control (SPF mice), ampicillin-treated mice, colistin-treated mice, neomycin-treated mice, and metronidazole-treated mice, these bacteria were attached to their stomach contents. The amount of nucleic acid sequences of the bacteria was determined using Miseq (Illumina). As a result of the analysis, it was confirmed that sequences possessed by bacteria belonging to the S24-7 family satisfied this condition (11 types of sequences were determined). A nucleic acid sequence (SEQ ID NO: 1) common to these 11 types of sequences was determined. A probe was created by labeling the 3' end of SEQ ID NO: 1 with a fluorescent protein (A555). The results are shown in FIG.

As shown in FIG. 9, S24-7 was shown to be present in the gastric epithelium. This result also strongly suggests that S24-7 engrafts in the stomach, forms the bacterial flora, and has a great influence on the induction of ILC2 in the stomach.

[Example 8] Study on the correlation between the number of ILC2 cells in the stomach and the population of S24-7 Mice in SPF condition were given ampicillin (0.1 g/L), colistin (1 g/L), and neomycin in their drinking water. (1 g/L), metronidazole (1 g/L), or vancomycin (0.5 g/L), and the mice were given orally for 3 weeks. The mouse stomach contents and feces were then collected and analyzed as shown in Examples 4 and 6. In addition, lymphocytes collected from the stomach at the same time were analyzed by flow cytometry according to the procedure described above. A correlation diagram between bacterial species (family) and flow cytometry analysis results was created as a heat map and a scatter plot, with a value of 1% or more as the upper limit for each bacterial count obtained by bacterial flora analysis.

As shown in FIG. 10, there was a strong positive correlation between the number of bacteria in S24-7 and the number of ILC2 cells (r=0.8, p=0.03). On the other hand, no such relationship was observed between the number of bacteria other than the S24-7 family and the number of ILC2 cells. The present results showed that S24-7 bacteria induce an increase in ILC2 in the stomach.

[Example 9] Detailed study of immune response to Helicobacter pylori infection Germ-free mice were orally administered 1x10 ⁹ CFU/300 μl of PBS. The study was conducted at two weeks after infection, when the number of Helicobacter pylori is highest, and at nine weeks, when inflammatory symptoms appear in the stomach. Bacteria-bound IgA was detected using the method described above. Next, the stomachs were removed 2 weeks and 9 weeks after infection, the stomachs were opened, and the contents were gently washed with PBS, and the tissues were frozen and crushed. The powdered stomach was suspended in the Lysis buffer attached to the QIAGEN RNAeasy kit, and RNA was extracted according to the attached protocol. The extracted RNA was reverse transcribed using SuperScript III enzyme to obtain cDNA. The cDNA was analyzed by quantitative PCR using pIgR-specific primers. In addition, the stomachs 2 weeks after infection were compared with germ-free mice according to the intracellular cytokine production protocol described above. The results are shown in FIGS. 11-A to 11-C.

In order to confirm infection with Helicobacter pylori, we detected the CagA protein secreted by the bacterium, and while its presence was clearly confirmed at 2 weeks post-infection, the protein was detected at 9 weeks post-infection. was not detected, and CagA protein was not detected at any time in the small intestine (SI) ((Figure 11-A(a)). In addition, the number of ILC2 cells in the stomach in Helicobacter pylori-infected mice was It reached its maximum at 2 weeks after infection (Figure 11-A(b), Figure 11-B(a) and (b)).On the other hand, as shown in Figure 11-A(c), PMSS1 in the stomach CFU peaked at 1 week post-infection. B cells increased significantly at 2 weeks post-infection, and the cell number remained increased at 9 weeks post-infection. On the other hand, T cells did not increase until 3 weeks after infection (Figure 11-A(d), (e), (f) and Figure 11-B(c)). At 2 weeks post-infection, there was a significant increase in ILC2 expressing IL-5 or IL-13 (Figures 11-C(a) and (b)).At 2 weeks post-infection, IL -33 production was markedly increased (Figure 11-C(c)), strongly suggesting that cytokine production by ILC2 is important for response immunity to Helicobacter pylori. When GF 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 (Figure 11- C(d) and (e)), it was shown that there was a concomitant decrease in IgA secretion in the gastric tissue (Figure 11-B(d)). As shown in Figure 11-B(e), IgA binding to Helicobacter pylori was highest at 2 weeks post-infection and then decreased.The significant decrease in Helicobacter pylori at 9 weeks post-infection may be due to clearance of the bacterium by IgA. In addition, as shown in Figure 11-C(f), the expression level of pIgR, which is important in converting IgA into a secretory form, is enhanced in the stomach compared to the intestinal tract, and this is also the case. This is considered to be indirect evidence that IgA is involved in the elimination of Helicobacter pylori.

The above suggests that the following protective responses occur against Helicobacter pylori infection: (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 to and clearance of Helicobacter pylori by IgA.

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

As shown in Figure 12, the amount of IgA bound to bacteria was reduced in the gastric contents and gastric mucus (biofilm) of vancomycin-treated SPF mice compared to controls (vancomycin-untreated SPF mice). It has been shown. This means that vancomycin treatment reduces the number of S24-7 cells in the stomach and concomitantly reduces the number of ILC2 cells. A decrease in ILC2 reduces the amount of IL-5 and/or IL-13 produced and reduces B cell activation. As a result, it is thought that the amount of IgA secreted in the stomach decreased. Therefore, this example demonstrates that commensal bacteria of the S24-7 family can directly influence the amount of secreted IgA in the stomach.

[Example 11] Induction of gastric ILC2 and IgA by Muribaculum intestinale GF mice were orally infected once with 1x10 ⁸ CFU/mouse of the type strain Muribaculum intestinalale (also referred to as "YL27"), and 2 weeks later the infection was carried out. The mice were dissected and gastric cells were analyzed by flow cytometry. The outline of this experiment is shown in FIG. 13(a), and the experimental results are shown in FIGS. 13(b) to 13(d), respectively.

As shown in FIG. 13, ILC2 present in the stomach of GF mice infected with YL27 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 tissues of infected mice was confirmed by qPCR. At the same time, the amount of IgA contained in the stomach contents and feces was confirmed by ELISA. The results are shown in FIG.

As shown in FIG. 14, the expression level of plgR was significantly increased in the stomach tissue of mice infected with Y27 (FIG. 14(a)). Furthermore, the amount of IgA was increased in both the stomach contents and feces of mice infected with Y27.

[Example 12] Confirmation of the presence of YL27 in the stomach The presence of YL27 was confirmed in the stomachs of mice infected with YL27. A fluorescent dye (Alexa488) was coupled to a probe (EUB338 (5'-GCTGCCTCCCGTAGGAGT-3': SEQ ID NO: 29)) that detects 338-355 of 16s rRNA of Y27, and YL27 was isolated by FISH method (see above for protocol details). The presence of this substance in the stomach was confirmed. The results are shown in Figure 15.

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

[Example 13] Examination of the effect of YL27 on preventing Helicobacter pylori infection YL27 (1x10 ⁸ /mouse) was orally administered to germ-free mice. One week after the administration, YL27 (1×10 ⁸ /mouse) was administered again. The day after the second administration of YL27, the mice were orally infected with Helicobacter pylori. Infected mice were dissected two weeks after H. pylori infection, and (1) the amount of IgA produced in the stomach contents or feces of infected mice, (2) the amount of mRNA expression of pIgR in the stomach of infected mice, and (3) The amount of H. pylori in the stomach tissues of infected mice was analyzed. The analysis methods used in (1) and (2) are as described above. The outline of the analysis method (3) is as follows: Mice were sacrificed two weeks after infection with Helicobacter pylori, and their stomachs were collected. The contents were removed from the collected stomach, the weight of the stomach tissue was measured, and the tissue was cut into pieces with scissors and suspended in a liquid culture medium for Helicobacter pylori (PMSS1). The suspension was allowed to stand for a while, and then, after removing large tissues, it was cultured on an agar medium for Helicobacter pylori culture (BD BBLPlate) for 2 days.

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

As shown in FIG. 17, although there was no significant difference in the amount of IgA produced in the stomach contents, the amount of IgA increased, and the amount of IgA in feces increased significantly. Furthermore, as shown in FIG. 18, pIgR expression in the gastric tissue was also significantly induced by administration of YL27 before infection.

Furthermore, as shown in FIG. 19, the number of Helicobacter pylori present in the stomach was significantly reduced in mice pre-administered with YL27 compared to controls.

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

According to the present invention, expression of ILC2, which is a type of immunocompetent cell, can be induced 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 differentiate into antibody-producing cells, and as a result, IgA production in the subject's stomach is induced and enhanced, making it possible to treat and/or prevent oral infections including Helicobacter pylori. Therefore, the present invention is very useful in the medical field and the like.

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

Claims (8)

ILC2 inducer in the stomach comprising at least one bacterium with the following properties:
(1) Susceptibility to vancomycin, and
(2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . An inducer of IgA production in the stomach, comprising at least one type of bacteria having the following properties:
(1) Susceptibility to vancomycin, and
(2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . Agents for the treatment or prevention of oral infections, including bacteria with the following characteristics:
(1) Susceptibility to vancomycin, and
(2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . The therapeutic or preventive agent according to claim 3 , wherein the oral infection is at least one infection selected from the following group:
pylori infection, O:157 infection, and Salmonella infection. A method for inducing ILC2 in the stomach comprising orally administering to a subject (other than a human) at least one bacterium having the following properties:
(1) Susceptibility to vancomycin, and
(2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . A method for inducing IgA production in the stomach, comprising orally administering to a subject (excluding humans) at least one type of bacteria having the following characteristics:
(1) susceptibility to vancomycin, and (2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . A method of treating or preventing an oral infection comprising orally administering to a subject (other than a human) a bacterium that has the following characteristics:
(1) Susceptibility to vancomycin, and
(2) resistance to at least one drug selected from the group consisting of ampicillin, colistin, neomycin, and metronidazole ;
Here, the bacterium has the 16S rRNA nucleotide sequence shown in SEQ ID NO:28 . The method of treatment or prevention according to claim 7 , wherein the oral infection is at least one infection selected from the following group:
pylori infection, O:157 infection, and Salmonella infection.