WO2020071463A1 - Procédé pour induire la production d'iga ciblant l'ilc2 - Google Patents

Procédé pour induire la production d'iga ciblant l'ilc2

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Publication number
WO2020071463A1
WO2020071463A1 PCT/JP2019/039036 JP2019039036W WO2020071463A1 WO 2020071463 A1 WO2020071463 A1 WO 2020071463A1 JP 2019039036 W JP2019039036 W JP 2019039036W WO 2020071463 A1 WO2020071463 A1 WO 2020071463A1
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WIPO (PCT)
Prior art keywords
bacterium
nucleotide
stomach
seq
ilc2
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PCT/JP2019/039036
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English (en)
Japanese (ja)
Inventor
尚子 佐藤
大野　博司
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国立研究開発法人理化学研究所
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Priority to JP2020550524A priority Critical patent/JP7444459B2/ja
Publication of WO2020071463A1 publication Critical patent/WO2020071463A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to an IgA production inducer targeting ILC2 present in the stomach.
  • H. pylori also referred to as "H. pylori”, “H. pylori”, etc.
  • H. pylori 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.”
  • ILC Innate Lymphoid cells
  • Non-Patent Document 7 Non-Patent Document 7
  • 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.
  • 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.
  • 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.
  • [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.
  • 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.
  • 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.
  • [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.
  • [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.
  • [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]
  • ILC2 can be induced in the stomach of a mammal.
  • 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-33R ⁇ 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • FIG. 12 is a diagram showing the amount of bacterial-bound IgA in the stomach of vancomycin-treated mice.
  • FIG. 13 is a diagram showing various data relating to an immune response to infection with Muribaculum intestinale (YL27).
  • 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.
  • 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.
  • 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.
  • the bacterium formulated in the ILC2 inducer of the present invention has a nucleotide sequence represented by SEQ ID NO: 1.
  • the bacterium contained in the ILC2 inducer of the present invention is a bacterium belonging to the family S24-7.
  • 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.).
  • the term “thermophilic animal” refers to mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, humans, and the like.
  • thermophilic animal refers to mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, cows, horses, sheep, monkeys, humans, and the like.
  • 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.
  • 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.
  • the bacterium may be, but is not limited to, Muribulculum intestinale (also referred to as “YL27” or the like).
  • YL27 has 16s ⁇ rRNA 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).
  • 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.
  • 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.).
  • 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.
  • 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.
  • 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. .
  • 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.
  • 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.
  • 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.
  • the ILC2 inducer of the present invention may be provided in the form of 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.
  • examples of such food compositions include, but are not limited to, yogurt, jelly, beverages, and the like.
  • 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.
  • 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.
  • ILC2 inducer Can be the same as described above.
  • 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.
  • the IgA production inducer of the present invention can be provided in the form of a pharmaceutical or a food.
  • the food composition 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic or prophylactic agent of the present invention can be provided in the form of a medicament or food.
  • the food composition 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • prevention 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.
  • mice [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)), CD3 ⁇ - / - mice (M.Malissen et al . , Altered T cell development in mice with a targeted mutation of the CD3-epsilon gene.
  • the anti-mouse DCLK antibody was labeled with alexa488 according to the manufacturer's instructions (Thermo Fisher scientific).
  • isolated cells were incubated with PMA (50 ng / ml) and ionomycin (2 ⁇ g / 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.
  • 1 ⁇ permeabilization buffer included in Foxp3 / TF buffer set (Thermo Fisher Scientific)
  • 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).
  • 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 ⁇ l 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.
  • RNA total RNA was extracted from purified gastric or intestinal cell preparations using RNeasy micro kit (QIAGEN).
  • QIAGEN RNeasy micro kit
  • 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).
  • RT SYBR Green qPCR Master Mix TAKARA SYBR Premix ExTaq II
  • 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.
  • V4 variable region (515F-806R) was sequenced with the Illumina Miseq library prepared according to the manufacturer's (Takara Bio, Inc) protocol.
  • 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 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).
  • 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 ⁇ m 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.
  • 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.
  • a fluorescently labeled antibody was expressed with a fluorescently labeled antibody.
  • ⁇ 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.
  • Fixation / Permeabilization solution Fraxp3 / Transscription Factor Staining Buffer Set, ThermoFisher
  • 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.
  • Permeabilization buffer Foxp3 /
  • ILC2 was extremely predominant in the stomach (ILC 1: 5%, ILC2: 94%, ILC3: 0.6%) as compared to the small intestine.
  • 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 ⁇ m CellStrainer (BD Bioscience), centrifuged, and used for lymphocyte isolation.
  • SIGMA Collagenase
  • 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 ⁇ m 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.
  • 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.
  • SIGMA Collagenase
  • the collected supernatant was filtered using a 100 ⁇ m 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 ⁇ m 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.
  • 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).
  • 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.
  • 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).
  • 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.
  • T cells and B cells which are cells that mainly produce IL-5 and / or IL-13 other than ILC2 in the stomach. 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 ⁇ m Cell strainer (BD Bioscience) for a total of 3 ml. , And 300 ⁇ l 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.
  • 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.
  • Example 4 Analysis of bacterial flora by oral ingestion of gastric microbial flora
  • 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.
  • Tris-10 ⁇ EDTA (10 mmol / l Tris-HCl, 10 mmol / l EDTA, pH 8.0
  • a 10 mg stool sample suspending with vortex
  • 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 ⁇ l of TE.
  • 2 ⁇ l of RNase (10 mg / ml) was added and incubated at 37 ° C. for 1 hour.
  • 200 ⁇ l of 10% PEG6000-2.5M NaCl was added and incubated for 30 minutes.
  • washing with 1000 ⁇ l of 70% EtOH centrifuging again at 15000 rpm at 4 ° C.
  • Thermo Thermo
  • the extracted DNA was diluted to 5 ng / ⁇ l, 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).
  • 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 / ⁇ l, 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.
  • 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
  • ampicillin 0.1 g / L
  • colistin 1 g / L
  • neomycin 1 g / L
  • metronidazole 1 g / L
  • vancomycin 0.5 g / L
  • 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.
  • 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.
  • Carnoy's fixative 60% methanol, 30% chloroform, 10% acetic anhydride
  • 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 ⁇ m 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 ⁇ l 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.
  • a hybridization buffer 0.9 M NaCl, 20 mM pH 7.4 Tris-HCl, 10% formamide
  • 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.
  • 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.
  • Example 9 Detailed Examination of Immune Response to Helicobacter pylori Infection
  • mice were orally administered at 1 ⁇ 10 9 CFU / 300 ⁇ l 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • YL27 was shown to be a bacterium capable of inducing ILC2 in the stomach.
  • FIG. 14 shows the results.
  • 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).
  • EUB338 5′-GCTGCCTCCCGTAGGAGT-3 ′: SEQ ID NO: 29
  • 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).
  • PMSS1 H. pylori
  • 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.
  • the expression of pIgR in gastric tissue was also significantly induced by administration of YL27 before infection.
  • ILC2 a type of immunocompetent cell
  • 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.

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Abstract

L'invention concerne un agent pour induire l'ILC2 dans l'estomac, ledit agent comprenant au moins un type de bactérie ayant les caractéristiques suivantes : (1) une sensibilité à la vancomycine ; et (2) une tolérance à au moins un médicament choisi dans le groupe constitué par l'ampicilline, la colistine, la néomycine et le métronidazole.
PCT/JP2019/039036 2018-10-04 2019-10-03 Procédé pour induire la production d'iga ciblant l'ilc2 WO2020071463A1 (fr)

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