WO2021221110A1 - Petite bactérie intestinale induisant la prolifération ou l'activation de cellules th1 et/ou de cellules th17 - Google Patents

Petite bactérie intestinale induisant la prolifération ou l'activation de cellules th1 et/ou de cellules th17 Download PDF

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WO2021221110A1
WO2021221110A1 PCT/JP2021/017000 JP2021017000W WO2021221110A1 WO 2021221110 A1 WO2021221110 A1 WO 2021221110A1 JP 2021017000 W JP2021017000 W JP 2021017000W WO 2021221110 A1 WO2021221110 A1 WO 2021221110A1
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cells
bacterium
bacteria
disease
polynucleotide
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Japanese (ja)
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賢也 本田
学 永山
博徳 山本
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学校法人慶應義塾
国立研究開発法人理化学研究所
学校法人自治医科大学
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Publication of WO2021221110A1 publication Critical patent/WO2021221110A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • 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

Definitions

  • the present invention relates to a small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells (hereinafter also referred to as "Th1 cells and the like").
  • the present invention relates to a method for evaluating a disease caused by Th1 cells or the like using the bacterium as an index.
  • the present invention relates to a composition or a vaccine composition for inducing proliferation or activation of Th1 cells or the like containing the bacteria or the like as an active ingredient.
  • the present invention is for treating a composition for suppressing the proliferation or activation of Th1 cells or the like or a disease caused by the Th1 cells or the like, which contains a substance having an antibacterial action against the bacteria as an active ingredient.
  • the composition is for treating a composition for suppressing the proliferation or activation of Th1 cells or the like or a disease caused by the Th1 cells or the like, which contains a substance having an antibacterial action against the bacteria as an active ingredient.
  • the present invention relates to a method for screening a substance using the bacterium as an index. Furthermore, the present invention relates to a method for suppressing the proliferation or activation of Th1 cells or the like, or a method for treating a disease caused by Th1 cells or the like, which targets the bacterium.
  • CD Crohn's disease
  • Non-Patent Documents 4 and 5 The gut microbiota is a potent regulator of host immunity and has been reported to be associated with several diseases.
  • CD it is being shown that a change in the bacterial flora called intestinal bacterial flora symbiotic imbalance (disbiosis) affects the pathogenesis.
  • intestinal bacterial flora symbiotic imbalance affects the pathogenesis.
  • disbiosis intestinal bacterial flora symbiotic imbalance
  • a decrease in ⁇ -diversity of the microbial flora and a decrease in the abundance of Faecalibacterium prasnitzii have been frequently reported (Non-Patent Document 6).
  • An object of the present invention is to provide a composition for treating, improving or preventing a CD or the like that targets the identified bacterium, and a method for inspecting the CD or the like using the bacterium as an index.
  • a mucosal sample of jejunum and ileum was collected from a CD patient using a double-balloon endoscopy (DBE) system.
  • the DBE system includes a fiberscope and balloon pump system, which allows for more detailed examination of SI (distal jejunum and proximal ileum) compared to conventional push enteroscopy.
  • SI mucosal samples were collected from CD patients and non-CD patients by scraping or biopsy using such DBE, and comparative microbiome analysis was performed. As a result, it was revealed that the composition of the microbial flora of the SI mucosa in CD patients was different from that in non-CD patients, and that the population belonging to several families including Enterobacteriaceae, Ruminococcaceae and Bacteroidaceae was increasing. ..
  • Th1 cells in the intestinal tract was enhanced. Furthermore, the accumulation of Th17 cells was enhanced, though not as much as Th1 cells.
  • the E. The coli strain induced Th1 cells specifically for the strain and showed a high ability to induce intestinal inflammation.
  • the present inventors have found a small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells that can be involved in the onset of CD, etc., and have completed the present invention.
  • ⁇ 1> A small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells.
  • Bacterial bacterial group according to ⁇ 1> which is at least one bacterium selected from the following bacterial group: Bacteria having a polynucleotide having 95% or more identity to the DNA sequence set forth in SEQ ID NO: 1. Bacteria having a polynucleotide having 95% or more identity to the DNA sequence set forth in SEQ ID NO: 2. Bacteria having a polynucleotide having 95% or more identity to the DNA sequence set forth in SEQ ID NO: 3.
  • Bacteria to have ⁇ 3> Bacteria having a polynucleotide having 95% or more identity to the DNA sequence set forth in SEQ ID NO: 1 and 95% or more identity to the DNA sequence set forth in SEQ ID NO: 2.
  • the bacterium according to ⁇ 1> which is a combination with a bacterium having a polynucleotide.
  • a step of ingesting a test substance into a non-human germ-free animal (2) When the number or activity of Th1 cells and / or Th17 cells in the small intestine of the non-human animal is detected, and when suppression of proliferation or activation of the cells is detected in (3) step (2).
  • the step of determining that the test substance is a substance that suppresses the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine. How to include.
  • ⁇ 5> A method for screening a substance that suppresses the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine.
  • the test substance is a bacterium having a polynucleotide having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1 and / or 95% or more with respect to the DNA sequence set forth in SEQ ID NO: 2.
  • Ingestion of non-human animals colonized with a polynucleotide having a polynucleotide having the same identity as When (2) the step of detecting the number of the bacteria in the small intestine of the non-human animal and (3) the suppression of the growth of the bacteria are detected in the step (2), the test substance is Th1 cells in the small intestine. And / or the step of determining that the substance suppresses the proliferation or activation of Th17 cells. How to include.
  • ⁇ 6> The method according to ⁇ 4> or ⁇ 5>, wherein the substance is a bacterium or a bacteriophage.
  • ⁇ 7> A method for evaluating a disease caused by Th1 cells and / or Th17 cells. (1) For bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1 and / or the DNA sequence set forth in SEQ ID NO: 2 in the small intestinal mucosa of the subject.
  • Quantifying bacteria with polynucleotides having 95% or more identity (2) A step of comparing the value obtained by quantifying in step (1) with a corresponding value obtained by quantifying the bacteria in the mucosa of the small intestine of a human who does not suffer from the disease, and step (3) (3).
  • the quantitative value in the small intestinal mucosa of the subject is higher than the corresponding value, the step of determining that the subject is suffering from the disease is performed.
  • ⁇ 8> Induces proliferation or activation of Th1 cells and / or Th17 cells containing the bacterium according to any one of ⁇ 1> to ⁇ 3> or a physiologically active substance derived from the bacterium as an active ingredient.
  • composition to do The bacterium according to any one of ⁇ 1> to ⁇ 3> for producing a composition for inducing proliferation or activation of Th1 cells and / or Th17 cells, or the same. Regarding the use of bioactive substances derived from bacteria.
  • a vaccine composition comprising the bacterium according to any one of ⁇ 1> to ⁇ 3> or an antigen specific to the bacterium as an active ingredient.
  • the present invention also relates to the use of the bacterium according to any one of ⁇ 1> to ⁇ 3> or an antigen specific to the bacterium for producing a vaccine composition.
  • ⁇ 10> Suppresses the proliferation or activation of Th1 cells and / or Th17 cells containing a substance having an antibacterial action against the bacterium according to any one of ⁇ 1> to ⁇ 3> as an active ingredient.
  • Composition for. ⁇ 11> The composition according to ⁇ 10>, wherein the substance is a substance selected by the method according to any one of ⁇ 4> to ⁇ 6>.
  • the present invention also relates to the bacterium according to any one of ⁇ 1> to ⁇ 3> for producing a composition for suppressing the proliferation or activation of Th1 cells and / or Th17 cells.
  • the present invention relates to the use of a substance having an antibacterial activity, or a bacterium or bacteriophage obtained by the screening method according to any one of ⁇ 4> to ⁇ 6>.
  • ⁇ 12> The composition according to any one of ⁇ 9> to ⁇ 11>, which is a composition for treating, ameliorating or preventing a disease caused by Th1 cells and / or Th17 cells.
  • ⁇ 13> The composition according to ⁇ 12>, which is ingested by the subject determined to be suffering from the disease by the method according to ⁇ 7>.
  • ⁇ 14> The composition according to ⁇ 12> or ⁇ 13>, wherein the disease is Crohn's disease.
  • the present invention also comprises the production of compositions for treating, ameliorating or preventing said diseases.
  • the present invention relates to the use of a substance having a bacterium or a substance selected by the method according to any one of ⁇ 4> to ⁇ 6>.
  • ⁇ 16> The bacterium according to any one of ⁇ 1> to ⁇ 3> or an antigen specific to the bacterium is ingested by the subject, and the proliferation or activity of Th1 cells and / or Th17 cells in the subject.
  • ⁇ 17> A substance having an antibacterial action against the bacterium according to any one of ⁇ 1> to ⁇ 3> is ingested by a subject, and the proliferation or activity of Th1 cells and / or Th17 cells in the subject.
  • ⁇ 18> The method according to ⁇ 17>, wherein the substance is a substance selected by the method according to any one of ⁇ 4> to ⁇ 6>.
  • the present invention also relates to the bacterium according to any one of ⁇ 1> to ⁇ 3> for suppressing the proliferation or activation of Th1 cells and / or Th17 cells in a subject, or a bacterium specific to the bacterium.
  • the antigen the substance having an antibacterial action against the bacterium according to any one of ⁇ 1> to ⁇ 3>, or the method according to any one of ⁇ 4> to ⁇ 6>.
  • ⁇ 19> A disease caused by Th1 cells and / or Th17 cells in the subject by ingesting the bacterium according to any one of ⁇ 1> to ⁇ 3> or an antigen specific to the bacterium. How to treat, improve or prevent.
  • ⁇ 20> A disease caused by Th1 cells and / or Th17 cells in a subject by ingesting a substance having an antibacterial action against the bacterium according to any one of ⁇ 1> to ⁇ 3>. How to treat, improve or prevent.
  • ⁇ 21> The method according to ⁇ 20>, wherein the substance is a substance selected by the method according to any one of ⁇ 4> to ⁇ 6>.
  • ⁇ 22> The item according to any one of ⁇ 19> to ⁇ 21>, wherein the subject is the subject determined to have the disease by the method according to ⁇ 7>.
  • Method. ⁇ 23> The method according to any one of ⁇ 19> to ⁇ 22>, wherein the disease is Crohn's disease.
  • the present invention also comprises the bacterium according to any one of ⁇ 1> to ⁇ 3> or an antigen specific to the bacterium for treating, ameliorating or preventing the disease, ⁇ 1> to ⁇ 3.
  • the present invention by targeting a small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells, it becomes possible to suppress the proliferation or activation of the cells, and thus to the cells. It is possible to treat, improve or prevent the resulting disease. Further, according to the present invention, it is also possible to test for diseases caused by the cells by using the amount of bacteria in the small intestine as an index.
  • FIG. 6 is a schematic diagram outlining a method for obtaining a sample from the small intestine (SI) via double-balloon enteroscopy.
  • SI small intestine
  • CD Crohn's disease
  • non-CD 16S rRNA gene sequence analysis
  • Each graph shows the results of analysis of the number of OTUs (Operational Taxonomy Units), ACE (Abundance-based Coverage Estimator), Chao1 and Shannon index.
  • Each bar graph represents the average value of the group.
  • Each point represents an individual sample. Error bars represent standard deviation (SD).
  • SD standard deviation
  • the P value obtained by analyzing with the unpaired Student's t-test is added. It is a two-dimensional plot figure which shows the result of having analyzed the structural similarity of the bacterial flora between the SI sample derived from a CD patient and that derived from a non-CD patient.
  • FIG. 5 is a graph showing the relative abundance of bacterial taxa at the Family level of the SI flora.
  • the horizontal axis shows the results for each bacteriaceae (the left side shows the sample analysis results derived from non-CD patients, and the right side shows the sample analysis results derived from CD patients).
  • Each thick bar represents the average value of the group.
  • Each point represents an individual sample. Error bars represent SD.
  • FIG. 5 is a graph showing the relative abundance of bacterial taxa at the family level of the SI flora.
  • the horizontal axis shows the results for each bacteriological family (the left side shows the sample analysis results derived from non-CD patients, and the middle shows the sample analysis results of CD patients without intestinal strictures (trictures).
  • Each right side shows the results of sample analysis from a CD patient with intestinal stenosis).
  • Each thick bar represents the average value of the group.
  • Each point represents an individual sample. Error bars represent SD.
  • Each asterisk indicates that the value obtained by multiple t-test by the FDR approach is *** ⁇ 0.05; ** less than 0.01; *** ⁇ 0.001; *** ⁇ 0.0001.
  • 6 is a histogram of LDA scores showing the results of calculations for the differentially abundant taxa between CD patients (“CD” in the figure) and non-CD patients (“non-CD” in the figure). In the figure, in the LDA score, the analysis result for non-CD patients is shown on the negative side, and the analysis result for CD patients is shown on the positive side. It is a histogram of the misjudgment rate which shows the result calculated by comparing the sample derived from a CD patient (“CD” in the figure) with that of a non-CD patient (“non-CD” in the figure).
  • FDR farnesoid ⁇ 0.05
  • the analysis result for non-CD patients is shown on the minus side
  • the analysis result for CD patients is shown on the plus side.
  • the three types of computational analysis are linear discriminant analysis (LDA) (LEfSe) combined with effect size measurement, multiple t-test method (Multiple t-tests) using false judgment rate, and CD-related SI mucous membrane [SI of CD patients].
  • each asterisk indicates that the value obtained by the unpaired Student's t-test is * P ⁇ 0.05; ** P ⁇ 0.01.
  • E. coli in saliva (“Saliva” in the figure), SI and feces (“Faces” in the figure). colli and R.
  • It is a graph which shows the relative abundance of gnavus.
  • the horizontal axis shows the results for each sample (the left side shows the sample analysis result derived from a non-CD patient, and each right side shows the sample analysis result derived from a CD patient).
  • the vertical axis represents the relative abundance.
  • Each thick bar represents the average of the group.
  • Each point represents an individual sample. Error bars represent SD.
  • Each asterisk indicates that the value obtained by the unpaired Student's t-test is * P ⁇ 0.05; ** P ⁇ 0.01.
  • It is a band graph which shows the SI bacterial flora composition in each sample determined by 16S rRNA sequencing.
  • OTUs significantly concentrated in CD patients are marked in warm colors, those concentrated in non-CD patients (non-CD patients) are blue, and those with no significant difference are those. Shown in gray.
  • nine kinds of strains isolated from the SI sample of the CD patient are shown.
  • CD4 + TCR ⁇ + T cells CD4T cells
  • SLP lamina propria of the small intestine
  • CLP lamina propria of the colon
  • GF Aseptic (GF) C57BL / 6 (B6) mice were orally inoculated with a mixture of the above 9 types of CD concentrates (9-mix) and euthanized after 3 weeks.
  • GF + 9mix (“+9mix” in FIGS. 3B and 3D) indicates the analysis result of the mouse.
  • GF indicates the analysis result of GF mice not inoculated with the CD concentrate.
  • SPF indicates the analysis result of a specific pathogen-free mouse. It is a graph which shows the result of having analyzed the ratio of IFN- ⁇ + cell and IL-17 + cell about the CD4T cell of the small intestine laminalitis and the colon laminalitis. Each point represents an individual mouse. Each thick bar represents the average of the group.
  • FIG. 3 is a flow cytometric plot showing representative results of analyzing the expression of IFN- ⁇ , IL-17 and DR3 for CD4T cells in the colon lamina intestinal. The notation in the figure is the same as in FIG. 3A. It is a graph which shows the result of having analyzed the expression of IFN- ⁇ , IL-17 and DR3 about the CD4T cell of the colon lamina propria.
  • FIG. 3 is a flow cytometric plot showing representative results of analyzing the expression of IFN- ⁇ and IL-17 for CD4T cells in the colon lamina basement.
  • “GF + 131A1” and “GF + 35A1” are referred to as R.I. gnavus strain and E. coli.
  • the analysis result of the GF mouse which was orally inoculated with each of the colli strains is shown.
  • "GF + 2mix” shows the analysis results of GF mice that were orally inoculated with a mixture of these two strains. It is a graph which shows the result of having analyzed the expression of IFN- ⁇ and IL-17 about the CD4T cell of the colon lamina intestinal.
  • "GF” indicates the analysis result of GF mice not inoculated with the strain.
  • “+ 131A1” and “+ 35A1” are R.I. gnavus strain and E. coli.
  • the analysis result of the GF mouse which orally inoculated each of the colli strains is shown.
  • “+ 2-mix” indicates the analysis result of GF mice orally inoculated with a mixture of these two strains. Representative results of analysis of the proportions of IFN- ⁇ + cells and IL-17 + cells for CD4T cells in the lamina intestinal of the small intestine (“SILP” in the upper middle of the figure) and the lamina intestinal of the colon (“CLP” in the lower middle of the figure) are shown. It is a flow cytometry plot figure which shows. In GF mice, CD patient-derived E. E. coli strain (E. coli 35A1 strain, E. coli LF82 strain, or E. coli MG1655 strain) was orally inoculated and euthanized 3 weeks later.
  • E. E. coli strain E. coli 35A1 strain, E. coli LF82 strain, or E. coli MG1655 strain
  • the coli strain was established, followed by intraperitoneal injection of anti-mouse IL-10 receptor (IL-10R) antibody (1 mg / individual) weekly from day 1 to the end of the experiment to assess intestinal inflammation.
  • IL-10R anti-mouse IL-10 receptor
  • "GF + 35A1" and "GF” are E.I.
  • the analysis results of the mouse in which the colli 35A1 strain is colonized and the mouse in which the colli 35A1 strain is not colonized are shown.
  • the "+” and "-" of anti-IL10R indicate the presence or absence of anti-mouse IL-10R antibody injection, respectively.
  • Each E. It is a graph which shows the histological colitis score in the cecum of the GF mouse which established the coli strain. Each point represents an individual mouse. Each thick bar represents the average of the group.
  • CD shows the result of analyzing a sample derived from a Crohn's disease patient
  • non-CD shows the result of analyzing a sample derived from a non-Crohn's disease patient.
  • Each graph shows the results of analysis for the number of observed OTUs, ACE, Chao1 and Shannon index.
  • “Antegrada” and “Retrograda” indicate that they are the results of analysis of SI mucosal samples obtained by antegrade and prograde insertion, respectively.
  • Each bar graph represents the average value of the group. The P value obtained by analyzing with the unpaired Student's t-test is added.
  • the analysis results for each bacterial phylum are shown in order from the left, SI samples derived from non-CD patients obtained by anterograde insertion, and SIs derived from CD patients obtained by anterograde insertion.
  • the sample, the SI sample derived from a non-CD patient obtained by retrograde insertion, and the SI sample derived from a CD patient obtained by retrograde insertion are shown.
  • Each bar graph represents the average value of the group. Error bars represent SD.
  • FIG. 5 is a graph showing the relative abundance of bacterial taxa at the Family level in the mucosa of the small intestine. The notation in the figure is the same as that in FIG. 5C. It is a graph which shows the relative abundance of a bacterial taxon at the OTU level in the small intestinal mucosa. The notation in the figure is the same as that in FIG. 5C. It is a graph which shows the ⁇ diversity index of SI bacterial flora.
  • CD shows the result of analyzing a sample derived from a Crohn's disease patient
  • non-CD shows the result of analyzing a sample derived from a non-Crohn's disease patient.
  • Each graph shows the results of analysis for the number of observed OTUs, ACE, Chao1 and Shannon index.
  • Biopsy and “Scrape” indicate that they are the results of analysis of SI mucosal samples obtained by biopsy and scraping, respectively.
  • Each bar graph represents the average value of the group. The P value obtained by analyzing with the unpaired Student's t-test is added.
  • FIG. 5 is a graph showing the relative abundance of bacterial taxa at the Family level in the mucosa of the small intestine. The notation in the figure is the same as that in FIG. 5H. It is a graph which shows the relative abundance of a bacterial taxon at the OTU level in the small intestinal mucosa. The notation in the figure is the same as that in FIG. 5H.
  • FIG. 1 It is a graph which shows the relative abundance in a family-level bacterial taxon in the SI mucosal flora.
  • the horizontal axis shows the results for each bacteriological family (each left side shows the sample analysis results from non-CD patients, and each right side shows the sample analysis from CD patients receiving anti-TNF- ⁇ antibody therapy. The results are shown, and the results of sample analysis from other CD patients are shown in the middle).
  • Each thick bar represents the average of the group.
  • Each triangle represents an individual sample. Error bars represent 95% confidence intervals. For each asterisk, the value obtained by the multiple t-test using the misjudgment rate is *** ⁇ 0.05; *** ⁇ 0.01; *** ⁇ 0.001; *** ⁇ 0.0001. Show that.
  • each left side shows a sample analysis result (non-CD) derived from a non-CD patient
  • each right side shows a sample analysis result (CD) derived from a CD patient.
  • Each thick bar represents the average of the group.
  • Each point represents an individual sample. Error bars represent 95% confidence intervals.
  • the value obtained by the unpaired Student's t-test is *** ⁇ 0.05; *** ⁇ 0.01; *** ⁇ 0.001; *** ⁇ 0.0001.
  • the horizontal axis shows the results for each OTU (each left side shows the sample analysis result derived from a non-CD patient, and each right side shows the sample analysis result derived from a CD patient).
  • Each bar shows the average for the group. Error bars represent SD.
  • E. 3 is a graph showing the DNA concentration of the strain in SI, cecum (“Cecum” in the figure) and feces (“Feces” in the figure) of mice in which only the coli 35A1 strain has been established.
  • DNA was extracted from the fecal pellets of the mice and the lumen contents of the cecum and SI, and the DNA concentration of the fungus was determined by qPCR. Each point represents an individual mouse. Each thick bar represents the average of the group. Error bars represent SD. For each asterisk, the value obtained by one-way analysis of variance (one-way ANOVA) used in combination with Tukey's post-test is * P ⁇ 0.05; ** P ⁇ 0.01; *** P ⁇ 0. Indicates that it is 001. It is a graph which shows the ratio of IFN- ⁇ + cell in CD4T cell in the colon laminalitis. E. The E.
  • the coli 35A1 strain was administered to GF mice on the first day, and the anti-mouse IL-10R antibody (1 mg / individual) was intraperitoneally administered every week from the first day to the end of the experiment. The results of the mouse are shown in "GF + 35A1 + anti-IL10R". "GF” is E.I. The results of GF mice to which the colli 35A1 strain and the anti-mouse IL-10R antibody were not administered are shown, and "GF + 35A1" was not administered with the anti-mouse IL-10R antibody. The results of GF mice in which the colli 35A1 strain was established are shown, and "GF + anti-IL10R" is described in E. coli.
  • the results of the GF mouse to which the anti-mouse IL-10R antibody was administered without administering the coli 35A1 strain are shown. It is a graph which shows the result of having analyzed the histological Escherichia coli inflammation score in the proximal colon (Proximal colon) of a mouse.
  • the coli MG1655 strain was administered to GF mice on the first day, and the anti-mouse IL-10R antibody (1 mg / individual) was intraperitoneally administered every week from the first day to the end of the experiment.
  • mice The results of these mice are shown in "+ anti-IL10R + 35A1", “+ anti-IL10R + LF82" and “+ anti-IL10R + MG1655", respectively.
  • "GF” is E.I. The results of GF mice to which the colli strain and the anti-mouse IL-10R antibody were not administered are shown, and "GF + 35A1" was not administered with the anti-mouse IL-10R antibody. The results of GF mice in which the colli 35A1 strain was established are shown, and "+ anti-IL10R GF” is described in E. coli. The results of the GF mouse to which the anti-mouse IL-10R antibody was administered without the administration of the coli strain are shown. Each point represents an individual mouse.
  • Each thick bar represents the average of the group. Error bars represent SD. Each asterisk indicates that the values obtained by one-way ANOVA in combination with Tukey's post-test are * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001. .. It is a photograph showing a representative result of analysis of the distal colon of a mouse by hematoxylin and eosin staining. The notation in the figure is the same as in FIG. 11C. It is a graph which shows the result of having analyzed the histological Escherichia coli inflammation score in the distal colon of a mouse. The notation in the figure is the same as in FIG. 11D.
  • ⁇ Small intestinal bacteria of the present invention As shown in Examples below, nine strains of small intestinal bacteria isolated from patients with Crohn's disease were found to be significantly associated with Crohn's disease, and Th1 cells and / or Th17. It has become clear that it can induce proliferation or activation of cells (Th1 cells, etc.).
  • the present invention provides a small intestinal bacterium that induces proliferation or activation of Th1 cells and the like, and preferably has a DNA sequence encoding 16S rRNA (V1-V2 region) of these nine bacterial strains. It provides a bacterium having 95% or more identity.
  • small intestinal bacteria means bacteria that can colonize the human small intestine.
  • the site of the small intestine in which the bacterium is colonized is not particularly limited, and may be the ileum or the jejunum.
  • the tissue of the small intestine to which the bacteria colonize is not particularly limited, but is usually the mucous membrane of the small intestine.
  • Th1 cell is a subgroup of CD4 positive helper T cells (Th cells) and means a cell that enhances cell-mediated immunity.
  • Th1 cell activity refers to the production of Th1 cytokines (IFN- ⁇ , etc.) by the cells, the activation of cells such as macrophages and cytotoxic T cells (CTL) by the cytokines, and the cells due to the activation. It means that it includes enhancement of sexual immunity.
  • induction of proliferation or activation of Th1 cells means that induction of differentiation from naive T cells to Th1 cells leading to proliferation or activation of Th1 cells is also included. It also includes the accumulation (accumulation) of Th1 cells.
  • Th17 cells is a subgroup of CD4 positive helper T cells (Th cells) and means cells that induce inflammation.
  • the activity of Th17 cells means that the cells produce inflammatory cytokines (IL-17, IL-21, IL-22, TNF- ⁇ , etc.) and induce inflammation by the cytokines.
  • induction of Th17 cell proliferation or activation means to include induction of differentiation from naive T cells to Th17 cells, which leads to proliferation or activation of Th17 cells. It also includes the accumulation (accumulation) of Th17 cells.
  • the small intestinal bacterium of the present invention may be a single strain of bacteria or a mixture of bacterial strains composed of a plurality of strains of bacteria.
  • the bacterial strains When composed of a plurality of strains of bacteria, it is desirable that at least one of the bacterial strains has an activity of inducing proliferation or activation of Th1 cells or the like.
  • the plurality of strains of bacteria have an action of enhancing the activity of the bacterial strain having the inducing activity, even if the bacterial strain does not have the inducing activity.
  • Examples of the small intestinal bacterium of the present invention include a bacterium having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in any one of SEQ ID NOs: 1 to 9.
  • the DNA sequences shown in SEQ ID NOs: 1 to 1, respectively, are Eschericia coli (strain ID: 35A1), Ruminococcus gnavus (strain ID: 131A1), Bacteroides doriei (strain ID: 131H4), and Klebsiella, respectively, which are shown in Examples described later. pneumoniae (strain ID: 39F4), Streptococcus pasteurianus (strain ID: 133A7), Parabacteroides distasonis (strain ID: 134F1), Bacteroides fragilis (strain ID: 32E9), Erym : 131A11) 16SrRNA (V1-V2 region) encoding DNA sequence.
  • the "identity" with respect to the DNA sequence defined by each SEQ ID NO: is at least 95%, preferably 96% or more, more preferably 97% or more, still more preferably 98% or more, more preferably 99%. As mentioned above, it is particularly preferably 100%. Further, “identity” can be determined by using an alignment program such as BLAST. For example, nucleotide sequence identity includes identity between nucleotide sequences calculated using blastn, and more specifically, blastn with default parameters (ie, with default parameters). ) The calculated identity between the nucleotide sequences can be mentioned.
  • such a bacterium may be a bacterium having a nucleotide having at least 95% identity with respect to the DNA sequence defined by each SEQ ID NO:, and their species are not specified.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 1 are preferably bacteria belonging to Escherichia coli.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 2 are preferably bacteria belonging to Ruminococcus gnavus.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 3 are preferably bacteria belonging to Bacteroides doriei.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 4 are preferably bacteria belonging to Klebsiella pneumoniae.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 5 are preferably bacteria belonging to Streptococcus pasteurianus.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 6 are preferably bacteria belonging to Parabacteroides distasonis.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 7 are preferably bacteria belonging to Bacteroides fragilis.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 8 are preferably bacteria belonging to the Erysiperatoclastridium ramosom.
  • Bacteria having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 9 are preferably bacteria belonging to Bacteroides uniformis.
  • the bacteria in the small intestine of the present invention may be at least one of the above nine types, but the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine. From the viewpoint of being easier to induce, preferably with respect to a bacterium having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1, and the DNA sequence set forth in SEQ ID NO: 2.
  • the small intestinal bacteria of the present invention may be 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types or all combinations of the above 9 types, which will be described later.
  • the small intestinal bacteria of the present invention may be 2 types, 3 types, 4 types, 5 types, 6 types, 7 types, 8 types or all combinations of the above 9 types, which will be described later.
  • the same DNA sequence as shown in SEQ ID NO: 1 is used. It is a combination of a bacterium having a polynucleotide having sex and a bacterium having a polynucleotide having 95% or more identity with respect to the DNA sequence shown in SEQ ID NO: 2.
  • the present invention provides the first screening method shown below.
  • a method of screening for substances that suppress the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine (1) A step of ingesting a test substance into a non-human germ-free animal, (2) When the number or activity of Th1 cells and / or Th17 cells in the small intestine of the non-human animal is detected, and when suppression of proliferation or activation of the cells is detected in (3) step (2).
  • a method including a step of determining that the test substance is a substance that suppresses the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine.
  • the present inventors have clarified that the number or activity of Th1 cells and the like in the small intestine is enhanced by colonizing the above-mentioned small intestinal bacteria. ..
  • the present invention also provides the following second screening method.
  • the test substance is a bacterium having a polynucleotide having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1 and / or 95% or more with respect to the DNA sequence set forth in SEQ ID NO: 2.
  • the test substance is Th1 cells in the small intestine. And / or a method comprising the step of determining that the substance suppresses the proliferation or activation of Th17 cells.
  • test substance used in these screening methods of the present invention is not particularly limited as long as it can contain a substance that suppresses the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine, but for example. , Cells (bacteria, plant cells, animal cells, etc.), phages, and extracts, cultures (culture supernatants, etc.), secretory products, and metabolites thereof. Furthermore, it may be in the form of a library composed of a plurality of types of cells or phages.
  • synthetic small molecule compounds for example, synthetic small molecule compounds, antibodies, polypeptides, polynucleotides, lipids, sugars (monosaccharides, disaccharides, oligosaccharides, sugar chains, etc.), marine organisms, plant or animal-derived extracts, soil, and these substances.
  • synthetic small molecule compounds include a library composed of (chemical library, random peptide library, phage display library, etc.).
  • the "bacterium” used for the screening of the present invention is not particularly limited and may be a wild type or a mutant (genetical recombinant or the like), but it is established in the small intestine. Bacteria present in the small intestine of animals can be mentioned from the viewpoint that it is desirable to obtain them. Examples of such animals include humans and non-human animals (non-human animals) described below.
  • the test substance may be an isolated bacterium or a mixture of a plurality of types of bacteria. More specifically, the mixture includes a sample in the small intestine (for example, the contents of the small intestine of the animal, the small intestine lavage fluid of the animal, or a culture thereof).
  • the "bacteriophage” means a virus that infects and dissolves bacteria, and may be a lytic bacteriophage or a lysogenic bacteriophage, but a lytic bacteriophage is preferable. It is a phage. Further, it may be a mutant such as a genetically modified bacteriophage. Furthermore, the test bacteriophage may be an isolated bacteriophage or a mixture of multiple bacteriophages. More specifically, the mixture includes a sample in the small intestine (for example, the contents of the small intestine of the animal, the small intestine lavage fluid of the animal, or a culture thereof).
  • a phage against Escherichia coli is preferably used as a test bacteriophage. More specifically, T-type phages (T1, T2, T3, T4, T5, T6, T7, etc.), ⁇ X-174, ⁇ , ⁇ X80, Q ⁇ , P1 and the like can be mentioned.
  • a phage against Ruminococcus gnavus is preferably used as a test bacteriophage. More specifically, ⁇ Ra02, ⁇ Ra04 and the like can be mentioned.
  • Non-human germ-free animal to ingest the test substance means a non-human animal that is born and growing under sterile conditions.
  • Non-human animals include, but are not limited to, for example, mice, rats, monkeys, pigs, cows, horses, sheep, goats, chickens, ducks, ostriches, ducks, dogs, cats, rabbits, hamsters and the like. ..
  • a mouse is preferably used.
  • a non-human animal carrying the fungus may be used instead of the non-human germ-free animal.
  • Such non-human animals include, for example, specific pathogen-free (SPF) non-human animals.
  • a bacterium having a polynucleotide having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1 and / or the DNA sequence set forth in SEQ ID NO: 2.
  • a non-human animal in which a bacterium having a polynucleotide having a polynucleotide having 95% or more of the same identity (hereinafter, also referred to as “a bacterium defined by SEQ ID NO: 1 etc.”) has been established is as shown in Examples described later.
  • it can be prepared by administering (for example, oral administration) the bacteria defined by SEQ ID NO: 1 and the like to the non-human germ-free animal and colonizing the animal.
  • step (1) there is no particular limitation on the method of "ingesting" the test substance to a non-human animal, and it is usually carried out by oral administration, but it is parenteral administration (for example, administration into the intestinal tract). May be good.
  • the non-human animal is a non-human animal in which the bacteria defined by SEQ ID NO: 1 etc. have been established, the ingestion of the test substance and the bacteria defined by SEQ ID NO: 1 etc. is simultaneous.
  • the test substance may be ingested by a non-human germ-free animal and then the animal specified by SEQ ID NO: 1 etc. may be ingested, and the bacteria defined by SEQ ID NO: 1 etc. may be ingested by non-human germ-free animals.
  • the test substance may be ingested by the animal after being ingested by the animal.
  • the "detection of the number of Th1 cells and / or Th17 cells" in the small intestine in the step (2) does not have to be the number (amount) of these cells itself, and reflects, for example, the amount (number) of the cells. This can be done by measuring the amount of cell-specific substances. Examples of such a substance include the cell-specific oligonucleotide (for example, a transcript specific to each helper T cell).
  • Th1 cell and / or Th17 cell activity can be performed by measuring the amount of cytokine produced by the bacterium.
  • cytokines produced by Th1 cells include Th1 cytokines such as IFN- ⁇ , and examples of cytokines produced by Th17 cells include inflammatory cytokines (IL-17, IL-21, IL-22, TNF-). ⁇ , etc.).
  • detection of the number of bacteria defined by SEQ ID NO: 1 etc. in the small intestine does not have to be the amount (number) of the bacteria itself, as in the detection of the cell number, for example, the amount of the bacteria.
  • This can be done by measuring the amount of the bacterium-specific substance that reflects the (number).
  • Such substances include the bacterium-specific oligonucleotide (for example, 16SrRNA gene and its transcript), the bacterium-specific constituent substance (peptides, nucleic acids, sugars, lipids, and complexes thereof that constitute the bacterium). Etc.), products of the bacterium (eg, secretory products of the bacterium, metabolites of the bacterium).
  • oligonucleotide for example, PCR (RT-PCR, real-time PCR, quantitative PCR), DNA microarray analysis method, Northern blotting, next-generation sequencing method (synthetic sequencing).
  • Method sequencing-by-synthesis, eg, sequencing by Illumina Solexa genome analyzer or Hiseq® 2000), pyrosequencing method (eg, sequencer GSLX or FLX by Roche Diagnostics (454)). Sequencing by (so-called 454 sequencing)), rigase reaction sequencing method (for example, SoliD® manufactured by Life Technology Co., Ltd. or sequencing by 5500 xl), T-RFLP method, bead array method, in sit hybridization. , Dot blot, RNase protection assay, mass analysis, genomic PCR, and southern blotting can be used for quantification.
  • antibodies such as ELISA method, immunoblotting, antibody array analysis method, immunohistochemical staining method, flow cytometry, imaging cytometry, radioimmunoassay, and immunoprecipitation method are used. It can be quantified using a method of detection (immunological method).
  • the value obtained by quantifying in this way may be a relative value as well as an absolute quantity.
  • the relative value include a quantitative ratio (so-called numerical value expressed in an arbitrary unit (AU)) based on the measuring method or measuring device used for detection.
  • the relative value may be, for example, a value (relative abundance, occupancy) indicating the proportion of the bacterium in the entire small intestinal bacterial flora.
  • a value calculated based on the amount of reference small intestinal bacteria may be used.
  • the "reference small intestinal bacterium” according to the present invention may be a bacterium that is stably present in the small intestine and has a small difference in amount between different biological samples.
  • the timing of detection is not particularly limited, and a person skilled in the art can appropriately adjust the timing according to the type of animal used.
  • “Inhibition” of proliferation or activation of Th1 cells or the like in step (3) means that these values in non-human animals fed with the test substance are compared with those in non-human animals not fed with the sample. This means that it is significantly reduced.
  • the "growth suppression" of the bacterium defined by SEQ ID NO: 1 etc. means that the number of bacteria defined by SEQ ID NO: 1 etc. in the non-human animal ingested the test substance ingests the sample. It means that it is significantly reduced compared to those in non-human animals that have not been fed.
  • Statistical analysis methods include, for example, t-test, Wilcoxon signed rank test, analysis of variance (ANOVA), Kruskal-Wallis test, Mann-Whitney U test (Wilcoxon rank sum test), odds ratio, hazard ratio, etc. Fisher's exact test, receiver operating characteristic analysis (ROC analysis), classification tree and decision tree analysis (CART analysis) can be mentioned. Normalized or standardized and normalized data can also be used for comparison.
  • the screening method of the present invention if it is not possible to select a bacterium or bacteriophage that suppresses the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine by one execution, it is obtained.
  • the bacterium or bacteriophage can be isolated by ingesting a new non-human animal as the next test substance and performing the above-mentioned screening a plurality of times. can.
  • the present invention also provides a third screening method shown below, as shown in Examples described later.
  • a method of screening for bacteria that induce the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine (1) The process of ingesting the test substance into non-human germ-free animals, (2) A step of detecting the number or activity of Th1 cells and / or Th17 cells in the small intestine of the non-human animal. (3) A method comprising a step of isolating bacteria from a sample in the small intestine of a non-human animal in which proliferation or activation of Th1 cells and / or Th17 cells was detected in step (2).
  • the present invention also provides a fourth screening method shown below.
  • a method of screening for bacteria that induce the proliferation or activation of Th1 cells and / or Th17 cells in the small intestine (1) A step of ingesting a physiologically active substance derived from a small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells into a non-human germ-free animal. (2) A step of detecting the number or activity of Th1 cells and / or Th17 cells in the small intestine of the non-human animal. (3) When proliferation or activation of Th1 cells and / or Th17 cells is detected in step (2), the physiologically active substance is used to proliferate or activate Th1 cells and / or Th17 cells in the small intestine. A method comprising a step of determining that the substance is a physiologically active substance to be induced.
  • test substance "non-human germ-free animal”, “ingestion”, “detection of helper T cell proliferation (number) or activity” and “isolation of bacteria” in the third and fourth screening methods. Is the same as the first and second screening methods described above. Further, the “physiologically active substance” is as described later.
  • the small intestinal bacterium of the present invention is associated with the etiology of diseases caused by Th1 cells and the like, such as Crohn's disease. Therefore, the present invention A method for assessing diseases caused by Th1 cells and / or Th17 cells.
  • the present invention A method for assessing diseases caused by Th1 cells and / or Th17 cells.
  • Quantifying bacteria with polynucleotides having 95% or more identity (2) A step of comparing the value obtained by quantifying in step (1) with a corresponding value obtained by quantifying the bacteria in the mucosa of the small intestine of a human who does not suffer from the disease, and step (3) (3).
  • the "disease caused by Th1 cells and / or Th17 cells” means a disease induced by proliferation or activation of Th1 cells and / or Th17 cells, and is an inflammatory bowel disease (Clone's disease (CD). ), Chronic inflammatory bowel diseases such as ulcerative bowel disease and inflammatory bowel disease), type 1 diabetes, rheumatoid arthritis, experimental immune encephalitis (EAE), multiple sclerosis, autoimmune diseases such as systemic erythematosus, Chronic inflammatory diseases can be mentioned, and among them, Crohn's disease is a suitable subject of the present invention.
  • CD inflammatory bowel disease
  • Chronic inflammatory bowel diseases such as ulcerative bowel disease and inflammatory bowel disease
  • type 1 diabetes rheumatoid arthritis
  • EAE experimental immune encephalitis
  • multiple sclerosis autoimmune diseases such as systemic erythematosus
  • Chronic inflammatory diseases can be mentioned, and among them, Crohn's disease is a suitable subject of the present invention.
  • Crohn's disease is classified into small intestine type, small intestine large intestine type, and large intestine type according to the site where the lesion is present, but in the present invention, small intestine type Crohn's disease can be a more suitable target.
  • Examples of the "subject" in the evaluation method of the present invention include humans suspected of having or recurring a disease caused by Th1 cells and / or Th17 cells.
  • step (1) The quantification of small intestinal bacteria of the present invention in step (1) can be carried out in the same manner as in step (2) of the above-mentioned "screening method".
  • the bacteria can be collected from the mucosa of the small intestine using, for example, a double-balloon endoscopy (DBE) system as shown in Examples described later.
  • DBE double-balloon endoscopy
  • the bacterium to be quantified in the step (1) is a bacterium having a polynucleotide having 95% or more identity with respect to the DNA sequence set forth in SEQ ID NO: 1 and / or the bacterium described in SEQ ID NO: 2.
  • it is a bacterium having a polynucleotide having 95% or more identity with respect to the DNA sequence, it may be at least one of the nine small intestinal bacteria of the present invention.
  • the “human who does not suffer from a disease induced by proliferation or activation of Th1 cells and / or Th17 cells” to be compared in step (2) is not particularly limited as long as it does not suffer from the disease. , Age, gender, nationality and medical history are preferably consistent with the subject.
  • “comparison” with the corresponding value obtained by quantifying the bacterium in the small intestinal mucosa of a human not suffering from the disease can be performed by a person skilled in the art in the same manner as in step (3) of the above-mentioned "screening method". For example, it can be appropriately performed based on the above statistical analysis method.
  • the quantitative value obtained in the step (1) is higher than the corresponding value, and the difference is considered to be statistically significant (for example, P ⁇ 0.05).
  • the quantitative value may be twice or more (preferably 5 times or more and 10 times or more) the corresponding value.
  • the “determination” in step (3) includes not only the presence or absence of the onset of the disease but also the risk of its onset. That is, when the quantitative value in the small intestinal mucosa of the subject is higher than the corresponding value, it is determined that the subject has developed the disease or is at high risk of developing the disease.
  • the method of the present invention includes a method of collecting data on the above-mentioned quantitative values for diagnosis by a doctor, a method of presenting the data to a doctor, a method of comparing and analyzing the quantitative values and corresponding values, and a method of comparing and analyzing the corresponding values of a patient. It can also be described as a method for assisting a doctor's diagnosis in consideration of clinical symptoms and / or other test results.
  • the present invention provides a composition used in the above-mentioned inspection method. More specifically, the test composition of the following aspects is provided.
  • a composition for examining a disease caused by Th1 cells and / or Th17 cells which comprises an antibody that specifically recognizes the small intestinal bacteria of the present invention.
  • a composition for examining a disease caused by Th1 cells and / or Th17 cells which comprises a polynucleotide for detecting a nucleotide sequence specific to the small intestinal bacterium of the present invention.
  • the "antibody that specifically recognizes the small intestinal bacterium of the present invention” may be a polyclonal antibody, a monoclonal antibody, or an antibody as long as the bacterium can be specifically recognized.
  • Functional fragments eg, Fab, Fab', F (ab') 2, variable region fragment (Fv), disulfide-bound Fv, single-stranded Fv (scFv), sc (Fv) 2, diabodies, multispecific antibodies , Or a polymer of these).
  • an immune animal is immunized with an antigen (polypeptide, polynucleotide, sugar chain, lipid, etc.
  • the monoclonal antibody can be produced by a hybridoma method or a recombinant DNA method.
  • an antibody to which a labeling substance is bound can be used.
  • detecting the labeling substance it is possible to directly measure the amount of antibody bound to the small intestinal bacterium of the present invention or a substance derived from the bacterium.
  • the labeling substance is not particularly limited as long as it can bind to an antibody and can be detected by a chemical or optical method.
  • a fluorescent dye (GFP or the like), an enzyme (HRP or the like), or a radioactive substance is used. Substances are mentioned.
  • the test composition of the present invention may contain other components permitted as a composition in addition to the antibody component.
  • Such other components include, for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspensions, stabilizers, preservatives, preservatives, physiological salts, labeling substances, secondary antibodies. ..
  • a labeled substance for example, secondary antibody, protein G, protein A, etc.
  • the kit for testing for diseases caused by Th1 cells and the like can include an instruction manual for the kit.
  • test composition of the present invention can be combined with an apparatus for detecting the antibody of the present invention.
  • a device include a flow cytometry device and a microplate reader.
  • the "polynucleotide for detecting the nucleotide sequence specific to the bacterium of the present invention is not particularly limited as long as the sequence specific to the bacterium is detected, and for example, at least 15 nucleotides.
  • the polynucleotide of the present invention has a base sequence complementary to the nucleotide sequence of the small intestinal bacterium of the present invention.
  • “complementary” does not have to be completely complementary as long as it hybridizes.
  • These polynucleotides are usually 80% or more, preferably 90% or more, more preferably 95% or more (for example, 96% or more, 97% or more, 98% or more, 99% or more) with respect to the nucleotide sequence. Particularly preferably, it has 100% homology.
  • the "chain length" of the polynucleotide of the present invention is usually 15 to 100 nucleotides, preferably 17 to 30 nucleotides, and more preferably 20 to 25 nucleotides.
  • the "chain length" of the polynucleotide of the present invention is usually 15 to 1000 nucleotides, preferably 20 to 100 nucleotides.
  • the polynucleotide of the present invention may be DNA or RNA, and in part or all thereof, LNA (registered trademark, crosslinked nucleic acid), ENA (registered trademark, 2'-O, 4'- Nucleotides may be substituted with artificial nucleic acids such as C-Ethylene-bridged nucleic acids), GNA (glycerol nucleic acids), TNAs (treose nucleic acids), and PNAs (peptide nucleic acids).
  • LNA registered trademark, crosslinked nucleic acid
  • ENA registered trademark, 2'-O, 4'- Nucleotides
  • artificial nucleic acids such as C-Ethylene-bridged nucleic acids
  • GNA glycerol nucleic acids
  • TNAs treose nucleic acids
  • PNAs peptide nucleic acids
  • the polynucleotide of the present invention can be chemically synthesized using a commercially available nucleotide automatic synthesizer or the like. Further, as the polynucleotide used for the test of the present invention, a polynucleotide to which a labeling substance is bound can be used.
  • the labeling substance is not particularly limited as long as it can bind to a polynucleotide and can be detected by a chemical or optical method.
  • a fluorescent dye (DEAC, FITC, R6G, TexRed, Cy5, etc.)
  • Dyes such as DAB (chromogen)
  • enzymes radioactive substances in addition to fluorescent dyes.
  • test composition of the present invention may contain other pharmacologically acceptable components in addition to the above-mentioned polynucleotide.
  • examples of such other components include buffers, emulsifiers, suspending agents, stabilizers, preservatives, physiological salts and the like.
  • a sample and the present invention are combined by combining a substrate necessary for detecting a labeling substance added to a polynucleotide, a positive control or a negative control, and a buffer solution used for diluting or washing the sample.
  • a tube or plate used for the reaction with the polynucleotide of No. 1 can be combined, and a kit for testing a disease caused by Th1 cells can also be used.
  • the kit for testing for diseases caused by such Th1 cells can include instructions for use of the kit.
  • test composition of the present invention can be combined with an apparatus for detecting a nucleotide sequence specific to the small intestinal bacterium of the present invention.
  • a device for detecting a nucleotide sequence specific to the small intestinal bacterium of the present invention examples include a thermal cycler, a sequencer, and a microarray.
  • the present invention is a composition for inducing proliferation or activation of Th1 cells and / or Th17 cells, which comprises the small intestinal bacterium of the present invention or a physiologically active substance derived from the bacterium as an active ingredient, or a method thereof. Can be provided.
  • the small intestinal bacterium of the present invention which can be a pathogen of the disease, has an immunostimulatory action.
  • Th1 cells are CD4-positive helper T cells that produce IFN- ⁇ , play an important role in defense against pathogen infections such as Mycobacterium tuberculosis and Listeria monocytogenes, and monitor and eliminate cancerous cells. It also plays an important role in.
  • the presence of bacteria in the intestinal tract such as Bacteroides and Bifidobacterium and the induction of Th1 cells by such bacteria have been one of the immunotherapies for cancer, such as immune checkpoint inhibitors (anti-CTLA-4 antibody and anti-PD-L1).
  • the present invention can provide a composition for activating immunity or a method thereof.
  • the "immunity" activated or suppressed in the present invention includes not only mucosal immunity (intestinal immunity, etc.) but also systemic immunity. Moreover, not only cell-mediated immunity but also humoral immunity is included.
  • the "small intestinal bacterium of the present invention” contained as an active ingredient in the composition of the present invention is as described in the above ⁇ small intestinal bacterium of the present invention>, but it may be a live bacterium or a dead bacterium. It may be.
  • Examples of the "killed cell” include a heat-treated body, a fire-treated body, a steamed body, a radiation ( ⁇ -ray, etc.) irradiation-treated body, an enzyme-treated body, a drug (antibiotic, etc.) treated body, and a chemical substance (formaline, etc.). )
  • a processed body and an ultrasonic processed body can be mentioned.
  • the form of the small intestinal bacterium of the present invention is not particularly limited and may be either liquid or solid, but the form is in a dry form (for example, powder form, bacterial powder form, etc.) during production or storage. It is suitable from the viewpoint of easy handling.
  • the dried product can be prepared by drying a suspension in which the cells are dispersed in a solvent (water or the like).
  • the drying method is not particularly limited, and examples thereof include a spray drying method and a freeze drying method.
  • the sterilization method is not particularly limited, and the retort sterilization method, UHT sterilization method, air sterilization method, pressure sterilization method, high pressure steam sterilization method, dry heat sterilization method, distribution steam sterilization method, electromagnetic wave sterilization method, electron beam sterilization method, High-frequency sterilization method, radiation sterilization method, ultraviolet sterilization method, ethylene oxide gas sterilization method, hydrogen peroxide plasma sterilization method, chemical sterilization method (alcohol sterilization method, formalin fixation method, electrolyzed water treatment method) and the like can be mentioned.
  • surfactant treatment, grinding / crushing treatment, enzyme treatment, fractionation treatment, etc. can be performed before and after the sterilization treatment by heating or the like, or before and after the drying treatment.
  • surfactant treatment, grinding / crushing treatment, enzyme treatment, fractionation treatment, etc. can be performed.
  • the obtained product can also be contained in the composition of the present invention.
  • the bacterium may be modified (gene modification, etc.) according to the use of the contained active ingredient.
  • modifications are not particularly limited, and when the composition of the present invention is a vaccine adjuvant described later, modifications such that the action of inducing proliferation or activation of Th1 cells or the like is enhanced can be mentioned.
  • the active ingredient contained in the composition of the present invention may be a culture of small intestinal bacteria of the present invention.
  • the culture may be one containing the bacterium (culture solution containing the grown bacterium, solid medium, etc.), and the form of the culture is not particularly limited and may be either liquid or solid.
  • a dry form (for example, a dried culture product) is suitable for this form from the viewpoint of easy handling during production and storage. Those skilled in the art can appropriately prepare such a dried culture product or the like by using the above-mentioned drying method.
  • composition of the present invention may contain one strain of the small intestinal bacterium of the present invention, but may contain a plurality of types of strains.
  • the compositions can be used in combination, and as a result, when ingested or absorbed in combination (in the case of a combination composition), the plurality of strains can be present in two or more kinds of compositions. (For example, they can be in separate compositions).
  • the "physiologically active substance” is not particularly limited, but includes a substance contained in the bacterium, a secreted product of the bacterium, and a metabolite of the bacterium, and more specifically, the bacterium or the metabolite. Examples thereof include a polypeptide fraction, a polynucleotide fraction, a sugar chain fraction, a lipid fraction, and a low molecular weight metabolite fraction of the culture supernatant.
  • Such a physiologically active substance can be obtained from, for example, the above-mentioned screening method from the small intestinal bacterium of the present invention, the culture supernatant thereof, the small intestinal sample of a non-human animal in which the bacterium has settled, the human small intestine sample, and the like. As described above, it is possible to identify by purifying the active ingredient using the proliferation or activation of Th1 cells or the like as an index.
  • composition of the present invention can be in the form of a pharmaceutical composition, food or drink (including animal feed), or a reagent used for research purposes (eg, in vitro or in vivo experiments).
  • the active ingredient of the composition of the present invention induces the proliferation or activation of Th1 cells and the like, and activates immunity.
  • Pharmaceutical composition for treatment, prevention or improvement of infectious diseases such as tuberculosis pharmaceutical composition for enhancing anticancer activity as food and drink, and in combination with anticancer agent or immune checkpoint inhibitor , As a food and drink, and also as a pharmaceutical composition (vaccine adjuvant) for enhancing its immune response action when used in combination with a vaccine.
  • “treatment” also includes assistance for treatment.
  • composition of the present invention can be formulated by a known pharmaceutical method.
  • a known pharmaceutical method for example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chewing agents, buccal agents, pastes, syrups, suspensions, Oral and parenteral (eg, intestinal tract) as elixirs, emulsions, coatings, ointments, plasters, paps, transdermal formulations, lotions, inhalants, aerosols, injections, suppositories, etc.
  • carriers that are pharmacologically or acceptable as foods and drinks, specifically, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, etc.
  • the composition of the present invention is used in the small intestine. It may be combined with a composition that enables efficient delivery to the inside and the like.
  • the composition capable of such delivery into the small intestine or the like is not particularly limited, and a known composition can be appropriately adopted, for example, a pH-sensitive composition, suppression of release to the small intestine or the like.
  • compositions cellulose-based polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.
  • bioadhesive compositions that specifically adhere to the small intestinal mucosa
  • protease inhibitor-containing compositions in the small intestine Examples thereof include compositions that are specifically degraded by an enzyme.
  • composition that induces proliferation or activation of Th1 cells or the like of the present invention is used as a pharmaceutical composition
  • a known substance antiviral agent, antiviral agent, used for treatment, prevention or amelioration of infectious diseases and cancers, etc.
  • Antibacterial agents, anticancer agents, immune checkpoint inhibitors, etc. may be further contained, or may be used in combination with such substances.
  • a vaccine adjuvant in addition to the antigen that is the active ingredient of the vaccine (for example, a bacterial or virus-specific antigen, a cancer-specific antigen), other known vaccine adjuvants and immunopotentiators should be used.
  • the compositions of the invention may be included or may be used in combination with those substances.
  • the food or drink may be, for example, a health food, a functional food, a food for specified health use, a food with a nutritional function, a food with a functional claim, a nutritional supplement, a food for the sick, or It can be an animal feed.
  • Functional foods are usually classified into four categories, probiotics, biogenics, prebiotics, and symbiotics, based on their mechanism of action. In the present invention, probiotics, biogenics, and symbiotics are classified. It can take the form of tics or prebiotics.
  • foods and drinks include fermented beverages, oil-containing products, soups, dairy beverages, soft drinks, tea beverages, alcoholic beverages, drinks, jelly-like beverages and other liquid foods; carbohydrate-containing foods; processed livestock foods. Processed marine foods; processed vegetable foods; semi-solid foods; fermented foods; confectionery; retort products; microwave-compatible foods and the like. Further, healthy foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly can also be mentioned.
  • the food and drink according to the present invention can be produced by a production technique known in the art. In the food and drink, ingredients (for example, nutrients, etc.) effective for improving or preventing infectious diseases and cancer may be added. In addition, it may be a multifunctional food or drink by combining it with other ingredients or other functional foods that exhibit functions other than the improvement.
  • compositions of the present invention induce the proliferation or activation of Th1 cells and the like, or treat, improve or prevent diseases such as infectious diseases and cancer. It may be labeled as being used for this purpose.
  • the act of "displaying” includes all acts that inform the consumer of the use, and constitutes an expression that can directly recognize the use or an expression that can recall or infer the use. It may be attached to the product itself, or it may be attached to the container, packaging material or package insert containing the composition. Further, “display” refers to information related to the composition of the present invention, such as leaflets, pamphlets, pops, catalogs, posters, books, storage media such as DVDs, advertisements such as electronic bulletin boards and the Internet, and the like. It may be something that displays / advertises that it is effective.
  • composition of the present invention may be in the form of a kit.
  • kits include, for example, the small intestinal bacterium of the present invention or a physiologically active substance derived from the bacterium, an antiviral agent, an antibacterial agent, an anticancer agent, an immune checkpoint inhibitor, an antigen, another vaccine adjuvant, and the like.
  • an antiviral agent for example, an antiviral agent, an antibacterial agent, an anticancer agent, an immune checkpoint inhibitor, an antigen, another vaccine adjuvant, and the like.
  • the present invention requires that a composition for inducing proliferation or activation of Th1 cells or the like, or an active ingredient thereof, the small intestinal bacterium of the present invention or a physiologically active substance derived from the bacterium, be ingested. It also provides a method of inducing proliferation or activation of Th1 cells or Th17 cells in a subject, or a method of activating immunity in the subject.
  • composition of the present invention or an active ingredient thereof can be usually used for humans, but may be animals other than humans, and various livestock, poultry, pets, laboratory animals and the like are targeted. be able to.
  • an ingestion target of the composition for inducing the proliferation or activation of Th1 cells and the like of the present invention or the active ingredient thereof
  • animals infected with viruses, bacteria, etc. regardless of their onset, are used.
  • an animal that has not been infected with a virus, a bacterium, or the like or is suspected of being infected with the virus may be allowed to ingest the composition or the like of the present invention.
  • the composition of the present invention can be suitably used for animals that carry viruses, bacteria, etc. that do not have the symptom.
  • the composition of the present invention can be suitably used not only for animals suffering from cancer, but also for animals suspected of suffering from cancer and animals after anticancer therapy.
  • the method of ingesting the composition or the like of the present invention is not particularly limited and may be oral administration or parenteral administration (for example, administration into the small intestine), but in the case of oral administration.
  • the subject of ingestion of the composition of the present invention should reduce the production of gastric acid by ingesting a proton pump inhibitor (PPI) or the like. , Or it is preferable to take antibiotics.
  • PPI proton pump inhibitor
  • the ingestion amount depends on the age, weight, disease symptom, health condition, type of composition (pharmaceuticals, foods and drinks, etc.), intake method, etc. of the subject. Those skilled in the art can appropriately select it.
  • the small intestinal bacterium of the present invention induces a disease caused by Th1 cells or the like such as Crohn's disease by inducing proliferation or activation of Th1 cells or the like. Therefore, the disease can be treated, ameliorated or prevented by removing the bacteria from the small intestine or the like by inducing an immune response by targeting the bacteria in the small intestine of the present invention.
  • the present invention can provide a small intestinal bacterium of the present invention, a vaccine composition containing an antigen specific to the bacterium as an active ingredient, or a method for inducing an immune response against the bacterium.
  • the "small intestinal bacterium of the present invention” contained as an active ingredient in the composition of the present invention is as described above, and may be a live bacterium or a dead bacterium (for the dead bacterium). As mentioned above).
  • the small intestinal bacterium of the present invention may be a bacterium that has been modified (gene modification or the like) depending on the use of the active ingredient contained therein. The modification is not particularly limited, and when the composition of the present invention is a vaccine composition, the action of inducing the proliferation or activation of Th1 cells or the like in the small intestine or the like is suppressed so as not to induce inflammation or the like. Modifications can be mentioned.
  • the active ingredient contained in the composition of the present invention may be a culture of the bacteria in the small intestine of the present invention (the culture is as described above).
  • the composition of the present invention may contain at least one small intestinal bacterium of the present invention, but a plurality of strains (for example, 2 types, 3 types, 4 types, 5 types, 6 types, 7 types). , 8 types or 9 types).
  • the compositions can be used in combination, and as a result, when ingested or absorbed in combination (in the case of a combination composition), the plurality of strains can be present in two or more kinds of compositions. ..
  • the "antigen specific to the small intestinal bacterium of the present invention” is a substance (polypeptide, polynucleotide, sugar chain, lipid, etc.) contained in the bacterium, and is antigenic or immunogenic. It means a substance to have.
  • immunogenicity means that a primary immune response or a memory immune response can be activated.
  • the “immune response” includes responses of CD4 positive (CD4 +) T lymphocytes, CD8 positive (CD8 +) T lymphocytes and B lymphocytes.
  • T lymphocytes such responses are proliferative and / or cytokines (eg, IL-2, IL-3, IL-4, IL-5, IL-6, IL-12, IL-13, IL. -15, TNF- ⁇ , IFN- ⁇ ) production type may be used.
  • these responses may result in the production of cytotoxic T lymphocytes (CTL).
  • CTL cytotoxic T lymphocytes
  • the B lymphocyte reaction may result in antibody production by the reacting B lymphocytes.
  • "antigenic" means that it can be recognized by an antibody molecule or an antigen-specific T cell receptor (TCR) on activated effector T cells (eg, cytokine-producing T cells, CTL, etc.). ..
  • the "antigen specific to the small intestinal bacterium of the present invention” is a substance that can be recognized and bound to the antibody that specifically recognizes the bacterium or the like, or an appropriate antigen-presenting cell (APC). ), And after binding to a suitable major histocompatibility complex (MHC) molecule, contains a substance that can be recognized and bound to the TCR on the effector T cells induced in response to the bacteria or the like. Means.
  • MHC major histocompatibility complex
  • such a bacterial-specific antigen can be obtained by a person skilled in the art using a known screening method (for example, Paul WE editing, Fundamental Immunology) using the reactivity with the antigen-specific antiserum and / or T lymphocyte as an index. , 1993, 3rd edition, pp. 243-247, Harlow and Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, 1998).
  • the amino acid sequence of the bacterial-specific antigen (polypeptide) is analyzed using a computer program (for example, MHC-THREAD, EpiPredict, HLA-DR4 binding, ProPred, BIMAS, SVMHC, NetMHC, PREDICT, LpPep, SYFPEITHI, RankPe. ) Can also be inferred.
  • the vaccine composition of the present invention can be formulated by a known pharmaceutical method.
  • inhalants aerosols, injections, powders, granules, fine granules, liquids, capsules, tablets, pills, film coatings, pellets, lozenges, sublinguals, chewing agents, buccal agents, pastes.
  • Oral and parenteral eg, intestinal tract as agents, syrups, suspensions, elixirs, emulsions, coatings, ointments, ointments, paps, transdermal formulations, lotions, suppositories, etc.
  • carriers that are pharmacologically or acceptable as foods and drinks, specifically, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, etc.
  • the vaccine composition of the present invention may contain a known vaccine adjuvant and immunopotentiator.
  • the vaccine adjuvant include aluminum hydroxide, KLH, MPL, QS21, complete Freund's adjuvant, incomplete Freund's adjuvant, aluminum phosphate, BCG, myoban, TLR agonist such as CpG DNA, or a combination thereof.
  • an auxiliary agent such as albumin, a wetting agent, and an emulsifier may be added.
  • examples of the immunopotentiator include various cytokines (for example, IL-12, IL-18, GM-CSF, IFN- ⁇ , IFN- ⁇ , IFN- ⁇ , Flt3 ligand).
  • the product (pharmaceutical product, vaccine) of the composition of the present invention or its description induces an immune response against a bacterium that induces proliferation or activation of Th1 cells or the like in the small intestine or the like, and treats a disease caused by Th1 cells or the like.
  • It may be labeled as being used for improvement or prevention.
  • "marked on a product or instruction manual” means that a label is attached to the main body, container, packaging, etc. of the product, or a manual, package insert, advertisement, or other printed matter that discloses product information. It means that the display is attached to.
  • composition of the present invention may be in the form of a kit.
  • a kit for example, the small intestinal bacterium of the present invention or an antigen, a vaccine adjuvant, an immunopotentiator or the like specific to the bacterium usually exists as two or more kinds of substances (compositions and the like). Examples thereof include an embodiment in which one composition can be prepared by mixing or the like before ingesting the subject.
  • the present invention also comprises ingesting a vaccine composition, a bacterium as an active ingredient thereof, or an antigen specific to the bacterium into a subject, or a method for inducing an immune response against the bacterium in the subject. It also provides a method for treating, ameliorating or preventing a disease caused by Th1 cells or the like in a subject.
  • composition of the present invention or an active ingredient thereof can be used for animals including humans, but the animals other than humans are not particularly limited, and various livestock, poultry, pets, laboratory animals and the like are targeted. Can be.
  • a subject determined to be suffering from the disease by the above-mentioned test method of the present invention may be a suitable target.
  • the vaccine composition of the present invention or an active ingredient thereof is a bacterium that induces proliferation or activation of Th1 cells or the like in the small intestine or the like regardless of the onset of a disease caused by Th1 cells or the like.
  • examples include animals that possess. From the viewpoint of prevention, an animal that does not possess or is suspected of possessing the bacterium may be allowed to ingest the composition of the present invention or the like.
  • the method of ingesting the composition or the like of the present invention is not particularly limited, and may be oral administration or parenteral administration.
  • the amount of the ingestion may be appropriately determined by those skilled in the art depending on the age, body weight, symptoms of the disease, health condition, dosage form of the composition, ingestion method, etc. You can choose.
  • composition for suppressing proliferation or activation of Th1 cells and the like of the present invention The small intestinal bacterium of the present invention induces proliferation or activation of Th1 cells and the like, and enhances the immune action, thereby inducing diseases caused by Th1 cells and the like such as Crohn's disease. Therefore, if the bacterium is removed from the small intestine or the like, the induction of Th1 cells or the like is suppressed, and the immune action is suppressed, which leads to the treatment of the disease or the like.
  • the present invention is a composition for suppressing proliferation or activation of Th1 cells and / or Th17 cells, which contains a substance having an antibacterial effect against small intestinal bacteria of the present invention as an active ingredient, and suppresses immunity.
  • a composition for treating, ameliorating or preventing a disease caused by Th1 cells and / or Th17 cells is provided.
  • treatment means complete recovery from the disease
  • improvement includes alleviation or improvement of the symptoms of the disease, suppression of its progression, and suppression of its recurrence.
  • prevention includes suppression of the onset of the disease, delay, and suppression of its recurrence.
  • the "antibacterial action” means an action of suppressing the growth of bacteria and / or a bactericidal action.
  • the "substance having an antibacterial action against the bacteria in the small intestine of the present invention” contained as an active ingredient in the composition of the present invention is not particularly limited as long as it has the action, but for example, an antibiotic or a lytic substance ( Bacteriophage, lytic enzyme, etc.), antibodies that specifically recognize the bacterium, and the like. Further, it may be a substance that binds to a physiologically active substance derived from the small intestinal bacterium of the present invention, and examples of such a substance include an antibody that binds to the physiologically active substance and a low molecular weight compound that binds to the physiologically active substance. Can be mentioned. Furthermore, it may be a substance selected by the screening method of the present invention described above.
  • the composition of the present invention may contain a plurality of types of substances having an antibacterial action against the small intestinal bacteria of the present invention.
  • the compositions can be used in combination, and as a result, when ingested or absorbed in combination (in the case of a combination composition), the plurality of substances can be present in two or more kinds of compositions. ..
  • composition of the present invention can be in the form of a pharmaceutical composition, food or drink (including animal feed), or a reagent used for research purposes (eg, in vitro or in vivo experiments). Further, the composition of the present invention may be in the form of a kit, if necessary.
  • the composition of the present invention is a pharmaceutical composition for treating, preventing or improving the above-mentioned diseases caused by Th1 cells and the like, eating and drinking. It is preferably used as a product.
  • composition of the present invention can be formulated by a known pharmaceutical method.
  • a known pharmaceutical method for example, capsules, tablets, pills, liquids, powders, granules, fine granules, film coatings, pellets, lozenges, sublinguals, chewing agents, buccal agents, pastes, syrups, suspensions, Oral and parenteral (eg, intestinal tract) as elixirs, emulsions, coatings, ointments, plasters, paps, transdermal formulations, lotions, inhalants, aerosols, injections, suppositories, etc.
  • carriers that are pharmacologically or acceptable as foods and drinks, specifically, sterile water, physiological saline, vegetable oils, solvents, bases, emulsifiers, suspending agents, surfactants, stabilizers, etc.
  • the composition of the present invention is used in the small intestine. It may be combined with a composition that enables efficient delivery to the inside and the like.
  • the composition capable of such delivery into the small intestine or the like is not particularly limited, and a known composition can be appropriately adopted, for example, a pH-sensitive composition, suppression of release to the small intestine or the like.
  • compositions cellulose-based polymers, acrylic acid polymers and copolymers, vinyl acid polymers and copolymers, etc.
  • bioadhesive compositions that specifically adhere to the small intestinal mucosa
  • protease inhibitor-containing compositions in the small intestine Examples thereof include compositions that are specifically degraded by an enzyme.
  • composition that suppresses the proliferation or activation or immunity of Th1 cells or the like of the present invention is used as a pharmaceutical composition
  • a known substance used for the treatment, prevention or improvement of diseases caused by Th1 cells or the like for example, an anti-inflammatory agent, an immunosuppressant
  • an anti-inflammatory agent, an immunosuppressant may be further contained, or may be used in combination with such a substance.
  • the food or drink may be, for example, a health food, a functional food, a food for specified health use, a food with a nutritional function, a food with a functional claim, a nutritional supplement, a food for the sick, or It can be an animal feed.
  • Functional foods are usually classified into four categories, probiotics, biogenics, prebiotics, and symbiotics, based on their mechanism of action. In the present invention, probiotics, biogenics, and symbiotics are classified. It can take the form of tics or prebiotics.
  • foods and drinks include fermented beverages, oil-containing products, soups, dairy beverages, soft drinks, tea beverages, alcoholic beverages, drinks, jelly-like beverages and other liquid foods; carbohydrate-containing foods; livestock. Processed foods; processed marine products; processed vegetable foods; semi-solid foods; fermented foods; confectionery; retort products; microwave-compatible foods and the like. Further, healthy foods and drinks prepared in the form of powder, granules, tablets, capsules, liquid, paste or jelly can also be mentioned.
  • the food and drink according to the present invention can be produced by a production technique known in the art.
  • an ingredient for example, nutrients, etc.
  • it may be a multifunctional food or drink by combining it with other ingredients or other functional foods that exhibit functions other than the improvement.
  • the product (pharmaceutical product, food or drink, reagent) of the composition of the present invention or a description thereof suppresses the proliferation or activation of Th1 cells or the like, suppresses immunity, or treats or improves a disease caused by Th1 cells or the like. Alternatively, it may be labeled as being used for prevention.
  • the act of "displaying” includes all acts that inform the consumer of the use, and constitutes an expression that can directly recognize the use or an expression that can recall or infer the use. It may be attached to the product itself, or it may be attached to the container, packaging material or package insert containing the composition. Further, “display” refers to information related to the composition of the present invention, such as leaflets, pamphlets, pops, catalogs, posters, books, storage media such as DVDs, advertisements such as electronic bulletin boards and the Internet, and the like. It may be something that displays / advertises that it is effective.
  • the present invention has an antibacterial action against the composition for suppressing the proliferation or activation of Th1 cells and the like, the composition for suppressing immunity, or the small intestinal bacterium of the present invention which is an active ingredient thereof.
  • composition of the present invention or an active ingredient thereof can be used for animals including humans, but the animals other than humans are not particularly limited, and various livestock, poultry, pets, laboratory animals and the like are targeted. Can be.
  • the subject according to the present invention may be a person suffering from a disease caused by Th1 cells or the like, but it is not necessary to be diagnosed with these diseases, for example, a person who may have suffered from these diseases, these. Some people feel that they have a disease. Furthermore, even a healthy person can take it on a daily basis for the purpose of preventing these diseases.
  • a subject determined to be suffering from the disease by the above-mentioned test method of the present invention may be a suitable target.
  • the method of ingesting the composition or the like of the present invention is not particularly limited and may be oral administration or parenteral administration (for example, administration into the small intestine), but in the case of oral administration.
  • the subject of ingestion of the composition of the present invention should reduce the production of gastric acid by ingesting a proton pump inhibitor (PPI) or the like. Is preferable.
  • PPI proton pump inhibitor
  • the ingestion amount depends on the age, weight, disease symptom, health condition, type of composition (pharmaceuticals, foods and drinks, etc.), intake method, etc. of the subject. Those skilled in the art can appropriately select it.
  • SI mucosal samples were biopsied or biopsied from 27 Crohn's disease (CD) patients and 17 non-CD patients who received ongoing clinical care using a double-balloon endoscopy (DBE) system. Collected by scraping. Patients who underwent retrograde endoscopy underwent intestinal lavage from the day before the examination. Midazolam and pethidine were given intravenously for sedation and timepidium bromide hydrate or glucagon was used to reduce intestinal peristalsis. Based on the patient's condition, DBE insertion pathway (antegrade or prograde), endoscope type (EN-580 T, EN-450 T 5 or EN-450 P 5 (Fujifilm, Tokyo)) and The sampling method was decided.
  • endoscopic biopsy forceps (Radial Jaw 4P, Boston Scientific, Massachusetts, USA) or endoscopy search net (Roth net, US endoscopy, Ohio, USA) US) was used and obtained from the central SI of each patient.
  • SI solution about 50 cc was aspirated through the endoscope before collecting the mucosal specimen.
  • saliva and stool were collected one day before endoscopy.
  • the biopsy sample is suspended in TE10 (10 mM Tris-HCl, 10 mM EDTA) buffer, stored at -80 ° C, the mucosal scrapes and intestinal juice samples are centrifuged, and the pellet is placed in TE10 buffer. It was dissolved and stored at ⁇ 80 ° C.
  • biopsy samples were suspended in 20% glycerol / PBS, frozen in liquid nitrogen and then stored at -80 ° C. Mucosal scraping samples were centrifuged, pellets were dissolved in 20% glycerol / PBS, frozen in liquid nitrogen and stored at -80 ° C.
  • Frozen samples were thawed and mixed with 800 ⁇ l TE10 buffer containing RNaseA (final concentration 100 ⁇ gml- 1 , Invitrogen, MA, USA) and lysozyme (final concentration 15 mgml- 1, Sigma, MO, USA). The suspension was incubated at 37 ° C. for 1 hour with gentle mixing. Purified acromopeptidase (Wako, Osaka, Japan) was added at a final concentration of 2,000 units ml-1 , and the sample was incubated for an additional 30 minutes at 37 ° C.
  • RNaseA final concentration 100 ⁇ gml- 1 , Invitrogen, MA, USA
  • lysozyme final concentration 15 mgml- 1, Sigma, MO, USA
  • PCR was performed using primer sets of 27Fmod 5'-AGRGTTTTGATYMTGCTCAG-3'(SEQ ID NO: 10) and 338R 5'-TGCTGCCTCGTGGT-3' (SEQ ID NO: 11) targeting the V1-V2 region of the 16S rRNA gene. ..
  • the amplicon produced from each sample (about 330 bp) was purified using APPure XP (Beckman Coulter, CA, USA). DNA was quantified using a Quant-iT Picogreen ds DNA assay kit (Invitrogen) and a TBS-380 mini fluorometer (Turner Biosystems, CA, USA).
  • 16S metagenomic sequencing was performed using MiSeq according to the Illumina protocol. Two opposite-end reads were merged using a fastq-join program based on overlapping sequences. Reads with an average quality value of ⁇ 25 and inaccurate matches with both universal primers were excluded. The filtered reads were used for further analysis after trimming both primer sequences. For each sample, reads that have passed through the 3,000 quality filters are sorted in descending order according to their quality value and have a 97% pair-wise-identity cutoff using UCLUST program version 5.2.32. Clustered into OTUs having (https://www.drive5.com).
  • Taxonomic attribution of each OTU was made using the GLSEARCH program based on similarity to RDP and the National Center for Biotechnology Information (NCBI) genomic database. Taxa with relative amount> 0.1% were considered positive to determine bacterial prevalence.
  • Frozen samples are thawed, serially diluted with PBS and seeded on non-selective and selective agar plates (for aerobic culture: trypticase soiagar, for anaerobic culture: EG, BHK, MRS, CM 0619 + SR 0107).
  • EG and BHK are non-selective media for anaerobic bacteria.
  • MRS is a selective isolation medium for lactic acid bacteria.
  • CM 0619 supplemented with SR 0107 is a selective isolation medium for non-spore-forming anaerobes.
  • strains Individual isolates in the culture collection were classified as "strains" if their 16S rRNA sequences showed> 98% identity.
  • the obtained strain sequence was compared with the sequence of the NCBI database and the OTU observed by 16S rRNA analysis, and the closely related species and the corresponding OTU were determined.
  • TSI slope medium BD, NJ, USA
  • LIM medium LIM medium
  • SIM medium Eiken Chemical, Tokyo, Japan
  • API 20E BioMerieux, Marcy-l'Etoile
  • each E.I. coli strain 35A1, LF82 and MG1655 were cultured overnight aerobically in TS broth 37 ° C., gavage aliquots of 200 ⁇ l containing 7 ⁇ 9 ⁇ 10 8 colony forming units (CFU) in GF mice bottom.
  • the CD-related AIEC strain LF82 used in this example was originally isolated from the ileal mucosa of a CD patient by Darfeile-Michaud et al. (Reference 16).
  • the gnavus 131A1 strain was anaerobically cultured in EG broth or on an EG plate. The colonies on the plate were rubbed and resuspended in EG broth.
  • coli 35A1 strain and R Seven strains other than gnavus 131A1 strain were anaerobically cultured in EG or HK medium. Then, equal amounts of bacterial suspension were mixed to prepare 2-mix or 9-mix. A mixture of isolates or a single bacterial suspension was orally administered to GF mice and colonization was evaluated by Gram stain of the fecal suspension.
  • mice administered a mixture of specific bacterial strains or a single bacterial suspension were maintained on a single notobiotic isolator with a 12 hour / 12 hour light / dark cycle.
  • GFB6 mice (7-14 weeks old) were analyzed 3 weeks after the first gavage. All animal experiments were conducted with the approval of the Keio University Animal Experiment Committee.
  • the GF and SPF mice are all B6 mice obtained from CLEA Japan (Tokyo, Japan).
  • Intestinal lymphocytes were isolated as follows. First, the intestine was opened vertically and washed with PBS to remove the luminal contents. All samples were placed in 20 ml of 5 mM EDTA-containing Hanks Balanced Salt Solution (HBSS) and incubated in a shaking water bath at 37 ° C. for 20 minutes to remove enterocytes (IEC). The muscle layer and adipose tissue were then manually removed using forceps.
  • HBSS Hanks Balanced Salt Solution
  • the remaining LP layer was cut into small pieces and in 10 ml RPMI 1640, 4% fetal bovine serum, 0.5 mgml -1 collagenase D (Roche), 0.5 mgml -1 dispase (Gibco) and 40 ⁇ gml -1 DNase I ( Roche) was contained and incubated in a shaking water bath at 37 ° C. for 45 minutes.
  • Digested tissue was washed with HBSS containing 10 ml 5 mM EDTA, resuspended in 5 ml 40% Percoll (GE Healthcare, Illinois, USA) and in a 15 ml Falcon tube, 2.5 ml 80%. Percoll was layered. Percoll gradient separation was performed by centrifugation at 900 xg for 30 minutes at 25 ° C. Fractions containing lymphocytes were collected from the interface between the two layers and washed with RPMI 1640 containing 10% FBS.
  • cytokine detection cells were stimulated with 50 ngml -1 PMA and 750 ngml -1 ionomycin (both purchased from Sigma) at 37 ° C. for 3.5 hours in the presence of GolgiStop (BD).
  • the cells were permeabilized and treated with anti-TCR ⁇ (BV605; BioLegend, California, USA), anti-CD4 antibody (BV510; BioLegend), anti-TCR ⁇ antibody (BV510; biolegend).
  • anti-TCR ⁇ BV605; BioLegend, California, USA
  • anti-CD4 antibody BV510; BioLegend
  • anti-TCR ⁇ antibody BV510; biolegend
  • CD4T cells were defined as CD4 + TCR ⁇ + cell population within the live cell gate.
  • a model of colitis mediated by anti-IL-10R antibody A mouse model of colitis induced by an anti-IL-10 receptor (IL-10R) antibody was prepared as described in Schiering et al. (Reference 17). That is, on the first day, E.I. The coli 35A1, LF82 or MG1655 strains were established, followed by weekly intraperitoneal injection of anti-mouse IL-10R antibody (1 mg / individual) (BioXcell, New Hampshire, USA) from day 1 to the end of the experiment. Mice were then analyzed 5 weeks after initial colonization.
  • IL-10R anti-IL-10 receptor
  • RNA isolation and qPCR Total RNA was isolated from the scraped colonic epithelium using TRIzol reagent (Invitrogen) according to the manufacturer's instructions. The cDNA was synthesized using Revertra Ace qPCR RT Master Mix (Toyobo, Osaka, Japan). Then, using cDNA as a template, the following primer set and Thunderbird SYBR qPCR Mix (Toyobo) were used, and qPCR was performed with LightCyclor480 (Roche).
  • SI mucosal samples were obtained from 27 CD patients and 17 non-CD patients (patients with diseases other than SI, hamartomatous diseases, etc., see Table 1 below) by biopsy or scraping during the DBE method.
  • Fig. 1A Of the CD patients, 23 had intestinal stenosis (Montreal classification B2 or B3) and 19 were treated with anti-tumor necrosis factor- ⁇ (TNF- ⁇ ) antibody (Table 1).
  • the insertion route and sampling method of DBE were determined by the endoscopist based on the patient's condition. If the antegrade (oral) insertion route was selected, the distal jejunal mucosa was harvested. For retrograde (transanal) insertion, the proximal ileal mucosa was collected. Small intestinal samples were obtained from the mucosa outside the active ulcer or stenosis.
  • the small intestinal mucosal flora of CD patients contains more taxa within Proteobacteria and Bacteroidetes at the portal level and more taxa within Enterobacteriae, Ruminococcaceae and Bacteroidetes at the family level compared to those of non-CD patients.
  • Was FIGS. 1D and 1E.
  • the Firmicutes and Streptococcaceae taxa were reduced in CD patients (FIGS. 1D and 1E).
  • Table 2 shows 18 types of bacteria that are abundant in CD patients. For each group, the most closely related species or strain, National Center for Biotechnology Information (NCBI) genome database percent similarity (%), 9-mix strain ID, average abundance (Avenance), abundance (Abundance) Prevalence) is shown. The abundance rate was calculated by dividing the number of subjects in which a relative amount of bacteria exceeding 0.1% was detected in the SI mucosal sample by the total number of subjects.
  • NCBI National Center for Biotechnology Information
  • E. Colli, Ruminococcus gnavus, Bacteroides dorie, Klebsiella pneumoniae, Streptococcus pasteurianus, Parabacteroides merdee, Parabacteroides merdee, Parabacteroides merdae, Parabacteroides colli and R. gnavus was found to be significantly associated with CD SI mucosa in all three analyzes for abundance (Fig. 2C).
  • E. colli and R. gnavus was concentrated in the CD sample regardless of the insertion route or sampling method (FIGS. 5C-5E and 5H-5J).
  • strain ID: 39F4 S.A. pasteurianus
  • strain ID: 133A7 P. et al. Disstasonis
  • strain ID: 134F1 Bacteroides fragilis
  • strain ID: 32E9 Bacteroides fragilis
  • Erysiperatoclastridium ramosum strain ID: 131A10
  • Bacteroides fragilis strain ID: 131A10
  • Bacteroides uniformis strain ID: 131A11
  • the gut microbiota is recognized as a potent regulator of the host immune system (Reference 14). Therefore, the present inventors analyzed the ability of GF mice to stimulate immune cells in vivo with respect to the selected 9 strains. That is, each of the 9 strains was individually cultured, and the mixture was mixed to prepare a bacterial mixture (9-mix), which was orally administered to GF C57BL / 6 (B6) mice by oral forced gastric administration (Fig. 2G). Then, the mice were bred in a notobioto vinyl isolator for 3 weeks, and the lymphocytes and colon laminalitis (LP) in SI were examined by flow cytometry (Fig. 10).
  • Th17 cells in addition to Th1 cells 2-mix and E.I.
  • the coli 35A1 strain induced a significant increase in the Th cell frequency to the same extent as 9-mix. However, it was not as good as that in SPF mice (comparison between FIGS. 3B and 3F).
  • E. coli strain. colli LF82 strain or E.I. E. et al., A non-pathogenic laboratory strain derived from colli K-12.
  • a mouse in which the coli MG1655 strain has been established has been produced (Reference 16). Therefore, the degree of induction of Th1 cells and Th17 cells in the intestines of these mice was detected, and E.I. Compared with those of colli 35A1 colonized mice.
  • Th1 cell-induced E.I. in the onset of enteritis such as CD Th1 cell-induced E.I. in the onset of enteritis such as CD.
  • a colitis model induced by an anti-IL-10R antibody was used (Reference 17). Specifically, GF wild-type B6 mice were treated with E. coli on the first day. The coli 35A1 strain, the LF82 strain, or the MG1655 strain was established. Subsequently, anti-IL-10R antibody was injected intraperitoneally weekly and the intestines of those mice were analyzed.
  • CD is associated with intestinal flora disbiosis (decreased bacterial diversity) characterized by decreased ⁇ -diversity and increased potential inflammatory bacteria (Reference 11). However, few bacterial strains have been shown to be isolated from the CD-related flora and activate the host immune system.
  • the present inventors evaluated the SI bacterial flora of CD patients and screened for immunostimulatory bacteria.
  • the screening we obtained SI samples from CD patients using the DBE method, which is different from standard colonoscopy and is thorough from distal and proximal SI. It enables various tests and bacterial isolation.
  • E.I. The coli LF82 strain is a clinically relevant strain previously isolated from CD patients.
  • E. Escherichia coli LF82 strain is called intestinal adhesive invasive Escherichia coli (AIEC) because it adheres to and invades intestinal epithelial cells (EC) and can replicate in macrophages (References 16, 18, and 19).
  • AIEC intestinal adhesive invasive Escherichia coli
  • it involves mucin degradation (Vat-AIEC protease), adhesion to CEACAM6 via type 1 pili (expressed on the apical surface of EC) and activation of Toll-like receptors (TLRs) 5 via flagella.
  • E. The coli 35A1 strain is an inflammatory E. coli associated with the SI mucosa of CD patients. Another example of colli is shown. Interestingly, E.I. The coli 35A1 strain was used in the induction of Th1 cells by E. coli. It was stronger than the coli LF82 strain. Bifidobacterium longum and K. Considering that the pneumoniae strain is also associated with intestinal Th1 cell induction (References 25 and 26), E. coli. The colli 35A1 strain is B.I. longum and K. It may have factors similar to those of pneumoniae.
  • R. gnavus is also associated with CD.
  • R. gnavus has been reported to be increased in CD and thereby contributes to its pathogenesis (Reference 27).
  • R. gnavus comprises at least two lineage groups, one of which is predominant in CD patients and produces inflammation-inducing polysaccharides that induce the production of TNF- ⁇ by dendritic cells via TLR4 (References 28, 29). ).
  • R. The immunostimulatory effect of the gnavus 131A1 strain was weak, but R. gnavus may promote colonization of other bacteria associated with CD pathogenesis. In fact, R.
  • gnavus is a mucin-degrading bacterium that degrades the mucin layer, thereby promoting the colonization of other bacteria in the vicinity of the epithelial cell layer.
  • R. gnavus produces sialic acid (Reference 30), which is described in E. coli. It may promote the growth and colonization of other sialic acid-utilizing pathogens, including coli (Reference 31).
  • E.I. colli or R. It is noteworthy that the induction of Th17 cells by 2-mix colonization was significantly enhanced compared to gnavus alone (Fig. 3F).
  • the present invention by targeting a small intestinal bacterium that induces proliferation or activation of Th1 cells and / or Th17 cells, it becomes possible to suppress the proliferation or activation of the cells. As a result, it becomes possible to treat, improve or prevent the disease caused by the cells. Further, according to the present invention, it is also possible to test for diseases caused by the cells by using the amount of bacteria in the small intestine as an index.
  • the present invention is extremely useful in the development, treatment, improvement, prevention and diagnosis of pharmaceuticals relating to diseases such as Crohn's disease caused by Th1 cells and the like.

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Abstract

Afin de fournir une composition pour traiter, améliorer ou prévenir la maladie de Crohn ou similaire, un procédé de test pour cette maladie, etc., les présents inventeurs ont réussi à isoler une bactérie de l'intestin grêle, ladite bactérie induisant la prolifération ou l'activation des lymphocytes Th1 et/ou des lymphocytes Th17 liés à la maladie de Crohn, à partir d'une flore bactérienne dans la muqueuse de l'intestin grêle d'un patient atteint de cette maladie.
PCT/JP2021/017000 2020-04-29 2021-04-28 Petite bactérie intestinale induisant la prolifération ou l'activation de cellules th1 et/ou de cellules th17 WO2021221110A1 (fr)

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