ISOLATED BACTERIAL STRAIN FOR INDUCING PROLIFERATION OR ACCUMULATION OF REGULATORY T-CELLS

Abstract

The present invention relates to methods of using bacterial strain 1687A6 in detecting, diagnosing and treating disease or disorders of the GI tract. The present invention also relates to modulating the immune responses of an individual by inducing Th17 cell differentiation, proliferation, or accumulation. Further, the invention relates to therapeutic compositions containing strain 1687A6 or compounds derived from it and methods for treating disease in a subject using such compositions.

Claims

1. What is claimed is: A method of detecting strain 1687A6 in a patient comprising: obtaining a fecal sample from a human patient and screening for the presence of strain 1687A6 specific polynucleotides or polypeptides in the sample.

2. The method of claim 1 further comprising isolating nucleic acid sequences present in the fecal sample; and screening for the presence of strain 1687A6 specific sequences in the sample.

3. The method of claim 1 further comprising contacting the sample with an anti-strain 1687A6 antibody; and detecting whether strain 1687A6 is present in the sample by detecting binding between strain 1687A6 and the antibody.

4. The method of claim 1 further comprising detecting strain 1687A6 specific RNA sequences in the sample.

5. A method of determining the levels of strain 1687A6 in the gastrointestinal tract of a patient with inflammatory bowel disease comprising: obtaining a fecal sample from a human patient; isolating RNA from the sample; detecting strain 1687A6 specific RNA in the sample; and comparing the amount of strain 1687A6 specific RNA in the sample to a predetermined level of strain 1687A6 specific RNA.

6. A method of diagnosing IBD in a patient comprising: obtaining a fecal sample from a human patient; isolating nucleotides from the sample; detecting strain 1687A6 specific nucleotides in the sample; and comparing the amount of strain 1687A6 specific nucleotides in the sample to a predetermined level of strain 1687A6 specific nucleotides.

7. A method of identifying a therapeutic composition for treating IBD comprising: (a) determining the amount of strain 1687A6 in the GI of an individual; (b) administering a therapeutic composition to the individual; (c) assaying the effect of the therapeutic composition on the levels of strain 1687A6 or of Th17 cells in the GI of the individual; (c) comparing the levels of strain 1687A6 in the treated individual with predetermined levels of strain 1687A6 or of Th17 cells in the treated individual with predetermined levels of Th17 cells; and (d) determining whether the therapeutic agent reduces the levels of strain 1687A6 or of Th17 cells in the individual below the predetermined level.

8. The method of claim 7 wherein the method is limited to detecting and determining the levels of Th17.

9. The method of claim 7 wherein the method is limited to detecting and determining the levels of strain 1687A6.

10. A method for modulating an immune response in a subject by administering to a subject a therapeutic composition comprising: (a) the bacterial strain 1687A6; (b) at least one physiologically active substance derived from strain 1687A6; or (c) a fecal sample containing strain 1687A6.

11. The method of claim 10 wherein the immune response comprises inducing differentiation, proliferation, or accumulation of Th17 cells in the subject.

12. A method for identifying a physiologically active substance derived from strain 1687A6 that induces Th17 cell differentiation, proliferation, or accumulation in a subject comprising: (a) sequencing the genome of strain 1687A6; (b) comparing the genome of strain 1687A6 with the genome of at least one E. coli strain that does not normally induce Th17 cell proliferation, accumulation, or differentiation; (c) identifying the differences in nucleotide sequences between the two strains; (d) use recombinant technology to express strain 1687A6 specific sequences in an E. coli strain that does not normally induce Th17 cell proliferation, accumulation, or differentiation; and (e) testing whether the recombinant E. coli induces Th17 cell proliferation, accumulation, or differentiation in a subject.

13. A therapeutic composition containing strain 1687A6 or at least one physiologically active substance derived from strain 1687A6, wherein the composition comprises a: vaccine; adjuvant; biological; pharmaceutical composition, probiotic; food; beverage; fecal transplant; or a reagent used in an animal model, or a combination of such ingredients.

14. A method for treating a disease in a subject by administering a therapeutic composition containing strain 1687A6 or at least one physiologically active substance derived from strain 1687A6, wherein the composition comprises a: vaccine; adjuvant; biological; pharmaceutical composition, probiotic; food; beverage; fecal transplant; a reagent used in an animal model; or a combination of such ingredients.

15. The method of claim 14 wherein the disease is an infectious disease.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0048] FIG. 1 shows the induction of Intestinal Th17 cells in germ-free mice by human fecal microbiotas. Germ free C57B1/6 mice were colonized with human fecal microbiota from 18 different donors. After 5-8 weeks, the proportion of IL-17A.sup.+ (A,B) and RORγt.sup.+ (C,D) CD4.sup.+ T cells in the colon (A,C) and ileum (B,D) lamina propria was measured by flow cytometry. IL-17A.sup.+ T cells were measured following ex vivo restimulation for 4 hours with PMA and ionomycin in the presence of monensin. Each point represents one mouse and black bars show the arithmetic mean +/− the standard error of the mean for each group of mice. The color of the symbols represents the disease status of the donor (healthy, ulcerative colitis, or Crohn's disease) and the symbol shape represents if the microbiota tested was a total fecal microbiota or a cultured collection of microbes from that donor.

[0049] FIG. 2 Combinatorial gnotobiotics identifies a strain of E. coli that induces colonic Th17 cells and worsens experimental colitis. The figure shows the results of high throughput anaerobic culturing combined with gnotobiotic mouse screening to identify a strain of E. coli that induces colonic Th17 cells and worsens experimental colitis. In pane 2A, the cultured fraction of one donor's microbiota (donor 87 ) recapitulates the Th17 phenotype observed with the total fecal microbiota.

[0050] FIG. 2B Combinatorial gnotobiotics identifies a strain of E. coli that induces colonic Th17 cells and worsens experimental colitis (in pane 2F-H). The figure shows the capacity of 16 isolates from a single donor with Crohn's disease to induce Th17 cells. The 16 isolates from donor 87 were recombined into 8 sub-communities and the capacity of each to induce Th17 cells was tested. The table shows which isolates were included in each sub-community (+). The graph on the right of the table shows the proportion of IL-17A.sup.+ CD4.sup.+ T cells in mice colonized with each sub-community. The graph under the table shows the p-value of the association between each microbe and the colon Th17 cells. Data is pooled from two independent experiments.

[0051] FIGS. 2C, 2D show E. coli_A6 is strongly and uniquely associated with colonic IL-17A+ and RORγt+ T cells. Data in C and D is pooled from two independent experiments and each point represents data from one mouse. Data in C and D are not normally distributed. P values are calculated using Mann-Whitney test. FIG. 2C shows the association of a specific isolate (E. coli_A6 ) with colonic IL-17A.sup.+ cells. FIG. 2D shows the association of a specific isolate (E. coli_A6 ) with colonic RORγt.sup.+ T cells.

[0052] FIG. 2E Combinatorial gnotobiotics identifies a strain of E. coli that induces colonic Th17 cells and worsens experimental colitis. The figure shows that a community of bacteria isolated from a single donor (donor 87 ) but that lack E. coli A6 fails to induce colonic Th17 cells. It also shows that another strain of E. coli isolated from donor 87 (E. coli E2) is not associated with colonic Th17 cells. A community of all donor 87 isolates but lacking E. coli_A6 fails to induce colonic Th17 cells. Another strain of E. coli isolated from donor 87 (E. coli_E2) is not associated with Th17 cells. Each point represents one mouse and is data pooled from two independent experiments. * p<0.01.

[0053] FIG. 2F, G, H show that mice colonized with E. coli_A6 exhibit more severe intestinal inflammation induced by 2.5% DSS in drinking water. Combinatorial gnotobiotics identifies a strain of E. coli that induces colonic Th17 cells and worsens experimental colitis. FIG. 2F shows that mice colonized with E. coli_A6 exhibit more severe intestinal inflammation induced by 2.5% DSS in drinking water—by mean change in body mass +/− SEM. Representative of two independent experiments with similar results. * p=0.04, ** p=0.0014 at day 7, as calculated by ANOVA with Tukey's correction for multiple comparisons. FIG. 2G shows that mice colonized with E. coli_A6 exhibit more severe intestinal inflammation induced by 2.5% DSS in drinking water—he colon length of individual mice. Pooled from two independent experiments, each point represents the length of the colon of one mouse at day 7 after start of DSS treatment. ** p=0.0014, *** p=0.0007 calculated by ANOVA with Tukey's correction for multiple comparisons. FIG. 2H shows that mice colonized with E. coli_A6 exhibit more severe intestinal inflammation induced by 2.5% DSS in drinking water—by colon histopathology score. Scored by a pathologist blinded to the experimental conditions * p=0.04.

BRIEF DESCRIPTION OF THE SEQUENCES

[0054] Escherichia coli strain 1687A6 is defined by a 4.9 megabase genome. This genome sequence can be used to track the strain using metagenomics sequencing or cultured isolate sequencing.

DETAILED DESCRIPTION

[0055] Note that the term “individual” in the present invention is not particularly limited, and examples thereof include humans, mice, rats, cattle, horses, pigs, sheep, monkeys, dogs, and cats.

[0056] The term “therapeutic composition” according to the present invention may be in the form of a vaccine, adjuvant, biological, pharmaceutical composition, probiotic, food, beverage, fecal transplant, complex microbial or fecal sample, a reagent used in an animal model, or a combination of such ingredients. The vaccine, adjuvant, biological, pharmaceutical composition, probiotic, food, beverage, or reagent, or combinatorial product can have the effect of reducing or eliminating strain 1687A6 or of physiologically active substances derived from strain 1687A6 in the GI of a subject. The therapeutic compositions according to the present invention can also have the effect of stimulating or enhancing the differentiation, accumulation, or proliferation of Th17 cells in a subject or of stimulating the immune response in a subject. Administration of such therapeutic compositions may be oral, buccal, parenteral, rectal, or via fecal transplantation.

[0057] The term “IBD” in the present invention includes gastrointestinal disorders such as inflammatory bowel disease (IBD), ulcerative colitis, and Crohn's disease.

[0058] The term “Th17” or “Th17 cells” means CD4.sup.+ TH17 cells that express the transcription factor RORγt.sup.+ T, produce the cytokine IL-17, and play a critical role in promoting homeostasis at the mucosal barrier in the gut.

[0059] The phrase “reducing or eliminating differentiation, accumulation, or proliferation of Th17 cells” in the present invention includes an effect of reducing or inhibiting the differentiation of immature T cells into Th17 cells, which differentiation leads to the proliferation or the accumulation of Th17 cells. In addition, the meaning of the “reducing or eliminating differentiation, accumulation, or proliferation of Th17 cells” in the present invention includes in-vivo effects, in vitro effects, and ex vivo effects. Accordingly, all of the following effects are included: reducing or inhibiting in vivo proliferation or accumulation of Th17 cells in the gut through administration or ingestion of a therapeutic composition that inhibits strain 1687A6 or inhibits a physiologically active substance derived from strain 1687A6; reducing or inhibiting proliferation or accumulation of Th17 cells by preventing strain 1687A6 or a physiologically active substance derived from the bacteria to act on the cultured Th17 cells; and reducing or inhibiting proliferation or accumulation of Th17 cells that are collected from a living organism and that are intended to be subsequently reintroduced into that organism or introduced into another organism, by preventing strain 1687A6 or a physiologically active substance derived from the bacteria to act on the Th17 cells.

[0060] The effect of reducing or inhibiting differentiation, proliferation, or accumulation of Th17 cells can be evaluated, for example, by: orally administering strain 1687A6 to an experimental animal such as a germ-free mouse, allowing strain 1687A6 to proliferate in the GI of the animal, administering a therapeutic composition to the animal, isolating CD4-positive cells from the GI, measuring by flow cytometry the ratio of Th17 cells contained in the CD4-positive cells, and comparing the post-administration Th17 ratio to the pre-administration ratio or a predetermined level of Th17 cells.

[0061] One can determine whether the “reducing or eliminating differentiation, accumulation, or proliferation of Th17 cells” is occurring, for example, by assaying the ratio of Th17 cells in the T cell group of the colon, a function of Th17 cells in the colon, or expression of a marker of Th17 cells in the colon, such as RORγt.sup.+.

[0062] Methods of detecting Th17 cell RNA expression markers include, for example, high throughput RNA screening, northern blotting, dot blotting, and RT-PCR. Examples of methods for detecting protein markers include, for example, ELISA, radioimmunoassay, immunoblotting, immunoprecipitation, and flow cytometry.

[0063] Methods of screening for strain 1687A6 specific nucleotides include, for example, high throughput DNA or RNA screening, Southern blotting, northern blotting, dot blotting, recombinant DNA expression, and RT-PCR. Examples of methods for detecting strain 1687A6 specific protein markers include, for example, ELISA, radioimmunoassay, immunoblotting, and immunoprecipitation.

[0064] The meaning of “physiologically active substance derived from strain 1687A6 and “physiologically active substance derived from the bacteria” of the present invention includes metabolites of the bacteria and substances: contained in the bacteria; secreted by the bacteria; or affixed to the surface of the bacteria. Such a physiologically active substance can be identified by purifying an active component from the bacteria or supernatants of bacterial cultures or from the intestinal tract of a mouse colonized only by strain 1687A6. It can also be identified by screening for strain 1687A6 specific nucleotide sequences that lead to the differentiation, proliferation, or accumulation of Th17 cells in a subject.

[0065] The present invention can provide methods for determining the effects of a therapeutic composition by measuring the absolute amount or the ratio of strain 1687A6 in a microbiota of an individual diagnosed with IBD, treating the individual with a therapeutic composition, and evaluating whether the absolute amount or ratio of strain 1687A6 is reduced in comparison with a base line value obtained by performing a similar evaluation on a healthy individual.

[0066] One embodiment of the present invention provides a method for predicting a patient's response to a therapeutic composition and provide a prognosis. The method comprises measuring the percentage or absolute amounts strain 1687A6 in the microbiota in a subject diagnosed with IBD. Comparing it to a baseline value for those amounts in a healthy subject. Combining the results of the comparison with additional diagnostic information and medical history data related to the patient. Evaluating whether the patient may show a reduction in IBD after administration of a particular therapeutic composition.

EXAMPLES

[0067] Provided below are select examples of certain embodiments of the present invention; however, the invention is not limited to these examples or the specific embodiments recited above.

[0068] The inventors have identified a single bacterial strain that is a component of human gut microbiotas and that regulates different aspects of intestinal T cell function. To identify this bacterial strain, the inventors colonized germ free mice (which are born and raised under sterile conditions and have no pre-existing microbiota) with human gut microbiotas. The inventors used flow cytometry to study how these microbiotas changed the tone of the T cell response in the gut tissue of the mice.

[0069] Stool samples from inflammatory bowel disease patients and healthy controls were processed under anaerobic conditions. Using a wide range of solid media and culture conditions, a diverse selection of microbes were isolated from each sample and cultured in multiwell formats. Each isolate was identified using a combination of MALDI-TOF mass spectrometry, 16S rRNA and whole-genome sequencing. Microbes were pooled or selectively recombined and introduced to germ-free mice to generate personalized, humanized gnotobiotic mice.

[0070] Microbiotas were obtained from healthy donors and donors with ulcerative colitis (UC) or Crohn's disease (CD). Eighteen (18) different complex fecal microbiotas from each human donor were screened. Of these, microbiotas from two donors, both individuals with Crohn's disease, induced a greater proportion of IL-17A-secreting Th17 cells in the colon and small intestine of mice than all other donors tested. (See FIG. 1.)

[0071] The inventors subjected the complex fecal microbiota of one donor with Crohn's disease (“donor 87”) to high throughput anaerobic culturing and generated a diverse cultured collection of bacteria from this donor. Using 16s rDNA and whole genome sequencing, the inventors identified 16 unique isolates from the microbiota of donor 87 . The inventors subsequently colonized germ-free mice with a pool of the isolates and surprisingly discovered that this culturable fraction of the microbiota from donor 87 recapitulated the elevated Th17 cell response observed with the complex fecal microbiota from the donors. (See FIG. 2A.) At the time of sampling, the Crohn's disease in donor 87 was in remission.

[0072] The inventors recombined the isolates into 8 new sub-communities using an orthogonal screen design. Each sub-community comprised four microbes, with each microbe appearing in two subcommunities. The inventors subsequently colonized groups of germ-free mice with each of the 8 sub-communities and assessed Th17 cell induction in each group. Th17 cells were induced by two of the subcommunities. One strain of Escherichia coli (“E. coli_A6”) featured in both communities and in no other. Linear modeling confirmed that E. coli_A6 was the only isolate that had a significant positive correlation with the proportion of colonic Th17 cells induced. (See FIGS. 2B, 2C, and 2D.)

[0073] The inventors discovered that E. coli_A6 was necessary for Th17 induction by colonizing mice with either the entire cultured collection of microbes, the collection of microbes without E. coli_A6, or the collection of microbes lacking a different strain of E. coli isolated from donor 87 (referred to as E. coli E2). E. coli E2 showed no significant correlation with Th17 cells in the combinatorial screen. In agreement with the screening data, the collection of microbes lacking E. coli_A6 induced a lower proportion of colonic Th17 cells than either the entire cultured collection or the microbe collection lacking E. coli_E2. (See FIG. 2E.)

[0074] The inventors further discovered that the increased proportion of Th17 cells induced by donor 87, and specifically by E. coli_A6, renders mice more susceptible to intestinal inflammation. The inventors colonized germ free mice with either (a) the cultured collection of microbes from donor 87 or (b) the collections lacking either strain of E. coli. The inventors induced intestinal inflammation using dextran sodium sulfate, administered in drinking water. Colitis was less severe in mice lacking E. coli_A6, as measured by reduced weight loss, increased colon length, and less severe histological pathology. (See FIGS. 2F, 2G, and 2H).