WO2022155443A1 - Compositions et méthodes de traitement et de prévention de maladies ou de troubles au moyen d'interactions entre espèces - Google Patents

Compositions et méthodes de traitement et de prévention de maladies ou de troubles au moyen d'interactions entre espèces Download PDF

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WO2022155443A1
WO2022155443A1 PCT/US2022/012472 US2022012472W WO2022155443A1 WO 2022155443 A1 WO2022155443 A1 WO 2022155443A1 US 2022012472 W US2022012472 W US 2022012472W WO 2022155443 A1 WO2022155443 A1 WO 2022155443A1
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strain
species
disease
subject
allobaculum
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PCT/US2022/012472
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Noah PALM
Tyler RICE
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Yale University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
    • A23K10/16—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions
    • A23K10/18—Addition of microorganisms or extracts thereof, e.g. single-cell proteins, to feeding-stuff compositions of live microorganisms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • 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
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales

Definitions

  • pathogenic immunostimulatory bacteria can play potentially causal roles in inflammatory bowel disease (IBD), autoimmunity, and malnutrition, while beneficial immunostimulatory species have been employed to treat metabolic syndrome and as adjuncts for cancer immunotherapy (Atarashi et al., 2015, Cell, 163:367-380; Baruch et al., 2020, Science, eabb5920; Plovier et al., 2017, Nat. 25 Med.
  • IBD inflammatory bowel disease
  • beneficial immunostimulatory species have been employed to treat metabolic syndrome and as adjuncts for cancer immunotherapy
  • a method of preventing or treating a disease or disorder induced by a first gut microbe species or strain thereof in a subject in need thereof comprises administering to the subject a composition comprising a second gut microbe species or strain thereof, wherein the level of the second gut microbe species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by the first gut microbe species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • the first gut microbe species or strain thereof is an Allobaculum species or strain thereof.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the second gut microbe species or strain thereof is an Akkermansia species or strain thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
  • the first gut microbe species or strain thereof inhibits systemic antibody responses directed against the second gut microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the second gut microbe species or strain thereof.
  • the disease or disorder is an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof.
  • the at least one compound that reduces the level of the first gut microbe species or strain thereof comprises a probiotic, antibiotic, antimicrobe, prebiotic of the second gut microbe species or strain thereof, or any combination thereof.
  • the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof.
  • the at least one compound that increases the level of the second gut microbe species or strain thereof comprises a probiotic, prebiotic of the second gut microbe species or strain thereof, antibiotic of the first gut microbe species or strain thereof, antimicrobe of the first gut microbe species or strain thereof, or any combination thereof.
  • the method further comprises the step of administering to the subject a composition comprising at least one compound that reduces the level of the first gut microbe species or strain thereof prior to the step of administering to the subject the composition comprising the second gut microbe species or strain thereof. In some cases, the method further comprises detecting the presence of the first gut microbe species or strain thereof in the subject prior to the administration of the composition.
  • a method of preventing or treating a disease or disorder induced by a first gut microbe species or strain thereof in a subject in need thereof comprises administering to the subject a composition comprising an active agent isolated from conditioned culture media harvested from a culture of a second gut microbe species or strain thereof, wherein the active agent reduces or inhibits at least one pathogenic effect produced by the first gut microbe species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • the first gut microbe species or strain thereof is an Allobaculum species or strain thereof.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the second gut microbe species or strain thereof is an Akkermansia species or strain thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
  • the first gut microbe species or strain thereof inhibits systemic antibody responses directed against the second gut microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the second gut microbe species or strain thereof.
  • the disease or disorder is an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof.
  • the at least one compound that reduces the level of the first gut microbe species or strain thereof comprises a probiotic, antibiotic, antimicrobe, prebiotic of the second gut microbe species or strain thereof, or any combination thereof.
  • the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof.
  • the at least one compound that increases the level of the second gut microbe species or strain thereof comprises a probiotic, prebiotic of the second gut microbe species or strain thereof, antibiotic of the first gut microbe species or strain thereof, antimicrobe of the first gut microbe species or strain thereof, or any combination thereof.
  • the method further comprises the step of administering to the subject a composition comprising at least one compound that reduces the level of the first gut microbe species or strain thereof prior to the step of administering to the subject the composition comprising the second gut microbe species or strain thereof.
  • the method further comprises detecting the presence of the first gut microbe species or strain thereof in the subject prior to the administration of the composition.
  • the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
  • a method of preventing or treating a disease or disorder induced by an Allobaculum species or strain thereof in a subject in need thereof comprises the steps of: (a) detecting the presence of the Allobaculum species or strain thereof in the subject; and (b) administering a composition comprising at least one Akkermansia species or strain thereof to the subject, wherein the level of the at least one Akkermansia species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by Axe Allobaculum species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • the method further comprises the step of administering to the subject a composition comprising at least one compound that reduces the level of the Allobaculum species or strain thereof prior to the step of administering the composition comprising the at least one.
  • Akkermansia species or strain thereof to the subject.
  • the at least one compound that reduces the level of Axe Allobaculum species or strain thereof comprises a probiotic, antibiotic, antimicrobe, prebiotic of at least one Akkermansia species or strain thereof, nucleic acid molecule encoding at least one Akkermansia species or strain thereof, or any combination thereof.
  • the presence of the Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • the presence of the Allobaculum species or strain thereof is detected in a biological sample of the subject.
  • Axe Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (EEC) activation.
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
  • a method of preventing or treating a disease or disorder induced by an Allobaculum species or strain thereof in a subject in need thereof comprises the steps of: (a) detecting the presence of the Allobaculum species or strain thereof in the subject; and (b) administering a composition comprising an active agent isolated from conditioned culture media harvested from a culture of at least one Akkermansia species or strain thereof to the subject, wherein the active agent reduces or inhibits at least one pathogenic effect produced by the Allobaculum species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • the method further comprises the step of administering to the subject a composition comprising at least one compound that reduces the level of the Allobaculum species or strain thereof prior to the step of administering the composition comprising the at least one.
  • Akkermansia species or strain thereof to the subject.
  • the at least one compound that reduces the level of Axe Allobaculum species or strain thereof comprises a probiotic, antibiotic, antimicrobe, prebiotic of at least one Akkermansia species or strain thereof, nucleic acid molecule encoding at least one Akkermansia species or strain thereof, or any combination thereof.
  • the presence of the Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • the presence of the Allobaculum species or strain thereof is detected in a biological sample of the subject.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (EEC) activation.
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
  • the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
  • a method of predicting the effectiveness of a composition comprising an Akkermansia species or strain thereof for treating or preventing a disease or disorder in a subject, wherein the method comprises the steps of: (a) detecting the level of at least one Allobaculum species or strain thereof in a subject suffering from a disease or disorder; and (b) comparing the level of the at least one Allobaculum species or strain thereof in the subject to a comparator. In some cases, the level of the at least one Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO. : 2 or a fragment thereof.
  • a method of predicting the effectiveness of a composition comprising an active agent isolated from conditioned culture media harvested from a culture of an Akkermansia species or strain thereof for treating or preventing a disease or disorder in a subject, wherein the method comprises the steps of: (a) detecting the level of at least one Allobaculum species or strain thereof in a subject suffering from a disease or disorder; and (b) comparing the level of the at least one Allobaculum species or strain thereof in the subject to a comparator. In some cases, the level of the at least one Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
  • a composition comprising a beneficial gut microbe species or strain thereof, wherein the level of the beneficial gut microbe species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by a pathogenic species or strain thereof that induces a disease or disorder.
  • the composition modulates an immune response toward the disease or disorder.
  • the composition further comprises at least one additional agent selected from the group consisting of a probiotic, prebiotic, antibiotic, antimicrobe, and any combination thereof.
  • the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof.
  • the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the disease or disorder is an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an inflammatory bowel disease, colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
  • IEC intestinal epithelial cell
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • compositions comprising an active agent isolated from conditioned culture media harvested from a culture of a beneficial gut microbe species or strain thereof, wherein the active agent reduces or inhibits at least one pathogenic effect produced by a pathogenic species or strain thereof that induces a disease or disorder.
  • the composition modulates an immune response toward the disease or disorder.
  • the composition further comprises at least one additional agent selected from the group consisting of a probiotic, prebiotic, antibiotic, antimicrobe, and any combination thereof.
  • the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof.
  • AXQ Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof.
  • the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the disease or disorder is an inflammatory disease or disorder.
  • the inflammatory disease or disorder is an inflammatory bowel disease, colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
  • IEC intestinal epithelial cell
  • the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
  • the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • FIGs 1A-1N illustrate that a novel Allobaculum species from an ulcerative colitis patient exacerbates acute and chronic colitis in gnotobiotic mice.
  • FIG. 1 A Identification and Isolation of IgA-coated Allobaculum sp. 128 from an ulcerative colitis patient.
  • FIG. IB Scanning electron micrographs of Allobaculum sp. 128 in vitro. Scale bars, 2pm (top), 10m (bottom).
  • FIGs 1C-H Germ-free WT mice were inoculated by oral gavage and equilibrated for seven days before treatment with 2% DSS-H2O ad libitum.
  • FIG. 1C Fecal microbiota on dO (first bar) and d7 (bars 2-5) of DSS treatment.
  • FIGs 1D-E Colons at euthanasia on d7 and representative H&E-stained colon sections. Scale bars, 1mm.
  • FIG. IF Colon length on d7.
  • FIG. 1G Fecal lipocalin (LCN2) on d2.
  • FIG. 1H Histopathology scoring of blinded colon sections.
  • FIG. 1J lamina limbal growth factor + Ly6G + neutrophils
  • FIG. IL cytokines from colon explant cultures
  • FIGs 2A-2I illustrate that Allobaculum sp. 128 elicits mucosal and systemic immunity at steady state.
  • FIG. 2G Micrographs of colon sections stained with bacterial FISH probe EUB338 and DAPI. Each scale bar is 25pm. Solid yellow line marks the epithelial brush border and white dashed line marks the inner mucus layer.
  • FIGs 3A-3F illustrates that Allobaculum sp. 128 is inversely correlated with Akkermansia muciniphila in human microbiota-associated gnotobiotic mice.
  • FIG. 3 A Experimental workflow.
  • FIG. 3C Each genus-level OTU was tested for Spearman correlation to Allobaculum sp. 128 abundance.
  • FIG. 3D XY plot of data shown in (FIG. 3B).
  • FIGs 4A-4R illustrates that A. muciniphila attenuates Allobaculum sp. 128-mediated intestinal epithelial cell activation and colitis.
  • FIGs 4B-D Inflammation as assessed by d2 fecal lipocalin (FIG.
  • FIG. 4B colon length at euthanasia
  • FIG. 4C-D colon length at euthanasia
  • FIG. 4K Experimental design for assessing intestinal epithelial cell activation by RNAseq.
  • FIG. 40 Schematic depicting the experimental approach wherein GF mice were either colonized with live commensal microbes or gavaged daily with sterile conditioned culture supernatant for 10 days before harvesting RNA from intestinal epithelial cells.
  • FIGs 4P-R Ileal IEC expression of key Allobaculum-induced genes by qRT-PCR. Error bars show mean ⁇ SEM.
  • FIGs 5A-5E illustrates that A. muciniphila-colomzadon protects against Allobaculum sp. 128-induced colitis in the context of a complex human microbiota.
  • FIG. 5A Experimental design: Homogenized healthy control stool (HC19) prepared under anaerobic conditions was gavaged into GF mice along with either or both immunostimulatory strains of interest and colitis was induced via administration of 2% DSS-H2O.
  • FIG. 5B Fecal microbiota composition on d3 of colitis.
  • FIG. 5C Fecal lipocalin assessed on d2-3.
  • FIGs 6A-6D illustrates that Allobaculum sp. 128 blunts antigen-specific serum antibody responses to A. muciniphila and oral vaccination.
  • FIG. 6A Schematic shows the experimental workflow for analyzing week 6 serum antibody binding to cultured bacterial cells.
  • FIGs 6C-D Cholera toxin (CT)-vaccinated mice colonized with MC or MC+Allo were bled after 5 weeks and CT-specific serum IgG responses were measured by ELISA.
  • CT Cholera toxin
  • FIGs 7A-7J illustrates that Allobaculum sp. 128 and A. muciniphila induce context-dependent transcriptomic reprogramming in mucosal lymphoid tissues.
  • FIGs 7A-B Annotated UMAP dimensionality reduction plots of single-cell gene expression libraries, pooled by tissue (FIG. 7A, Mesenteric lymph nodes (MLN); FIG. 7B, Peyer’s patches (PP)). Right, heatmap of each cell lineage relative frequency normalized to mock community (MC).
  • FIG. 7C TCR repertoire diversity.
  • FIG. 7D Top 12 most expanded clonotypes in MC+A.m.
  • FIGs 7E,7H MLN Tfh+Tfr and MigrDC were examined in isolation, re-clustered, and highlighted by microbiome.
  • FIG. 7F Expression of key genes within Tfh+Tfr shown across microbiome groups.
  • FIG. 7G Prominent TCR clonotypes within MLN Tfh+Tfr induced by MC+A.m.
  • FIG. 7H MLN MigrDC UMAP clustering.
  • FIG. 71 Expression of key MigrDC antigen presentation genes, including Cd74 (li, invariant chain).
  • FIGs 8A-8K illustrates that Allobaculum sp. 128 does not bloom during inflammation, and a second UC patient Allobaculum isolate is colitogenic in gnotobiotic mice.
  • FIG. 8B Time course of fecal lipocalin (LCN-2) overlaid with Allobaculum sp. 128 abundance.
  • FIG. 8C H&E-stained colon sections from Ragl -/- mice on d7 of DSS administration. Scale bars, 200pm.
  • FIG. 8H Phylogenetic tree constructed using 16S rRNA gene sequences from members of family Erysipelotrichaceae, using maximum likelihood estimation, bootstrapped (BS) to 1,000 replicates. BS values are shown along branches.
  • FIG. 81 Experimental schematic.
  • FIG. 8J Second Allobaculum isolate (Allo2) was used for acute colitis model in WT gnotobiotic mice as in FIGs 1C-1H. Three of six mice colonized with Allo2 were found dead (3 F.D.) prior to endpoint.
  • FIGs 9A-9G illustrate an unremarkable histopathology and total serum Ig of untreated Allobaculum sp. 128-colonized WT mice.
  • FIG. 9A Bouin’s-fixed H&E-stained colon sections from WT mice colonized with MC bacteria or MC+ Allobaculum sp. 128, euthanized 12 weeks later. Scale bars, 200pm.
  • FIG. 9B Blinded scoring for colitis.
  • FIGs 10A-10F illustrates that microbial diversity cannot explain the relationship between Allobaculum and A. muciniphila, A. muciniphila and Allobaculum co-colonize the ileal mucosa, and co-colonization has minimal impacts on A. muciniphila and Allobaculum gene expression.
  • FIG. 10C GF WT mice were mono- or bi-colonized as shown for 10 days. Terminal ilea were fixed, and sections stained with bacterial FISH probes EUB338 (to stain Allobaculum) and VP403 (to stain A. muciniphila). Scale bars, 10pm.
  • FIG. 10D In vivo bacterial transcriptomes from the ileum and colon were compared for differential expression of ORFs across single colonization or co- colonization conditions (MC+Allo vs MC+Both, and MC+Akk vs. MC+Both).
  • FIG. 10E Allobaculum sp. 128 and
  • FIGs 11A-11H illustrates that type strain A. muciniphila attenuates Allobaculum sp. 128- mediated colitis and Allobaculum sp. 128 blunts A. muciniphila-induced dendritic cell responses in MLN.
  • FIG. 11 A Experimental schematic for acute DSS colitis in WT gnotobiotic mice colonized with MC, MC+ Allobaculum sp. 128, MC+4. muciniphila T (type strain ATCC BAA- 835), or MC+ Allobaculum sp. 128+A. muciniphilcA (ATCC B
  • FIG. 11C Fecal microbiota profiling
  • FIG. 11 ID Colon length
  • FIG. 1 ID d2 fecal lipocalin
  • FIG. 1 IE gross colon pathology.
  • FIG. 1 IF Representative gating strategy for analysis of MLN cells performed in FlowJo after > 100,000 events per sample were collected on a BD LSRII cytometer.
  • FIG. 11H Quantification of DCs (Live B220-TCRb'CDl lb + CDl lc + MHCII + ).
  • FIGs 12A-12B illustrate an approach for profiling microbiota-dependent mucosal immune landscape using single cell RNAseq.
  • FIG. 12 A Schematic depicting single cell RNAseq (scRNAseq) experiment.
  • FIG. 12B Quality control metrics used for filtration of scRNAseq data before proceeding to clustering and differential expression analyses.
  • FIG. 13 illustrate that expression of marker genes mapped to MLN cell clusters. Violin plots showing expression of marker genes across MLN cell clusters, numbered to match clusters shown in FIG. 14A-14G.
  • FIGs 14A-14G illustrate epistatic reversal of A. muciniphila-induced MLN immune cell clusters by co-colonization with Allobaculum sp. 128, and direct assessment of MLN DC function in co-colonized gnotobiotic mice.
  • FIGs 14A-E Graph-based probabilistic analysis of MLN scRNAseq data, comparing two microbiota groups at a time.
  • FIGs 14A-B MC+Allo relative to MC+Both or relative to MC.
  • FIGs 14C-E MC+Akk relative to MC+Both reveals strong reversal (high MELD score) of cell clusters induced by A.
  • FIG. 13 The marker genes that define each cluster are displayed in FIG. 13.
  • CTV CellTrace Violet
  • FIG. 15 is a schematic outlining the process of reciprocal epistasis between Allobaculum sp. 128 and A. muciniphila.
  • FIG. 16A depicts representative results for quantification of confocal micrographs of ileum cryosections, one representative image of which is shown in FIG. 16B. Welch’s t-test was used to compare microbiota groups at each time point. *** P ⁇ 0.001, **** P ⁇ 0.0001, n.s. not significant.
  • FIG. 16B and 16C depict representative results demonstrating that Allobaculum penetrates terminal ileum crypts more so than IgA-neg bacteria.
  • FIG. 16B depicts representative confocal microscopy of ileum cryosections, from left to right: AF488 pan- bacterial cell wall (Green); AF647 Allobaculum nanobody (Red); DAPI (Blue); and crypt border (dotted yellow outline). Scale bars, 10 pm. All micrographs were analyzed in Imaged, blinded to color of bacterial cells, measuring distance from each cell to the crypt base as demonstrated.
  • FIGs 17A through FIGs 17C depicts representative results demonstrating that Akkermansia-specfic Peyer’s Patch T cells are blunted by Allobaculum.
  • FIG. 17B depicts representative quantification of PP CD4 + T cells, both Akkermansia-specfic, left, and bulk follicular T helper (Tfh) cells, right.
  • FIG. 17C depicts representative FACS plots of PP T cells stained with Akkermansia tetramers (gated on singlets > FSC 10 lymphocytes > CD4 + TCRb + ).
  • FIG. 18A through 18D depict representative results demonstrating Allobaculum spontaneously translocates to mesenteric lymph nodes (mLN) of IL10-deficient mice.
  • FIG. 18A depicts representative qPCR results for Allobaculum gDNA.
  • FIG. 18B depicts representative qPCR results for universal bacterial 16S rRNA from 8 week mLN samples from IL10 -/- mice colonized with MC or MC + Allobaculum.
  • FIG. 18C depicts representative results demonstrating the Allobaculum-specific serum IgA and IgG.
  • FIG. 18D depicts representative results demonstrating the fecal microbiota profile by 16S amplicon sequencing.
  • FIG. 19A through 19F depict representative results for competition between Allobaculum and commensal bacteria from diverse human gut microbiota.
  • FIG. 19A depicts a schematic representation of general description of workflow.
  • FIG. 19B depicts representative fecal microbiota profiling of human microbiota-colonized mice, sorted by Allobaculum abundance.
  • FIG. 19C depicts representative tabulated OTUs that are most positively and most negatively correlated with Allobaculum.
  • FIG. 19D depicts representative receiver operating curves (ROC) for the eight logistic regressions corresponding to the OTUs shown in FIG. 19C.
  • FIG. 19E depicts representative volcano plot of each OTUs Spearman R vs. P-values of the regression’s log likelihood ratio (LLR).
  • FIG. 19F depicts representative three microbiome datasets, plotted as relative abundance Akkermansia vs Allobaculum.
  • the term “a” or “an” can refer to one or more of that entity, i.e., can refer to a plural referents. As such, the terms “a” or “an”, “one or more” and “at least one” can be used interchangeably herein.
  • reference to “an element” by the indefinite article “a” or “an” does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there is one and only one of the elements.
  • patient refers to any animal, or cells thereof whether in vitro or in vivo, amenable to the methods described herein.
  • patient, subject or individual is, by way of non-limiting examples, a human, a dog, a cat, a horse, or other domestic mammal.
  • a “disease” is a state of health of an animal wherein the animal cannot maintain homeostasis, and wherein if the disease is not ameliorated then the animal’s health continues to deteriorate.
  • a “disorder” in an animal is a state of health in which the animal is able to maintain homeostasis, but in which the animal’s state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the animal’s state of health.
  • a disease or disorder is “alleviated” if the severity of a sign or symptom of the disease or disorder, the frequency with which such a sign or symptom is experienced by a patient, or both, is reduced.
  • treating a disease or disorder means reducing the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a patient.
  • Immuno response means a process involving the activation and/or induction of an effector function in, by way of non-limiting examples, a T cell, B cell, natural killer (NK) cell, and/or antigen-presenting cells (APC).
  • an immune response includes, but is not limited to, any detectable antigen- specific activation and/or induction of a helper T cell or cytotoxic T cell activity or response, production of antibodies, antigen presenting cell activity or infiltration, macrophage activity or infiltration, neutrophil activity or infiltration, and the like.
  • GI gastrointestinal tract
  • gut refers to the entire alimentary canal, from the oral cavity to the rectum.
  • the term encompasses the tube that extends from the mouth to the anus, in which the movement of muscles and release of hormones and enzymes digest food.
  • the gastrointestinal tract starts with the mouth and proceeds to the esophagus, stomach, small intestine, large intestine, rectum and, finally, the anus.
  • microbiota refers to the population of microorganisms present within or upon a subject.
  • the microbiota of a subject includes commensal microorganisms found in the absence of disease and may also include pathobionts and disease-causing microorganisms found in subjects with or without a disease or disorder.
  • microbiome refers to the totality of microbes (bacteria, fungae, protists), their genetic elements (genomes) in a defined environment.
  • the microbiome is a gut microbiome (e.g., intestinal microbiome).
  • gut microbiome as used herein can refer to the totality of microorganisms, bacteria, viruses, protozoa and fungi and their collective genetic material present in the gastrointestinal tract (GIT).
  • microbe refers to an intact or whole microbe or any matter or component that is derived, originated or secreted from a microbe. Any matter or component that is derived, originated or secreted from a microbe is also referred to as “microbial matter” herein.
  • gut microbe can refer to any commensal or pathogenic microorganisms, bacteria, viruses, protozoa and fungi that colonize the gastrointestinal tract (GIT) or gut.
  • gut microbiota as used herein can refer to the collection or population of microorganisms, bacteria, viruses, protozoa and fungi , commensal and pathogenic, residing in the GIT.
  • gut microbes that make up the gut microbiota and gut microbiome can include, but not be limited to bacteria selected from Segmented Filamentous Bacteria (SFB), Helicobacter flexispira, Lactobacillus, Helicobacter, S24-7, Erysipelotrichaceae, Prevotellaceae, Paraprevotella, Prevotella, Acidaminococcus spp., Actinomyces spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides acidifaciens, Bacteroides coprophilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Bamesiellaceae spp., Bifidobacterium adolescentis, Bifidobacterium Other, Bif
  • microbe-binding and “microbe-targeting” are used interchangeably and refer to an ability of a molecule or composition to not only bind and/or capture a microbe and/or microbial matter, but also to provide high sensitivity in detecting the microbe and/or microbial matter when the molecule or composition is used as a detection agent.
  • the microbe-binding molecules disclosed herein can bind/capture and also detect an intact or whole microbe or microbial matter derived, originated or secreted from the microbe.
  • Exemplary microbial matter that can bind to the microbe-targeting molecule can include, but is not limited to, a cell wall component, an outer membrane, a plasma membrane, a ribosome, a microbial capsule, a pili or flagella, any fragments of the aforementioned microbial components, any nucleic acid (e.g., DNA, including 16S ribosomal DNA, and RNA) derived from a microbe, microbial endotoxin (e.g., lipopolysaccharide), and the like.
  • microbial matter can encompass nonviable microbial matter that can cause an adverse effect (e.g., toxicity) to a host or an environment.
  • pathobiont or “pathogenic microbe” are used interchangeably and refer to potentially disease-or disorder-causing members of the microbiota that are present in the microbiota of a non-diseased or a diseased subject, and which has the potential to contribute to the development or progression of a disease or disorder.
  • the term “beneficial microbe”, as used herein, refers to members of the microbiota that are present in the microbiota of a non-diseased or a diseased subject, and which has the potential to contribute to the reduction of the severity and/or frequency with which a sign or symptom of the disease or disorder is experienced by a subject having a disease or disorder.
  • isolated means altered or removed from the natural state.
  • a microbe naturally present in its normal context in a living animal is not “isolated,” but the same microbe partially or completely separated from the coexisting materials of its natural context is “isolated.”
  • An isolated microbe can exist in substantially purified form, or can exist in a non-native environment such as, for example, a gastrointestinal tract.
  • an “effective amount” or “therapeutically effective amount” of a compound is that amount of a compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • a “therapeutic” treatment is a treatment administered to a subject who exhibits signs or symptoms of a disease or disorder, for the purpose of diminishing or eliminating those signs or symptoms.
  • the term “pharmaceutical composition” refers to a mixture of at least one compound useful within the invention with a pharmaceutically acceptable carrier.
  • the pharmaceutical composition facilitates administration of the compound to a patient or subject. Multiple techniques of administering a compound exist in the art including, but not limited to, intravenous, oral, rectal, aerosol, parenteral, ophthalmic, pulmonary and topical administration.
  • the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively non-toxic, i.e., the material may be administered to an individual without causing an undesirable biological effect or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • regulating can mean any method of altering the level or activity of a substrate (e.g., microbiome).
  • a substrate e.g., microbiome
  • Non-limiting examples of regulating with regard to a microbiome or microbiota further include affecting the microbiome or microbiota activity.
  • regulator refers to a molecule whose activity includes affecting the level or activity of a substrate (e.g., microbiome).
  • a regulator can be direct or indirect.
  • a regulator can function to activate or inhibit or otherwise modulate its substrate (e.g., microbiome).
  • the terms “silence”, “silencing”, “inhibit”, and “inhibition,” as used herein, means to reduce, suppress, diminish, or block an activity or function relative to a control value.
  • the activity is suppressed or blocked by at least about 10% relative to a control value.
  • the activity is suppressed or blocked by at least about 50% compared to a control value.
  • the activity is suppressed or blocked by at least about 75%.
  • the activity is suppressed or blocked by at least about 95%.
  • homology refers to a degree of complementarity. There may be partial homology or complete homology (i.e., identity). Homology is often measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705). Such software matches similar sequences by assigning degrees of homology to various substitutions, deletions, insertions, and other modifications.
  • sequence analysis software e.g., Sequence Analysis Software Package of the Genetics Computer Group. University of Wisconsin Biotechnology Center. 1710 University Avenue. Madison, Wis. 53705.
  • a “fragment” of a peptide sequence or a nucleic acid sequence that encodes an antigen may be 100% identical to the full length except missing at least one amino acid or at least one nucleotide from the 5’ and/or 3’ end, in each case with or without sequences encoding signal peptides and/or a methionine at position 1.
  • Fragments may comprise 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more percent of the length of the particular full length coding sequence, excluding any heterologous signal peptide added.
  • the fragment may comprise a fragment that encode a polypeptide that is 95% or more, 96% or more, 97% or more, 98% or more or 99% or more identical to the antigen and additionally optionally comprise sequence encoding an N terminal methionine or heterologous signal peptide which is not included when calculating percent identity.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting there from.
  • a gene encodes a protein if transcription and translation of mRNA corresponding to that gene produces the protein in a cell or other biological system.
  • nucleotide sequence encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence.
  • nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).
  • a “probiotic” refers live, non-pathogenic microorganisms, e.g., bacteria, which can confer health benefits to a host organism that contains an appropriate amount of the microorganism.
  • the host organism is a mammal.
  • the host organism is a human.
  • Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • probiotic bacteria examples include, but are not limited to, Bifidobacteria, Escherichia coli, Lactobacillus, and Saccharomyces, e.g., Bifidobacterium bifidum, Enterococcus faecium, Escherichia coli strain Nissle, Lactobacillus acidophilus, Lactobacillus bulgaricus, Lactobacillus paracasei, Lactobacillus plantarum, and Saccharomyces boulardii (Dinleyici et al., 2014; U.S. Pat. Nos. 5,589,168; 6,203,797; 6,835,376).
  • the probiotic may be a variant or a mutant strain of bacterium (Arthur et al., 2012; Cuevas-Ramos et al., 2010; Olier et al., 2012; Nougayrede et al., 2006).
  • Non-pathogenic bacteria may be genetically engineered to enhance or improve desired biological properties, e.g., survivability.
  • Non-pathogenic bacteria may be genetically engineered to provide probiotic properties.
  • Probiotic bacteria may be genetically engineered to enhance or improve probiotic properties.
  • a prebiotic refers to an ingredient that allows specific changes both in the composition and/or activity in the gastrointestinal microbiota that may (or may not) confer benefits upon the host.
  • a prebiotic can be a comestible food or beverage or ingredient thereof.
  • Prebiotics may include complex carbohydrates, amino acids, peptides, minerals, or other essential nutritional components for the survival of the bacterial composition.
  • Prebiotics include, but are not limited to, amino acids, biotin, fructooligosaccharide, galactooligosaccharides, hemicelluloses (e.g.
  • inulin chitin, lactulose, mannan oligosaccharides, oligofructose-enriched inulin, gums (e.g. , guar gum, gum arabic and carregenaan), oligofructose, oligodextrose, tagatose, resistant maltodextrins (e.g., resistant starch), trans- galactooligosaccharide, pectins (e.g.
  • xylogal actouronan citrus pectin, apple pectin, and rhamnogalacturonan-I
  • dietary fibers e.g. , soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber
  • xylooligosaccharides e.g. , soy fiber, sugarbeet fiber, pea fiber, corn bran, and oat fiber
  • antibody refers to an immunoglobulin molecule which is able to specifically bind to a specific epitope on an antigen.
  • Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • the antibodies in the present invention may exist in a variety of forms including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies (“intrabodies”), Fv, Fab and F(ab)2, as well as single chain antibodies (scFv), heavy chain antibodies, such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York;
  • synthetic antibody as used herein, is meant an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • the term “nanobody”, “heavy chain antibody” or “heavy chain antibodies” comprises immunoglobulin molecules derived from camelid species, either by immunization with a peptide and subsequent isolation of sera, or by the cloning and expression of nucleic acid sequences encoding such antibodies.
  • the term “heavy chain antibody” or “heavy chain antibodies” further encompasses immunoglobulin molecules isolated from an animal with heavy chain disease, or prepared by the cloning and expression of VH (variable heavy chain immunoglobulin) genes from an animal.
  • antigen or “Ag” as used herein is defined as a molecule that provokes an adaptive immune response. This immune response may involve either antibody production, or the activation of specific immunogenically-competent cells, or both.
  • antigens can be derived from recombinant or genomic DNA or RNA.
  • any DNA or RNA which comprises a nucleotide sequences or a partial nucleotide sequence encoding a protein that elicits an adaptive immune response therefore encodes an “antigen” as that term is used herein.
  • an antigen need not be encoded solely by a full-length nucleotide sequence of a gene. It is readily apparent that the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene and that these nucleotide sequences are arranged in various combinations to elicit the desired immune response. Moreover, a skilled artisan will understand that an antigen need not be encoded by a “gene” at all. It is readily apparent that an antigen can be generated synthesized or can be derived from a biological sample. Such a biological sample can include, but is not limited to a microbiota sample, tissue sample, a tumor sample, a cell or a biological fluid.
  • an “immunoassay” refers to any binding assay that uses an antibody capable of binding specifically to a target molecule to detect and quantify the target molecule.
  • an antibody that specifically binds to an antigen from one species may also bind to that antigen from one or more species. But, such cross-species reactivity does not itself alter the classification of an antibody as specific.
  • an antibody that specifically binds to an antigen may also bind to different allelic forms of the antigen. However, such cross reactivity does not itself alter the classification of an antibody as specific.
  • the terms “specific binding” or “specifically binding,” can be used in reference to the interaction of an antibody, a protein, or a peptide with a second chemical species, to mean that the interaction is dependent upon the presence of a particular structure (e.g., an antigenic determinant or epitope) on the chemical species; for example, an antibody recognizes and binds to a specific protein structure rather than to proteins generally.
  • a particular structure e.g., an antigenic determinant or epitope
  • nucleotide as used herein is defined as a chain of nucleotides.
  • nucleic acids are polymers of nucleotides.
  • nucleic acids and polynucleotides as used herein are interchangeable.
  • nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides.
  • polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • recombinant means i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.
  • peptide As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably, and refer to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide must contain at least two amino acids, and no limitation is placed on the maximum number of amino acids that can comprise a protein’s or peptide’s sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • biological sample as used herein, is intended to include any sample comprising a cell, a tissue, feces, or a bodily fluid in which the presence of a microbe, nucleic acid or polypeptide is present or can be detected.
  • Bio fluids Samples that are liquid in nature are referred to herein as “bodily fluids.”
  • Biological samples may be obtained from a patient by a variety of techniques including, for example, by scraping or swabbing an area of the subject or by using a needle to obtain bodily fluids. Methods for collecting various body samples are well known in the art.
  • the term “obesity” means the condition of excess body fat (adipose tissue), including by way of example in accordance with the National Institutes of Health Federal Obesity Clinical Guidelines for adults, whereby body mass index (“BMI”) calculated by dividing body mass in kilograms by height in meters squared is equal to or greater than twenty-five (25), and further including an overweight condition and comparable obesity and overweight condition in children.
  • BMI body mass index
  • dietary supplement refer to any product that is added to the diet.
  • nutritional supplements are taken by mouth and often contain one or more dietary ingredients, including but not limited to vitamins, minerals, herbs, amino acids, enzymes, and cultures of organisms.
  • food product or “foodstuff’ refers an edible product, e.g. a food or a beverage.
  • Ranges throughout this disclosure, various aspects of the invention can be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.
  • the present invention relates to compositions and methods for preventing or treating a disease or disorder, such as inflammatory bowel disease (IBD), in a subject using inter-species interactions between members of the gut microbiome, either direct or indirect, as well as methods of identifying said inter-species interactions.
  • a disease or disorder such as inflammatory bowel disease (IBD)
  • the present invention is based, in part, on the unexpected discovery that the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic member of the gut microbiome can induce a disease or disorder such as inflammatory bowel disease (IBD) in a subject.
  • the present invention is also based, in part, on the unexpected discovery that the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic member of the gut microbiome can be inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of a commensal or non-pathogenic member of the gut microbiome.
  • the pathogenic member of the gut microbiome can be a pathogenic gut microbe.
  • the commensal or non-pathogenic member of the gut microbiome can be a commensal gut microbe.
  • the pathogenic gut microbe is arx Allobaculum species.
  • the commensal or non-pathogenic gut microbe is an Akkermansia species.
  • the Akkermansia species is an Akkermansia sp. comprising a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the level e.g., activity, expression, concentration, level, etc.
  • arx Allobaculum sp the level (e.g., activity, expression, concentration, level, etc.) of arx Allobaculum sp.
  • a gut microbiota or gut microbiome in a gut microbiota or gut microbiome is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of an Akkermansia species (e.g., Akkermansia sp. comprising a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof) in the gut microbiota or gut microbiome.
  • an Akkermansia species e.g., Akkermansia sp. comprising a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof
  • the present invention also provides a method for diagnosing or assessing the risk of developing a disease or disorder that is induced by at least one microbe (e.g., pathogenic microbe, such as an Allobaculum sp.) in a subject.
  • the method comprises detecting an increased amount of at least one microbe (e.g., pathogenic microbe, such as an Allobaculum sp.) that induces the disease or disorder in a biological sample of the subject.
  • the method comprises detecting a decreased amount of at least one microbe (e.g., beneficial microbe, such as an Akkermansia sp.) that is inversely correlated to the microbe (e.g., pathogenic microbe, such as zn Allobaculum sp.) that induces the disease or disorder in a biological sample of the subject.
  • beneficial microbe such as an Akkermansia sp.
  • pathogenic microbe such as zn Allobaculum sp.
  • the present invention provides methods for identifying at least one microbe or strain thereof that induces a disease or disorder (e.g., pathogenic microbe or strain thereof) in a subject.
  • the present invention provides methods of identifying direct or indirect inter-species interactions.
  • the method comprises using in vivo microbial ecology experiments in gnotobiotic mice, as well as data mining of publicly available human microbiome data, to identify key microbes that provided context-dependent cues that modified the immune responses elicited by individual immunostimulatory bacteria.
  • the specific microbe whose relative abundance is inversely correlated with a specific disease-driving microbe or strain thereof, is critical in modulating immunological outcomes and disease in subjects.
  • the method further comprises an IgA- SEQ technology to enable the identification of both potential disease-driving microbe or strain thereof, as well as potential ‘precision probiotics’ that are likely to protect against the pathogenic effects of these specific microbe or strain thereof.
  • the present invention provides methods for the developing of improved microbiome-based prognostics that predict phenotypic outcomes and/or potential responsiveness to microbiome-targeted therapeutics (e.g., potential responsiveness to probiotics or fecal microbiota transplantation) based on the combination of immunomodulatory strains present in a given individual’s microbiome.
  • microbiome-targeted therapeutics e.g., potential responsiveness to probiotics or fecal microbiota transplantation
  • the present invention relates, in part, to methods for the prediction and discovery of many new potent host-microbiome interactions that are relevant to human health.
  • the method leverages “humanization” of gnotobiotic mice with human stool samples to represent the microbial ecology of the human microbiome in a mouse gut.
  • the method comprises identifying a specific pair of commensal bacteria whose levels, abundance or carriage are inversely correlated across many different “humanized” mice microbiome samples, indicative of an in vivo ecology where either bacteria have a powerful effect upon the host.
  • the method comprises examining the immune responses of mice colonized with defined communities including one or the other bacteria.
  • the method comprises examining publicly available human data from thousands of human microbiomes.
  • the immunostimulatory bacteria from the genus Allobaculum induces the initiation or progression of inflammatory bowel disease (IBD).
  • the Allobaculum abundance is inversely correlated with another immunostimulatory microbe from the genus Akkermansia (e.g., Akkermansia muciniphila').
  • the co- colonization with both taxa potently alters the immune responses elicited by each taxon on its own.
  • the Akkermansia ameliorates Allobaculum-induced pathogenic colonic inflammation, while Allobaculum severely blunts potentially-beneficial AkkermansiaAn&iceA immune responses.
  • the Akkermansia ameliorates Allobaculum-induced pathogenic colonic inflammation and intestinal epithelial cell (IEC) activation (see FIG. 4A-4R and FIG. 5A-5E), while Allobaculum severely blunts systemic antibody response against Akkermansia (see FIG. 6A-6D).
  • co-localization of Akkermansia and Allobaculum in the gut of a subject as achieved using the methods provided herein reshapes the immunological landscape in lymphoid tissues (e.g., PPs and MLNs) of the subject as compared to immunological landscape of the gut of the subject by either Akkermansia ox Allobaculum alone (see FIG. 7A-7J).
  • the present invention provides methods for identifying “precision probiotics” that block the pathogenic effects of specific microbe species and can be paired with a microbiome-based diagnostic to target patients that harbor such pathogenic species.
  • the present invention provides methods of identifying specific taxa whose presence or absence are likely to predict responsiveness to a live-biotherapeutic (e.g., a probiotic strain, such as Akkermansia, or group of beneficial bacteria as in fecal microbiome transplantation).
  • the present invention relates, in part, to methods of identifying discrete inter-species interactions that dictate divergent impacts of individual gut microbes on immunity and disease, as exemplified by the discovery of a unique relationship between Allobaculum species (sp.) and Akkermansia sp. As outlined in FIG. 15, these discrete inter-species interactions can manifest in a reciprocal epistasis between the species.
  • the prevent invention enables the unbiased identification of key microbial taxa that shape host immunity and provide contextual cues that can impact immune and disease outcomes induced by other immunomodulatory gut microbes.
  • the prevent invention enables identifying ‘precision probiotics’ that counteract specific pathogenic species, to improve microbiome-based diagnostics and prognostics, and to predict individual responses to microbiome-target therapeutics based on the combination of immunomodulatory strains present in an individual.
  • the understanding of the specific microbes that contribute to disease dictate responses to specific therapeutic treatments (e.g., specific probiotics), or predict disease trajectory that can be useful for the development of precision medicine-based approaches to treat microbiota- modulated diseases, or as companion diagnostics to determine treatment selection.
  • the present invention relates to a method of identifying a combination of two gut microbe species or strains thereof that modulates an immune response.
  • the method comprises the steps of identifying a first gut microbe species (e.g., pathogenic gut microbe species, such as Allobaculum sp.) or strain thereof; and identifying a second gut microbe species (e.g., beneficial gut microbe species, such as Akkermansia sp.) or strain thereof that is inversely correlated to the first gut microbe species or strain thereof.
  • the first gut microbe species or strain thereof induces a disease or disorder, such as an inflammatory disease or disorder.
  • the identification of inversely correlated microbe species or strains, as described herein, can thus be used to provide predictions of treatment efficacy as well as be used to develop unique treatment plans based on the level of each inversely correlated microbe species or strain in a subject.
  • the present invention relates to a method of preventing or treating a disease or disorder induced by a first gut microbe species (e.g., pathogenic gut microbe species, such as Allobaculum sp.) or strain thereof in a subject in need thereof.
  • the method comprises administering to the subject a composition comprising a second gut microbe species (e.g., beneficial gut microbe species, such as Akkermansia sp.) or strain thereof that is inversely correlated to the first gut microbe species or strain thereof.
  • a second gut microbe species e.g., beneficial gut microbe species, such as Akkermansia sp.
  • the method comprises administering to the subject culture media (e.g., conditioned culture media) or an active agent isolated therefrom harvested from a culture of a second gut microbe species (e.g., beneficial gut microbe species, such as Akkermansia sp.) or strain thereof whose level, abundance or carriage is inversely correlated to the first gut microbe species or strain thereof.
  • a second gut microbe species e.g., beneficial gut microbe species, such as Akkermansia sp.
  • strain thereof whose level, abundance or carriage is inversely correlated to the first gut microbe species or strain thereof.
  • the method comprises administering a composition comprising at least one compound that reduces the level, activity, or concentration of the first gut microbe species (e.g., pathogenic gut microbe species, such as Allobaculum sp.) or strain thereof to the subject prior to the step of administering the composition comprising the second gut microbe species (e.g., beneficial gut microbe species, such as Akkermansia sp.) or strain thereof or the culture media (e.g., conditioned culture media) or the active agent isolated therefrom harvested from a culture of the second gut microbe species (e.g., beneficial gut microbe species, such as Akkermansia sp.) or strain thereof to the subject.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the present invention relates to a method of predicting the effectiveness of a treatment of a disease or disorder (e.g., an inflammatory disease or disorder) in a subject, the treatment comprising administering to the subject having the disease or disorder, a composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof that whose level (e.g., activity, expression, concentration, level, etc.) has been found to be or is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof that induces the disease or disorder.
  • a beneficial gut microbe species e.g., Akkermansia sp.
  • a pathogenic gut microbe species e.g., Allobaculum sp.
  • a method of predicting the effectiveness of a treatment of a disease or disorder comprising administering to the subject having the disease or disorder, a composition comprising culture media (e.g., conditioned culture media) or an active agent isolated therefrom harvested or derived from a culture of a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof whose level (e.g., activity, expression, concentration, level, etc.) has been found to be or is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof that induces the disease or disorder.
  • a beneficial gut microbe species e.g., Akkermansia sp.
  • a pathogenic gut microbe species e.g., Allobaculum sp.
  • the method comprises the steps of detecting the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject. In various embodiments, the method comprises the steps of comparing the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof to a comparator.
  • the level e.g., activity, expression, concentration, level, etc.
  • the pathogenic gut microbe species e.g., Allobaculum sp.
  • the method comprises the step of determining that the composition is effective when the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof is higher when compared to a comparator.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the present invention relates to a method of predicting the effectiveness of a treatment of a disease or disorder (e.g., cancer or obesity) in a subject, the treatment comprising administering a composition to the subject having the disease or disorder (e.g., cancer or obesity), the composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof or culture media (e.g., conditioned culture media) or an active agent isolated therefrom harvested from a culture of a beneficial gut microbe species or strain thereof.
  • a beneficial gut microbe species e.g., Akkermansia sp.
  • culture media e.g., conditioned culture media
  • the method comprises the steps of detecting the level (e.g., activity, expression, concentration, level, etc.) of at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject that is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of the beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof.
  • the method comprises the steps of comparing the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof to a comparator.
  • the method comprises the step of determining that the composition is ineffective, or would be less effective, when the level (e.g., activity, expression, concentration, level, etc.) of the at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject is higher when compared to a comparator.
  • the level e.g., activity, expression, concentration, level, etc.
  • the at least one pathogenic gut microbe species e.g., Allobaculum sp.
  • the method comprises administering to the subject at least one compound that decreases the level (e.g., activity, expression, concentration, level, etc.) of the at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof prior to administering to the subject a composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the present invention relates, in part, to methods of identifying and/or screening a microbe (e.g., microbe species) or strain thereof that induces a disease or disorder.
  • a microbe e.g., microbe species
  • the present invention relates, in part, to methods of identifying and/or screening a microbe or strain thereof that is inversely correlated to a microbe or strain thereof that induces a disease or disorder.
  • the present invention provides methods of identifying inter-species interactions.
  • the present invention relates, in part, to methods of identifying and/or screening an inter-species relationship between a first microbe or strain thereof that induces a disease or disorder and a second microbe or strain thereof that is inversely correlated to the first microbe or strain thereof.
  • the inter-species relationship between the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response.
  • the inter-species relationship between the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response toward the disease or disorder induced by the first microbe or strain thereof.
  • the inter- species relationship between the first microbe or strain thereof and the second microbe or strain thereof ameliorates a pathogenic effect of the first microbe or strain thereof.
  • the pathogenic effect can be intestinal epithelial cell (TEC) activation.
  • the amelioration of IEC activation can be characterized by a decrease in expression of inflammatory genes in the lECs of the subject as shown in FIGs 4O-4R.
  • the inflammatory genes can be selected from the group consisting of rag3b, saal and saa3.
  • the first gut microbe species or strain thereof inhibits systemic antibody responses directed against the second gut microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the second gut microbe species or strain thereof.
  • DCs intestinal dendritic cells
  • the present invention relates, in part, to methods of identifying and/or screening a pair or a combination of a first microbe or strain thereof that induces a disease or disorder and a second microbe or strain thereof that is inversely correlated to the first microbe or strain thereof.
  • the pair or the combination of the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response.
  • the pair or the combination of the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response toward the disease or disorder induced by the first microbe or strain thereof.
  • the method comprises the steps of identifying a first gut microbe species or strain thereof and identifying a second gut microbe species or strain thereof.
  • the first gut microbe species or strain thereof induces at least one disease or disorder.
  • the level of the second gut microbe species or strain thereof is inversely correlated to the level of the first gut microbe species or strain thereof.
  • Modulation of the immune response can entail amelioration of IEC activation caused by the first gut microbe species or strain thereof.
  • the amelioration of the IEC can be characterized by a decrease in expression of inflammatory genes in the lECs of the subject as shown in FIGs 4O-4R.
  • the inflammatory genes can be selected from the group consisting of rag3b, saal and saa3.
  • modulation of the immune response can entail inhibition of the systemic antibody responses directed against the second gut microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the second gut microbe species or strain thereof.
  • modulation of the immune response can entail inhibition of the systemic antibody responses directed against the second gut microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the second gut microbe species or strain thereof as well as amelioration of IEC activation caused by the first gut microbe species or strain thereof.
  • the method comprises identifying a second gut microbe species or strain thereof that is inversely correlated to a first gut microbe species or strain thereof.
  • the first gut microbe species or strain thereof can be any particular species or strain of interest.
  • the first gut microbe species or strain thereof is a pathogenic species or strain that is known to, or predicted to, cause a disease or disorder (e.g., an inflammatory disease or disorder) in a subject.
  • a pathogenic species or strain, used as the first gut microbe species or strain in the present methods is identified using IgA-SEQ or related methodology, as described elsewhere herein.
  • the first gut microbe species or strain thereof is a beneficial species or strain that is known to, or predicted to, have beneficial effects in the overall health a subject or for treatment of a specific disease or disorder in a subject.
  • a beneficial species or strain, used as the first gut microbe species or strain in the present methods is identified using IgA-SEQ or related methodology, as described elsewhere herein.
  • the method comprises the steps of colonizing a subject with the first microbe or strain thereof that induces the disease or disorder; and subsequently identifying in the subject a second microbe or strain thereof that is inversely correlated to the first microbe or strain thereof.
  • the subject is a healthy subject.
  • the subject is a non-human mammal.
  • the subject is a healthy non- human mammal humanized with human microbiota.
  • the subject is a healthy non-human mammal humanized with human gut microbiota.
  • the method comprises evaluating potential competition between the first microbe or strain thereof and the second microbe or strain thereof from diverse human gut microbiota.
  • the method comprises individually- housed germ-free mice monocolonized with the first microbe or strain thereof for 24 hours before gavaging each monocolonized mouse with different healthy human stool samples.
  • the method comprises evaluating the microbial community composition after a defined interval (e.g., 7 days) in all mice via 16S rRNA gene sequencing.
  • the defined interval could be at least, at most or exactly 1 day, 2, days, 3 days, 4 days, 5 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or any day or half-day intervals in between.
  • the method comprises identifying a taxon of interest that exist in human-relevant pairwise relationships.
  • the method comprises obtaining Spearman correlation coefficients for all genus-level OTUs across all microbiome samples paired with the first microbe or strain thereof abundance.
  • the method comprises identifying the relative abundance of the first microbe or strain thereof and the second microbe or strain thereof in subjects having a disease or disorder (e.g., pediatric ulcerative colitis patients) and healthy subjects from publicly available large-scale human microbiome datasets (e.g., American Gut Project data) through the QIITA repository and analysis suite.
  • the method comprises obtaining Spearman correlation coefficients for all genus-level OTUs across all microbiome samples paired with the first microbe or strain thereof abundance.
  • the microbe or strain thereof is identified as a microbe or strain thereof that induces a disease or disorder when the level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof is increased in the biological sample when compared to a comparator.
  • the level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof is determined to be increased when the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.) are differentially expressed when compared to a comparator.
  • the level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof is determined to be increased when the level of microbe or strain thereof (e.g., activity, expression, concentration, level, etc.) in the biological sample is increased by at least 0.1%, by at least 1%, by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%,
  • the level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof is determined to be increased when the level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof in the biological sample is determined to be increased by at least 1 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8
  • the comparator may be a predetermined threshold.
  • the comparator may be a predetermined threshold level of the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.).
  • the comparator may be the level of the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.) in a subject not having a disease or disorder associated with increased level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof (e.g., an inflammatory disease or disorder), a subject not at risk of developing a disease or disorder associated with increased level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof (e.g., an inflammatory disease or disorder), a population not having a disease or disorder associated with increased level (e.g., activity, expression, concentration, level, etc.) of microbe or strain thereof (e.g., an inflammatory disease or disorder), or a population not having a risk of developing a disease or disorder associated with increased level (e.g., activity, activity, expression, concentration, level, etc.) in a subject not having
  • the comparator may be the level of the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.) in a subject having a disease or disorder (e.g., an inflammatory disease or disorder), a subject at risk of developing a disease or disorder (e.g., an inflammatory disease or disorder), a population having a disease or disorder (e.g., an inflammatory disease or disorder), or a population having a risk of developing a disease or disorder (e.g., an inflammatory disease or disorder).
  • a disease or disorder e.g., an inflammatory disease or disorder
  • a subject at risk of developing a disease or disorder e.g., an inflammatory disease or disorder
  • a population having a disease or disorder e.g., an inflammatory disease or disorder
  • a population having a risk of developing a disease or disorder e.g., an inflammatory disease or disorder
  • the comparator may be the level of the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.) in a subject not having a disease or disorder (e.g., cancer or obesity), a subject not at risk of developing a disease or disorder (e.g., cancer or obesity), a population not having a disease or disorder (e.g., cancer or obesity), or a population not having a risk of developing a disease or disorder (e.g., cancer or obesity).
  • a disease or disorder e.g., cancer or obesity
  • a subject not at risk of developing a disease or disorder e.g., cancer or obesity
  • a population not having a disease or disorder e.g., cancer or obesity
  • a population not having a disease or disorder e.g., cancer or obesity
  • a population not having a risk of developing a disease or disorder e.g., cancer or obesity
  • the comparator may be the level of the relevant biomarkers (e.g., the activity of microbe or strain thereof, expression of microbe or strain thereof, concentration of microbe or strain thereof, level of microbe or strain thereof, etc.) in a subject having a disease or disorder (e.g., cancer or obesity), a subject at risk of developing a disease or disorder (e.g., cancer or obesity), a population having a disease or disorder (e.g., cancer or obesity), or a population having a risk of developing a disease or disorder (e.g., cancer or obesity).
  • a disease or disorder e.g., cancer or obesity
  • a subject at risk of developing a disease or disorder e.g., cancer or obesity
  • a population having a disease or disorder e.g., cancer or obesity
  • a population having a risk of developing a disease or disorder e.g., cancer or obesity
  • the microbe or strain thereof that induces a disease or disorder comprises at least one microbe or strain thereof of Erysipelotrichaceae family. In one embodiment, the microbe or strain thereof that induces a disease or disorder comprises a pathogenic microbe species or strain thereof. In one embodiment, the microbe or strain thereof that induces a disease or disorder comprises a pathogenic gut microbe species or strain thereof. For example, in one embodiment, the microbe or strain thereof that induces a disease or disorder comprises an Allobaculum sp. or strain thereof. In various embodiments, the Allobaculum sp. or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • the microbe or strain thereof that induces a disease or disorder comprises one or more bacterium from one or more bacterial strains, wherein the one or more bacterial strains comprise a 16S rRNA sequence comprising a nucleic acid sequence having homology to a nucleic acid sequence selected from SEQ ID NOs: 1 and 3.
  • the microbe or strain thereof that induces a disease or disorder comprises one or more bacterium from one or more bacterial strains, wherein the one or more bacterial strains comprise a 16S rRNA sequence comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to a nucleic acid sequence selected from SEQ ID NOs: 1 and 3.
  • the microbe or strain thereof whose level is inversely correlated to a microbe or strain thereof that induces a disease or disorder comprises a beneficial microbe or strain thereof.
  • the microbe or strain thereof whose level is inversely correlated to a microbe or strain thereof that induces a disease or disorder is a beneficial microbe or strain thereof.
  • the beneficial microbe or strain thereof comprises a beneficial gut microbe species or strain thereof.
  • the microbe or strain thereof that is inversely correlated to a microbe or strain thereof that induces a disease or disorder comprises an Akkermansia sp. or strain thereof.
  • the Akkermansia sp. or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • the microbe or strain thereof that is inversely correlated to a microbe or strain thereof that induces a disease or disorder comprises one or more bacterium from one or more bacterial strains, wherein the one or more bacterial strains comprise a 16S rRNA sequence comprising a nucleic acid sequence having homology to a nucleic acid sequence selected from SEQ ID NO: 2.
  • the microbe or strain thereof that is inversely correlated to a microbe or strain thereof that induces a disease or disorder comprises one or more bacterium from one or more bacterial strains, wherein the one or more bacterial strains comprise a 16S rRNA sequence comprising a nucleic acid sequence having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology to a nucleic acid sequence selected from SEQ ID NO: 2.
  • identifying and/or screening of the microbe or strain thereof there are many methods known in the art for the identifying and/or screening of the microbe or strain thereof.
  • identifying and/or screening of the microbe or strain thereof that induces a disease or disorder and/or the microbe or strain thereof that is inversely correlated to the microbe or strain thereof that induces a disease or disorder can be performed using methods described in U.S. Patent Application Publications No. 20190083599 Al and 20200370098 Al and U. S. Pat. Nos. 9,758,838 B2 and 10,428,392 B2; which are incorporated herein by reference.
  • the present invention relates, in part, to methods of identifying and/or screening a microbe (e.g., microbe species) or strain thereof that induces a disease or disorder (e.g., pathogenic microbes or strains thereof), methods of identifying and/or screening a microbe or strain thereof that is inversely correlated to a microbe or strain thereof that induces a disease or disorder (e.g., beneficial microbes or strains thereof), and/or methods of identifying and/or screening an inter-species relationship between a first microbe or strain thereof that induces a disease or disorder (e.g., a pathogenic microbe or strain thereof) and a second microbe e.g., a beneficial microbe or strain thereof) or strain thereof that is inversely correlated to the first microbe or strain thereof using a human microbiota-associated gnotobiotic mouse-based pipeline.
  • a microbe e.g., microbe species
  • a microbe or strain thereof that induces a disease or disorder (e
  • the method comprises evaluating potential competition between a potentially pathogenic microbe (e.g., Allobaculum) or strain thereof and commensal bacteria from diverse human gut microbiota.
  • a potentially pathogenic microbe e.g., Allobaculum
  • the method comprises individually-housed germ-free mice monocolonized with the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp. 128 (Allot) or Allo2) for 24 hours before gavaging each monocolonized mouse with a myriad of different healthy human stool samples.
  • the method comprises evaluating the microbial community composition after a defined period of time (e.g., 7 days) in all mice via 16S rRNA gene sequencing.
  • the defined period of time could be at least, at most or exactly 1 day, 2, days, 3 days, 4 days, 5 days, 7 days, 8 days, 9, days, 10 days, 11 days, 12 days, 13 days, 14 days, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months or any day or half-day intervals in between.
  • the method comprises identifying a taxon of interest that exist in human- relevant pairwise relationships.
  • the method comprises obtaining Spearman correlation coefficients for all genus-level OTUs across all microbiome samples paired with the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp. 128) abundance.
  • the method comprises identifying the relative abundance of these two taxa in subjects having a disease or disorder (e.g., pediatric ulcerative colitis patients) and healthy subjects from publicly available large-scale human microbiome datasets (e.g., American Gut Project data) through the QIITA repository and analysis suite.
  • the method comprises obtaining Spearman correlation coefficients for all genus- level OTUs across all microbiome samples paired with the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp. 128) abundance.
  • the method further comprises transferring the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp.
  • the present invention also relates, in part, to methods of detecting, identifying, and determining the absolute number or relative proportions of the pathogenic microbe or strain thereof and the inversely correlated beneficial microbe or strain thereof of a subject’s microbiota, to determine whether a subject’s microbiota is an altered microbiota associated with a disease or disorder, such as an inflammatory disease or disorder.
  • the methods of the invention combine a flow cytometry-based microbial cell sorting and genetic analyses to detect, to isolate and to identify the pathogenic microbe or strain thereof and the inversely correlated beneficial microbe or strain thereof from the microbiota of a subject.
  • Pathobionts, as well as other disease-causing microbes, present in the microbiota of the of the subject are recognized by the subject’s immune system, which triggers an immune response, including antibody production and secretion, directed against the pathobionts, and disease-causing microbes.
  • secretory antibodies e.g., IgA, IgM
  • secretory antibodies e.g., IgA, IgM
  • the secretory antibody is IgA (i.e., IgAl, IgA2), or IgM, or any combination thereof.
  • the microbiota of the subject can be any microbiota present on any mucosal surface of subject where antibody is secreted, including the gastrointestinal tract, the respiratory tract, genitourinary tract, and mammary gland.
  • the present invention relates to the isolation and identification of constituents of the microbiota of a subject that influence the development and progression of a disease or disorder, such as an inflammatory disease and disorder.
  • the invention relates to compositions and methods for detecting and determining the identity of the pathogenic microbe or strain thereof and/or the inversely correlated beneficial microbe or strain thereof of a subject’s microbiota to determine whether the pathogenic microbe or strain thereof and/or the inversely correlated beneficial microbe or strain thereof of a subject’s microbiota form an altered microbiota associated with an inflammatory disease or disorder.
  • the relative proportions of the pathogenic microbe or strain thereof and the inversely correlated beneficial microbe or strain thereof of a subject’s microbiota are indicative of an altered microbiota associated with an inflammatory disease or disorder.
  • the detection and identification of the pathogenic microbe or strain thereof and/or the inversely correlated beneficial microbe or strain thereof of the microbiota of the subject are used to diagnose the subject as having, or as at risk of developing, a disease or disorder, such as an inflammatory disease or disorder.
  • the detection and identification of the pathogenic microbe or strain thereof and/or the inversely correlated beneficial microbe or strain thereof of the microbiota of the subject are used to diagnose the subject as having, or as at risk of developing, a recurrence or flare of a disease or disorder, such as an inflammatory disease or disorder.
  • the detection and identification of the pathogenic microbe or strain thereof and/or the inversely correlated beneficial microbe or strain thereof of the microbiota of the subject are used to diagnose the subject as having, or as likely to have, remission or a disease or disorder, such as an inflammatory disease or disorder.
  • the inflammatory diseases and disorders associated with altered microbiota having the pathogenic microbe or strain thereof include, but are not limited to, at least one of inflammatory bowel disease, celiac disease, colitis, irritable bowel syndrome, intestinal hyperplasia, metabolic syndrome, obesity, diabetes, rheumatoid arthritis, liver disease, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).
  • inflammatory bowel disease include, but are not limited to, at least one of inflammatory bowel disease, celiac disease, colitis, irritable bowel syndrome, intestinal hyperplasia, metabolic syndrome, obesity, diabetes, rheumatoid arthritis, liver disease, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), or non-alcoholic steatohepatitis (NASH).
  • the present invention relates to the isolation and identification of constituents (e.g., pathogenic microbe or strain thereof) of the microbiota of a subject that are not associated with the development and progression of a disease or disorder, such as an inflammatory disease and disorder.
  • the invention relates to compositions and methods for detecting and determining the identity of constituents (e.g., pathogenic microbe or strain thereof) of the subject’s microbiota that are not substantially bound by secretory antibodies.
  • the level of the pathogenic microbe or strain thereof present in a subject’s microbiota are indicative of an altered microbiota associated with an inflammatory disease or disorder.
  • the invention is a method for determining the relative proportions of the types of microbes or strains thereof (e.g., a pathogenic microbe or strain thereof and/or an inversely correlated beneficial microbe or strain thereof) of a subject’s microbiota, to identify the microbes or strains thereof of a subject’s microbiota that are, and are not, associated with the development or progression of a disease or disorder, such as an inflammatory disease or disorder.
  • a disease or disorder such as an inflammatory disease or disorder.
  • the detection of particular types of microbes or strains thereof (e.g., pathogenic microbes or strains thereof) of the subject’s microbiota is used to diagnose the subject as having, or as at risk of developing, a disease or disorder, such as an inflammatory disease or disorder.
  • the microbe of the subject s microbiota associated with the development or progression, including inhibition and/or alleviation, of a disease or disorder, such as an inflammatory disease or disorder, in the subject is at least one strain of Segmented Filamentous Bacteria (SFB), Helicobacter flexispira, Lactobacillus, Helicobacter, S24-7, Erysipelotrichaceae, Prevotellaceae, Paraprevotella, Prevotella, Acidaminococcus spp., Actinomyces spp., Akkermansia muciniphila, Allobaculum spp., Anaerococcus spp., Anaerostipes spp., Bacteroides spp., Bacteroides Other, Bacteroides acidifaciens, Bacteroides coprophilus, Bacteroides fragilis, Bacteroides ovatus, Bacteroides uniformis, Barnesiellaceae spp.,
  • SFB Segmented Filament
  • the pathogenic microbe or strain thereof that induces a disease or disorder comprises an Allobaculum sp. or strain thereof and the inversely correlated beneficial microbe or strain thereof comprises an Akkermansia sp. or strain thereof.
  • the present invention provides a method of identifying the type or types of microbes or strains thereof (e.g., a beneficial microbe or strain thereof) that are inversely correlated to a disease- or disorder-inducing microbe or strain thereof (e.g., pathogenic microbe or strain thereof) in the microbiota of a subject that contribute to the inhibition or alleviation of the disease or disorder (i.e., the disease or disorder induced by the pathogenic microbe or strain thereof) in the subject.
  • the identified type or types of the beneficial microbes or strains thereof may be used to treat a subject having a disease or disorder induced by a pathogenic microbe or strain thereof.
  • the identified type or types of the beneficial microbes or strains may be used to prevent the development of a disease or disorder induced by a pathogenic microbe or strain thereof in a subject at risk.
  • microbiota including the presence of the pathogenic microbes or strains thereof and/or the presence of the beneficial microbes or strains thereof, can be detected using various methods, including without limitation quantitative PCR or high-throughput sequencing methods which detect relative proportions of microbial genetic markers in a total heterogeneous microbial population.
  • the microbial genetic marker is a bacterial genetic marker.
  • the bacterial genetic marker is at least some portion of thel6S rRNA.
  • the relative proportion of particular constituent bacterial phyla, classes, orders, families, genera, and species present in the microbiota of a subject is determined.
  • the relative proportion of pathogenic and/or beneficial bacterial phyla, classes, orders, families, genera, and species present in the microbiota of a subject is determined. In some embodiments, the relative proportion of particular pathogenic and/or beneficial bacterial phyla, classes, orders, families, genera, and species present in the microbiota of a subject is determined and compared with that of a comparator normal microbiota.
  • the comparator normal microbiota is, by way of non-limiting examples, a microbiota of a subject known to be free of a disease or disorder induced by the pathogenic microbe or strain thereof, free of a pathogenic microbe or strain thereof inducing a disease or disorder, or a historical norm, or a typical microbiota of the population of which the subject is a member.
  • the present invention relates to methods of diagnosing a subject as having, or assessing the risk of a subject for developing, a disease or disorder.
  • the present invention provides a method of diagnosing a disease or disorder (e.g., an inflammatory disease or disorder), in a subject by identifying a type or types of microbes or strains thereof (e.g., a pathogenic microbe or strain thereof and a beneficial microbe or strain thereof such that level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof) in the microbiota of the subject that contribute to the development or progression of the disease or disorder.
  • a type or types of microbes or strains thereof e.g., a pathogenic microbe or strain thereof and a beneficial microbe or strain thereof such that level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of the
  • the method of the invention is a diagnostic assay for diagnosing a disease or disorder associated with an altered microbiota, such as an inflammatory disease or disorder associated with an altered microbiota, in a subject in need thereof, by determining the absolute or relative abundance of particular types of pathogenic microbes or strains thereof and beneficial microbes or strains thereof of the subject’s microbiota present in a biological sample derived from the subject.
  • the subject is diagnosed as having a disease or disorder associated with a specific pathogenic microbe or strain thereof when the specific pathogenic microbe (e.g., Allobaculum sp.) or strains thereof are determined to be present in the biological sample derived from the subject with increased relative abundance.
  • the amount of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof in a sample of a subject is indicative of a disease or disorder.
  • the detection of an increased amount of Allobaculum sp. or strain thereof, as compared to a control or comparator as provided herein is used to diagnose the subject as having, or as at risk of developing, a disease or disorder.
  • the detection of a decreased amount of Akkermansia sp. or strain thereof in a sample of the subject, as compared to a control or comparator as provided herein is used to diagnose the subject as having, or as at risk of developing, a disease or disorder.
  • the detection of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof is used to assess the progression of a disease or disorder, or to assess the efficacy of a treatment method.
  • a subject is diagnosed as having, or at risk for developing, a disease or disorder induced by Allobaculum sp. or strain thereof when Allobaculum sp. or strain thereof is detected at a level that is increased by at least 10%, by at least 20%, by at least 30%, by at least 40%, by at least 50%, by at least 60%, by at least 70%, by at least 80%, by at least 90%, by at least 100%, by at least 125%, by at least 150%, by at least 175%, by at least 200%, by at least 250%, by at least 300%, by at least 400%, by at least 500%, by at least 600%, by at least 700%, by at least 800%, by at least 900%, by at least 1000%, by at least 1500%, by at least 2000%, by at least 2500%, by at least 3000%, by at least 4000%, or by at least 5000%, in the biological sample when compared with a comparator control.
  • a subject is diagnosed as having, or at risk for developing, a disease or disorder induced by Allobaculum sp. or strain thereof when Allobaculum sp. or strain thereof are detected at a level that is increased by at least 1 fold, at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.1 fold, at least 2.2 fold, at least 2.3 fold, at least 2.4 fold, at least 2.5 fold, at least 2.6 fold, at least 2.7 fold, at least 2.8 fold, at least 2.9 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 5.5 fold, at least 6 fold, at least 6.5 fold, at least 7 fold, at least 7.5 fold, at least 8 fold, at least 8.5 fold, at least 9
  • the amount of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof can be detected using various methods, including without limitation quantitative PCR or high-throughput sequencing methods.
  • the amount of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof can be assessed by detecting a bacterial genetic marker.
  • the bacterial genetic marker is at least some portion of thel6S rRNA.
  • the method of the invention is a diagnostic assay for diagnosing a disease or disorder induced by Allobaculum sp. or strain thereof, by determining the absolute or relative abundance of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof in a biological sample derived from the subject.
  • the subject is diagnosed as having a disease or disorder induced by Allobaculum sp. or strain thereof when Allobaculum sp. or strain thereof are determined to be presented at an increased abundance, relative to a comparator control.
  • the method comprises detecting the level of microbes or strains thereof (e.g.., Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof) in a test sample of a subject.
  • the test sample is a biological sample (e.g., fluid, tissue, cell, cellular component, etc.) of the subject.
  • the biological sample is blood, serum, plasma, saliva, sweat, stool, vaginal fluid, or urine.
  • a biological sample can be obtained by appropriate methods, such as, by way of examples, blood draw, fluid draw, or biopsy.
  • a biological sample can be used as the test sample; alternatively, a biological sample can be processed to enhance access to the antibodies and the processed biological sample can then be used as the test sample.
  • methods of detecting a microbe or strain thereof may be carried out using any assay or methodology known in the art.
  • methods of measuring a microbe or strain thereof in a biological sample include, but are not limited to, an immunochromatography assay, an immunodot assay, a Luminex assay, an ELISA assay, an ELISPOT assay, a protein microarray assay, a ligand-receptor binding assay, an immunostaining assay, a Western blot assay, a mass spectrophotometry assay, a radioimmunoassay (RIA), a radioimmunodiffusion assay, a liquid chromatography -tandem mass spectrometry assay, an ouchterlony immunodiffusion assay, reverse phase protein microarray, a rocket immunoelectrophoresis assay, an immunohistostaining assay, an immunoprecipitation assay, a complement fixation assay
  • test biological sample from a subject is assessed for the absolute or relative abundance of pathogenic microbes or strains thereof and beneficial microbes or strains thereof of the microbiota.
  • the test biological sample can be an in vitro sample or an in vivo sample.
  • the subject is a human subject, and may be of any race, sex and age.
  • Representative subjects include those who are suspected of having an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), those who have been diagnosed with an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), those whose have an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), those who have had an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), those who at risk of a recurrence of an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), those who at risk of a flare of an altered microbiota associated with a disease or disorder (e.g., an inflammatory disease or disorder), and those who are at risk of developing an altered microbiota associated with a disease or disorder (e.
  • the test biological sample is prepared from a biological sample obtained from the subject.
  • a heterogeneous population of microbes will be present in the biological samples.
  • Enrichment of a microbial population for microbes (e.g., bacteria) bound by secretory antibody (e.g., IgA, IgM) may be accomplished using separation technique.
  • microbes of interest may be enriched by separation the microbes of interest from the initial population using affinity separation techniques.
  • Techniques for affinity separation may include magnetic separation using magnetic beads conjugated with an affinity reagent, affinity chromatography, “panning” with an affinity reagent attached to a solid matrix, e.g., plate, or other convenient technique.
  • affinity reagent useful in the methods of the invention is an antibody, such as anti-species antibody or anti-isotype (e.g., anti-IgA, anti-IgM) antibody.
  • anti-species antibody or anti-isotype e.g., anti-IgA, anti-IgM
  • labeled antibodies are used as affinity reagents. Conveniently, these antibodies are conjugated with a label for use in separation.
  • Labels include magnetic beads, which allow for direct separation; biotin, which can be removed with avidin or streptavidin bound to a support; fluorochromes, which can be used with a fluorescence activated cell sorter; or the like, to allow for ease of separation of the particular cell type.
  • the initial population of microbes is contacted with one or more affinity reagent(s) and incubated for a period of time sufficient to permit the affinity reagent to specifically bind to its target.
  • the microbes in the contacted population that become labeled by the affinity reagent are selected for by any convenient affinity separation technique, e.g., as described elsewhere herein or as known in the art.
  • Compositions highly enriched for a microbe of interest e.g., secretory antibody -bound bacteria
  • the affinity enriched microbes will be about 70%, about 75%, about 80%, about 85% about 90%, about 95% or more of the composition.
  • the enriched composition can be a substantially pure composition of the microbes of interest.
  • the test biological sample is a sample containing at least a fragment of a microbial nucleic acid.
  • fragment indicates that the portion of a nucleic acid (e.g., DNA, RNA) that is sufficient to identify it as comprising a microbial nucleic acid.
  • the biological sample can be a sample from any source which contains a microbial nucleic acid (e.g., DNA, RNA), such as a bodily fluid or fecal sample, or a combination thereof.
  • a biological sample can be obtained by any suitable method.
  • a biological sample containing bacterial DNA is used.
  • a biological sample containing bacterial RNA is used.
  • the biological sample can be used as the test sample; alternatively, the biological sample can be processed to enhance access to nucleic acids, or copies of nucleic acids, and the processed biological sample can then be used as the test sample.
  • a nucleic acid is prepared from a biological sample, for use in the methods.
  • an amplification method can be used to amplify nucleic acids comprising all or a fragment of an RNA or DNA in a biological sample, for use as the test biological sample in the assessment of the presence, absence and proportion of particular types of microbes present in the sample.
  • hybridization methods such as Southern analysis, Northern analysis, or in situ hybridizations, can be used (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, including all supplements).
  • the presence of nucleic acid from a particular type of microbe can be determined by hybridization of nucleic acid to a nucleic acid probe.
  • a “nucleic acid probe,” as used herein, can be a DNA probe or an RNA probe.
  • the nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate target RNA or DNA.
  • the hybridization sample is maintained under conditions which are sufficient to allow specific hybridization of the nucleic acid probe to RNA or DNA. Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, as appropriate. In a preferred embodiment, the hybridization conditions for specific hybridization are high stringency. More than one nucleic acid probe can also be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of the presence of the particular type of bacteria of interest, as described herein.
  • RNA such as unprocessed, partially processed or fully processed rRNA.
  • a test sample comprising RNA is prepared from a biological sample from the subject by appropriate means. Specific hybridization of a nucleic acid probe, as described above, to RNA from the biological sample is indicative of the presence of the particular type of bacteria of interest, as described herein.
  • a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described herein.
  • PNA is a DNA mimic having a peptide-like, inorganic backbone, such as N-(2-aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, 1994, Nielsen et al., Bioconjugate Chemistry 5:1).
  • the PNA probe can be designed to specifically hybridize to a particular microbial nucleic acid sequence. Hybridization of the PNA probe to a nucleic acid sequence is indicative of the presence of the particular type of bacteria of interest.
  • Direct sequence analysis can also be used to detect a microbial nucleic acid of interest.
  • a sample comprising DNA or RNA can be used, and PCR or other appropriate methods can be used to amplify all or a fragment of the nucleic acid, and/or its flanking sequences, if desired.
  • the microbial nucleic acid, or a fragment thereof, is determined, using standard methods.
  • arrays of oligonucleotide probes that are complementary to target microbial nucleic acid sequences can be used to detect and identify microbial nucleic acids.
  • an oligonucleotide array can be used.
  • Oligonucleotide arrays typically comprise a plurality of different oligonucleotide probes that are coupled to a surface of a substrate in different known locations. These oligonucleotide arrays, also known as “Genechips,” have been generally described in the art, for example, U.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and 92/10092.
  • arrays can generally be produced using mechanical synthesis methods or light directed synthesis methods which incorporate a combination of photolithographic methods and solid phase oligonucleotide synthesis methods. See Fodor et al., Science, 251 :767-777 (1991), Pirrung et al., U.S. Pat. No. 5,143,854 (see also PCT Application No. WO 90/15070) and Fodor et al., PCT Publication No. WO 92/10092 and U.S. Pat. No. 5,424,186. Techniques for the synthesis of these arrays using mechanical synthesis methods are described in, e.g., U.S. Pat. No. 5,384,261.
  • a nucleic acid of interest is hybridized with the array and scanned for particular microbial nucleic acids.
  • Hybridization and scanning are generally carried out by methods described herein and also in, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186, the entire teachings of which are incorporated by reference herein.
  • a target microbial nucleic acid sequence is amplified by well-known amplification techniques, e.g., PCR. Typically, this involves the use of primer sequences that are complementary to the target sequence.
  • Amplified target generally incorporating a label
  • the array is scanned to determine the position on the array to which the target sequence hybridizes.
  • the hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.
  • nucleic acid analysis can be used to detect microbial nucleic acids of interest.
  • Representative methods include direct manual sequencing (1988, Church and Gilbert, Proc. Natl. Acad. Sci. USA 81 : 1991-1995; 1977, Sanger et al., Proc. Natl. Acad. Sci. 74:5463-5467; Beavis et al. U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single- stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (1981, Sheffield et al., Proc. Natl. Acad. Sci.
  • the methods of assessing a biological sample for the presence or absence of a particular nucleic acid sequence are used to detect, identify or quantify particular constituents (e.g., a pathogenic microbe or strain thereof and/or inversely correlated beneficial microbe or strain thereof) of a subject’s microbiota, and to aid in the diagnosis of an altered microbiota associated with a disease or disorder, such as an inflammatory disease or disorder, in a subject in need thereof.
  • particular constituents e.g., a pathogenic microbe or strain thereof and/or inversely correlated beneficial microbe or strain thereof
  • the probes and primers according to the invention can be labeled directly or indirectly with a radioactive or nonradioactive compound, by methods well known to those skilled in the art, in order to obtain a detectable and/or quantifiable signal; the labeling of the primers or of the probes according to the invention is carried out with radioactive elements or with nonradioactive molecules.
  • radioactive isotopes mention may be made of 32 P, 33 P, 35 S or 3 H.
  • the nonradioactive entities are selected from ligands such as biotin, avidin, streptavidin or digoxigenin, haptens, dyes, and luminescent agents such as radioluminescent, chemoluminescent, bioluminescent, fluorescent or phosphorescent agents.
  • Nucleic acids can be obtained from the biological sample using known techniques.
  • Nucleic acid herein refers to RNA, including mRNA, and DNA, including genomic DNA.
  • the nucleic acid can be double-stranded or single-stranded (i.e., a sense or an antisense single strand) and can be complementary to a nucleic acid encoding a polypeptide.
  • the nucleic acid content may also be an RNA or DNA extraction performed on a fresh or fixed biological sample.
  • Routine methods also can be used to extract DNA from a biological sample, including, for example, phenol extraction.
  • genomic DNA can be extracted with kits such as the QIAampTM. Tissue Kit (Qiagen, Chatsworth, Calif.), the WizardTM Genomic DNA purification kit (Promega, Madison, Wis.), the Puregene DNA Isolation System (Gentra Systems, Inc., Minneapolis, Minn.), and the A.S.A.P.TM Genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.).
  • the detection of hybridization to the duplex form is a Southern blot technique.
  • a nucleic acid sample is separated in an agarose gel based on size (molecular weight) and affixed to a membrane, denatured, and exposed to (admixed with) the labeled nucleic acid probe under hybridizing conditions. If the labeled nucleic acid probe forms a hybrid with the nucleic acid on the blot, the label is bound to the membrane.
  • the nucleic acid probe is preferably labeled with a tag.
  • That tag can be a radioactive isotope, a fluorescent dye or the other well-known materials.
  • Another type of process for the specific detection of nucleic acids of exogenous organisms in a body sample known in the art are the hybridization methods as exemplified by U.S. Pat. No. 6,159,693 and No. 6,270,974, and related patents.
  • a nucleic acid probe of at least 10 nucleotides, preferably at least 15 nucleotides, more preferably at least 25 nucleotides, having a sequence complementary to a desired region of the target nucleic acid of interest is hybridized in a sample, subjected to depolymerizing conditions, and the sample is treated with an ATP/luciferase system, which will luminesce if the nucleic sequence is present.
  • an ATP/luciferase system which will luminesce if the nucleic sequence is present.
  • levels of the target nucleic acid can be determined.
  • a further process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • nucleic acid primers complementary to opposite strands of a nucleic acid amplification target nucleic acid sequence, are permitted to anneal to the denatured sample.
  • a DNA polymerase typically heat stable
  • the process is repeated to amplify the nucleic acid target. If the nucleic acid primers do not hybridize to the sample, then there is no corresponding amplified PCR product.
  • the PCR primer acts as a hybridization probe.
  • the nucleic acid probe can be labeled with a tag as discussed before.
  • the detection of the duplex is done using at least one primer directed to the target nucleic acid.
  • the detection of the hybridized duplex comprises electrophoretic gel separation followed by dye-based visualization.
  • DNA amplification procedures by PCR are well known and are described in U.S. Pat. No. 4,683,202. Briefly, the primers anneal to the target nucleic acid at sites distinct from one another and in an opposite orientation. A primer annealed to the target sequence is extended by the enzymatic action of a heat stable DNA polymerase. The extension product is then denatured from the target sequence by heating, and the process is repeated. Successive cycling of this procedure on both DNA strands provides exponential amplification of the region flanked by the primers.
  • Amplification is then performed using a PCR-type technique, that is to say the PCR technique or any other related technique.
  • Two primers, complementary to the target nucleic acid sequence are then added to the nucleic acid content along with a polymerase, and the polymerase amplifies the DNA region between the primers.
  • the expression “specifically hybridizing in stringent conditions” refers to a hybridizing step in the process of the invention where the oligonucleotide sequences selected as probes or primers are of adequate length and sufficiently unambiguous so as to minimize the amount of non-specific binding that may occur during the amplification.
  • the oligonucleotide probes or primers herein described may be prepared by any suitable methods such as chemical synthesis methods.
  • Hybridization is typically accomplished by annealing the oligonucleotide probe or primer to the DNA under conditions of stringency that prevent non-specific binding but permit binding of this DNA which has a significant level of homology with the probe or primer.
  • the melting temperature (Tm) for the amplification step using the set of primers which is in the range of about 55 °C to about 70 °C.
  • the Tm for the amplification step is in the range of about 59 °C to about 72 °C.
  • the Tm for the amplification step is about 60 °C.
  • Typical hybridization and washing stringency conditions depend in part on the size (i.e., number of nucleotides in length) of the DNA or the oligonucleotide probe, the base composition and monovalent and divalent cation concentrations (Ausubel et al., 1997, eds Current Protocols in Molecular Biology).
  • the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the amplifications are real-time amplifications performed using a labeled probe, preferably a labeled hydrolysis-probe, capable of specifically hybridizing in stringent conditions with a segment of a nucleic acid sequence, or polymorphic nucleic acid sequence.
  • the labeled probe is capable of emitting a detectable signal every time each amplification cycle occurs.
  • the real-time amplification such as real-time PCR, is well known in the art, and the various known techniques will be employed in the best way for the implementation of the present process.
  • hydrolysis probes such as hydrolysis probes, hybridization adjacent probes, or molecular beacons.
  • the techniques employing hydrolysis probes or molecular beacons are based on the use of a fluorescence quencher/reporter system, and the hybridization adjacent probes are based on the use of fluorescence acceptor/donor molecules.
  • Hydrolysis probes with a fluorescence quencher/reporter system are available in the market and are for example commercialized by the Applied Biosystems group (USA).
  • Many fluorescent dyes may be employed, such as FAM dyes (6-carboxy-fluorescein), or any other dye phosphoramidite reagents.
  • the Tm which is in the range of about 65°C to 75°C.
  • the Tm for any one of the hydrolysis-probes of the present invention is in the range of about 67 °C to about 70 °C.
  • the Tm applied for any one of the hydrolysis-probes of the present invention is about 67 °C.
  • the process for determining the quantitative and qualitative profile according to the present invention is characterized in that the amplification products can be elongated, wherein the elongation products are separated relative to their length.
  • the signal obtained for the elongation products is measured, and the quantitative and qualitative profile of the labeling intensity relative to the elongation product length is established.
  • the elongation step also called a run-off reaction, allows one to determine the length of the amplification product.
  • the length can be determined using conventional techniques, for example, using gels such as polyacrylamide gels for the separation, DNA sequencers, and adapted software. Because some mutations display length heterogeneity, some mutations can be determined by a change in length of elongation products.
  • the invention includes a primer that is complementary to a target microbial nucleic acid, and more particularly the primer includes 12 or more contiguous nucleotides substantially complementary to the sequence flanking the nucleic acid sequence of interest.
  • a primer featured in the invention includes a nucleotide sequence sufficiently complementary to hybridize to a nucleic acid sequence of about 12 to 25 nucleotides. More preferably, the primer differs by no more than 1, 2, or 3 nucleotides from the target flanking nucleotide sequence.
  • the length of the primer can vary in length, preferably about 15 to 28 nucleotides in length (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides in length).
  • the level (e.g., activity, expression, concentration, level, etc.) of the microbe or strain thereof that induces a disease or disorder modulates the level (e.g., activity, expression, concentration, level, etc.) of the inversely correlated microbe or strain thereof.
  • the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof that induces a disease or disorder inhibits the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof.
  • the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof inhibits the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof.
  • the beneficial microbe or strain thereof ameliorates the pathogenic colonic inflammation and intestinal epithelial cell (IEC) activation of the pathogenic microbe or strain thereof.
  • the pathogenic microbe or strain thereof reduces the systemic antibody response against the beneficial microbe or strain thereof.
  • co-localization of the beneficial microbe or strain thereof and the pathogenic microbe or strain thereof in the gut of a subject as achieved using the methods provided herein reshapes the immunological landscape in lymphoid tissues (e.g., PPs and MLNs) of the subject as compared to immunological landscape of the gut of a subject with either the beneficial microbe or strain thereof or the pathogenic microbe or strain thereof alone.
  • the amelioration of the IEC can be characterized by a decrease in expression of inflammatory genes in the lECs of the subject as shown in FIGs 4O-4R.
  • the inflammatory genes can be selected from the group consisting of rag3b, saal and saa3.
  • the present invention comprises a composition comprising a beneficial microbe or strain thereof.
  • the present invention comprises a composition comprising culture media (e.g., conditioned culture media) or an active agent isolated therefrom, harvested, prepared from or derived from a beneficial microbe or strain thereof.
  • the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic microbe or strain thereof.
  • the pathogenic microbe or strain thereof is any microbe or strain thereof described herein that induces a disease or disorder.
  • the pathogenic microbe or strain thereof is a Allobaculum sp. or strain thereof.
  • the beneficial microbe or strain thereof is any microbe or strain thereof described herein that is inversely correlated to any of the microbe or strain thereof described herein that induces a disease or disorder.
  • the beneficial microbe or strain thereof is a Akkermansia sp. or strain thereof.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the composition modulates the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof, the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof, or any combination thereof.
  • the composition increases the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof.
  • the composition decreases the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof.
  • the level is the level of intestinal epithelial cell (IEC) activation.
  • the level of IEC activation can be determining by measuring the gene expression of any inflammatory genes in the lECs of the subject, such as, for example, the genes shown in FIGs 4O-4R.
  • the inflammatory genes can be selected from the group consisting of rag 3b, saal and saa3.
  • the composition increases the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof and decreases the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof.
  • the level is the level of intestinal epithelial cell (IEC) activation.
  • IEC intestinal epithelial cell
  • the level of IEC activation can be determining by measuring the gene expression of any inflammatory genes in the lECs of the subject, such as, for example, the genes shown in FIGs 4O-4R.
  • the composition modulates an immune response toward the disease or disorder.
  • the composition increases an immune response toward the disease or disorder.
  • Modulation of the immune response can entail amelioration of IEC activation caused by the pathogenic microbe species or strain thereof.
  • the amelioration of the IEC can be characterized by a decrease in expression of inflammatory genes in the lECs of the subject as shown in FIGs 4O-4R.
  • the inflammatory genes can be selected from the group consisting of rag3b, saal and saa3.
  • modulation of the immune response can entail inhibition of the systemic antibody responses directed against the beneficial microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the beneficial microbe species or strain thereof.
  • modulation of the immune response can entail inhibition of the systemic antibody responses directed against the beneficial microbe species or strain thereof and/or activation of intestinal dendritic cells (DCs) by the beneficial microbe species or strain thereof as well as amelioration of IEC activation caused by the pathogenic microbe species or strain thereof.
  • the composition further comprises at least one probiotic, prebiotic, antibiotic, antimicrobe, or any combination thereof.
  • the composition comprises at least one probiotic, prebiotic of the beneficial microbe or strain thereof, antibiotic of the pathogenic microbe or strain thereof, antimicrobe of the pathogenic microbe or strain thereof, or any combination thereof.
  • the prebiotic, probiotic, antibiotic, antimicrobe, or any combination thereof reduces or inhibits the level of the pathogenic microbe or strain thereof.
  • the composition comprises a probiotic.
  • the composition comprises a probiotic composition that comprises one or more bacterium.
  • the one or more bacterium are indigenous members of the human gut microbiome.
  • the composition comprises one or more bacterium from one or more bacterial species of: Akkermansia sp., Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium sp., Bifidobacterium infantis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium adelocentis, Bifidobacterium lactis, Bifidobacterium pseudocatenulatum, Eggerthella lenta, Bacteroides sarotrii, Bacteroides fragilis, Bacteroides uniformis, Lactobacillus sp., Bifidobacterium sp.,
  • compositions may include bacterium from multiple strains of a particular species.
  • the composition is a probiotic composition for use as a food or drink additive.
  • the composition is a probiotic beverage or drink.
  • the composition is soluble or suspendable in a liquid medium.
  • the composition comprises probiotic microorganisms in about 1 x 10 9 cfu/g, about 2x 10 9 cfu/g, about 3 x 10 9 cfu/g, about 4x 10 9 cfu/g, about 5x 10 9 cfu/g, about 6x 10 9 cfu/g, about 7x 10 9 cfu/g, about 8x 10 9 cfu/g, about 9x 10 9 cfu/g, about 1 x 10 10 cfu/g, about 2x 10 10 cfu/g, about 3x 10 10 cfu/g, about 4x 10 10 cfu/g, about 5x 10 10 cfu/g, about 6x 10 10 cfu/g, about 7x 10 10 cfu/g, about 8x 10 10 cfu/g, about 9 x 10 10 cfu/g, or about 1 x 10 11 cfu/g.
  • the probiotic composition comprises about 1 x 10 10 cfu of probiotic microorganisms in each gram of bulk, dried raw powder where each gram contains about 60% or less of bacterial mass and about 40% carrier system.
  • each gram contains about 70% or less of bacterial mass and about 30% carrier system, about 80% or less of bacterial mass and about 20% carrier system, about 90% or less of bacterial mass and about 10% carrier system, about 50% or less of bacterial mass and about 50% carrier system, about 40% or less of bacterial mass and about 60% carrier system, about 30% or less of bacterial mass and about 70% carrier system, about 20% or less of bacterial mass and about 80% carrier system, or about 10% or less of bacterial mass and about 90% carrier system.
  • the compositions do not include bacterial species or strains that are resistant to one or more antibiotics. It should be appreciated that in certain instances, it may be desirable to have a mechanism to remove the bacterial compositions provided herein from the body of the subject after administration. One such mechanism is to remove the bacterial compositions by antibiotic treatment. Thus, in some embodiments, the compositions do not include bacterial species or strains that are resistant to one or more antibiotics.
  • the compositions do not include bacterial species or strains that are resistant to one or more antibiotics selected from the group consisting of penicillin, benzylpenicillin, ampicillin, sulbactam, amoxicillin, clavulanate, tazobactam, piperacillin, cefmetazole, vancomycin, imipenem, meropenem, metronidazole and clindamycin.
  • the compositions include bacterial species or strains that are susceptible to at least four antibiotics that are efficacious in humans.
  • the compositions include bacterial species or strains that are susceptible to at least three antibiotics that are efficacious in humans.
  • the compositions include bacterial species or strains that are susceptible to at least two antibiotics that are efficacious in humans. In some embodiments, the compositions include bacterial species or strains that are susceptible to at least one antibiotic that is efficacious in humans.
  • an “antibiotic that is efficacious in a human” refers to an antibiotic that has been used to successfully treat bacterial infections in a human.
  • the compositions described herein comprise spore forming and non-spore forming bacterial species or strains. In some embodiments, the compositions described herein comprise spore forming bacterial species or strains. In some embodiments, the compositions described herein comprise only spore forming bacterial species or strains. In some embodiments, the compositions described herein comprise only non-spore forming bacterial species or strains.
  • the spore-forming bacteria can be in spore form (i.e., as spores) or in vegetative form (i.e., as vegetative cells). In spore form, bacteria are generally more resistant to environmental conditions, such as heat, acid, radiation, oxygen, chemicals, and antibiotics.
  • bacteria are more susceptible to such environmental conditions, compared to in the spore form.
  • bacterial spores are able to germinate from the spore form into a vegetative/actively growing state, under appropriate conditions. For instance, bacteria in spore format may germinate when they are introduced in the intestine.
  • the bacterial species or strains are purified.
  • the bacterial species or strains are isolated. Any of the bacterial species or strains described herein may be isolated and/or purified, for example, from a source such as a culture or a microbiota sample (e.g., fecal matter).
  • the bacterial strains used in the compositions provided herein generally are isolated from the microbiome of healthy individuals. However, bacterial strains can also be isolated from individuals that are considered not to be healthy.
  • the compositions include strains originating from multiple individuals.
  • the term “isolated” bacteria that have been separated from one or more undesired component, such as another bacterium or bacterial species or strain, one or more component of a growth medium, and/or one or more component of a sample, such as a fecal sample.
  • the bacteria are substantially isolated from a source such that other components of the source are not detected.
  • the term “purified” refers to a bacterial species or strain or composition comprising such that has been separated from one or more components, such as contaminants.
  • the bacterial species or strain is substantially free of contaminants.
  • one or more bacterial species or strains of a composition may be independently purified from one or more other bacteria produced and/or present in a culture or a sample containing the bacterial species or strain.
  • a bacterial species or strain is isolated or purified from a sample and then cultured under the appropriate conditions for bacterial replication, e.g., under anaerobic culture conditions. The bacteria that is grown under appropriate conditions for bacterial replication can subsequently be isolated/purified from the culture in which it is grown.
  • the one or more of the bacterium of the compositions provided herein colonize or recolonize the intestinal tract or parts of the intestinal tract (e.g., the colon or the cecum) of a subject. Such colonization or recolonization may also be referred to as grafting.
  • the one or more of the bacterium of the compositions recolonize the intestinal tract (e.g., the colon or the cecum) of a subject after the naturally present microbiome has been partially or completely removed, e.g., because of administration of antibiotics.
  • the one or more of the bacterium of the compositions colonize a dysbiotic gastrointestinal tract.
  • the bacterial species or strains used in the compositions provided herein generally are isolated from the microbiome of healthy individuals.
  • the compositions include bacteria from species or strains originating from a single individual.
  • the compositions include bacteria from species or strains originating from multiple individuals.
  • the bacterial strains are obtained from multiple individuals, isolated and grown up individually. The bacterial compositions that are grown up individually may subsequently be combined to provide the compositions of the disclosure. It should be appreciated that the origin of the bacterial species or strains of the compositions provided herein is not limited to the human microbiome from a healthy individual. In some embodiments, the bacterial species or strains originate from a human with a microbiome in dysbiosis.
  • the bacterial species or strains originate from non-human animals or the environment (e.g., soil or surface water). In some embodiments, the combinations of bacterial species or strains provided herein originate from multiple sources (e.g., human and non-human animals).
  • compositions described herein may contain one or more bacterium in any form, for example in an aqueous form, such as a solution or a suspension, embedded in a semi-solid form, in a powdered form or freeze dried form.
  • the composition or the one or more bacterium of the composition are lyophilized.
  • a subset of the bacteria in a composition is lyophilized.
  • the bacteria may be lyophilized as a combination and/or the bacteria may be lyophilized separately and combined prior to administration.
  • One or more bacterium may be combined with a pharmaceutical excipient prior to combining it with the other bacterial or multiple lyophilized bacteria may be combined while in lyophilized form and the mixture of bacteria, once combined may be subsequently be combined with a pharmaceutical excipient.
  • the bacteria is a lyophilized cake.
  • the compositions comprising the one or more bacterium are a lyophilized cake.
  • the bacterial species or strains of the composition can be manufactured using fermentation techniques well known in the art.
  • the active ingredients are manufactured using anaerobic fermenters, which can support the rapid growth of anaerobic bacterial species.
  • the anaerobic fermenters may be, for example, stirred tank reactors or disposable wave bioreactors.
  • Culture media such as BL media and EG media, or similar versions of these media devoid of animal components, can be used to support the growth of the bacterial species.
  • the bacterial product can be purified and concentrated from the fermentation broth by traditional techniques, such as centrifugation and filtration, and can optionally be dried and lyophilized by techniques well known in the art.
  • the composition may further comprise one or more additional therapeutic compositions.
  • the composition further comprises a corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti- leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (preferably vaccines used for vaccination where the amount of an allergen is gradually increased), anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, go
  • the composition may be formulated for administration as a pharmaceutical composition.
  • the formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.
  • composition means a product that results from the mixing or combining of at least one active ingredient, such as any two or more purified bacterial strains described herein, and one or more inactive ingredients, which may include one or more pharmaceutically acceptable excipient.
  • an “acceptable” excipient refers to an excipient that must be compatible with the active ingredient and not deleterious to the subject to which it is administered.
  • the pharmaceutically acceptable excipient is selected based on the intended route of administration of the composition, for example a composition for oral or nasal administration may comprise a different pharmaceutically acceptable excipient than a composition for rectal administration.
  • excipients include sterile water, physiological saline, solvent, a base material, an emulsifier, a suspending agent, a surfactant, a stabilizer, a flavoring agent, an aromatic, an excipient, a vehicle, a preservative, a binder, a diluent, a tonicity adjusting agent, a soothing agent, a bulking agent, a disintegrating agent, a buffer agent, a coating agent, a lubricant, a colorant, a sweetener, a thickening agent, and a solubilizer.
  • compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art (see e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co. 20th ed. 2000).
  • the pharmaceutical compositions described herein may further comprise any carriers or stabilizers in the form of a lyophilized formulation or an aqueous solution.
  • Acceptable excipients, carriers, or stabilizers may include, for example, buffers, antioxidants, preservatives, polymers, chelating reagents, and/or surfactants.
  • pharmaceutical compositions are manufactured under GMP conditions.
  • compositions can be used orally, nasally or parenterally, for instance, in the form of capsules, tablets, pills, sachets, liquids, powders, granules, fine granules, film-coated preparations, pellets, troches, sublingual preparations, chewables, buccal preparations, pastes, syrups, suspensions, elixirs, emulsions, liniments, ointments, plasters, cataplasms, transdermal absorption systems, lotions, inhalations, aerosols, injections, suppositories, and the like.
  • the composition comprising a beneficial microbe or strain thereof and/or culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof is formulated for delivery to the intestines (e.g., the small intestine and/or the colon).
  • the composition is formulated with an enteric coating that increases the survival of the bacteria through the harsh environment in the stomach.
  • the enteric coating is one which resists the action of gastric juices in the stomach so that the bacteria which are incorporated therein will pass through the stomach and into the intestines.
  • the enteric coating may readily dissolve when in contact with intestinal fluids, so that the bacteria enclosed in the coating will be released in the intestinal tract.
  • Enteric coatings may consist of polymer and copolymers well known in the art, such as commercially available EUDRAGIT (Evonik Industries). (See e.g., Zhang, AAPS PharmSciTech, (2016) 17 (1), 56-67).
  • EUDRAGIT Evonik Industries
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the composition comprising a beneficial microbe or strain thereof and/or culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof is formulated for rectal delivery to the intestine (e.g., the colon).
  • culture media e.g., conditioned culture media
  • the compositions may be formulated for delivery by suppository, colonoscopy, endoscopy, sigmoidoscopy or enema.
  • a pharmaceutical preparation or formulation and particularly a pharmaceutical preparation for oral administration may include an additional component that enables efficient delivery of the compositions of the disclosure to the intestine (e.g., the colon).
  • a variety of pharmaceutical preparations that allow for the delivery of the compositions to the intestine (e.g., the colon) can be used. Examples thereof include pH sensitive compositions, more specifically, buffered sachet formulations or enteric polymers that release their contents when the pH becomes alkaline after the enteric polymers pass through the stomach.
  • the pH sensitive composition is a polymer whose pH threshold of the decomposition of the composition is between about 6.8 and about 7.5.
  • Such a numeric value range is a range in which the pH shifts toward the alkaline side at a distal portion of the stomach, and hence is a suitable range for use in the delivery to the colon.
  • each part of the intestine e.g., the duodenumjejunum, ileum, cecum, colon and rectum
  • parts of the intestines have different pHs, allowing for targeted delivery by compositions that have a specific pH sensitivity.
  • compositions provided herein may be formulated for delivery to the intestine or specific parts of the intestine (e.g., the duodenumjejunum, ileum, cecum, colon and rectum) by providing formulations with the appropriate pH sensitivity.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • a pharmaceutical preparation useful for delivery of the compositions to the intestine is one that ensures the delivery to the colon by delaying the release of the contents (e.g., the beneficial microbe or strain thereof) by approximately 3 to 5 hours, which corresponds to the small intestinal transit time.
  • a hydrogel is used as a shell. The hydrogel is hydrated and swells upon contact with gastrointestinal fluid, with the result that the contents are effectively released (released predominantly in the colon). Delayed release dosage units include drug-containing compositions having a material which coats or selectively coats a drug or active ingredient to be administered.
  • Examples of such a selective coating material include in vivo degradable polymers, gradually hydrolyzable polymers, gradually water- soluble polymers, and/or enzyme degradable polymers.
  • a wide variety of coating materials for efficiently delaying the release is available and includes, for example, cellulose-based polymers such as hydroxypropyl cellulose, acrylic acid polymers and copolymers such as methacrylic acid polymers and copolymers, and vinyl polymers and copolymers such as polyvinylpyrrolidone.
  • compositions that allow for the delivery to the intestine (e.g., the colon) include bioadhesive compositions which specifically adhere to the colonic mucosal membrane (for example, a polymer described in the specification of U.S. Pat. No. 6,368,586) and compositions into which a protease inhibitor is incorporated for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.
  • bioadhesive compositions which specifically adhere to the colonic mucosal membrane
  • a protease inhibitor for protecting particularly a biopharmaceutical preparation in the gastrointestinal tracts from decomposition due to an activity of a protease.
  • a system enabling the delivery to the intestine is a system of delivering a composition to the colon by pressure change in such a way that the contents are released by utilizing pressure change caused by generation of gas in bacterial fermentation at a distal portion of the stomach.
  • a system is not particularly limited, and a more specific example thereof is a capsule which has contents dispersed in a suppository base and which is coated with a hydrophobic polymer (for example, ethyl cellulose).
  • a further example of a system enabling the delivery of a composition to the intestine is a composition that includes a coating that can be removed by an enzyme present in the gut (e.g., the colon), such as, for example, a carbohydrate hydrolase or a carbohydrate reductase.
  • a composition that includes a coating that can be removed by an enzyme present in the gut (e.g., the colon), such as, for example, a carbohydrate hydrolase or a carbohydrate reductase.
  • Such a system is not particularly limited, and more specific examples thereof include systems which use food components such as non-starch polysaccharides, amylose, xanthan gum, and azopolymers.
  • compositions provided herein can also be delivered to specific target areas, such as the intestine, by delivery through an orifice (e.g., a nasal tube) or through surgery.
  • an orifice e.g., a nasal tube
  • the compositions provided herein that are formulated for delivery to a specific area may be administered by a tube (e.g., directly into the small intestine).
  • a tube e.g., directly into the small intestine.
  • Combining mechanical delivery methods such as tubes with chemical delivery methods such as pH specific coatings allow for the delivery of the compositions provided herein to a desired target area (e.g., the cecum or the colon).
  • compositions are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., the prophylactic or therapeutic effect).
  • the dosage form of the composition is a tablet, pill, capsule, powder, granules, solution, or suppository.
  • the pharmaceutical composition is formulated for oral administration. In some embodiments, the pharmaceutical composition is formulated such that the bacteria of the composition, or a portion thereof, remain viable after passage through the stomach of the subject. In some embodiments, the pharmaceutical composition is formulated for rectal administration, e.g. as a suppository.
  • the pharmaceutical composition is formulated for delivery to the intestine or a specific area of the intestine (e.g., the colon) by providing an appropriate coating (e.g., a pH specific coating, a coating that can be degraded by target area specific enzymes, or a coating that can bind to receptors that are present in a target area).
  • an appropriate coating e.g., a pH specific coating, a coating that can be degraded by target area specific enzymes, or a coating that can bind to receptors that are present in a target area.
  • Dosages of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired pharmaceutical response for a particular subject, composition, and mode of administration, without being toxic or having an adverse effect on the subject.
  • the selected dosage level depends upon a variety of factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors.
  • a physician, veterinarian or other trained practitioner can start doses of the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect (e.g., treatment of a disease or disorder, weight loss, decreased blood glucose, etc.) is achieved.
  • effective doses of the compositions of the present invention for the prophylactic treatment of groups of people as described herein vary depending upon many different factors, including routes of administration, physiological state of the subject, whether the subject is human or an animal, other medications administered, and the therapeutic effect desired. Dosages need to be titrated to optimize safety and efficacy.
  • the dosing regimen entails oral administration of a dose of any of the compositions described herein.
  • the dosing regimen entails oral administration of multiple doses of any of the compositions described herein.
  • the composition is administered orally the subject once, twice, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, or at least 10 times.
  • compositions including the pharmaceutical compositions disclosed herein, include compositions with a range of active ingredients (e.g., live bacteria, bacteria in spore format).
  • the amount of bacteria in the compositions may be expressed in weight, number of bacteria and/or CFUs (colony forming units).
  • the pharmaceutical compositions disclosed herein contain about 10, about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 or more of each of the bacteria of the composition per dosage amount.
  • the pharmaceutical compositions disclosed herein contain about 10, about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 or more total bacteria per dosage amount. It should further be appreciated that the bacteria of the compositions may be present in different amounts. In some embodiments, the pharmaceutical compositions disclosed herein contain about 10, about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 or more CFUs of each of the bacteria in the composition per dosage amount.
  • the pharmaceutical compositions disclosed herein contain about 10 1 , about 10 2 , about 10 3 , about 10 4 , about 10 5 , about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , about 10 11 , about 10 12 , about 10 13 or more CFUs in total for all of the bacteria combined per dosage amount.
  • bacteria of the compositions may be present in different amounts.
  • the pharmaceutical compositions disclosed herein contain about 10 -7 , about 10 -6 , about 10“ 5 , about 10 -4 , about 10“ 3 , about 10 -2 , about 10 -1 or more grams of each of the bacteria in the composition per dosage amount.
  • the pharmaceutical compositions disclosed herein contain about 10 -7 , about 10 -6 , about 10“ 5 , about 10 -4 , about 10“ 3 , about 10 -2 , about 10 -1 or more grams in total for all of the bacteria combined per dosage amount.
  • the dosage amount is one administration device (e.g., one table, pill or capsule).
  • the dosage amount is the amount that is administered in a particular period (e.g., one day or one week).
  • the pharmaceutical compositions disclosed herein contain between 10 and 10 13 , between 10 2 and 10 13 , between 10 3 and 10 13 , between 10 4 and 10 13 , between 10 5 and 10 13 , between 10 6 and 10 13 , between 10 7 and 10 13 , between 10 8 and 10 13 , between 10 9 and 10 13 , between 10 10 and 10 13 , between 10 u and 10 13 , between 10 12 and 10 13 , between 10 and 10 12 , between 10 2 and 10 12 , between 10 3 and 10 12 , between 10 4 and 10 12 between 10 5 and 10 12 , between 10 6 and 10 12 , between 10 7 and 10 12 , between 10 8 and 10 12 between 10 9 and 10 12 , between 10 10 and
  • the pharmaceutical compositions disclosed herein contain between 10 and 10 13 , between 10 2 and 10 13 , between 10 3 and 10 13 , between 10 4 and 10 13 , between 10 5 and 10 13 , between 10 6 and 10 13 , between 10 7 and 10 13 , between 10 8 and 10 13 , between 10 9 and 10 13 , between 10 10 and 10 13 , between 10 u and 10 13 , between 10 12 and 10 13 , between 10 and 10 12 , between 10 2 and 10 12 , between 10 3 and 10 12 , between 10 4 and 10 12 between 10 5 and 10 12 , between 10 6 and 10 12 , between 10 7 and 10 12 , between 10 8 and 10 12 between 10 9 and 10 12 , between 10 10 and
  • 10 12 between 10 x and 10 2 , between 10 and 10 11 , between 10 2 and 10 11 , between 10 3 and 10 13 , between 10 4 and 10 13 , between 10 5 and 10 13 , between 10 6 and 10 13 , between 10 7 and 10 13 , between 10 8 and 10 13 , between 10 9 and 10 11 , between 10 10 and 10 11 , between 10 and 10 10 , between 10 2 and 10 10 , between 10 3 and 10 10 , between 10 4 and 10 10 , between 10 5 and 10 10 , between 10 6 and 10 10 , between 10 7 and 10 10 , between 10 and 10 10 , between 10 9 and 10 10 , between
  • the pharmaceutical compositions disclosed herein contain between 10 -7 and 10 -1 , between 10 -6 and 10 -1 , between 10 -5 and 10 -1 , between 10 -4 and 10 -1 , between 10 -3 and 10 -1 , between 10 -2 and 10 -1 , between 10 -7 and 10 -2 , between 10 -6 and 10 -2 , between 10 -5 and 10 -2 , between 10 -4 and 10 -2 , between 10 -3 and 10 -2 , between 10 -7 and 10 -3 between 10 -6 and 10 -3 , between 10 -5 and 10 -3 , between 10 -4 and 10 -3 , between 10 -7 and 10 -4 between 10 -6 and 10 -4 , between 10 -5 and 10 -4 , between 10 -7 and 10 -5 , between 10 -6 and 10 -5 , or between 10 -7 and 10 -6 grams of each of the bacteria in the composition per dosage amount.
  • the pharmaceutical compositions disclosed herein contain between 10 -7 and 10 -1 , between 10 -6 and 10 -1 , between 10 -5 and 10 -1 , between 10 -4 and 10 -1 , between 10 -3 and 10 -1 , between 10 -2 and 10 -1 , between 10 -7 and 10 -2 , between 10 -6 and 10 -2 , between 10 -5 and 10 -2 , between 10 -4 and 10 -2 , between 10 -3 and 10 -2 , between 10 -7 and 10 -3 , between 10 -6 and 10 -3 , between 10 -5 and 10 -3 , between 10 -4 and 10 -3 , between 10 -7 and 10 -4 , between 10 -6 and 10 -4 , between 10 -5 and 10 -4 , between 10 -7 and 10 -5 , between 10 -6 and 10 -5 , or between 10 -7 and 10 -6 grams of all of the bacteria combined per dosage amount.
  • Food products comprising any of the prebiotics and/or bacterial species or strains described herein and a nutrient.
  • Food products are, in general, intended for the consumption of a human or an animal. Any of the prebiotics and/or bacterial species or strains described herein may be formulated as a food product.
  • the one or more bacterium are formulated as a food product in spore form. In some embodiments, the one or more bacterium are formulated as a food product in vegetative form. In some embodiments, the food product comprises both vegetative bacteria and bacteria in spore form.
  • compositions disclosed herein can be used in a food or beverage, such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food or beverage such as a health food or beverage, a food or beverage for infants, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • Non-limiting examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products such as Western confectionery products including biscuits, cookies, and the like, Japanese confectionery products including steamed bean-jam buns, soft adzuki -bean jellies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups;
  • Food products containing the prebiotics and/or bacterial species or strains described herein may be produced using methods known in the art and may contain the same amount of prebiotic or bacteria (e.g., by weight, amount or CFU) as the pharmaceutical compositions provided herein. Selection of an appropriate amount of prebiotic or bacteria in the food product may depend on various factors, including for example, the serving size of the food product, the frequency of consumption of the food product, the specific prebiotic or bacteria contained in the food product, the amount of water in the food product, and/or additional conditions for survival of the bacteria in the food product.
  • Examples of food products which may be formulated to contain any of the prebiotic and/or bacterial species or strains described herein include, without limitation, a beverage, a drink, a bar, a snack, a dairy product, a confectionery product, a cereal product, a ready-to-eat product, a nutritional formula, such as a nutritional supplementary formulation, a food or beverage additive.
  • Suitable routes of administration may, for example, include topical, oral, rectal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intranasal, or intraocular injections.
  • compositions disclosed herein may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tableting processes.
  • compositions for use in accordance with the present disclosure thus may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. Any of the well-known techniques, carriers, and excipients may be used as suitable and as understood in the art; e.g., in Remington‘s Pharmaceutical Sciences, above.
  • the agents disclosed herein may be formulated in aqueous solutions, preferably in physiologically compatible buffers, such as Hank‘s solution, Ringer‘s solution, or physiological saline buffer.
  • physiologically compatible buffers such as Hank‘s solution, Ringer‘s solution, or physiological saline buffer.
  • penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
  • oral administration either solid or fluid unit dosage forms can be prepared.
  • the compound of Formula (I) or derivatives thereof, disclosed above herein is mixed into formulations with conventional ingredients, such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers.
  • conventional ingredients such as talc, magnesium stearate, dicalcium phosphate, magnesium aluminum silicate, calcium sulfate, starch, lactose, acacia, methylcellulose, and functionally similar materials as pharmaceutical diluents or carriers.
  • the compounds can be also formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art.
  • Such carriers enable the compounds disclosed herein to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a subject to be treated.
  • compositions for oral use can be obtained by mixing one or more solid excipient with pharmaceutical combination disclosed herein, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores.
  • suitable excipients are, in particular, fillers, such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
  • Capsules are prepared by mixing the compound with an inert pharmaceutical diluent and filling the mixture into a hard gelatin capsule of appropriate size.
  • Soft gelatin capsules are prepared by machine encapsulation of slurry of the compound with an acceptable vegetable oil, light liquid petrolatum or other inert oil.
  • Fluid unit dosage forms for oral administration such as syrups, elixirs, and suspensions, can be prepared.
  • the water-soluble forms can be dissolved in an aqueous vehicle together with sugar, aromatic flavoring agents and preservatives to form syrup.
  • An elixir is prepared by using a hydro alcoholic (e. g., ethanol) vehicle with suitable sweeteners, such as sugar and saccharin, together with an aromatic flavoring agent.
  • Suspensions can be prepared with an aqueous vehicle with the aid of a suspending agent, such as acacia, tragacanth, methylcellulose, and the like.
  • Dragee cores are provided with suitable coatings.
  • suitable coatings For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
  • Starch microspheres can be prepared by adding a warm aqueous starch solution, e. g., of potato starch, to a heated solution of polyethylene glycol in water with stirring to form an emulsion.
  • the mixture is then cooled to room temperature under continued stirring whereupon the inner phase is converted into gel particles. These particles are then filtered off at room temperature and slurred in a solvent, such as ethanol, after which the particles are again filtered off and laid to dry in air.
  • a solvent such as ethanol
  • the micro spheres can be hardened by well-known cross-linking procedures, such as heat treatment or by using chemical cross-linking agents.
  • Suitable agents include dialdehydes, including glyoxal, malondialdehyde, succinic aldehyde, adipaldehyde, glutaraldehyde and phthalaldehyde, diketones, such as butadione, epichlorohydrin, polyphosphate, and borate.
  • Dialdehydes are used to crosslink proteins, such as albumin, by interaction with amino groups, and diketones form schiff bases with amino groups.
  • Epichlorohydrin activates compounds with nucleophiles, such as amino or hydroxyl, to an epoxide derivative.
  • compositions which can be used orally, include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol.
  • the push-fit capsules can contain the active ingredients in admixture with filler, such as lactose, binders, such as starches, and/or lubricants, such as talc or magnesium stearate, and, optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • suitable liquids such as fatty oils, liquid paraffin, or liquid polyethylene glycols.
  • stabilizers and/or antioxidants may be added. All formulations for oral administration should be in dosages suitable for such administration.
  • Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents, such as suspending, stabilizing, and/or dispersing agents.
  • compositions of the present invention also include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • Aqueous suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the preparation of highly, concentrated solutions.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
  • a suitable vehicle e.g., sterile pyrogen-free water
  • the present invention further relates to a method of modulating a level of microbe or strain thereof.
  • the invention relates, in part, to a method of modulating the level of a pathogenic microbe or strain thereof in a subject in need thereof.
  • the method comprises decreasing the level of a pathogenic microbe or strain thereof in a subject in need thereof.
  • the invention relates, in part, to a method of modulating the level of a beneficial microbe or strain thereof in a subject in need thereof.
  • the method comprises increasing the level (e.g., activity, expression, level, etc.) of a beneficial microbe or strain thereof in a subject in need thereof.
  • the method comprises decreasing the systemic antibody response against the beneficial microbe or strain thereof in a subject in need thereof.
  • the method comprises administering a therapeutically effective amount of at least one composition described herein. In one embodiment, the method comprises administering a therapeutically effective amount of an inhibitor of the pathogenic microbe or strain thereof to the subject.
  • the method comprises reducing the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof in the subject by modulating the pH. In some embodiments, the method comprises reducing the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof in the subject by increasing the pH. In some embodiments, the method comprises reducing the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof in the subject by decreasing the pH.
  • the present invention also relates, in part, to a method of preventing or treating a disease or disorder induced by a pathogenic microbe (e.g., Allobaculum sp. or strain thereof) or strain thereof in a subject in need thereof.
  • a pathogenic microbe e.g., Allobaculum sp. or strain thereof
  • the disease or disorder induced the pathogenic microbe or strain thereof is an inflammatory bowel disease, celiac disease, colitis, irritable bowel syndrome, intestinal hyperplasia, metabolic syndrome, obesity, diabetes, rheumatoid arthritis, liver disease, hepatic steatosis, fatty liver disease, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or any combination thereof.
  • the method prevents or treats a disease or disorder induced by Allobaculum sp. or strain thereof in a subject in need thereof.
  • the method comprises administering to the subject any of the composition of the present invention.
  • the method comprises administering to the subject a composition comprising a beneficial microbe (e.g., Akkermansia sp. or strain thereof) or strain thereof and/or culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the method comprises the steps of detecting the presence of the pathogenic microbe or strain thereof in the subject; identifying a beneficial microbe or strain thereof; and administering to the subject a composition comprising the beneficial microbe species or strain thereof culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof.
  • the beneficial microbe or strain thereof is identified as being one whose level (e.g., activity, expression, level, etc.) is inversely correlated to the pathogenic microbe or strain thereof.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the method further comprises the step of administering to the subject a composition comprising at least one compound that reduces the level of the pathogenic microbe or strain thereof prior to the step of administering to the subject the composition comprising the beneficial microbe or strain thereof.
  • the method comprises increasing the level (e.g., activity, expression, level, etc.) of the beneficial microbe or strain thereof.
  • the method comprises administering to the subject at least one compound that increases the level (e.g., activity, expression, level, etc.) of the beneficial microbe or strain thereof.
  • compounds include, but are not limited to, a probiotic, prebiotic of the beneficial microbe or strain thereof, antibiotic of the pathogenic microbe or strain thereof, antimicrobe of the pathogenic microbe or strain thereof, or any combination thereof.
  • the method comprises reducing the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof.
  • the method comprises administering to the subject at least one compound that reduces the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof.
  • compounds include, but are not limited to, a probiotic, prebiotic of the beneficial microbe or strain thereof, antibiotic of the pathogenic microbe or strain thereof, antimicrobe of the pathogenic microbe or strain thereof, a nucleic acid molecules comprising a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof, or any combination thereof.
  • the method comprises modification of the altered microbiota having over-represented pathogenic microbe or strain thereof that is achieved by administering to a subject in need thereof a therapeutically effective amount of a vaccine to induce an immune response against the over-represented constituent (e.g., pathogenic microbe or strain thereof), wherein the administered vaccine and ensuing immune response diminishes the number or pathogenic effects of at least one type (e.g., genus, species, strain, sub-strain, etc.) of the pathogenic microbe or strain thereof that is over-represented in the altered microbiota, as compared with a normal microbiota.
  • a therapeutically effective amount of a vaccine to induce an immune response against the over-represented constituent (e.g., pathogenic microbe or strain thereof), wherein the administered vaccine and ensuing immune response diminishes the number or pathogenic effects of at least one type (e.g., genus, species, strain, sub-strain, etc.) of the pathogenic microbe or strain thereof that is over-represented in the altered microbio
  • the term “vaccine” refers to a substance that induces immunity upon inoculation into animals.
  • the vaccine of the invention can be used to inducing immunity to one or more bacteria types of the over-represented constituent (e.g., pathogenic microbe or strain thereof).
  • modification of the altered microbiota having over- represented pathogenic microbe or strain thereof is achieved by administering to a subject in need thereof a therapeutically effective amount of a passive immunotherapy or passive vaccine, such as by the administration of immunoglobulin (e.g., IgA) against the over-represented constituent (e.g., pathogenic microbe or strain thereof), wherein the administered passive vaccine and ensuing immune response diminishes the number or pathogenic effects of at least one type (e.g., genus, species, strain, sub-strain, etc.) of pathogenic microbe or strain thereof that is over- represented in the altered microbiota, as compared with a normal microbiota.
  • the immunoglobulin is administered orally.
  • the immunoglobulin can be administered rectally or by enema.
  • modification of the altered microbiota having over- represented pathogenic microbe or strain thereof is achieved by administering to a subject in need thereof a therapeutically effective amount of antibiotic composition comprising an effective amount of at least one antibiotic, or a combinations of several types of antibiotics, wherein the administered antibiotic diminishes the number or pathogenic effects of at least one type (e.g., genus, species, strain, sub-strain, etc.) of pathogenic microbe or strain thereof that is over- represented in the altered microbiota, as compared with a normal microbiota.
  • at least one type e.g., genus, species, strain, sub-strain, etc.
  • the type and dosage of the administered antibiotic will vary widely, depending upon the nature of the inflammatory disease or disorder, the character of subject’s altered microbiota, the subject’s medical history, the frequency of administration, the manner of administration, and the like.
  • the initial dose may be larger, followed by smaller maintenance doses.
  • the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level.
  • the administered antibiotic is at least one of lipopeptide, fluoroquinolone, ketolide, cephalosporin, amikacin, gentamicin, kanamycin, neomycin, netilmicin, paromomycin, streptomycin, tobramycin, cefacetrile, cefadroxil, cefalexin, cefaloglycin, cefalonium, cefaloridine, cefalotin, cefapirin, cefatrizine, cefazaflur, cefazedone, cefazolin, cefradine, cefroxadine, ceftezole, cefaclor, cefamandole, cefmetazole, cefonicid, cefotetan, cefoxitin, cefprozil, cefuroxime, cefuzonam, cefcapene, cefdaloxime, cefdinir, cefditoren, cefetamet, cefprozil
  • modification of the altered microbiota is achieved by administering to a subject in need thereof a therapeutically effective amount of a probiotic composition comprising an effective amount of at least one type (e.g., genus, species, strain, sub- strain, etc.) of bacteria, or a combinations of several types of bacteria, wherein the administered bacteria supplements the number of the types of bacteria which are under-represented in the altered microbiota, as compared with a normal microbiota.
  • the probiotic is a surgical probiotic.
  • the invention is a method of treating an inflammatory disease or disorder of a subject in need thereof, including the step of administering to the subject at least one type (e.g., genus, species, strain, sub-strain, etc.) of bacteria, or a combinations of several types of bacteria, that is desired, preferred, neutral, beneficial, and/or under-represented in the subject’s microbiota.
  • a type e.g., genus, species, strain, sub-strain, etc.
  • the at least one type of bacteria is at least one bacterium of a species of bacteria identified from a healthy subject that does not have the disease.
  • the species or strain of bacteria is a secretory antibody -bound bacteria identified from a healthy subject.
  • administration of secretory antibody -bound bacteria from a healthy subject can treat or prevent an inflammatory disease or disorder.
  • Bacteria administered according to the methods of the present invention can comprise live bacteria.
  • One or several different types of bacteria can be administered concurrently or sequentially.
  • Such bacteria can be obtained from any source, including being isolated from a microbiota and grown in culture using known techniques.
  • the administered bacteria used in the methods of the invention further comprise a buffering agent.
  • buffering agents include sodium bicarbonate, milk, yogurt, infant formula, and other dairy products.
  • Administration of a bacterium can be accomplished by any method suitable for introducing the organisms into the desired location.
  • the bacteria can be mixed with a carrier and (for easier delivery to the digestive tract) applied to a liquid or to food.
  • the carrier material should be non-toxic to the bacteria as wells as the subject.
  • the carrier contains an ingredient that promotes viability of the bacteria during storage.
  • the formulation can include added ingredients to improve palatability, improve shelf-life, impart nutritional benefits, and the like.
  • the dosage of the administered bacteria will vary widely, depending upon the nature of the inflammatory disease or disorder, the character of subject’s altered microbiota, the subject’s medical history, the frequency of administration, the manner of administration, the clearance of the agent from the host, and the like.
  • the initial dose may be larger, followed by smaller maintenance doses.
  • the dose may be administered as infrequently as weekly or biweekly, or fractionated into smaller doses and administered daily, semi-weekly, etc., to maintain an effective dosage level. It is contemplated that a variety of doses will be effective to achieve colonization of the gastrointestinal tract with the desired bacteria.
  • the dose ranges from about 10 6 to about 10 10 CFU per administration. In other embodiments, the dose ranges from about 10 4 to about 10 6 CFU per administration.
  • the present invention relates to a method for modifying an altered microbiota comprising administering to a subject in need of such treatment, an effective amount of at least one gastric, esophageal, or intestinal bacterium, or combinations thereof.
  • the bacteria are administered orally.
  • bacteria can be administered rectally or by enema.
  • the organisms contemplated for administration to modify the altered microbiota include any of the bacteria identified herein as under-represented in an altered microbiota.
  • the bacteria administered in the therapeutic methods of the invention comprise administration of a combination of organisms.
  • a bacteria for therapy While it is possible to administer a bacteria for therapy as is, it may be preferable to administer it in a pharmaceutical formulation, e.g., in admixture with a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • a suitable pharmaceutical excipient, diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice.
  • the excipient, diluent and/or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof. Acceptable excipients, diluents, and carriers for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington: The Science and Practice of Pharmacy. Lippincott Williams & Wilkins (A. R. Gennaro edit. 2005).
  • the choice of pharmaceutical excipient, diluent, and carrier can be selected with regard to the intended route of administration and standard pharmaceutical practice.
  • oral delivery is preferred for delivery to the digestive tract because of its ease and convenience, and because oral formulations readily accommodate additional mixtures, such as milk, yogurt, and infant formula.
  • additional mixtures such as milk, yogurt, and infant formula.
  • bacteria can be also administered rectally or by enema.
  • modification of the altered microbiota is achieved by both administering at least one type (e.g., genus, species, strain, sub-strain, etc.) of bacteria to supplement the numbers of at least one type (e.g., genus, species, strain, sub-strain, etc.) of bacteria that is under-represented in the altered microbiota, and administering at least one antibiotic to diminish the numbers of at least one type (e.g., genus, species, strain, sub-strain, etc.) of bacteria that is over-represented in the altered microbiota.
  • at least one type e.g., genus, species, strain, sub-strain, etc.
  • compositions of the invention can be administered singly or in any combination. Further, the compositions of the invention can be administered singly or in any combination in a temporal sense, in that they may be administered concurrently, or before, and/or after each other.
  • compositions of the invention can be used to prevent or to treat a disease or disorder, and that the composition can be used alone or in any combination with another modulator to affect a therapeutic result.
  • any of the compositions of the invention described herein can be administered alone or in combination with other modulators of other molecules associated with the diseases and disorders described herein.
  • compositions of the invention can be administered in combination with an additional therapeutic composition selected from the group consisting of corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, anti- leukotrienes, anti-cholinergic drugs for rhinitis, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines (preferably vaccines used for vaccination where the amount of an allergen is gradually increased), anti-TNF inhibitors such as infliximab, adalimumab, certolizumab pegol, golimumab,
  • an additional therapeutic composition selected from
  • the invention includes a method comprising administering a combination of compositions described herein.
  • the method has an additive effect, wherein the overall effect of the administering a combination of compositions is approximately equal to the sum of the effects of administering each individual composition.
  • the method has a synergistic effect, wherein the overall effect of administering a combination of compositions is greater than the sum of the effects of administering each individual composition.
  • the method comprises administering a combination of compositions in any suitable ratio.
  • the method comprises administering two individual compositions at a 1 : 1 ratio.
  • the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
  • the regimen of administration may affect what constitutes an effective amount.
  • the therapeutic formulations may be administered to the subject either prior to or after a diagnosis of disease. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.
  • the method of treatment comprises monitoring the biomarker levels (e.g., the level of a pathogenic microbe or strain thereof) during the course of treatment of a disease or disorder.
  • the method of treatment comprises an assessment of the effectiveness of the treatment regimen for a disease or disorder, such as cancer, by detecting one or more biomarkers (e.g., the level of a pathogenic microbe or strain thereof) in an effective amount from samples obtained from a subject over time and comparing the amount of biomarker or biomarkers detected.
  • a first sample is obtained prior to the subject receiving treatment and one or more subsequent samples are taken after or during treatment of the subject.
  • changes in biomarker levels over time provide an indication of effectiveness of the therapy.
  • the present invention relates to a method of predicting the effectiveness of a treatment of a disease or disorder (e.g., an inflammatory disease or disorder) in a subject, the treatment comprising administering to the subject having the disease or disorder, a composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof and/or culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof that is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of a pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof that induces the disease or disorder.
  • a beneficial gut microbe species e.g., Akkermansia sp.
  • culture media e.g.,
  • the method comprises the steps of detecting the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject. In various embodiments, the method comprises the steps of comparing the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof to a comparator.
  • the level e.g., activity, expression, concentration, level, etc.
  • the pathogenic gut microbe species e.g., Allobaculum sp.
  • the method comprises the step of determining that the composition is effective when the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof is higher when compared to a comparator.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • the present invention relates to a method of predicting the effectiveness of a treatment of a disease or disorder (e.g., cancer or obesity) in a subject, the treatment comprising administering a composition to the subject having the disease or disorder (e.g., cancer or obesity), the composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof and/or culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain and/or an active agent isolated or purified from culture media (e.g., conditioned culture media) harvested from a culture of a beneficial microbe or strain thereof.
  • a beneficial gut microbe species e.g., Akkermansia sp.
  • culture media e.g., conditioned culture media
  • the method comprises the steps of detecting the level (e.g., activity, expression, concentration, level, etc.) of at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject that is inversely correlated to the level (e.g., activity, expression, concentration, level, etc.) of the beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof.
  • the method comprises the steps of comparing the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof to a comparator.
  • the method comprises the step of determining that the composition is ineffective, or would be less effective, when the level (e.g., activity, expression, concentration, level, etc.) of the at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof in the subject is higher when compared to a comparator.
  • the level e.g., activity, expression, concentration, level, etc.
  • the at least one pathogenic gut microbe species e.g., Allobaculum sp.
  • the method comprises administering to the subject at least one compound that decreases the level (e.g., activity, expression, concentration, level, etc.) of the at least one pathogenic gut microbe species (e.g., Allobaculum sp.) or strain thereof prior to administering to the subject a composition comprising a beneficial gut microbe species (e.g., Akkermansia sp.) or strain thereof.
  • the active agent can be selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • Example 1 Inter-Species Gut Commensal Rivalry Dictated Mucosal and Systemic Immune Responses
  • ‘pathogenic’ immunostimulatory bacteria can play potentially causal roles in inflammatory bowel disease (IBD), autoimmunity, and malnutrition, while ‘beneficial’ immunostimulatory species have been employed to treat metabolic syndrome and to augment cancer immunotherapy (Atarashi et al., 2015; Atarashi et al., 2017; Brown et al., 2015; Kau et al., 2015; Plovier et al., 2017; Routy et al., 2018; Zegarra-Ruiz et al., 2019).
  • IBD inflammatory bowel disease
  • beneficial immunostimulatory species have been employed to treat metabolic syndrome and to augment cancer immunotherapy
  • muciniphila ameliorated Allobaculum-induced intestinal epithelial cell (IEC) activation and colitis, while Allobaculum blunted the antigen-specific T and B cell responses typically elicited by A. muciniphila.
  • IEC Allobaculum-induced intestinal epithelial cell
  • these studies defined a unique interaction (i.e., a reciprocal ‘epistatic’ interaction) between two immunostimulatory gut commensals that directed divergent immunological outcomes and began to decode the specific contextual cues that underlied population-level variability in responses to individual immunostimulatory strains.
  • Allobaculum sp. 128 a highly immunoglobulin A- coated strain from the genus Allobaculum (i.e., Allobaculum sp. 128) from the gut microbiota of an ulcerative colitis (UC) patient (FIG. 1A; Palm et al., 2014, Cell, 158: 1000-1010).
  • This isolate hereafter referred to as Allobaculum sp. 128, is culturable under strict anaerobic conditions, and is nonmotile and non-spore-forming (FIG. IB).
  • this strain was a member of an unnamed species from the genus Allobaculum and the prevalent, yet poorly characterized, family Erysipelotrichaceae (Greetham et al., 2004, Anaerobe, 10:301-307; Ha et al., 2020, Cell, 183:666-683 e617;
  • Allobaculum sp. 128 Elicited Mucosal and Systemic Antibody Responses at Steady State
  • WT gnotobiotic mice colonized with Allobaculum sp. 128 showed no apparent intestinal inflammation in the absence of DSS treatment up to 12 weeks after colonization (FIGs 9A-9B).
  • this strain was identified based on high levels of coating with IgA, experiments in this Example focused on directly interrogating the ability of Allobaculum to induce antigen-specific antibody responses in gnotobiotic mice in the absence of overt pathology.
  • 128-specific nanobody was engineered using directed evolution, which enabled tracking of anP-Allobaculum IgA responses directly from fecal samples using flow cytometry -As expected, WT gnotobiotic mice colonized with MC + Allobaculum sp. 128 mounted a potent Allobaculum sp. 128-specific IgA response (see FIG. 2A-2C).
  • mice colonized with different human samples harbored distinct microbial communities. Furthermore, a range of Allobaculum sp. 128 colonization levels across these 19 unique community contexts was observed (FIG. 3B; Table 1). This variation in Allobaculum sp. 128 abundance was not due to variation in overall microbial diversity as there were no significant differences in richness or evenness between samples containing Allobaculum sp. 128 and those lacking Allobaculum sp. 128 (FIG. 10A).
  • Table 1 Relative Abundance across Nineteen Community Contexts.
  • Table 2 Tabulated Spearman and Pearson Correlation Coefficients Calculated for all Genus-Level OTUs across all Microbiome Samples Paired with Allobaculum sp. 128.
  • Table 3 Logistic Regression Analysis in Prism.
  • A. muciniphila protects against Allobaculum-mediated exacerbation of DSS colitis
  • A. muciniphila protects against Allobaculum-induced colitis in gnotobiotic mice colonized with a complete human gut microbial community and A. muciniphila-mediated protection is consistent across multiple A. muciniphila strains
  • a muciniphila could protect against Allobaculum- induced colitis in the context of a complex human gut microbial community. Germ-free mice were colonized with homogenized stool from a healthy human donor plus either Allobaculum sp. 128, A. muciniphila, or both immunogenic strains and then induced colitis using DSS (FIG. 5A- 5B). Consistent with previous observations in the context of a simplified mock community, co- colonized mice exhibited significantly less severe colitis as compared to mice colonized with Allobaculum sp. 128 in the absence of A. muciniphila (FIG. 5A-5E). Finally, to test whether the protective effects of A.
  • muciniphila are consistent across strains, the impacts of type strain A muciniphila (ATCC BAA-835) on Allobaculum-induced colitis was assessed and significant A muciniphila-mediated amelioration of Allobaculum-mediated disease was observed (FIG. 11A- 1 IE). Together, these data demonstrate that A. muciniphila ameliorates pathological intestinal immune responses incited by Allobaculum sp. 128 in multiple ecological contexts and across multiple independent strains.
  • Allobaculum sp. 128 blunts antigen-specific systemic antibody responses to A. muciniphila and oral vaccination
  • Follicular T helper cells are critical for the generation high-affinity antigen- specific antibodies to gut commensals. Recent studies revealed that A muciniphila was a potent inducer of Peyer’s patch Tfh cells in mice colonized with a simplified commensal community, yet conventional mice with complex microbial communities exhibited variable Tfh and antibody responses (Ansaldo et al., 2019, Science, 364: 1179-1184). Because Allobaculum sp. 128 blunted the systemic IgG response to A.
  • muciniphila and although not bound by any particular theory, it was hypothesized that it is also likely to reduce the differentiation or survival of A. muciniphila- specific Tfh cells.
  • Using a custom-generated A. muciniphila-specific I-Ab tetramer it was found that co-colonization with Allobaculum sp. 128 significantly reduced the A. muciniphila-s ⁇ ectfdc Tfh response in the Peyer’s patches (FIG. 17B and FIG. 17C).
  • Allobaculum sp. 128 blunted both T and B cell responses to A. muciniphila.
  • Allobaculum sp. 128 and A. muciniphila elicit unique alterations in the immunological landscape in mucosal lymphoid organs, which are reciprocally reprogrammed by co- colonization
  • muciniphila is T cell-dependent (Ansaldo et al., 2019), the activation and clonal expansion of T cells in individually colonized and co-colonized mice was examined, with a specific focus on T follicular helper (Tfh) cells.
  • Tfh T follicular helper
  • muciniphila-specific T cells in the MLN for example by blocking A. muciniphi la-induced activation or migration of professional antigen-presenting cells such as dendritic cells (DCs).
  • DCs dendritic cells
  • A. muciniphila colonization elicited a unique population of migratory DCs (MigDC) in the MLN that exhibited enhanced expression of transcripts encoding antigen presentation machinery and activation markers, and the appearance of these cells was completely abrogated by co-colonization with Allobaculum (FIG. 7H-7J; cluster 10 in FIGs 13 & 14C-14E).
  • DCs isolated from the MLNs of individually colonized and co-colonized gnotobiotic mice were co- cultured with naive OT-II T cells and ovalbumin and tracked T cell proliferation. It was observed that DCs isolated from A. muciniphila colonized mice elicited increased T cell proliferation as compared to Allobaculum sp. 128 colonized mice and that this increase was blunted by co- colonization (FIGs 14F-14G). Overall, these data suggest that Allobaculum may blocks. muciniphila-specific adaptive immune responses by preventing A. muciniphi la-induced activation of intestinal dendritic cells.
  • lECs from Allobaculum sp. 128-colonized mice displayed similar gene expression patterns to lECs from human ulcerative colitis patients, including changes in serum amyloid A, guanylate cyclase, cathepsins, and claudins (Parikh et al., 2019).
  • Allobaculum and related taxa may be important drivers of pathological intestinal inflammation in humans and potential therapeutic targets for the treatment of inflammatory disease.
  • IgA coating can be used as a marker to identify potentially pathogenic immunostimulatory strains in IBD (Palm & de Zoete et al., 2014; Viladomiu et al., 2017). Indeed, Allobaculum sp. 128 was originally identified as a putative disease-driving microbe in IBD based on its high level of coating with secretory immunoglobulin IgA. However, highly IgA-coated taxa can also exhibit beneficial and immunoregulatory effects (Peterson et al., 2007; Kawamoto et al., 2014; Kubinak et al., 2015; Donaldson et al., 2018).
  • highly IgA-coated bacteria from healthy humans can protect against the pathogenic effects of IgA-coated taxa from undernourished children (Kau et al., 2015).
  • A. muciniphila which can protect against diet-induced obesity and is associated with enhanced responses to immunotherapy, is the most prevalent highly IgA-coated taxon in healthy humans (Png et al., 2010; Everard et al., 2013; Palm & de Zoete et al., 2014; Bajer et al., 2017; Routy et al., 2018).
  • High IgA-coating thus marks microbes that elicit diverse adaptive immune responses at steady state.
  • muciniphila elicits Tfh responses in mice colonized with Altered Schaedler flora (ASF), but induces a mixture of Th cell types, including Thl, Thl7, and Tregs, in the context of a complex microbiota (Ansaldo et al., 2019).
  • the magnitude of the antigen-specific IgG response to A muciniphila was also highly variable in the presence of a complex microbiota and some animals even lacked detectable A. muciniphila-induced T cell responses in these settings (Ansaldo et al., 2019).
  • the present studies described herein suggest that Allobaculum or other phylogenetically- or functionally-related taxa may explain this context-dependence of the adaptive immune response to A. muciniphila.
  • the data presented herein underscores the importance of microbial context in dictating immune responses elicited by individual commensal organisms and suggest that immunostimulatory strains, in particular, may provide critical contextual cues that alter the magnitude, specificity, or polarization of intestinal immune responses.
  • the composite effects of the specific immunostimulatory strains present in each person may determine individual immunological outcomes and susceptibility to immune-related diseases.
  • muciniphila dramatically altered the immune responses evoked by each microbe on its own by ameliorating Allobaculum-induced colitis while also blunting A Allobaculum-induced B and T cell responses.
  • ‘pathogenic’ immunostimulatory bacteria can play potentially causal roles in IBD, autoimmunity, and malnutrition, while ‘beneficial’ immunostimulatory species have been employed to treat metabolic syndrome and as adjuncts for cancer immunotherapy (Routy et al., 2018, Science 359, 91-97; Baruch et al., 2020, Science, eabb5920). Nonetheless, potentially disease-driving bacteria were also found in apparently healthy individuals and the effects of putative beneficial strains on host physiology often vary widely between subjects. Thus, the predictive power of strain carriage alone remains limited even for microbes with well-characterized disease-modulating activities, which severely hampers the accuracy of microbiome-based prognostics and constrains the overall efficacy of existing and emerging live bio-therapeutics.
  • microbiome-based prognostics that predicted phenotypic outcomes and/or potential responsiveness to microbiome-targeted therapeutics (e.g., potential responsiveness to probiotics or fecal microbiota transplantation) based on the combination of immunomodulatory strains present in a given individual’s microbiome.
  • the present invention relates, in part, to an approach that leveraged “humanization” of gnotobiotic mice with human stool samples to represent the microbial ecology of the human microbiome in a mouse gut. It was found that a specific pair of commensal bacteria were inversely correlated across many different “humanized” mice microbiome samples, indicative of an in vivo ecology where either bacteria had a powerful effect upon the host. In follow-up experiments examining the immune responses of mice colonized with defined communities including one or the other bacteria, this pair of hits were evaluated to be robust under further mechanistic study. Surprisingly, this data was essentially mirrored in publicly available human data from thousands of human microbiomes. For this reason, the present invention is a useful approach for prediction and discovery of many new potent host-microbiome interactions that are relevant to human health.
  • this approach can be used to identify “precision probiotics” that block the pathogenic effects of specific microbial species, and can be paired with a microbiome-based diagnostic to target patients that harbor such pathogenic species.
  • this technology also enabled the identification of specific taxa whose presence or absence are likely to predict responsiveness to a live-biotherapeutic (e.g., a probiotic strain, such as Akkermansia, or group of beneficial bacteria as in fecal microbiome transplantation).
  • a live-biotherapeutic e.g., a probiotic strain, such as Akkermansia, or group of beneficial bacteria as in fecal microbiome transplantation.
  • One major novelty was, in part, the ability to identify discrete inter-species interactions that dictated divergent impacts of individual gut microbes on immunity and disease, as exemplified by the discovery of a unique relationship between Allobaculum sp. and Akkermansia sp.
  • the herein-described approach enabled the unbiased identification of key microbial taxa that shaped host immunity and provided contextual cues that impacted immune and disease outcomes induced by other immunomodulatory gut microbes.
  • This approach is useful in identifying ‘precision probiotics’ that counteract specific pathogenic species, to improve microbiome-based diagnostics and prognostics, and to predict individual responses to microbiome-target therapeutics based on the combination of immunomodulatory strains present in an individual.
  • this understanding of the specific microbes that contributed to disease, dictated responses to specific therapeutic treatments (e.g, specific probiotics), or predicted disease trajectory is very useful for the development of precision medicine-based approaches to treat microbiota-modulated diseases, or as companion diagnostics to determine treatment selection.
  • V4_F GTGCCAGCMGCCGCGGTAA - SEQ ID NO.: 4
  • V4_R GGACTACHVGGGTWTCTAAT - SEQ ID NO.: 5
  • 16S rRNA gene using published primer sequences 8F: 5’-AGAGTTTGATCCTGGCTCAG-3’ - SEQ ID NO.: 6 and 1391R: 5’-GACGGGCGGTGTGTRCA-3’ - SEQ ID NO.: 7). Sequences were queried against NCBI and RDP databases.
  • mice BL/6, RAG1-/-, IL 10-/- were maintained in flexible film isolators (CBC) with all bedding, chow (Teklad 2018S), and water being autoclaved before import. All germ-free breeding isolators were regularly monitored for the presence of bacteria (both culture-dependent and -independent techniques). All experiments were conducted by transferring mice to positive pressure ventilated microisolator cages (Techniplast #ISO72P) and inoculating each mouse by oral gavage immediately upon transfer. Inocula were previously prepared in anaerobic culture and frozen at -80 °C in media+20% glycerol in gasket-sealed airtight glass vials (Wheaton).
  • Freshly defecated fecal samples were collected into sterile 2 mL screw-cap tubes and rehydrated in 1 mL sterile PBS, disrupted by 10 sec bead beating (Lysing matrix D beads, MP Biomedicals) in a Biospec bead beater, then centrifuged 5 min at 50 xg to gently pellet large debris. Bacterial cell suspension was then transferred to sterile 2 mL deep-well plates for downstream processing. Fecal bacteria were pelleted at 10,000 xg for 10 min, and clarified fecal water was removed for evaluation of Lipocalin-2 content by ELISA (R&D Systems DY1857).
  • the 16S rRNA gene V4 region was amplified from each bacterial gDNA sample by PCR according to a dual-index multiplexing strategy (Kozich et al., 2013, Appl. Environ. Microb., 79:5112-5120), then amplicons were normalized and cleaned (Agencourt AMPure XP purification beads; Beckman Coulter #A63881). Samples were pooled and libraries were quantified by qPCR (KAPA Biosystems KK4835; Applied Biosystems ABI 7500 instrument) then sequenced on an Illumina Miseq (500 cycle V2 reagent kit #MS- 102-2003).
  • Colon tissues were opened longitudinally and washed thoroughly in sterile PBS until no visible fecal debris remained, then finely minced with a razor blade and transferred to 2 mL screw-cap tubes with ImL ice-cold TRI Reagent (Sigma Aldrich #T9424) and nuclease-free 0.1 mm glass beads, thoroughly bead beating for 20 sec *3, resting on ice in between.
  • Bulk RNA samples were cleaned using Qiagen RNeasy Mini columns, DNase I digested, and quality checked on an Agilent Bioanalyzer RNA 6000 Nano Kit (#5067-1511). Sequencing libraries were prepared by Yale Center for Genome Analysis staff and run using Illumina Hiseq 2 x 75 chemistry.
  • Intestinal epithelial cell and bacterial RNAseq libraries were prepared using 60ng total RNA input into Illumina Total Stranded RNA Prep Kit with Ribo-zero Plus (#20040529) and sequenced using Illumina NovaSeq (2x150).
  • Bacterial probe EUB-338 ([Cy3 ]-5 ’ - GCTGCCTCCCGTAGGAGT-3’-[Cy3]; SEQ ID NO: 8) and VP403 ([biotin]-5’- CGAAGACCTTATCCTCCACG-3 ’-[biotin]; SEQ ID NO: 9) were used for staining at Ipg/mL in hybridization buffer (0.9M NaCl + 0.02M Tris, pH 7.5 + 20% Formamide + 0.05% SDS) in a humidified chamber for 2h at 46 °C. After washing, slides were counterstained with DAPI and mounted in ProlongGold Antifade mounting media with overnight curing. Images were acquired on a Leica SP8 confocal microscope running LAS-X software version 3.1.5.
  • Fecal bacterial cell suspensions were transferred to sterile LB +20% Glycerol and frozen at -80 °C until further analysis. Bacteria were thawed on ice, then aliquoted 10 4 - 10 5 CFU per well of 2 mL 96-deep-well plate (pellet not visible)(Moor et al., 2016, Nat. Protoc., 11 : 1531- 1553). Each staining reaction was blocked with normal rat serum for 15 min, then washed in sterile PBS/0.1% BSA.
  • Serum was washed out three times with 500 pl PBS then stained with secondary detection antibody (AF647 anti-Ms-IgG at 1 :800, Biolegend #405322) for 30 min RT.
  • Cells washed three times in 500pl PBS, then transferred to LlmL microdilution tubes (VWR 20901-013) for analysis on a BD FACS Calibur instrument, including control tubes for sterile buffer (log FSC, log SSC), unstained cells, and secondary only cells to set appropriate gates.
  • a minimum of 50,000 events/sample were collected and analyzed using FlowJo v9.
  • Bacterial ELISAs Bacterial ELISAs
  • Ileum and colon tissue was harvested into 25 °C complete RPMI 1640 medium (supplemented with 10% FBS, Pen-Strep, L-Glutamine, HEPES). After gentle cleaning to remove large fecal debris, tissues were shaken in strip buffer (HBSS + 1.5 mM EDTA + 0.145 mg/ml DTT) at 37 °C 225 rpm for 20 min x 2 to remove mucus and epithelial layers. Epithelial cells were filtered through stainless steel mesh, then centrifuged 10min 400xg, and resuspended in Trizol for RNA extraction.
  • Remaining lamina limbal tissue was shaken in strip buffer a second time, then minced and transferred to cRPMI + 0.5mg/mL DNase + Img/mL Collagenase D for 45min shaken at the same speed. Then cells were filtered twice through stainless steel mesh and lymphocytes enriched in a 40%-70% Percoll interface (20min at 600xg, brake off). Cells were aliquoted to round-bottom polystyrene microplates for Fc Blocking, fluor-conjugated antibody staining (see Table 1) and washing.
  • Ex vivo cell restimulations were performed for 4h with 50ng/mL PMA + IpM ionomycin, in the presence of brefeldin A (GolgiStop reagent, BD #554724), before surface staining, fixation, permeabilization, and intracellular staining.
  • brefeldin A GolgiStop reagent, BD #554724
  • Mucosal lymphoid tissues were dissected and gently washed in sterile PBS, transferred to digestion media (serum -free RPMI 1640 supplemented with, Pen-Strep, L- Glutamine, HEPES, 2-mercaptoehtanol, NEAA, Sodium Pyruvate, DNase I, and Collagenase D) in 30 mL beaker with a small magnetic stir bar and stirred at 400 rpm in 5% CO2 incubator for 15 min. After stirring, beakers were transferred to ice and triturated with media containing 3% FBS, filtered through stainless steel mesh, centrifuged 350xg 10 min 4 oC. Cells were washed twice more in media to remove large debris chunks, then resuspended in PBS + 0.04%BSA and filtered again through 40 pm nylon.
  • digestion media serum -free RPMI 1640 supplemented with, Pen-Strep, L- Glutamine, HEPES, 2-
  • MLNs were digested to generate single cell suspensions as described above and MLN DCs were isolated from each mouse using EasySep Mouse Pan-DC Enrichment Kit (Stem Cell Technologies #19763), counted by hemacytometer, normalized for cell concentration, and plated at 7.5e4 cells/well of a round-bottom TC plate.
  • OT-II cells were isolated from pooled spleens and peripheral lymph nodes of OT-II mice using EasySep Mouse Naive CD4+ T cell isolation kit (Stem Cell Tech #19765), labeled for 20min in 5pM CellTraceViolet (Biolegend #425101), and plated at 2e5 cells/well with 10Ong/mL OVA. On day 3 of co-culture, T cell proliferation was assessed by flow cytometry.
  • Microbiota profiling 16S rRNA amplicon sequencing data were processed and analyzed using QIME (vl.9), including rarefaction to 1000 reads/sample, elimination of reads below a frequency of 0.0001, open reference OTU picking, and filtering out contaminating OTUs known to originate from water control PCRs (Caporaso, et al., 2010; Lozupone et al., 2012; McDonald et al., 2012). Bulk RNAseq sequencing data were trimmed, aligned, and gene counts quantified using Partek Flow (v6.0). Gene lists were analyzed for GO enrichment using Panther vl4 available at geneontology.org (Mi et al., 2019).
  • Mucosal lymphoid tissues were dissected and gently washed in sterile PBS, transferred to digestion media (serum -free RPMI 1640 supplemented with, Pen-Strep, L- Glutamine, HEPES, 2-mercaptoethanol, NEAA, Sodium Pyruvate, DNase I, and Collagenase D) in 30 mL beaker with a small magnetic stir bar and stirred at 400 rpm in 5% CO2 incubator for 15 min. After stirring, beakers were transferred to ice and triturated with media containing 3% FBS, filtered through stainless steel mesh, centrifuged 350 xg 10 min 4 °C. Cells were washed twice more in media to remove large debris chunks, then resuspended in PBS+0.04% BSA and filtered again through 40 pm nylon.
  • digestion media serum -free RPMI 1640 supplemented with, Pen-Strep, L- Glutamine, HEPES, 2-mercaptoethanol,
  • Microbiota sequencing data was processed and analyzed using QIIME (vl.9), including rarefaction to 1000 reads/sample, elimination of reads below a frequency of 0.0001, open reference OTU picking and filtering out contaminating OTUs known to originate from water control PCRs (Caporaso et al., 2010, Nature Methods, 7:335-336; Lozupone et al., 2012, Nature, 489:220-230; McDonald et al., 2012, Isme Journal, 6:610-618). RNAseq data was trimmed, aligned, and gene counts quantified using Partek Flow (v6.0).
  • Example 2 A Human Microbiota- Associated Gnotobiotic Mouse-Based Pipeline Methodology to Evaluate Potential Competition between Allobaculum and Commensal Bacteria from Diverse Human Gut Microbiota
  • a human microbiota-associated gnotobiotic mouse-based pipeline was established to evaluate potential competition between Allobaculum and commensal bacteria from diverse human gut microbiota. Briefly, individually-housed germ-free mice were monocolonized with Allobaculum sp. 128 for 24 hours before gavaging each monocolonized mouse with one of nineteen different healthy human stool samples. After seven days, microbial community composition was evaluated in all mice via 16S rRNA gene sequencing. To identify taxa of interest that exist in human-relevant pairwise relationships, Spearman correlation coefficients were calculated for all genus-level OTUs across all microbiome samples paired with Allobaculum sp. 128 abundance.
  • Table 7 SEQUENCES OF THE DISCLOSURE WITH SEQ ID NO IDENTIFIERS
  • a method of preventing or treating a disease or disorder induced by a first gut microbe species or strain thereof in a subject in need thereof comprises administering to the subject a composition comprising a second gut microbe species or strain thereof, wherein the level of the second gut microbe species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by the first gut microbe species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • a method of preventing or treating a disease or disorder induced by a first gut microbe species or strain thereof in a subject in need thereof comprises administering to the subject a composition comprising an active agent isolated from conditioned culture media harvested from a culture of a second gut microbe species or strain thereof, wherein the active agent reduces or inhibits at least one pathogenic effect produced by the first gut microbe species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO. : 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • IBD inflammatory bowel disease
  • colitis Crohn’s disease
  • ulcerative colitis Clostridium difficile colitis
  • composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof.
  • composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof.
  • the at least one compound that increases the level of the second gut microbe species or strain thereof comprises a probiotic, prebiotic of the second gut microbe species or strain thereof, antibiotic of the first gut microbe species or strain thereof, antimicrobe of the first gut microbe species or strain thereof, or any combination thereof.
  • Allobaculum species or strain thereof in a subject in need thereof comprises the steps of: (a) detecting the presence of the Allobaculum species or strain thereof in the subject; and (b) administering a composition comprising at least one Akkermansia species or strain thereof to the subject, wherein the level of the at least one Akkermansia species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by the Allobaculum species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • Allobaculum species or strain thereof in a subject in need thereof comprises the steps of: (a) detecting the presence of the Allobaculum species or strain thereof in the subject; and (b) administering a composition comprising an active agent isolated from conditioned culture media harvested from a culture of at least one Akkermansia species or strain thereof to the subject, wherein the active agent reduces or inhibits at least one pathogenic effect produced by the Allobaculum species or strain that induces the disease or disorder, thereby treating the disease or disorder.
  • the at least one compound that reduces the level of the Allobaculum species or strain thereof comprises a probiotic, antibiotic, antimicrobe, prebiotic of at least one Akkermansia species or strain thereof, nucleic acid molecule encoding at least one Akkermansia species or strain thereof, or any combination thereof.
  • Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • Allobaculum species or strain thereof is detected in a biological sample of the subject.
  • Akkermansia species or strain thereof for treating or preventing a disease or disorder in a subject wherein the method comprises the steps of: (a) detecting the level of at least one Allobaculum species or strain thereof in a subject suffering from a disease or disorder; and (b) comparing the level of the at least one Allobaculum species or strain thereof in the subject to a comparator.
  • a method of predicting the effectiveness of a composition comprising an active agent isolated from conditioned culture media harvested from a culture of an Akkermansia species or strain thereof for treating or preventing a disease or disorder in a subject, wherein the method comprises the steps of: (a) detecting the level of at least one Allobaculum species or strain thereof in a subject suffering from a disease or disorder; and (b) comparing the level of the at least one Allobaculum species or strain thereof in the subject to a comparator.
  • Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
  • a composition comprising a beneficial gut microbe species or strain thereof, wherein the level of the beneficial gut microbe species or strain thereof in the composition is sufficient to reduce or inhibit at least one pathogenic effect produced by a pathogenic species or strain thereof that induces a disease or disorder.
  • composition comprising an active agent isolated from conditioned culture media harvested from a culture of a beneficial gut microbe species or strain thereof, wherein the active agent reduces or inhibits at least one pathogenic effect produced by a pathogenic species or strain thereof that induces a disease or disorder.
  • composition of embodiment 38 or 39, wherein the composition modulates an immune response toward the disease or disorder.
  • composition of embodiment 38 or 39 further comprising at least one additional agent selected from the group consisting of a probiotic, prebiotic, antibiotic, antimicrobe, and any combination thereof.
  • composition of embodiment 39, wherein the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
  • composition of embodiment 38 or 39, wherein the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof.
  • composition of embodiment 43 wherein the Allobaculum species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 1 or a fragment thereof, a nucleotide sequence as set forth in SEQ ID NO.: 3 or a fragment thereof, or any combination thereof.
  • composition of embodiment 45, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
  • composition of embodiment 47, wherein the inflammatory disease or disorder is an inflammatory bowel disease, colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
  • composition of any one of embodiments 38-48, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
  • IEC intestinal epithelial cell
  • IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.

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Abstract

La présente invention concerne des compositions et des méthodes de traitement d'une maladie ou d'un trouble, tel qu'une maladie intestinale inflammatoire, à l'aide d'interactions entre espèces.
PCT/US2022/012472 2021-01-15 2022-01-14 Compositions et méthodes de traitement et de prévention de maladies ou de troubles au moyen d'interactions entre espèces WO2022155443A1 (fr)

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US20130224155A1 (en) * 2012-02-29 2013-08-29 The General Hospital Corporation D/B/A Massachusetts General Hospital Compositions of microbiota and methods related thereto
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