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WO2022155443A1 - Compositions and methods for treating and preventing diseases or disorders using inter-species interactions - Google Patents

Compositions and methods for treating and preventing diseases or disorders using inter-species interactions Download PDF

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Publication number
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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Prior art keywords
strain
species
disease
subject
allobaculum
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PCT/US2022/012472
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French (fr)
Inventor
Noah PALM
Tyler RICE
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Yale University
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Publication of WO2022155443A1 publication Critical patent/WO2022155443A1/en

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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

The present invention provides compositions and methods for treating a disease or disorder, such as inflammatory bowel disease, using inter-species interactions.

Description

IN THE UNITED STATES PATENT & TRADEMARK RECEIVING OFFICE
INTERNATIONAL PCT PATENT APPLICATION
Compositions and Methods for Treating and Preventing Diseases or Disorders using Inter-Species Interactions
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of priority to U.S. Provisional Application No. 63/137,880 filed January 15, 2021, which is hereby incorporated by reference in its entirety for all purposes.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT
[0002] This invention was made with government support under AI137935 and AI123477 awarded by the National Institutes of Health. The government has certain rights in the invention.
STATEMENT REGARDING SEQUENCE LISTING
[0003] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 047162_5304_00WO_SeqList_ST25.txt. The text file is ~ 6,087 bytes, was created on January 14, 2022, and is being submitted electronically via EFS-Web.
BACKGROUND
[0004] Indigenous gut microbial strains that shape intestinal and systemic immune responses can impact diverse host phenotypes, yet the understanding of the specific human commensal species that play causal roles in health and disease remains limited. Studies have leveraged antigen specific mucosal and systemic antibody responses to the commensal microbiota to identify individual commensal species that shape local and systemic immune responses and exacerbate or ameliorate mouse models of human disease. These studies have revealed that a small subset of bacterial strains from the human gut microbiota induce antigen-specific adaptive immune responses and may critically shape disease susceptibility. For example, 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. 23: 107-113; Routy et al., 2018, Science, 359:91-97; Atarashi et al., 2017, Science 358, 359-365; Brown et al., 2015, Nat Commun 6, 7806.; Kau et al., 2015, Sci Transl Med 7, 276ra224; Zegarra-Ruiz et al., 2019, Cell Host & Microbe 25, 113-127). 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 (McDonald et al., 2018, Isme Journal, 6:610-618; Ji et al., 2020, Nature Microbiology 5, 768-775; Buffle et al., 2015, Nature 517, 205-208). Thus, the predictive power of strain carriage alone remains poor even for microbes with well-characterized disease-modulating activities, which severely hamper the accuracy of microbiome-based prognostics and constrain the population-wide efficacy of existing and emerging live bio- therapeutics.
[0005] Thus, there is a need in the art for improving the predictive power of commensal strain carriage for manifesting in gastrointestinal diseases or disorders as well as improved compositions and methods for treating and preventing diseases or disorders. The present invention satisfies these unmet needs.
SUMMARY
[0006] In one aspect, provided herein is 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, wherein the method 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. In some cases, the first gut microbe species or strain thereof is an Allobaculum species or strain thereof. In some cases, 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. In some cases, the second gut microbe species or strain thereof is an Akkermansia species or strain thereof. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. In some cases, the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. In some cases, 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. In some cases, the disease or disorder is an inflammatory disease or disorder. In some cases, the inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof. In some cases, the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof. In some cases, 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. In some cases, the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof. In some cases, 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. In some cases, 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.
[0007] In another aspect, provided herein is 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, wherein the method 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. In some cases, the first gut microbe species or strain thereof is an Allobaculum species or strain thereof. In some cases, 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. In some cases, the second gut microbe species or strain thereof is an Akkermansia species or strain thereof. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. In some cases, the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. In some cases, 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. In some cases, the disease or disorder is an inflammatory disease or disorder. In some cases, the inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof. In some cases, the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof. In some cases, 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. In some cases, the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof. In some cases, 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. In some cases, 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. In some cases, the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[0008] In one aspect, provided herein is a method of preventing or treating a disease or disorder induced by an Allobaculum species or strain thereof in a subject in need thereof, wherein the method 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. In some cases, 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. In some cases, 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. In some cases, the presence of the Allobaculum species or strain thereof is detected in the gut microbiota of the subject. In some cases, the presence of the Allobaculum species or strain thereof is detected in a biological sample of the subject. In some cases, 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. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (EEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. In some cases, the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. [0009] In another aspect, provided herein is a method of preventing or treating a disease or disorder induced by an Allobaculum species or strain thereof in a subject in need thereof, wherein the method 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. In some cases, 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. In some cases, 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. In some cases, the presence of the Allobaculum species or strain thereof is detected in the gut microbiota of the subject. In some cases, the presence of the Allobaculum species or strain thereof is detected in a biological sample of the subject. In some cases, 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. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (EEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. In some cases, the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. In some cases, the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[0010] In one aspect, provided herein is 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. In some cases, 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. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO. : 2 or a fragment thereof.
[0011] In another aspect, provided herein is 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. In some cases, 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. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[0012] In one aspect, provided herein is 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. In some cases, the composition modulates an immune response toward the disease or disorder. In some cases, the composition further comprises at least one additional agent selected from the group consisting of a probiotic, prebiotic, antibiotic, antimicrobe, and any combination thereof. In some cases, the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof. In some cases, 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. In some cases, the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the disease or disorder is an inflammatory disease or disorder. In some cases, the inflammatory disease or disorder is an inflammatory bowel disease, colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
[0013] In another aspect, provided herein is a 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. In some cases, the composition modulates an immune response toward the disease or disorder. In some cases, the composition further comprises at least one additional agent selected from the group consisting of a probiotic, prebiotic, antibiotic, antimicrobe, and any combination thereof. In some cases, the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof. In some cases, 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. In some cases, the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof. In some cases, the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In some cases, the disease or disorder is an inflammatory disease or disorder. In some cases, the inflammatory disease or disorder is an inflammatory bowel disease, colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof. In some cases, the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. In some cases, the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. In some cases, the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and any combination thereof.
BRIEF DESCRIPTION OF THE FIGURES
[0014] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.
[0015] 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. (FIGs 1I-L) Acute DSS colitis in Ragl-/- gnotobiotic mice (n=4-9 mice/group), assessed by colon length (FIG. II), d2 fecal lipocalin (FIG. 1 J), lamina propria CD45+Ly6G+ neutrophils (FIG. IK), and cytokines from colon explant cultures (FIG. IL). (FIGs 1M-N) Development of spontaneous colitis in ll10-/- gnotobiotic mice (n=5 mice/group), exhibited by fecal lipocalin (FIG. IM) and colon histopathology scores (FIG. IN). Welch’s t-test was used to compare microbiota groups. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Error bars show mean ± SEM. (FIs 1C-H) show one of N=6 independent experiments, n=5-6 mice per group. (FIGs 1I-L) show one of N=4 independent experiments. (FIGs 1M-N) show one of N=3 independent experiments.
[0016] FIGs 2A-2I illustrate that Allobaculum sp. 128 elicits mucosal and systemic immunity at steady state. (FIGs 2A-B) GF WT mice colonized with MC bacteria or MC+Allobaculum sp. 128 and fecal bacteria were analyzed for IgA coating by flow cytometry at week 3 post-colonization (n=4 per group). (FIG. 2C) Fecal water was analyzed by ELISA for Allobaculum sp. 128-specific IgA. Data shown in (FIGs 2A-C) are representative of N=2 independent experiments. (FIGs 2D- F) GF WT mice colonized with MC bacteria or MC+Allo were bled at several time points and heat-inactivated sera were assayed against Allobaculum sp. 128 or MC bacteria by ELISA for determination of bacteria-specific serum IgA and IgG (n=3-6 mice per group). Welch’s t-test was used to compare microbiota groups at week 7. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. (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. N=2 independent microscopy experiments were conducted with n=2 mice/group. (FIG. 2H) WT mice were euthanized at week 4 and colon RNA was prepared for bulk RNAseq (n=2-3 RNA samples submitted per group). Volcano plot of a subset of significantly differentially expressed genes and (FIG. 21) Gene ontology pathway enrichment for MC+Allo vs MC mice. Data shown in (FIGs 2H-I) is from N=1 experiment.
[0017] 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. 3B) GF WT mice were gavaged with live Allobaculum sp. 128 culture, then with suspension of healthy human stool 24h later. One week after colonization, fecal pellets were collected for gDNA extraction and 16S rRNA V4 sequencing (n=19). Bacterial OTU legend shown in Figure S4B. (FIG. 3C) Each genus-level OTU was tested for Spearman correlation to Allobaculum sp. 128 abundance. Allobaculum sp128 abundance was binary-transformed and logistic regressions were also fit for each OTU pair, allowing for calculation of log-likelihood ratios and P-values. (FIG. 3D) XY plot of data shown in (FIG. 3B). (FIGs 3E-F) Meta-analysis of human microbiome datasets (see also Tables 2-4): American Gut Project (McDonald, et al. 2018; QIITA study IDs: 48742, 51570, 52698, 53379, & 54454) and pediatric ulcerative colitis (UC)(Schirmer, et al. 2018). Data shown in (FIGs 3A-D) are from N=1 experiment.
[0018] FIGs 4A-4R illustrates that A. muciniphila attenuates Allobaculum sp. 128-mediated intestinal epithelial cell activation and colitis. (FIG. 4A) Experimental schematic. GF WT mice were colonized with MC+Allobaculum sp. 128, MC+A muciniphila (in-house isolate 2G4), or MC+Both Allobaculum sp. 128 and A. muciniphila and allowed to equilibrate for 1 week before initiation of 2% DSS colitis (n=4-5 mice per group). (FIGs 4B-D) Inflammation as assessed by d2 fecal lipocalin (FIG. 4B) and colon length at euthanasia (FIGs 4C-D). (FIGs 4E-J) GF WT mice were colonized with 'MC+Allobaculum sp. 128, MC+A muciniphila, or MC+Both. Fecal microbiota composition was profiled over four weeks by absolute quantitative amplicon sequencing (FIG. 4E; n=3-4 mice/group) and the ileal mucosal microbiota was profiled after euthanasia at week 5 (FIGs 4H-J). (FIG. 4K) Experimental design for assessing intestinal epithelial cell activation by RNAseq. (FIG. 4L) Principal component analysis (PCA) of gene expression in ileal and colonic intestinal epithelial cell (IEC) after 2 weeks of colonization. (FIGs 4M-N) Log2 fold change of the top differentially expressed genes from IEC transcriptomes. (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. Welch’s t-test was used to compare across MC+Allo+A.m.supe vs MC+Allo; * P<0.05, ** P<0.01. Data shown in (FIGs 4A-D) are one representative of N=2 experiments. Data shown in (FIGs 4E-J), (FIGs 4L-N), and (FIGs 40-R) are each from separate N=1 experiments.
[0019] 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 5D-E) Colon length at d7 euthanasia. Data shown are from N=1 experiment.
[0020] 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. (FIG. 6B) Dilution curves show A muciniphila-veacdve and Allobaculum sp. 128-reactive serum IgA & IgGl from mice colonized with each microbiota (n=3-5). (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. Welch’s t-test was used to compare MC+A.m. to MC+Both at each dilution. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown in (FIG. 6B) are representative from one of N=4 independent experiments. Data shown in (FIG. 6D) are from N=1 experiment.
[0021] FIGs 7A-7J illustrates that Allobaculum sp. 128 and A. muciniphila induce context- dependent transcriptomic reprogramming in mucosal lymphoid tissues. Experimental design for single cell RNA sequencing shown in FIGs 14A-14G. (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. colonized mice and their corresponding silencing. (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). (FIG. 7J) Top 100 differentially expressed genes within MLN MigrDC transcriptomes. Welch’s t-test was used to compare gene expression across microbiome groups. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. Data shown from N=1 experiment with n=3 mice/group, pooled.
[0022] 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. 8A) Time course of fecal microbiota profiling of MC+Allo colonized mice across 7 days of 2% DSS administration (n=4 mice/group). (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. (FIGs 8D-E) Colon lamina propria CD4+IL-17A+ T cells from ll10-/- mice after 8 weeks colonization (n=5 mice/group). (FIGs 8F-G) GF mice were monocolonized with either Ery47 (control bacteria) or Allobaculum sp. 128 for 7 days before the induction of acute colitis via 2% DSS administration. Colonic inflammation was assessed by fecal lipocalin on d3 (FIG. 8F) and colon length on d7 (FIG. 8G) (n=5 mice/group). (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. (FIG. 8K) Fecal lipocalin, LCN2, was measured longitudinally by ELISA. Error bars show Mean ± SEM. Data shown in (FIGs 8A-B) from N=1 experiment. Data in (FIGs 8D-E) from one of N=2 experiments. Data in (FIGs 8F-G) from N=1 experiment. Data shown in (FIGs 8J-K) from one of N=2 independent experiments, n=5-6 mice per group. [0023] 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. One experiment shown is representative of N=2 independent experiments. (FIGs 9C-D) Total serum IgA and IgG content at 7 weeks (n=3-4 mice/group). (FIG. 9E) FITC-dextran concentration in serum Ih after oral gavage (n=4-8 mice/group). (FIG. 9F) Allobaculum sp. 128-specific IgG in fecal water at 7 weeks post- colonization with MC or MC+Allo (n=5 mice/group). (FIG. 9G) Thickness of the colonic inner mucus layer after 2 weeks of colonization (tissues fixed in Camoy’s solution to preserve the inner mucus layer; n=10-12 mice/group). Welch’s t-test was used to compare microbiota groups at each time point. ** P<0.01, * P<0.05. Data shown in (FIGs 9A-B) are from one representative of N=3 experiments. Data shown in (FIGs 9C-D) show one of N=2 experiments. Data in (FIG. 9E) are from N=1 experiments. Data in (FIG. 9F) show one of N=2 experiments. Data in (FIG. 9G) are compiled from N=2 experiments.
[0024] 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. 10 A) Genus-level richness, Simpson’s diversity index, Shannon’s diversity index, and evenness of each microbiome that contained Allobaculum (n=14) or lacked Allobaculum sp. 128 (n=5). (FIG. 10B) Legend accompanying FIG. 4B. Data shown from N=1 experiment. (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 (FIG. 10F) A. muciniphila ORFs that were differentially expressed in the ileum, along with their fold changes, P-values, and annotations (Prokka). Data are from N=1 experiment.
[0025] 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. muciniphilaT (type strain ATCC BAA- 835), or MC+ Allobaculum sp. 128+A. muciniphilcA (ATCC BAA-835) (n=4-6 mice/group). (FIG. 1 IB) Fecal microbiota profiling, (FIG. 11C) Colon length, (FIG. 1 ID) d2 fecal lipocalin (LCN2), and (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. 11G) Immunophenotyping of MLN cell populations (% of viable cells), n=4-6 mice/group. (FIG. 11H) Quantification of DCs (Live B220-TCRb'CDl lb+CDl lc+MHCII+). Welch’s t-test was used to compare microbiota groups. *P<0.05. Data shown in (FIGs 11 A-E) are from N=1 experiment. Data shown in (FIGs 11F-H) are from one of N=2 independent experiments. [0026] 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. Only cells with 500-5000 RNA features were retained (between blue dashed lines), as well as cells with <8% genes of mitochondrial (mt) origin. Welch’s t-test was used to compare each cell lineage across microbiota groups; * P<0.05, ** P<0.01, *** P<0.001. N=1 experiment.
[0027] 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.
[0028] 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. muciniphila after co-colonization with Allobaculum sp. 128. The marker genes that define each cluster are displayed in FIG. 13. (FIGs 14F-G) Ex vivo co-cultures of purified MLN DCs from gnotobiotic mice were examined for their capacity to prime CellTrace Violet (CTV)-labeled naive OT-II T cells, by proliferation and dilution of CTV. Data shown are from N=1 experiment.
[0029] FIG. 15 is a schematic outlining the process of reciprocal epistasis between Allobaculum sp. 128 and A. muciniphila. [0030] 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. GF WT mice were colonized with IgA-neg bacteria +/- Allobaculum and euthanized two weeks after colonization. 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.
FIG. 16C depicts representative data for distance from cypt base of three individual mice (n = 5- 9 crypts per mouse). Welch’s t-test was used to compare mean distance per crypt after removing blind. * P<0.05, ** P<0.01, **** P<0.0001. Error bars show mean ± SD. Data representative of N = 2 independent experiments.
[0031] FIGs 17A through FIGs 17C, depicts representative results demonstrating that Akkermansia-specfic Peyer’s Patch T cells are blunted by Allobaculum. FIG. 17A depicts representative fecal microbiota profiling of WT gnotobiotic mice at week 2 and week 3 post- colonization (n = 3 mice per * 2 time pts). 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 > FSC10 lymphocytes > CD4+TCRb+).
[0032] 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.
[0033] 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
Figure imgf000018_0001
Akkermansia vs Allobaculum.
DETAILED DESCRIPTION
Definitions
[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
[0035] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.
[0036] As used herein, 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. In addition, 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.
[0037] Unless the context requires otherwise, throughout the present specification and claims, the word “comprise” and variations thereof, such as, “comprises” and “comprising” are to be construed in an open, inclusive sense that is as “including, but not limited to”.
[0038] Reference throughout this specification to “one embodiment” or “an embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in one embodiment” or “in an embodiment” in various places throughout this specification may not necessarily all referring to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
[0039] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate to perform the disclosed methods.
[0040] The terms “patient,” “subject,” “individual,” and the like are used interchangeably herein, and refer to any animal, or cells thereof whether in vitro or in vivo, amenable to the methods described herein. In certain non-limiting embodiments, the patient, subject or individual is, by way of non-limiting examples, a human, a dog, a cat, a horse, or other domestic mammal.
[0041] 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.
[0042] In contrast, 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.
[0043] 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.
[0044] As used herein, “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.
[0045] “Immune response,” as the term is used herein, 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). Thus, an immune response, as would be understood by the skilled artisan, 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.
[0046] The term “dysbiosis,” as used herein, refers to imbalances in quality, absolute quantity, or relative quantity of members of the microbiota of a subject, which is sometimes, but not necessarily, associated with the development or progression of a disease or disorder. [0047] As used herein, the term “gastrointestinal tract” (“GI”) or “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.
[0048] The term “microbiota,” as used herein, 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.
[0049] As used herein, the term “microbiome” refers to the totality of microbes (bacteria, fungae, protists), their genetic elements (genomes) in a defined environment. In one embodiment, the microbiome is a gut microbiome (e.g., intestinal microbiome). The term “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).
[0050] The term “microbe” as used herein 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.
[0051] The term “gut microbe” as used herein can refer to any commensal or pathogenic microorganisms, bacteria, viruses, protozoa and fungi that colonize the gastrointestinal tract (GIT) or gut. The term “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.
[0052] Examples of 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, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia producta, Blautia Other, Blautia spp., Bulleidia spp., Catenibacterium spp., Citrobacter spp., Clostridiaceae spp., Clostridiales Other, Clostridiales spp., Clostridium perfringens, Clostridium spp., Clostridium Other, Collinsella aerofaciens, Collinsella spp., Collinsella stercoris, Coprococcus catus, Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp., Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other, Eggerthella lenta, Enterob acteriaceae Other, Enterob acteriaceae spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp., Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp., Leuconostocaceae spp., Megamonas spp., Megasphaera spp., Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp., Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp., Parabacteroides distasonis, Parabacteroides spp., Paraprevotella spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp., Pediococcus Other, Peptococcus spp., Peptoniphilus spp., Peptostreptococcus anaerobius, Peptostreptococcus Other, Phascolarctobacterium spp., Prevotella copri, Prevotella spp., Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other, Ruminococcaceae spp., Ruminococcus bromii,, Ruminococcus gnavus, Ruminococcus spp., Ruminococcus Other, Ruminococcus torques, Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus, Streptococcus luteciae, Streptococcus spp., Streptococcus Other, Sutterella spp., Turicibacter spp., UC Bulleidia, UC Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Varibaculum spp., Veillonella spp., Sutterella, Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella dispar and Weissella.
[0053] The terms “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. Thus, 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. In addition, microbial matter can encompass nonviable microbial matter that can cause an adverse effect (e.g., toxicity) to a host or an environment.
[0054] The terms “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.
[0055] 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.
[0056] “Isolated” means altered or removed from the natural state. For example, 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.
[0057] 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.
[0058] 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.
[0059] As used herein, 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. [0060] As used herein, 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.
[0061] The term “regulating” or “modulating” as used herein can mean any method of altering the level or activity of 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.
[0062] The term “regulator” or “modulator” 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).
[0063] 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. For example, in one embodiment, the activity is suppressed or blocked by at least about 10% relative to a control value. In some embodiments, the activity is suppressed or blocked by at least about 50% compared to a control value. In some embodiments, the activity is suppressed or blocked by at least about 75%. In some embodiments, the activity is suppressed or blocked by at least about 95%.
[0064] The term “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.
[0065] 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.
[0066] “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. Thus, 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. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and the non-coding strand, used as the template for transcription of a gene or cDNA, can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “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. The phrase 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).
[0067] As used herein, 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. In some embodiments, the host organism is a mammal. In some embodiments, 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. Some species, strains, and/or subtypes of non-pathogenic bacteria are currently recognized as probiotic bacteria. Examples of probiotic bacteria 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.
[0068] As used herein, 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. In some embodiments, 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. , arabinoxylan, xylan, xyloglucan, and glucomannan), 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) and xylooligosaccharides.
[0069] The term “antibody,” as used herein, 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;
Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).
[0070] By the term “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.
[0071] As used herein, 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.
[0072] The term “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. The skilled artisan will understand that any macromolecule, including virtually all proteins or peptides, can serve as an antigen. Furthermore, antigens can be derived from recombinant or genomic DNA or RNA. A skilled artisan will understand that 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. Furthermore, one skilled in the art will understand that 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.
[0073] The term “adjuvant” as used herein is defined as any molecule to enhance an antigen- specific adaptive immune response. [0074] As used herein, 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. [0075] By the term “specifically binds,” as used herein with respect to an antibody, is meant an antibody which recognizes and binds to a specific antigen, but does not substantially recognize or bind other molecules in a sample. For example, 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. In another example, 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.
[0076] In some instances, 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.
[0077] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art has the general knowledge that nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein 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.
[0078] 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. As used herein, 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. [0079] The phrase “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. 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.
[0080] 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. [0081] As used herein, the terms “nutritional supplement” and “dietary supplement” refer to any product that is added to the diet. In some particularly preferred embodiments, 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. [0082] As used herein “food product” or “foodstuff’ refers an edible product, e.g. a food or a beverage.
[0083] 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.
Overview
[0084] 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. 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. Furthermore, 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. In some cases, the pathogenic member of the gut microbiome can be a pathogenic gut microbe. In some cases, the commensal or non-pathogenic member of the gut microbiome can be a commensal gut microbe. In some cases, the pathogenic gut microbe is arx Allobaculum species. In one embodiment, the Allobaculum species in an Allobaculum sp. comprising a strain having a nucleotide sequence as set forth in SEQ ID NO.: 1 and/or a strain having a nucleotide sequence as set forth in SEQ ID NO.: 3). In some cases, the commensal or non-pathogenic gut microbe is an Akkermansia species. In some cases, the Akkermansia species is an Akkermansia sp. comprising a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. In one embodiment, the level (e.g., activity, expression, concentration, level, etc.) of arx Allobaculum sp. 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.
[0085] In certain aspects, 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. In one embodiment, 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. In one embodiment, 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.
[0086] In certain aspects, 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. In one embodiment, the present invention provides methods of identifying direct or indirect inter-species interactions. In various embodiments, 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. In some embodiments, 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. In some embodiments, 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.
[0087] Thus, in certain aspects, 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.
[0088] In other aspects, 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. In various embodiments, the method leverages “humanization” of gnotobiotic mice with human stool samples to represent the microbial ecology of the human microbiome in a mouse gut. In some embodiments, 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. In some embodiments, the method comprises examining the immune responses of mice colonized with defined communities including one or the other bacteria. In some embodiments, the method comprises examining publicly available human data from thousands of human microbiomes.
[0089] In some embodiments, the immunostimulatory bacteria from the genus Allobaculum induces the initiation or progression of inflammatory bowel disease (IBD). In some embodiments, the Allobaculum abundance is inversely correlated with another immunostimulatory microbe from the genus Akkermansia (e.g., Akkermansia muciniphila'). In some embodiments, the co- colonization with both taxa potently alters the immune responses elicited by each taxon on its own. In one embodiment, the Akkermansia ameliorates Allobaculum-induced pathogenic colonic inflammation, while Allobaculum severely blunts potentially-beneficial AkkermansiaAn&iceA immune responses. In one embodiment, 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). In one embodiment, 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). Thus, in certain aspects, 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. In other aspects, 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).
[0090] In certain aspects, 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. In other aspects, 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. In other aspects, 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. In various embodiments, 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.
[0091] In one aspect, the present invention relates to a method of identifying a combination of two gut microbe species or strains thereof that modulates an immune response. In one embodiment, 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. In some embodiments, 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.
[0092] In another aspect, 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. In one embodiment, 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. In another embodiment, 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. In one embodiment, 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.
[0093] In another aspect, 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. Alternatively, provided herein is 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 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. In various embodiments, 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. In one embodiment, 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.
[0094] In another aspect, 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. In various embodiments, 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. 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. In some embodiment, 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. Thus, in some embodiments, 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.
Methods of Identifying and Screening Beneficial and Pathogenic Microbes
[0095] In certain aspects, 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. In certain aspects, 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.
[0096] In one aspect, the present invention provides methods of identifying inter-species interactions. In one embodiment, 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. In one embodiment, the inter-species relationship between the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response. In one embodiment, 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. In one embodiment, 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. In some cases, 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.
[0097] In another aspect, 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. In one embodiment, the pair or the combination of the first microbe or strain thereof and the second microbe or strain thereof modulates an immune response. In one embodiment, 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. For example, in some embodiments, 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. In one embodiment, the first gut microbe species or strain thereof induces at least one disease or disorder. In one embodiment, 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. In some cases, 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. In some cases, 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.
[0098] In one embodiment, 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. For example, in certain embodiments, 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. In one embodiment, 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. In certain embodiments, 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. In one embodiment, 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.
[0099] In various embodiments, 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. In one embodiment, the subject is a healthy subject. In one embodiment, the subject is a non-human mammal. For example, in one embodiment, the subject is a healthy non- human mammal humanized with human microbiota. In one embodiment, the subject is a healthy non-human mammal humanized with human gut microbiota.
[00100] For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In one embodiment, the method comprises identifying a taxon of interest that exist in human-relevant pairwise relationships. In one embodiment, 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. In some embodiments, the method further comprises transferring the first microbe or strain thereof relative abundance into binary values (Absent=0, Present=l) so that logistic regressions can be fit for each OTU test pairing (performed using software package GraphPad Prism v9).
[00101] In other embodiments, 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. In one embodiment, 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. In some embodiments, the method further comprises transferring the first microbe or strain thereof relative abundance into binary values (Absent=0, Present=l) so that logistic regressions can be fit for each OTU test pairing (performed using software package GraphPad Prism v9).
[00102] In one embodiment, 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. In various embodiments of the methods of the invention, 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. [00103] In various embodiments of the methods of the invention, 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%, when compared with a comparator.
[00104] In various embodiments of the methods of the invention, 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 fold, at least 8.5 fold, at least 9 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 200 fold, at least 250 fold, at least 500 fold, or at least 1000 fold, or at least 10000 fold, when compared with a comparator.
[00105] The comparator, as used herein, may be a predetermined threshold. In certain embodiments, the comparator, as used herein, 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.). [00106] For example, in some embodiments, the comparator, as used herein, 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, expression, concentration, level, etc.) of microbe or strain thereof (e.g., an inflammatory disease or disorder). [00107] In other embodiments, the comparator, as used herein, 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).
[00108] In other embodiments, the comparator, as used herein, 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).
[00109] In other embodiments, the comparator, as used herein, 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).
[00110] In one embodiment, 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.
[00111] In one embodiment, 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. In one embodiment, 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.
[00112] In one embodiment, 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. In one embodiment, 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. In one embodiment, the beneficial microbe or strain thereof comprises a beneficial gut microbe species or strain thereof. For example, in one embodiment, 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. In various embodiments, the Akkermansia sp. or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
[00113] In one embodiment, 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. In one embodiment, 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.
[00114] There are many methods known in the art for the identifying and/or screening of the microbe or strain thereof. For example, in various embodiments, 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.
[00115] In certain aspects, 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.
[00116] For example, in some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In one embodiment, the method comprises identifying a taxon of interest that exist in human- relevant pairwise relationships. In one embodiment, 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. In some embodiments, the method further comprises transferring the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp. 128) relative abundance into binary values (Absent=0, Present=l) so that logistic regressions can be fit for each OTU test pairing (performed using software package GraphPad Prism v9).
[00117] In other embodiments, 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. In one embodiment, 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. In some embodiments, the method further comprises transferring the potentially pathogenic microbe or strain thereof (e.g., Allobaculum sp. 128) relative abundance into binary values (Absent=0, Present=l) so that logistic regressions can be fit for each OTU test pairing (performed using software package GraphPad Prism v9). [00118] In one aspect, 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. In some embodiments, 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. Thus, in some embodiments of the methods of the invention, specifically binding secretory antibodies (e.g., IgA, IgM) produced by the subject and secreted through the mucosa of the subject, serve as a marker and a means for isolating and identifying putative pathobionts, pathobionts, and other disease-causing bacteria, that are the targets of the subject’s immune response. In various embodiments of the methods of the invention, 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.
[00119] In various embodiments, 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. In one embodiment, 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. In various embodiments, 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. In some embodiments, 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. In other embodiments, 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. In other embodiments, 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. In various embodiments, 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).
[00120] In other various embodiments, 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. In one embodiment, 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. In various embodiments, 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.
[00121] In one embodiment, 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. In some embodiments, 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.
[00122] In some embodiments, 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., Bifidobacterium adolescentis, Bifidobacterium Other, Bifidobacterium spp., Bilophila spp., Blautia obeum, Blautia producta, Blautia Other, Blautia spp., Bulleidia spp., Catenibacterium spp., Citrobacter spp., Clostridiaceae spp., Clostridiales Other, Clostridiales spp., Clostridium perfringens, Clostridium spp., Clostridium Other, Collinsella aerofaciens, Collinsella spp., Collinsella stercoris, Coprococcus catus, Coprococcus spp., Coriobacteriaceae spp., Desulfovibrionaceae spp., Dialister spp., Dorea formicigenerans, Dorea spp., Dorea Other, Eggerthella lenta, Enterob acteriaceae Other, Enterob acteriaceae spp., Enterococcus spp., Erysipelotrichaceae spp., Eubacterium biforme, Eubacterium biforme, Eubacterium dolichum, Eubacterium spp., Faecalibacterium prausnitzii, Fusobacterium spp., Gemellaceae spp., Haemophilus parainfluenzae, Haemophilus Other, Helicobacter spp., Helicobacter Lachnospiraceae Other, Lachnospiraceae spp., Lactobacillus reuteri, Lactobacillus mucosae, Lactobacillus zeae, Lactobacillus spp., Lactobacillaceae spp., Lactococcus spp., Leuconostocaceae spp., Megamonas spp., Megasphaera spp., Methanobrevibacter spp., Mitsuokella multacida, Mitsuokella spp., Mucispirillum schaedleri, Odoribacter spp., Oscillospira spp., Parabacteroides distasonis, Parabacteroides spp., Paraprevotella spp., Paraprevotellaceae spp., Parvimonas spp., Pediococcus spp., Pediococcus Other, Peptococcus spp., Peptoniphilus spp., Peptostreptococcus anaerobius, Peptostreptococcus Other, Phascolarctobacterium spp., Prevotella copri, Prevotella spp., Prevotella stercorea, Prevotellaceae, Proteus spp., Rikenellaceae spp., Roseburia faecis, Roseburia spp., Ruminococcaceae Other, Ruminococcaceae spp., Ruminococcus bromii,, Ruminococcus gnavus, Ruminococcus spp., Ruminococcus Other, Ruminococcus torques, Slackia spp., S24-7 spp., SMB53 spp., Streptococcus anginosus, Streptococcus luteciae, Streptococcus spp., Streptococcus Other, Sutterella spp., Turicibacter spp., UC Bulleidia, UC Enterobacteriaceae, UC Faecalibacterium, UC Parabacteroides, UC Pediococcus, Varibaculum spp., Veillonella spp., Sutterella, Turicibacter, UC Clostridiales, UC Erysipelotrichaceae, UC Ruminococcaceae, Veillonella parvula, Veillonella spp., Veillonella dispar, Weissella, or any combination thereof. [00123] For example, in one embodiment, 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. [00124] In certain aspects, 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. In certain embodiments, 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. In certain embodiments, 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.
[00125] Specific alterations in a subject’s 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. In some embodiments, the microbial genetic marker is a bacterial genetic marker. In particular embodiments, the bacterial genetic marker is at least some portion of thel6S rRNA. In some embodiments, the relative proportion of particular constituent bacterial phyla, classes, orders, families, genera, and species present in the microbiota of a subject is determined. In other embodiments, 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. In various embodiments, 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.
Methods of Diagnosis
[00126] In various embodiments, 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.
[00127] In certain aspects, 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.
[00128] In one embodiment, 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. For example, in some embodiments, 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.
[00129] For example, in various embodiments, 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. In some embodiments, 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. In other embodiments, 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.
[00130] In various embodiments, 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.
[00131] In various embodiments of the method of the invention, 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.
[00132] In various embodiments of the method of the invention, 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 fold, at least 9.5 fold, at least 10 fold, at least 11 fold, at least 12 fold, at least 13 fold, at least 14 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 75 fold, at least 100 fold, at least 200 fold, at least 250 fold, at least 500 fold, or at least 1000 fold, in the biological sample when compared with a comparator control.
[00133] 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. In some embodiments, the amount of Allobaculum sp. or strain thereof and/or Akkermansia sp. or strain thereof can be assessed by detecting a bacterial genetic marker. In particular embodiments, the bacterial genetic marker is at least some portion of thel6S rRNA.
[00134] In one embodiment, 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. In some embodiments, 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.
[00135] In one embodiment, 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. In various embodiments, the test sample is a biological sample (e.g., fluid, tissue, cell, cellular component, etc.) of the subject. In some embodiments, 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.
[00136] The methods of detecting a microbe or strain thereof may be carried out using any assay or methodology known in the art. In various embodiments of the invention, 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, FACS, an enzyme-substrate binding assay, an enzymatic assay, an enzymatic assay employing a detectable molecule, such as a chromophore, fluorophore, or radioactive substrate, a substrate binding assay employing such a substrate, a substrate displacement assay employing such a substrate, and a protein chip assay (see also, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007).
[00137] The results of the diagnostic assay can be used alone, or in combination with other information from the subject, or from the biological sample derived from the subject. [00138] In the assay methods of the invention, a 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.
[00139] In various embodiments, 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.g., an inflammatory disease or disorder).
[00140] In some embodiments, the test biological sample is prepared from a biological sample obtained from the subject. In some instances, 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. For example, 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. Other techniques providing separation include fluorescence activated cell sorting, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. One example of an 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. The details of the preparation of such antibodies and their suitability for use as affinity reagents are well-known to those skilled in the art. In some embodiments, 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.
[00141] In various embodiments, 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) are achieved in this manner. The affinity enriched microbes will be about 70%, about 75%, about 80%, about 85% about 90%, about 95% or more of the composition. In other words, the enriched composition can be a substantially pure composition of the microbes of interest.
[00142] In one embodiment, the test biological sample is a sample containing at least a fragment of a microbial nucleic acid. The term, “fragment,” as used herein, indicates that the portion of a nucleic acid (e.g., DNA, RNA) that is sufficient to identify it as comprising a microbial nucleic acid. [00143] In some embodiments, 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. In some embodiments, a biological sample containing bacterial DNA is used. In other embodiments, 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. For example, in various embodiments, a nucleic acid is prepared from a biological sample, for use in the methods. Alternatively, or in addition, if desired, 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.
[00144] In some embodiments, 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). For example, 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.
[00145] 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.
[00146] In Northern analysis (see Current Protocols in Molecular Biology, Ausubel, F. et al., eds., John Wiley & Sons, supra), the hybridization methods described above are used to identify the presence of a sequence of interest in an RNA, such as unprocessed, partially processed or fully processed rRNA. For Northern analysis, 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.
[00147] Alternatively, 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.
[00148] 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.
[00149] In another embodiment, arrays of oligonucleotide probes that are complementary to target microbial nucleic acid sequences can be used to detect and identify microbial nucleic acids. For example, in one embodiment, 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. These 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. [00150] After an oligonucleotide array is prepared, 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. In brief, 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, is then hybridized with the array under appropriate conditions. Upon completion of hybridization and washing of the array, 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.
[00151] Other methods of 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. USA 86:232-236), mobility shift analysis (1989, Orita et al., Proc. Natl. Acad. Sci. USA 86:2766-2770; 1987, Rosenbaum and Reissner, Biophys. Chem. 265: 1275; 1991, Keen et al., Trends Genet. 7:5); restriction enzyme analysis (1978, Flavell et al., Cell 15:25; 1981, Geever, et al., Proc. Natl. Acad. Sci. USA 78:5081); heteroduplex analysis; chemical mismatch cleavage (CMC) (1985, Cotton et al., Proc. Natl. Acad. Sci. USA 85:4397-4401); RNase protection assays (1985, Myers, et al., Science 230: 1242); use of polypeptides which recognize nucleotide mismatches, such as E. coli mutS protein (see, for example, U.S. Pat. No. 5,459,039); Luminex xMAP™ technology; high-throughput sequencing (HTS) (2011, Gundry and Vijg, Mutat Res, doi: 10.1016/j.mrfmmm.2011.10.001); next-generation sequencing (NGS) (2009, Voelkerding et al., Clinical Chemistry 55:641-658; 2011, Su et al., Expert Rev Mol Diagn.
11 :333-343; 2011, Ji and Myllykangas, Biotechnol Genet Eng Rev 27: 135-158); ion semiconductor sequencing (2011, Rusk, Nature Methods doi: 10.1038/nmeth.f.330; 2011, Rothberg et al., Nature 475:348-352) and/or allele-specific PCR, for example. These and other methods can be used to identify the presence of one or more microbial nucleic acids of interest, in a biological sample derived from a subject. In various embodiments of the invention, the methods of assessing a biological sample for the presence or absence of a particular nucleic acid sequence, as described herein, 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.
[00152] 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. Among the radioactive isotopes used, mention may be made of 32P, 33P, 35 S or 3H. 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.
[00153] 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.
[00154] Routine methods also can be used to extract DNA from a biological sample, including, for example, phenol extraction. Alternatively, genomic DNA can be extracted with kits such as the QIAamp™. Tissue Kit (Qiagen, Chatsworth, Calif.), the Wizard™ Genomic DNA purification kit (Promega, Madison, Wis.), the Puregene DNA Isolation System (Gentra Systems, Inc., Minneapolis, Minn.), and the A.S.A.P.™ Genomic DNA isolation kit (Boehringer Mannheim, Indianapolis, Ind.).
[00155] There are many methods known in the art for the detection of specific nucleic acid sequences and new methods are continually reported. A great majority of the known specific nucleic acid detection methods utilize nucleic acid probes in specific hybridization reactions. Preferably, the detection of hybridization to the duplex form is a Southern blot technique. In the 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.
[00156] In the Southern blot, 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. To briefly summarize one of those methods, 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. In quantitative Southern blotting, levels of the target nucleic acid can be determined.
[00157] A further process for the detection of hybridized nucleic acid takes advantage of the polymerase chain reaction (PCR). The PCR process is well known in the art (U.S. Pat. No. 4,683,195, No. 4,683,202, and No. 4,800,159). To briefly summarize PCR, 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) extends the DNA duplex from the hybridized primer. 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. In this case, the PCR primer acts as a hybridization probe. [00158] In PCR, the nucleic acid probe can be labeled with a tag as discussed before. Most preferably the detection of the duplex is done using at least one primer directed to the target nucleic acid. In yet another embodiment of PCR, the detection of the hybridized duplex comprises electrophoretic gel separation followed by dye-based visualization.
[00159] 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.
[00160] 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.
[00161] 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.
[00162] 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.
[00163] Among the conditions of stringency is 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. Preferably, the Tm for the amplification step is in the range of about 59 °C to about 72 °C. Most preferably, the Tm for the amplification step is about 60 °C.
[00164] 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).
[00165] In one embodiment, 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. [00166] 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. These techniques are performed using various categories of 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.
[00167] 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.
[00168] Among the stringent conditions applied for any one of the hydrolysis-probes of the present invention is the Tm, which is in the range of about 65°C to 75°C. Preferably, 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. Most preferably, the Tm applied for any one of the hydrolysis-probes of the present invention is about 67 °C.
[00169] In one embodiment, 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.
[00170] 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.
[00171] In one aspect, 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. Preferably, 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. In another aspect, 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).
[00172] In various embodiments, 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. In some embodiments, 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. In some embodiments, 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. In one embodiment, the beneficial microbe or strain thereof ameliorates the pathogenic colonic inflammation and intestinal epithelial cell (IEC) activation of the pathogenic microbe or strain thereof. In another embodiment, the pathogenic microbe or strain thereof reduces the systemic antibody response against the beneficial microbe or strain thereof. In one embodiment, 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.
Compositions
[00173] In one aspect, the present invention comprises a composition comprising a beneficial microbe or strain thereof. In another aspect, 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. In various embodiments, 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. In some embodiments, the pathogenic microbe or strain thereof is any microbe or strain thereof described herein that induces a disease or disorder. For example, in one embodiment, the pathogenic microbe or strain thereof is a Allobaculum sp. or strain thereof. In some embodiments, 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. For example, in one embodiment, 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.
[00174] In various embodiments, 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. In one embodiment, the composition increases the level (e.g., activity, expression, concentration, level, etc.) of the beneficial microbe or strain thereof. In one embodiment, the composition decreases the level (e.g., activity, expression, concentration, level, etc.) of the pathogenic microbe or strain thereof. In some cases, 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.
[00175] In one embodiment, 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. In some cases, 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.
[00176] In some embodiments, the composition modulates an immune response toward the disease or disorder. In some embodiments, 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. In some cases, 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. In some cases, 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.
[00177] In various embodiments, the composition further comprises at least one probiotic, prebiotic, antibiotic, antimicrobe, or any combination thereof. For example, in some embodiments, 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. In some embodiments, the prebiotic, probiotic, antibiotic, antimicrobe, or any combination thereof reduces or inhibits the level of the pathogenic microbe or strain thereof.
[00178] In one embodiment, the composition comprises a probiotic. For example, in certain embodiments, the composition comprises a probiotic composition that comprises one or more bacterium. In certain embodiments the one or more bacterium are indigenous members of the human gut microbiome. In one embodiment, 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., Lactococcus sp., Lactobacillus acidophilus, Lactobacillus rhamnosus, Lactobacillus fermentum, Lactobacillus casei, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus helveticus, Lactobacillus johnsonii, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus salivarius, Lactobacillus paracasei, Parabacteroides distasonis, Dorea longicatena, Ruminococcus faecis, Blautia producta, Clostridium citroniae, Anaerostipes hadrus, Coprococcus comes, Roseburia faecis, Oscillospira plautii, and Clostridium spiroforme .
[00179] It should be appreciated that the compositions may include bacterium from multiple strains of a particular species.
[00180] In various embodiments, the composition is a probiotic composition for use as a food or drink additive. In some embodiments, the composition is a probiotic beverage or drink. In some embodiments, the composition is soluble or suspendable in a liquid medium.
[00181] In various embodiments, the composition comprises probiotic microorganisms in about 1 x 109 cfu/g, about 2x 109 cfu/g, about 3 x 109 cfu/g, about 4x 109 cfu/g, about 5x 109 cfu/g, about 6x 109 cfu/g, about 7x 109 cfu/g, about 8x 109 cfu/g, about 9x 109 cfu/g, about 1 x 1010 cfu/g, about 2x 1010 cfu/g, about 3x 1010 cfu/g, about 4x 1010 cfu/g, about 5x 1010 cfu/g, about 6x 1010 cfu/g, about 7x 1010 cfu/g, about 8x 1010 cfu/g, about 9 x 1010 cfu/g, or about 1 x 1011 cfu/g. In some embodiments, the probiotic composition comprises about 1 x 1010 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. In some embodiments, 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.
[00182] In some embodiments of the compositions provided herein, 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. In some embodiments, 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. [00183] In some embodiments, the compositions include bacterial species or strains that are susceptible to at least four antibiotics that are efficacious in humans. In some embodiments, the compositions include bacterial species or strains that are susceptible to at least three antibiotics that are efficacious in humans. In some embodiments, 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. As used herein, an “antibiotic that is efficacious in a human” refers to an antibiotic that has been used to successfully treat bacterial infections in a human.
[00184] In some embodiments, 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. In contrast, in the vegetative state or actively growing state, bacteria are more susceptible to such environmental conditions, compared to in the spore form. In general, 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.
[00185] In any of the compositions provided herein, in some embodiments, the bacterial species or strains are purified. In any of the compositions provided herein, in some embodiments, 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. In some embodiments, the compositions include strains originating from multiple individuals.
[00186] As used herein, 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. In some embodiments, the bacteria are substantially isolated from a source such that other components of the source are not detected.
[00187] As also used herein, 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. In some embodiments, the bacterial species or strain is substantially free of contaminants. In some embodiments, 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. In some embodiments, 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.
[00188] In some embodiments, 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. In some embodiments, 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. In some embodiments, the one or more of the bacterium of the compositions colonize a dysbiotic gastrointestinal tract.
[00189] The bacterial species or strains used in the compositions provided herein generally are isolated from the microbiome of healthy individuals. In some embodiments, the compositions include bacteria from species or strains originating from a single individual. In some embodiments, the compositions include bacteria from species or strains originating from multiple individuals. In some embodiments, 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. In some embodiments, 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).
[00190] Any of the compositions described herein, including the pharmaceutical compositions and food products comprising the compositions, 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. In some embodiments, the composition or the one or more bacterium of the composition are lyophilized. In some embodiments, a subset of the bacteria in a composition is lyophilized. Methods of lyophilizing compositions, specifically compositions comprising bacteria, are well known in the art. See, e.g., U.S. Pat. Nos. 3,261,761; 4,205,132; PCT Publications WO 2014/029578 and WO 2012/098358, herein incorporated by reference in their entirety. 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. In some embodiments, the bacteria is a lyophilized cake. In some embodiments, the compositions comprising the one or more bacterium are a lyophilized cake.
[00191] The bacterial species or strains of the composition can be manufactured using fermentation techniques well known in the art. In some embodiments, 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.
[00192] In some embodiments, the composition may further comprise one or more additional therapeutic compositions. For example, in some embodiments, 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, golimumab, etanercept, or combinations thereof. Pharmaceutical Compositions
[00193] In some embodiments, 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.
[00194] The term “pharmaceutical composition” as used herein 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.
[00195] 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. In some embodiments, 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. Examples of 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.
[00196] Pharmaceutical 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. In certain instances, pharmaceutical compositions are manufactured under GMP conditions. The pharmaceutical 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.
[00197] In some embodiments, 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). In some embodiments, 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). 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.
[00198] In some embodiments, 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). Thus, in some embodiments, 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. In certain embodiments, when a pH sensitive composition is used for formulating the pharmaceutical preparation, 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. It should further be appreciated that each part of the intestine (e.g., the duodenumjejunum, ileum, cecum, colon and rectum), has different biochemical and chemical environment. For instance, parts of the intestines have different pHs, allowing for targeted delivery by compositions that have a specific pH sensitivity. Thus, the 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. (See e.g., Villena et al., IntJPharm 2015, 487 (1-2): 314-9). 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.
[00199] Another embodiment of a pharmaceutical preparation useful for delivery of the compositions to the intestine (e.g., the colon) 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. In one embodiment of a pharmaceutical preparation for delayed release, 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.
[00200] Additional examples of pharmaceutical 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.
[00201] Another example of a system enabling the delivery to the intestine (e.g., the colon) 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. Such 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).
[00202] A further example of a system enabling the delivery of a composition to the intestine (e.g., the colon), 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. 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.
[00203] The 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. In addition, the compositions provided herein that are formulated for delivery to a specific area (e.g., the cecum or the colon), may be administered by 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).
[00204] The 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). In some embodiments, the dosage form of the composition is a tablet, pill, capsule, powder, granules, solution, or suppository. In some embodiments, 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. In some embodiments, 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).
[00205] 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.
[00206] 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. In general, 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. In some embodiments, the dosing regimen entails oral administration of a dose of any of the compositions described herein. In some embodiments, the dosing regimen entails oral administration of multiple doses of any of the compositions described herein. In some embodiments, 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.
[00207] The 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). In some embodiments, the pharmaceutical compositions disclosed herein contain about 10, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013 or more of each of the bacteria of the composition per dosage amount. In some embodiments, the pharmaceutical compositions disclosed herein contain about 10, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013 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 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013 or more CFUs of each of the bacteria in the composition per dosage amount. In some embodiments, the pharmaceutical compositions disclosed herein contain about 101, about 102, about 103, about 104, about 105, about 106, about 107, about 108, about 109, about 1010, about 1011, about 1012, about 1013 or more CFUs in total for all of the bacteria combined per dosage amount. As discussed above, bacteria of the compositions may be present in different amounts. In some embodiments, 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. In some embodiments, 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. In some embodiment, the dosage amount is one administration device (e.g., one table, pill or capsule). In some embodiment, the dosage amount is the amount that is administered in a particular period (e.g., one day or one week).
[00208] In some embodiments, the pharmaceutical compositions disclosed herein contain between 10 and 1013, between 102 and 1013, between 103 and 1013, between 104 and 1013, between 105 and 1013, between 106 and 1013, between 107 and 1013, between 108 and 1013, between 109 and 1013, between 1010and 1013, between 10uand 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012 between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012 between 109and 1012, between 1010and
1012, between 10nand 1012, between 10 and 1011, between 102and 1011, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1011, between 108and 1011, between 109and 1011, between 1010and 1011, between 10 and 1010, between 102and 1010, between 103and 1010, between 104and 1010, between 105and 1010, between 106and 1010, between 107and 1010, between 108and 1010, between 109and 1010, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between 105and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 10, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between 105and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and 105, between 102and 105, between 103and 105, between 104and 105, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102 of each of the bacteria of the composition per dosage amount. In some embodiments, the pharmaceutical compositions disclosed herein contain between 10 and 1013, between 102and
1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between 10uand 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012 between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012 between 109and 1012, between 1010and 1012, between 10xand 102, between 10 and 1011, between 102and 1011, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 101, between 108and 1012, between 109and 1011, between 1010and 1011, between 10 and 1010, between 102and 1010, between 103and 1010, between 104and 1010, between 105and 1010, between 106and 1010, between 107and 1010, between 108and 1010, between 109and 1010, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between 105and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between 105and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and 105, between 102and 105, between 103and 105, between 104and 105, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102 total bacteria per dosage amount.
[00209] In some embodiments, the pharmaceutical compositions disclosed herein contain between 10 and 1013, between 102and 1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between 10uand 1013, between 1012and 1013, between 10 and 1012, between 102and 1012, between 103and 1012, between 104and 1012 between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012 between 109and 1012, between 1010and
1012, between 10xand 102, between 10 and 1011, between 102and 1011, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1011, between 1010and 1011, between 10 and 1010, between 102and 1010, between 103and 1010, between 104and 1010, between 105and 1010, between 106and 1010, between 107and 1010, between 10 and 1010, between 109and 1010, between
10 and 109, between 102and 109, between 103and 109, between 104and 109, between 105and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between 105and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and 105, between 102and 10, between 103and 105, between 104and 105, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102 CFUs of each of the bacteria of the composition per dosage amount. In some embodiments, the pharmaceutical compositions disclosed herein contain between 10 and 1013, between 102and
1013, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1013, between 109and 1013, between 1010and 1013, between 10uand 1013, between 1012and 1013, between 10 and 1011, between 10 and 1012, between 103and 1012, between 104and 1012, between 105and 1012, between 106and 1012, between 107and 1012, between 108and 1012, between 109and 1012, between 1010and 1012, between l°and 102, between 10 and 1011, between 102and 1011, between 103and 1013, between 104and 1013, between 105and 1013, between 106and 1013, between 107and 1013, between 108and 1011, between 109and 1011, between 1010and 1011, between 10 and 1010, between 102and 1010, between 103and 1010, between 104and 1010, between 105and 1010, between 106and 1010, between 107and 1010, between 108and 1010, between 109and 1010, between 10 and 109, between 102and 109, between 103and 109, between 104and 109, between 105and 109, between 106and 109, between 107and 109, between 108and 109, between 10 and 108, between 102and 108, between 103and 108, between 104and 108, between 105and 108, between 106and 108, between 107and 108, between 10 and 107, between 102and 107, between 103and 107, between 104and 107, between 105and 107, between 106and 107, between 10 and 106, between 102and 106, between 103and 106, between 104and 106, between 105and 106, between 10 and 105, between 102and 105, between 103and 105, between 104and 105, between 10 and 104, between 102and 104, between 103and 104, between 10 and 103, between 102and 103, or between 10 and 102 total CFUs per dosage amount.
[00210] In some embodiments, the pharmaceutical compositions disclosed herein contain between 10-7and 10-1, between 10-6and 10-1, between 10-5and 10-1, between 10-4and 10-1, between 10-3and 10-1, between 10-2and 10-1, between 10-7and 10-2, between 10-6and 10-2, between 10-5and 10-2, between 10-4and 10-2, between 10-3and 10-2, between 10-7and 10-3 between 10-6and 10-3, between 10-5and 10-3, between 10-4and 10-3, between 10-7and 10-4 between 10-6and 10-4, between 10-5and 10-4, between 10-7and 10-5, between 10-6and 10-5, or between 10-7 and 10-6 grams of each of the bacteria in the composition per dosage amount. In some embodiments, the pharmaceutical compositions disclosed herein contain between 10-7and 10-1, between 10-6and 10-1, between 10-5and 10-1, between 10-4and 10-1, between 10-3and 10-1, between 10-2and 10-1, between 10-7and 10-2, between 10-6and 10-2, between 10-5and 10-2, between 10-4and 10-2, between 10-3and 10-2, between 10-7and 10-3, between 10-6and 10-3, between 10-5and 10-3, between 10-4and 10-3, between 10-7and 10-4, between 10-6and 10-4, between 10-5and 10-4, between 10-7and 10-5, between 10-6and 10-5, or between 10-7 and 10-6 grams of all of the bacteria combined per dosage amount.
[00211] Also with the scope of the present disclosure are 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. In some embodiments, 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. The 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. 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; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.
[00212] 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.
[00213] 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.
[00214] 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.
[00215] Alternatively, one may administer the compound in a local rather than systemic manner, for example, via injection of the compound directly into the area of pain, often in a depot or sustained release formulation. Furthermore, one may administer the drug in a targeted drug delivery system, for example, in a liposome coated with a tissue-specific antibody. The liposomes will be targeted to and taken up selectively by the organ.
[00216] The 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.
[00217] 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.
[00218] For injection, 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. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [00219] For oral administration, either solid or fluid unit dosage forms can be prepared. For preparing solid compositions, such as tablets, 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. For oral administration, 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. Pharmaceutical preparations 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). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate.
[00220] 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.
[00221] Dragee cores are provided with 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. [00222] 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. When the two-phase system has formed (with the starch solution as the inner phase) 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. 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.
[00223] Pharmaceutical preparations, 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. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers and/or antioxidants may be added. All formulations for oral administration should be in dosages suitable for such administration.
[00224] 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.
[00225] Slow or extended-release delivery systems, including any of a number of biopolymers (biological-based systems), systems employing liposomes, colloids, resins, and other polymeric delivery systems or compartmentalized reservoirs, can be utilized with the compositions described herein to provide a continuous or long-term source of therapeutic compound. Such slow-release systems are applicable to formulations for delivery via topical, intraocular, oral, and parenteral routes. [00226] 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. Optionally, 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.
[00227] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use.
Methods of Modulating Bacterium and/or Microbiota
[00228] The present invention further relates to a method of modulating a level of microbe or strain thereof. In one aspect, 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. In one embodiment, the method comprises decreasing the level of a pathogenic microbe or strain thereof in a subject in need thereof. In one aspect, 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. In one embodiment, 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. In one embodiment, the method comprises decreasing the systemic antibody response against the beneficial microbe or strain thereof in a subject in need thereof.
[00229] In various embodiments, 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.
[00230] 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 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. Methods of Preventing or Treating Diseases or Disorders
[00231] 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. In various embodiments, 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. For example, in some embodiments, the method prevents or treats a disease or disorder induced by Allobaculum sp. or strain thereof in a subject in need thereof.
[00232] In various embodiments, the method comprises administering to the subject any of the composition of the present invention. For example, in one embodiment, 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. [00233] In some embodiments, 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. In one embodiment, 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.
[00234] In some embodiments, 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.
[00235] In some embodiments, the method comprises increasing the level (e.g., activity, expression, level, etc.) of the beneficial microbe or strain thereof. In some embodiments, 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. Examples of such 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.
[00236] In some embodiments, the method comprises reducing the level (e.g., activity, expression, level, etc.) of the pathogenic microbe or strain thereof. In some embodiments, 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. Examples of such 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.
[00237] In some embodiments, 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.
[00238] In the context of the present invention, the term “vaccine” (also referred to as an immunogenic composition) refers to a substance that induces immunity upon inoculation into animals. In some instances, 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). [00239] In other embodiments, 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. In some embodiments, the immunoglobulin is administered orally. Alternatively, the immunoglobulin can be administered rectally or by enema.
[00240] In other embodiments, 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.
[00241] 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. In various embodiments, 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, cefixime, cefmenoxime, cefodizime, cefotaxime, cefpimizole, cefpodoxime, cefteram, ceftibuten, ceftiofur, ceftiolene, ceftizoxime, ceftriaxone, cefoperazone, ceftazidime, cefclidine, cefepime cefluprenam, cefoselis, cefozopran, cefpirome, cefquinome, cefaclomezine, cefaloram, cefaparole, cefcanel, cefedrolor, cefempidone, cefetrizole, cefivitril, cefmatilen, cefmepidium, cefovecin, cefoxazole, cefrotil, cefsumide, ceftaroline, ceftioxide, cefuracetime, imipenem, primaxin, doripenem, meropenem, ertapenem, flumequine, nalidixic acid, oxolinic acid, piromidic acid pipemidic acid, rosoxacin, ciprofloxacin, enoxacin, lomefloxacin, nadifloxacin, norfloxacin, ofloxacin, pefloxacin, rufloxacin, balofloxacin, gatifloxacin, grepafloxacin, levofloxacin, moxifloxacin, pazufloxacin, sparfloxacin, temafloxacin, tosufloxacin, clinafloxacin, gemifloxacin, sitafloxacin, trovafloxacin, prulifloxacin, azithromycin, erythromycin, clarithromycin, dirithromycin, roxithromycin, telithromycin, amoxicillin, ampicillin, bacampicillin, carbenicillin, cioxacillin, dicloxacillin, flucioxacillin, mezlocillin, nafcillin, oxacillin, penicillin g, penicillin v, piperacillin, pivampicillin, pivmecillinam, ticarcillin, sulfamethizole, sulfamethoxazole, sulfisoxazole, trimethoprim-sulfamethoxazole, demeclocycline, doxycycline, minocycline, oxytetracycline, tetracycline, linezolid, clindamycin, metronidazole, vancomycin, vancocin, mycobutin, rifampin, nitrofurantoin, chloramphenicol, or derivatives thereof.
[00242] In some embodiments, 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. In some embodiments, the probiotic is a surgical probiotic.
[00243] In one embodiment, 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.
[00244] In some embodiments, 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. For example, in one embodiment, the species or strain of bacteria is a secretory antibody -bound bacteria identified from a healthy subject. As described herein, administration of secretory antibody -bound bacteria from a healthy subject can treat or prevent an inflammatory disease or disorder.
[00245] 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.
[00246] In certain embodiments, the administered bacteria used in the methods of the invention further comprise a buffering agent. Examples of useful buffering agents include sodium bicarbonate, milk, yogurt, infant formula, and other dairy products.
[00247] 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. Preferably, 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.
[00248] The dosage of the administered bacteria (e.g., probiotic, surgical probiotic) 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. In some embodiments, the dose ranges from about 106 to about 1010 CFU per administration. In other embodiments, the dose ranges from about 104 to about 106 CFU per administration.
[00249] In certain embodiments, 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. In a preferred embodiment, the bacteria are administered orally. Alternatively, bacteria can be administered rectally or by enema. [00250] The organisms contemplated for administration to modify the altered microbiota include any of the bacteria identified herein as under-represented in an altered microbiota. In certain embodiments, the bacteria administered in the therapeutic methods of the invention comprise administration of a combination of organisms.
[00251] 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. 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.
[00252] Although there are no physical limitations to delivery of the formulations of the present invention, 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. For delivery to colon, bacteria can be also administered rectally or by enema.
[00253] In a further embodiment, 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.
[00254] One of skill in the art will appreciate that the 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. One of ordinary skill in the art will appreciate, based on the disclosure provided herein, that the 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. In various embodiments, 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. For example, in certain embodiments, the 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, or etanercept, and combinations thereof.
[00255] In one embodiment, the invention includes a method comprising administering a combination of compositions described herein. In some embodiments, 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. In other embodiments, 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.
[00256] The method comprises administering a combination of compositions in any suitable ratio. For example, in one embodiment, the method comprises administering two individual compositions at a 1 : 1 ratio. However, the method is not limited to any particular ratio. Rather any ratio that is shown to be effective is encompassed.
[00257] 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.
[00258] In some embodiments, 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. In some embodiments, 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. In some embodiments, 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. In some embodiments, changes in biomarker levels over time provide an indication of effectiveness of the therapy.
[00259] In another aspect, 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. In various embodiments, 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. In one embodiment, 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.
[00260] In another aspect, 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. In various embodiments, 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. 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. In some embodiment, 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. Thus, in some embodiments, 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.
EXAMPLES
[00261] The invention is further described in detail by reference to the following experimental examples. These examples are provided for purposes of illustration only and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather, should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.
[00262] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure. Example 1 : Inter-Species Gut Commensal Rivalry Dictated Mucosal and Systemic Immune Responses
[00263] Individual gut microbiota strains can influence diverse human phenotypes, but our understanding of the particular commensals that play causal roles in human health and disease remains limited. The antigen-specific mucosal and systemic antibody responses to the commensal microbiota have been leveraged to identify specific commensal taxa that induce adaptive immune responses and critically exacerbate or ameliorate susceptibility to mouse models of human diseases (Kau et al., 2015; Palm & de Zoete et al., 2014; Viladomiu et al. 2017). For example, ‘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). Nonetheless, potentially disease-driving bacteria are also found in apparently healthy individuals, and the effects of putative beneficial strains vary widely between subjects (Buffie et al., 2015; Ji et al., 2020; McDonald et al., 2018). Thus, the predictive power of strain carriage alone remains poor even for microbes with well-characterized disease-modulating activities, and this can severely hamper the accuracy of microbiome-based prognostics and constrain the efficacy of existing and emerging live bio-therapeutics.
[00264] One key contributor to the ‘incomplete penetrance’ of microbial impacts on disease was that the effects of individual strains on host immunity can differ dramatically depending on the surrounding microbial community context (Belkaid et al., 2017; Buffie et al., 2013; Externest et al., 2000; Gould et al. 2018). However, the specific contextual rules dictating these differential outcomes in humans remain almost entirely unclear.
[00265] Here, by studying a novel immunogenic and colitogenic taxon isolated from an inflammatory bowel disease patient, a reciprocal ‘epistatic’ interaction between two phylogenetically distinct immunostimulatory taxa that dictated divergent immunological and disease outcomes was uncovered. The study identified a unique IBD-associated strain (SEQ ID NO.: 1 : ATAACCTGCCCGTACCCGGGGGATACGCTTTGGAAACGAAGTCTAAAACCCCATAG GAAAGAAGAAGGCATCTTCTTCTTTTGAAACAAGCTTTTGCCTGGGGGACGGATGGA TCTGCGGTGCATTAGTTAGTTGGTGAGGCAAAAGCTCACCAAGACGATGATGCATA GCCGGCCTGAGAGGGCGAACGGCCACACTGGGACTGAGACACGGCCCAAACTTCTG CGGGAGGCAGCAGTAGGGAATTTTCGTCAATGGGCGCAAGCCTGAACGAGCAATGC CGCGTGAGTGAGGAAGGTCTTCGGATCGTAAAGCTCTGTTGCGGGGGAAAAAGGAA GCAGAAAGGAAATGGTCTGCTTTTGATGGTACCCCGCCAGAAAGTCACGGCTAACT ACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCGAGCGTTATCCGGAATGATTGGG CGTAAAGGGTGCGCAGGCGGCGCGTCAAGTCTGAAGTAAAAGGTACAGGCTCAACC TGTGCAGGCTTTGGAAACTGGCGCGCTCGAGGACAGGAGAGGGCGGTGGAACTCCA TGTGTAGCGGTAAAATGCGTAGATATATGGAAGAACACCAGTTGCGAAGGCGGCCG CCTGGACTGTTACTGACGCTGAGGCACGAAAGCGTGGGGAGCAAATAGGATTAGAT ACCCTAGTAGTCCACGCCCTAAACGATGAGGAGCAGGTGTCGGAGGGAGTACCCCG GTGCCGAAGCTAACGCAATGACTCCTCCGCCTGGGGAGTATGCACGCAAGTGTGAA ACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGTATGTGGTTTAATTCGA AGCAACGCGAAGAACCTTACCAGGCCTTGACATCGGATGCGAAGACTCAGAGATGA GTTGGAGGCTATCATCCAGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGGGAG ATGTTCAGTTAAGTCTGGCAACGA) from the genus Allobaculum that elicited mucosal and systemic antibody responses at steady-state and exacerbated colitis in simplified gnotobiotic mouse models, implying a potentially causal role for this taxon in IBD. However, this taxon was also present in healthy humans, suggesting the potential existence of additional taxa that modulate the host response to Allobaculum. Using human microbiota-associated mice and meta- analyses of human microbiome data, it was found that Allobaculum relative abundance was strongly inversely correlated or anti-correlated with the well-known immunostimulatory commensal Akkermansia muciniphila in both mice and humans. Finally, co-colonization with these two unique immunogenic strains (SEQ ID NO.: 1 and SEQ ID NO.: 2:
AACGAACGCTGGCGGCGTGGATAAGACATGCAAGTCGAACGAGAGAATTGCTAGCT TGCTAATAATTCTCTAGTGGCGCACGGGTGAGTAACACGTGAGTAACCTGCCCCCGA GAGCGGGATAGCCCTGGGAAACTGGGATTAATACCGCATAGAATCGCAAGATTAAA GCAGCAATGCGCTTGGGGATGGGCTCGCGGCCTATTAGTTAGTTGGTGAGGTAACG GCTCACCAAGGCGATGACGGGTAGCCGGTCTGAGAGGATGTCCCGCCACACTGGAA CTGAGACACGGTCCAGACACCTACGGGTGGCAGCAGTCGAGAATCATTCACAATGG GGGAAACCCTGATGGTGCGACGCCGCGTGGGGGAATGAAGGTCTTCGGATTGTAAA CCCCTGTCATGTGGGAGCAAATTAAAAAGATAGTACCACAAGAGGAAGAGACGGCT AACTCTGTGCCAGCAGCCGCGGTAATACAGAGGTCTCAAGCGTTGTTCGGAATCACT GGGCGTAAAGCGTGCGTAGGCTGTTTCGTAAGTCGTGTGTGAAAGGCGCGGGCTCA ACCCGCGGACGGCACATGATACTGCGAGACTAGAGTAATGGAGGGGGAACCGGAAT TCTCGGTGTAGCAGTGAAATGCGTAGATATCGAGAGGAACACTCGTGGCGAAGGCG GGTTCCTGGACATTAACTGACGCTGAGGCACGAAGGCCAGGGGAGCGAAAGAGATT AGATACCCCTGTAGTCCTGGCAGTAAACGGTGCACGCTTGGGGGGGGGGGAATCGA CCCCCCGGCGCCCCGGAGTAAACCGGTAAAGCGTCCCCGCCGGGGGGGAGTTCGGT CCGCAAAGATTAAAACTTAAAAAAGAAATGGACGGGGACCCCCCCCAAAGCGGTG GAAGTTATTGTGGTTTAATTTAGATGCAACGAGAAGAACCTTACCTGGGCTTGACAT GTAATGAACAACATGTGAAAGCATGCGACTCTTCGGAGGCGTTACACAGGTGCTGC ATGGCCGTCGTCAGCTCGTGTCGTGAGATGTTTGGTTAAGTCCAGCAACGAGCGCAA CCCCTGTTGCCAGTTACCAGCACGTGAAGGTGGGGACTCTGGCGAGACTGCCCAGA TCAACTGGGAGGAAGGTGGGGACGACGTCAGGTCAGTATGGCCCTTATGCCCAGGG CTGCACACGTACTACAATGCCCAGTACAGAGGGGGCCGAAGCCGCGAGGCGGAGG AAATCCTGAAAACTGGGCCCAGTTCGGACTGTAGGCTGCAACCCGCCTACACGAAG CCGGAATCGCTAGTAATGGCGCATCAGCTACGGCGCCGTGAATACGTTCCCGGGTCT TGC AC A) dramatically altered the immune responses elicited by each strain on its own -A. 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. Thus, 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. More broadly, they suggested that the combination of immunostimulatory strains present in an individual’s gut may predict immunological outcomes and established a generalizable framework for identifying specific inter-species interactions that dictated the context-dependent impacts of individual microbes on human health and disease.
Immunostimulatory Allobaculum Strains from IBP Patients Exacerbated Colitis in Simplified Gnotobiotic Mouse Models
[00266] Previously, studies identified and isolated a highly immunoglobulin A (IgA)- 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). Based on full-length 16S rRNA gene sequence similarly and whole-genome sequencing, 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;
Miyauchi et al., 2020, Nature, 585:102-106). Because IgA coating was previously found to mark potentially colitogenic strains in human IBD patients (Palm et al., 2014, Cell, 158: 1000-1010), this portion of this Example set out to establish a reductionist gnotobiotic mouse model to examine the individual impact of this unique immunogenic strain on immunity and colitis. [00267] As described in the materials and methods section for this Example, germ-free wild-type C57B1/6 mice were colonized with a non-colitogenic mock community (MC) of nine (9) human gut bacteria representing the three major phyla in the human gut microbiota (see Palm et al., 2014, Cell, 158: 1000-1010) with or without Allobaculum sp. 128 and induced colitis by administering dextran sodium sulfate (DSS).
[00268] It was found that WT mice colonized with MC plus Allobaculum sp. 128 exhibited severe, sometimes lethal, inflammation characterized by reduced colon length, elevated fecal lipocalin, and increased leukocyte infiltration, while mice colonized with MC alone showed only limited disease (FIG. 1C-1H). Notably, Allobaculum sp. 128 abundance remained stable during colitis, suggesting that Allobaculum sp. 128 does not “bloom” in response to inflammation (FIG. 8A-8B). Allobaculum sp. 128 colonization also exacerbated DSS colitis in gnotobiotic Ragl-/- mice, demonstrating that the colitogenic activities of Allobaculum do not strictly require T or B cells in the acute DSS model (FIG. II- IL and FIG. 8C). Nonetheless, Allobaculum sp. 128 colonization also drove spontaneous disease development and enhanced effector T cell responses in IL10-deficient mice (FIG. 8D-8E). Finally, monocolonization with Allobaculum sp. 128 was sufficient to exacerbate acute DSS colitis as compared to a non- immunogenic strain from the family Erysipelotrichaceae (FIGs 8F-8G).
[00269] To test whether other immunostimulatory strains from the genus Allobaculum also exhibit colitogenic activities, a related highly IgA-coated Allobaculum strain was identified in the microbiota of a UC patient from a separate IBD cohort and isolated via high-throughput anaerobic culturomics and massively-parallel 16S rRNA gene sequencing. The full-length 16S rRNA sequence of this novel Allobaculum isolate (Allo2; SEQ ID NO.: 3 GGCGCAAGCCTGAACGAGCAATGCCGCGTGAGCGAAGAAGGTCTTCGGATCGTAAA ACTCTGTTGCGGGGGAAAAAGGAAGGGAAGAGGAAATGCTTTTCTTTTGATGGTAC CCCGCCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGG CAAGCGTTATCCGGAATGATTGGGCGTAAAGGGTGCGCAGGCTGCGCGTCAAGTCT GAAGTGAAAGGTACGGGCTTAACCGGTACAGGCTTTGGAAACTGGCACGCTAGAGG ACAGGAGAGGGCGGTGGAACTCCATGTGTAGCGGTAAAATGCGTAGATATATGGAA GAACACCAGTTGCGAAGGCGACCGCCTGGACTGTTGCTGACGCTCAGGCACGAAAG CGTGGGGAGCAAATAGGATTAGATACCCTAGTAGTCCACGCCCTAAACGATGAGGA GCAGGTGTCGGAGGGAGGACCCCGGTGCCGAAGCTAACGCAGTGACTCCTCCGCCT GGGGAGTATGCACGCAAGTGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGC GGTGGAGTATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGCCTTGACAT AGGACGCGAAGACTTAGAGAAAAGTTGGAGGTTACCGTCCATACAGTGGTGCATGG TTGTCGTCAGCTC) was 95.2% similar to Allobaculum sp. 128, with a genome-level pairwise average nucleotide identity of 0.80, indicating that these were two different species within genus Allobaculum (see FIG. 8H). Nonetheless, similar to the initial Allobaculum isolate (i.e., Allot or Allobaculum sp. 128), this second isolate (i.e., Allo2) also exacerbated DSS colitis in simplified gnotobiotic mouse models (FIG. 8I-FIG. 8K).
[00270] Overall, two unique immunostimulatory Allobaculum strains were identified and isolated from UC patients that confer enhanced susceptibility to mouse models of IBD.
Allobaculum sp. 128 Elicited Mucosal and Systemic Antibody Responses at Steady State [00271] 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). However, because 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. An Allobaculum sp. 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). [00272] Select highly IgA-coated commensal strains, including gamma-Proteobacteria and Akkermansia miiciniphila. also sometimes elicited systemic IgA and IgG responses at steady state (Ansaldo et al., 2019; Castro-Dopico et al. 2019; Wilmore et al., 2018; Zeng et al., 2016). Similarly, Allobaculum sp. 128 colonization elicited robust antigen-specific serum IgA and IgG responses at steady state (FIG. 2D-2E). However, systemic antibody responses to other strains in the mock community remained unchanged in the presence or absence of Allobaculum sp. 128 (FIG. 2F), as did total serum Ig titers and gut barrier permeability (FIG. 9C-9E). Nonetheless, a slight increase in Allobaculum sp. 128-specific fecal IgG was observed and a modest decrease in colonic mucus thickness in Allobaculum sp. 128 colonized mice (FIG. 9F-9G). Finally, consistent with the ability of Allobaculum to induce antigen-specific antibody responses, the presence of bacteria closely opposed to the colonic epithelium in MC + Allobaculum sp. 128 colonized mice (FIG. 2G) was observed. Allobaculum sp. 128 was likely to selectively trigger mucosal and systemic immune responses through invasion of the mucus layer or translocation across the epithelial barrier. Using the HZ/ Allobaculum-specific nanobody described above, the localization of Allobaculum sp. 128 relative to the base of the intestinal crypts via confocal microscopy was examined. The present studies found that Allobaculum sp. 128 cells localized closer to the base of the crypts in the terminal ileum as compared to control MC bacteria (FIGs 16A-16B).
[00273] Finally, to examine the Allobaculum sp. 128-induced response in an unbiased manner, bulk RNA-seq were performed on colon tissue isolated from gnotobiotic mice colonized with MC or MC+Allobaculum sp. 128. Gene ontology analysis of differentially expressed genes revealed an enrichment in genes involved in cytokine production, adaptive immune activation, and leukocyte proliferation (FIG. 2H-2I) in mice colonized with Allobaculum sp. 128, underscoring the immunostimulatory effects of this strain at steady state. Together, these data demonstrate that Allobaculum sp. 128 evokes antigen-specific mucosal and systemic antibody responses, as well as low-level intestinal inflammation, but is insufficient to trigger a wholesale disruption of the epithelial barrier or cause overt intestinal pathology on its own in wild-type mice.
Allobaculum Relative Abundance was Inversely Correlated with phylogenetically- divergent Immunostimulatory Commensal Akkermansia muciniphila
[00274] The gnotobiotic mouse data as described previously herein suggested that immunostimulatory Allobaculum strains may play potentially causal roles in inflammatory bowel disease (IBD). However, related Allobaculum strains were detected in a meta-analysis of microbiome data from ostensibly healthy humans (American Gut Project; Table l).One potential explanation for this observation is that specific microbial taxa present in healthy humans may protect against the colitogenic effects of Allobaculum. To begin to examine this hypothesis, a human microbiota-associated gnotobiotic mouse-based screen to reveal potential relationships between Allobaculum and diverse bacterial taxa from the human gut microbiota was established. Briefly, individually housed germ-free mice were mono-colonized with Allobaculum sp. 128 for 24 hours before gavaging each mono-colonized mouse with one of 19 different healthy human stool samples. After seven days, microbial community composition was evaluated in all mice via 16S rRNA gene sequencing (FIG. 3A-3B). As expected, 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). Thus, it was hypothesized that specific microbial taxa may impact Allobaculum sp. 128 carriage or abundance. To identify such taxa, Spearman correlation coefficients were calculated for all genus-level OTUs paired with Allobaculum sp. 128 abundance and tabulated log likelihood ratios for each pairing. Remarkably, the well-known immunogenic mucinophile Akkermansia muciniphila (OTU 363731; SEQ ID NO.: 2) exhibited the lowest Spearman coefficient (R = - 0.52) and the most significant likelihood ratio (FIG. 3B-3D, S3B, and Table 2 and Table 3). This suggested that A. muciniphila is likely to compete with Allobaculum sp. 128 for a shared immunogenic niche. -To test whether this relationship between Allobaculum sp. 128 and A. muciniphila is generalizable to humans with naturally acquired microbiomes, the relative abundance of these two taxa in publicly available large-cohort studies of pediatric ulcerative colitis patients (n = 1,212) and healthy human volunteers (n = 19,524) was assessed (McDonald et al., 2018; Schirmer et al., 2018; Table 1). It was found that A muciniphila ox Allobaculum exhibited a broadly similar anti correlation to what was observed in the human microbiota- associated gnotobiotic mice (FIG. 3E-3F; Table 4). Overall, these data reveal an inverse relationship between two phylogenetically distinct immunostimulatory commensal taxa and raise the possibility that A muciniphila may influence Allobaculum-induced immune responses. In other words, in subjects where the level, carriage or abundance of Allobaculum (e.g., Allobaculum sp. 128 or Allo2) is high relative to a control subject, the level, carriage or abundance of A. muciniphila is low as relative to the control subject. The opposite appears to be also true.
[00275] Table 1 : Relative Abundance across Nineteen Community Contexts.
Figure imgf000096_0001
Figure imgf000097_0001
Figure imgf000098_0001
Figure imgf000099_0001
Figure imgf000100_0001
[00276] Table 2: Tabulated Spearman and Pearson Correlation Coefficients Calculated for all Genus-Level OTUs across all Microbiome Samples Paired with Allobaculum sp. 128.
Figure imgf000100_0002
Figure imgf000101_0001
[00277] Table 3: Logistic Regression Analysis in Prism.
Figure imgf000102_0001
Figure imgf000103_0001
[00278] Table 4: Correlation Matrix for all OTUs.
Figure imgf000103_0002
Figure imgf000104_0001
A. muciniphila protects against Allobaculum-mediated exacerbation of DSS colitis
[00279] To test the potential effects of A. muciniphila on Allobacuhmi-mduced immune responses, groups of WT gnotobiotic mice were colonized with either Allobaculum sp. 128, A. muciniphila (in-house human isolate 2G4), or both Allobaculum sp. 128 and A. muciniphila in the MC background and treated them with DSS (FIG. 4A). Importantly, A. muciniphila and Allobaculum sp. 128 durably co-colonized in the context of this mock community, allowed for the examination of the impacts of both taxa on immunity concurrently. As expected, Allobaculum-colonized mice exhibited severe colitis after DSS treatment, as measured by fecal lipocalin and gross colon pathology. However, both A muciniphila- and co-colonized mice displayed significantly lower levels of intestinal inflammation (FIG. 4B-4D).
[00280] To examine whether this protection was mediated by changes in Allobaculum sp. 128 colonization or localization, Allobaculum sp. 128 abundance in the feces and ileal mucosa was assessed via absolute quantitative amplicon sequencing and FISH. It was observed that co- colonization had no appreciable effect on the absolute abundance of Allobaculum sp. 128 in the feces or ileal mucosa and that both Allobaculum sp. 128 and A. muciniphila durably co-colonized ileal mucosa (FIG. 4E-4J and FIG. 10C). Co-colonization also had only modest impacts on Allobaculum sp. 128 and A. muciniphila gene expression as measured by bacterial transcriptomics (FIG. 10D-10F). Together, these data suggest that the impacts of co-colonization on colitis are not due to alterations in Allobaculum density or direct inter-bacterial interactions. [00281] The effects of Allobaculum sp. 128, A. muciniphila, or co-colonization on IEC activation was tested and observed that Allobaculum sp. 128 colonization elicited increased inflammatory gene expression in both ileal and colonic lECs, but this effect was blocked by co- colonization with A. muciniphila (FIG. 4K-4N). Daily gavage of germ-free mice with sterile Allobaculum sp. 128 culture supernatant for 10 days was insufficient to induce key inflammatory genes in lECs (FIG. 4O-4R). However, gavage with A. muciniphila supernatant profoundly suppressed the expression of Allobaculum-induced genes in MC+Allo colonized mice (FIG. 40- 4R).
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
[00282] It was then tested whether 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
[00283] In addition to testing the effects of co-colonization on local intestinal inflammation, immunophenotyping of the mesenteric lymph nodes (MLN) after DSS treatment was performed to assess potential inflammatory signatures outside the gut lamina propria (FIG. 11F-11G). However, unlike in the colon, where A. muciniphila ameliorated Allobaculum-induced responses, co-colonization blunted putative A. muciniphila-induced immune responses. For example, MLNs from mice colonized with MC + A. muciniphila contained elevated levels of dendritic cells (DC) compared to those colonized by MC + Allobaculum sp. 128, and this effect was abrogated in mice co-colonized with both Allobaculum sp. 128 and A muciniphila (FIG. 11H). These data imply that Allobaculum sp. 128 colonization may alter A. muciniphila-induced immune responses outside the colon. Based on this observation, it was hypothesized that co- colonization may affect the development of Allobaculum sp. 128-specific and A muciniphila- specific immune responses in a bi-directional manner.
[00284] Because both Allobaculum sp. 128 and A muciniphila induce potent systemic IgG responses, the effects of co-colonization on systemic antibody responses at steady state was examined next (FIG. 6A). As expected, colonization with A. muciniphila or Allobaculum sp. 128 in the MC background elicited potent bacterial-specific serum IgG and IgA responses (FIG. 6B). Despite the protective effects of A. muciniphila on Allobaculum-induced colitis, Allobaculum- specific antibody responses were unaltered after co-colonization. By contrast, Allobaculum sp. 128 co-colonization almost completely blocked the induction of A muciniphila-specific serum IgA and IgGl responses (FIG. 6B). Furthermore, colonization with Allobaculum sp. 128 significantly decreased systemic IgG responses to oral vaccination with cholera toxin (CT; FIG. 6C-6D). Overall, these data show that Allobaculum sp. 128 blunts systemic antibody responses to both endogenous A. muciniphila antigens and an exogenous vaccine antigen. Thus, co- colonization with Allobaculum sp. 128 and A. muciniphila reciprocally altered the immune responses elicited by each organism in isolation.
Allobaculum sp. 128 Reduced A muciniphila-specific Follicular T Helper Cell Responses [00285] Follicular T helper cells (Tfh) 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). Thus, 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
[00286] To further explore the individual and epistatic impacts of Allobaculum sp. 128 and A. muciniphila on host immune responses, single-cell RNA sequencing was performed (scRNA-seq) and simultaneous repertoire sequencing on mesenteric lymph node (MLN) and Peyer’s patch (PP) cells from gnotobiotic mice colonized for four weeks with either MC alone, MC with Allobaculum sp. 128 or A. muciniphila. or MC with both Allobaculum sp. 128 and 4. muciniphila. 4,391-10,306 cells per microbiota group were captured, with 77.3-93.6% cells passing quality filters set to retain only viable cells with high-quality transcriptomes (FIGs 12A- 12B). After data scaling, dimensionality reduction, and manual annotation of clusters based on conserved marker genes, significant microbiome-dependent alterations were observed in the relative abundance of diverse immune cell populations (FIGs 7A-7B and FIG. 13).
[00287] At baseline, Allobaculum sp. 128 colonization induced only subtle alterations in the immunological milieu in PP and MLN compared to mice colonized with MC alone, including slight increases in activated B and T cells, plasmacytoid dendritic cells, and lymphoid tissue inducer (LTi) cells in the MLN, and increased Tfh/Tfr, dendritic cell, and LTi in the PP (FIG. 7A-7B and FIG. 14A). However, A. muciniphila colonization induced an even more dramatic immunological restructuring, particularly in the MLN. This reprogramming was characterized by increases in activated CD4+ T cells and B cells, as well as increases in plasma cells, macrophages, and B cell zone reticular cells. Remarkably, most A muciniphila-induced changes in the MLN were severely blunted upon co-colonization with Allobaculum sp. 128, while Allobaculum-induced alterations were either unaltered or enhanced upon co-colonization (FIG. 7A and 14B-14E). A. muciniphila- and Allobaculum-induced alterations in PP cellularity were less dramatic overall and were characterized mainly by an increase in Tfh/Tfr cells, which was unaltered by co-colonization. Together, these data suggest that co-colonization with Allobaculum sp. 128 and A muciniphila reprograms the immune responses elicited by each organism on its own.
[00288] Finally, scRNA-seq and TCR repertoire sequencing data were leveraged to dissect the cellular mechanisms by which co-colonization with Allobaculum sp. 128 blunts the A. muciniphi la-induced systemic antibody response. As expected, it was observed that A muciniphila-induced alterations in B cell clusters in the MLN were similarly blunted by co- colonization (FIG. 14C-14E, clusters 6, 11, & 18). Since the systemic antibody response to A. 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. A. muciniphila colonization alone was associated with the expansion of global TCR repertoire clonality, emergence of specific clonotypes in both the MLN and PP, and the appearance of a unique population of Tfh cells in the MLN (FIG. 7C-7G). However, these responses were nearly completely blocked by co-colonization with Allobaculum (FIG. 7C- 7G). These data suggest that Allobaculum may prevent the initial priming of A. 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). Indeed, it was found that 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). To directly assess the impact of different microbial colonization conditions on DC function, 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.
Conclusions
[00289] Accumulating evidence suggests that individual immunostimulatory strains can play potentially causal roles in human health and disease, but the full spectrum of human gut microbes that shape human immunity remains to be defined. Furthermore, the presence or absence of such ‘causal’ strains alone remains a poor prognostic marker for phenotypic outcomes in humans, suggesting that additional factors may critically alter the responses elicited by specific microbial strains in different individuals. Here, a novel immunostimulatory strain from the genus Allobaculum isolated from an ulcerative colitis patient that can elicit both local and systemic antibody responses and exacerbate intestinal inflammation in simplified gnotobiotic mouse models of IBD was described. Using human microbiota-associated mice, an inverse correlation was uncovered between Allobaculum and the taxonomically divergent immunostimulatory strain A. muciniphila. Co-colonization with both immunostimulatory strains protected against Allobaculum-induced IEC activation and colitis while simultaneously blunting A. muciniphi Za-induced adaptive immune responses. Furthermore, using scRNA-seq, it was found that co-colonization reshaped the unique immunological landscapes in the PP and MLN induced by colonization with either Allobaculum or A. muciniphila in isolation. These findings provide insights into the microbiota-dependent contextual cues that dictate divergent immune responses to colonization with individual commensal strains and support a model whereby the impacts of specific immunostimulatory strains are determined by the presence or absence of other immunomodulatory taxa.
[00290] Two independent Allobaculum strains (i.e., Allot (Allobaculum sp. 128) and Allo2) from IBD patients that exacerbated colitis in gnotobiotic mouse models were identified, implying that immunogenic Allobaculum species may play causal roles in disease in a subset of IBD patients. Notably, recent studies in mice and humans have identified phylogenetically related taxa as potential drivers of autoimmunity in mice and Crohn’s disease in humans. A mouse-associated strain of Allobaculum drove intestinal T cell responses and facilitated the induction of autoimmunity (Miyauchi et al., 2020), while Erysipleotrichaceae strains were enriched in creeping fat in Crohn’s disease patients (Ha et al., 2020). Although the specific mechanisms by which Allobaculum and its relatives induce intestinal inflammation remain unclear, an ability to invade the mucus layer and trigger inflammatory responses in lECs, including the production of serum amyloid A, may contribute to the pathogenic impacts of this genus (Atarashi et al., 2015; Lee et al., 2020; Miyauchi et al., 2020; van Muijlwijk et al., 2021). Notably, 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). Overall, Allobaculum and related taxa may be important drivers of pathological intestinal inflammation in humans and potential therapeutic targets for the treatment of inflammatory disease.
[00291] It has been previously demonstrated that 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). For example, highly IgA-coated bacteria from healthy humans can protect against the pathogenic effects of IgA-coated taxa from undernourished children (Kau et al., 2015). Furthermore, 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. Prior studies of IgA coating of the human gut microbiota suggested that each individual harbors only a handful of potent immunostimulatory strains (less than ten), which may compete for a limited number of unique immunogenic niches. These niches likely share specific features that facilitate active sampling by the immune system and thus initiation of innate and adaptive immune responses.
[00292] A growing body of work demonstrates that the specific immune responses induced by individual immunostimulatory commensal species may often be highly dependent on the inflammatory and microbial context. For example, murine Helicobacter species that drive colitis in mouse models of IBD primarily trigger Treg responses in healthy animals, but the same microbial antigens elicit pathogenic effector T cells upon induction of colitis (Chai et al., 2017; Xu et al., 2018). Furthermore, A. 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.
[00293] Conversely, while A. muciniphila co-colonization had no appreciable effect on Allobaculum-induced systemic IgG responses, both co-colonization and feeding with A. muciniphila supernatants ameliorated Allobaculum-induced IEC activation and colitis. Additional studies will be necessary to determine if these activities are conserved across diverse A. muciniphila strains and additional microbial contexts, as well as whether they are mediated by previously described A. muciniphila-derived immunomodulatory products (Plovier et al., 2017; Becken et al., 2021; Liu et al., 2021). Nonetheless, these observations may at least partially explain the relatively common detection of putative disease-driving taxa such as Allobaculum sp. in ostensibly healthy human subjects. In this model, the pathogenic effects of disease-driving taxa may be blunted by ‘epistatic’ interactions with phylogenetically divergent immunostimulatory taxa such as A. muciniphila. Notably, A. muciniphila is significantly more prevalent and abundant in healthy humans as compared to IBD patients, and carriage of Allobaculum alone is rare among healthy humans. Taken together, 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. Thus, the composite effects of the specific immunostimulatory strains present in each person (the immunostimulatory gut microbiota composition code) may determine individual immunological outcomes and susceptibility to immune-related diseases.
[00294] Using scRNA-seq, it was found that both Allobaculum sp. 128 and A. muciniphila dramatically reshaped the immunological milieu in the PP and MLN at homeostasis and that co- colonization reprogrammed the immune responses elicited by each microbe on its own (see FIG. 15). These data thus demonstrate that immunostimulatory commensals critically shape the immunological environment in the gut, which may also impact the initiation or polarization of immune responses to intestinal antigens beyond the commensal microbiota, including food- and self-antigens, as well as mucosal vaccines. Indeed, it was found that Allobaculum colonization also blunted systemic IgG responses to oral CT immunization. Although the precise molecular mechanisms by which A. muciniphila and Allobaculum sp. 128 reshape steady-state and pathophysiological immune responses remain unclear, the data presented herein suggests that impacts on critical cell types such as lECs and dendritic cells may explain at least some of the epistatic interactions between these gut commensals.
[00295] These proof-of-concept studies presented herein represent a critical first step towards establishing a microbiota composition code that more accurately predicts the impacts of immunogenic species/strains on human physiological trajectories. Furthermore, they provide a generalizable experimental framework to define the specific microbes and microbial interactions that dictate these divergent outcomes. The ability to predict the epistatic impacts of immunostimulatory taxa has potentially profound therapeutic implications, even beyond improving microbiome-based diagnostics and prognostics. For example, the approaches described herein can potentially be used to identify specific microbial taxa that neutralize the pathogenic activities of detrimental immunostimulatory species/strains. These “precision probiotics” may be particularly useful for treating or preventing disease in individuals harboring “matched” disease-driving taxa. Conversely, carriage of specific taxa that blunt the beneficial effects of particular strains may predict non-responsiveness to live bio-therapeutics (e.g., proposed microbial adjuncts for cancer immunotherapy, including^, muciniphila), which may be useful as a gating strategy for patient selection or to identify patients that would benefit from a specific pre-treatment regimen (e.g., antibiotic pre-treatment prior to fecal microbiota transplantation). Overall, these studies begin to uncover the key contextual features that contribute to the incomplete penetrance of strain-specific microbial impacts on human disease and may eventually enable the development of improved microbiota-targeted interventions tailored to the specific microbial context of each individual.
[00296] In summary, the impacts of individual commensal microbes on immunity and disease can differ dramatically depending on the surrounding microbial context, yet the specific inter-species interactions that dictate these divergent outcomes remain largely undefined. The above-described studies isolated a novel immunostimulatory Allobaculum strain from an IBD patient that exacerbated colitis in gnotobiotic mice and likely played causal roles in IBD. Using human microbiota-associated gnotobiotic mouse models, the studies uncovered a remarkable inverse correlation between Allobaculum and the well-known immunostimulatory species Akkermansia muciniphila, which was confirmed in two large-scale human cohorts. Co- colonization with Allobaculum and A. 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. These studies began to explain the incomplete penetrance of microbial impacts on human health and disease and are facilitating the development of improved microbiome-based diagnostics and therapeutics.
[00297] Overall, indigenous gut microbial strains that shape intestinal and systemic immune responses can have dramatic impacts on diverse host phenotypes, yet the understanding of the specific human commensal species that play causal roles in health and disease remains limited. Studies have leveraged antigen-specific antibody responses to the commensal microbiota to identify individual commensal species that shape local and systemic immune responses and exacerbate or ameliorate mouse models of human disease. These studies have revealed that a small subset of bacterial strains from the human gut microbiota induce antigen-specific adaptive immune responses and may critically shape disease susceptibility. For example, ‘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.
[00298] Using in vivo microbial ecology experiments in gnotobiotic mice, as well as data mining of publicly available human microbiome data, an approach to identify key microbial taxa that provided context-dependent cues that modified the immune responses elicited by individual immunostimulatory bacteria were developed. In essence, it was found that specific taxa whose relative abundance inversely correlated with a specific disease-driving bacterial strain critically modulated immunological outcomes and disease in animal models. When combined with the previously patented technology (IgA-SEQ), this enabled the identification of both potential disease-driving taxa, as well as potential ‘precision probiotics’ that are likely to protect against the pathogenic effects of these specific taxa. Furthermore, it enabled the development of improved 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.
[00299] Thus, 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.
[00300] In the embodiment described above, it was shown that immunostimulatory bacteria from the genus Allobaculum played potentially causal roles in the initiation or progression of inflammatory bowel disease. Using the herein-described methodologies, it was found that Allobaculum abundance was inversely correlated with another immunostimulatory microbe that is best known for its beneficial effects (Akkermansia muciniphila). Furthermore, co- colonization with both taxa potently altered the immune responses elicited by each taxon on its own. Akkermansia ameliorated Allobaculum-induced pathogenic colonic inflammation, while Allobaculum severely blunted potentially-beneficial Akkermansia-induced immune responses. Thus, 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. On the other hand, 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).
[00301] 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 discovery that the pathogenic impacts of IBD-driving Allobaculum sp. were ameliorated by A. muciniphila and that some of the potentially beneficial immunomodulatory effects of A. muciniphila were blunted by Allobaculum was also novel and useful.
[00302] 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. Moreover, 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.
Materials and Methods
Bacterial Strains
[00303] Frozen stocks of each strain were streaked on Gut Microbiota Media agar (Goodman et al., 2011, Proceedings of the National Academy of Sciences of the United States of America, 108:6252-6257) or Gifu Anaerobic Media agar (HyServe #05422) and incubated 48 hours at 37 °C. Unless otherwise noted, all bacteria were grown in anaerobic conditions (gas composition: 4% Hz, 10% CO2, 86% N2). Single colonies were picked into sterile broth and grown overnight at 37 °C without shaking. 10 pL aliquots of overnight broths were removed for alkaline lysis with boiling to extract genomic DNA, then identities of these monocultures were confirmed by PCR amplification of V4 region of the 16S rRNA gene and Sanger sequencing (V4_F: GTGCCAGCMGCCGCGGTAA - SEQ ID NO.: 4, V4_R: GGACTACHVGGGTWTCTAAT - SEQ ID NO.: 5) (or full length 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.
Human Fecal Sample
[00304] Human study protocols were approved by the Institutional Review Board (HIC # 1607018104) of Yale School of Medicine. Informed consent was obtained from all participants and/or their legal guardians and all methods were performed according to relevant guidelines and regulations. Healthy subjects were recruited via advertisements on the Yale medical campus and in the New Haven Public Library. All fecal samples in this study were collected at home and stored on ice packs at -20 °C before either overnight shipment or direct laboratory drop-off the day following collection in an insulated container. Samples were then stored at -80 °C until use.
Animal Experiments
[00305] Germ-free 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). The day of inoculation, Wheaton vials were thawed to 25 °C and 0.1 mL gavaged per mouse. All animal protocols were approved by Yale University Institutional Animal Care and Use Committee (IACUC Protocol 2018-11513). Dextran Sodium Sulfate (DSS; MP Biomedical) was dissolved in sterile H2O to 2% w/v and passed through a 0.2 pm vacuum filter before ad libitum administration. To assess gut permeability, mice were fasted for 4h before gavage with 600 mg/kg FITC-Dextran (Sigma Aldrich #46944). For oral vaccinations, 10pg of cholera toxin (List Biological Laboratories #100B) was administered by gavage weekly. Serum was collected under isoflurane anesthesia, by retro-orbital puncture.
Fecal Sample Processing
[00306] 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). Bacterial pellet was resuspended in Qiagen PowerBead buffer, sonicated for 5 min in sonicating water bath, lysis buffer was added, then complete lysis achieved by 0.1 mm bead beating followed by genomic DNA isolation (Qiagen DNeasy Ultraclean Microbial; cat #12224). For absolute quantification of microbial strains in vivo, ZymoBIOMICS Spike-in Control I reagent was spiked into all sample wells before DNA extraction, according to manufacturer’s instructions (Zymo Research #D6320).
Microbiota Profiling
[00307] 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).
Whole Genome Sequencing
[00308] Overnight bacterial cultures were harvested by centrifugation, cells were lysed for high molecular weight gDNA extraction (Quick-DNA HMW MagBead Kit; Zymo Research #D6060). Genomic DNA was used to prepare two different types of sequencing libraries. Illumina’s Nextera XT kit (#FC-131-1024) was used to prepare short-read libraries, which were sequenced on Illumina Miseq (2x250), while Oxford Nanopore Technologies Ligation Sequencing kit (#SQK-LSK109) was used to prepare long-read libraries, which were sequenced using ONT MinlON (Flow Cell R9.4.1; #FLO-MIN106D).
Histology
[00309] Whole mouse colons were placed in plastic histology cassettes and immersed in Bouin’s fixative fluid for 24h before transfer to 70% ethanol, paraffin embedding, sectioning, and H & E staining. Blinded slides were scored by a board-certified pathologist. For assessment of colonic mucus thickness, colon tissues were fixed in Carnoy’s solution for four hours, embedded in paraffin, sectioned, and stained with periodic acid Schiff (PAS). The thickness of the inner mucus layer) was quantified using Imaged (Johansson et al. 2008; Kamphuis et al., 2017).
RNA-Seq
[00310] 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).
Fluorescence in situ hybridization
[00311] 1 cm segments of mouse tissue were excised and fixed in Carnoy’s solution (1 acetic Acid : 3 Acetic Acid : 6 Ethanol) for no more than 2 hours. Fixed tissues were embedded in paraffin for 5 pm cryosectioning. Slides were deparaffinized in xylenes, rinsed in ethanol, and dried thoroughly before hybridization. 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.
Bacterial Flow Cytometry and Serum-Binding Assays
[00312] 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 104 - 105 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. Staining for endogenous coating by mouse IgA was performed at 1 : 100 with PE-conjugated eBioscience clone mA-6El (Thermo Fisher #12420482). Detection of Allobaculum using 1 pg/mL species-specific nanobody (developed in-house) was performed similarly, for 45 min on ice, then washed x 3 in PBS/0.1% BSA before incubation with an AlexaFluor647-conjugated anti-HuFc secondary (clone HP6017; Biolegend). Negative control nanobody stains were performed with a HA-tag-specific nanobody. For serum-binding assays, freshly cultured bacterial strains (CFSE-labeled Allobaculum, unlabeled Akkermansia) were fixed 5 min room temperature (RT) in 4% PF A, then washed three times in PBS, mixed 1 : 1 and diluted for aliquoting into deep-well plates, as above. Bacteria were spun 10 min 5000 xg, supernatant flicked off and gently blotted on a clean paper towel, then cells resuspended in 50 pl PBS/0.1% BSA. 50 pl of heat-inactivated mouse serum samples (30 min at 55 °C) over a range of dilutions was added to each well of bacterial suspension and incubated 1 hr at RT. 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
[00313] Overnight broth cultures of bacterial strains of interest were washed three times in sterile PBS, then normalized to an OD600 of 0.1. Many 100 pl aliquots were prepared and snap frozen in liquid nitrogen. To prepare ELISA plates, bacterial aliquots were thawed on ice, diluted further 1 : 10 in PBS, then coated 50pl/well of Nunc Maxisorp Immunoplates overnight at 4 °C. The next day plates were spun 15 min at 5000 xg before discarding supernatant and confirming bacterial adhesion by light microscopy. Plates were blocked with 1% BSA in PBS before serially diluting serum or fecal water. After 2 h incubation at RT, plates were washed four times with TBS-T, then mouse IgG was detected using HRP-conj. Goat anti-Ms-IgG (Thermo Fisher Scientific #31430; 1 :6,000 dilution), or mouse IgA using HRP-conj . Goat anti-Ms-IgA (Sigma Aldrich A4789; 1 :6,000 dilution). Plates were washed four times before detection with TMB (Pierce), stopped with 2N H2SO4, and read at Abs 450 nm (Molecular Devices SpectraMax i3x).
Intestinal Epithelial and Lamina Propria Cell Isolation
[00314] 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 propria 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.
MLN & PP cell isolation
[00315] 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.
Single-cell RNA sequencing
[00316] Single cell suspensions were counted by hemacytometer and normalized to le6/mL for submission to Yale Center for Genome Analysis staff for droplet generation and gel bead encapsulation using 10X Genomics Controller. Cell lysis, barcoding and reverse transcription were performed using Chromium 5’ V2 chemistry according to manufacturer’s instructions. PCR-amplified gene expression libraries were quantified and evaluated for QC by Agilent Bioanalyzer and sequenced on Illumina NovaSeq 6000 at a depth of 175M read pairs per library.
Dendritic cell T cell co-culture
[00317] 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.
Bioinformatic analyses
[00318] Phylogenetic analysis of bacterial taxa belonging to family laysipelolrichaceae: 16S rRNA gene sequences from NCBI Genbank were aligned using Clustal Omega and alignments imported into MEGA v10.2.6. Phylogenetic trees were constructed using both neighbor-joining method and maximum likelihood estimation method, in each case bootstrapping for 1,000 replicates, both of which resulted in the same overall phylogeny. Trees were visualized using interactive Tree of Life (Letunic and Bork 2021). Whole genome assemblies: long-read fastq files were used to finish assembling remaining contigs using Unicycler v0.4.9b (Wick et al., 2017). 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). Single cell sequencing data were demultiplexed then processed using 10X Genomics cellranger count. Count matrices were imported into Seurat (v3.2.1) within R (v4.0.3) (Butler et al., 2018), paired with microbiome metadata, filtered for nFeature_RNAs<500 & <6000 and percent mitochondrial genes <8%. Clusters were generated by UMAP with resolution = 0.8, manually annotated based on expression of conserved marker genes, then analyzed for differential expression across microbiome groups using FindMarkers. MLN cell clusters were analyzed for pairwise enrichment in microbiome conditions via MELD, using default parameters (Burkhardt et al., 2021). TCR repertoires were analyzed using Immunarch (Immunomind Team 2019).
Quanti fication And Statiscal Analysis
[00319] Statistical analysis was conducted in GraphPad Prism v9. Unless otherwise noted, data are plotted as mean + SEM. Each figure legend describes the sample sizes of the data shown in that figure, as well as the specific statistical test applied.
MLN & PP Cell Isolation
[00320] 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.
Single-Cell RNA Sequencing
[00321] Single cell suspensions were counted by hemacytometer and normalized to le6/ mL for submission to Yale Center for Genome Analysis staff for droplet generation and gel bead encapsulation using 10X Genomics Controller. Cell lysis, barcoding, and reverse transcription was performed using Chromium 5prime V2 chemistry according to manufacturer’s instructions. PCR-amplified gene expression libraries were quantified and evaluated for QC by Agilent Bioanalyzer and sequenced on Illumina NovaSeq 6000 at a depth of 175M read pairs per library.
Bioinformatic Analyses
[00322] 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). Gene lists were analyzed for GO enrichment using Panther vl4 available at geneontology.org (Mi et al., 2019, Nucleic Acids Research, 47:D419-D426). Single cell sequencing data was demultiplexed then processed using 10X Genomics cellranger count. Count matrices were imported into Seurat (v3.2.1) within R (v4.0.3) (Butler, et al. 2018), paired with microbiome metadata, filtered for nFeature RNAs <500 & <6000 and percent mitochondrial genes <10%. Clusters were generated by UMAP with resolution = 0.8, manually annotated based on expression of conserved marker genes, then analyzed for differential expression across microbiome groups using FindMarkers.
Immunofluorescence microscopy
[00323] 1 cm segments of mouse tissue were excised and fixed in Carnoy’s solution (1
Acetic Acid : 3 Chloroform : 6 Ethanol). Fixed tissues were then transferred to cryomolds, immersed in OCT, snap frozen in a shallow bath of 2-Methylbutane on dry ice and stored at -20 °C until 5 pm cryosectioning. Slides were thawed and permeabilized in 0.1% Triton-X-100 and blocked in PBS+5%BSA+Mouse FcBlock (ThermoFisher cat # 14-0161-82) before staining with various combinations of primary and secondary antibodies (Table 5). Images were captured on a Leica SP8 confocal microscope running LAS-X software version 3.1.5.
Histology
[00324] Whole mouse colons were placed in plastic histology cassettes and immersed in Bouin’s fixative fluid for 24 h before transfer to 70% ethanol, paraffin embedding, sectioning, and H&E staining. Blinded slides were scored by a board-certified pathologist.
[00325] Table 5: Key Resources
Figure imgf000122_0001
Figure imgf000123_0001
Figure imgf000124_0001
Figure imgf000125_0001
Figure imgf000126_0001
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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
Mouse
[00386] 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. To further examine this relationship in the data, Allobaculum sp. 128 relative abundance was transformed into binary values (Absent=0, Present=l) so that logistic regressions could be fit for each OTU test pairing (performed using software package GraphPad Prism v9). Raw data and calculations for these mouse data are found in Table 6.
Attorney Docket No. 047162-5304-00WO
Table 6: Diversity Calculations.
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Human
[00387] From publicly available large-scale human microbiome datasets, the relative abundance of these two taxa were examined in pediatric ulcerative colitis patients and healthy human volunteers (Schirmer et al., 2018, Cell Host Microbe, 24:600-610; included in their supplementary material an OTU table of relative abundances, which was ready for analysis). American Gut Project data was accessed in various forms (raw fastq; pre-processed but not OTU-picked; biom tables) through the QIITA repository and analysis suite: qiita.ucsd.edu/). The same analysis methods were used as the ones that were employed for the gnotobiotic mouse data (Spearman’s correlation and logistic regression to binary -transformed Allobaculum values) to assess whether bacterial taxa of interest exhibit any significant relationships.
Table 7: SEQUENCES OF THE DISCLOSURE WITH SEQ ID NO IDENTIFIERS
Figure imgf000145_0001
Figure imgf000146_0001
[00388] Numbered Embodiments of the Disclosure
[00389] Other subject matter contemplated by the present disclosure is set out in the following numbered embodiments:
[00390] 1. 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, wherein the method 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.
[00391] 2 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, wherein the method 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.
[00392] 3. The method of embodiment lor 2, wherein the first gut microbe species or strain thereof is an Allobaculum species or strain thereof.
[00393] 4. The method of embodiment 3, 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.
[00394] 5. The method of any one of the above embodiments, wherein the second gut microbe species or strain thereof is an Akkermansia species or strain thereof.
[00395] 6. The method of embodiment 5, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. [00396] 7 The method of any one of the above embodiments, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
[00397] 8. The method of embodiment 7, wherein the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
[00398] 9. The method of embodiment 8, wherein the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
[00399] 10. The method of any one of the above embodiments, wherein 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.
[00400] 11. The method of any one of the above embodiments, wherein the disease or disorder is an inflammatory disease or disorder.
[00401] 12. The method of embodiment 11, wherein the inflammatory disease or disorder is an inflammatory bowel disease (IBD), colitis, Crohn’s disease, ulcerative colitis, Clostridium difficile colitis, or any combination thereof.
[00402] 13. The method of embodiment 1 or 2, wherein the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof.
[00403] 14. The method of embodiment 13, wherein 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.
[00404] 15. The method of embodiment 1 or 2, wherein the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof.
[00405] 16. The method of embodiment 15, wherein 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.
[00406] 17. The method of embodiment 1 or 2, wherein 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.
[00407] 18. The method of embodiment 1 or 2, further comprising detecting the presence of the first gut microbe species or strain thereof in the subject prior to the administration of the composition.
[00408] 19. The method of any one of embodiments 2-18, wherein the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[00409] 20. A method of preventing or treating a disease or disorder induced by an
Allobaculum species or strain thereof in a subject in need thereof, wherein the method 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.
[00410] 21. A method of preventing or treating a disease or disorder induced by an
Allobaculum species or strain thereof in a subject in need thereof, wherein the method 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.
[00411] 22. The method of embodiment 20 or 21, wherein 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.
[00412] 23. The method of embodiment 22, wherein 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.
[00413] 24. The method of any one of embodiments 20-23, wherein the presence of the
Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
[00414] 25. The method of any one of embodiments 20-23, wherein the presence of the
Allobaculum species or strain thereof is detected in a biological sample of the subject.
[00415] 26. The method of any one of embodiments 20-25, 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.
[00416] 27. The method of any one of embodiments 20-26, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
[00417] 28. The method of any one of embodiments 20-27, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
[00418] 29. The method of embodiment 28, wherein the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. [00419] 30. The method of embodiment 29, wherein the inflammatory genes are selected from the group consisting of rag3b, saal and saa3.
[00420] 31. The method of any one of embodiments 21-30, wherein the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[00421] 32. 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.
[00422] 33. 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.
[00423] 34. The method of embodiment 32 or 33, wherein the level of the at least one
Allobaculum species or strain thereof is detected in the gut microbiota of the subject.
[00424] 35. The method of any one of embodiments 32-34, 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.
[00425] 36. The method of any one of embodiments 32-34, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. [00426] 37. The method of any one of embodiments 33-36, wherein the active agent is selected from the group consisting of a protein, an amino acid, a metabolite, a nucleic acid and combinations thereof.
[00427] 38. 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.
[00428] 39. A 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.
[00429] 40. The composition of embodiment 38 or 39, wherein the composition modulates an immune response toward the disease or disorder.
[00430] 41. The 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.
[00431] 42. The 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.
[00432] 43. The composition of embodiment 38 or 39, wherein the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof.
[00433] 44. The 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. [00434] 45. The composition of any one of embodiments 38-44, wherein the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof.
[00435] 46. The 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.
[00436] 47. The composition of any one of embodiments 38-46, wherein the disease or disorder is an inflammatory disease or disorder.
[00437] 48. The 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.
[00438] 49. The composition of any one of embodiments 38-48, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation.
[00439] 50. The composition of embodiment 49, wherein the reduction or inhibition of
IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
[00440] The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent application, foreign patents, foreign patent application and non-patent publications referred to in this specification and/or listed in the Application Data Sheet are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, application and publications to provide yet further embodiments.
[00441] These and other changes can be made to the embodiments in light of the above- detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.
INCORPORATION BY REFERENCE [00442] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

CLAIMS What is claimed:
1. 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, wherein the method 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.
2. 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, wherein the method 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.
3. The method of claim lor 2, wherein the first gut microbe species or strain thereof is an Allobaculum species or strain thereof.
4. The method of claim 3, 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.
5. The method of claim 1 or 2, wherein the second gut microbe species or strain thereof is an Akkermansia species or strain thereof.
6. The method of claim 5, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof.
7. The method of claim 1 or 2, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. The method of claim 7, wherein the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. The method of claim 8, wherein the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. The method of claim 1 or 2, wherein 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 method of claim 1 or 2, wherein the disease or disorder is an inflammatory disease or disorder. The method of claim 11, wherein 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 method of claim 1 or 2, wherein the composition further comprises at least one compound that reduces the level of the first gut microbe species or strain thereof. The method of claim 13, wherein 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 method of claim 1 or 2, wherein the composition further comprises at least one compound that increases the level of the second gut microbe species or strain thereof. The method of claim 15, wherein 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 of claim 1 or 2, wherein 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 of claim 1 or 2, further comprising detecting the presence of the first gut microbe species or strain thereof in the subject prior to the administration of the composition. The method of claim 2 wherein 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, wherein the method 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. A method of preventing or treating a disease or disorder induced by an Allobaculum species or strain thereof in a subject in need thereof, wherein the method 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 of claim 20 or 21, wherein 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 method of claim 22, wherein 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. The method of claim 20 or 21, wherein the presence of the Allobaculum species or strain thereof is detected in the gut microbiota of the subject. The method of claim 20 or 21, wherein the presence of the Allobaculum species or strain thereof is detected in a biological sample of the subject. The method of claim 20 or 21, 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. The method of claim 20 or 21, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. The method of claim 20 or 21, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. The method of claim 28, wherein the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject. The method of claim 29, wherein the inflammatory genes are selected from the group consisting of rag3b, saal and saa3. The method of claim 21, wherein 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. 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. The method of claim 32 or 33, wherein the level of the at least one Allobaculum species or strain thereof is detected in the gut microbiota of the subject. The method of claim 32 or 33, 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. The method of claim 32 or 33, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. The method of claim 32 or 33, wherein 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. A 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. The composition of claim 38 or 39, wherein the composition modulates an immune response toward the disease or disorder. The composition of claim 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. The composition of claim 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. The composition of claim 38 or 39, wherein the pathogenic gut microbe species or strain thereof is an Allobaculum species or strain thereof. The composition of claim 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. The composition of claim 38 or 39, wherein the beneficial gut microbe species or strain thereof is an Akkermansia species or strain thereof. The composition of claim 45, wherein the Akkermansia species or strain thereof comprises a nucleotide sequence as set forth in SEQ ID NO.: 2 or a fragment thereof. The composition of claim 38 or 39, wherein the disease or disorder is an inflammatory disease or disorder. The composition of claim 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. The composition of claim 38 or 39, wherein the at least one pathogenic effect comprises intestinal epithelial cell (IEC) activation. The composition of claim 49, wherein the reduction or inhibition of IEC activation is characterized by a decrease in expression of inflammatory genes in the lECs of the subject.
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