Compositions and Methods for Treating Disease II

Abstract

Therapeutic compositions and methods comprising bacterial strains for the treatment or prevention of disease are disclosed. More specifically, compositions comprising bacterial strains isolated from the human gastrointestinal tract and their use in the treatment or prevention of inflammatory and autoimmune disorders are disclosed. Also disclosed are novel bacterial strains and isolated populations of therapeutic importance.

Description

[Technical Field]

[0001] Related Applications This application claims priority to Australian Provisional Application No. 2021 / 902122 entitled "Compositions and Methods for Treating Diseases", filed on July 10, 2021, Australian Provisional Application No. 2021 / 902960 entitled "Compositions and Methods for Treating Diseases", filed on September 13, 2021, and Australian Provisional Application No. 2022 / 900261 entitled "Compositions and Methods for Treating Diseases", filed on February 9, 2022, the entire contents of which are incorporated herein by reference.

[0002] The present invention relates generally to the field of bacterial strains and therapeutic compositions and methods comprising the bacterial strains for the treatment or prevention of disease. More particularly, the present invention relates to compositions comprising bacterial strains isolated from the human gastrointestinal tract and their use in the treatment or prevention of inflammatory and autoimmune disorders. [Background technology]

[0003] The human gut microbiota comprises over 500–1000 distinct phylotypes belonging to several bacterial phyla, including Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and Verrucomicrobia. Two major phyla, Bacteroidetes and Firmicutes, generally account for over 90% of the gut microbiota (Arumugam et al., 2011). Symbiotic relationships resulting from bacterial colonization of the human gut produce a wide variety of metabolic, structural, defensive, and other beneficial functions. Gut bacteria are key regulators of digestion along the gastrointestinal (GI) tract, with commensal bacteria playing crucial roles in the extraction, synthesis, and absorption of many nutrients and metabolites, including bile acids, lipids, amino acids, vitamins, and short-chain fatty acids (SCFAs). Recently, the immunological importance of the gut microbiota and its products in regulating the development, homeostasis, and function of innate and adaptive immune cells has been recognized (Brestoff and Atris, 2013).

[0004] It is increasingly recognized that the gut microbiota regulates the host's intestinal mucosal immunity and predisposition to inflammation ( Geva-Zatorsky et al., 2017 ; Kabat et al., 2014 ), opening new avenues for novel therapeutic interventions.

[0005] Dramatic changes in microbiota composition have been reported in many inflammatory and autoimmune disorders, including inflammatory bowel disease (IBD). Recognizing the potential positive effects of certain bacterial strains on the animal intestine, various strains have been proposed for use in treating various diseases. Certain strains, including Lactobacillus and Bifidobacterium strains, have been proposed for use in treating various extraintestinal inflammatory and autoimmune disorders (see Goldin & Gorbach, 2008; Azad et al., 2013). However, the precise effects of specific bacterial strains locally in the gastrointestinal tract and systemically have not been elucidated. As a result, the relationship between different diseases and different bacterial strains in the human gastrointestinal tract has not yet been clearly elucidated.

[0006] IBD (which includes two major disease subtypes, Crohn's disease (CD) and ulcerative colitis (UC)) is characterized by paroxysmal and disruptive inflammation of the gastrointestinal tract. In 2017, IBD was estimated to affect 6.8 million people worldwide, with the highest prevalence in the United States and Europe (GBD 2017; Inflammatory Bowel Disease Collaborative, 2019). Up to 20% of patients are diagnosed before the age of 16, and pediatric-onset IBD (PIBD) is associated with a more complex and aggressive disease that adversely affects growth and psychosocial development.

[0007] There is currently no cure for IBD, and long-term clinical management requires effective therapeutic agents with excellent safety profiles. However, existing treatments exhibit a range of deficiencies, and remissions are generally short-lived. Furthermore, IBD therapeutic agents are ineffective when early onset and more aggressive disease coincide, leading to progressive intestinal damage and the need for surgery. There is an urgent need to develop more effective and safer treatments to improve patients' quality of life, maintain remission over the long term, reduce surgery, and reduce personal and public health costs.

[0008] Existing treatments for IBD are suboptimal, with strong adverse effects, low compliance (average non-adherence rate of 50% (see Chan et al., 2017)), and high costs. Furthermore, there are no effective solutions for maintaining long-term disease-free remission. Methalmine is one of the most widely used first-line drugs for relapse and maintenance of remission in mild to moderate ulcerative colitis, with response rates of 40–70% and remission rates of 15–20% (Karagozian & Burakoff, 2007).

[0009] There is a need in the art for new methods to treat inflammatory and autoimmune disorders. In order to develop new therapies using gut bacteria, there is also a need to characterize the potential effects of gut bacteria. Summary of the Invention [Problem to be solved by the invention]

[0010] The present invention is based, in part, on the inventors' identification of several bacterial strains, including Alistipes shahii, Gemmiger formicilis, and Colonithrix sana, to enhance or improve intestinal barrier function. Based on this observation, it is proposed that strains of these and closely related species are particularly suitable for therapeutic use for the treatment and prevention of inflammatory and autoimmune disorders, as described below. [Means for solving the problem]

[0011] The present inventors have developed new compositions comprising viable bacterial strains of species selected from Alistipes shahii, Gemmiger formicilis, and Colonithrix sana that can be used to treat and prevent inflammatory and autoimmune disorders.

[0012] Thus, in one aspect, the present invention provides a cell of the Alistipes shahii strain deposited under any one of accession numbers V21 / 014432, V21 / 014433 or V21 / 014434 or a derivative thereof.

[0013] In some embodiments, the cells are at least partially isolated.

[0014] In another aspect, the present invention provides a biologically pure culture of the Alistipes shahii strain deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, or a derivative thereof.

[0015] In another aspect, the invention provides a composition comprising a cell or culture as described above or elsewhere herein.

[0016] In yet another aspect, the invention provides a composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 1-3 or 44, or having a 16S rRNA sequence represented by any one of SEQ ID NOs: 1-3 or 44. In some embodiments, the bacterial strain comprises two or more copies (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies) of the 16S rRNA sequence in its genome.

[0017] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0018] In yet another aspect, the present invention provides a pharmaceutical composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Alistipes shahii, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0019] In a related aspect, the present invention provides pharmaceutical compositions comprising an effective amount of a bacterial strain that is a phylogenetic descendant of the most recent common ancestor (MRCA) of A. timonensis and A. shahii, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 35262 of the bac120 phylogenetic tree from the Genome Taxonomy Database (GTDB). In some embodiments, the phylogenetic tree is generated by release 89 of the GTDB, although any subsequent release is believed to provide equally applicable results. In some preferred embodiments of this type, the bacterial strain is of the species A. shahii.

[0020] Typically, the bacterial strain is at least partially isolated.

[0021] In some embodiments, the bacterial strain is live. In some alternative embodiments, the bacterial strain is dead.

[0022] In some embodiments, the composition further comprises a prebiotic.

[0023] In some embodiments, the composition is formulated in a dry form. Typically, the composition is dried using a technique selected from freeze drying, spray drying, fluidized bed drying, vacuum drying, or a combination thereof.

[0024] In some embodiments, the composition is formulated for oral administration.

[0025] In some embodiments, the bacterial strain produces an agent that attenuates or impairs intracellular signal transduction and activator of transcription 3 (STAT3) signaling.

[0026] In some embodiments of this type, the agent is a small molecule, a peptide, or a nucleotide. Typically, the agent is released by the bacterial strain.

[0027] In some embodiments, the agent specifically binds to any one of STAT3, JAK2, TYK2, or IL-23.

[0028] In some embodiments, A. shahii includes as a carbon source or metabolizes one or more agents selected from the group consisting of starch, glucose, fructose, gluconate, lactose, trehalose, and lactaldehyde.

[0029] In another aspect, the present invention provides a method of restoring or improving intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of the A. shahii species, thereby restoring or improving intestinal barrier function.

[0030] In some preferred embodiments, the restoration or improvement of intestinal barrier function is characterized by at least one of: (i) an increase in the quality and / or quantity of mucin; (ii) an improvement in the integrity of tight junction proteins; (iii) a reduction in the translocation of luminal contents into the systemic circulation; or (iv) a reduction in intestinal ulcers and / or wounds.

[0031] In some embodiments, the luminal contents comprise lipopolysaccharide (LPS).

[0032] In some embodiments, restoration or improvement of intestinal barrier function results in a decrease in systemic inflammation in the subject. In some embodiments of this type, the systemic inflammation is characterized by elevated levels of inflammatory cytokines (e.g., IL-1β, IL-8, IL-6, and TNF) in the subject compared to the levels of inflammatory cytokines in healthy subjects.

[0033] In yet another aspect, the present invention provides a method of maintaining intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of the A. shahii species, thereby maintaining intestinal barrier function in the subject.

[0034] In yet another aspect, the present invention provides a method of reducing inflammation in a subject, the method comprising administering to the subject a bacterial strain of A. shahii, thereby reducing inflammation in the subject.

[0035] In some embodiments, the inflammation is local to the intestinal environment or is systemic inflammation.

[0036] In another aspect, the present invention provides a method of inducing or enhancing mucosal healing in a subject, the method comprising administering to the subject a bacterial strain of the A. shahii species in an amount sufficient to induce epithelial cell migration, proliferation, and / or differentiation, thereby inducing mucosal healing in the subject.

[0037] In some embodiments, mucosal healing in a subject can be measured using one or more fecal or serum markers. As illustrative examples, one or more fecal markers can be selected from the group including calprotectin, lactoferrin, metalloproteinase (MMP)-9, and lipocalin-2.

[0038] In some embodiments, the bacterial strain reduces inflammation by attenuating the NFκB pathway, hi some embodiments of this type, the bacterial strain inhibits the production of one or more transcription factors, cytokines, or chemokines selected from the group including NFκB, TNF, IFN-γ, IL-1β, IL-8, and MCP-1.

[0039] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a target cell, the method comprising contacting the cell with at least a soluble component of a bacterial cell preparation of A. shahii to block or otherwise inhibit STAT3 signaling in the cell. Typically, the method of this aspect is performed in vitro.

[0040] In some embodiments, the target cell is selected from a reporter cell (e.g., a HEK cell), an immune cell (e.g., a Th17 immune cell), an epithelial cell, or an endothelial cell. In some embodiments, the target cell is a mammalian cell, preferably a human cell.

[0041] In some embodiments, the bacterial cell preparation is a bacterial cell culture. Thus, the soluble components can comprise, consist of, or consist essentially of the soluble fraction of the bacterial cell culture (e.g., cell culture supernatant). The soluble components can further comprise some insoluble components of the bacterial cell culture. For example, the soluble components can comprise substantially all of the bacterial culture. Preferably, the soluble components are substantially depleted of bacterial cells.

[0042] In some alternative embodiments, the bacterial cell preparation is a bacterial cell lysate. In exemplary embodiments of this type, the soluble component can refer to the soluble fraction of the cell lysate. The soluble fraction can be suitably achieved by any method, including by centrifugation.

[0043] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a cell, the method comprising administering to a subject a bacterial strain of the A. shahii species, thereby blocking or otherwise inhibiting STAT3 signaling in the cell. Typically, the method of this aspect is performed in vivo.

[0044] In some embodiments, the cell is an immune cell (eg, a Th17 immune cell) or an epithelial cell.

[0045] In some embodiments, the cells are epithelial cells and the bacterial strain or a metabolite produced by the bacterial strain increases the production of IL-22 in the subject.

[0046] In some embodiments, the bacterial strain produces a molecule that is a direct or indirect inhibitor of STAT3. For example, the bacterial strain may produce a metabolite that directly inhibits at least one of an IL-23 polypeptide, a JAK2 polypeptide, a TYK2 polypeptide, or a STAT3 polypeptide.

[0047] In some embodiments, the bacterial strains used in the methods described above and elsewhere herein produce one or more metabolites selected from butyrate, acetate, ethanol, and fumarate.

[0048] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to the 16S rRNA sequence of an A. shahii bacterial strain.

[0049] In some alternative embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 1-3 or 44, or the bacterial strain has a 16S rRNA gene sequence represented by any one of SEQ ID NOs: 1-3 or 44. In some embodiments, the bacterial strain comprises two or more independently selected copies (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies) of the 16S rRNA sequence set forth in any one of SEQ ID NOs: 1-3 or 44.

[0050] In some embodiments, the bacterial strain is an A. shahii strain deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, or a derivative thereof.

[0051] Preferably, the bacterial strain is at least partially isolated.

[0052] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is a dry composition. In some embodiments, the dry composition is selected from the group consisting of particles, granules, and powders. As illustrative examples, the pharmaceutical composition may be freeze-dried, spray-dried, fluid-bed dried, vacuum-dried, or a combination thereof.

[0053] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0054] In still yet another aspect, the present invention provides a method of treating an inflammatory or autoimmune disorder in a subject, the method comprising administering to the subject an effective amount of a bacterial strain of A. shahii, thereby treating or preventing the inflammatory or autoimmune disorder.

[0055] In some embodiments, the inflammatory or autoimmune disorder is selected from the group comprising inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and vasculitis. Preferably, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0056] In some embodiments, the bacterial strain blocks or otherwise inhibits STAT3 signaling in at least one cell of the subject. Typically, the cell is an epithelial cell, an endothelial cell, or an immune cell (e.g., a Th17 immune cell).

[0057] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of an A. shahii bacterial strain.

[0058] Alternatively, in some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 1-4 or 44, or the bacterial strain has a 16S rRNA gene sequence represented by any one of SEQ ID NOs: 1-3 or 44.

[0059] Preferably, the bacterial strain is at least partially isolated.

[0060] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the composition is a dry composition selected from the group consisting of particles, granules, and powders. For example, the composition may be lyophilized. Alternatively, the composition may be spray-dried, fluid-bed dried, or vacuum-dried.

[0061] In some embodiments, the composition is formulated for oral administration.

[0062] In one aspect, the present invention provides a composition comprising a bacterial strain of the genus Alistipes for use in therapy.

[0063] In another aspect, the invention provides a composition comprising a bacterial strain of A. shahii for use in therapy. In some of the same and some other embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of the bacterial strain of A. shahii.

[0064] Alternatively, the bacterial strain may have a 16S rRNA sequence that is at least about 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 1-3 or 44, or where the bacterial strain has a 16S rRNA gene sequence represented by any one of SEQ ID NOs: 1-3 or 44.

[0065] In yet another aspect, the present invention provides a composition comprising a bacterial strain of the genus Alistipes for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0066] In yet another aspect, the present invention provides a composition comprising a bacterial strain of A. shahii for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0067] In some embodiments, the bacterial strain is an A. shahii strain deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, or a derivative thereof.

[0068] In some embodiments, the inflammatory or autoimmune disorder is selected from inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and diabetes mellitus type 1. In some preferred embodiments, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0069] In one aspect, the invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of A. shahii and an adjunctive therapeutic agent.

[0070] In some embodiments, the adjunctive therapeutic agent is an anti-inflammatory agent. As illustrative examples, the anti-inflammatory agent is selected from the group including 5-aminosalicylates, corticosteroids, azathioprine, or combinations thereof. In some other embodiments, the adjunctive therapy is an antibody (e.g., a monoclonal antibody). As illustrative examples, the antibody may be selected from infliximab, adalimumab, golimumab, certolizumab pegol, natalizumab, and vedolizumab.

[0071] In another aspect, the present invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of A. shahii and a dietary supplement, in this type of embodiment, the dietary supplement improves bacterial stain engraftment.

[0072] In another aspect, the present invention provides a cell of the C. sana strain deposited under accession number V21 / 019213 or V21 / 019214 or a derivative thereof.

[0073] In some embodiments, the cells are at least partially isolated.

[0074] In another aspect, the present invention provides a biologically pure culture of the C. sana strain deposited under accession number V21 / 019213 or V21 / 019214 or a derivative thereof.

[0075] In another aspect, the invention provides a composition comprising a cell or culture as described above or elsewhere herein.

[0076] In yet another aspect, the present invention provides a composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 11-18, or having a 16S rRNA sequence represented by any one of SEQ ID NOs: 11-18. In some embodiments, the bacterial strain comprises two or more copies of the 16S rRNA sequence in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, or eight copies). For example, the bacterial strain may comprise four copies of the 16S rRNA sequence in its genome (e.g., each of SEQ ID NOs: 11-14 or each of SEQ ID NOs: 15-18).

[0077] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0078] In yet another aspect, the present invention provides a pharmaceutical composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a bacterial strain of C. sana, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0079] In a related aspect, the present invention provides pharmaceutical compositions comprising an effective amount of a bacterial strain that is a phylogenetic descendant of the most recent common ancestor (MRCA) of C. sana and C. sp002437735, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23879 of the bac120 phylogenetic tree from the Genome Taxonomy Database (GTDB). In some embodiments, the phylogenetic tree is generated by release 89 of the GTDB, although any subsequent release is believed to provide equally applicable results. In some preferred embodiments of this type, the bacterial strain is C. sana.

[0080] Typically, the bacterial strain is at least partially isolated.

[0081] In some embodiments, the bacterial strain is live. In some alternative embodiments, the bacterial strain is dead.

[0082] In some embodiments, the composition further comprises a prebiotic.

[0083] In some embodiments, the composition is formulated in a dry form. Typically, the composition is dried using a technique selected from freeze drying, spray drying, fluidized bed drying, vacuum drying, or a combination thereof.

[0084] In some embodiments, the composition is formulated for oral administration.

[0085] In some embodiments, the bacterial strain produces an agent that attenuates or impairs intracellular signal transduction and activator of transcription 3 (STAT3) signaling.

[0086] In some embodiments of this type, the agent is a small molecule, a peptide, or a nucleotide. Typically, the agent is released by the bacterial strain.

[0087] In some embodiments, the agent specifically binds to any one of STAT3, JAK2, TYK2, or IL-23.

[0088] In some embodiments, C. sana includes as a carbon source or metabolizes one or more agents selected from the group consisting of starch, glucose, fructose, gluconate, lactose, trehalose, and lactaldehyde.

[0089] In another aspect, the present invention provides a method for restoring or improving intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of the C. sana species, thereby restoring or improving intestinal barrier function.

[0090] In some preferred embodiments, the restoration or improvement of intestinal barrier function is characterized by at least one of: (i) an increase in the quality and / or quantity of mucin; (ii) an improvement in the integrity of tight junction proteins; (iii) a reduction in the translocation of luminal contents into the systemic circulation; or (iv) a reduction in intestinal ulcers and / or wounds.

[0091] In some embodiments, the luminal contents comprise lipopolysaccharide (LPS).

[0092] In some embodiments, restoration or improvement of intestinal barrier function results in a decrease in systemic inflammation in the subject. In some embodiments of this type, the systemic inflammation is characterized by elevated levels of inflammatory cytokines (e.g., IL-1β, IL-8, IL-6, and TNF) in the subject compared to the levels of inflammatory cytokines in healthy subjects.

[0093] In yet another aspect, the present invention provides a method for maintaining intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of the C. sana species, thereby maintaining intestinal barrier function in the subject.

[0094] In yet another aspect, the present invention provides a method of reducing inflammation in a subject, the method comprising administering to the subject a bacterial strain of C. sana, thereby reducing inflammation in the subject.

[0095] In some embodiments, the inflammation is local to the intestinal environment or is systemic inflammation.

[0096] In another aspect, the present invention provides a method for inducing or enhancing mucosal healing in a subject, the method comprising administering to the subject a bacterial strain of the C. sana species in an amount sufficient to induce epithelial cell migration, proliferation, and / or differentiation, thereby inducing mucosal healing in the subject.

[0097] In some embodiments, mucosal healing in a subject can be measured using one or more fecal or serum markers. As illustrative examples, one or more fecal markers can be selected from the group including calprotectin, lactoferrin, metalloproteinase (MMP)-9, and lipocalin-2.

[0098] In some embodiments, the bacterial strain reduces inflammation by attenuating the NFκB pathway, hi some embodiments of this type, the bacterial strain inhibits the production of one or more transcription factors, cytokines, or chemokines selected from the group including NFκB, TNF, IFN-γ, IL-1β, IL-8, and MCP-1.

[0099] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a target cell, the method comprising contacting the cell with at least a soluble component of a bacterial cell preparation of C. sana to block or otherwise inhibit STAT3 signaling in the cell. Typically, the method of this aspect is performed in vitro.

[0100] In some embodiments, the target cell is selected from a reporter cell (e.g., a HEK cell), an immune cell (e.g., a Th17 immune cell), an epithelial cell, or an endothelial cell. In some embodiments, the target cell is a mammalian cell, preferably a human cell.

[0101] In some embodiments, the bacterial cell preparation is a bacterial cell culture. Thus, the soluble components can comprise, consist of, or consist essentially of the soluble fraction of the bacterial cell culture (e.g., cell culture supernatant). The soluble components can further comprise some insoluble components of the bacterial cell culture. For example, the soluble components can comprise substantially all of the bacterial culture. Preferably, the soluble components are substantially depleted of bacterial cells.

[0102] In some alternative embodiments, the bacterial cell preparation is a bacterial cell lysate. In exemplary embodiments of this type, the soluble component can refer to the soluble fraction of the cell lysate. The soluble fraction can be suitably achieved by any method, including by centrifugation.

[0103] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a cell, the method comprising administering to a subject a bacterial strain of the species C. sana, thereby blocking or otherwise inhibiting STAT3 signaling in the cell. Typically, the method of this aspect is performed in vivo.

[0104] In some embodiments, the cell is an immune cell (eg, a Th17 immune cell) or an epithelial cell.

[0105] In some embodiments, the cells are epithelial cells and the bacterial strain or a metabolite produced by the bacterial strain increases the production of IL-22 in the subject.

[0106] In some embodiments, the bacterial strain produces a molecule that is a direct or indirect inhibitor of STAT3. For example, the bacterial strain may produce a metabolite that directly inhibits at least one of an IL-23 polypeptide, a JAK2 polypeptide, a TYK2 polypeptide, or a STAT3 polypeptide.

[0107] In some embodiments, the bacterial strains used in the methods described above and elsewhere herein produce one or more metabolites selected from butyrate, acetate, ethanol, and fumarate.

[0108] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to the 16S rRNA sequence of a bacterial strain of C. sana.

[0109] In some alternative embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 11-18, or has a 16S rRNA sequence represented by any one of SEQ ID NOs: 11-18. In some embodiments, the bacterial strain comprises two or more copies of the 16S rRNA sequence in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, or eight copies). For example, the bacterial strain may comprise four copies of the 16S rRNA sequence in its genome (e.g., each of SEQ ID NOs: 11-14 or each of SEQ ID NOs: 15-18).

[0110] In some embodiments, the bacterial strain is the C. sana strain deposited under accession number V21 / 019213 or V21 / 019214, or a derivative thereof.

[0111] Preferably, the bacterial strain is at least partially isolated.

[0112] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is a dry composition. In some embodiments, the dry composition is selected from the group consisting of particles, granules, and powders. As illustrative examples, the pharmaceutical composition may be freeze-dried, spray-dried, fluid-bed dried, vacuum-dried, or a combination thereof.

[0113] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0114] In still yet another aspect, the present invention provides a method of treating an inflammatory or autoimmune disorder in a subject, the method comprising administering to the subject an effective amount of a bacterial strain of C. sana, thereby treating or preventing the inflammatory or autoimmune disorder.

[0115] In some embodiments, the inflammatory or autoimmune disorder is selected from the group comprising inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and vasculitis. Preferably, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0116] In some embodiments, the bacterial strain blocks or otherwise inhibits STAT3 signaling in at least one cell of the subject. Typically, the cell is an epithelial cell, an endothelial cell, or an immune cell (e.g., a Th17 immune cell).

[0117] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to a 16S rRNA sequence of a bacterial strain of the genus C. sana.

[0118] Alternatively, in some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% identical to any one of SEQ ID NOs: 11-18, or has a 16S rRNA sequence represented by any one of SEQ ID NOs: 11-18. In some embodiments, the bacterial strain comprises two or more copies of the 16S rRNA sequence in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, or eight copies). For example, the bacterial strain may comprise four copies of the 16S rRNA sequence in its genome (e.g., each of SEQ ID NOs: 11-14, or each of SEQ ID NOs: 15-18).

[0119] Preferably, the bacterial strain is at least partially isolated.

[0120] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the composition is a dry composition selected from the group consisting of particles, granules, and powders. For example, the composition may be lyophilized. Alternatively, the composition may be spray-dried, fluid-bed dried, or vacuum-dried.

[0121] In some embodiments, the composition is formulated for oral administration.

[0122] In one aspect, the present invention provides a composition comprising a bacterial strain of the genus C. sana for use in therapy.

[0123] In another aspect, the present invention provides a composition comprising a bacterial strain of C. sana for use in therapy.

[0124] In yet another aspect, the present invention provides a composition comprising a bacterial strain of the genus C. sana for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0125] In yet another aspect, the present invention provides a composition comprising a bacterial strain of C. sana for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0126] In some embodiments, the bacterial strain is the C. sana strain deposited under accession number V21 / 019213 or V21 / 019214, or a derivative thereof.

[0127] In some embodiments, the inflammatory or autoimmune disorder is selected from inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and diabetes mellitus type 1. In some preferred embodiments, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0128] In one aspect, the present invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of C. sana and an anti-inflammatory agent.

[0129] In some embodiments, the anti-inflammatory agent is selected from the group including 5-aminosalicylates, corticosteroids, azathioprine, infliximab, and adalimumab.

[0130] In another aspect, the present invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of C. sana and a dietary supplement, in this type of embodiment, the dietary supplement improves bacterial staining survival.

[0131] In still yet another aspect, therefore, in one aspect, the present invention provides a cell of the Gemmiger formicilis strain deposited under accession number V21 / 011520 or a derivative thereof.

[0132] In some embodiments, the cells are at least partially isolated.

[0133] In another aspect, the present invention provides a biologically pure culture of the Gemmiger formicilis strain deposited under accession number V21 / 011520 or a derivative thereof.

[0134] In another aspect, the invention provides a composition comprising a cell or culture as described above or elsewhere herein.

[0135] In yet another aspect, the invention provides a composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97.5%, 98%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 27 or one or more of SEQ ID NOs: 45-47, or having a 16S rRNA sequence represented by SEQ ID NO: 27 or one or more of SEQ ID NOs: 45-47. In some embodiments, the bacterial strain comprises two or more copies (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies) of the 16S rRNA sequence in its genome.

[0136] In some embodiments, the composition further comprises a pharmaceutically acceptable excipient, diluent, or carrier.

[0137] In yet another aspect, the present invention provides a pharmaceutical composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a bacterial strain of Gemmiger formicilis, together with a pharmaceutically acceptable carrier, diluent, or excipient.

[0138] In a related aspect, the present invention provides pharmaceutical compositions comprising an effective amount of a bacterial strain that is a phylogenetic descendant of the most recent common ancestor (MRCA) of G. variabile and G. sp002306375, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23818 of the bac120 phylogenetic tree from the Genome Taxonomy Database (GTDB). In some embodiments, the phylogenetic tree is generated by release 89 of the GTDB, although any subsequent release is believed to provide equally applicable results. In some preferred embodiments of this type, the bacterial strain is G. formicilis.

[0139] Typically, the bacterial strain is at least partially isolated.

[0140] In some embodiments, the bacterial strain is live. In some alternative embodiments, the bacterial strain is dead.

[0141] In some embodiments, the composition further comprises a prebiotic.

[0142] In some embodiments, the composition is formulated in a dry form. Typically, the composition is dried using a technique selected from freeze drying, spray drying, fluidized bed drying, vacuum drying, or a combination thereof.

[0143] In some embodiments, the composition is formulated for oral administration.

[0144] In some embodiments, the bacterial strain produces an agent that attenuates or impairs intracellular signal transduction and activator of transcription 3 (STAT3) signaling.

[0145] In some embodiments of this type, the agent is a small molecule, a peptide, or a nucleotide. Typically, the agent is released by the bacterial strain.

[0146] In some embodiments, the agent specifically binds to any one of STAT3, JAK2, TYK2, or IL-23.

[0147] In some embodiments, G. formicilis includes as a carbon source or metabolizes one or more agents selected from the group consisting of starch, glucose, fructose, gluconate, lactose, trehalose, and lactaldehyde.

[0148] In another aspect, the present invention provides a method of restoring or improving intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of G. formicilis, thereby restoring or improving intestinal barrier function.

[0149] In some preferred embodiments, the restoration or improvement of intestinal barrier function is characterized by at least one of: (i) an increase in the quality and / or quantity of mucin; (ii) an improvement in the integrity of tight junction proteins; (iii) a reduction in the translocation of luminal contents into the systemic circulation; or (iv) a reduction in intestinal ulcers and / or wounds.

[0150] In some embodiments, the luminal contents comprise lipopolysaccharide (LPS).

[0151] In some embodiments, restoration or improvement of intestinal barrier function results in a decrease in systemic inflammation in the subject. In some embodiments of this type, the systemic inflammation is characterized by elevated levels of inflammatory cytokines (e.g., IL-1β, IL-8, IL-6, and TNF) in the subject compared to the levels of inflammatory cytokines in healthy subjects.

[0152] In yet another aspect, the present invention provides a method of maintaining intestinal barrier function in a subject, the method comprising administering to the subject a bacterial strain of the G. formicilis species, thereby maintaining intestinal barrier function in the subject.

[0153] In yet another aspect, the present invention provides a method of reducing inflammation in a subject, the method comprising administering to the subject a bacterial strain of G. formicilis, thereby reducing inflammation in the subject.

[0154] In some embodiments, the inflammation is local to the intestinal environment or is systemic inflammation.

[0155] In another aspect, the present invention provides a method of inducing or enhancing mucosal healing in a subject, the method comprising administering to the subject a bacterial strain of the G. formicilis species in an amount sufficient to induce epithelial cell migration, proliferation, and / or differentiation, thereby inducing mucosal healing in the subject.

[0156] In some embodiments, mucosal healing in a subject can be measured using one or more fecal or serum markers. As illustrative examples, one or more fecal markers can be selected from the group including calprotectin, lactoferrin, metalloproteinase (MMP)-9, and lipocalin-2.

[0157] In some embodiments, the bacterial strain reduces inflammation by attenuating the NFκB pathway, hi some embodiments of this type, the bacterial strain inhibits the production of one or more transcription factors, cytokines, or chemokines selected from the group including NFκB, TNF, IFN-γ, IL-1β, IL-8, and MCP-1.

[0158] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a target cell, the method comprising contacting the cell with at least a soluble component of a bacterial cell preparation of G. formicilis to block or otherwise inhibit STAT3 signaling in the cell. Typically, the method of this aspect is performed in vitro.

[0159] In some embodiments, the target cell is selected from a reporter cell (e.g., a HEK cell), an immune cell (e.g., a Th17 immune cell), an epithelial cell, or an endothelial cell. In some embodiments, the target cell is a mammalian cell, preferably a human cell.

[0160] In some embodiments, the bacterial cell preparation is a bacterial cell culture. Thus, the soluble components can comprise, consist of, or consist essentially of the soluble fraction of the bacterial cell culture (e.g., cell culture supernatant). The soluble components can further comprise some insoluble components of the bacterial cell culture. For example, the soluble components can comprise substantially all of the bacterial culture. Preferably, the soluble components are substantially depleted of bacterial cells.

[0161] In some alternative embodiments, the bacterial cell preparation is a bacterial cell lysate. In exemplary embodiments of this type, the soluble component can refer to the soluble fraction of the cell lysate. The soluble fraction can be suitably achieved by any method, including by centrifugation.

[0162] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a cell, the method comprising administering to a subject a bacterial strain of the species G. formicilis, thereby blocking or otherwise inhibiting STAT3 signaling in the cell. Typically, the method of this aspect is performed in vivo.

[0163] In some embodiments, the cell is an immune cell (eg, a Th17 immune cell) or an epithelial cell.

[0164] In some embodiments, the cells are epithelial cells and the bacterial strain or a metabolite produced by the bacterial strain increases the production of IL-22 in the subject.

[0165] In some embodiments, the bacterial strain produces a molecule that is a direct or indirect inhibitor of STAT3. For example, the bacterial strain may produce a metabolite that directly inhibits at least one of an IL-23 polypeptide, a JAK2 polypeptide, a TYK2 polypeptide, or a STAT3 polypeptide.

[0166] In some embodiments, the bacterial strains used in the methods described above and elsewhere herein produce one or more metabolites selected from butyrate, acetate, ethanol, and fumarate.

[0167] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to the 16S rRNA sequence of a bacterial strain of G. formicilis.

[0168] In some alternative embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 97.5%, 98.5%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to SEQ ID NO:27 or one or more of SEQ ID NOs:45-47, or where the bacterial strain has a 16S rRNA gene sequence represented by SEQ ID NO:27 or one or more of SEQ ID NOs:45-47. In some embodiments, the bacterial strain comprises two or more independently selected copies (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies) of the 16S rRNA sequence set forth in SEQ ID NO:27 or one or more of SEQ ID NOs:45-47.

[0169] In some embodiments, the bacterial strain is the G. formicilis strain deposited under accession number V21 / 011520, or a derivative thereof.

[0170] Preferably, the bacterial strain is at least partially isolated.

[0171] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, diluent, or excipient. In some embodiments, the pharmaceutical composition is a dry composition. In some embodiments, the dry composition is selected from the group consisting of particles, granules, and powders. As illustrative examples, the pharmaceutical composition may be freeze-dried, spray-dried, fluid-bed dried, vacuum-dried, or a combination thereof.

[0172] In some embodiments, the pharmaceutical composition is formulated for oral administration.

[0173] In yet another aspect, the present invention provides a method of blocking or otherwise inhibiting STAT3 signaling in a cell, the method comprising administering to a subject a bacterial strain of the species Gemmiger sp. MD158, thereby blocking or otherwise inhibiting STAT3 signaling in the cell. Typically, the method of this aspect is performed in vivo.

[0174] In still yet another aspect, the present invention provides a method of treating an inflammatory or autoimmune disorder in a subject, the method comprising administering to the subject an effective amount of a bacterial strain of G. formicilis, thereby treating or preventing the inflammatory or autoimmune disorder.

[0175] In some embodiments, the inflammatory or autoimmune disorder is selected from the group comprising inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and vasculitis. Preferably, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0176] In some embodiments, the bacterial strain blocks or otherwise inhibits STAT3 signaling in at least one cell of the subject. Typically, the cell is an epithelial cell, an endothelial cell, or an immune cell (e.g., a Th17 immune cell).

[0177] In some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to a 16S rRNA sequence of a bacterial strain of the genus Gemmiger.

[0178] Alternatively, in some embodiments, the bacterial strain has a 16S rRNA sequence that is at least about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical to SEQ ID NO:27 or one or more of SEQ ID NOs:45-47, or where the bacterial strain has a 16S rRNA gene sequence represented by SEQ ID NO:27 or one or more of SEQ ID NOs:45-47.

[0179] Preferably, the bacterial strain is at least partially isolated.

[0180] In some embodiments, the bacterial strain is formulated as a pharmaceutical composition together with a pharmaceutically acceptable carrier, diluent, and / or excipient. In some embodiments, the composition is a dry composition selected from the group consisting of particles, granules, and powders. For example, the composition may be lyophilized. Alternatively, the composition may be spray-dried, fluid-bed dried, or vacuum-dried.

[0181] In some embodiments, the composition is formulated for oral administration.

[0182] In one aspect, the present invention provides a composition comprising a bacterial strain of the genus Gemmiger for use in therapy.

[0183] In another aspect, the present invention provides a composition comprising a bacterial strain of G. formicilis for use in therapy.

[0184] In another aspect, the present invention provides a composition comprising the bacterial strain of Gemmiger sp. MD158 for use in therapy.

[0185] In yet another aspect, the present invention provides a composition comprising a bacterial strain of the genus Gemmiger for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0186] In yet another aspect, the present invention provides a composition comprising a bacterial strain of G. formicilis for use in the treatment or prevention of an inflammatory or autoimmune disorder.

[0187] In some embodiments, the bacterial strain is the G. formicilis strain deposited under accession number V21 / 011520, or a derivative thereof.

[0188] In some embodiments, the bacterial strain is Gemmiger sp. strain MD158, which has a genome sequence comprising at least one, two, three, four, or all of the sequences set forth in SEQ ID NOs: 34-38.

[0189] In some embodiments, the inflammatory or autoimmune disorder is selected from inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, and diabetes mellitus type 1. In some preferred embodiments, the inflammatory or autoimmune disorder is inflammatory bowel disease (IBD).

[0190] In one aspect, the present invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of G. formicilis and an anti-inflammatory agent.

[0191] In some embodiments, the anti-inflammatory agent is selected from the group including 5-aminosalicylates, corticosteroids, azathioprine, infliximab, and adalimumab.

[0192] In another aspect, the present invention provides a composition for use in treating an inflammatory or autoimmune disorder, the composition comprising a bacterial strain of Gemmiger sp. and a dietary supplement. In embodiments of this type, the dietary supplement improves bacterial staining survival. In some embodiments of this type, the bacterial strain is of the G. formicilis species. In some other embodiments, the bacterial strain is of the Gemmiger sp. MD158 species.

[0193] In some related aspects, the technology described above and / or elsewhere herein provides bacterial species and compositions comprising them in the form of probiotics. Preferably, such probiotics are effective in improving gut microbial ecology, alleviating symptoms of microbial dysbiosis, promoting health, and / or treating and / or preventing inflammatory and / or autoimmune disorders.

[0194] The following figures form part of the present specification and are included to further demonstrate certain aspects of the present disclosure. The present disclosure may be better understood by reference to one or more of these figures in combination with the detailed description of specific embodiments presented herein. [Brief explanation of the drawings]

[0195] [Figure 1] A representative phylogenetic tree (r89, as described in Parks et al. 2018) of GTDB species is shown, focusing on the phylogenetic neighborhood of A. shahii. The most recent common ancestor node of the health-associated species A. shahii, with high stability (bootstrap value >90%), is highlighted.

[0196] [Figure 2-1]This figure provides a graphical representation of the association between Alistipes bacteria and representative inflammatory and / or autoimmune disorders. (A) Using high-resolution gut metagenomic data from 6,020 subjects, we identified A. shahii, A. finegoldii, A. senegalensis, A. onderdonkii, and A. sp000434235 as prevalent in healthy individuals (dark bars) but depleted in Crohn's disease and ulcerative colitis (striped bars). (B) These observations were validated in an independent IBD cohort previously published by Harvard (Franzosa et al., 2019). (C) A. shahii, (D) A. finegoldii, and (E) A. senegalensis were significantly less prevalent in a range of inflammatory and autoimmune disorders (striped bars) (P<0.05, Fisher's exact test). [Figure 2-2] This figure provides a graphical representation of the association between Alistipes bacteria and representative inflammatory and / or autoimmune disorders. (A) Using high-resolution gut metagenomic data from 6,020 subjects, we identified A. shahii, A. finegoldii, A. senegalensis, A. onderdonkii, and A. sp000434235 as prevalent in healthy individuals (dark bars) but depleted in Crohn's disease and ulcerative colitis (striped bars). (B) These observations were validated in an independent IBD cohort previously published by Harvard (Franzosa et al., 2019). (C) A. shahii, (D) A. finegoldii, and (E) A. senegalensis were significantly less prevalent in a range of inflammatory and autoimmune disorders (striped bars) (P<0.05, Fisher's exact test). [Figure 2-3]This figure provides a graphical representation of the association between Alistipes bacteria and representative inflammatory and / or autoimmune disorders. (A) Using high-resolution gut metagenomic data from 6,020 subjects, we identified A. shahii, A. finegoldii, A. senegalensis, A. onderdonkii, and A. sp000434235 as prevalent in healthy individuals (dark bars) but depleted in Crohn's disease and ulcerative colitis (striped bars). (B) These observations were validated in an independent IBD cohort previously published by Harvard (Franzosa et al., 2019). (C) A. shahii, (D) A. finegoldii, and (E) A. senegalensis were significantly less prevalent in a range of inflammatory and autoimmune disorders (striped bars) (P<0.05, Fisher's exact test).

[0197] [Figure 3] Graphical representation of the morphology and phylogeny of A. shahii strains is provided. (A) Gram stain of A. shahii isolate MH21-1 showing cell morphology. (B) Genome tree constructed from the alignment of 120 bacterial-specific single-copy marker genes from a high-quality reference genome (GTDB r89). Chips are labeled with the NCBI species name and NCBI GenBank ID. Clades are labeled with the GTDB r89 taxonomy. Nonparametric bootstrap values ​​were calculated from 1000 replicates.

[0198] [Figure 4]This figure provides a graphical representation of the lack of effect of A. shahii on healthy intestinal function in naive C57Bl / 6 mice. (A) Overview of the model used to evaluate the effects of A. shahii MH21-1 on naive C57Bl / 6 mice. (B) Treatment with A. shahii MH21-1 has little effect on body weight in naive animals. (C)-(D) Treatment with A. shahii MH21-1 has no effect on colon length or colon weight / length ratio compared to vehicle-treated controls in naive animals. (E)-(G) Treatment with A. shahii MH21-1 has no effect on epithelial damage, inflammation, or hypervascularization compared to vehicle-treated controls in naive animals. (H) Treatment with A. shahii MH21-1 has no effect on intestinal histology compared to vehicle-treated controls in naive animals. All data are reported as mean and standard deviation. ns, not significant.

[0199] [Figure 5]A graphic representation of A. shahii's ability to restore intestinal barrier function is provided. (A) Overview of the DSS model used to evaluate the therapeutic effects of A. shahii. (B) Effect of daily treatment with vehicle, prednisone, and A. shahii on body weight in healthy and DSS-treated mice. All treatment groups were compared to the DSS + vehicle group. Significance was determined using two-way anova with Tukey's multiple comparison test. (C) Representative images of intestinal tissue from C57Bl / 6 mice treated with vehicle, prednisone, or A. shahii. (D) DSS treatment results in an increase in histopathological scores, which are ameliorated by treatment with prednisone, F. prausnitzii A2-165, and A. shahii. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group, and significance was determined using one-way anova with Dunnett's multiple comparison test. (E) DSS treatment results in increased epithelial damage, which is ameliorated by treatment with prednisone or A. shahii. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group using the Kruskal-Wallis test with Dunn's multiple comparisons correction. (F) DSS treatment results in a significant decrease in goblet cells, which is ameliorated by treatment with prednisone or A. shahii. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group using the Kruskal-Wallis test with Dunn's multiple comparisons correction. (G) DSS treatment results in a significant decrease in intraepithelial lymphocytes, which is ameliorated by treatment with A. shahii. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group using the Kruskal-Wallis test with Dunn's multiple comparisons correction. (H) Overview of the SKG model used to evaluate the therapeutic effects of A. shahii. (I) Curdlan treatment results in an increase in histopathological scores that are ameliorated by treatment with anti-IL-23 antibody and A. shahii. All data are presented as mean and standard deviation. Anova was used with Dunnett's multiple comparison test to compare all groups with the curdlan + vehicle group.(J)-(M) Curdlan treatment results in increases in IL-23, IL-12p70, IL-6, and GM-CSF, which are ameliorated by treatment with A. shahii. All data are presented as means and standard deviations. For IL-23, IL-12p70, and GM-CSF, all groups were compared to the DSS + vehicle group using the Kruskal-Wallis test with Dunn's multiple comparisons correction. For IL-6, all groups were compared to the curdlan + vehicle group using an Anova with Dunnett's multiple comparisons test. For all data, ns: not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001.

[0200] [Figure 6] Graphical representations of A. shahii suppressing STAT3 in vitro are provided. (A)-(C) STAT3 signaling was inhibited by treating HEKBlue IL-23 reporter cells with cell-free or <3 kDa fractionated culture supernatants from A. shahii strains MH21-1 (t-test, n = 9; ****p < 0.0001; ***p = 0.0004), A. shahii MH21-3, and A. shahii MH21-6. (D) A. shahii culture supernatants were size-fractionated and heat-treated at 37°C or 97°C before testing using the HEKBlue IL-23 reporter cell line. After heat treatment, all fractions were able to suppress STAT3 signaling (t-test, n = 9; ****p < 0.0001; *p = 0.0362; ****p < 0.0001). (E) Cell-free supernatant from A. shahii MH21-1 grown in PYG exhibits potent STAT3 inhibitory activity, but not significant activity when grown in WCB or MCM (one-way ANOVA, n = 9; ns p = 0.4896 and 0.7489; ***p < 0.0001 and < 0.0001, ns p = 0.3677 and 0.4524). (F) STAT3 signaling is inhibited by treatment of HEKBlue IL-6 reporter cells with cell-free supernatant or <3 kDa fraction culture supernatant from A. shahii strain MH21-2 and A. shahii strain MH21-3 (t-test, ****p < 0.0001).

[0201] [Figure 7-1]A graphic representation shows that A. shahii promotes the migration of human intestinal epithelial cells. (A) A Transwell migration assay was used to study the effect of a sterile culture supernatant extract from A. shahii on the migration of HCT116 colon cancer cells. Under serum-starved conditions (0.5% FBS), the addition of 1x A. shahii extract to the bottom of the chamber significantly increased the basolateral migration of HCT116 cells compared with the TYG-M medium extract control. (TYG-M, untreated, n = 6 technical replicates; A. shahii, n = 4 technical replicates, each with 3 biological replicates; unpaired t-test, two-tailed P = 0.0047). (B) As a second readout for cell migration, an Incucyte scratch wound assay was performed. After scratching the HCT116 cell monolayer, relative wound confluence was measured every 2 h. Ninety hours after scratching, serum-starved HCT116 cells incubated in 0.6x extract from A. shahii MH21-1 showed significantly higher wound confluence compared to cells treated with TYG-M medium extract (TYG-M, untreated, n = 16 technical replicates; A. shahii, n = 16 technical replicates, 3 biological replicates each); unpaired t-test, two-tailed p = 0.0069). (C) TEER across T84 cell monolayers was assessed during and after 48 hours of basolateral treatment with the barrier-disrupting factor IFN-γ. T84 cells treated apically with culture supernatant without A. shahii cells exhibited faster recovery after 48 hours of treatment after IFN-γ induced changes to barrier integrity, as assessed by higher TEER values ​​(ohms) compared to PYG medium controls (n = 4 replicates per condition). (D) T84 cells apically treated with A. shahii cell-free culture supernatant show significantly greater TEER improvement after 7 days of IFN-γ-induced mediated loss of barrier integrity compared to PYG medium controls, as assessed by TEER (n = 4 replicates per condition). Samples were compared, and statistical significance was determined using an unpaired t-test. ****p < 0.0001. (E) TEER across T84 cell monolayers was assessed during and after 48 hours of basolateral treatment with the barrier disruption factor IFN-γ.T84 cells apically treated with culture supernatant without A. shahii cells show a faster recovery from IFN-γ-induced changes in barrier integrity loss after 48 hours of treatment compared to PYG medium controls, as assessed by TEER. All samples were normalized to untreated cells, and percentage differences in TEER compared to control are presented. (F) T84 cells apically treated with culture supernatant without A. shahii cells show a significantly greater TEER improvement after 7 days of IFN-γ-induced changes in barrier integrity compared to PYG medium controls, as assessed by TEER. All samples were normalized to untreated cells. Samples were compared using an unpaired t-test. ****p<0.0001. (G) Overview of the gating strategy used to isolate A. shahii MH21-6. [Figure 7-2]A graphic representation shows that A. shahii promotes the migration of human intestinal epithelial cells. (A) A Transwell migration assay was used to study the effect of a sterile culture supernatant extract from A. shahii on the migration of HCT116 colon cancer cells. Under serum-starved conditions (0.5% FBS), the addition of 1x A. shahii extract to the bottom of the chamber significantly increased the basolateral migration of HCT116 cells compared with the TYG-M medium extract control. (TYG-M, untreated, n = 6 technical replicates; A. shahii, n = 4 technical replicates, each with 3 biological replicates; unpaired t-test, two-tailed P = 0.0047). (B) As a second readout for cell migration, an Incucyte scratch wound assay was performed. After scratching the HCT116 cell monolayer, relative wound confluence was measured every 2 h. Ninety hours after scratching, serum-starved HCT116 cells incubated in 0.6x extract from A. shahii MH21-1 showed significantly higher wound confluence compared to cells treated with TYG-M medium extract (TYG-M, untreated, n = 16 technical replicates; A. shahii, n = 16 technical replicates, 3 biological replicates each); unpaired t-test, two-tailed p = 0.0069). (C) TEER across T84 cell monolayers was assessed during and after 48 hours of basolateral treatment with the barrier-disrupting factor IFN-γ. T84 cells treated apically with culture supernatant without A. shahii cells exhibited faster recovery after 48 hours of treatment after IFN-γ induced changes to barrier integrity, as assessed by higher TEER values ​​(ohms) compared to PYG medium controls (n = 4 replicates per condition). (D) T84 cells apically treated with A. shahii cell-free culture supernatant show significantly greater TEER improvement after 7 days of IFN-γ-induced mediated loss of barrier integrity compared to PYG medium controls, as assessed by TEER (n = 4 replicates per condition). Samples were compared, and statistical significance was determined using an unpaired t-test. ****p < 0.0001. (E) TEER across T84 cell monolayers was assessed during and after 48 hours of basolateral treatment with the barrier disruption factor IFN-γ.T84 cells apically treated with culture supernatant without A. shahii cells show a faster recovery from IFN-γ-induced changes in barrier integrity loss after 48 hours of treatment compared to PYG medium controls, as assessed by TEER. All samples were normalized to untreated cells, and percentage differences in TEER compared to control are presented. (F) T84 cells apically treated with culture supernatant without A. shahii cells show a significantly greater TEER improvement after 7 days of IFN-γ-induced changes in barrier integrity compared to PYG medium controls, as assessed by TEER. All samples were normalized to untreated cells. Samples were compared using an unpaired t-test. ****p<0.0001. (G) Overview of the gating strategy used to isolate A. shahii MH21-6.

[0202] [Figure 8] We provide a graphical representation that Colonithrix species are significantly less prevalent in the gut microbiota of humans with inflammatory and autoimmune diseases compared to healthy controls, using high-resolution gut metagenomic data from 6,020 subjects.

[0203] [Figure 9] A graphical representation of the phylogeny and morphology of C. sana strains is provided. (A) Gram staining of a C. sana isolate showing morphology. (B) Genome tree constructed with an alignment of 120 bacterial-specific single-copy marker genes from a high-quality reference genome (GTDB r89, method described in Parks et al., 2018). The chips are labeled with the NCBI species name and NCBI GenBank ID. Clades are labeled with the GTDB r89 taxonomy. Nonparametric bootstrap values ​​were calculated from 1,000 replicates. This tree focuses on the phylogenetic neighborhood of the genus C. sana (i.e., around node 23879). Ancestral nodes with high stability (bootstrap values ​​>90%) of health-associated C. sana species are labeled up to the most recent common ancestor of the genus Colonithrix.

[0204] [Figure 10] A graphic representation is provided showing that C. sana restores intestinal barrier function. (A) Overview of the DSS model used to evaluate the therapeutic effects of Colonithrix species. (B) DSS treatment results in an increase in histopathological scores, which are ameliorated by treatment with C. sana MH35-1 and C. sana MH35-2. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group, and significance was determined using an unpaired t-test with Welch's correction. (C) DSS treatment results in an increase in epithelial damage, which is ameliorated by treatment with C. sana MH35-1 and C. sana MH35-2. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group using Kruskal-Wallis. For all data, ns: not significant; *, p<0.05.

[0205] [Figure 11] Figure 1 provides a graphic representation of C. sana suppressing STAT3 activation in vitro. STAT3 signaling is inhibited when the HEKBlue IL23 reporter cell line is treated with cell-free supernatants of (A) C. sana MH35-1 and (B) C. sana MH35-2 or filtered at <3 KDa (t-test, n=9, A ****p<0.0001; B ****p<0.0001).

[0206] [Figure 12]Graphical representation of C. sana suppressing IL-6-mediated STAT3 activation in vitro. (A-B) STAT3 signaling is inhibited by treating HEKBlue IL-6 reporter cells with cell-free or <3 kDa fractionated culture supernatants of C. sana MH35-1 (A) and C. sana MH35-2 (B) at 10% v / v (unpaired t-test, n=18, ****p<0.0001). (C) STAT3 signaling is inhibited by treating HEKBlue IL-6 reporter cells with 1x extracts of C. sana MH35-1 and C. sana MH35-2 (unpaired t-test, n=24, ****p<0.0001).

[0207] [Figure 13]A graphic representation is provided showing that C. sana promotes migration of human intestinal epithelial cells. (A) A Transwell migration assay was used to study the effect of sterile culture supernatant extracts from C. sana MH35-1 and C. sana MH35-2 on the migration of HCT116 colon cancer cells. Under serum-starved conditions (0.5% FBS), untreated HCT116 cells and cells treated with 0.5x bacterial media extract exhibited comparable background levels of migration toward the basolateral side of the Transwell chamber. Addition of 0.5× C. sana MH35-1 and C. sana MH35-2 extracts to the bottom of the chamber significantly increased basolateral migration of HCT116 cells compared with the medium control (untreated n = 6 technical replicates; medium control n = 6 technical replicates; C. sana MH35-1 n = 4 technical replicates (2 biological replicates each); C. sana MH35-2 n = 4 technical replicates (3 biological replicates each); unpaired t-test, two-tailed; ***P = 0.0003 for C. sana MH35-1, ***P = 0.0009 for C. sana MH35-2). (B) As a second readout for cell migration, an Incucyte scratch wound assay was performed. After scratching the HCT116 cell monolayer, relative wound confluence was measured every 2 h. Untreated (n = 18 technical replicates); medium control (n = 18 technical replicates); C. sana MH35-1 (n = 36 technical replicates, 2 biological replicates); C. sana MH35-2 (n = 36 technical replicates, 3 biological replicates). 24 hours after scratching, serum-starved HCT116 cells incubated in 0.5x extracts from C. sana MH35-1 and C. sana MH35-2 showed significantly higher wound confluence than cells treated with bacterial medium extract. Unpaired t-test: ***P = 0.0008 (C. sana MH35-1) and ***P = 0.0004 (C. sana MH35-2).

[0208] [Figure 14-1]This figure provides a graphical representation of the significantly lower prevalence of Gemmiger species in the gut microbiota of humans with inflammatory and autoimmune diseases compared with healthy controls. Using high-resolution gut metagenomic data from 6,020 subjects, we identified (A) Gemmiger formicilis, (B) Gemmiger species_C, (C) Gemmiger species_A, and (D) Gemmiger sp003476825 as prevalent in healthy individuals (dark bars) but depleted in a range of inflammatory and autoimmune disorders (striped bars). (E) The strongest effect was observed in IBD, including the major subtypes of both ulcerative colitis and Crohn's disease. (F) These observations were validated in an independent IBD cohort previously published by Harvard. All associations shown are P<0.05 (Fisher's exact test). [Figure 14-2] This figure provides a graphical representation of the significantly lower prevalence of Gemmiger species in the gut microbiota of humans with inflammatory and autoimmune diseases compared with healthy controls. Using high-resolution gut metagenomic data from 6,020 subjects, we identified (A) Gemmiger formicilis, (B) Gemmiger species_C, (C) Gemmiger species_A, and (D) Gemmiger sp003476825 as prevalent in healthy individuals (dark bars) but depleted in a range of inflammatory and autoimmune disorders (striped bars). (E) The strongest effect was observed in IBD, including the major subtypes of both ulcerative colitis and Crohn's disease. (F) These observations were validated in an independent IBD cohort previously published by Harvard. All associations shown are P<0.05 (Fisher's exact test).

[0209] [Figure 15]Graphical representation of the phylogeny and morphology of G. formicilis strains is presented. (A) Gram stain of G. formicilis isolate MH32-1 showing morphology. (B) Upper panel: Photograph of G. formicilis MH32-2 on a plate, showing round, generally raised colonies after 48 hours on TY medium. Lower panel: Gram stain of G. formicilis isolate MH32-2 showing morphology. (C) Genome tree constructed from the alignment of 120 bacterial-specific single-copy marker genes from a high-quality reference genome (GTDB r89). Chips are labeled with the NCBI species name and NCBI GenBank ID. Clades are labeled with the GTDB r89 taxonomy. Nonparametric bootstrap values ​​were calculated from 1,000 replicates. (D) A representative tree of GTDB species (r89, method described in Parks et al. 2018) focused on the phylogenetic neighborhood of the genus Gemmiger (around node 23818). Ancestral nodes with high stability (bootstrap values ​​>90%) of health-associated G. variabile species are labeled up to the most recent common ancestor of the genus Gemmiger.

[0210] [Figure 16] This figure provides a graphic representation of the lack of effect of G. formicilis on healthy intestinal function in naive C57Bl / 6 mice. (A) Overview of the model used to evaluate the effect of G. formicilis MH32-1 on naive C57Bl / 6 mice. (B) Treatment with MH32-1 has little effect on body weight in naive animals. (C)-(D) Treatment with MH32-1 has no effect on colon length or colon weight / length ratio compared to vehicle-treated controls in naive animals. (E)-(F) Treatment with G. formicilis MH32-1 has no effect on epithelial damage or hypervascularization compared to vehicle-treated controls in naive animals. (G) Treatment with G. formicilis MH32-1 has no effect on intestinal histology compared to vehicle-treated controls in naive animals. All data are reported as mean and standard deviation. ns, not significant; *p<0.05.

[0211] [Figure 17]A graphic representation of G. formicilis' ability to restore intestinal barrier function is provided. (A) Overview of the DSS model used to evaluate the therapeutic effects of G. formicilis MH32-1. (B) Effects of daily treatment with vehicle, prednisone, and G. formicilis MH32-1 in healthy and DSS-treated mice. All treatment groups were compared to the DSS + vehicle group. Significance was determined using a two-way anova with Tukey's multiple comparisons test. (C) Endoscopic assessment of colitis assessed on days 1, 2, and 6. Each group was compared to the DSS + vehicle group on each individual day using the Kruskal-Wallis test with Dunn's multiple comparisons correction (day 1) or one-way anova with Dunnett's multiple comparisons correction (days 2 and 6), as appropriate. All data are presented as means and standard deviations. (D) DSS treatment results in a reduction in colon length, which is ameliorated by F. prausnitzii A2-165 and G. formicilis MH32-1, but not prednisone. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group, and significance was determined using one-way anova with Dunnett's multiple comparison test. (E) Representative images of intestinal histology from C57Bl / 6 mice treated with vehicle, prednisone, or G. formicilis MH32-1. (F) DSS treatment results in an increase in histopathological scores, which is ameliorated by treatment with prednisone, F. prausnitzii A2-165, and G. formicilis MH32-1. All data are presented as means and standard deviations. All groups were compared to the DSS + vehicle group, and significance was determined using one-way anova with Dunnett's test for multiple comparisons. (G) DSS treatment results in increased epithelial damage that is ameliorated by treatment with prednisone or G. formicilis MH32-1. All data are presented as mean and standard deviation. All groups were compared to the DSS + vehicle group using the Kruskal-Wallis test with Dunn's correction for multiple comparisons.(H) Fecal lipocalin-2 concentrations in C57Bl / 6 mice treated with vehicle, prednisone, F. prausnitzii, or G. formicilis MH32-1. Significance was determined using one-way anova with Dunnett's test for multiple comparisons. (I) Overview of the SKG model used to evaluate the therapeutic effects of G. formicilis MH32-1. (J) Curdlan treatment results in an increase in histopathological scores, which are ameliorated by treatment with anti-IL-23 antibody and G. formicilis MH32-1. All data are presented as means and standard deviations. All groups were compared to the curdlan + vehicle group using anova with Dunnett's test for multiple comparisons. For all data, ns: not significant; *, p<0.05; **, p<0.01; ****, p<0.0001.

[0212] [Figure 18-1]A graphic representation of G. formicilis suppressing STAT3 activation in vitro is provided. (A) STAT3 signaling is inhibited when the HEKBlue IL-23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-1 or filtered to <3 kDa (t-test, n = 9; p < 0.0001, p = 0.0001). (B) Cell-free supernatant from G. formicilis MH32-1 was size-fractionated, heat-treated at 37°C or 97°C, and then tested on the HEKBlue IL-23 reporter cell line. After heat treatment, all fractions were still able to inhibit STAT3 signaling (t-test, n = 9; p = 0.0421, p < 0.0001, p = 0.0009). (C) Cell-free supernatants from G. formicilis MH32-1 were size-fractionated and treated with proteinase K for 30 min at 37°C, then tested on the HEKBlue IL-23 reporter cell line. After proteinase K treatment, all fractions were still able to inhibit STAT3 signaling (t-test, n = 9; p < 0.0001, p < 0.0001). (D) Cell-free supernatants from MH32-1 grown in TY exhibit potent STAT3 inhibitory activity, but show minimal activity when grown in PYG, WCB, and MCM (t-test, n = 9; p < 0.0001, p = 0.0027, p = 0.0203, p = 0.0114). (E-F) STAT3 signaling is inhibited when the HEKBlue IL23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-2 and G. formicilis ATCC 27749 or filtered to <3 kDa (t-test, n=9, p<0.0001, p=0.0001). (G) STAT3 signaling is inhibited when the HEKBlue IL23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-2 or G. formicilis ATCC 27749 or filtered to <3 kDa (unpaired t-test was used to compare samples). ***, p<0.001; ****, p<0.0001). (H) PBMCs show decreased secretion of IL-6 when pretreated with cell-free supernatant from G. formicilis MH32-1 and then stimulated with PIM compared to TY medium control.Normally distributed data were analyzed using ordinary one-way anova with Sidak's multiple comparison test (n = 7 per treatment, ns = not significant; * = p < 0.05; *** = p < 0.001; **** = p < 0.0001). All data are presented as means and standard deviations. (I) G. formicilis MH32-1 significantly suppresses IL-6 secretion from LPS-stimulated T84 intestinal epithelial cells compared to TY medium control. Inhibitory activity is retained in the <3 kDa fraction of G. formicilis culture supernatant. All data are presented as means and standard deviations. T-test ****p < 0.0001. (J) STAT3 signaling is inhibited by treatment of the HEKBlue IL-6 reporter cell line with <3 kDa filtered cell-free supernatant from G. formicilis MH32-1 (t-test, n = 9; ***p < 0.0009). [Figure 18-2]A graphic representation of G. formicilis suppressing STAT3 activation in vitro is provided. (A) STAT3 signaling is inhibited when the HEKBlue IL-23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-1 or filtered to <3 kDa (t-test, n = 9; p < 0.0001, p = 0.0001). (B) Cell-free supernatant from G. formicilis MH32-1 was size-fractionated, heat-treated at 37°C or 97°C, and then tested on the HEKBlue IL-23 reporter cell line. After heat treatment, all fractions were still able to inhibit STAT3 signaling (t-test, n = 9; p = 0.0421, p < 0.0001, p = 0.0009). (C) Cell-free supernatants from G. formicilis MH32-1 were size-fractionated and treated with proteinase K for 30 min at 37°C, then tested on the HEKBlue IL-23 reporter cell line. After proteinase K treatment, all fractions were still able to inhibit STAT3 signaling (t-test, n = 9; p < 0.0001, p < 0.0001). (D) Cell-free supernatants from MH32-1 grown in TY exhibit potent STAT3 inhibitory activity, but show minimal activity when grown in PYG, WCB, and MCM (t-test, n = 9; p < 0.0001, p = 0.0027, p = 0.0203, p = 0.0114). (E-F) STAT3 signaling is inhibited when the HEKBlue IL23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-2 and G. formicilis ATCC 27749 or filtered to <3 kDa (t-test, n=9, p<0.0001, p=0.0001). (G) STAT3 signaling is inhibited when the HEKBlue IL23 reporter cell line is treated with cell-free supernatant from G. formicilis MH32-2 or G. formicilis ATCC 27749 or filtered to <3 kDa (unpaired t-test was used to compare samples). ***, p<0.001; ****, p<0.0001). (H) PBMCs show decreased secretion of IL-6 when pretreated with cell-free supernatant from G. formicilis MH32-1 and then stimulated with PIM compared to TY medium control.Normally distributed data were analyzed using ordinary one-way anova with Sidak's multiple comparison test (n = 7 per treatment, ns = not significant; * = p < 0.05; *** = p < 0.001; **** = p < 0.0001). All data are presented as means and standard deviations. (I) G. formicilis MH32-1 significantly suppresses IL-6 secretion from LPS-stimulated T84 intestinal epithelial cells compared to TY medium control. Inhibitory activity is retained in the <3 kDa fraction of G. formicilis culture supernatant. All data are presented as means and standard deviations. T-test ****p < 0.0001. (J) STAT3 signaling is inhibited by treatment of the HEKBlue IL-6 reporter cell line with <3 kDa filtered cell-free supernatant from G. formicilis MH32-1 (t-test, n = 9; ***p < 0.0009).

[0213] [Figure 19]A graphic representation is provided showing that G. formicilis promotes the migration of human intestinal epithelial cells. (A) A Transwell migration assay was used to study the effect of sterile culture supernatant extract from G. formicilis MH32-1 on the migration of HCT116 colon cancer cells. Under serum-starved conditions (0.5% FBS), untreated HCT116 cells and cells treated with TY medium extract exhibited comparable background levels of migration toward the basolateral side of the Transwell chamber. Addition of G. formicilis MH32-1 extract to the bottom of the chamber significantly increased the basolateral migration of HCT116 cells compared to the TY control (TY, C n = 6 technical replicates; MH32-1 n = 4 technical replicates (3 biological replicates each); Dunnett's multiple comparison test ****P < 0.0001). (B) Representative brightfield images at 10x magnification of a Transwell® migration experiment. Image size is 570 μm x 570 μm. (C) As a second readout for cell migration, an Incucyte scratch wound assay was performed. After scratching HCT116 cell monolayers, relative wound confluence was measured every 2 h. TY, C n = 27 technical replicates; G. formicilis MH32-1 n = 18 technical replicates (3 biological replicates each). (D) 18 h after scratching, serum-starved HCT116 cells incubated in 0.3x extract from G. formicilis MH32-1 showed significantly higher wound confluence compared to cells treated with TY medium extract. TY, C n = 27 technical replicates; G. formicilis MH32-1 n = 18 technical replicates (3 biological replicates each); unpaired t-test **p = 0.0028.

[0214] [Figure 20]A graphic representation is provided showing that G. formicilis restores intestinal barrier integrity. The effect of treatment with G. formicilis MH32-1 and vehicle control on IFN-γ-stimulated T84 cells, assessed through transepithelial electrical resistance (TEER) using the Maestro Pro system. T84 cells were grown as monolayers in 96-well CytoView-Z plates, and once stable TEER values ​​were established, they were stimulated with IFNγ (100 ng / mL). After 72 hours, cells were washed, and the medium was replaced with fresh medium alone or supplemented with 1× G. formicilis MH32-1 extract or vehicle control. 45 hours after treatment, TEER measurements were recorded, and the percentage difference in TEER values ​​compared to the control (untreated T84 cells) was visualized in a histogram as the mean ± standard deviation. Statistical significance was determined by unpaired t-test. **p<0.01; ****p<0.0001. (B) Overview of the gating strategy used to isolate Gemmiger sp. MD158. [Table 1-1] [Table 1-2] DETAILED DESCRIPTION OF THE INVENTION

[0215] 1.Definition Unless otherwise defined, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For purposes of the present invention, the following terms are defined below.

[0216] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0217] As used herein, the term "about" refers to the normal error range for each value, which is readily known to one of ordinary skill in the art. Reference to "about" a value or parameter herein includes (and describes) embodiments directed to the value or parameter itself.

[0218] As used herein, the term "administering" refers to placing an agent (e.g., a bacterium) as disclosed herein into a subject by a method or route that results in at least partial delivery of the agent at a desired site. Compositions including compounds disclosed herein can be administered by any suitable route that results in an effective biological activity or therapeutic effect in a subject. In some embodiments, administration involves physical activity (e.g., injection, the act of ingestion, the act of application, and / or the operation of a delivery device or machine). Such activity can be performed (e.g., by a medical professional and / or the subject being treated).

[0219] Specifically, "administering" and "administration" as used herein encompass embodiments in which one person instructs another person to consume live bacteria, killed bacteria, spent media derived from bacteria, bacterial cell pellets, purified metabolites produced by bacteria, purified proteins produced by bacteria, prebiotics, small molecules, or combinations thereof in a manner and / or for a purpose, independent of or different from instructions received from the second person. Non-limiting examples of embodiments include situations in which one person instructs another person to consume live bacteria, dead bacteria, spent media derived from bacteria, bacterial cell pellets, purified metabolites produced by bacteria, purified proteins produced by bacteria, prebiotics, small molecules, or combinations thereof in a particular manner and / or for a purpose independent of or different from instructions received from the second person; or when a physician prescribes a course of action and / or treatment to a patient; when a parent instructs a minor user (e.g., a child) to ingest such a product; when a trainer advises a user (e.g., an athlete) to follow instructions for a particular course of action and / or treatment; or when a manufacturer, distributor, or marketer recommends conditions of use to an end user, for example, through advertising or labeling on packaging or other materials provided in connection with the sale or marketing of the product. In some embodiments, the disclosed compositions can be administered orally, intravenously, intramuscularly, intrathecally, subcutaneously, sublingually, buccally, rectally, intravaginally, ocularly, optically, nasally, by inhalation, by spray, skin, transdermally, or a combination thereof, and can be formulated for delivery with a pharmaceutically acceptable excipient, carrier, or diluent. It is noteworthy that while the disclosed compositions encompass multiple formulations and delivery modes for treatments to ameliorate dysplasia and its sequelae, live biological therapeutic products such as probiotics are not typically administered intravenously, intramuscularly, or intraperitoneally. These delivery modes may be advantageous for small molecule products of bacterial metabolism.

[0220] The terms "concurrent administration," "administering simultaneously," "co-administration," and the like refer to administration of a single composition containing two or more active agents, or administration of each active agent as a separate composition, and / or delivery by separate routes, contemporaneously, simultaneously, or sequentially, within a sufficient time period such that effective results are comparable to those obtained when all such active agents are administered as a single composition. "Concurrently" means that the active agents are administered together at substantially the same time, preferably in the same formulation. "Concurrently" means that the active agents are administered closely in time, e.g., one agent is administered before or after the next, within about one minute to about one day. Any contemporaneity is useful. However, when not administered simultaneously, agents are often administered within about one minute to about eight hours, suitably within about one hour to about four hours. When administered simultaneously, agents are suitably administered at the same site on a subject. The term "same site" includes the exact location, but may be within about 0.5 to about 15 centimeters, preferably within about 0.5 to about 5 centimeters. As used herein, the term "separately" means that the agents are administered at intervals of, for example, about one day to several weeks or months. The active agents can be administered in any order. As used herein, the term "sequentially" means that the agents are administered consecutively, for example, at intervals of minutes, hours, days, or weeks. Where appropriate, the active agents can be administered in regular, repeated cycles.

[0221] The term "agent" includes compounds that induce a desired pharmacological and / or physiological effect. This term also encompasses pharmaceutically acceptable, pharmacologically active components of the compounds specifically described herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, analogs, and the like. It should be understood that when using the above term, it includes not only the active agent itself, but also pharmaceutically acceptable, pharmacologically active salts, esters, amides, prodrugs, metabolites, analogs, and the like. The term "agent" should not be construed narrowly and encompasses small molecules, proteinaceous molecules such as peptides, polypeptides, and proteins, and compositions containing them, as well as RNA, DNA, and mimetics and their chemical analogs, and cellular agents. The term "agent" includes cells capable of producing and secreting the polypeptides referred to herein, as well as polynucleotides containing nucleotide sequences encoding the polypeptides. Thus, the term "agent" encompasses vectors, such as viral or non-viral vectors, expression vectors, and nucleic acid constructs, including plasmids for expression and secretion in a range of cells.

[0222] The "amount" or "level" of a biomarker is the detectable level in a sample. These can be measured by methods known to those of skill in the art and also disclosed herein. The expression level or amount of the assessed biomarker can be used to determine response to treatment.

[0223] As used herein, "and / or" refers to and includes any possible combination of one or more of the associated listed items, and the lack of combination when interpreted as alternatives (or).

[0224] The term "anaerobic" means not requiring oxygen for growth. Anaerobic strains include obligate anaerobes (i.e., strains that are harmed by the presence of oxygen), aerobic anaerobes (i.e., strains that cannot use oxygen for growth but can tolerate its presence), and facultative anaerobes (i.e., strains that can grow without oxygen but can use oxygen if it is present).

[0225] "Anaerobic conditions" are defined as conditions in which the oxygen concentration in the fermentation medium is too low for the microorganism to use it as a terminal electron acceptor. "Anaerobic conditions" can be further defined as conditions in which no or little oxygen is added to the medium at a rate of less than 3 mmol / L / h, preferably less than 2.5 mmol / L / h, more preferably less than 2 mmol / L / h, and most preferably less than 1.5 mmol / L / h. "Anaerobic conditions" specifically refer to a complete absence of oxygen (=0 mmol / L / h oxygen) or the addition of little oxygen to the medium, for example, at a rate of <0.5 to <1 mmol / L / h. "Anaerobic metabolism" refers to a biochemical process in which NADH is not the final acceptor for electrons. Anaerobic metabolism can be divided into anaerobic respiration, in which compounds other than oxygen are the final electron acceptor, and substrate-level phosphorylation, in which electrons from NADH are used to generate reduced products via fermentation pathways.

[0226] The term "carbon source" generally refers to a substrate or compound suitable for supporting microbial growth. Carbon sources can be in a variety of forms, including, but not limited to, polymers, carbohydrates, alcohols, acids, aldehydes, ketones, amino acids, peptides, and the like. For example, they can include monosaccharides (glucose, fructose, xylose, etc.), oligosaccharides (i.e., sucrose, lactose), polysaccharides (i.e., starch, cellulose, hemicellulose), lignocellulosic materials, fatty acids (i.e., succinate, lactate, acetate), glycerol, and the like, or mixtures thereof. Carbon sources can also be products of photosynthesis, such as glucose or cellulose.

[0227] Monosaccharides used as carbon sources can be products of hydrolysis of polysaccharides, such as acid or enzymatic hydrolysates of cellulose, starch, and pectin. The term "energy source" may be used interchangeably herein with carbon source, since in chemoorganotrophic metabolism, carbon sources are used as electron donors during catabolism and as carbon sources during cell growth.

[0228] Throughout this specification, unless the context indicates otherwise, the terms "comprise," "comprising," and "comprising" will be understood to mean the inclusion of a stated step, element, or group of steps or elements, but the exclusion of other steps or elements, or group of steps or elements, or group of elements. Thus, use of the term "comprise," etc., indicates that the listed elements are required or mandatory, while other elements are optional and may or may not be present. "Consisting of" means including, and is limited to, what follows the phrase "consisting of." Thus, the phrase "consisting of" indicates that the listed elements are required or essential, and that no other elements are present. "Consisting essentially of" means including the elements listed after the phrase, and is limited to other elements that do not interfere with or contribute to the activity or behavior specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, while other elements are optional and may or may not be present depending on whether they affect the activity or behavior of the listed elements.

[0229] As used herein, "culturing," "culture," and the like refer to a set of procedures used in vitro in which a population of cells (or single cells) are incubated under conditions shown to support the growth or maintenance of cells in vitro. The art recognizes a wide range of formats, media, temperature ranges, gas concentrations, and the like, which must be defined in the culture system. Parameters will vary based on the format selected and the particular needs of the individual practicing the methods disclosed herein. However, it is recognized that determining culture parameters is essentially routine.

[0230] The terms "lower," "reduce," "decrease," "reduction," "inhibition," "suppression," "attenuation," and the like are all used herein to refer to a decrease by a statistically significant amount. In some embodiments, these terms typically refer to a decrease of at least 10% compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, a decrease of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, or at least about 90%. They can also include a decrease of at least about 95%, at least about 98%, at least about 99% or more. As used herein, "reduce," "suppress," and "inhibition" do not require complete inhibition or reduction compared to a reference level. "Complete inhibition," etc., is 100% inhibition compared to a reference level. The decrease can preferably be down to a level that is accepted as within the normal range (eg, for an individual without a given disorder).

[0231] The terms "increased," "increase," "enhancement," or "activation" are all used herein to mean to increase by a statistically significant amount. In some embodiments, the terms "increased," "increase," "enhancement," or "activation" can refer to an increase of at least 10% as compared to a reference level (e.g., in the absence of a given treatment or agent), and can include, for example, an increase of at least about 10%, e.g., at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or up to (including) a 100% increase as compared to the reference level, or a 10-100% increase as compared to the reference level, or at least about 2-fold, or at least about 3-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold or more increase as compared to the reference level. With reference to a marker or symptom, an "increase" is a statistically significant increase in such level.

[0232] As used herein, the term "isolated" encompasses bacteria or other entities or substances that (1) have been separated from at least some of the components with which they were originally produced (whether in nature, such as human stool, or in a laboratory setting, such as a Petri plate containing an artificial growth medium), and / or (2) have been produced, prepared, purified, and / or manufactured by the hand of man. Isolated bacteria, proteins, metabolites, or combinations thereof can be separated from at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the other components with which they were originally associated. In some embodiments, isolated bacteria, proteins, metabolites, or combinations thereof have a purity of about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components (such as other bacterial species). The terms "purify," "purifying," and "purified" refer to bacteria or other substances that have been separated from the bacteria or other substances at the time of initial production or generation (e.g., in nature or in a laboratory setting) or at any time after initial production, as recognized by those skilled in the art of bacterial culture, or at least in part by associated techniques (e.g., chemistry). A bacterium or bacterial population is isolated from materials or environments containing the bacteria or bacterial population at the time or after its production, e.g., and a purified bacterium or bacterial population can contain about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more than about 90% other substances and still be considered "isolated." In some embodiments, purified bacteria and bacterial populations have a purity of about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or greater than about 99%. In the case of the bacterial compositions provided herein, one or more bacterial types present in the composition can be purified independently from one or more other bacteria produced and / or present in the material or environment containing the bacterial types. In some embodiments, a bacterium or bacterial population is "isolated" if it comprises a single strain of bacteria.In some embodiments, such isolated bacteria can be admixed with or administered with other isolated bacteria (e.g., a defined community of isolated bacteria). Bacterial compositions and their bacterial components are generally purified from residual habitat products.

[0233] As used herein, the term "genome" includes the DNA comprising the genes (coding nucleic acid sequences) and non-coding nucleic acid sequences of a microorganism, and thus includes, for example, the introduction of nucleic acids into the coding and non-coding DNA of a microorganism.

[0234] The term "Gram variable" refers to giving a positive and / or negative result in the Gram strain test (i.e., retaining the color of the crystal violet stain). Retention of crystal violet stain by bacteria is related to the thickness of the peptidoglycan layer of the bacterial cell wall. Gram-positive bacteria have a thicker peptidoglycan layer. Gram staining is commonly used to aid in the classification of bacterial strains in the field of microbiology.

[0235] As used herein, the term "gut" is understood to refer to the human digestive tract, also known as the gastrointestinal tract. The intestine includes the oral cavity, pharynx, esophagus, stomach, small intestine (duodenum, jejunum, ileum), large intestine (cecum and colon), and rectum. Although the entire gastrointestinal tract can be colonized by a variety of microorganisms, the majority of the gut microbiota, both in terms of species number and biomass, resides in the small intestine (small intestine and large intestine).

[0236] The terms "marker," "biomarker," and the like refer to any compound that can be measured as an indicator of the physiological state of a biological system. Markers can be biomarkers including amino acid sequences, nucleic acid sequences, and fragments thereof. Exemplary biomarkers include, but are not limited to, cytokines, chemokines, growth and angiogenesis factors, metastasis-associated molecules, cancer antigens, apoptosis-associated proteins, enzymes, proteases, adhesion molecules, cell signaling molecules, and hormones. In some embodiments, a marker can also be a sugar that is not significantly metabolized in a biological system. The sugar can be, for example, mannitol, lactulose, sucrose, sucralose, and any combination of the above.

[0237] "Measuring" or "measuring" means assessing the presence, absence, amount or quantity (which may be an effective amount) of a given substance in a sample (including deriving a qualitative or quantitative concentration level of such substance), or otherwise assessing the value or classification of a clinical parameter of interest. Alternatively, the terms "assaying," "detecting," or "detection" may be used to refer to all measurements or measurements described herein.

[0238] The term "mucosal healing" as used herein refers to the improvement of one or more characteristics that indicate a damaged mucosal layer.Such characteristics are usually determined by colonoscopy, and include, but are not limited to, erythema, loss of vascular pattern, fragility, bleeding, erosion and ulcer.In some situations, mucosal healing refers to the complete improvement of the adverse effects that characterize the damage of the mucosal layer.Alternatively, mucosal healing can refer to the reduction or improvement of one or more negative effects that characterize the damage of the mucosal layer.

[0239] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). The phrase "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without undue toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. In some embodiments of any aspect, a pharmaceutically acceptable carrier can be a carrier other than water. In some embodiments, any aspect of a pharmaceutically acceptable carrier can be a cream, emulsion, gel, liposome, nanoparticle, and / or ointment. In some embodiments of any aspect, a pharmaceutically acceptable carrier can be an artificial or engineered carrier (e.g., a carrier in which the active ingredient would be found in nature or non-naturally occurring).

[0240] The term "phylogenetic tree" refers to a graphical representation of the evolutionary relationships of one gene sequence to another, generated using a defined set of phylogenetic reconstruction algorithms (e.g., parsimony, maximum likelihood, or Bayesian). Nodes in the tree represent different ancestral sequences, and the reliability of any node is given by bootstrap or Bayesian posterior probabilities, which measure the uncertainty of the branches.

[0241] In some embodiments, the term "strain" refers to a terminal leaf of a phylogenetic tree and is defined by a specific gene sequence, which may be a concatenated alignment of 120 ubiquitous single-copy proteins (Parks et al., 2018) extracted from genome assemblies using GTDB-tk (Chaumeil et al., 2020) or other tools known in the art.

[0242] The term "clade" refers to a set of phylogenetic tree members downstream of a stable node in the phylogenetic tree (bootstrap value >90%). A clade is a group of related organisms representing all phylogenetic descendants of a common ancestor. A clade comprises a series of terminal leaves in a phylogenetic tree, which are distinct monophyletic evolutionary units.

[0243] As used herein, "prebiotics" is understood to mean ingredients that enable specific changes in both composition and / or activity in the gut microbiota that can benefit (or not benefit) the host. Preferred prebiotics will promote the growth or beneficial functions of the probiotic composition, but will not promote the growth or virulence-related genes (e.g., toxins) of pathogens.

[0244] As used herein, "probiotic" is understood to mean "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host," as currently defined by the WHO.

[0245] The term "species" is defined as a collection of closely related organisms that have greater than 97% 16S ribosomal RNA (rRNA) sequence homology and greater than 70% genomic hybridization, and that are sufficiently distinct from all other organisms to be recognized as a distinct unit. Species and other phylogenetic designations follow classifications known to those skilled in the art of microbiology.

[0246] As used herein, "subject" refers to a human or an animal. Typically, an animal is a vertebrate such as a primate, rodent, livestock, or game animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques (e.g., rhesus monkeys). Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Livestock and game animals include cattle, horses, pigs, deer, bison, buffalo, feline species (e.g., house cats), canine species (e.g., dogs, foxes, wolves), avian species (e.g., chickens, emperors, ostriches), and fish (e.g., trout, catfish, and salmon). In some embodiments, the subject is a mammal (e.g., a primate (e.g., a human)). The terms "individual," "patient," and "subject" are used interchangeably herein.

[0247] Preferably, the subject is a mammal. The mammal may be, but is not limited to, a human, a non-human primate, a mouse, a rat, a dog, a cat, a horse, or a cow. Non-human mammals can be advantageously used as subjects representing animal models of inflammatory and autoimmune disorders (e.g., models of intestinal barrier function). The subject may be male or female.

[0248] As used herein, the terms "treat," "treatment," "treating," and the like refer to a therapeutic procedure in which a subject reverses, alleviates, relieves, suppresses, slows, or halts the progression or severity of a condition associated with a disease or disorder (e.g., an inflammatory or autoimmune disorder). The term "treatment" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease, or disorder associated with an inflammatory or autoimmune disorder. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least slowing of the progression or worsening of symptoms compared to that expected in the absence of treatment. Beneficial or desirable clinical results include, but are not limited to, alleviation of one or more symptoms, diminished progression of the disease, stabilization of the disease state (i.e., not worsening), delay or slowing of disease progression, improvement or palliation of the disease state, remission (whether partial or total), and / or reduced mortality. The term "treating" a disease also includes providing relief from the symptoms or side effects of the disease (including palliative treatment). It is not necessary to cure the disorder (i.e., complete reversal or absence of disease) for treatment to be effective.

[0249] In some embodiments, sequencing involves 16S rRNA gene sequencing, which may also be referred to as "16S ribosomal RNA sequencing," "16S rDNA sequencing," or "16S rRNA sequencing." Sequencing of the 16S rRNA gene can be used for genetic studies because it is highly conserved among different bacterial species but is absent in eukaryotes. In addition to highly conserved regions, the 16S rRNA gene also contains nine hypervariable regions (V1-V9) that differ between species. 16S rRNA gene sequencing typically involves PCR amplification of a bacterial 16S rRNA gene region (including the hypervariable region) using multiple universal primers that bind to conserved regions of the 16S rRNA gene, and sequencing the amplified 16S rRNA gene using next-generation sequencing technology as described herein (see, e.g., U.S. Pat. Nos. 5,654,418, 6,344,316, and 8,889,358, and U.S. Patent Publication Nos. 2013 / 157,265 and 2018 / 195,111, each of which is incorporated by reference in its entirety).

[0250] Each embodiment described in this specification applies mutatis mutandis to each embodiment and each embodiment unless otherwise specified.

[0251] 2. Bacterial Strains - Alistipes Species The compositions of the present invention comprise bacterial strains of the genus Alistipes. These examples demonstrate that bacteria of this genus are useful for treating or preventing diseases associated with impaired intestinal barrier function. A preferred bacterial strain is the species A. shahii.

[0252] Alistipes is a genus of bacteria in the Clostridia class. The scientific classification is as follows: Bacteria (kingdom), Bacteroidetes (phylum), Bacteroidia (class), Bacteroidales (order), Rikenellaceae (family), and Alistipes (genus). Bacteria within the Alistipes genus are short, rod-shaped, Gram-negative, non-motile bacteria and are obligate anaerobes. These criteria are important because they can inform the phylogenetic classification of bacterial strains.

[0253] A. shahii typically grows in circular colonies of approximately 0.5 mm to 1.0 mm and is chromogenic and non-fluorescent when grown on rabbit blood agar.

[0254] The species A. shahii was first described in Song et al., 2006. The A. shahii type strain WAL 8301 (=ATCC BAA-1179) was isolated from human appendix tissue (Song et al., 2006). The GenBank accession number for the 16S rRNA gene sequence of A. shahii type strain ATCC 27749 is NR_104846.

[0255] The breadth of the genus Alistipes and the species A. shahii may be as defined by the Genome Taxonomy Database reference tree, a taxonomic classification system as described in Parks et al., 2018.

[0256] The A. shahii bacterium deposited under accession number V21 / 014432 (i.e., A. shahii MH21-1) was tested in the Examples and is one of the preferred strains of the present invention. The A. shahii strain MH21-1 was deposited by Microba IP Pty Ltd (388 Queen Street, Brisbane, Queensland 4000, Australia) with the International Depository National Measurement Institute (NMI, 1 / 153 Bertie Street, Port Melbourne, Victoria, 3207, Australia) on July 22, 2021, under the name "Alistipes shahii MH21-1," and assigned accession number V21 / 014432.

[0257] An exemplary 16S rRNA sequence for the tested A. shahii strain MH21-1 is set forth in SEQ ID NO: 1. A bacterial strain of the A. shahii species may contain a single 16S rRNA sequence within its genome, or may contain two or more 16S rRNA sequences within its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some embodiments, a bacterial strain may be identified as A. shahii strain MH21-1 by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 1 by any method known in the art. The chromosomal sequence of A. shahii strain MH211-1 is provided in SEQ ID NO: 4. This sequence was generated using the Illumina NovSeq6000 platform.

[0258] Bacterial strains closely related to strain MH21-1 are also shown in the Examples to be effective in treating or preventing inflammatory and autoimmune disorders through their beneficial effects on restoring intestinal barrier function.

[0259] For example, the A. shahii bacterium deposited under accession number V21 / 014433 (i.e., A. shahii MH23-2) was tested in the Examples and is another of the preferred strains of the present invention. An exemplary 16S rRNA sequence of the tested A. shahii strain MH212 is set forth in SEQ ID NO: 2. In some embodiments, a bacterial strain may be identified as A. shahii strain MH21-2 by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 2 by any method known in the art. A. shahii strain MH21-2 was deposited by Microba IP Pty Ltd (388 Queen Street, Brisbane, QLD 4000, Australia) with the International Depository National Metrology Institute (NMI, 1 / 153 Bertie Street, Port Melbourne, Victoria, 3207, Australia) on July 22, 2021, as "Alistipes shahii MH21-2" and assigned accession number V21 / 014433. The genome of A. shahii strain MH21-2 contains a chromosome having the sequence shown in SEQ ID NO:5.

[0260] For example, the A. shahii bacterium deposited under accession number V21 / 014434 (i.e., A. shahii MH23-3) was tested in the Examples and is another of the preferred strains of the present invention. An exemplary 16S rRNA sequence of the tested A. shahii MH21-3 strain is set forth in SEQ ID NO: 3. In some embodiments, a bacterial strain may be identified as A. shahii MH21-3 by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 3 by any method known in the art. A. shahii strain MH21-3 was deposited by Microba IP Pty Ltd (388 Queen Street, Brisbane, QLD 4000, Australia) with the International Depository National Metrology Institute (NMI, 1 / 153 Bertie Street, Port Melbourne, Victoria, 3207, Australia) on July 22, 2021, as "Alistipes shahii MH21-3" and assigned accession number V21 / 014434. The genome of A. shahii strain MH21-3 contains a chromosome having the sequence shown in SEQ ID NO:6.

[0261] An exemplary 16S rRNA sequence for the A. shahii strain MH21-6 tested in the Examples is set forth in SEQ ID NO: 44. In some embodiments, a bacterial strain may be identified as A. shahii strain MH21-6 by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 44 by any method known in the art. The genome of A. shahii strain MH21-6 comprises a chromosome having a sequence set forth in one or more of SEQ ID NOs: 38-43.

[0262] In some embodiments, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a bacterial strain of A. shahii. Preferably, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOS: 1-3 and 44. In some preferred embodiments, the bacterial strains of the invention have a 16S rRNA sequence represented by one or more of SEQ ID NOS: 1-3 and 44.

[0263] The genome of the bacterial strain may comprise a 16S rRNA sequence set forth in any one of SEQ ID NOs: 1-3 and 44.

[0264] In some embodiments, bacterial strains of the invention have a chromosome with sequence identity to SEQ ID NO: 4. In preferred embodiments, bacterial strains of the invention have a chromosome with at least 90% sequence identity to SEQ ID NO: 4 (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 4. For example, a bacterial strain of the invention may have at least 90% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or may have a chromosome having at least 95% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 98% sequence identity to SEQ ID NO:4 across 100% of SEQ ID NO:4. A particularly preferred strain of the invention is the A. shahii strain deposited under accession number V21 / 014432. This is an exemplary strain MH21-1 that has been tested in the DSS mouse model shown in the Examples and has been shown to be effective for treating disease. Accordingly, the invention provides cells, such as isolated cells, of the A. shahii strain deposited under accession number V21 / 014432 or derivatives thereof. The present invention also provides a composition comprising cells of the A. shahii strain deposited under accession number V21 / 014432 or a derivative thereof.The present invention also provides a biologically pure culture of A. shahii strain MH21-1 deposited under accession number V21 / 014432.

[0265] In some embodiments, bacterial strains of the invention have a chromosome with sequence identity to SEQ ID NO: 5. In preferred embodiments, bacterial strains of the invention have a chromosome with at least 90% sequence identity to SEQ ID NO: 5 (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 5. For example, a bacterial strain of the invention may have at least 90% sequence identity to SEQ ID NO:5 across 70% of SEQ ID NO:5, or at least 90% sequence identity to SEQ ID NO:5 across 80% of SEQ ID NO:5, or at least 90% sequence identity to SEQ ID NO:5 across 90% of SEQ ID NO:5, or at least 90% sequence identity to SEQ ID NO:5 across 100% of SEQ ID NO:5, or at least 95% sequence identity to SEQ ID NO:5 across 70% of SEQ ID NO:5, or at least 95% sequence identity to SEQ ID NO:5 across 80% of SEQ ID NO:5, or may have a chromosome having at least 95% sequence identity to SEQ ID NO:5 across 90% of SEQ ID NO:5, or at least 95% sequence identity to SEQ ID NO:5 across 100% of SEQ ID NO:5, or at least 98% sequence identity to SEQ ID NO:5 across 70% of SEQ ID NO:5, or at least 98% sequence identity to SEQ ID NO:5 across 80% of SEQ ID NO:5, or at least 98% sequence identity to SEQ ID NO:5 across 90% of SEQ ID NO:5, or at least 98% sequence identity to SEQ ID NO:5 across 100% of SEQ ID NO:5. A particularly preferred strain of the invention is the A. shahii strain deposited under accession number V21 / 014433. This is an exemplary MH21-2 strain that was tested in the DSS mouse model shown in the Examples and has been shown to be effective for treating disease. Accordingly, the invention provides cells, such as isolated cells, of the A. shahii strain deposited under accession number V21 / 014433 or derivatives thereof. The present invention also provides a composition comprising cells of the A. shahii strain deposited under accession number V21 / 014433 or a derivative thereof.The present invention also provides a biologically pure culture of A. shahii strain MH21-2 deposited under accession number V21 / 01443.

[0266] In some embodiments, bacterial strains of the invention have a chromosome with sequence identity to SEQ ID NO: 6. In preferred embodiments, bacterial strains of the invention have a chromosome with at least 90% sequence identity to SEQ ID NO: 6 (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 6. For example, a bacterial strain of the invention may have at least 90% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or may have a chromosome having at least 95% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6. A particularly preferred strain of the invention is the A. shahii strain deposited under accession number V21 / 014434. This is an exemplary strain MH21-3 that was tested in the DSS mouse model shown in the Examples and has been shown to be effective for treating disease. Accordingly, the invention provides cells, such as isolated cells, of the A. shahii strain deposited under accession number V21 / 014434 or derivatives thereof. The present invention also provides a composition comprising cells of the A. shahii strain deposited under accession number V21 / 014434 or a derivative thereof.The present invention also provides a biologically pure culture of A. shahii strain MH21-3 deposited under accession number V21 / 01444.

[0267] In some embodiments, bacterial strains of the invention have a chromosome that has sequence identity to one or more of SEQ ID NOs: 38 to 43. In preferred embodiments, bacterial strains of the invention have a chromosome that has at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to one or more of SEQ ID NOs: 38 to 43 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of one or more of SEQ ID NOs: 38 to 43.For example, the bacterial strain of the invention may have at least 90% sequence identity to one or more of SEQ ID NOs: 38-43 across 70% of one or more of SEQ ID NOs: 38-43, or at least 90% sequence identity to one or more of SEQ ID NOs: 38-43 across 80% of one or more of SEQ ID NOs: 38-43, or at least 90% sequence identity to one or more of SEQ ID NOs: 38-43 across 90% of one or more of SEQ ID NOs: 38-43, or at least 90% sequence identity to one or more of SEQ ID NOs: 38-43 across 100% of one or more of SEQ ID NOs: 38-43, or at least 95% sequence identity to one or more of SEQ ID NOs: 38-43 across 70% of one or more of SEQ ID NOs: 38-43, or at least 95% sequence identity to one or more of SEQ ID NOs: 38-43 across 80% of one or more of SEQ ID NOs: 38-43, or may have a chromosome having at least 95% sequence identity to one or more of SEQ ID NOs: 38-43 across 90% of one or more of SEQ ID NOs: 38-43, or at least 95% sequence identity to one or more of SEQ ID NOs: 38-43 across 100% of one or more of SEQ ID NOs: 38-43, or at least 98% sequence identity to one or more of SEQ ID NOs: 38-43 across 70% of one or more of SEQ ID NOs: 38-43, or at least 98% sequence identity to one or more of SEQ ID NOs: 38-43 across 80% of one or more of SEQ ID NOs: 38-43, or at least 98% sequence identity to one or more of SEQ ID NOs: 38-43 across 90% of one or more of SEQ ID NOs: 38-43, or at least 98% sequence identity to one or more of SEQ ID NOs: 38-43 across 100% of one or more of SEQ ID NOs: 38-43. In preferred embodiments of this type, the bacterial strain has the recited sequence identity across each of SEQ ID NOs: 38-43.

[0268] Derivatives of the strains deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 may be daughter strains (progeny) or strains cultured (subcloned) from the original strain. Derivatives of the strains of the present invention may be modified, for example, at the genetic level, without eliminating biological activity. In particular, derivative strains of the present invention are therapeutically active. Derivative strains have activity equivalent to that of the original V21 / 014432, V21 / 014433, or V21 / 014434 strain from which they are derived. Specifically, derivative strains elicit equivalent effects in at least one disease model (e.g., colitis), as shown in the Examples, which can be identified using the culture and administration protocols described in the Examples. A derivative of any one of strains V21 / 014432, V21 / 014433, or V21 / 014434 is generally a biotype of strain V21 / 014432, V21 / 014433, or V21 / 014434, respectively.

[0269] Reference to cells of the A. shahii strain deposited under accession number V21 / 014432 includes any cells that have the same safety and therapeutic efficacy characteristics as the strain deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, and such cells are encompassed by the present invention.

[0270] 2.1 Bacterial biotypes Bacterial strains that are biotypes of the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 are also expected to be effective for treating or preventing inflammatory and autoimmune disorders. Biotypes are closely related strains that have identical or very similar physiological and biochemical properties.

[0271] Strains suitable for use in the present invention that are biotypes of the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 can be identified by sequencing other nucleotide sequences of the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434. For example, substantially the entire genome can be sequenced, and biotype strains of the present invention can have at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity across at least 80% of their entire genome (e.g., at least 85%, 90%, 95%, or 99%, or across their entire genome). Other sequences suitable for use in identifying biotype strains include repeat sequences such as hsp60 or BOX, ERIC, (GTG)5, or REP (Masco et al., 2003; Kim et al., 2019). Biotype strains can have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity to the corresponding sequences of bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434.

[0272] Alternatively, a strain that is a bacterial biotype deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, and restriction fragment analysis and / or PCR analysis, for example, by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23S rRNA sequencing. In some preferred embodiments, such techniques can be used to identify other suitable A. shahii strains.

[0273] In certain embodiments, a strain that is a biotype of the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 and that is suitable for use in the present invention is a strain that, when analyzed by amplified ribosomal DNA restriction analysis (ARDRA), for example, using the Sau3AI restriction enzyme (see, e.g., Srutkova et al., 2011 for exemplary methods and guidance), provides the same pattern as the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434. Alternatively, a biotype strain is identified as a strain that has the same carbohydrate fermentation pattern as the bacteria deposited under any one of accession numbers V21 / 014432, V21 / 014433, or V21 / 014434.

[0274] In some embodiments, bacterial strains useful in the present invention can be identified by routinely profiling the production and consumption of metabolites by the bacterial strain. The bacterial strains described above and elsewhere herein are predicted to affect the production of acetate, butyrate, ethanol, and fumarate. Thus, in some embodiments, the bacterial strains of the present invention induce the in vivo production of one or more of the metabolites acetate, ethanol, butyrate, and fumarate. Furthermore, in some embodiments, the bacterial strains of the present invention do not produce butyrate.

[0275] Other Alistipes strains useful in the compositions and methods of the present invention, such as the bacterial biotypes deposited under accession numbers V21 / 014432, V21 / 014433, or V21 / 014434, can be identified using any suitable method or strategy, including the assays described in the Examples. For example, strains for use in the present invention can be identified by culturing them in anaerobic TY or PYG medium and / or administering them to a DSS-induced intestinal barrier function model, followed by assessing cytokine / chemokine levels as described in the Examples. In particular, bacterial strains with similar growth patterns, metabolic types, and / or surface antigens to the bacteria deposited under accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 may be useful in the present invention. Useful strains have immunomodulatory activity comparable to that of strains V21 / 014432, V21 / 014433, or V21 / 014434. In particular, biovar strains induce comparable effects on host intestinal function. Furthermore, it is expected that biovars will have similar effects in disease models (e.g., colitis, asthma, arthritis, multiple sclerosis, and uveitis disease models), have comparable effects on cytokine / chemokine levels, and can be identified using the culture and administration protocols described in the Examples.

[0276] In some embodiments, the bacterial strain may be the A. finegoldii species. The A. finegoldii species was first described in Rautio et al., 2003. The A. shahii type strain CCUG 406020(T) (=AHN2437) was isolated from human appendix tissue from a child (Rautio et al., 2003). The GenBank accession number for the 16S rRNA gene sequence of A. finegoldii type strain AHN 2437 is NR_043064.

[0277] An exemplary 16S rRNA sequence of a tested A. finegoldii strain is set forth in SEQ ID NO: 7. A bacterial strain of the A. finegoldii species may contain a single 16S rRNA sequence in its genome, or may contain two or more 16S rRNA sequences in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some embodiments, a bacterial strain may be identified as an A. finegoldii strain by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 7 by any method known in the art. The chromosomal sequence of an A. finegoldii strain is provided in SEQ ID NO: 9. This sequence was obtained from NCBI Genbank accession number CP003274, which is associated with A. finegoldii strain DSM 17242.

[0278] Bacterial strains of the A. finegoldii species and closely related strains of A. finegoldii are believed to be effective for treating or preventing inflammatory and autoimmune disorders (e.g., IBD) through their beneficial effects on restoring intestinal barrier function.

[0279] The species A. onderdonkii was first described in Song et al., 2006. The A. onderdonkii type strain WAL 8169 (=ATCC BAA-1178) was isolated from human appendix tissue (Song et al., 2006). The GenBank accession number for the 16S rRNA gene sequence of A. onderdonkii type strain WAL 8169 is NR_043318.1.

[0280] The A. shahii onderdonkii species breadth can be as defined by the Genome Taxonomic Database Reference Tree, a taxonomic classification system as described in Parks et al., 2018.

[0281] An exemplary 16S rRNA sequence of an A. onderdonkii strain is set forth in SEQ ID NO: 8. A bacterial strain of the A. shahii species may contain a single 16S rRNA sequence in its genome, or may contain two or more 16S rRNA sequences in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some embodiments, a bacterial strain may be identified as an A. onderdonkii strain by determining whether the strain contains a 16S rRNA sequence that highly corresponds to SEQ ID NO: 8 by any method known in the art. The chromosomal sequence of an A. onderdonkii strain is provided in SEQ ID NO: 10. This sequence was obtained from NCBI Genbank accession number NR_043318.1, which is associated with A. onderdonkii strain WAL 8169.

[0282] Bacterial strains of the A. onderdonkii species and closely related strains of A. onderdonkii are believed to be effective for treating or preventing inflammatory and autoimmune disorders (e.g., IBD) through their beneficial effects on restoring intestinal barrier function.

[0283] 2.2 Viability of bacterial strains. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable and can partially or completely colonize the intestine. In some preferred embodiments, the bacterial strain in the composition of the present invention is live. For example, the bacterial strain in the composition of the present invention is not heat-killed. The bacteria of the present invention can have immunomodulatory effects that would not be exhibited by non-viable bacteria, for example, because non-viable bacteria cannot produce metabolites and cannot interact with the immune system in a different manner. The cell surface of live bacteria also appears to be significantly different from that of dead bacteria, especially heat-killed bacteria.

[0284] In some alternative embodiments, the bacteria are non-viable, for example, in some embodiments, the bacteria are heat-killed.

[0285] In some preferred embodiments, bacterial strains for use in the present invention are naturally occurring, for example, the bacterial strains are isolated from the digestive tract of a mammal.

[0286] In some preferred embodiments, bacterial strains for use in the present invention are not genetically engineered, e.g., they are not transformed with recombinant DNA.

[0287] 3. Composition - Alistipes species The compositions provided herein are compositions comprising, consisting of, or consisting essentially of a therapeutically effective amount of the bacterial strain or strains described above and / or elsewhere herein. In some embodiments, the bacteria in the composition can be identified by strain, species, operational taxonomic unit (OTU), whole genome sequence, 16S rRNA sequence, or other method known in the art for defining different types of bacteria.

[0288] 3.1 Most Recent Common Ancestor (MRCA) In some embodiments, the composition comprises an effective amount of a bacterial strain that is a phylogenetic descendant of the MRCA of A. timonensis and A. sp000434235 (see Figure 3b). Preferably, the phylogenetic classification is as defined by the GTDB (Parks et al., 2018). In some embodiments, the phylogenetic classification is as defined in release 89 (r89) of the GTDB.

[0289] In some embodiments, determining whether a bacterial strain is a descendant of the MRCA of A. timonensis and A. sp000434235 can be performed using phylogenetic grouping procedures known in the art. In some embodiments, a rooted phylogenetic tree containing A. timonensis and A. sp000434235 and a third taxon of interest (e.g., a classified taxon) can be used, and the following analytical packages can be applied to determine whether the taxon of interest is useful in the compositions of the invention: Analysis of Phylogenetic and Evolution ("ape"; https: / / cran.r-project.org / web / packages / ape / index) and Phylogenetic Tools for Comparative Biology ("phytools"; http: / / cran.r-project.org / web / packages / phytools / index.html). Both ape and phylogenomics are packages written in the R programming language and are used in molecular evolution and phylogenetic studies. The apes and phylogenetic tools package provides methods for phylogenetic and evolutionary analysis, the use of which is known to those skilled in the art. In some embodiments, the following scripts can be used: library("ape") library("phytools") input.tree=read.tree(file=”tree_file”) alistip c(“s__Alistipes sp900083545”,“s__Alistipes sp000434235”) alistip.node=getMRCA(input.tree,alistip) alistip.tree=extract.clade(input.tree,alisip.node) print(alistip.tree$tip.label)

[0290] In some embodiments, after the script is run, if the taxon of interest is in the printed list, it is the descendant of the MRCA of the two species.

[0291] In other embodiments, different phylogenetic grouping methods known in the art can be used to determine whether a bacterial strain is descended from the MRCA of A. timonensis and A. sp000434235 using different analytical packages, including methods based on different programming languages ​​(see Figure 3B).

[0292] In some other embodiments, the bacterial strain, together with a pharmaceutically acceptable carrier, diluent, or excipient, is a phylogenetic descendant of the MRCA of A. timonensis and A. sp000434235. Preferably, the MRCA is defined at node 35260 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated by release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0293] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of A. timonensis and A. finegoldii, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 35261 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0294] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of A. timonensis and A. shahii, along with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 35261 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0295] In some preferred embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of A. shahii and A. finegoldii, along with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined as a node of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0296] In some embodiments, the compositions disclosed herein are substantially free of any bacteria of the A. senegalensis species. In some embodiments, the bacterial strain is not of the A. senegalensis species.

[0297] 3.2 16S rRNA sequence identity. In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the genus Alistipes. In some embodiments, the query 16S rRNA sequence is compared to the 16S rRNA sequence set forth in SEQ ID NO: 1. In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the genus Alistipes. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the genus Alistipes. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of the genus Alistipes.

[0298] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 97.5%. In some embodiments, the threshold sequence identity is 99.0%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0 ... 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0299] In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the Rikenellaceae family (including those set forth in any one of SEQ ID NOS: 1-3). In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the Rikenellaceae family. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the Rikenellaceae family. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of that family.

[0300] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 98.7%. In some embodiments, the threshold sequence identity is 94.8%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98 ...1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0301] In some embodiments, the composition comprises an at least partially isolated bacterial strain of A. shahii described above and / or elsewhere herein.

[0302] In some embodiments, the bacterial strains of the present invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of an A. shahii bacterial strain. Preferably, the bacterial strains of the present invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOS: 1-3. In some preferred embodiments, the bacterial strains of the present invention have a 16S rRNA sequence represented by any one of SEQ ID NOS: 1-3.

[0303] The genome of the bacterial strain may comprise a 16S rRNA sequence shown in any one of SEQ ID NOs: 1-3.

[0304] 3.3 Genomic sequence identity. In some embodiments, the bacterial strain of the present invention has a chromosome having sequence identity to any one of SEQ ID NOs: 4 to 6. In preferred embodiments, the bacterial strain of the present invention has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to any one of SEQ ID NOs: 4 to 6 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NOs: 4 to 6. For example, the bacterial strain of the present invention may have at least 90% sequence identity to any one of SEQ ID NOs: 4 to 6 across 70% of SEQ ID NOs: 4 to 6, or at least 90% sequence identity to any one of SEQ ID NOs: 4 to 6 across 80% of SEQ ID NOs: 4 to 6, or at least 90% sequence identity to any one of SEQ ID NOs: 4 to 6 across 90% of SEQ ID NOs: 4 to 6, or at least 90% sequence identity to any one of SEQ ID NOs: 4 to 6 across 100% of SEQ ID NOs: 4 to 6, or at least 95% sequence identity to any one of SEQ ID NOs: 4 to 6 across 70% of SEQ ID NOs: 4 to 6, or at least 95% sequence identity to any one of SEQ ID NOs: 4 to 6 across 80% of SEQ ID NOs: 4 to 6, or The strain may have a chromosome having at least 95% sequence identity to any one of SEQ ID NOS: 4-6 across 90% of SEQ ID NOS: 4-6, or at least 95% sequence identity to any one of SEQ ID NOS: 4-6 across 100% of SEQ ID NOS: 4-6, or at least 98% sequence identity to any one of SEQ ID NOS: 4-6 across 70% of SEQ ID NOS: 4-6, or at least 98% sequence identity to any one of SEQ ID NOS: 4-6 across 80% of SEQ ID NOS: 4-6, or at least 98% sequence identity to any one of SEQ ID NOS: 4-6 across 90% of SEQ ID NOS: 4-6, or at least 98% sequence identity to any one of SEQ ID NOS: 4-6 across 100% of SEQ ID NOS: 4-6. A particularly preferred strain of the present invention is the A. shahii strain deposited under accession number V21 / 014432.This is an exemplary A. shahii strain MH21-1 that has been tested in the DSS mouse model shown in the Examples and shown to be effective for treating the disease. Accordingly, the present invention provides cells, such as isolated cells, of the A. shahii strain deposited under accession number V21 / 014432 or a derivative thereof. The present invention also provides compositions comprising cells of the A. shahii strain deposited under accession number V21 / 014432 or a derivative thereof. The present invention also provides biologically pure cultures of the A. shahii strain MH21-1 deposited under accession number V21 / 014432.

[0305] The present invention also provides a composition comprising cells of the A. shahii strain deposited under accession number V21 / 014433 or a derivative thereof. The present invention also provides a biologically pure culture of the A. shahii strain MH21-1 deposited under accession number V21 / 014433.

[0306] The present invention also provides a composition comprising cells of the A. shahii strain deposited under accession number V21 / 014434 or a derivative thereof. The present invention also provides a biologically pure culture of the A. shahii strain MH21-1 deposited under accession number V21 / 014434.

[0307] In certain embodiments, compositions of the invention comprise at least a partially purified strain of A. finegoldii. In some embodiments, the strain of A. finegoldii has a chromosome having sequence identity to SEQ ID NO: 9. In preferred embodiments, the bacterial strain of A. finegoldii has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to SEQ ID NO: 9 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 9. For example, a bacterial strain of A. finegoldii may have at least 90% sequence identity to SEQ ID NO:9 across 70% of SEQ ID NO:9, or at least 90% sequence identity to SEQ ID NO:9 across 80% of SEQ ID NO:9, or at least 90% sequence identity to SEQ ID NO:9 across 90% of SEQ ID NO:9, or at least 90% sequence identity to SEQ ID NO:9 across 100% of SEQ ID NO:5, or at least 95% sequence identity to SEQ ID NO:9 across 70% of SEQ ID NO:9, or at least 95% sequence identity to SEQ ID NO:9 across 80% of SEQ ID NO:9. or at least 95% sequence identity to SEQ ID NO:9 across 90% of SEQ ID NO:9, or at least 95% sequence identity to SEQ ID NO:9 across 100% of SEQ ID NO:9, or at least 98% sequence identity to SEQ ID NO:9 across 70% of SEQ ID NO:9, or at least 98% sequence identity to SEQ ID NO:9 across 80% of SEQ ID NO:9, or at least 98% sequence identity to SEQ ID NO:9 across 90% of SEQ ID NO:9, or at least 98% sequence identity to SEQ ID NO:9 across 100% of SEQ ID NO:9.

[0308] In certain embodiments, compositions of the invention comprise at least a partially purified strain of A. onderdonkii. In some embodiments, the strain of A. onderdonkii has a chromosome having sequence identity to SEQ ID NO: 10. In preferred embodiments, the bacterial strain of A. onderdonkii has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to SEQ ID NO: 10 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 10. For example, a bacterial strain of A. onderdonkii may have at least 90% sequence identity to SEQ ID NO:10 across 70% of SEQ ID NO:10, or at least 90% sequence identity to SEQ ID NO:10 across 80% of SEQ ID NO:10, or at least 90% sequence identity to SEQ ID NO:10 across 90% of SEQ ID NO:10, or at least 90% sequence identity to SEQ ID NO:10 across 100% of SEQ ID NO:10, or at least 95% sequence identity to SEQ ID NO:10 across 70% of SEQ ID NO:10, or at least 95% sequence identity to SEQ ID NO:10 across 80% of SEQ ID NO:10. or at least 95% sequence identity to SEQ ID NO:10 across 90% of SEQ ID NO:10, or at least 95% sequence identity to SEQ ID NO:10 across 100% of SEQ ID NO:10, or at least 98% sequence identity to SEQ ID NO:10 across 70% of SEQ ID NO:10, or at least 98% sequence identity to SEQ ID NO:10 across 80% of SEQ ID NO:10, or at least 98% sequence identity to SEQ ID NO:10 across 90% of SEQ ID NO:10, or at least 98% sequence identity to SEQ ID NO:10 across 100% of SEQ ID NO:10.

[0309] 4. Bacterial strain - Colonithrix sana The compositions of the present invention comprise bacterial strains of the genus Colonithrix. These examples demonstrate that bacteria of this genus are useful for treating or preventing diseases associated with impaired intestinal barrier function. A preferred bacterial strain is the species Colonithrix sana.

[0310] Colonithrix is ​​a genus of bacteria in the Clostridia class. The scientific classification is as follows: Bacteria (kingdom), Firmicutes_A (phylum), Clostridia (class), Oscillospirales (order), Oscillospiraceae (family), and Colonithrix (genus). Bacteria within the Colonithrix genus are Gram-negative to Gram-variable, non-motile, long, thin, rod-shaped, and obligate anaerobes. These criteria are important because they can inform the phylogenetic classification of bacterial strains. For example, the bacterial species C. sana has previously been classified as belonging to the genus ER and / or Ruminococcaceae based on these criteria, among others.

[0311] The breadth of the genus Colonithrix and the species C. sana is as defined by the Genome Taxonomic Database Reference Tree, a taxonomic classification system as described in Parks et al., 2018.

[0312] The C. sana bacterium deposited under accession number V21 / 019213 (i.e., C. sana MH35-1) was tested in the Examples and is one of the preferred strains of the present invention. C. sana strains MH35-1 and MH35-2 were deposited by Microba IP Pty Ltd (388 Queen Street, Brisbane, QLD 4000, Australia) with the International Depository National Measurement Institute (NMI, 1 / 153 Bertie Street, Port Melbourne, Victoria, 3207, Australia) on September 28, 2021 as "Colonithrix sana MH35-1" and "Colonithrix sana MH35-2," and were assigned accession numbers V21 / 019213 and V21 / 019214, respectively.

[0313] An exemplary 16S rRNA sequence of the tested C. sana MH35-1 strain is set forth in any one of SEQ ID NOS: 11-14. A bacterial strain of the species may contain a single 16S rRNA sequence within its genome, or may contain two or more 16S rRNA sequences within its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some preferred embodiments, a bacterial strain of the C. sana species contains four copies of a 16S rRNA sequence within its genome. In some embodiments, a bacterial strain may be identified as C. sana MH35-1 by determining whether the strain contains a 16S rRNA sequence corresponding to any one of SEQ ID NOS: 11-14 by any method known in the art. The chromosomal sequence of the C. sana MH35-1 strain is provided in one or more of SEQ ID NOS: 19-22. In some embodiments, the chromosomal sequence of the C. sana MH35-1 strain includes each of the sequences set forth in SEQ ID NOs: 19 to 22. These sequences were generated using the Illumina NovSeq6000 platform.

[0314] An exemplary 16S rRNA sequence of the tested C. sana MH35-2 strain is set forth in any one of SEQ ID NOS: 15-18. A bacterial strain of the species may contain a single 16S rRNA sequence within its genome, or may contain two or more 16S rRNA sequences within its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some preferred embodiments, a bacterial strain of the C. sana species contains four copies of a 16S rRNA sequence within its genome. In some embodiments, a bacterial strain may be identified as C. sana MH35-2 by determining whether the strain contains a 16S rRNA sequence corresponding to any one of SEQ ID NOS: 15-18 by any method known in the art. The chromosomal sequence of the C. sana MH35-1 strain is provided in one or more of SEQ ID NOS: 23-26. In some embodiments, the chromosomal sequence of the C. sana MH35-2 strain includes each of the sequences set forth in SEQ ID NOs: 23 to 26. These sequences were generated using the Illumina NovSeq6000 platform.

[0315] Bacterial strains closely related to C. sana strains MH35-1 and MH35-2 are also believed to be effective for treating or preventing inflammatory and autoimmune disorders through their beneficial effects on restoring intestinal barrier function.

[0316] In some embodiments, the bacterial strains of the present invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a C. sana bacterial strain. Preferably, the bacterial strains of the present invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOS: 11-18. In some of these same preferred embodiments, the bacterial strains of the present invention have four copies of the 16S rRNA sequence represented by at least one, two, three, or all of SEQ ID NOS: 11-14. In some other preferred embodiments, the bacterial strain of the invention has four copies of the 16S rRNA sequence represented by at least one, two, three, or all of SEQ ID NOs: 15-18.

[0317] The genome of the bacterial strain may comprise each of the 16S rRNA sequences shown in SEQ ID NOs: 11 to 14. Alternatively, the genome of the bacterial strain may comprise each of the 16S rRNA sequences shown in SEQ ID NOs: 15 to 18.

[0318] In some embodiments, the bacterial strain of the present invention has a chromosome having sequence identity to any one of SEQ ID NOs: 19-22 or 23-26. In a preferred embodiment, the bacterial strain of the present invention has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to any one of SEQ ID NOs: 19-22 or 23-26 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of any one of SEQ ID NOs: 19-22 or 23-26. For example, the bacterial strain of the present invention may have at least 90% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 70% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 90% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 80% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 90% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 90% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 90% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 100% of any one of SEQ ID NOs: 19-22 or 23-26. 0% sequence identity, or at least 95% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 70% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 95% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 80% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 95% sequence identity to any one of SEQ ID NOs: 15-18 across 90% of any one of SEQ ID NOs: 19-22 or 23-26, or at least 95% sequence identity to any one of SEQ ID NOs: 19-22 or 23-26 across 100% of any one of SEQ ID NOs: 19-22 or 23-26,or a chromosome having at least 98% sequence identity to any one of SEQ ID NOS: 19-22 or 23-26 across 70% of any one of SEQ ID NOS: 19-22 or 23-26, or at least 98% sequence identity to any one of SEQ ID NOS: 19-22 or 23-26 across 80% of any one of SEQ ID NOS: 19-22 or 23-26, or at least 98% sequence identity to any one of SEQ ID NOS: 19-22 or 23-26 across 90% of any one of SEQ ID NOS: 19-22 or 23-26, or at least 98% sequence identity to any one of SEQ ID NOS: 19-22 or 23-26 across 100% of any one of SEQ ID NOS: 19-22 or 23-26. Particularly preferred strains of the present invention are C. sana strains deposited under accession numbers V21 / 019213 and / or V21 / 019214. These are exemplary C. sana MH35-1 and C. sana MH35-2 strains that have been tested in the DSS mouse model shown in the Examples and shown to be effective for treating the disease. Accordingly, the present invention provides cells, such as isolated cells, of the C. sana strain deposited under accession numbers V21 / 019213 and / or V21 / 019214 or derivatives thereof. The present invention also provides compositions comprising cells of the C. sana strain deposited under accession numbers V21 / 019213 and / or V21 / 019214 or derivatives thereof. The present invention also provides biologically pure cultures of the C. sana MH35-1 and MH35-2 strains deposited under accession numbers V21 / 019213 and V21 / 019214, respectively.

[0319] Derivatives of the strains deposited under accession numbers V21 / 019213 or V21 / 019214 may be daughter strains (progeny) or strains cultured (subcloned) from the original strain. Derivatives of the strains of the present invention may be modified, for example, at the genetic level, without eliminating biological activity. In particular, derivative strains of the present invention are therapeutically active. Derivative strains have activity equivalent to that of the original V21 / 019213 or V21 / 019214 strain from which they are derived. Specifically, derivative strains elicit equivalent effects in at least one disease model (e.g., colitis), as shown in the Examples, which can be identified using the culture and administration protocols described in the Examples. Derivatives of any one of the V21 / 019213 or V21 / 019214 strains are generally biotypes of the V21 / 019213 and V21 / 019214 strains, respectively.

[0320] Reference to cells of the C. sana strain deposited under accession number V21 / 019213 or V21 / 019214 includes any cells that have the same safety and therapeutic efficacy characteristics as the strain deposited under accession number V21 / 019213 or V21 / 019214, and such cells are encompassed by the present invention.

[0321] 4.1 Bacterial biotypes Bacterial strains that are biotypes of the bacteria deposited under accession numbers V21 / 019213 or V21 / 019214 are also expected to be effective for treating or preventing inflammatory and autoimmune disorders. Biotypes are closely related strains that have identical or very similar physiological and biochemical properties.

[0322] Strains suitable for use in the present invention that are biotypes of the bacteria deposited under accession numbers V21 / 019213 or V21 / 019214 can be identified by sequencing other nucleotide sequences of the bacteria deposited under accession numbers V21 / 019213 or V21 / 019214. For example, substantially the entire genome can be sequenced, and biotype strains of the present invention can have at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity across at least 80% of their entire genome (e.g., at least 85%, 90%, 95%, or 99%, or across their entire genome). Other sequences suitable for use in identifying biotype strains include repeat sequences such as hsp60 or BOX, ERIC, (GTG)5, or REP (Masco et al., 2003; Kim et al., 2019). A biotype strain may have a sequence that has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number V21 / 019213 or V21 / 019214.

[0323] Alternatively, strains that are biotypes of the bacteria deposited under accession numbers V21 / 019213 or V21 / 019214, and restriction fragment analysis and / or PCR analysis, for example, by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23S rRNA sequencing. In some preferred embodiments, such techniques can be used to identify other suitable C. sana strains.

[0324] In certain embodiments, strains that are biotypes of the bacteria deposited under accession number V21 / 019213 or V21 / 019214 and that are suitable for use in the present invention are strains that provide the same pattern as the bacteria deposited under accession number V21 / 019213 or V21 / 019214 when analyzed by amplified ribosomal DNA restriction analysis (ARDRA), e.g., using the Sau3AI restriction enzyme (see, e.g., Srutkova et al., 2011, for exemplary methods and guidance). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation pattern as the bacteria deposited under accession number V21 / 019213 or V21 / 019214.

[0325] In some embodiments, bacterial strains useful in the present invention can be identified by routinely profiling the production and consumption of metabolites by the bacterial strain. The bacterial strains described above and elsewhere herein are predicted to affect the production of acetate, butyrate, ethanol, and fumarate. Thus, in some embodiments, the bacterial strains of the present invention induce the in vivo production of one or more of the metabolites acetate, ethanol, butyrate, and fumarate. Furthermore, in some embodiments, the bacterial strains of the present invention do not produce butyrate.

[0326] Other Colonithrix strains useful in the compositions and methods of the present invention, such as the bacterial biotypes deposited under accession numbers V21 / 019213 and / or V21 / 019214, can be identified using any suitable method or strategy, including the assays described in the Examples. For example, strains for use in the present invention can be identified by culturing them in anaerobic TY or PYG medium and / or administering them to a DSS-induced intestinal barrier function model and then assessing cytokine / chemokine levels as described in the Examples. In particular, bacterial strains with similar growth patterns, metabolic types, and / or surface antigens to the bacteria deposited under accession numbers V21 / 019213 and / or V21 / 019214 may be useful in the present invention. Useful strains have immunomodulatory activity equivalent to that of the V21 / 019213 and / or V21 / 019214 strains. In particular, biotype strains cause equivalent effects on host intestinal function. Furthermore, it is expected that biotypes having similar effects in disease models (e.g., colitis, asthma, arthritis, multiple sclerosis, and uveitis disease models) and comparable effects on cytokine / chemokine levels to those shown in the Examples can be identified by using the culture and administration protocols described in the Examples.

[0327] 4.2 Viability of bacterial strains. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable and can partially or completely colonize the intestine. In some preferred embodiments, the bacterial strain in the composition of the present invention is live. For example, the bacterial strain in the composition of the present invention is not heat-killed. The bacteria of the present invention can have immunomodulatory effects that would not be exhibited by non-viable bacteria, for example, because non-viable bacteria cannot produce metabolites and cannot interact with the immune system in a different manner. The cell surface of live bacteria also appears to be significantly different from that of dead bacteria, especially heat-killed bacteria.

[0328] In some alternative embodiments, the bacteria are non-viable, for example, in some embodiments, the bacteria are heat-killed.

[0329] In some preferred embodiments, bacterial strains for use in the present invention are naturally occurring, for example, the bacterial strains are isolated from the digestive tract of a mammal.

[0330] In some preferred embodiments, bacterial strains for use in the present invention are not genetically engineered, e.g., they are not transformed with recombinant DNA.

[0331] 5. Composition - Colonithrix species The compositions provided herein are compositions comprising, consisting of, or consisting essentially of a therapeutically effective amount of the bacterial strain or strains described above and / or elsewhere herein. In some embodiments, the bacteria in the composition can be identified by strain, species, operational taxonomic unit (OTU), whole genome sequence, 16S rRNA sequence, or other method known in the art for defining different types of bacteria.

[0332] 5.1 Most Recent Common Ancestor (MRCA) In some embodiments, the composition comprises an effective amount of a bacterial strain that is a phylogenetic descendant of the MRCA of C. sp003522105 and C. sp002437735 (see Figure 8). Preferably, the phylogenetic classification is as defined by the GTDB (Parks et al., 2018). In some embodiments, the phylogenetic classification is as defined in release 89 (r89) of the GTDB.

[0333] In some embodiments, determining whether a bacterial strain is a descendant of the MRCA of C. sp003522105 and C. sp002437735 can be performed using phylogenetic grouping procedures known in the art. In some embodiments, a rooted phylogenetic tree containing C. sp003522105, C. sp002437735, and a third taxon of interest (e.g., a classified taxon) can be used, and the following analytical packages can be applied to determine whether the taxon of interest is useful in the compositions of the invention: Phylogenetic and Evolutionary Analysis (ape: https: / / cran.r-project.org / web / packages / ape / index.html) and Phylogenetic Tools for Comparative Biology ("phytools"; http: / / cran.r-project.org / web / packages / phytools / index.html). Both ape and phylogenetic are packages written in the R programming language and are used in molecular evolution and phylogenetic studies. The apes and phylogenetic tools package provides methods for phylogenetic and evolutionary analysis, the use of which is known to those skilled in the art. In some embodiments, the following scripts can be used: library("ape") library("phytools") input.tree=read.tree(file=”tree_file”) colonithrix=c(“s__C.sp003522105”,“s__C.sp002437735”)) colonithrix.node=getMRCA(input.tree, colonithrix) colonithrix.tree=extract.clade(input.tree, colonithrix.node) print(colonithrix.tree$tip.label)

[0334] In some embodiments, after the script is run, if the taxon of interest is in the printed list, it is the descendant of the MRCA of the two species.

[0335] In other embodiments, different phylogenetic grouping methods known in the art can be used to determine whether a bacterial strain is descended from the MRCA of C.sp003522105 and C.sp002437735 using different analytical packages, including methods based on different programming languages ​​(see Figure 8).

[0336] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of C.sp003522105 and C.sp002437735, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23879 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0337] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of C.sp003522105 and C.sp002437735, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23820 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0338] In other embodiments, a bacterial species is a member of the family Oscillospiraceae if the species has a 16S rRNA sequence that has sequence identity to a 16S rRNA sequence from a species previously identified as a member of the family Oscillospiraceae. In some embodiments, identifying whether a bacterial species is a member of the family Oscillospiraceae is performed using the methods described in Yarza et al., 2014, Nature Reviews Microbiology 12:635-645 and Stackebrandt, E. & Ebers, J., 2006, Microbiol. Today 8:6-9, which are incorporated herein by reference.

[0339] 5.2 16S rRNA sequence identity In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the genus Colonithrix. In some embodiments, the query 16S rRNA sequence is compared to the 16S rRNA sequence set forth in any one of SEQ ID NOS: 11-18. In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the genus Colonithrix. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the genus Colonithrix. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of the genus Colonithrix.

[0340] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 97.5%. In some embodiments, the threshold sequence identity is 99.0%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0 ... 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0341] In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the Oscillospiraceae family. In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the Oscillospiraceae family. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the Oscillospiraceae family. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of the Oscillospiraceae family.

[0342] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 98.7%. In some embodiments, the threshold sequence identity is 94.8%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98 ...1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100%.

[0343] 6. Bacterial Strains - Gemmiger Species The compositions of the present invention comprise bacterial strains of the genus Gemmiger. These examples demonstrate that bacteria of this genus are useful for treating or preventing diseases associated with impaired intestinal barrier function. Some of the preferred bacterial strains are the species G. formicilis.

[0344] Gemmiger is a genus of bacteria in the Clostridia class. The scientific classification is as follows: Bacteria (kingdom), Firmicutes (phylum), Clostridia (class), Clostridiales (order), Ruminococcaceae (family), and Gemmiger (genus). Bacteria within the genus Gemmiger are Gram-negative to Gram-variable, non-motile, have a "budding" morphology, and are obligate anaerobes. These criteria are important because they can inform the phylogenetic classification of bacterial strains. For example, the bacterial species G. formicilis has previously been classified as belonging to the genus Subdoligranulum based on these criteria, among others.

[0345] G. formicilis strains (previously characterized as Subdoligranulum formicilis) were described in Gossling and Moore, 1975. The G. formicilis type strain, Virginia Polytechnic Institute strain X2-56 (=ATCC 27749), was isolated from human feces (Gossling and Moore, 1975). The GenBank accession number for the 16S rRNA gene sequence of G. formicilis type strain ATCC 27749 is NR_104846.

[0346] The breadth of the genus Gemmiger and the species G. formicilis is as defined by the Genome Taxonomic Database Reference Tree, a taxonomic classification system as described in Parks et al., 2018.

[0347] The G. formicilis bacterium deposited under accession number V21 / 011520 (i.e., G. formicilis MH32-1) was tested in the Examples and is one of the preferred strains of the present invention. G. formicilis strain MH32-1 was deposited by Microba IP Pty Ltd (388 Queen Street, Brisbane, QLD 4000, Australia) with the International Depository National Measurement Institute (NMI, 1 / 153 Bertie Street, Port Melbourne, Victoria, 3207, Australia) on June 11, 2021, as "Gemmiger formicilis MH32-1," and was assigned accession number V21 / 011520.

[0348] An exemplary 16S rRNA sequence of the tested strain MH321 is shown in SEQ ID NO: 27. A bacterial strain of the G. formicilis species may contain a single 16S rRNA sequence in its genome, or may contain two or more 16S rRNA sequences in its genome (e.g., two copies, three copies, four copies, five copies, six copies, seven copies, eight copies, or more than eight copies). In some embodiments, a bacterial strain may be identified as G. formicilis strain MH32-1 by determining whether the strain contains a 16S rRNA sequence corresponding to SEQ ID NO: 27 by any method known in the art. The chromosomal sequence of strain MH32-1 is provided in SEQ ID NO: 28. This sequence was generated using the Illumina NovSeq6000 platform.

[0349] Bacterial strains closely related to G. formicilis strain MH32-1 and / or G. formicilis MH32-2 are also believed to be effective for treating or preventing inflammatory and autoimmune disorders through their beneficial effects on restoring intestinal barrier function.

[0350] In some embodiments, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of a G. formicilis bacterial strain. Preferably, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 27 or one or more of SEQ ID NOs: 45-47. In some preferred embodiments, the bacterial strains of the invention have a 16S rRNA sequence represented by SEQ ID NO: 27 or one or more of SEQ ID NOs: 45-47.

[0351] The genome of the bacterial strain may comprise the 16S rRNA sequence set forth in SEQ ID NO: 27. Alternatively, the genome of the bacterial strain may comprise the 16S rRNA sequence set forth in one or more of SEQ ID NOs: 45-47.

[0352] In some embodiments, bacterial strains of the invention have a chromosome with sequence identity to SEQ ID NO: 28. In preferred embodiments, bacterial strains of the invention have a chromosome with at least 90% sequence identity to SEQ ID NO: 28 (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of SEQ ID NO: 28. For example, a bacterial strain of the invention may have at least 90% sequence identity to SEQ ID NO:28 across 70% of SEQ ID NO:28, or at least 90% sequence identity to SEQ ID NO:28 across 80% of SEQ ID NO:28, or at least 90% sequence identity to SEQ ID NO:28 across 90% of SEQ ID NO:28, or at least 90% sequence identity to SEQ ID NO:28 across 100% of SEQ ID NO:28, or at least 95% sequence identity to SEQ ID NO:28 across 70% of SEQ ID NO:28, or at least 95% sequence identity to SEQ ID NO:28 across 80% of SEQ ID NO:28, or may have a chromosome having at least 95% sequence identity to SEQ ID NO:28 across 90% of SEQ ID NO:28, or at least 95% sequence identity to SEQ ID NO:28 across 100% of SEQ ID NO:28, or at least 98% sequence identity to SEQ ID NO:28 across 70% of SEQ ID NO:28, or at least 98% sequence identity to SEQ ID NO:28 across 80% of SEQ ID NO:28, or at least 98% sequence identity to SEQ ID NO:28 across 90% of SEQ ID NO:28, or at least 98% sequence identity to SEQ ID NO:28 across 100% of SEQ ID NO:28. A particularly preferred strain of the invention is the G. formicilis strain deposited under accession number V21 / 011520. This is an exemplary G. formicilis strain MH32-1 that was tested in the DSS mouse model shown in the Examples and shown to be effective for treating disease. Accordingly, the invention provides cells, such as isolated cells, of the G. formicilis strain deposited under accession number V21 / 011520 or derivatives thereof.The present invention also provides a composition comprising cells of the G. formicilis strain deposited under accession number V21 / 011520 or a derivative thereof. The present invention also provides a biologically pure culture of the G. formicilis strain MH32-1 deposited under accession number V21 / 011520.

[0353] In some embodiments, the bacterial strain of the invention has a chromosome having sequence identity to any one of SEQ ID NOs: 29 to 33. In preferred embodiments, the bacterial strain of the invention has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to any one of SEQ ID NOs: 29 to 33 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of any one of SEQ ID NOs: 29 to 33.For example, the bacterial strain of the present invention may have a sequence identity of at least 90% to any one of SEQ ID NOs: 29 to 33 across 70% of any one of SEQ ID NOs: 29 to 33, or at least 90% to any one of SEQ ID NOs: 29 to 33 across 80% of any one of SEQ ID NOs: 29 to 33, or at least 90% to any one of SEQ ID NOs: 29 to 33 across 90% of any one of SEQ ID NOs: 29 to 33, or at least 90% to any one of SEQ ID NOs: 29 to 33 across 100% of any one of SEQ ID NOs: 29 to 33, or at least 95% to any one of SEQ ID NOs: 29 to 33 across 70% of any one of SEQ ID NOs: 29 to 33, or at least 95% to any one of SEQ ID NOs: 29 to 33 across 80% of any one of SEQ ID NOs: 29 to 33, or The present invention may have a chromosome having at least 95% sequence identity to any one of SEQ ID NOs: 29-33 across 90% of any one of SEQ ID NOs: 29-33, or at least 95% sequence identity to any one of SEQ ID NOs: 29-33 across 100% of any one of SEQ ID NOs: 29-33, or at least 98% sequence identity to any one of SEQ ID NOs: 29-33 across 70% of any one of SEQ ID NOs: 29-33, or at least 98% sequence identity to any one of SEQ ID NOs: 29-33 across 80% of any one of SEQ ID NOs: 29-33, or at least 98% sequence identity to any one of SEQ ID NOs: 29-33 across 90% of any one of SEQ ID NOs: 29-33, or at least 98% sequence identity to any one of SEQ ID NOs: 29-33 across 100% of any one of SEQ ID NOs: 29-33.

[0354] In some embodiments, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to the 16S rRNA sequence of the bacterial strain Gemmiger sp. MD158. Preferably, the bacterial strains of the invention have a 16S rRNA sequence that is at least 97.5%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 48. In some preferred embodiments, the bacterial strains of the invention have a 16S rRNA sequence set forth in SEQ ID NO: 48.

[0355] The genome of the bacterial strain may comprise the 16S rRNA sequence shown in SEQ ID NO:48.

[0356] In some other embodiments, the bacterial strain of the present invention is a bacterial strain of the species Gemmiger sp. MD158. As an example, the bacterial strain may have a chromosome having sequence identity to any one of SEQ ID NOs: 34-38. In a preferred embodiment, the bacterial strain of the present invention has a chromosome having at least 90% sequence identity (e.g., at least 92%, 94%, 95%, 96%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence identity) to any one of SEQ ID NOs: 29-33 over at least 60% (e.g., at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%, or 100%) of any one of SEQ ID NOs: 34-38.For example, the bacterial strain of the present invention may have at least 90% sequence identity to any one of SEQ ID NOs: 34 to 38 across 70% of any one of SEQ ID NOs: 34 to 38, or at least 90% sequence identity to any one of SEQ ID NOs: 34 to 38 across 80% of any one of SEQ ID NOs: 34 to 38, or at least 90% sequence identity to any one of SEQ ID NOs: 34 to 38 across 90% of any one of SEQ ID NOs: 34 to 38, or at least 90% sequence identity to any one of SEQ ID NOs: 34 to 38 across 100% of any one of SEQ ID NOs: 29 to 33, or at least 95% sequence identity to any one of SEQ ID NOs: 34 to 38 across 70% of any one of SEQ ID NOs: 34 to 38, or at least 95% sequence identity to any one of SEQ ID NOs: 34 to 38 across 80% of any one of SEQ ID NOs: 34 to 38, or The present invention may have a chromosome having at least 95% sequence identity to any one of SEQ ID NOs: 34-38 across 90% of any one of SEQ ID NOs: 34-38, or at least 95% sequence identity to any one of SEQ ID NOs: 34-38 across 100% of any one of SEQ ID NOs: 34-38, or at least 98% sequence identity to any one of SEQ ID NOs: 34-38 across 70% of any one of SEQ ID NOs: 34-38, or at least 98% sequence identity to any one of SEQ ID NOs: 34-38 across 80% of any one of SEQ ID NOs: 34-38, or at least 98% sequence identity to any one of SEQ ID NOs: 34-38 across 90% of any one of SEQ ID NOs: 34-38, or at least 98% sequence identity to any one of SEQ ID NOs: 34-38 across 100% of any one of SEQ ID NOs: 34-38.

[0357] Derivatives of the strain deposited under accession number V21 / 011520 may be daughter strains (progeny) or strains cultured (subcloned) from the original strain. Derivatives of the strains of the present invention may be modified, for example, at the genetic level, without eliminating biological activity. In particular, derivative strains of the present invention are therapeutically active. Derivative strains have activity equivalent to that of the original V21 / 011520 strain from which they are derived. Specifically, derivative strains elicit equivalent effects in at least one disease model (e.g., colitis), as shown in the Examples, which can be identified using the culture and administration protocols described in the Examples. Derivatives of any one of the V21 / 011520 strains are generally biotypes of the respective V21 / 011520 strains.

[0358] Reference to cells of the G. formicilis strain deposited under accession number V21 / 011520 includes any cells that have the same safety and therapeutic efficacy characteristics as the strain deposited under accession number V21 / 011520, and such cells are encompassed by the present invention.

[0359] 6.1 Bacterial Biotypes Bacterial strains that are biotypes of the bacterium deposited under accession number V21 / 011520 are also expected to be effective for treating or preventing inflammatory and autoimmune disorders. Biotypes are closely related strains that have identical or very similar physiological and biochemical properties.

[0360] Strains suitable for use in the present invention that are biotypes of the bacteria deposited under accession number V21 / 011520 can be identified by sequencing other nucleotide sequences of the bacteria deposited under accession number V21 / 011520. For example, substantially the entire genome can be sequenced, and biotype strains of the present invention can have at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity across at least 80% of their entire genome (e.g., at least 85%, 90%, 95%, or 99%, or across their entire genome). Other sequences suitable for use in identifying biotype strains include repeat sequences such as hsp60 or BOX, ERIC, (GTG)5, or REP (Masco et al., 2003; Kim et al., 2019). A biotype strain may have a sequence that has at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number V21 / 011520.

[0361] Alternatively, strains that are the bacterial biotype deposited under accession number V21 / 011520, and restriction fragment analysis and / or PCR analysis, for example, by using fluorescent amplified fragment length polymorphism (FAFLP) and repetitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23S rRNA sequencing. In some preferred embodiments, such techniques can be used to identify other suitable G. formicilis strains.

[0362] In one embodiment, a strain that is a biotype of the bacterium deposited under accession number V21 / 011520 and is suitable for use in the present invention is one that provides the same pattern as the bacterium deposited under accession number V21 / 011520 when analyzed by amplified ribosomal DNA restriction analysis (ARDRA), e.g., using the Sau3AI restriction enzyme (see, e.g., Srutkova et al., 2011, for exemplary methods and guidance). Alternatively, a biotype strain is identified as one that has the same carbohydrate fermentation pattern as the bacterium deposited under accession number V21 / 011520.

[0363] In some embodiments, bacterial strains useful in the present invention can be identified by routinely profiling the production and consumption of metabolites by the bacterial strain. The bacterial strains described above and elsewhere herein are predicted to affect the production of acetate, butyrate, ethanol, and fumarate. Thus, in some embodiments, the bacterial strains of the present invention induce the in vivo production of one or more of the metabolites acetate, ethanol, butyrate, and fumarate. Furthermore, in some embodiments, the bacterial strains of the present invention do not produce butyrate.

[0364] Other Gemmiger strains useful in the compositions and methods of the present invention, such as the biotype of the bacterium deposited under accession number V21 / 011520, can be identified using any suitable method or strategy, including the assays described in the Examples. For example, strains for use in the present invention can be identified by culturing them in anaerobic TY or PYG medium and / or administering them to a DSS-induced intestinal barrier function model, followed by assessing cytokine / chemokine levels as described in the Examples. In particular, bacterial strains with similar growth patterns, metabolic types, and / or surface antigens to the bacterium deposited under accession number V21 / 011520 may be useful in the present invention. Useful strains have immunomodulatory activity equivalent to that of the V21 / 011520 strain. In particular, biotype strains cause equivalent effects on host intestinal function. Furthermore, it is expected that biotypes having similar effects in disease models (e.g., colitis, asthma, arthritis, multiple sclerosis, and uveitis disease models) and comparable effects on cytokine / chemokine levels to those shown in the Examples can be identified by using the culture and administration protocols described in the Examples.

[0365] Viability of bacterial strains. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable. In a preferred embodiment, the bacterial strain in the composition of the present invention is viable and can partially or completely colonize the intestine. In some preferred embodiments, the bacterial strain in the composition of the present invention is live. For example, the bacterial strain in the composition of the present invention is not heat-killed. The bacteria of the present invention can have immunomodulatory effects that would not be exhibited by non-viable bacteria, for example, because non-viable bacteria cannot produce metabolites and cannot interact with the immune system in a different manner. The cell surface of live bacteria also appears to be significantly different from that of dead bacteria, especially heat-killed bacteria.

[0366] In some alternative embodiments, the bacteria are non-viable, for example, in some embodiments, the bacteria are heat-killed.

[0367] In some preferred embodiments, bacterial strains for use in the present invention are naturally occurring, for example, the bacterial strains are isolated from the digestive tract of a mammal.

[0368] In some preferred embodiments, bacterial strains for use in the present invention are not genetically engineered, e.g., they are not transformed with recombinant DNA.

[0369] 7. Composition - Gemmiger species The compositions provided herein are compositions comprising, consisting of, or consisting essentially of a therapeutically effective amount of the bacterial strain or strains described above and / or elsewhere herein. In some embodiments, the bacteria in the composition can be identified by strain, species, operational taxonomic unit (OTU), whole genome sequence, 16S rRNA sequence, or other method known in the art for defining different types of bacteria.

[0370] 7.1 Most Recent Common Ancestor (MRCA) In some embodiments, the composition comprises an effective amount of a bacterial strain that is a phylogenetic descendant of the MRCA of G. variabile and G. sp002306375 (see Figure 15). Preferably, the phylogenetic classification is as defined by the GTDB (Parks et al., 2018). In some embodiments, the phylogenetic classification is as defined in release 89 (r89) of the GTDB.

[0371] In some embodiments, determining whether a bacterial strain is a descendant of the MRCA of G. variabile and G. sp002306375 can be performed using phylogenetic grouping procedures known in the art. In some embodiments, a rooted phylogenetic tree containing G. variabile, G. sp002306375, and a third taxon of interest (e.g., a classified taxon) can be used, and the following analytical packages can be applied to determine whether the taxon of interest is useful in the compositions of the invention: Phylogenomics and Evolutionary Analysis ("ape"; https: / / cran.r-project.org / web / packages / ape / index.html) and Phylogenetic Tools for Comparative Biology ("phytools";). Both ape and phylogenetic tools are packages written in the R language and have been used in molecular evolution and phylogenetic studies. The ape and phylogenetic tools packages provide methods for phylogenetic and evolutionary analysis, and their use is known to those skilled in the art. In some embodiments, the following scripts can be used: library("ape") library("phytools") input.tree=read.tree(file=”tree_file”) gemmi c(“s__Gemmiger sp900554145”,“s__Gemmiger sp004555405”) gemmi.node=getMRCA(input.tree,gemmi) gemmi.tree=extract.clade(input.tree,gemmi.node) print(gemmi.tree$tip.label)

[0372] In some embodiments, after the script is run, if the taxon of interest is in the printed list, it is the descendant of the MRCA of the two species.

[0373] In other embodiments, different phylogenetic grouping methods known in the art can be used to determine whether a bacterial strain is a descendant of the MRCA of G. variabile and G. sp002306375, using different analytical packages and including methods based on different programming languages ​​(see Figure 15B, C).

[0374] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of G. variabile and G. sp003476825, together with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23819 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated using release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0375] In some other embodiments, the bacterial strain is a phylogenetic descendant of the MRCA of G. variabile and G. formicilis, along with a pharmaceutically acceptable carrier, diluent, or excipient. Preferably, the MRCA is defined at node 23820 of the bac120 phylogenetic tree from the GTDB. In some embodiments, the phylogenetic tree is generated by release 89 of the GTDB, although any suitable subsequent release is believed to provide equally applicable results.

[0376] In other embodiments, a bacterial species is a member of the family Ruminococcaceae if the species has a 16S rRNA sequence that has sequence identity to a 16S rRNA sequence from a species previously identified as a member of the family Ruminococcaceae. In some embodiments, identifying whether a bacterial species is a member of the family Ruminococcaceae is performed using the methods described in Yarza et al., 2014, Nature Reviews Microbiology 12:635-645 and Stackebrandt, E. & Ebers, J., 2006, Microbiol. Today 8:6-9, which are incorporated herein by reference.

[0377] 7.2 16S rRNA sequence identity In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the genus Gemmiger. In some embodiments, the query 16S rRNA sequence is compared to the 16S rRNA sequence set forth in SEQ ID NO: 27. In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the genus Gemmiger. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the genus Gemmiger. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of the genus Gemmiger.

[0378] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 97.5%. In some embodiments, the threshold sequence identity is 99.0%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0 ... 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0379] In some embodiments, a 16S rRNA sequence is obtained or determined for the bacterial species to be classified. This query 16S rRNA sequence is compared to 16S rRNA sequences from bacterial species already classified as members of the Ruminococcaceae family. In some embodiments, the query 16S rRNA sequence is compared to all known 16S rRNA sequences for bacterial species already classified as members of the Ruminococcaceae family. In other embodiments, the query 16S rRNA sequence is compared to a subset of all known 16S rRNA sequences for bacterial species already classified as members of the Ruminococcaceae family. The percent identity between the query sequence and the comparison sequence is determined. If the percent identity of the query sequence is determined to exceed a defined threshold, the bacterial species to be classified is classified as a member of the Ruminococcaceae family.

[0380] In some embodiments, the threshold sequence identity is 95%. In some embodiments, the threshold sequence identity is 98.7%. In some embodiments, the threshold sequence identity is 94.8%. In some embodiments, the threshold sequence identity is 94.5%, 94.6%, 94.7%, 94.8%, 94.9%, 95.0%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96.0%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97.0%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98 ...1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 0.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.0%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99.0%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%.

[0381] 8. Functional Characterization of Bacterial Strains Intestinal barrier dysregulation is a typical pathway leading to systemic inflammation. As shown in the examples, the bacterial strains of the present invention and compositions comprising the strains are effective in enhancing intestinal barrier function.

[0382] Any inflammatory or autoimmune disorder mediated by intestinal barrier dysregulation that causes systemic inflammation in a subject is amenable to treatment with the bacterial strains mentioned above and / or elsewhere herein.

[0383] 8.1 Intestinal barrier function. Intestinal barrier (also known as the gastrointestinal barrier) function regulates transport and host defense mechanisms at the mucosal interface with the external environment. Intracellular and extracellular fluxes are tightly controlled by membrane pumps, ion channels, and tight junctions, regulating permeability to physiological needs.

[0384] The translocation of foreign (i.e., non-host) substances, such as lipopolysaccharide (LPS) and other inflammatory compounds, from the intestinal lining to the circulatory system is inhibited by the epithelial barrier. One function of this epithelial barrier is the tight junction. Tight junctions, or zonula occludens, are closely associated regions of two epithelial cells whose membranes join together to form a virtually impermeable barrier to fluids, thereby separating the vasculature from the lumen of the gastrointestinal tract. Impairments at either level, particularly those that inhibit bacterial translocation due to increased permeability or disrupt oral tolerance due to impaired epithelial-T cell interactions, can lead to inflammation and tissue damage. Thus, impaired tight junction barrier function has been shown to increase the translocation of unwanted substances, such as LPS, from the intestinal lumen to the circulatory system.

[0385] The present invention provides methods for restoring or improving intestinal barrier function in a subject, comprising administering to the subject a composition comprising a bacterial strain as described above and / or elsewhere herein, thereby restoring or improving intestinal barrier function in the subject. In some specific embodiments, the present invention provides methods for restoring or improving intestinal barrier function in a subject, comprising administering to the subject a composition comprising one or more bacterial strains of A. shahii, C. sana, and G. formicilis, thereby restoring or improving intestinal barrier function in the subject. As used herein, intestinal barrier integrity refers to a measure of intestinal barrier function. High intestinal barrier integrity may be associated with a lack of intestinal or intestinal permeability, with high levels of intestinal permeability indicating low intestinal barrier integrity. In related embodiments, the present invention also provides methods for maintaining healthy or normal intestinal barrier function. Such methods can be used to prevent subjects considered at high risk for intestinal barrier dysregulation (e.g., subjects in remission of IBD). In some embodiments, the composition comprises an A. shahii bacterial strain. In some other embodiments, the composition comprises a C. sana bacterial strain. In yet some other embodiments, the composition comprises a bacterial strain of G. formicilis.

[0386] In some embodiments, at least one biomarker measured in a sample (and particularly a biological sample) is used to assess changes, particularly improvements, in the intestinal barrier integrity of a subject.

[0387] In some embodiments of the methods and uses provided herein, a composition comprising a bacterial strain of A. shahii may increase or decrease the level of one or more biomarkers of intestinal barrier integrity in a sample from a subject.

[0388] In some embodiments of the methods and uses provided herein, a composition comprising a bacterial strain of C. sana may increase or decrease the level of one or more biomarkers of intestinal barrier integrity in a sample from a subject.

[0389] Furthermore, in some embodiments of the methods and uses provided herein, a composition comprising a bacterial strain of G. formicilis may increase or decrease the level of one or more biomarkers of intestinal barrier integrity in a sample from a subject.

[0390] In some embodiments, depending on the particular biomarker, either an increase or decrease in the level of the marker indicates increased intestinal barrier integrity and / or decreased intestinal permeability. In some embodiments, the biomarker is selected from a cytokine, a chemokine, a growth factor, an angiogenic factor, an enzyme, a protease, an adhesion molecule, a cell signaling molecule, a hormone, or a sugar. In some embodiments, the biomarker comprises a cytokine. In some embodiments, the marker comprises a chemokine. In some embodiments, the marker comprises a growth factor. In some embodiments, the marker comprises an angiogenic factor. In some embodiments, the marker comprises an enzyme. In some embodiments, the marker comprises a protease. In some embodiments, the marker comprises an adhesion molecule. In some embodiments, the marker comprises a cell signaling molecule. In some embodiments, the marker comprises a hormone. In some embodiments, the marker comprises a sugar.

[0391] This specification provides assays for biomarkers of intestinal permeability. Using a subject-derived biological sample, such as blood (plasma or serum) or tissue, levels of any suitable biomarker can be measured, including, but not limited to, one or more of LPS, lipopolysaccharide-binding protein (LPSBP), intestinal fatty acid-binding protein (IFABP), zonulin, bacteria, and / or 16S rRNA. LPS, I-FABP, and zonulin can be measured by enzyme-linked immunosorbent assay (ELISA). Techniques and kits for ELISA are well known to those skilled in the art. In some embodiments, elevated LPS, I-FABP, and / or zonulin compared to controls in blood, serum, saliva, urine, and / or plasma are used as indicators of increased intestinal permeability and, therefore, lower intestinal barrier integrity.

[0392] LPSBP can also be measured by ELISA. In some embodiments, a significant change in LPSBP, either higher or lower compared to a control, can be used as an indicator of increased intestinal permeability and can confirm a decrease in intestinal barrier integrity.

[0393] In some embodiments, an increase in bacterial 16S rRNA is used as an indicator of increased intestinal permeability and therefore decreased intestinal barrier integrity. Bacterial 16S rRNA can be purified from blood, serum, organ tissue, or urine using standard nucleic acid isolation protocols. These are, for example, commercially available. The isolated nucleic acid can be detected by qPCR amplification using primers specific to the bacterial 16S rRNA sequence, or by amplification using primers specific to bacterial 16S rRNA and sequencing the resulting amplicon.

[0394] Tight junction proteins, which are expressed by intestinal epithelial cells and regulate intestinal permeability, can also be used as biomarkers of intestinal permeability. In some embodiments, tight junction proteins are assayed to determine changes in intestinal permeability and intestinal barrier integrity. In some embodiments, measured proteins may include, but are not limited to, claudins, occludin, ZO-1, and E-cadherin (adherens junction) proteins. Other tight junction proteins can also be assayed. In some embodiments, tight junction proteins are measured using immunohistochemical staining. In some embodiments, tight junction proteins are measured using ELISA.

[0395] In some embodiments, plasma citrulline is assayed to determine changes in intestinal permeability and intestinal barrier integrity. A decrease in plasma citrulline concentration corresponds to a decrease in epithelial cell mass and indicates increased intestinal barrier permeability.

[0396] In some embodiments, the method involves oral administration of an insoluble sugar, such as sucralose, collecting a bodily fluid, such as urine or blood, after one or more specified time periods, and measuring the amount of insoluble sugar in the bodily fluid via standard clinical analytical techniques. Insoluble sugars include, but are not limited to, mannitol, lactulose, sucrose, sucralose, and combinations of any of the above.

[0397] In some embodiments, intestinal barrier integrity is measured using an in vitro assay.A particularly preferred in vitro assay for measuring intestinal barrier function is by transepithelial electrical resistance (TEER).Such assays are well known in the art (e.g., Srinivasan, 2015; Lea, 2015).

[0398] 8.2 Mucosal healing Mucosal healing has become an important endpoint for evaluating the efficacy of treatment in inflammatory and autoimmune disorders. The definition of complete mucosal healing currently used in IBD (e.g., CD and UC) clinical trials is "complete absence of all inflammatory and ulcerative lesions," but this definition lacks validation and does not include grading of mucosal improvement and mucosal healing.

[0399] Mucosal healing is primarily defined by endoscopic assessment of enteritis. Various endoscopic scoring systems have been developed to assess the presence or absence of mucosal healing during endoscopy. These indices allow for the determination of endoscopic improvement of lesions even when the rather limited endpoint of mucosal healing, i.e., complete disappearance of all mucosal ulcers, is not achieved. The endoscopic component of the clinical Mayo score, introduced in 1987, is currently the most commonly used score for the mucosal layer in clinical practice (see Schroeder et al., 1987). This includes erythema, loss of vascular pattern, friability, bleeding, erosion, and ulcers, and ranges from 0 to 3. Mucosal healing is classically considered to be a score of 0 (normal mucosa) or 1 (mucosal erythema, diminished vascular pattern, and mild friability) (D'Haens, 2007).

[0400] In some other embodiments, mucosal healing is determined to have occurred when a patient is determined to have an endoscopic subscore, as assessed by flexible sigmoidoscopy, of 0 or 1. In certain such embodiments, a patient who experiences mucosal healing is determined to have an endoscopic subscore of 0.

[0401] Corticosteroids and aminosalicylates have both been used for decades and are among the most commonly prescribed medications for repairing mucosal linings (e.g., in patients with UC) (Carvalho and Cotter, 2017). The mechanisms by which they reduce mucosal inflammation include regulating nuclear factor (NF)-kB expression and proinflammatory cytokines, which directly regulate cell migration and proliferation of epithelial cell lines. Anti-TNF drugs (e.g., infliximab, adalimumab, and golimumab) act at several stages of mucosal injury, limiting inflammatory infiltration and T cell proliferation within the lamina propria (Baert, 1999) and downregulating the expression of metalloproteinases and proinflammatory molecules (Baert, 1999). They also restore the mucosal protective capacity and affect the regenerative process by enhancing intestinal permeability and mucosal secretion, activating fibroblasts, and maintaining epithelial regeneration (Suenaert, 2002).

[0402] Other measures for assessing mucosal healing are known in the art, including measurement of the biomarkers C-reactive protein and calprotectin. An advantage of using in vitro biomarker assays for assessing mucosal healing is that such assays are typically much less invasive to the subject. Histopathology is another measure of inflammation and has been cited as being particularly informative for mucosal healing.

[0403] 8.3 STAT3 Signaling Pathway Cytokine pathways mediate a wide range of biological functions, including many aspects of inflammation and immunity. Janus kinases (JAKs), including JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), are cytoplasmic tyrosine kinases that associate with type I and type II cytokine receptors and regulate cytokine signaling. Cytokine binding to its cognate receptor triggers activation of receptor-associated JAKs, which leads to JAK-mediated tyrosine phosphorylation of signal transducer and activator of transcription (STAT) proteins and ultimately to transcriptional activation of specific sets of genes (Schindler et al., 2007, J. Biol. Chem. 282:20059-63). Cytokine receptors typically function as heterodimers, and as a result, two or more JAK kinases are usually associated with the cytokine receptor complex. The specific JAKs associated with various cytokine receptor complexes are often determined by genetic studies and supported by other experimental evidence.

[0404] STAT3 plays a key role in the activation of several autoimmune and inflammatory disorders, including IBD. The bacterial strains of the present invention significantly suppress IL-23-mediated STAT3 activation. Accordingly, the present invention provides a method of suppressing or otherwise inhibiting STAT3 signaling (i.e., IL-23-mediated STAT3 signaling) in a subject, comprising administering to the subject a composition comprising a bacterial strain described above and / or elsewhere herein. Thus, in some embodiments, the bacterial strains described herein directly or indirectly suppress STAT3 activity. In some embodiments, the A. shahii strain produces a bioactive molecule that directly binds to a STAT3 polypeptide. In some alternative embodiments, the bacterial strain is an indirect inhibitor of STAT3 activation, for example, by binding to a molecule upstream of STAT3 in the IL-23-mediated STAT3 signaling pathway or by binding to a molecule that regulates STAT3 activity (e.g., ubiquitination). As illustrative examples, the bioactive agent may directly bind to or antagonize any one of IL23, JAK2, or TYK2 to suppress the IL-23-mediated STAT3 signaling pathway.

[0405] 8.4 Th17 inflammatory response Some bacterial compositions of the present invention are effective in reducing Th17 inflammatory responses. In particular, the compositions described above and treatments described elsewhere herein can modulate Th17 pathway cytokines (including TNF, IL-22, IL-21, and IL-17) and result in clinical improvement in animal models of conditions mediated by the Th17 pathway. Thus, compositions of the present invention may be useful for treating or preventing inflammatory and autoimmune disorders, and in some embodiments, diseases or conditions mediated by Th17. In particular, compositions of the present invention may be useful for reducing or preventing elevated Th17 inflammatory responses.

[0406] Th17 cells are a subset of T helper cells that produce, among other cytokines, IL17A, IL17F, IL-21, and IL-22. Th17 cell differentiation can be driven by IL-23. These cytokines and others form an important part of the Th17 pathway, a well-established inflammatory signaling pathway that contributes to and underlies many inflammatory and autoimmune disorders (see, e.g., Ye, 2015; Fabro, 2015; Yin, 2014; Cheluvappa, 2014; Schieck, 2014; Balato, 2014). Some Th17-mediated diseases can be improved or alleviated by suppressing the Th17 pathway, potentially through reduced differentiation of Th17 cells, their activity, or reduced levels of Th17 pathway cytokines. Diseases mediated by the Th17 pathway may be characterized by increased levels of cytokines produced by Th17 cells, such as IL-17A, IL-17F, IL-21, IL-22, IL-26, and IL-9 (reviewed in Monteleone, 2011). Diseases mediated by the Th17 pathway may be characterized by increased expression of Th17-associated genes, such as STAT3 or IL-23 receptor. Diseases mediated by the Th17 pathway may be associated with increased levels of Th17 cells.

[0407] IL-17 is a key cytokine that links T cell activation to neutrophil activation and recruitment, and thus plays a central role in innate immunity. However, due to its role in neutrophil activation, IL-17 may contribute to inflammatory autoimmune diseases such as inflammatory bowel disease, psoriasis, and rheumatoid arthritis. As used herein, IL-17 can refer to any member of the IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17-mediated diseases and conditions are characterized by high expression of IL-17 and / or the accumulation or presence of IL-17-positive cells in tissues affected by the disease or condition. Similarly, IL-17-mediated diseases and conditions are those that are exacerbated by elevated IL-17 levels or increased IL-17 levels and are alleviated by reduced IL-17 levels or decreased IL-17 levels. IL-17 inflammatory responses can be local or systemic.

[0408] Examples of diseases and conditions that may be mediated by the Th17 pathway include, but are not limited to, inflammatory bowel disease (such as Crohn's disease and ulcerative colitis), multiple sclerosis, arthritis, osteoarthritis, psoriatic arthritis, and juvenile idiopathic arthritis, neuromyelitis optica (Devic's disease), ankylosing spondylitis, arthritis, psoriasis, systemic lupus erythematosus, celiac disease, asthma (such as allergic asthma or neutrophilic asthma), asthma, chronic obstructive pulmonary disease (such as COPD), cancer (such as breast cancer, colon cancer, lung cancer, or ovarian cancer), uveitis, scleritis, vasculitis, Behcet's disease, atherosclerosis, atopic dermatitis, emphysema, periodontitis, allergic rhinitis, and allograft rejection. Accordingly, in some embodiments, the present invention provides methods of treating or preventing one or more of these conditions or diseases by administering a composition described above and / or elsewhere herein. In further preferred embodiments, these conditions or diseases are mediated by the STAT3 signaling pathway. In further preferred embodiments, these conditions or diseases are mediated via the Th17 pathway.

[0409] In one embodiment, the present invention relates to the use of a method composition of the present invention in a method for reducing Th17 cell differentiation in the treatment or prevention of a disease or condition mediated by the Th17 pathway. In one embodiment, the composition of the present invention is for use in the treatment or prevention of an inflammatory or autoimmune disorder, wherein the treatment or prevention is achieved by reducing or preventing an elevated Th17 inflammatory response. In one embodiment, the composition of the present invention is used to treat a patient with an inflammatory or autoimmune disorder, wherein the patient has elevated IL-17 levels or elevated Th17 cells or exhibits a Th17 inflammatory response. In one embodiment, the patient may have been diagnosed with a chronic inflammatory or autoimmune disorder or condition, or the composition of the present invention may be used to prevent an inflammatory or autoimmune disorder or condition from progressing to a chronic inflammatory or autoimmune disorder or condition. In one embodiment, the disease or condition may be unresponsive to treatment with a TNF inhibitor. These uses of the present invention can be applied to any of the specific diseases or conditions listed in the previous section.

[0410] Because the Th17 pathway is often associated with chronic inflammatory and autoimmune disorders, the compositions of the present invention may be particularly useful for treating or preventing such chronic diseases or conditions. In some embodiments, the compositions are used in patients with chronic diseases. In some embodiments, the compositions are used to prevent the onset of chronic diseases.

[0411] The compositions of the present invention may be useful for treating diseases and conditions mediated by the Th17 pathway and for addressing Th17 inflammatory responses. Thus, the compositions of the present invention may be particularly useful for treating or preventing chronic diseases, treating or preventing diseases in patients who have not responded to other treatments (such as treatment with TNF inhibitors), and / or treating or preventing tissue damage and symptoms associated with Th17 cells. For example, IL-17 is known to activate matrix destruction in cartilage and bone tissue, and because IL-17 has an inhibitory effect on matrix production in chondrocytes and osteoblasts, the compositions of the present invention may be useful for treating or preventing bone erosion or cartilage damage.

[0412] In certain embodiments, treatment with the compositions of the invention provides a reduction or prevention of elevated IL-17 levels, particularly IL-17A levels. In certain embodiments, treatment with the compositions of the invention provides a reduction or prevention of elevated IFN-γ or IL-6 levels. Such reduction or prevention of elevated levels of these cytokines may be useful in treating or preventing inflammatory and autoimmune disorders and conditions, particularly disorders and conditions mediated by the Th17 pathway.

[0413] 8.5 Th1 inflammatory response CD4 + T cells play a key role in the pathogenesis of inflammatory diseases / disorders, and many CD4 + T cell subsets have been identified as drivers of chronic intestinal inflammation (see Imam et al., 2018). For example, T helper type 1 (Th1) cells accumulate in the intestine of IBD patients and are directly associated with the disease. Interferon-γ (IFN-γ) is the defining cytokine produced by Th1 cells. During intestinal inflammation, IFN-γ in combination with TNF has been proposed to drive β-catenin signaling in intestinal epithelial cells, limiting their differentiation and proliferation (Imam et al., 2018).

[0414] 9.Treatment method In some embodiments, the present invention provides a method of treating or preventing an inflammatory or autoimmune disorder in a subject, the method comprising administering to the subject a bacterial strain as described above and / or elsewhere herein.

[0415] Suitably, the inflammatory or autoimmune disorder is selected from the group comprising inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjogren's syndrome, vasculitis, and type 1 diabetes.

[0416] 9.1 Inflammatory bowel disease (IBD) The examples demonstrate that the compositions of the present invention have a beneficial restorative effect on intestinal barrier function and that they have anti-inflammatory properties and therefore may be useful in the treatment of IBD.

[0417] Thus, in some embodiments, the present invention provides a composition comprising a bacterial strain of the genus Alistipes for use in a method for treating or preventing inflammatory bowel disease. The inventors have determined that treatment with an Alistipes strain reduces the severity of colitis in a mouse disease model. Thus, the compositions of the present invention may be useful for treating inflammatory diseases. In some embodiments, the compositions of the present invention are used in the treatment or prevention of IBD. In some embodiments, the present invention provides a method for treating or preventing ulcerative colitis. In some embodiments, the present invention provides a method for treating or preventing Crohn's disease. In certain embodiments, the present invention provides a method for treating or preventing ulceration and / or bleeding in the treatment of IBD, particularly colitis and ulcerative colitis. In a preferred embodiment, the present invention provides a method for treating or preventing IBD in a subject, comprising administering to the subject a composition comprising a bacterial strain of the A. shahii species. In a further preferred embodiment, the present invention provides a method for treating or preventing colitis (particularly ulcerative colitis) in a subject, comprising administering to the subject a composition comprising a bacterial strain of the A. shahii species. In a further preferred embodiment, the present invention provides a method for reducing at least one side effect of colitis (particularly ulcerative colitis), including ulceration and / or bleeding.

[0418] Additionally, in some embodiments, the present invention provides a composition comprising a bacterial strain of the genus Colonithrix for use in a method for treating or preventing inflammatory bowel disease. The inventors have determined that treatment with a Colonithrix strain reduces the severity of colitis in a mouse disease model. Thus, the compositions of the present invention may be useful for treating inflammatory diseases. In some embodiments, the compositions of the present invention are used in the treatment or prevention of IBD. In some embodiments, the present invention provides a method for treating or preventing ulcerative colitis. In some embodiments, the present invention provides a method for treating or preventing Crohn's disease. In certain embodiments, the present invention provides a method for treating or preventing ulceration and / or bleeding in the treatment of IBD, particularly colitis and ulcerative colitis. In a preferred embodiment, the present invention provides a method for treating or preventing IBD in a subject, comprising administering to the subject a composition comprising a bacterial strain of the C. sana species. In a further preferred embodiment, the present invention provides a method for treating or preventing colitis (particularly ulcerative colitis) in a subject, comprising administering to the subject a composition comprising a bacterial strain of the C. sana species. In a further preferred embodiment, the present invention provides a method for reducing at least one side effect of colitis (particularly ulcerative colitis), including ulceration and / or bleeding.

[0419] Furthermore, in some embodiments, the present invention provides a composition comprising a bacterial strain of the genus Gemmiger for use in a method for treating or preventing inflammatory bowel disease. The inventors have determined that treatment with a Gemmiger strain reduces the severity of colitis in a mouse disease model. Thus, the compositions of the present invention may be useful for treating inflammatory diseases. In some embodiments, the compositions of the present invention are used in the treatment or prevention of IBD. In some embodiments, the present invention provides a method for treating or preventing ulcerative colitis. In some embodiments, the present invention provides a method for treating or preventing Crohn's disease. In certain embodiments, the present invention provides a method for treating or preventing ulceration and / or bleeding in the treatment of IBD, particularly colitis and ulcerative colitis. In a preferred embodiment, the present invention provides a method for treating or preventing IBD in a subject, the method comprising administering to the subject a composition comprising a bacterial strain of the G. formicilis species. In a further preferred embodiment, the present invention provides a method for treating or preventing colitis (particularly ulcerative colitis) in a subject, the method comprising administering to the subject a composition comprising a bacterial strain of the G. formicilis species. In a further preferred embodiment, the present invention provides a method for reducing at least one side effect of colitis (particularly ulcerative colitis), including ulceration and / or bleeding.

[0420] IBD is a complex disease caused by multiple environmental and genetic factors. Factors contributing to the development of IBD include diet, microbiota, intestinal permeability, and genetic susceptibility to increased inflammatory responses to intestinal infections. Symptoms of inflammatory bowel disease include abdominal pain, vomiting, diarrhea, rectal bleeding, severe pelvic cramps / muscle cramps, weight loss, and anemia. In one embodiment, the composition is used to reduce one or more symptoms associated with IBD. In one embodiment, the composition of the present invention is used to prevent one or more symptoms of IBD.

[0421] IBD may be accompanied by other diseases or conditions, such as cardiovascular disease, neuropsychiatric disorders, and metabolic syndrome. In certain embodiments, the compositions of the invention are used to treat or prevent one or more diseases or conditions associated with IBD.

[0422] IBD is generally diagnosed by biopsy or colonoscopy. Measuring fecal calprotectin is useful for preliminary diagnosis of IBD. Other laboratory tests for diagnosing IBD include complete blood count, erythrocyte sedimentation rate, comprehensive metabolic panel, fecal occult blood test, or C-reactive protein test. Typically, a combination of laboratory tests and biopsy / colonoscopy confirms the diagnosis of IBD. In one embodiment, the composition of the present invention is used in a subject diagnosed with IBD.

[0423] In one embodiment, the IBD is Crohn's disease and / or ulcerative colitis. As broadly described above, studies have shown that several inflammatory cytokines, including but not limited to STAT3 signaling and NFκB signaling pathway-mediated cytokines (e.g., IL-17, TNF, IL-21, IL-22), are upregulated in the inflamed fascia of patients with Crohn's disease and ulcerative colitis. Therefore, inhibiting cytokine activity via the STAT3 signaling pathway and / or cytokine activity via the NFκB signaling pathway may be useful in treating Crohn's disease and ulcerative colitis. In one embodiment, the compositions of the present invention are used to treat or prevent Crohn's disease and / or ulcerative colitis.

[0424] Crohn's disease and ulcerative colitis are complex diseases with a range of possible causes, including genetic risk factors, diet, other lifestyle factors such as smoking and alcohol consumption, and microbiome composition. Crohn's disease can affect any part of the gastrointestinal tract, while ulcerative colitis commonly affects the large intestine and colon.

[0425] Gastrointestinal symptoms of IBD range from mild to severe and include abdominal pain, diarrhea, bloody stool, ileitis, increased bowel movements, increased flatulence, intestinal stricture, vomiting, perianal discomfort, etc. The compositions of the present invention can be used for the prophylactic treatment of one or more gastrointestinal symptoms of Crohn's disease and / or ulcerative colitis.

[0426] Systemic symptoms of Crohn's disease and ulcerative colitis include growth disorders such as failure to thrive during adolescence, loss of appetite, fever, and weight loss. Extraintestinal features of Crohn's disease include uveitis, photodioderma, episcleritis, gallstones, seronegative spondyloarthropathy, arthritis, arthritis, adhesions, erythema nodosum, pyoderma gangrenosum, deep vein thrombosis, pulmonary embolism, autoimmune hemolytic anemia, clubbing, and osteoporosis. Extraintestinal features are additional conditions associated with Crohn's disease and / or ulcerative colitis that manifest outside the gastrointestinal tract. Patients with Crohn's disease also exhibit increased susceptibility to neurological complications such as seizures, stroke, myopathy, peripheral neuropathy, headache, and depression. In one embodiment, the compositions of the present invention are used to treat or prevent one or more systemic symptoms of Crohn's disease and / or ulcerative colitis. In one embodiment, the compositions of the present invention are used to treat or prevent one or more extraintestinal features of Crohn's disease and / or ulcerative colitis.

[0427] Diagnosis of Crohn's disease and ulcerative colitis typically involves performing multiple tests and surgical procedures, such as gastroscopy and / or colonoscopy and / or biopsies, typically of the ileum, radiological tests, complete blood counts, C-reactive protein tests, and erythrocyte sedimentation rates. In certain embodiments, the compositions of the invention are for use in subjects diagnosed with Crohn's disease or ulcerative colitis. In some embodiments, the compositions of the invention are used to treat subjects diagnosed with Crohn's disease or ulcerative colitis.

[0428] Crohn's disease and ulcerative colitis are classified by the extent of the region of the digestive tract affected (Gasche et al., 2000). Crohn's disease of both the ileum and colon is classified as ileocolonic Crohn's disease. In some embodiments, the composition is for use in the treatment or prevention of ileocolonic Crohn's disease. In some embodiments, the composition is for use in a subject diagnosed with ileocolonic Crohn's disease / Crohn's ileitis when only the ileum is affected. Crohn's colitis is classified when only the colon is affected. In some embodiments, the composition is used to treat or prevent Crohn's ileitis. In some embodiments, the composition is used in a subject diagnosed with Crohn's ileitis. In some embodiments, the composition is used in the treatment or prevention of Crohn's col ... colitis.

[0429] Crohn's disease and ulcerative colitis can be treated with a number of therapeutic agents, such as corticosteroids such as prednisone, immunosuppressants such as azathioprine, or biologics such as infliximab, adalimumab, and golimumab, vedolizumab, and etrolizumab. In certain embodiments, the compositions of the invention are for use in combination with an additional therapeutic agent, including but not limited to those listed above, for the treatment or prevention of Crohn's disease or ulcerative colitis. In certain embodiments, the additional therapeutic agent is used in the treatment or prevention of Crohn's disease and / or ulcerative colitis.

[0430] 9.2 Autoimmune disorders In humans, signs of intestinal inflammation are detected before the clinical onset of many autoimmune disorders, such as type 1 diabetes (T1D) (Bosi, 2006). Similarly, increased intestinal permeability appears before the onset of insulitis in diabetes-prone rats compared with diabetes-resistant rats (Meddings, 1999; Neu, 2005). These findings indicate that disruption of intestinal barrier integrity and the subsequent increased antigen transport and development of low-grade intestinal inflammation precede the onset of T1D and are directly related to its pathogenesis, rather than being secondary to diabetes-induced metabolic changes (i.e., hyperglycemia). The gastrointestinal barrier is a fundamental gatekeeper preventing contact between luminal contents and the human body. The barrier is composed of the mucus layer and the intestinal epithelial barrier (IEB), both of which are crucial for preventing the passage of commensal bacteria, pathogens, and food antigens from the intestinal lumen into the intestinal tissue and the systemic circulation. The IEB is a single layer of epithelial cells held together by a complex junctional system consisting of tight junction adhesion molecules (JAMs), tricellulin, and angulin. Interactions between them and with intracellular scaffolding proteins, namely, tight junction proteins (ZOs), are essential for maintaining tight junction integrity and regulating paracellular transport. Changes in IEB have been reported in patients and rat models of T1D associated with intestinal inflammation (Meddings, 1999; Sapone, 2006). Furthermore, the importance of the intestinal mucus layer, a key intestinal barrier containing antimicrobial peptides and immunomodulatory molecules such as mucins, has recently been demonstrated (see Sorini et al., 2019).

[0431] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of asthma, such as allergic asthma or neutrophilic asthma. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of asthma in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of asthma.

[0432] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of GVHD. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of GVHD in a subject. In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of GVHD.

[0433] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of arthritis in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of asthma.

[0434] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of multiple sclerosis. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of multiple sclerosis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of multiple sclerosis.

[0435] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of psoriasis. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of psoriasis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of psoriasis.

[0436] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of systemic lupus erythematosus (SLE). In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of SLE in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the A. shahii species for use in the treatment or prevention of SLE.

[0437] In some embodiments, bacterial strains derived from the A. shahii species may provide therapeutic benefits in the treatment or prevention of allograft rejection. In certain embodiments, compositions of the invention are used to treat or prevent allograft rejection in a subject. In certain embodiments, the invention provides compositions comprising bacterial strains of the A. shahii species for use in the treatment or prevention of allograft rejection.

[0438] In some embodiments, bacterial strains derived from the C. sana species may provide therapeutic benefits in the treatment or prevention of asthma, such as allergic asthma or neutrophilic asthma. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of asthma in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the C. sana species for use in the treatment or prevention of asthma.

[0439] In some embodiments, bacterial strains derived from the C. sana species may provide therapeutic benefits in the treatment or prevention of GVHD. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of GVHD in a subject. In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the C. sana species for use in the treatment or prevention of GVHD.

[0440] In some embodiments, bacterial strains derived from the C. sana species may provide therapeutic benefits in the treatment or prevention of arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of arthritis in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the C. sana species for use in the treatment or prevention of asthma.

[0441] In some embodiments, bacterial strains derived from the species C. sana may provide therapeutic benefits in the treatment or prevention of multiple sclerosis. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of multiple sclerosis in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species C. sana for use in the treatment or prevention of multiple sclerosis.

[0442] In some embodiments, bacterial strains derived from the species C. sana may provide therapeutic benefits in the treatment or prevention of psoriasis. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of psoriasis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the species C. sana for use in the treatment or prevention of psoriasis.

[0443] In some embodiments, bacterial strains derived from the species C. sana may provide therapeutic benefits in the treatment or prevention of systemic lupus erythematosus (SLE). In certain embodiments, the compositions of the invention are for use in the treatment or prevention of SLE in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species C. sana for use in the treatment or prevention of SLE.

[0444] In some embodiments, bacterial strains derived from the C. sana species may provide therapeutic benefits in the treatment or prevention of allograft rejection. In certain embodiments, compositions of the invention are used to treat or prevent allograft rejection in a subject. In certain embodiments, the invention provides compositions comprising bacterial strains of the C. sana species for use in the treatment or prevention of allograft rejection.

[0445] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of asthma, such as allergic asthma or neutrophilic asthma. In certain embodiments, the compositions of the invention are for use in the treatment or prevention of asthma in a subject. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species G. formicilis for use in the treatment or prevention of asthma.

[0446] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of GVHD. In one embodiment, the compositions of the present invention are for use in the treatment or prevention of GVHD in a subject. In a preferred embodiment, the present invention provides a composition comprising a bacterial strain of the species G. formicilis for use in the treatment or prevention of GVHD.

[0447] In some embodiments, bacterial strains derived from the G. formicilis species may provide therapeutic benefits in the treatment or prevention of arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis. In certain embodiments, the compositions of the present invention are for use in treating or preventing arthritis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the G. formicilis species for use in treating or preventing arthritis.

[0448] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of multiple sclerosis. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of multiple sclerosis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the species G. formicilis for use in the treatment or prevention of multiple sclerosis.

[0449] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of psoriasis. In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of psoriasis in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the species G. formicilis for use in the treatment or prevention of psoriasis.

[0450] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of systemic lupus erythematosus (SLE). In certain embodiments, the compositions of the present invention are for use in the treatment or prevention of SLE in a subject. In certain embodiments, the present invention provides a composition comprising a bacterial strain of the species G. formicilis for use in the treatment or prevention of SLE.

[0451] In some embodiments, bacterial strains derived from the species G. formicilis may provide therapeutic benefits in the treatment or prevention of allograft rejection. In certain embodiments, compositions of the invention are used to treat or prevent allograft rejection in a subject. In certain embodiments, the invention provides compositions comprising bacterial strains of the species G. formicilis for use in the treatment or prevention of allograft rejection.

[0452] 10. Preparation In some embodiments, the compositions of the present invention comprise fewer than 40 different bacterial strains. In some embodiments, the compositions comprise fewer than 30 different bacterial strains. In some embodiments, the compositions comprise fewer than 20 different bacterial strains. In some embodiments, the compositions comprise fewer than 10 different bacterial strains. In some embodiments, the compositions comprise fewer than 5 different bacterial strains. In some embodiments, the compositions comprise fewer than 3 different bacterial strains. In some preferred embodiments, the compositions comprise a single bacterial strain. In some embodiments, the compositions do not include bacteria of the genus Clostridium. In some of the same embodiments and in some other embodiments, the compositions do not include bacteria of the species Alistipes senegalensis.

[0453] The compositions of the present invention comprise bacteria (i.e., live and / or dead bacteria). In a preferred embodiment of the present invention, the compositions are formulated in lyophilized form. The compositions of the present invention may comprise granules or gelatin capsules, e.g., hard gelatin capsules, containing the bacterial strains of the present invention. Preferably, the compositions of the present invention comprise lyophilized bacteria. Freeze-drying of bacteria is an established procedure, and relevant guidance can be found in the literature (Miyamoto-Shinohara, 2008; and Day & Stacey, 2007).

[0454] The compositions of the present invention may contain live, active bacterial cultures. The examples show that the bacterial cultures of the present invention are therapeutically effective.

[0455] In some embodiments, the bacterial strains in the compositions of the present invention are not inactivated, e.g., not heat-inactivated. In some embodiments, the bacterial strains in the compositions of the present invention are not killed, e.g., not heat-killed. In some embodiments, the bacterial strains in the compositions of the present invention are not attenuated, e.g., not heat-attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention are not killed, inactivated, and / or attenuated. For example, in some embodiments, the bacterial strains in the compositions of the present invention are live. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable. For example, in some embodiments, the bacterial strains in the compositions of the present invention are capable of partially or fully colonizing the intestine. For example, in some embodiments, the bacterial strains in the compositions of the present invention are viable and capable of partially or fully colonizing the intestine.

[0456] In some embodiments, the composition comprises a mixture of killed and live bacterial strains and bacterial strains. In a preferred embodiment, the compositions of the present invention are encapsulated to enable delivery of the bacterial strains to the intestine. The encapsulation protects the composition from degradation until delivery at the target location by disrupting it using chemical or physical stimuli, such as pressure, enzymatic activity, or physical disruption, which can be triggered by a change in pH. Any suitable encapsulation method can be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption to a solid carrier surface, self-aggregation by aggregating or cross-linking agents, and mechanical containment behind a microporous membrane or microcapsules. Guidance on encapsulation that may be useful in preparing the compositions of the present invention is widely available in the art (e.g., Mitropoulou, 2013; and Kailasapathy, 2002).

[0457] The compositions can be administered orally and may be in the form of tablets, capsules or powders. Encapsulated products are preferred because bacteria of the genus Alistipes are obligate anaerobes.

[0458] The compositions of the present invention comprise a therapeutically effective amount of a bacterial strain of the present invention. The therapeutically effective amount of the bacterial strain is sufficient to exert a beneficial effect on a patient. The therapeutically effective amount of the bacterial strain may be sufficient to result in delivery and / or partial or complete colonization of the intestine of a patient.

[0459] A suitable daily dose of bacteria, for example, for an adult human, is about 1 x 10 3 ~Approx. 1×10 11 Colony forming units (CFU); e.g., approximately 1 x 10 7 ~Approx. 1×10 10 CFU; in another example, approximately 1 x 10 6 ~Approx. 1×10 10 CFU; in another example, approximately 1 x 10 7 ~Approx. 1×10 11 CFU; in another example, approximately 1 x 10 8 ~Approx. 1×10 10 CFU; in another example, approximately 1 x 10 8 ~Approx. 1×10 11 It may be CFU.

[0460] In one embodiment, the dose of bacteria is at least 10 per day 9 cells, e.g., at least 10 per day 10 , at least 10 11 , or at least 10 12 It is a cell.

[0461] In one embodiment, the dosage of the composition is about 1×10 by weight of the composition. 6 ~Approx. 1×10 11 The composition may contain a bacterial strain in an amount of colony forming units (CFU) / g. The dosage may be suitable for an adult. For example, the composition may contain about 1 x 10 3 ~Approx. 1×10 11 CFU / g; e.g., approximately 1 x 10 7 ~Approx. 1×10 10 CFU / g; in another example, approximately 1 x 10 6 ~Approx. 1×10 10 CFU / g; in another example, approximately 1 x 10 7 ~Approx. 1×10 11 CFU / g; in another example, approximately 1 x 108 ~Approx. 1×10 10 CFU / g; in another example, approximately 1 x 10 8 ~Approx. 1×10 11 CFU / g, approximately 1 x 10 8 ~Approx. 1×10 10 CFU / g, for example, about 1 x 10 8 ~Approx. 1×10 10 CFU / g. Doses can be, for example, up to 1 g, 3 g, 5 g, and 10 g or more.

[0462] In some embodiments, the compositions described above and / or elsewhere herein have a concentration of about 1×10 per gram by weight of the composition. 3 ~Approx. 1×10 11 It comprises, consists of, or consists essentially of a colony forming unit amount of a bacterial strain.

[0463] In some embodiments, the compositions described above and / or elsewhere herein comprise the bacterial strain in a dose of 500 mg to 1000 mg, 600 mg to 900 mg, 700 mg to 800 mg, 500 mg to 750 mg, or 750 mg to 1000 mg. In certain embodiments, the invention provides the pharmaceutical composition described above, wherein the lyophilized bacteria in the pharmaceutical composition is administered in a dose of 500 mg to 1000 mg, 600 mg to 900 mg, 700 mg to 800 mg, 500 mg to 750 mg, or 750 mg to 1000 mg.

[0464] The composition may be formulated as a probiotic, which is defined by FAO / WHO as a live microorganism that, when administered in adequate amounts, confers a health benefit on the host.

[0465] Typically, probiotics such as the compositions of the present invention are optionally combined with at least one suitable prebiotic compound.Prebiotic compounds are usually non-digestible carbohydrates, such as oligosaccharides or polysaccharides, or sugar alcohols, that are not broken down or absorbed in the upper gastrointestinal tract.Known prebiotics include commercially available products such as inulin and transgalactooligosaccharides.

[0466] Other prebiotic compounds (e.g., vitamin C) may be included as oxygen scavengers and to improve in vivo delivery and / or partial or total colonization and survival. Alternatively, the probiotic compositions of the present invention can be administered orally as a food or nutritional product, such as a milk or whey-based fermented dairy product, or as a pharmaceutical product.

[0467] In one embodiment, the probiotic compositions of the present invention contain prebiotic compounds in an amount of about 1 to about 30% by weight (e.g., 5 to 20% by weight) based on the total weight of the composition. Known prebiotics include commercially available products such as inulin and transgalactooligosaccharides.

[0468] In some embodiments, the prebiotic is a carbohydrate selected from the group consisting of fructooligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomal trigosaccharides, pectin, xylooligosaccharides (or XOS), chitosan oligosaccharides (or COS), β-glucan, modified araburu gum and resistant starch, polydextrose, tagatose, acacia fiber, carob, oats, and citrus fiber. In one aspect, the prebiotic is a short-chain fructooligosaccharide. Short-chain FOS is not a digestible carbohydrate. It is typically obtained by the conversion of beet sugar and contains a sugar molecule with three glucose molecules linked together.

[0469] The compositions of the present invention may contain pharmaceutically acceptable excipients or carriers, such as those listed in pharmaceutical excipient handbooks. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical arts and are described, for example, in Remington's Pharmaceutical Sciences. Examples of suitable carriers include lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, and the like. Examples of suitable diluents include ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent can be selected based on the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain one or more suitable binders, lubricants, suspending agents, coating agents, and / or solubilizing agents as, or in addition to, the carrier, excipient, or diluent. Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, gum tragacanth, or sodium alginate, carboxymethylcellulose, and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, etc. Preservatives, stabilizers, dyes, and even flavoring agents can also be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, cysteine, and esters of 4-hydroxybenzoic acid. For example, in some embodiments, the preservative is selected from sodium benzoate, sorbic acid, and esters of 4-hydroxybenzoic acid. Antioxidants and suspending agents can also be used. Another example of a suitable carrier is sugar. Another example of a suitable preservative is cysteine.

[0470] The compositions of the present invention can be formulated as food products. For example, foods can provide nutritional benefits in addition to the therapeutic effects of the present invention, such as dietary supplements. Similarly, foods can be formulated to enhance the taste of the compositions of the present invention or to make them more appealing to consume by resembling common food items rather than pharmaceutical compositions. In some embodiments, the compositions of the present invention are formulated as milk-based products. The term "milk-based products" refers to liquid or semi-solid milk- or whey-based products with various fat contents. Milk-based products can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, milk recombinantly prepared from powdered milk and unprocessed whey, or processed products such as yogurt, curd, sour milk, sour whole milk, buttermilk, and other sour milk products. Alternatively, the milk can be plant-based milk, including, for example, soy milk, oat milk, almond milk, coconut milk, or macadamia milk. Another important group includes milk-containing foods such as whey drinks, fermented milk, concentrated milk, infant or baby milk, flavored milk, milk drinks such as ice cream, and sweeteners.

[0471] In some embodiments, the compositions disclosed herein comprise one or more bacterial strains of the genus Alistipes, and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species. Thus, in some embodiments, the present invention provides compositions comprising one or more bacterial strains of the genus Alistipes (e.g., Alistipes shahii), and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species, for use in therapy.

[0472] In some embodiments, a composition comprises one or more bacterial strains of the genus Alistipes and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera. In some embodiments, a composition comprises one or more bacterial strains of the genus Alistipes (e.g., Alistipes shahii) and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera.

[0473] In some embodiments, the compositions disclosed herein contain a single bacterial species and are free of any other bacterial species. In some embodiments, the compositions disclosed herein contain a single bacterial strain and are free of other bacterial strains. For example, a composition of the present invention may contain only A. shahii strain bacteria. Such a composition may contain only A. de minimis or may contain biologically irrelevant amounts of other bacterial strains or species. Such a composition may be a culture that is substantially free of organisms from other species. In some embodiments, such a composition may be in a dried form and be substantially free of organisms from other species.

[0474] In some embodiments, the present invention provides compositions comprising a single bacterial strain of the genus Alistipes, for use in therapy, free of bacteria from any other strain, or containing only minimal or biologically irrelevant amounts of bacteria from other species. In some embodiments, the present invention provides compositions comprising a single bacterial strain of the species Alistipes shahii (e.g., Alistipes shahii MH21-1, Alistipes shahii MH21-2, or Alistipes shahii MH21-3), for use in therapy, free of bacteria from any other species, or containing only minimal or biologically irrelevant amounts of bacteria from other species.

[0475] In some embodiments, the compositions disclosed herein comprise one or more bacterial strains of the genus Colonithrix, and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species. Thus, in some embodiments, the present invention provides compositions comprising one or more bacterial strains of the genus Colonithrix (e.g., C. sana), and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species, for use in therapy.

[0476] In some embodiments, a composition comprises one or more bacterial strains of the genus Colonithrix and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera. In some embodiments, a composition comprises one or more bacterial strains of the genus Colonithrix and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera.

[0477] In some embodiments, the compositions disclosed herein contain a single bacterial species and are free of any other bacterial species. In some embodiments, the compositions disclosed herein contain a single bacterial strain and are free of other bacterial strains. For example, a composition of the present invention may contain only C. sana bacteria. Such a composition may contain only C. de minimis or may contain biologically irrelevant amounts of other bacterial strains or species. Such a composition may be a culture that is substantially free of organisms from other species. In some embodiments, such a composition may be in a dried form and be substantially free of organisms from other species.

[0478] In some embodiments, the present invention provides compositions comprising a single bacterial strain of the genus Colonithrix, and no bacteria from any other strain, or only minimal or biologically irrelevant amounts of bacteria from other strains, for use in therapy.

[0479] In some embodiments, the present invention provides compositions comprising a single bacterial strain of the species Colonithrix sana (e.g., C. sana MH35-1 or C. sana MH35-2) and no bacteria from any other strain, or only minimal or biologically irrelevant amounts of bacteria from other strains, for use in therapy.

[0480] In some embodiments, the compositions disclosed herein comprise one or more bacterial strains of the genus Gemmiger, and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species. Thus, in some embodiments, the present invention provides compositions comprising one or more bacterial strains of the genus Gemmiger (e.g., Gemmiger formicilis), and no bacteria from any other species, or only minimal or biologically irrelevant amounts of bacteria from other species, for use in therapy.

[0481] In some embodiments, a composition comprises one or more bacterial strains of the genus Gemmiger and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera. In some embodiments, a composition comprises one or more bacterial strains of the genus Gemmiger (e.g., Gemmiger formicilis) and no bacteria from any other genera, or only minimal or biologically irrelevant amounts of bacteria from other genera.

[0482] In some embodiments, the compositions disclosed herein contain a single bacterial species and are free of any other bacterial species. In some embodiments, the compositions disclosed herein contain a single bacterial strain and are free of other bacterial strains. For example, a composition of the present invention may contain only G. formicilis strain bacteria. Such a composition may contain only G. de minimis or may contain biologically irrelevant amounts of other bacterial strains or species. Such a composition may be a culture that is substantially free of organisms from other species. In some embodiments, such a composition may be in a dried form and be substantially free of organisms from other species.

[0483] In some embodiments, the present invention provides compositions comprising a single bacterial strain of the genus Gemmiger, free of bacteria from any other strain, or containing only minimal or biologically irrelevant amounts of bacteria from other species, for use in therapy. In some embodiments, the present invention provides compositions comprising a single bacterial strain of the species Gemmiger (e.g., Gemmiger formicilis MH32-1), free of bacteria from any other species, or containing only minimal or biologically irrelevant amounts of bacteria from other species, for use in therapy.

[0484] In some embodiments, the compositions of the present invention contain a single bacterial strain or species and no other bacterial strains or species. Such compositions may contain only a de minimis amount or may contain biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures that are substantially free of organisms from other species.

[0485] In certain embodiments, a composition of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 bacterial strains or species. In certain embodiments, a composition comprises 1 to 10, preferably 1 to 5, bacterial strains or species. In some embodiments, a composition disclosed herein comprises two or more strains from the same species (e.g., greater than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 strains), optionally excluding bacteria from any other species. In some embodiments, a composition disclosed herein comprises fewer than 50 strains from the same species (e.g., fewer than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 strains), optionally excluding bacteria from any other species. In some embodiments, the compositions disclosed herein comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from the same species, optionally excluding bacteria from other species. In some embodiments, the compositions disclosed herein comprise multiple species from the same genus (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25, 30, 35, or 40 species), optionally excluding bacteria from other genera. In some embodiments, the compositions disclosed herein include fewer than 50 species (e.g., fewer than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4, or 3 species) from the same genus, optionally excluding bacteria from other genera. In some embodiments, the compositions disclosed herein include 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species within the same genus. The present invention includes any combination of the above.

[0486] In some embodiments, compositions of the invention comprise two or more bacterial strains or species. For example, in some embodiments, compositions of the invention comprise multiple strains from the same species (e.g., more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, or 45 strains), optionally excluding bacteria from any other species. In some embodiments, compositions of the invention comprise fewer than 50 strains from the same species (e.g., fewer than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4, or 3 strains), optionally excluding bacteria from other species. In some embodiments, compositions of the invention include strains 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50. In some embodiments, compositions of the invention include multiple species from the same genus (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25, 30, 35, or 40 species), optionally excluding bacteria from other genera. In some embodiments, compositions of the invention include fewer than 50 species (e.g., fewer than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4, or 3 species) from within the same genus, optionally excluding bacteria from other genera. In some embodiments, compositions of the invention include 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains within the same genus, optionally excluding bacteria from other genera. The invention includes any combination of the above.

[0487] In one embodiment, the pharmaceutical composition of the invention comprises 1-50 different bacterial strains, such as 1-50, 1-40, 1-30, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or between two different bacterial strains. In one embodiment, the pharmaceutical composition of the invention comprises 1-50 different bacterial strains, such as 1-50, 1-40, 1-30, 1-20, 1-19, 1-18, 1-17, 1-16, 1-15, 1-14, 1-13, 1-12, 1-11, 1-10, 1-9, 1-8, 1-7, 1-6, 1-5, 1-4, 1-3 or between two different bacterial strains.

[0488] In some embodiments, the compositions of the invention additionally comprise bacterial strains that have the same safety and therapeutic efficacy characteristics as the strains deposited at NMI (Australia) under accession numbers V21 / 014432, V21 / 014433, and / or V21 / 014434.

[0489] In some embodiments, the compositions of the invention additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as the strain deposited at NMI (Australia) under accession numbers V21 / 019213 and / or V21 / 019214.

[0490] In some embodiments, the compositions of the invention additionally comprise a bacterial strain that has the same safety and therapeutic efficacy characteristics as the strain deposited at NMI (Australia) under accession number V21 / 011520.

[0491] In some embodiments, in which the compositions of the present invention comprise two or more bacterial strains, species, or genera, the individual bacterial strains, species, or genera may be for separate, simultaneous, or sequential administration. For example, the composition may include all of the two or more bacterial strains, species, or genera, or the bacterial strains, species, or genera may be stored separately and administered separately, simultaneously, or sequentially. In some embodiments, the two or more bacterial strains, species, or genera are stored separately but mixed together before use.

[0492] Preferably, the compositions disclosed herein should be administered to the gastrointestinal (GI) tract to allow delivery and / or partial or complete colonization of the intestinal tract by the bacterial strains of the present invention. In other words, the bacteria may colonize part or all of the GI tract, and such colonization may be transient or permanent. More specifically, the phrase "total intestinal colonization" means that the bacteria have colonized all parts of the intestine (i.e., the small intestine, large intestine, and rectum). Additionally or alternatively, the term "total colonization" may mean that the bacteria have permanently colonized part or all of the intestine.

[0493] Similarly, the phrase "partial colonization of the intestine" means that the bacteria have colonized some, but not all, of the intestine. Additionally, or alternatively, the term "partial colonization" means that the bacteria temporarily colonize some or all of the intestine.

[0494] Transience of bacterial engraftment can be determined by periodically (e.g., daily or weekly) assessing the abundance of the bacterial strains of the present invention after the end of the dosing interval and determining the washout period (e.g., in a fecal sample), i.e., the period between the end of the dosing interval and the absence of detectable levels of the bacterial strains of the present invention. In some embodiments, the washout period is 14 days or less, 12 days or less, 10 days or less, 7 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day or less.

[0495] In some embodiments, the bacteria described above or elsewhere herein colonize transiently in the large intestine.

[0496] In some embodiments, the bacterial strains of the present invention are obtained from adult human feces. In some embodiments in which the compositions of the present invention include two or more bacterial strains, all of the bacterial strains are obtained from adult human feces, or if other bacterial strains are present, they are present in minimal amounts. The bacteria can be obtained from these adult human feces and cultured after use in the compositions of the present invention.

[0497] In some embodiments, one or more Alistipes bacterial strains are the only therapeutically active agents in the compositions of the invention. In some embodiments, the bacterial strains in the compositions are the only therapeutically active agents in the compositions of the invention.

[0498] Compositions for use in accordance with the present invention may or may not require marketing approval.

[0499] In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is in a dried form. Optionally, the bacterial strain is reconstituted prior to administration. Optionally, dissolution is by use of a diluent as described herein. In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is spray-dried. In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and viable. In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and viable. In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilized or spray-dried and capable of partially or fully colonizing the intestine. In some embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is dried (e.g., lyophilized or spray-dried), viable, and capable of partially or fully colonizing the intestine. In some of the same embodiments and in some alternative embodiments, the bacterial strain transiently colonizes the intestine.

[0500] In some cases, freeze-dried or spray-dried strains are reconstituted prior to administration. In some cases, reconstitution is by use of a diluent as described herein.

[0501] The compositions of the present invention may include a pharmaceutically acceptable excipient, diluent or carrier.

[0502] In some embodiments, the present invention provides pharmaceutical compositions comprising the bacterial strain of the present invention and a pharmaceutically acceptable excipient, carrier, or diluent, wherein the bacterial strain is in an amount sufficient to treat or prevent an inflammatory or autoimmune disorder when administered to a subject in need thereof. In some preferred embodiments, the inflammatory or autoimmune disorder is selected from the group consisting of inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, vasculitis, and type 1 diabetes.

[0503] In one embodiment, the present invention provides a pharmaceutical composition comprising the bacterial strain of the present invention and a pharmaceutically acceptable excipient, carrier, or diluent, wherein the bacterial strain is in an amount sufficient to treat or prevent an inflammatory or autoimmune disorder mediated by the STAT3 signaling pathway. In a preferred embodiment, the disorder is selected from the group consisting of inflammatory bowel disease (such as Crohn's disease or ulcerative colitis), asthma (such as allergic asthma or neutrophilic asthma), arthritis (such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvenile idiopathic arthritis), fatty liver disease (such as non-alcoholic fatty liver disease (NAFLD)), ankylosing spondylitis, psoriasis, systemic lupus erythematosus (SLE), scleroderma, Sjögren's syndrome, vasculitis, and type 1 diabetes.

[0504] In one embodiment, the present invention provides the pharmaceutical composition described above, wherein the amount of the bacterial strain is about 1 x 10 per gram by weight of the composition. 3 ~Approx. 1×1011 Colony forming units (CFU).

[0505] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein the composition is administered in a dose of up to 1 g, 3 g, 5 g, or 10 g or more.

[0506] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

[0507] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol.

[0508] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, comprising a diluent selected from the group consisting of ethanol, glycerol, and water.

[0509] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free flow lactose, beta-lactose, corn sweeteners, acacia, gum tragacanth, sodium alginate, carboxymethylcellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0510] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, further comprising at least one of a preservative, an antioxidant, and a stabilizer.

[0511] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, further comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid, and esters of 4-hydroxybenzoic acid.

[0512] In certain embodiments, the present invention provides the above pharmaceutical composition, wherein the bacterial strain is in a dried form (e.g., freeze-dried, spray-dried, fluid-bed dried, etc.).

[0513] In certain embodiments, the present invention provides the aforementioned pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C, and the container is placed in an atmosphere having a relative humidity of 50%, at least 80% of the bacterial strains, as measured by colony forming units, remain after a period of at least about 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years, or 3 years.

[0514] In some embodiments, the compositions of the present invention are provided in a sealed container containing a composition described herein. In some embodiments, the sealed container is a pouch or bottle. In some embodiments, the compositions of the present invention are provided in a syringe containing a composition described herein.

[0515] In some embodiments, the compositions of the present invention may be provided as pharmaceutical formulations. For example, the compositions may be provided as tablets or capsules. In some embodiments, the capsules are gelatin capsules ("gel-caps"). The capsules may be hard or soft. In some embodiments, the formulations are soft capsules. Soft capsules are capsules that have a certain elasticity and flexibility due to the addition of softeners, such as glycerol, sorbitol, maltitol, and polyethylene glycol, present in the capsule shell. Soft capsules can be manufactured based on, for example, gelatin or starch. Gelatin-based soft capsules are commercially available from various suppliers. Depending on the method of administration, such as oral or rectal administration, soft capsules can have various shapes, such as round, oval, oval, or torpedo-shaped. Soft capsules can be manufactured by conventional methods, such as the Scherer method, the Acogel method, or the drop or blow method.

[0516] In some embodiments, the compositions disclosed herein are administered orally, where they may be swallowed and enter the digestive tract.

[0517] Pharmaceutical formulations suitable for oral administration include solid plugs, solid particulates, semi-solids and liquids (including multiphase or dispersion systems); soft or hard capsules containing multiparticulates, liquids (e.g., aqueous solutions), emulsions or powders; emulsions or powders; lozenges (including liquid-filled); chews; gels; fast-dispersing dosage forms; films; ovules; sprays; and buccal / mucoadhesive patches.

[0518] In some embodiments, the pharmaceutical formulation is an enteric formulation, i.e., a gastroresistant formulation (e.g., resistant to stomach pH) suitable for oral delivery of the compositions of the invention to the intestine. Enteric formulations may be particularly useful when the bacteria or other components of the composition are acid-sensitive (e.g., susceptible to degradation under stomach conditions).

[0519] In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or enteric-coated capsule, etc. The enteric coating may be a conventional enteric coating, such as a conventional coating for tablets, capsules, etc. for oral delivery. The formulation may also comprise a film coating, such as a thin layer of an enteric polymer (e.g., an acid-insoluble polymer).

[0520] In some embodiments, the enteric formulation is essentially enteric, e.g., gastric, without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not include an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thermogelling material is a cellulose material such as methylcellulose, hydroxymethylcellulose, or hydroxypropylmethylcellulose (HPMC). In some embodiments, the capsule comprises a shell that does not include a film-forming polymer. In some embodiments, the capsule comprises a shell, which includes hydroxypropylmethylcellulose and does not include a film-forming polymer (as described in U.S. Patent Publication No. 2016 / 0067188). In some embodiments, the formulation is essentially an enteric capsule (e.g., VCAPS® from Capsugel).

[0521] In some embodiments, the composition is a probiotic or medical food containing the A. shahii bacterial strain. The bacteria can be administered, for example, as a probiotic, in capsules, tablets, caplets, pills, troches, lozenges, powders, and / or granules. The strain can also be formulated as a dietary supplement, conventional food, medical food, or drug. The bacteria can also be administered as part of a fecal transplant or via a suppository. In some embodiments, the composition is formulated for delivery to the intestine, as further described herein. In some embodiments, the composition further comprises a prebiotic.

[0522] Coadministration with additional drugs In some embodiments, the methods described herein can further include co-administering a second agent and / or treatment to the subject (e.g., as part of a treatment). Combination therapies, when used, are tailored to the particular indication. For example, when administering a strain of A. shahii species to treat an inflammatory disorder (e.g., inflammatory bowel disease), it can be administered in combination with an anti-inflammatory agent or treatment known in the art that is approved for the clinical treatment of inflammatory disorders. Other indications can similarly be treated, for example, by combining a strain of A. shahii species as described herein with an agent known in the art or approved for the clinical treatment of those indications.

[0523] Similarly, when a strain of C. sana species is administered to treat an inflammatory disorder (e.g., inflammatory bowel disease), it can be administered in combination with an anti-inflammatory agent or therapy known in the art or approved for the clinical treatment of inflammatory disorders. Other indications can be treated similarly, for example, with a strain of C. sana species as described herein in combination with an agent known in the art or approved for the clinical treatment of those indications.

[0524] Similarly, when a strain of G. formicilis species is administered to treat an inflammatory disorder (e.g., inflammatory bowel disease), it can be administered in combination with an anti-inflammatory agent or therapy known in the art or approved for the clinical treatment of the inflammatory disorder. Other indications can similarly be treated using a strain of G. formicilis species, for example, as described herein, in combination with an agent known in the art or approved for the clinical treatment of those indications.

[0525] Suitable anti-inflammatory agents that can be used to treat inflammatory bowel disease include, but are not necessarily limited to, the group including 5-aminosalicylate, corticosteroids, azathioprine, infliximab, and adalimumab.

[0526] The present invention also includes the above-described compositions further comprising an anti-inflammatory agent. Such compositions may optionally be in the form of a single composition, or alternatively, may be in the form of two or more separate compositions.

[0527] 11. Screening Method The present invention also includes methods for identifying bacterial strains suitable for use in the methods of the present invention. Such methods typically involve screening for bacterial strains with specific functional activities. Suitable assays include those described in the Examples below, but any assay for measuring intestinal barrier function, mucosal healing, modulation of NF-κB activation, or modulation of STAT3 signaling is equally applicable.

[0528] In some embodiments, the screening method identifies the ability of an Alistipes bacterial strain to modulate the STAT3 signaling pathway. As an illustrative example, the invention provides a method of blocking or otherwise inhibiting activation of STAT3 signaling in a target cell, the method comprising contacting the target cell with at least a soluble component of a bacterial cell preparation of the species Alistipes shahii to block or otherwise inhibit activation of STAT3 signaling in the target cell.

[0529] In some embodiments, the screening method identifies the ability of a bacterial strain of Colonithrix to modulate the STAT3 signaling pathway. As an illustrative example, the present invention provides a method of blocking or otherwise inhibiting activation of STAT3 signaling in a target cell, the method comprising contacting the target cell with at least a soluble component of a bacterial cell preparation of the C. sana species to block or otherwise inhibit activation of STAT3 signaling in the target cell.

[0530] In some embodiments, the screening method identifies the ability of a bacterial strain of Gemmiger to modulate the STAT3 signaling pathway. As an illustrative example, the invention provides a method of blocking or otherwise inhibiting activation of STAT3 signaling in a target cell, the method comprising contacting the target cell with at least a soluble component of a bacterial cell preparation of the G. formicilis species to block or otherwise inhibit activation of STAT3 signaling in the target cell.

[0531] In some embodiments of this type, the target cells are selected from the group including screening bacterial strains for functional reporter cells (e.g., HEK cells), immune cells (e.g., Th17 immune cells), epithelial cells, and endothelial cells.

[0532] In some embodiments, the bacterial cell preparation comprises a bacterial cell culture. Preferably, the soluble components may comprise the supernatant of the bacterial cell culture. In some embodiments of this type, the soluble components are substantially depleted of bacterial cells.

[0533] In some alternative embodiments, the bacterial cell preparation comprises a bacterial cell pellet. Preferably, the bacterial cells of the cell pellet are lysed by any means known in the art. After cell lysis, the soluble fraction of the cell lysate is typically separated from the insoluble fraction. The cell lysate can be further processed (e.g., diluted in a buffer) or exposed to a treatment reagent before being used in a screening assay.

[0534] 12. Mode of administration Preferably, the compositions of the present invention should be administered to the gastrointestinal tract to allow delivery to the intestine along with the bacterial strains of the present invention. Preferably, the compositions of the present invention are formulated to be administered to the gastrointestinal tract to allow delivery to the intestine along with the bacterial strains of the present invention. In some embodiments, the compositions of the present invention are formulated to be administered to the gastrointestinal tract to allow delivery and partial or complete colonization of the intestine by the bacterial strains of the present invention.

[0535] In certain embodiments, the compositions of the present invention may be administered as a foam, as a spray, or as a gel.

[0536] In certain embodiments, the compositions of the present invention can be administered in the form of a suppository, such as theobroma oil (cocoa butter), synthetic hard fat (e.g., Suppocire®, WITEPSOL), glycerogelatin, polyethylene glycol, or soap glycerin composition.

[0537] In certain embodiments, the compositions of the present invention are administered to the gastrointestinal tract via a tube such as a nasogastric tube, an orogastric tube, a gastric tube, a jejunostomy tube, a jejunostomy tube (J-tube), a percutaneous endoscopic gastrostomy (PEG), or a port such as a chest wall port that provides access to the stomach, jejunum, and other suitable access ports.

[0538] The compositions of the present invention can be administered once or continuously as part of a treatment regimen. In some embodiments, the compositions of the present invention are administered daily (once or several times). In some embodiments, the compositions disclosed herein are administered periodically, such as daily, every other day, or weekly, for an extended period of time, such as at least one week, two weeks, one month, two months, six months, or one year.

[0539] In some embodiments, the compositions disclosed herein are administered for 7 days, 14 days, 16 days, 21 days, or 28 days, or for up to 7 days, 14 days, 16 days, 21 days, or 28 days. For example, in some embodiments, the compositions disclosed herein are administered for 16 days.

[0540] In one embodiment of the invention, treatment according to the invention is accompanied by an assessment of the patient's gut microbiota: if delivery and / or partial or complete colonization with the strains of the invention is not achieved and therefore efficacy is not observed, treatment can be repeated, or if delivery and / or partial or complete colonization is successful and efficacy is observed, treatment can be discontinued.

[0541] In certain embodiments, the compositions of the present invention can be administered to a pregnant animal, e.g., a mammal such as a human, to prevent inflammatory or autoimmune disorders (such as those disclosed herein) that occur in utero and / or after birth in the offspring.

[0542] The compositions of the invention can be administered to patients diagnosed with a disease or condition mediated by dysregulation of intestinal barrier function, or identified as being at risk for a disease or condition mediated by intestinal barrier dysfunction, a disease or condition mediated by the STAT3 signaling pathway, or a disease or condition mediated by the STAT3 signaling pathway, or an inflammatory or autoimmune disorder (such as those disclosed herein). The compositions can also be administered as a prophylactic measure to prevent the onset of a disease or condition mediated by the STAT3 signaling pathway in healthy patients.

[0543] The compositions disclosed herein can be administered to patients diagnosed with or identified as at risk for an inflammatory or autoimmune disorder, particularly an inflammatory or autoimmune disorder mediated by the microbiota-gut axis. The compositions can also be administered as a prophylactic measure to prevent the development of an inflammatory or autoimmune disorder, particularly an inflammatory or autoimmune disorder mediated by the microbiota-gut axis, in healthy patients.

[0544] The compositions of the present invention can be administered to patients identified as having an abnormal gut microbiota. For example, the patient may have reduced or absent colonization by bacteria of the genus Alistipes, particularly A. shahii. Alternatively, or in addition, the patient m...

Claims

**Claim 1** A composition comprising cells or a biologically pure culture of Alistipes shahii strain deposited under any one of the accession numbers V21 / 014432, V21 / 014433, or V21 / 014434 or a derivative thereof, or Gemmiger formicilis strain deposited under the accession number V21 / 011520 or a derivative thereof, or C. sana strain deposited under the accession numbers V21 / 019213 or V21 / 019214 or a derivative thereof. **Claim 2** A composition comprising a bacterial strain having a 16S rRNA sequence that is at least about 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to any one of SEQ ID NOs: 1-3, 11-18, 27, or 44-47, or having a 16S rRNA gene sequence represented by any one of SEQ ID NOs: 1-3, 11-18, 27, or 44-47. **Claim 3** A pharmaceutical composition comprising a bacterial strain that is a phylogenetic descendant of the MRCA of A. timonensis and A. sp000434235, or G. variable and G. sp002306375, or C. sana and C. sp002437735, together with a pharmaceutically acceptable carrier, diluent, or excipient, wherein the MRCA is defined by node 35260, or node 23818, or node 23867 of the bac120 phylogenetic tree from Genome Taxonomy Database (GTDB) release 89. **Claim 4** The composition according to any one of claims 1-3, wherein the bacterial strain is at least partially isolated. **Claim 5** The composition according to any one of claims 1-3, wherein the bacterial strain is viable or non-viable. **Claim 6** The composition according to any one of claims 1-3, further comprising prebiotics. **Claim 7** The composition according to any one of claims 1-3, further comprising one or more additional bacterial strains. **Claim 8** The composition according to any one of claims 1-3, wherein the bacterial strain produces an agent that attenuates or impairs intracellular signal transduction and activator of transcription 3 (STAT3), and / or produces one or more metabolites selected from butyrate, acetate, ethanol, and fumarate. **Claim 9** The composition according to claim 8, wherein the agent is a small molecule, a peptide, or a nucleotide.

10. The composition according to claim 8, wherein the agent specifically binds to any one of STAT3, JAK2, TYK, or IL-23.

11. Use of the composition according to claim 1 for the manufacture of a medicament for restoring or improving intestinal barrier function in a subject.

12. Restoring or improving intestinal barrier function is characterized by at least one of (i) an increase in the quality and / or quantity of mucin, (ii) an improvement in the integrity of tight junction proteins, (iii) a reduction in the translocation of luminal contents into the systemic circulation, or (iv) a reduction in intestinal ulcers and / or intestinal injuries, and the composition according to claim 10, wherein the luminal contents contain lipopolysaccharide (LPS).

13. The composition according to claim 10, wherein the restoration or improvement of intestinal barrier dysfunction results in a reduction in systemic inflammation in the subject, and systemic inflammation is identified in the subject when the levels of inflammatory cytokines (e.g., IL-1β, IL-8, IL-6, and TNF) in a sample derived from the subject exceed a predetermined threshold.

14. Use of the composition according to claim 1 for the manufacture of a medicament for inducing or enhancing mucosal healing in a subject.

15. The use according to claim 14, wherein mucosal healing is measured using one or more fecal markers or serum markers, and the one or more fecal markers are selected from the group consisting of calprotectin, lactoferrin, metalloproteinase (MMP)-9, and lipocalin-2.

16. Use of the composition according to claim 1 for the manufacture of a medicament for a method of reducing inflammation in a subject, wherein the inflammation is local to the intestinal environment or is systemic inflammation.

17. Use of the composition according to claim 1 for the manufacture of a medicament for a method of blocking or otherwise inhibiting the activation of STAT3 signaling in target cells, wherein the target cells are selected from the group consisting of reporter cells (e.g., HEK cells), immune cells (e.g., Th17 immune cells), epithelial cells, and endothelial cells.

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