Methods and compositions for immune system modulation

Abstract

Provided herein, inter alia, are compositions comprising one or more substantially pure bacterial strains and methods of making and using these compositions to treat and / or prevent one or more immune system-related disorders and modulate the production of one or more immune system function mediators in a subject in need thereof.

Description

Cross-references to related applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 578,003, filed August 22, 2023, and U.S. Provisional Patent Application No. 63 / 605,714, filed December 4, 2023, the disclosures of which are incorporated herein by reference in their entirety. By referencing and incorporating into the sequence list

[0002] According to 37 CFR The sequence list submitted as an XML file, 2412, is incorporated herein by reference. The XML file is named “NB42223WOPCT_Seq_List.xml”, created on August 12, 2024, and is 19,712 bytes in size. Technical Field

[0003] This article provides, in particular, bacterial compositions that can be used to modulate (i.e., enhance, stimulate, or downregulate) the immune system function and immune system function mediators of subjects, as well as methods for preparing and using these bacterial compositions. Background Technology

[0004] The human immune system is a complex network of organs, cells, and proteins that work together to maintain homeostasis within the body. The balanced interaction between human immune cells and symbiotic microorganisms is crucial not only for maintaining this homeostasis but also for infection prevention, lymphoid tissue maturation, and the functional development of immune cells.

[0005] Animal studies in germ-free mice have shown that the immune system cannot develop or function properly in the absence of a microbiome, and that immune dysregulation is associated with a variety of human diseases (Round and Mazmanian 2009). Probiotics have been found to enhance innate immunity and modulate pathogen-induced inflammation through various mechanisms, including toll-like receptor-regulated signaling pathways. First-generation probiotics are live microorganisms primarily derived from the genera *Lactobacillus* and *Bifidobacterium*, which are typically minor components of the digestive tract or obtained through use as dairy starter cultures. However, in most cases, the clinical efficacy and mechanisms of action of first-generation probiotics remain unclear. Unlike opportunistic pathogens that trigger immune responses leading to tissue damage during infection, some naturally occurring / symbiotic bacterial species have been shown to not only prevent inflammatory diseases during colonization but also induce inflammation under specific conditions.

[0006] Therefore, what is needed are additional microorganisms that are identified based on their natural presence in healthy individuals and selected based on their ability to maintain and optimize immune health and prevent disease.

[0007] The topics disclosed in this article address these needs and offer additional benefits. Summary of the Invention

[0008] This article provides, in particular, methods for treating and / or preventing one or more immune system-related disorders in subjects in need, including the administration of an effective amount of a composition comprising strains of one or more substantially pure bacterial species.

[0009] In some embodiments, the one or more substantially pure bacterial species are symbiotic bacterial species. In some embodiments, the one or more substantially pure bacterial species are selected from species of the genera Akkermansia, Alistipes, Bacteroides, Barnesiella, Intestinimonas, Oscillibacter, and Phocaeicola.

[0010] In some embodiments, the bacterial species is selected from species of the genus Akkermania.

[0011] In some embodiments, the bacterial species is an Akkermansia species. In some embodiments, the Akkermansia species is not (i) Akkermansia muciniphila; or (ii) Akkermansia glycaniphila. In some embodiments, the Akkermansia species shares at least about 95% genome-wide average nucleotide identity (gANI) with Akkermansia massiliensis. In some embodiments, the Akkermansia species includes Akkermansia massiliensis. In some embodiments, the Akkermansia species comprises an Akkermansia species deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of an Akkermansia species deposited in the DSM, numbered DSM 33459.

[0012] In some embodiments, the bacterial species is a species of the genus *Alistipes*. In some embodiments, the *Alistipes* species is *Alistipes inops* and / or *Alistipes onderdonkii*. In some embodiments, *Alistipes inops* is a strain deposited in the DSM, numbered DSM 34031, or a live strain possessing all the identifying characteristics of the *Alistipes inops* strain deposited in the DSM, numbered DSM 34031; and *Alistipes onderdonkii* is a strain deposited in the DSM, numbered DSM 34033, or a live strain possessing all the identifying characteristics of the *Alistipes onderdonkii* strain deposited in the DSM, numbered DSM 34033.

[0013] In some embodiments, the bacterial species is a species of the genus *Bacteroides*. In some embodiments, the *Bacteroides* species is *Bacteroides finegoldii*. In some embodiments, *Bacteroides finegoldii* is a strain deposited in DSM 34013 or a live strain possessing all the identifying characteristics of the *Bacteroides finegoldii* strain deposited in DSM 34013.

[0014] In some embodiments, the bacterial species is a species of the genus *Barnesiella*. In some embodiments, the *Barnesiella* species is *Barnesiella intestinihominis*. In some embodiments, the *Barnesiella intestinihominis* is a) a strain deposited in the DSM, numbered DSM 34012, or a live strain having all the identifying characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, numbered DSM 34012; and / or b) a strain deposited in the DSM, numbered DSM 34032, or a live strain having all the identifying characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, numbered DSM 34032.

[0015] In some embodiments, the bacterial species is an Enteromonas species. In some embodiments, the Enteromonas species is *Intestinimonas massiliensis*. In some embodiments, *Intestinimonas massiliensis* is a strain deposited in the DSM, numbered DSM 33460, or a live strain possessing all the identifying characteristics of the *Intestinimonas massiliensis* strain deposited in the DSM, numbered DSM 33460.

[0016] In some embodiments, the bacterial species is a species of the genus *Oscillatoria*. In some embodiments, the *Oscillatoria* species has at least about 95% genome-wide average nucleotide identity (gANI) with the *Oscillatoria* species deposited under DSM 34011. In some embodiments, the *Oscillatoria* strain is a strain deposited under DSM 34011 or a live strain possessing all the identifying characteristics of the *Oscillatoria* species deposited under DSM 34011.

[0017] In some embodiments, the bacterial species is a species of the genus *Phocaeicola*. In some embodiments, the *Phocaeicola* species is *Phocaeicola vulgatus*. In some embodiments, *Phocaeicola vulgatus* is a strain deposited in the DSM with the number DSM 34030 or a live strain possessing all the identifying characteristics of the *Phocaeicola* species deposited in the DSM with the number DSM 34030.

[0018] In some embodiments, the treatment and / or prevention includes stimulating an immune response in a subject. In some embodiments, the treatment and / or prevention includes downregulating an immune response in a subject. In some embodiments, the treatment and / or prevention includes modulating the production of one or more immune system functional mediators.

[0019] In some embodiments, the one or more immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both. In some embodiments, the one or more immune system functional mediators include IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β, and / or any combination thereof. In some embodiments, these immune system functional mediators are increased or decreased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the levels of immune system functional mediators in the subject prior to administration of the composition.

[0020] In some embodiments, the one or more immune system functional mediators induce immune enhancement or immune stimulation. In some embodiments, the one or more immune system functional mediators induce anti-inflammatory effects. In some embodiments, the one or more immune system-related disorders are selected from viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, oncological diseases, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders. In some embodiments, the one or more immune system-related disorders are not metabolic disorders.

[0021] In some embodiments, the one or more immune system-related disorders include acute inflammatory disorders or chronic inflammatory disorders. In some embodiments, the inflammatory-related disorder is selected from irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo.

[0022] In some embodiments, the composition comprises probiotics, prebiotics, post-biotics, human milk oligosaccharides, xylitol, betaine, and / or plant preparations. In some embodiments, the composition has been pasteurized or heat-treated. In some embodiments, the composition is lyophilized, freeze-dried, or spray-dried. In some embodiments, the composition is encapsulated or coated. In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated as tablets, lozenges, extended-release capsules, extended-release granules, powders, sachets, nasal sprays, ointments, serums, lotions, or adhesives. In some embodiments, the composition is formulated as a food product, nutritional product, food ingredient, dietary supplement, or pharmaceutical preparation.

[0023] In some embodiments, the composition contains at least about 1 x 10 4 CFU / g to at least approximately 1 x 10⁻⁶ 14 A substantially pure strain of any of the bacterial species provided herein, CFU / g. In some embodiments, the treatment and / or prevention comprises administering the composition for at least 1 day. In some embodiments, the treatment and / or prevention comprises administering the composition for at least 1 week. In some embodiments, the treatment and / or prevention comprises administering the composition for at least 1 month.

[0024] This article provides a method for modulating the production of one or more immune system functional mediators in a subject in need, the method comprising administering an effective amount of a composition comprising one or more substantially pure bacterial species.

[0025] In some embodiments, the one or more substantially pure bacterial species are symbiotic bacterial species. In some embodiments, the one or more substantially pure bacterial species are selected from Akkermania species and Alternaria species, Bacteroides species, Barnes species, Enteromonas species, Vibrio spirochetes species and Cyclosporium species.

[0026] In some embodiments, the bacterial species is selected from species of the genus Akkermania.

[0027] In some embodiments, the bacterial species is an Akkermansia species. In some embodiments, the Akkermansia species is not (i) Akkermansia myxophila; or (ii) Akkermansia polysaccharide. In some embodiments, the Akkermansia species shares at least about 95% genome-wide average nucleotide identity (gANI) with Akkermansia massiliensis. In some embodiments, the Akkermansia species includes Akkermansia massiliensis. In some embodiments, the Akkermansia species comprises an Akkermansia species deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of an Akkermansia species deposited in the DSM, numbered DSM 33459.

[0028] In some embodiments, the bacterial species is a species of the genus *Alistipes*. In some embodiments, the *Alistipes* species is *Alistipes inops* and / or *Alistipes onderdonkii*. In some embodiments, *Alistipes inops* is a strain deposited in the DSM, numbered DSM 34031, or a live strain possessing all the identifying characteristics of the *Alistipes inops* strain deposited in the DSM, numbered DSM 34031; and *Alistipes onderdonkii* is a strain deposited in the DSM, numbered DSM 34033, or a live strain possessing all the identifying characteristics of the *Alistipes onderdonkii* strain deposited in the DSM, numbered DSM 34033.

[0029] In some embodiments, the bacterial species is a species of the genus *Bacteroides*. In some embodiments, the *Bacteroides* species is *Bacteroides finegoldii*. In some embodiments, *Bacteroides finegoldii* is a strain deposited in DSM 34013 or a live strain possessing all the identifying characteristics of the *Bacteroides finegoldii* strain deposited in DSM 34013.

[0030] In some embodiments, the bacterial species is a species of the genus *Barnesiella*. In some embodiments, the *Barnesiella* species is *Barnesiella intestinihominis*. In some embodiments, *Barnesiella intestinihominis* is a) a strain deposited in the DSM, numbered DSM 34012, or a live strain having all the identifying characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, numbered DSM 34012; and / or b) a strain deposited in the DSM, numbered DSM 34032, or a live strain having all the identifying characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, numbered DSM 34032.

[0031] In some embodiments, the bacterial species is an Enteromonas species. In some embodiments, the Enteromonas species is *Intestinimonas massiliensis*. In some embodiments, *Intestinimonas massiliensis* is a strain deposited in the DSM, numbered DSM 33460, or a live strain possessing all the identifying characteristics of the *Intestinimonas massiliensis* strain deposited in the DSM, numbered DSM 33460.

[0032] In some embodiments, the bacterial species is a species of the genus *Oscillatoria*. In some embodiments, the *Oscillatoria* species has at least about 95% genome-wide average nucleotide identity (gANI) with the *Oscillatoria* species deposited under DSM 34011. In some embodiments, the *Oscillatoria* strain is a strain deposited under DSM 34011 or a live strain possessing all the identifying characteristics of the *Oscillatoria* species deposited under DSM 34011.

[0033] In some embodiments, the bacterial species is a species of the genus *Phocaeicola*. In some embodiments, the *Phocaeicola* species is *Phocaeicola vulgatus*. In some embodiments, *Phocaeicola vulgatus* is a strain deposited in the DSM with the number DSM 34030 or a live strain possessing all the identifying characteristics of the *Phocaeicola* species deposited in the DSM with the number DSM 34030.

[0034] In some embodiments, the one or more immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both. In some embodiments, the one or more immune system functional mediators include IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β, and / or any combination thereof. In some embodiments, the one or more immune system functional mediators are increased or decreased by 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the levels of immune system functional mediators in the subject prior to administration of the composition.

[0035] In some embodiments, the one or more immune system functional mediators induce immune enhancement or immune stimulation. In some embodiments, the one or more immune system functional mediators induce anti-inflammatory effects. In some embodiments, the one or more immune system functional mediators are associated with immune system-related disorders. In some embodiments, immune system-related disorders include non-inflammatory disorders, acute inflammatory disorders, and / or chronic inflammatory disorders.

[0036] In some embodiments, the composition comprises probiotics, prebiotics, post-biotics, human milk oligosaccharides, xylitol, betaine, and / or plant preparations. In some embodiments, the composition has been pasteurized or heat-treated. In some embodiments, the composition is lyophilized, freeze-dried, or spray-dried. In some embodiments, the composition is encapsulated or coated. In some embodiments, the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient. In some embodiments, the composition is formulated as tablets, lozenges, extended-release capsules, extended-release granules, powders, sachets, nasal sprays, ointments, serums, lotions, or adhesives. In some embodiments, the composition is formulated as a food product, food ingredient, dietary supplement, or pharmaceutical preparation. In some embodiments, the composition contains at least about 1 x 10 4 CFU / g to at least approximately 1 x 10⁻⁶ 14 A nearly pure strain of Akkermania species with a CFU / g concentration.

[0037] Each of the aspects and embodiments described herein can be used together unless explicitly or clearly excluded from the context of the embodiment or aspect.

[0038] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. For all purposes, the disclosures of all patents, patent applications, and other publications cited herein are incorporated herein by reference in their entirety. Attached Figure Description

[0039] Figure 1 A phylogenetic tree was constructed for 1000 single-copy genes from selected types of Akkermansia strains, including a new species of Akkermansia masai (Masai-P6666T) and a new species of Akkermansia timonensis (Akk0196T) (Ndongo et al., 2022). The phylogenetic tree was constructed using a neighbor-joining method with 100 bootstrap iterations. Numbers represent bootstrap values. Legend bars indicate Robinson-Foulds phylogenetic distances.

[0040] Figure 2 This study depicted the production of cytokines (in pg / ml) in the cell culture supernatant of macrophages stimulated with either *Akermansia myxophila* DSM22959 or *Akermansia masei* DSM33459 for 24 hours, with standard culture medium used as a negative control, and included standard error. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001.

[0041] Figure 3 This study depicted the production of cytokines (in pg / ml) in the cell culture supernatant of dendritic cells stimulated with either *Akermansia myxophila* DSM22959 or *Akermansia masei* DSM33459 over 24 hours, with standard culture medium used as a negative control, and included standard error. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001.

[0042] Figure 4A phylogenetic tree was constructed using 1000 single-copy genes from strains of selected types of the genus *Oscillatoria*, including type strains and other publicly available genomes, including a novel species of *Oscillatoria* (DSM34011). The phylogenetic tree was constructed using a neighbor-joining method with 100 bootstrap iterations. Numbers represent bootstrap values. Legend bars indicate Robinson-Foulds phylogenetic distances.

[0043] Figure 5 The logarithmic values ​​of cytokine production in the cell culture supernatant of macrophages stimulated with the following strains over 24 hours, relative to the negative control, were depicted. 10 Fold-change ratios: *Alternaria alternata* DSM 34031, *Alternaria onseti* DSM 34033, *Bacteroides cylindrica* DSM 34013, *Barnesia esculenta* DSM 34012, *Barnesia esculenta* DSM 34032, *Enteromonas masei* DSM 33460, *Oscillatoria* species DSM 34011, and *Cryptospira marineri* DSM 34030, with standard culture medium used as a negative control. Box plots show the median and whisker lines at the minimum and maximum values. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001.

[0044] Figure 6 The logarithmic values ​​of cytokine production in the cell culture supernatant of dendritic cells stimulated with the following strains over 24 hours, relative to the negative control, were depicted. 10 Fold-change ratios: *Alternaria alternata* DSM 34031, *Alternaria onseti* DSM 34033, *Bacteroides cylindrica* DSM 34013, *Barnesia esculenta* DSM 34012, *Barnesia esculenta* DSM 34032, *Enteromonas masei* DSM 33460, *Oscillatoria* species DSM 34011, and *Cryptospira marineri* DSM 34030, with standard culture medium used as a negative control. Box plots show the median and whisker lines at the minimum and maximum values. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001.

[0045] Figures 7A-7C This study describes the analytes tested against macrophage-like cells on a multiplex assay (Bio-Plex Pro human cytokine screening assay), including interleukin (IL) 1β (IL-1β), IL-6, IL-10, tumor necrosis factor α (TNFα), interferon-γ-induced protein 10 (IP-10, also known as CXCL10), and monocyte chemoattractant protein 1 (MCP-1, also known as CC motif chemokine 2 or CCL2). These macrophage-like cells were incubated alone in the presence of *Akermansia masei* DSM33459 (AmasDSM33459), *Akermansia masei* model strain CECT 30548 (Amas(TS)), or *Akermansia myxophilus* DSM 22959 (Amuc) strains (without stimulation, ( Figure 7A Alternatively, incubation can be performed in the presence of either of the following two compounds that are individually stimulating to macrophages: a combination of 15 µg / ml polyinosinic acid:polycytidylic acid (PolyIC) and 5 µM retinomod (R848) (PolyIC, Figure 7C ); or incubate with TNFα stimulation at a final concentration of 1 ng / ml ( ); Figure 7B ). Detailed Implementation

[0046] Various studies have demonstrated that probiotics (e.g., those from the genera *Lactobacillus* and *Bifidobacterium*) support the growth of beneficial gut bacterial colonies. Although the epithelial barrier ensures that microbes are largely confined to the gut, microbial metabolites can penetrate this barrier, allowing them to enter and accumulate in the host circulatory system, where they are sensed by immune cells. Therefore, symbiotic microbial organisms can modulate systemic immunity through their effects on several cell types, including epithelial cells, dendritic cells (DCs), and T cells, and have the potential to exert both pro-inflammatory and anti-inflammatory responses, contributing to the normal functioning of the immune system. For example, *Ackermania* is a common genus in the human gut, and although it is associated with beneficial effects on human health, most evidence to date has been evaluated for the *Ackermania myxophilus* species. *Ackermania masais* is a novel anaerobic bacterial strain isolated from feces from healthy human donors, and therefore its role in human immune health remains undetermined.

[0047] The inventors of this application have unexpectedly discovered that several microorganisms (including Akkermansia masais, Parvovirus DSM 34031, Parvovirus oncogenes DSM 34033, Bacteroides aureus DSM 34013, Barnesia rumenella DSM 34012, Barnesia rumenella DSM 34032, Enteromonas masais DSM 33460, Oscillatoria species DSM 34011, and Cyclosporium marinum DSM 34030) in addition to commonly used probiotics Lactobacillus and Bifidobacterium can successfully modulate immune system functional mediators in various cell types, and thus have potential applications in immune system-related disorders.

[0048] I. Definition

[0049] As used in this article, “microorganism (or microbe)” refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0050] As used herein, the term "probiotic" refers to a composition for animal consumption (i.e., as an animal feed or a component of animal feed) containing viable (i.e., live) microorganisms, that is, microorganisms capable of surviving and multiplying, which, when administered in adequate amounts, confer health benefits to a subject (see Hill et al., 2014 Nature Revs Gastro & Hep, 11, 506-514, which is incorporated herein by reference in its entirety). Probiotics may contain one or more of any of the microbial strains described herein (such as any of 1, 2, 3, or 4). Probiotics are distinguished from bacterial compositions that have been killed, for example, by pasteurization or heat treatment. In some embodiments of the methods disclosed herein, the administration of inactive bacterial compositions to treat one or more immune system-related disorders is also contemplated.

[0051] As used in this article, a bacterial "strain" refers to a bacterium that remains genetically unchanged during growth or reproduction. This includes many identical bacteria.

[0052] "At least one strain" means a single strain, but also a mixture of strains containing at least two microbial strains. "A mixture of at least two strains" means a mixture of two, three, four, five, six, or even more strains. In some embodiments of the strain mixture, the proportions can vary between 1% and 99%. When the mixture contains more than two strains, the strains may be present in substantially equal proportions or in different proportions in the mixture.

[0053] For the purposes of this disclosure, "substantially pure strain" means a strain that does not contain a quantity of other bacterial strains sufficient to interfere with the replication of the strain or detectable by normal bacteriological techniques. When used in conjunction with the organisms and cultures described herein, "isolation" includes not only substantially pure strains but also any culture of an organism that grows or is maintained other than those found in nature. In some embodiments, these strains are mutants, variants, or derivatives of strains of the genus *Ackermania* (including *Ackermania masae*), wherein the *Ackermania* species is not *Ackermania myxophilus* or *Ackermania polymorpha*, and these mutants, variants, or derivatives also provide benefits comparable to those provided by strains of the genus *Ackermania* (including *Ackermania masae*), wherein the *Ackermania* species is not *Ackermania myxophilus* or *Ackermania polymorpha*. In some embodiments, these strains are strains that have all the identifying characteristics of strains of the genus *Ackermania* (including *Ackermania masae*), wherein the *Ackermania* species is not *Ackermania myxophilus* or *Ackermania polymorpha*. In addition, each individual strain (Ackermania species, including Massey Ackermania) or any combination of these strains may provide one or more of the benefits described herein. In some embodiments, these strains are mutants, variants, or derivatives of the following strains: *Alternaria fusca*, *Alternaria onmosca*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masei*, *Cryptococcus marines*, and / or *Oscillatoria* species whose 16S ribosomal RNA sequence shows at least 97.0% sequence similarity. These mutants, variants, or derivatives also provide benefits comparable to those provided by the following strains: *Alternaria fusca*, *Alternaria onmosca*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas marines*, *Cryptococcus marines*, and / or *Oscillatoria* species whose 16S ribosomal RNA sequence shows at least 97.0% sequence similarity. In some embodiments, these strains are strains having all the identification characteristics of the following strains: *Alternaria fusca*, *Alternaria onmosca*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masei*, *Cryptospira marines*, and / or *Oscillatoria* species whose 16S ribosomal RNA sequence shows at least 97.0% sequence similarity to the *Oscillatoria* species deposited at the German Center for Microbiology and Cell Culture (DSM) with accession number DSM 34011.Furthermore, each individual strain (such as *Alternaria fusca*, *Alternaria oncogenes*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masei*, *Cryptospira marines*, and / or *Cryptospira* species deposited at the German Center for Microbiology and Cell Culture (DSM) with 16S ribosomal RNA sequences showing at least 97.0% sequence similarity) or any combination of these strains may also provide one or more of the benefits described herein. It will also be clear that the addition of other microbial strains, vectors, additives, enzymes, yeasts, etc., will also provide one or more benefits or improve one or more immune system-related disorders in subjects, and will not constitute substantially different bacterial strains.

[0054] The term "16S rRNA" or "16S ribosomal RNA" refers to the rRNA that makes up the small subunit of the ribosome in prokaryotes. In bacteria, this sequence can be used to identify and characterize operational taxonomic units.

[0055] As used herein, the terms “sequence identity” or “sequence similarity” mean that two polynucleotide sequences (candidate sequence and reference sequence) are identical in length (i.e., 100% sequence identity) or similar (i.e., on a nucleotide-by-nucleotide basis). When comparing a candidate sequence to a reference sequence, the candidate sequence may contain additions or deletions (i.e., vacancies) compared to a reference sequence used for optimal alignment of the two sequences (which does not contain additions or deletions). Optimal sequence alignment for determining sequence identity can be performed using any number of publicly available local alignment algorithms known in the art (such as ALIGN or Megalign (DNASTAR)) or by inspection.

[0056] As used herein, the terms “percentage (%) sequence identity” or “percentage (%) sequence similarity” with respect to a reference sequence are defined as the percentage of nucleotide residues in a candidate sequence that are identical to residues in a reference polynucleotide sequence after optimal alignment of the sequences and, where necessary, the introduction of gaps to achieve maximum percentage sequence identity.

[0057] As used herein, the terms “subject” or “patient” refer to a mammal (e.g., a human). In some embodiments, the subject has a relevant disease, disorder, or condition, such as, but not limited to, one or more immune system-related disorders. As used herein, immune system-related disorders include, but are not limited to, transient or permanent immunodeficiency, allergies, autoimmune diseases, infections, and cancer. In some embodiments, the subject has one or more of the following: viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, oncological diseases, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders. In some embodiments, the subject has one or more of the following: acute or chronic inflammatory disorders. In some embodiments, the subject has one or more inflammatory disorders selected from the following: irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage-nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo. In some embodiments, immune system-related disorders are not inflammatory disorders. In some embodiments, the subject is susceptible to a disease, disorder, or condition. In some embodiments, the subject exhibits one or more symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject does not exhibit any symptoms or characteristics of a disease, disorder, or condition. In some embodiments, the subject is an individual at risk of developing a disease, disorder, or condition. In some embodiments, the subject is an individual not at risk of developing a disease, disorder, or condition. In some embodiments, the subject is a person having one or more characteristics that indicate susceptibility to or risk of developing a disease, disorder, or condition. In some embodiments, the subject is a patient. In some embodiments, the subject is an individual who has received and / or has received diagnostic and / or therapeutic treatments.

[0058] As used in this article, “preventing” and its grammatical variations refer to methods of partially or completely delaying the occurrence or recurrence of one or more of a disorder or condition and / or its accompanying symptoms, reducing the risk of its occurrence or recurrence, or preventing its occurrence or recurrence, or preventing a subject from acquiring or regaining a disorder or condition, or reducing the risk of a subject acquiring or regaining one or more of a disorder or condition or its accompanying symptoms.

[0059] As used herein, the term “reduction” in relation to a particular trait, characteristic, property, biological process, or phenomenon means a reduction in that trait, characteristic, property, biological process, or phenomenon. A trait, characteristic, property, biological process, or phenomenon may be reduced by 0.5%, 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or greater than 100%.

[0060] As used herein, “administer” means, for example, the act of introducing one or more compositions containing one or more strains of microorganisms into a subject by feeding or oral administration, topical application, nasal administration, or a combination thereof. Compositions containing one or more strains of microorganisms may also be administered in one or more doses.

[0061] As used herein, "effective amount" means the amount by which a composition containing one or more strains of microorganisms improves one or more indicators in a subject. Improvement in one or more indicators in a subject can be measured as described herein or by other methods known in the art.

[0062] As used herein, “immune system-related disorder” means a condition, disease, or disorder characterized by alterations in the function of the immune system. As used herein, immune system-related disorders include, but are not limited to, transient or permanent immunodeficiency, allergies, autoimmune diseases, infections, and cancer. In some embodiments, an immune system-related disorder is one or more of viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, oncological diseases, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders. In some embodiments, an immune system-related disorder is an acute or chronic inflammatory disorder. In some embodiments, the immune system-related disorder is an inflammatory disorder, such as irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage-nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo. In some embodiments, the immune system-related disorder is not an inflammatory disorder. In some embodiments, the immune system-related disorder is not a metabolic disorder.

[0063] As used herein, “immune system functional mediator” means any protein, peptide, glycoprotein, lipid, and / or chemical produced by immune or non-immune cells that has an effect on the immune system of a subject. In some embodiments, immune system functional mediators are cytokines, interleukins, and / or chemokines. In some embodiments, immune system functional mediators are pro-inflammatory. In some embodiments, immune system functional mediators are anti-inflammatory. Examples of immune system functional mediators include, but are not limited to, IL-1β, IL-1α, IL-18, IL-2, IL-4, IL-7, IL-8, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-14, IL-16, IL-17, CSF, IFN-β, IFN-α, IFN-γ, IFN-λ, IFN-κ, IFN-ε, IFN-ω, TNF-α, TNF-β, TGF-α, and / or TGF-β.

[0064] Certain ranges are presented herein with the term "approximately" preceding the numerical value. The term "approximately" is used herein to provide textual support for the exact number that follows it, as well as for numbers that are close to or approximate to the number that follows the term. In determining whether a number is close to or approximate to a particular stated number, the close to or approximate unstated number may be a number that is substantially equivalent to the number in the context in which the particular statement is presented. For example, with respect to numerical values, the term "approximately" refers to the range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0065] Unless the context clearly indicates otherwise, as used herein, the singular terms “a / an” and “the” include plural indicators.

[0066] It should be further noted that claims can be drafted to exclude any optional elements. Therefore, this statement is intended as a basis for the use of exclusive terms such as “alone,” “only,” etc., or the use of “negative” to limit the description of the claim elements.

[0067] It should be noted that, as used herein, the term “consisting essentially of” refers to a composition in which the component following the term constitutes less than 30% by weight of the total composition in the presence of other known components, and does not affect or interfere with the function or activity of the components.

[0068] It should be further noted that, as used herein, the term “comprising” means, but is not limited to, the components following the term “comprising.” The components following the term “comprising” are essential or mandatory, but compositions comprising components may further include other non-mandatory or optional components.

[0069] It should also be noted that, as used herein, the term "composed of" means including but not limited to the components following the term "composed of". Therefore, the components following the term "composed of" are necessary or mandatory, and no other components are present in the composition.

[0070] Each maximum numerical limit given throughout this specification is intended to include each lower numerical limit, as such lower numerical limit is explicitly stated herein. Each minimum numerical limit given throughout this specification will include each higher numerical limit, as such higher numerical limit is explicitly stated herein. Each numerical range given throughout this specification will include each narrower numerical range falling within such a wider numerical range, as such narrower numerical range is explicitly stated in its entirety herein.

[0071] Unless otherwise defined herein, 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 pertains. Other definitions of terms may appear throughout this specification.

[0072] II. Composition

[0073] A. strain

[0074] Akkermania is a genus of Gram-negative anaerobic mucus-degrading bacteria that colonizes the intestinal mucosa of humans and rodents (Rodriguez et al. 2022. Front Immunol. [Immunology Frontiers] 13:934695). Until 2016, the genus contained only a single known species, Akkermania myxophilus. That year, Akkermania myxophilus was first isolated from the feces of reticulated pythons (Ouworkerk et al. 2016, International Journal of Systematic and Evolutionary Microbiology. [International Journal of Systematic and Evolutionary Microbiology] 66 (11):4614-4620). Recently, as described in WO / 2021 / 203083 (the contents of which are incorporated herein by reference in their entirety), a new Akkermania species (strain DSM 33459) was identified based on the genome-wide average nucleotide identity (gANI) between an isolated Akkermania species (strain DSM 33459) and Akkermania myxophilus. Subsequently, Ndongo et al. (2022) renamed this species Akkermania masaiensis, the contents of which are incorporated herein by reference in their entirety (Nature Scientific Reports. 12(21747)). It has been shown that Akkermania masaiensis synthesizes vitamin B12, a key cofactor of enzymes involved in propionate production, while Akkermania myxophilus requires chelation of vitamin B12 to produce propionate (Kumar et al., Microbiome Res Rep. 2024; 3:37).

[0075] The strains provided herein include strains of one or more substantially pure Akkermania species, such as *Ackermania marcescens*. In some embodiments, the Akkermania species are not *Ackermania myxophilia* or *Ackermania polymorphia*. The Akkermania species strain was deposited on March 4, 2020, at German Microbiology and Cell Culture GmbH (DSM), 7B Inhoffenstraße, 38124 Braunschweig, GERMANY, with accession number DSM 33459. The deposit was made in accordance with the provisions of the Budapest Treaty, which internationally recognizes the deposit of microorganisms for patent proceedings.

[0076] These compositions may include compositions containing one or more substantially pure strains of the genus *Akermansia* (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains). These compositions may include compositions containing one or more substantially pure strains of *Akermansia masae* (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) (e.g., *Akermansia masae* strain DSM 33459).

[0077] Microbial compositions (e.g., probiotic compositions) may include compositions containing one or more strains of an Akkermania species (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains), wherein the Akkermania species is Akkermania marcescens and has a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the Akkermania species deposited at the German Center for Microbiology and Cell Culture (DSM) with accession number DSM 33459. The compositions containing beneficial microorganisms disclosed herein may further include compositions containing one or more strains of Barnesella intestinalis, Paroxysmalgillus onychomycosis, Bacteroides aureus, Paroxysmalgillus niger, Enteromonas masei and / or Cyclosporium marinum and / or Oscillatoria species (e.g., any one of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains), and / or one or more strains disclosed herein (e.g., any one of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains).

[0078] Other strains provided in this article include two substantially pure strains of *Barnesia rumenella*, a substantially pure strain of *Alternaria ongolii*, a substantially pure strain of *Alternaria fusca*, a substantially pure strain of *Bacteroides aurea*, a substantially pure strain of *Enteromonas masei*, a substantially pure strain of *Cryptococcus marinum*, and a substantially pure bacterial strain with a 16S ribosomal RNA sequence showing at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of a *Cryptococcus marinum* species deposited at the German Collection of Microorganisms and Cell Cultures (DSM) with accession number DSM 34011. As used herein, “*Cryptococcus marinum*” can also be used interchangeably with “*Bacteroides marinum*” (Oren and Garrity, 2020, *International Journal of Systematic and Evolutionary Microbiology*, 70(5):2960-66, which is incorporated herein by reference).

[0079] The following strains of *Barnesium*, *Alternaria onychomycosis*, *Alternaria nigra*, *Bacteroides aurea*, *Enteromonas masei*, *Bacteroides vulgaris*, and *Oscillatoria* were deposited on September 8, 2021, at German Microbiology & Cell Culture GmbH (DSM) (7B Inhofenstrasse, Braunschweig 38124, Germany), with accession numbers DSM 34032, DSM 34012, DSM 34033, DSM 34031, DSM 34013, DSM 33460, DSM 34030, and DSM 34011, respectively. The deposit was made in accordance with the provisions of the Budapest Treaty, which internationally recognizes the deposit of microorganisms for patent proceedings. In a non-limiting embodiment, one or more strains provided herein can be used as probiotics.

[0080] Microbial compositions (such as probiotic compositions) may include compositions containing one or more strains of *Barnesia ulmoides* (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) (such as *Barnesia ulmoides* strain DSM 34032 and / or *Barnesia ulmoides* strain DSM 34012). *Barnesia ulmoides* is a Gram-negative, anaerobic, and non-spore-forming bacterium belonging to the genus *Barnesia*. The compositions containing beneficial microorganisms disclosed herein may further include those containing one or more strains of *Alternaria oncogenes*, *Alternaria nigra*, *Bacteroides aurea*, *Enteromonas masei*, *Cryptospira marinum*, and / or *Oscillatoria* species (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or one or more strains (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) and / or *Ackermania* species (e.g., *Ackermania masei* strain DSM 33459 and / or those disclosed in International Patent Application Publication No. WO 2021203083, which is incorporated herein by reference); and / or one or more strains disclosed herein (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0081] Microbial compositions (e.g., probiotic compositions) may include compositions containing one or more strains of *Alternaria oncogenes* (e.g., *Alternaria oncogenes* strain DSM 34033) and / or *Alternaria nigra* (e.g., *Alternaria nigra* strain DSM 34031) (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains). *Alternaria oncogenes* and *Alternaria nigra* are Gram-negative, rod-shaped, anaerobic bacteria belonging to the genus *Alternaria*. The compositions containing beneficial microorganisms disclosed herein may further comprise one or more strains of *Barnesium intestinale*, *Alternaria solani*, *Bacteroides aurea*, *Enteromonas masei*, *Cryptospira marines*, and / or *Oscillatoria* species (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) and / or *Ackermania* species (e.g., *Ackermania masei* strain DSM 33459, such as those disclosed in International Patent Application Publication No. WO 2021203083, which is incorporated herein by reference) (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or one or more strains disclosed herein (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0082] Microbial compositions (such as probiotic compositions) may include compositions containing one or more strains of Bacteroides aureus (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) (such as Bacteroides aureus strain DSM34013). Bacteroides aureus is a strictly anaerobic, Gram-negative bacillus commonly found in the gut. The compositions containing beneficial microorganisms disclosed herein may further include those containing one or more strains of the following species: Barnesella intestinalis, Paroxysmalgillus onychomycosis, Paroxysmalgillus niger, Enteromonas masei, Cyclosporium marinum, and / or Oscillatoria species (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or Akkermania species (e.g., Akkermania masei strain DSM 33459, such as those disclosed in International Patent Application Publication No. WO 2021203083, which is incorporated herein by reference) (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or one or more strains disclosed herein (e.g., any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0083] Compositions containing microorganisms (such as probiotic compositions) may include compositions containing one or more strains of *Monococcus marinum* (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains) (such as *Monococcus marinum* strain DSM 34030). *Monococcus marinum* is a Gram-negative obligate anaerobic bacterium. The compositions containing beneficial microorganisms disclosed herein may further include those containing one or more strains of the following species: Barnesella intestinalis, Paroxysmalgillus onychomycosis, Bacteroides aureus, Paroxysmalgillus niger, Enteromonas masei, and / or Oscillatoria species (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or Akkermania species (such as Akkermania masei strain DSM 33459, such as those disclosed in International Patent Application Publication No. WO 2021203083, which is incorporated herein by reference) (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains); and / or one or more strains disclosed herein (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains).

[0084] Microbial compositions (such as probiotic compositions) may include compositions containing one or more strains of a species of the genus *Oscillatoria* (such as any one of about 1, 2, 3, 4, 5, 6, 7, or 8 or more strains), wherein the *Oscillatoria* species has a 16S ribosomal RNA sequence that shows at least 97.0% sequence similarity to the 16S ribosomal RNA sequence of the *Oscillatoria* species deposited at the German Center for Microbiology and Cell Culture (DSM) with accession number DSM 34011. The compositions containing beneficial microorganisms disclosed herein may further include those containing one or more strains of Barnesella intestinalis, Paroxysmalgillus onychomycosis, Bacteroides aureus, Paroxysmalgillus niger, Enteromonas masei and / or Bacteroides vulgaris (such as any one of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains); and / or Akkermania species (such as Akkermania masei strain DSM 33459, such as those disclosed in International Patent Application Publication No. WO 2021203083, which is incorporated herein by reference) (such as any one of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains); and / or one or more strains disclosed herein (such as any one of about 1, 2, 3, 4, 5, 6, 7 or 8 or more strains).

[0085] The microbial compositions disclosed herein (such as probiotic compositions) may include one or more Barnesia enterica strains having a 16S ribosomal RNA sequence (such as Barnesia enterica strain DSM 34032 and / or Barnesia enterica strain DSM 34012), the 16S ribosomal RNA sequence showing at least about 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to a 16S ribosomal RNA sequence comprising SEQ ID NO:1 and / or SEQ ID NO:2. Compositions containing probiotics (such as probiotic compositions) may include one or more strains of *Osphaemonia oncogenes* having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO:3. Compositions containing probiotics (such as probiotic compositions) may include one or more strains of *Bacteroides zeylanica* having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO:4. Compositions containing probiotics (such as probiotic compositions) may include one or more strains of *Cryptospira marinum* having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO:5. Compositions containing probiotics (such as probiotic compositions) may include one or more strains of *Oscillatoria* having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity) to the 16S ribosomal RNA sequence comprising SEQ ID NO:6.

[0086] Compositions containing beneficial microorganisms (such as probiotic compositions) may include one or more Enteromonas masei strains having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity to the 16S ribosomal RNA sequence comprising SEQ ID NO:7 (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity).

[0087] Compositions containing beneficial microorganisms (such as probiotic compositions) may include one or more strains of idiopathic bacteria having a 16S ribosomal RNA sequence that shows at least about 97.0% sequence similarity to the 16S ribosomal RNA sequence comprising SEQ ID NO:8 (such as any one of about 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 100% sequence similarity).

[0088] The microbial compositions (e.g., probiotic compositions) disclosed herein may include one or more Akkermania masaiensis strains (e.g., Akkermania masaiensis strain DSM 33459), one or more Barnesia rumensis strains (e.g., Barnesia rumensis strain DSM 34032 and / or Barnesia rumensis strain DSM 34012), one or more Paroxysmaltella masaiensis strains (e.g., Paroxysmaltella rumensis strain DSM 34033), one or more Paroxysmaltella fusca strains (e.g., Paroxysmaltella fuscaensis strain DSM 34031), one or more Bacteroides cylindrica strains (e.g., Bacteroides cylindrica strain DSM 34013), one or more Enteromonas masaiensis strains (e.g., Enteromonas masaiensis strain DSM 33460), one or more Jovianella marineri strains (e.g., Jovianella marineri strain DSM 34030), and / or one or more Oscillatoria species strains (e.g., Oscillatoria species strain DSM 34011). These compositions may include actual bacteria (live or non-live) from these strains and / or one or more culture supernatants derived from culturing these strains (alone or co-cultured).

[0089] In some embodiments, the Akkermania species of the compositions disclosed herein is *Ackermania masaiensis*. In some embodiments, the Akkermania species of the compositions disclosed herein is *Ackermania masaiensis* strain DSM 33459. In some embodiments, the Akkermania species has at least 95% gANI compared to *Ackermania masaiensis*. In some embodiments, the Akkermania species is *Ackermania masaiensis* and has a gANI of less than 95%, for example, about 94%, 93%, 92%, 91%, 90%, 89%, or 88% (e.g., 87.58%) compared to the genome of *Ackermania myxophila*. In another embodiment, any Akkermania species (e.g., Akkermania masei) in the compositions disclosed herein has a gANI of less than 95%, for example about 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, or 71% (e.g., 70.17%) compared to the genome of Akkermania masei.

[0090] The compositions disclosed herein may include one or more Akkermania masae strains having a 16S ribosomal RNA sequence that shows at least about 75.0% sequence similarity (e.g., any one of about 75%, 80%, 85%, 88%, 89%, 90%, 95%, or 100% sequence similarity) to the 16S ribosomal RNA sequence of Akkermania masae.

[0091] The compositions disclosed herein can be used as supplements, food additives, and therapeutic agents for administration to subjects with and / or at risk of developing immune system-related disorders, or as part of a daily nutritional regimen for disease prevention. Probiotics are another term that can be used for these compositions containing viable microorganisms. The term "viable microorganisms" refers to microorganisms that are metabolically active or capable of differentiation. In some embodiments, the compositions disclosed herein comprise viable probiotic products and / or, in certain embodiments, compositions comprising non-viable bacteria (such as heat-treated or pasteurized compositions).

[0092] B. Preparations

[0093] Typically, the compositions disclosed herein contain bacteria, such as one or more bacterial strains. In some embodiments, the compositions are formulated into freeze-dried or lyophilized forms. For example, the compositions may contain granules or gelatin capsules, such as hard gelatin capsules, containing the bacterial strains disclosed herein.

[0094] In some embodiments, the compositions disclosed herein comprise lyophilized bacteria. Lyophilization of bacteria is a well-established procedure in the art. Alternatively, the compositions may comprise live bacterial cultures. In some embodiments, the compositions are lyophilized or spray-dried.

[0095] In some embodiments, any composition disclosed herein is encapsulated to enable the delivery of bacterial strains to the intestine. Encapsulation protects the composition from degradation before delivery to the target site, degradation pathways such as rupture due to chemical or physical stimuli (e.g., stress, enzyme activity, or physical disintegration, which may be triggered by pH changes). Any suitable encapsulation method can be used. Exemplary encapsulation techniques include retention within a porous matrix, attachment or adsorption on a solid carrier surface, self-aggregation by flocculation or with a crosslinking agent, and mechanical containment by a microporous membrane or microcapsule.

[0096] The compositions disclosed herein can be administered orally and may be in the form of tablets, capsules, lozenges, extended-release capsules, extended-release granules, powders, sachets, or adhesives. Other ingredients (such as vitamin C or minerals) may be included as oxygen scavengers and prebiotic substrates to improve delivery and / or partial or complete colonization and survival in vivo. Alternatively, the compositions disclosed herein may be administered orally as food or nutritional products (such as milk or whey-based fermented dairy products), food ingredients, dietary supplements, or as pharmaceutical products or agents.

[0097] The compositions disclosed herein can be administered nasally or through the respiratory system, and can be in the form of a nasal spray.

[0098] The compositions disclosed herein can be applied topically and can be in the form of ointment, serum or lotion.

[0099] The compositions disclosed herein can be formulated as probiotics. Alternatively, the compositions disclosed herein can be formulated as inactive bacterial compositions, such as pasteurized or heat-treated bacterial compositions. In some embodiments, the compositions disclosed herein can be formulated as prebiotics and / or postbiotics. In some embodiments, the composition may include human milk oligosaccharides, betaine, xylitol, and / or plant preparations. In some embodiments, the composition may contain vitamins, minerals, and trace elements.

[0100] Any composition disclosed herein may include a therapeutically effective amount of the disclosed bacterial strain. A therapeutically effective amount of the bacterial strain is sufficient to produce a beneficial effect on a patient. A therapeutically effective amount of the bacterial strain may be sufficient to result in partial or complete colonization of the subject's intestine and / or the subject's intestine.

[0101] For example, for adults, a suitable daily dose of bacteria could be approximately 1 x 10⁻⁶.4 To approximately 1 x 10 14 Colony forming units (CFU); for example, approximately 1 x 10⁻⁶. 7 To approximately 1 x 10 10 CFU; in another instance, approximately 1 x 10 4 To approximately 1 x 10 14 CFU; in another instance, approximately 1 x 10 7 To approximately 1 x 10 11 CFU; in another instance, approximately 1 x 10 8 To approximately 1 x 10 10 CFU; in another instance, approximately 1 x 10 6 To approximately 1 x 10 10 GPU; in another instance, approximately 1 x 10 7 To approximately 1 x 10 11 CFU; in another instance, approximately 1 x 10 8 To approximately 1 x 10 10 CFU; in another instance, approximately 1 x 10 8 To approximately 1 x 10 11 CFU. In some embodiments, the bacterial dose is at least 10. 9 Cells / day, such as at least 10 10 At least 10 11 Or at least 10 12 Cells / day.

[0102] In some embodiments, the composition contains about 1 x 10⁻⁶ units relative to its weight. 6 To approximately 1 x 10 11 The bacterial strain at a CFU / g level; for example, approximately 1 x 10⁻⁶. 8 To approximately 1 x 10 10 CFU / g. This dosage can be, for example, 1 g, 3 g, 5 g, and 10 g.

[0103] In some embodiments, the amount of bacterial strain is about 1 x 10⁻⁶ relative to the weight of the composition. 3 To approximately 1 x 10 11 Colony forming units / gram.

[0104] In some embodiments, the amount of bacterial strain is about 1 x 10⁻⁶ relative to the weight of the composition. 4 To approximately 1 x 10 14 Colony forming units / gram.

[0105] In some embodiments, any composition provided herein contains strains of one or more substantially pure Akkermania species. In some embodiments, any one or more substantially pure Akkermania species strains is substantially pure and comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% Akkermania species strains. In some embodiments, any one or more substantially pure Akkermania species strains is *Akkermania marcescens*. In some embodiments, any one or more substantially pure *Akkermania marcescens* strains is substantially pure and comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% *Akkermania marcescens* strains.

[0106] In some embodiments, any composition provided herein contains one or more substantially pure strains of: *Alternaria fusca*, *Alternaria onseti*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masae*, *Vibrio* species, or *C. marinum* species. In some embodiments, any one of one or more substantially pure strains of *Alternaria fusca*, *Alternaria onseti*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masae*, *Vibrio* species, or *C. marinum* is substantially pure and comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of *Alternaria fusca*, *Alternaria onseti*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masae*, *Vibrio* species, or *C. marinum* strains.

[0107] In some embodiments, any of the compositions disclosed herein are administered in doses between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg. In some embodiments, the lyophilized bacteria in any of the compositions disclosed herein are administered in doses between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700 mg and 800 mg, between 500 mg and 750 mg, or between 750 mg and 1000 mg.

[0108] Typically, probiotics are optionally combined with at least one suitable prebiotic compound. Prebiotic compounds are usually indigestible carbohydrates, such as oligosaccharides or polysaccharides or sugar alcohols, which are not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and trans-galacto-oligosaccharides.

[0109] In some embodiments, any composition disclosed herein is formulated to include a prebiotic compound in an amount of about 1% to about 30% by weight (e.g., 5% to 20% by weight) relative to the total weight of the composition. The carbohydrate may be selected from the group consisting of: fructooligosaccharides (or FOS), short-chain fructooligosaccharides, inulin, isomaltooligosaccharides, pectin, xylooligosaccharides (or XOS), chitosan oligosaccharides (or COS), human milk oligosaccharides (or HMOs), β-glucan, gum arabic modified starch and resistant starch, polydextrose, D-tagatose, gum arabic fiber, carob, oat, and citrus fiber. In one aspect, the prebiotic is a short-chain fructooligosaccharide (hereinafter referred to as FOSs-cc for simplicity); said FOSs-cc is not a digestible carbohydrate, is typically obtained by the conversion of beet sugar, and comprises a sucrose molecule bound with three glucose molecules. In some embodiments, any probiotic disclosed herein may be formulated together with other probiotics derived from (but not limited to) the genera *Lactobacillus* and *Bifidobacterium* (such as *Bifidobacterium lactis* B420).

[0110] In some embodiments, the compositions disclosed herein contain a single bacterial strain or species and contain no other bacterial strains or species. Such compositions may contain only trace amounts or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be cultures substantially free of other biological species. In some embodiments, the compositions of the invention consist of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 bacterial strains or species. In some embodiments, these compositions consist of 1 to 10 (e.g., 1 to 5) bacterial strains or species.

[0111] The compositions used according to the methods disclosed herein may or may not require market authorization.

[0112] Pharmaceutical preparations

[0113] Any composition disclosed herein may further comprise pharmaceutically acceptable excipients, diluents, or carriers. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical field. Examples of suitable carriers include, but are not limited to, lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, sorbitol, etc. Examples of suitable diluents include, but are not limited to, ethanol, glycerol, and water. The choice of pharmaceutical carrier, excipient, or diluent may be based on the intended route of administration and standard pharmaceutical practice. Pharmaceutical compositions may contain (or in addition to carriers, excipients, or diluents) any suitable binder, lubricant, suspending agent, coating agent (such as a gastrointestinal-resistant coating agent that does not dissolve or degrade before reaching the small or large intestine), or solubilizer. Examples of suitable binders include, but are not limited to, starch, gelatin, natural sugars such as glucose, anhydrous lactose, maltodextrin, free-flowing lactose, β-lactose, corn sweeteners, natural and synthetic gums such as gum arabic, tragacanth, or sodium alginate, carboxymethyl cellulose, and polyethylene glycol. Examples of suitable lubricants include, but are not limited to, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride. Preservatives, stabilizers, dyes, and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include, but are not limited to, esters of sodium benzoate, sorbic acid, and p-hydroxybenzoic acid. Antioxidants and suspending agents may also be used.

[0114] In some cases, the lyophilized bacterial strains are reconstituted prior to application. In other cases, reconstitution is achieved using the diluents described herein.

[0115] Pharmaceutically acceptable compositions or supports may be formulations or supports in the form of creams, foams, gels, lotions, and ointments, such as compressed tablets, tablets, capsules, ointments, suppositories, or drinkable solutions.

[0116] In some embodiments, this document provides a pharmaceutical composition comprising: a bacterial strain disclosed herein; and a pharmaceutically acceptable excipient, carrier, or diluent; wherein the amount of the bacterial strain is sufficient to treat a disorder when administered to a subject in need; and wherein the disorder is selected from the group consisting of immune system-related disorders, including but not limited to transient or permanent immunodeficiency, allergies, autoimmune diseases, infections, and cancer. In some embodiments, the subject suffers from an acute or chronic inflammatory disorder. In some embodiments, the disorder is selected from the group consisting of viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, oncological diseases, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders. In some embodiments, the disorder is selected from the group consisting of acute or chronic inflammatory disorders. In some embodiments, the disorder is selected from the group consisting of: irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage-nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo. In some embodiments, the immune system-related disorder is not an inflammatory disorder. In some embodiments, the immune system-related disorder is not a metabolic disorder.

[0117] In some embodiments, any of these pharmaceutical compositions may contain a carrier selected from the group consisting of lactose, starch, glucose, methylcellulose, magnesium stearate, mannitol, and sorbitol. In some embodiments, any of these pharmaceutical compositions may contain a diluent selected from the group consisting of ethanol, glycerol, and water.

[0118] In some embodiments, any of these pharmaceutical compositions may contain an excipient selected from the group consisting of: starch, gelatin, glucose, anhydrous lactose, free-flowing lactose, β-lactose, corn sweetener, gum arabic, tragacanth gum, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, and sodium chloride.

[0119] In some embodiments, any of these pharmaceutical compositions may contain at least one of a preservative, an antioxidant, and a stabilizer. In some embodiments, the preservative is selected from the group consisting of sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid.

[0120] In some embodiments, the above-described 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 with 50% relative humidity, at least 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the bacterial strains, as measured in colony-forming units, remain for 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.

[0121] The bacterial strains disclosed herein can be cultured using standard microbiological techniques, such as those described in the Examples section or those well known in the art.

[0122] Food products

[0123] The compositions disclosed herein can be formulated as food products. For example, in addition to the therapeutic effects of the invention, food products can also provide nutritional benefits, such as in nutritional supplements. Similarly, food products can be formulated to enhance the flavor of the compositions of the invention, or to make the compositions more consumer-appealing by making them more similar to ordinary food rather than pharmaceutical compositions. In some embodiments, the compositions are formulated as milk-based products. As used herein, the term "milk-based product" means any liquid or semi-solid milk-based or whey-based product with a different fat content. Milk-based products can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, unprocessed milk reconstituted from milk powder and whey, or processed products such as yogurt, curdled milk, curd, acidic milk, acidic whole milk, buttermilk, and other acidic milk products. Another important group includes milk beverages such as whey beverages, fermented milk, condensed milk, infant or toddler milk; flavored milk, ice cream; and milk-containing foods such as confectionery. As used herein, the term "food" is used broadly and covers both human food and animal food (i.e., feed). In the preferred aspect, food is provided for human consumption.

[0124] Foods may be in solution or as solids, depending on their use, and / or application method, and / or administration method. When used or in the preparation of foods (e.g., functional foods), any composition provided herein may be used in combination with one or more of the following substances: a nutritionally acceptable carrier, a nutritionally acceptable diluent, a nutritionally acceptable excipient, a nutritionally acceptable adjuvant, or a nutritionally active ingredient.

[0125] For example, any composition provided herein can be used as an ingredient in soft drinks, juices, or beverages containing: whey protein, health tea, cocoa beverages, milk beverages and lactic acid bacteria beverages, yogurt and drinking yogurt, cheese, ice cream, sorbets and desserts, pastries and confectionery, biscuits, cakes and cake mixes, snack foods, balanced foods and beverages, fruit fillings, health glazes, chocolate bread fillings, cheesecake-flavored fillings, fruit-flavored cake fillings, cake and donut icings, instant baking fillings, biscuit fillings, ready-to-eat bread fillings, low-calorie fillings, adult nutritional beverages, plant-based milk, acidified soy / juice beverages, aseptic / distilled chocolate beverages, bar mixes, beverage powders, calcium-fortified soy / yogurt and chocolate milk, and calcium-fortified coffee beverages.

[0126] Advantageously, when the product is a food product, the bacterial strain should remain valid up to the normal "latest sale" or "end" date for food products sold and supplied by retailers. Preferably, the validity period should extend beyond such a date until the normal freshness period ends when the food becomes noticeably spoiled. The desired length of time and normal shelf life will vary depending on the food, and those skilled in the art will recognize that shelf life will vary depending on the type of food, its size, storage temperature, processing conditions, packaging materials, and the age of the packaging equipment.

[0127] Food ingredients

[0128] The compositions of the present invention may be in the form of food ingredients and / or feed ingredients. As used herein, the terms "food ingredient" or "feed ingredient" include compositions that are functional foods or feeds or that can be added to functional foods or feeds as nutritional and / or health supplements for humans and animals. Depending on their use and / or manner of application and / or administration, food ingredients may be in liquid, suspension, or solid form.

[0129] Dietary supplements

[0130] Any composition provided herein may be in the form of a dietary supplement, or may be used alone in combination with dietary supplements, also referred to herein as food supplements. As used herein, the term "dietary supplement" means a product intended for ingestion containing a "dietary ingredient" designed to increase the nutritional value or health benefits of a diet (to complement the diet). A "dietary ingredient" may include (but is not limited to) one or any combination of the following substances: bacteria, probiotics (e.g., probiotics), vitamins, minerals, herbs or other plant products, amino acids, dietary substances, concentrates, metabolites, components, or extracts intended for use by people to supplement their diet by increasing total dietary intake.

[0131] Dietary supplements can exist in many forms, such as tablets, capsules, soft gels, gel caps, liquids, or powders. Some dietary supplements can help ensure that you get enough essential nutrients from your diet; others can help prevent or treat diseases.

[0132] Medical food

[0133] The compositions of the present invention may be in the form of medical foods. "Medical foods" means foods that are formulated for consumption or administration with or without medical supervision and are intended for specific dietary management or conditions for which unique nutritional requirements have been established through medical evaluation based on recognized scientific principles.

[0134] III. Methods

[0135] A. Methods used to treat or prevent disease

[0136] This document further provides a method for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Akermansia* species. This document further provides a method for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Alternaria rubra*, *Alternaria onseti*, *Bacteroides aureus*, *Barnesia medius*, *Enteromonas masei*, *Vibrio spirochetes* species, and / or *Cryptospira marinum* species. This document further provides a method for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Alternaria rubra* species. This document further provides a method for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Alternaria onseti* species. This document further provides methods for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Bacteroides aureus* species. This document further provides methods for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Barnesium enterica* species. This document further provides methods for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Enteromonas masei* species. This document further provides methods for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Cryptospira* species. This document further provides methods for treating and / or preventing one or more immune system-related disorders in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of *Cryptospira marinum* species. This document also provides methods for modulating the production of one or more immune system functional mediators in a subject of need, methods comprising administering an effective amount of a composition comprising one or more substantially pure strains of *Ackermania* species. This article also provides methods for modulating the production of one or more immune system functional mediators in subjects in need, methods comprising administering an effective amount of a composition comprising one or more substantially pure strains of a species of the genus *Bacteroides*.This article also provides methods for modulating the production of one or more immune system functional mediators in subjects in need, methods comprising administering an effective amount of a composition containing one or more substantially pure strains of the genus *Barnes*. This article also provides methods for modulating the production of one or more immune system functional mediators in subjects in need, methods comprising administering an effective amount of a composition containing one or more substantially pure strains of the genus *Enteromonas*. This article also provides methods for modulating the production of one or more immune system functional mediators in subjects in need, methods comprising administering an effective amount of a composition containing one or more substantially pure strains of the genus *Cryptococcus*. This article also provides methods for modulating the production of one or more immune system functional mediators in subjects in need, methods comprising administering an effective amount of a composition containing one or more substantially pure strains of the genus *Cryptococcus*.

[0137] The immune system is adept at communication and is designed to respond rapidly, specifically, and systemically to protect organisms from invading substances and diseases. The cytokine superfamily (a group of proteins that act as mediators of the immune system) is an essential part of the intercellular signaling network and is necessary for the generation and regulation of the immune system. These interacting signals can influence growth and development, hematopoiesis, lymphocyte recruitment, T cell subset differentiation, and inflammation. The exemplary signaling mechanism used by immune system modulators (such as IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, and interferons) begins with the dimerization of the appropriate receptor chain after ligand binding. Subsequently, different types of receptor-associated Janus family tyrosine kinases (Jaks) are activated, which phosphorylate the receptor chain and allow the recruitment and activation of other kinases and transcription factors, such as the signal transducer and activator of transcription (Stat) family. This facilitates the rapid translocation of these proteins to the cell nucleus and stimulates the transcription of target genes (Cameron et al. Cytokines, Chemokines and Their Receptors. [Cytokines, Chemokines and Their Receptors] in: Madame Curie Bioscience Database [Internet]. Austin (Texas): Landes Bioscience; 2000–2013).

[0138] The gut microbiome is a key factor in shaping and modulating immune system responses, with gut microbiota dysbiosis being associated with several autoimmune and immune-mediated diseases (Schirmer et al. Cell. 2016 Nov 3; 167(4):1125-1136.). However, symbiotic microbiota also regulate systemic immune responses (Lee et al. Nat. Chem. Biol. [Nature Chemical Biology] 2014; 10:416-424). Although the epithelial barrier ensures that the microbes are largely confined to the gut, microbial metabolites can penetrate the epithelial barrier, allowing them to enter and accumulate in the host circulatory system, where they are sensed by immune cells (Dorrestein et al. Immunity. [Immunity] 2014;40:824-832).

[0139] While responding to pathogenic organisms is a primary function of the immune system, the recognition and tolerance of symbiotic bacteria are equally important for host health (Kosiewicz et al., Front Microbiol. [Front Microbiology] 2011; 2:180). Symbiotic microbes calibrate innate and adaptive immune responses and influence the activation threshold of pathogenic stimuli in part by producing molecules that mediate host-microbe interactions (Donia et al., Science. [Science] 2015; 349:1254766). Other proposed modulatory approaches include activation of innate immune responses via Toll-like receptors (TLRs) and / or activation of free fatty acid receptors (FFARs) via microbial metabolites such as short-chain fatty acids (SCFAs), including acetate, propionate, and butyrate. Furthermore, these metabolites can induce naive T cells to differentiate into regulatory T cells (Tregs) or induce their migration to the gut (Rodrigues et al., Front Immunol. [Front Immunology] 2022 July 7; 13:934695).

[0140] Various clinical, epidemiological, and immunological studies have demonstrated that changes in the gut microbiota may be a significant factor in the incidence of many inflammatory disorders. Gut bacteria are a key component guiding the development and function of the immune system, and therefore, the absence of beneficial microbes that promote proper immune development due to dysbiosis can lead to inflammatory responses that are the cause of various immune diseases in humans. Important studies have shown that innate and adaptive immunosuppression exists during the control of disorders, including autoimmunity, asthma and allergies, cancer, and infectious diseases. (Round et al. Nat Rev Immunol. [Nature Immunology Review] May 2009; 9(5):313-23; Pabst et al. Muscosal Immunol. [Mucosal Immunology] May 2012; 5(3):232-9).

[0141] In some embodiments, the methods disclosed herein relate to the prevention, suppression, and treatment of immune system-related disorders. As used herein, immune system-related disorders include, but are not limited to, transient or permanent immunodeficiency, allergies, autoimmune diseases, infections, and cancer.

[0142] In some embodiments, any of the methods described herein are capable of reducing inflammation at a distal site in the subject. In some of any embodiments, the distal site is blood, skin, vagina, liver, spleen, fallopian tubes, uterus, or a combination thereof.

[0143] In some embodiments, any method described herein includes stimulating an immune response. In some embodiments, any method described herein includes stimulating an immune response in a subject. In some embodiments, any method described herein includes downregulating an immune response. In some embodiments, any method described herein includes downregulating an immune response in a subject.

[0144] In some embodiments, this document also provides a method for modulating the production of one or more immune system functional mediators in a subject of need by administering an effective amount of a composition comprising one or more substantially pure strains of Akkermania species.

[0145] In some embodiments, any method described herein includes modulating the production of one or more immune system functional mediators. In some embodiments, immune system functional mediators are cytokines, interleukins, and / or chemokines. In some embodiments, immune system functional mediators are pro-inflammatory. In some embodiments, immune system functional mediators are anti-inflammatory. Examples of immune system functional mediators include, but are not limited to, IL-1β, IL-1α, IL-18, IL-2, IL-4, IL-7, IL-8, IL-9, IL-13, IL-15, IL-3, IL-5, IL-6, IL-11, IL-12, IL-10, IL-14, IL-16, IL-17, CSF, IFN-β, IFN-α, IFN-λ, IFN-κ, IFN-ε, IFN-ω, IFN-γ, TNF-α, TNF-β, TGF-α, and / or TGF-β.

[0146] In some embodiments, immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both. In some embodiments, immune system functional mediators are cytokines or chemokines. In some embodiments, immune system functional mediators are a combination of cytokines and chemokines. In some embodiments, immune system functional mediators are cytokines. In some embodiments, immune system functional mediators are chemokines.

[0147] In some embodiments, immune system functional mediators comprise IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β, and / or any combination thereof. In some embodiments, immune system functional mediators comprise interferon-γ-induced protein 10 (IP-10, also known as CXCL10), monocyte chemokine 1 (MCP-1, also known as CC motif chemokine 2 or CCL2), and / or any combination thereof. In some embodiments, immune system functional mediators comprise IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β, interferon-γ-induced protein 10 (IP-10, also known as CXCL10), and monocyte chemokine 1 (MCP-1, also known as CC motif chemokine 2 or CCL2).

[0148] In some embodiments, compared with the levels of immune system functional mediators in the subject prior to administration of the composition, these immune system functional mediators are increased or decreased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%. In some embodiments, compared with the levels of immune system functional mediators in a subject who has not been administered the composition, these immune system functional mediators are increased or decreased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0149] In some embodiments, one or more immune system functional mediators induce immune enhancement or immune stimulation. Immune enhancement refers to an increase or stimulation of the immune system and / or its function.

[0150] In some embodiments, the one or more immune system functional mediators induce anti-inflammatory effects.

[0151] In some embodiments, the one or more immune system-related disorders are selected from viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, tumors, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders (including but not limited to immunosenescence).

[0152] In some embodiments, the one or more immune system-related disorders are not metabolic disorders.

[0153] In some embodiments, the one or more immune system-related disorders include acute inflammatory disorders or chronic inflammatory disorders.

[0154] In some embodiments, the inflammatory-related disorder is selected from irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo.

[0155] In some embodiments, any composition provided herein may be applied for at least 1 day. In some embodiments, any composition provided herein may be applied for between 1 and 2 days. In some embodiments, any composition provided herein may be applied for between 1 and 3 days. In some embodiments, any composition provided herein may be applied for between 1 and 4 days. In some embodiments, any composition provided herein may be applied for between 1 and 5 days. In some embodiments, any composition provided herein may be applied for between 1 and 6 days. In some embodiments, any composition provided herein may be applied for between 1 and 7 days. In some embodiments, any composition provided herein may be applied for between 1 and 8 days. In some embodiments, any composition provided herein may be applied for between 1 and 9 days. In some embodiments, any composition provided herein may be applied for between 1 and 10 days. In some embodiments, any composition provided herein may be applied for between 1 and 11 days. In some embodiments, any composition provided herein may be applied for between 1 and 12 days. In some embodiments, any composition provided herein may be applied for between 1 and 13 days. In some embodiments, any composition provided herein may be applied for between 1 and 14 days. In some embodiments, any composition provided herein may be applied for more than 1 day.

[0156] In some embodiments, any composition provided herein may be applied for at least 1 week. In some embodiments, any composition provided herein may be applied for between 1 and 2 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 3 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 4 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 5 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 6 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 7 weeks. In some embodiments, any composition provided herein may be applied for between 1 and 8 weeks. In some embodiments, any composition provided herein may be applied for more than 8 weeks.

[0157] In some embodiments, any composition provided herein may be applied for at least one month. In some embodiments, any composition provided herein may be applied for between one and two months. In some embodiments, any composition provided herein may be applied for between one and three months. In some embodiments, any composition provided herein may be applied for between one and four months. In some embodiments, any composition provided herein may be applied for between one and five months. In some embodiments, any composition provided herein may be applied for between one and six months. In some embodiments, any composition provided herein may be applied for between one and seven months. In some embodiments, any composition provided herein may be applied for between one and eight months. In some embodiments, any composition provided herein may be applied for between one and nine months. In some embodiments, any composition provided herein may be applied for between one and ten months. In some embodiments, any composition provided herein may be applied for between one and eleven months. In some embodiments, any composition provided herein may be applied for between one and twelve months. In some embodiments, any composition provided herein may be applied for more than twelve months.

[0158] In some embodiments, one or more (e.g., 1, 2, 3, 4, 5, 6, 7, or 8) Akkermania species strains are administered to the subject at the following ratios: at least about 1 × 10⁻⁶. 4 CFU / subject / day to at least approximately 1 x 10 14 CFU / subject / day, e.g., approximately 1 × 10 4 CFU / subject / day, 1 × 10 5 CFU / subject / day, 1 × 10 6 CFU / subject / day, 1 × 10 7 CFU / subject / day, 1 × 10 8 CFU / subject / day, 1 × 10 9 CFU / subject / day, 1 × 10 10 CFU / subject / day, 1 × 10 11 CFU / subject / day, 1 × 10 12 CFU / subject / day, 1 × 10 13 CFU / subject / day or 1 × 10 14 Any of CFU / subject / day, including all values ​​falling between these measurements.

[0159] B. Methods for preparing microbial compositions

[0160] This document also provides methods for preparing compositions comprising combining one or more substantially pure strains of an Akkermania species. In some embodiments, the Akkermania species is *Ackermania marcescens*, and said Akkermania species is not *Ackermania myxophilia* or *Ackermania polymorphia*. In some embodiments, *Ackermania marcescens* is a species deposited under DSM 33459, or a strain having all the identifiable characteristics of a strain deposited under DSM 33459. In some embodiments, the genome-wide average nucleotide identity (gANI) of the Akkermania species differs from that of other known Akkermania species by at least 1%, 2%, 3%, 4%, or 5%. The genome-wide average nucleotide identity (gANI) of the Akkermania species differs from that of other known Akkermania species by at least 5%. In some embodiments, the genome-wide average nucleotide identity (gANI) of the Akkermania species is similar to that of other known Akkermania species by at least 95%, 96%, 97%, 98%, or 99%. In some embodiments, the genome-wide average nucleotide identity (gANI) of this Akkermania species is at least 95% similar to other known Akkermania species.

[0161] This article also provides methods for preparing compositions comprising combining one or more substantially pure strains of *Ospora natans*, *Ospora natans*, *Bacteroides aurea*, *Barnesia medius*, *Enteromonas masei*, *Ospora spp.*, and / or *Cryptospira mariata*.

[0162] Furthermore, the method for preparing the composition may further include lyophilizing or freeze-drying the microbial composition. The method may also include additional steps for packaging the feed additive composition for storage or transportation.

[0163] IV. Reagent Kit

[0164] This document further provides kits containing one or more microbial strains derived from one or more microbial strains disclosed herein. The kits may include one or more strains (e.g., any of 1, 2, 3, or 4) derived from one or more microbial strains provided herein (including Akkermania species, wherein the Akkermania species is *Akkermania masae* rather than *Akkermania myxophilia* or *Akkermania polymorpha* (e.g., Akkermania strain DSM 33459)) and instructions for proper storage, maintenance, and use in administering the product to a subject for the treatment or prevention of one or more immune system-related disorders. In one embodiment, the kit may include Akkermania strain DSM33459.

[0165] This document further provides kits containing one or more microbial strains derived from one or more microbial strains disclosed herein. The kits may include one or more strains (e.g., any of 1, 2, 3, or 4) derived from one or more microbial strains provided herein (including *Alternaria rubra*, *Alternaria onmosae*, *Bacteroides aureus*, *Barnesia medius*, *Enteromonas masei*, *Vibrio spp.* species, and / or *C. marines* species (e.g., *Alternaria rubra* DSM 34031, *Alternaria onmosae* DSM 34033, *Bacteroides aureus* DSM 34013, *Barnesia medius* DSM 34012, *Barnesia medius* DSM 34032, *Enteromonas masei* DSM 33460, *Vibrio spp.* species DSM 34011, and *C. marines* DSM 34030) and instructions for proper storage, maintenance, and use in administering the medication to a subject for the treatment or prevention of one or more immune system-related disorders.

[0166] The invention can be further understood by referring to the following examples, which are provided for illustration and not for limitation.

[0167] Exemplary embodiments

[0168] 1. A method for treating and / or preventing one or more immune system-related disorders in subjects in need, the method comprising administering an effective amount of a composition comprising one or more substantially pure bacterial species.

[0169] 2. The method as described in Example 1, wherein the one or more substantially pure bacterial species comprises symbiotic bacterial species.

[0170] 3. The method as described in Example 1 or Example 2, wherein the one or more substantially pure bacterial species are selected from Akkermania species and Alternaria species, Bacteroides species, Barnes species, Enteromonas species, Vibrio parahaemolyticus species and Cyclosporium species.

[0171] 4. The method as described in Example 3, wherein the Akkermania species is not (i) Akkermania myxophilus; or (ii) Akkermania polysaccharide.

[0172] 5. The method as described in Example 3 or Example 4, wherein the Akkermania species and Akkermania masei comprise at least about 95% of the whole genome average nucleotide identity (gANI).

[0173] 6. The method as described in any one of Examples 3-5, wherein the Akkermania species comprises Akkermania marsiegei.

[0174] 7. The method as described in any one of Examples 3-6, wherein the Akkermania species comprises a strain deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of the Akkermania species deposited in the DSM, numbered DSM 33459.

[0175] 8. The method as described in any one of Examples 3-7, wherein the Alternaria species comprises *Alternaria fusca* and / or *Alternaria ongrylloides*.

[0176] 9. The method as described in Example 8, wherein the useless synodontia comprises a strain deposited in DSM, numbered DSM 34031, or a live strain having all the identification characteristics of the useless synodontia deposited in DSM, numbered DSM 34031; and the oncogenes comprises a strain deposited in DSM, numbered DSM 34033, or a live strain having all the identification characteristics of the oncogenes deposited in DSM, numbered DSM 34033.

[0177] 10. The method as described in any one of Examples 3-10, wherein the Bacteroides species comprises Bacteroides cylindrica.

[0178] 11. The method as described in Example 10, wherein the Bacteroides cylindrica comprises a strain deposited in DSM, numbered DSM 34013, or a live strain having all the identifying characteristics of the Bacteroides cylindrica strain deposited in DSM, numbered DSM 34013.

[0179] 12. The method as described in any one of Examples 3-11, wherein the Barnes species comprises Enterobacteriaceae.

[0180] 13. The method as described in Example 12, wherein the Barnesia ileus comprises a) a strain deposited in the DSM, designated DSM 34012, or a live strain having all the identification characteristics of the Barnesia ileus strain deposited in the DSM, designated DSM 34012; and / or b) a strain deposited in the DSM, designated DSM 34032, or a live strain having all the identification characteristics of the Barnesia ileus strain deposited in the DSM, designated DSM 34032.

[0181] 14. The method as described in any one of Examples 3-13, wherein the Enteromonas species comprises Enteromonas masei.

[0182] 15. The method as described in Example 14, wherein the Enteromonas masei comprises a strain deposited in the DSM with the number DSM33460 or a live strain having all the identifying characteristics of the Enteromonas masei conserved in the DSM with the number DSM33460.

[0183] 16. The method as described in any one of Examples 3-15, wherein the *Oscillatoria* species and the *Oscillatoria* species deposited in DSM 34011 share at least about 95% whole-genome average nucleotide identity (gANI).

[0184] 17. The method as described in Example 16, wherein the *Vibrio* strain comprises a strain deposited in the DSM with the number DSM34011 or a live strain having all the identifying characteristics of the *Vibrio* species deposited in the DSM with the number DSM34011.

[0185] 18. The method as described in any one of Examples 3-17, wherein the *Cryptococcus* species comprises *Cryptococcus variabilis*.

[0186] 19. The method as described in Example 18, wherein the common marine cephalococcus comprises a strain deposited in the DSM, numbered DSM 34030, or a live strain having all the identifying characteristics of the marine cephalococcus species deposited in the DSM, numbered DSM 34030.

[0187] 20. The method as described in any one of Examples 1-19, wherein the treatment and / or prevention comprises stimulating an immune response in the subject.

[0188] 21. The method as described in any one of Examples 1-19, wherein the treatment and / or prevention comprises downregulating the immune response in the subject.

[0189] 22. The method as described in any one of Examples 1-21, wherein the treatment and / or prevention comprises modulating the production of one or more immune system functional mediators.

[0190] 23. The method as described in Example 22, wherein the one or more immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both.

[0191] 24. The method as described in Example 22 or Example 23, wherein the one or more immune system functional mediators comprise IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β and / or any combination thereof.

[0192] 25. The method of any one of Examples 22-24, wherein, compared with the level of immune system functional mediators in the subject prior to administration of the composition, these immune system functional mediators are increased or decreased by at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0193] 26. The method as described in any one of Examples 22-25, wherein the one or more immune system functional mediators induce immune enhancement or immune stimulation.

[0194] 27. The method as described in any one of Examples 22-26, wherein the one or more immune system functional mediators induce an anti-inflammatory effect.

[0195] 28. The method of any one of Examples 1-27, wherein the one or more immune system-related disorders are selected from viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, tumors, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders.

[0196] 29. The method as described in any one of Examples 1-28, wherein the one or more immune system-related disorders are not metabolic disorders.

[0197] 30. The method as described in any one of Examples 1-29, wherein the one or more immune system-related disorders include acute inflammatory disorders or chronic inflammatory disorders.

[0198] 31. The method as described in Example 30, wherein the acute or chronic inflammatory disorder is selected from irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo.

[0199] 32. The method of any one of Examples 1-31, wherein the composition comprises probiotics, prebiotics, post-biotics, human milk oligosaccharides, xylitol, betaine and / or plant preparations.

[0200] 33. The method as described in any one of Examples 1-32, wherein the composition has been pasteurized or heat-treated.

[0201] 34. The method of any one of Examples 1-33, wherein the composition is lyophilized, freeze-dried, or spray-dried.

[0202] 35. The method as described in any one of Examples 1-34, wherein the composition is encapsulated or coated.

[0203] 36. The method of any one of Examples 1-35, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0204] 37. The method of any one of Examples 1-36, wherein the composition is formulated as tablets, lozenges, extended-release capsules, extended-release granules, powders, sachets, nasal sprays, ointments, serums, lotions, or adhesives.

[0205] 38. The method of any one of Examples 1-37, wherein the composition is formulated as a food product, nutritional product, food ingredient, dietary supplement, or pharmaceutical preparation.

[0206] 39. The method as described in any one of Examples 1-38, wherein the composition comprises at least about 1 x 10 4 CFU / g to at least approximately 1 x 10⁻⁶ 14 The CFU / g is essentially a pure bacterial species.

[0207] 40. The method as described in any one of Examples 1-39, wherein the treatment and / or prevention comprises administering the composition for at least 1 day.

[0208] 41. The method as described in any one of Examples 1-40, wherein the treatment and / or prevention comprises applying the composition for at least 1 week.

[0209] 42. The method as described in any one of Examples 1-41, wherein the treatment and / or prevention comprises applying the composition for at least one month.

[0210] 43. A method for modulating the production of one or more immune system functional mediators in a subject in need, the method comprising administering an effective amount of a composition comprising one or more substantially pure bacterial species.

[0211] 44. The method as described in Example 43, wherein the one or more substantially pure bacterial species comprises symbiotic bacterial species.

[0212] 45. The method as described in Example 43 or Example 44, wherein the one or more substantially pure bacterial species are selected from Akkermania species and Alternaria species, Bacteroides species, Barnes species, Enteromonas species, Vibrio parahaemolyticus species and Cyclosporium species.

[0213] 46. ​​The method as described in Example 45, wherein the Akkermania species is not (i) Akkermania myxophilus; or (ii) Akkermania polysaccharide.

[0214] 47. The method as described in Example 45 or Example 46, wherein the Akkermania species and Akkermania masei comprise at least about 95% whole-genome average nucleotide identity (gANI).

[0215] 48. The method of any one of Examples 45-47, wherein the Akkermania species comprises Akkermania marsiegei.

[0216] 49. The method of any one of Examples 45-48, wherein the Akkermania species comprises an Akkermania species deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of the Akkermania species deposited in the DSM, numbered DSM 33459.

[0217] 50. The method of any one of Examples 45-49, wherein the Alternaria species comprises *Alternaria fusca* and / or *Alternaria ongrylloides*.

[0218] 51. The method as described in Example 50, wherein the useless synodontia comprises a strain deposited in DSM, numbered DSM 34031, or a live strain having all the identification characteristics of the useless synodontia deposited in DSM, numbered DSM 34031; and the oncogenes comprises a strain deposited in DSM, numbered DSM 34033, or a live strain having all the identification characteristics of the oncogenes deposited in DSM, numbered DSM 34033.

[0219] 52. The method as described in any one of Examples 45-51, wherein the Bacteroides species comprises Bacteroides cylindrica.

[0220] 53. The method as described in Example 52, wherein the Bacteroides cylindrica comprises a strain deposited in DSM, numbered DSM 34013, or a live strain having all the identifying characteristics of the Bacteroides cylindrica strain deposited in DSM, numbered DSM 34013.

[0221] 54. The method as described in any one of Examples 45-53, wherein the Barnes species comprises Enterobacteriaceae.

[0222] 55. The method of Example 54, wherein the Barnesia ileus comprises a) a strain deposited in the DSM, designated DSM 34012, or a live strain having all the identification characteristics of the Barnesia ileus strain deposited in the DSM, designated DSM 34012; and / or b) a strain deposited in the DSM, designated DSM 34032, or a live strain having all the identification characteristics of the Barnesia ileus strain deposited in the DSM, designated DSM 34032.

[0223] 56. The method of any one of Examples 45-55, wherein the Enteromonas species comprises Enteromonas masei.

[0224] 57. The method as described in Example 56, wherein the Enteromonas masei comprises a strain deposited in the DSM with the number DSM33460 or a live strain having all the identifying characteristics of the Enteromonas masei conserved in the DSM with the number DSM33460.

[0225] 58. The method as described in any one of Examples 45-57, wherein the *Oscillatoria* species and the *Oscillatoria* species deposited in DSM34011 share at least about 95% whole-genome average nucleotide identity (gANI).

[0226] 59. The method as described in Example 58, wherein the *Vibrio* strain comprises a strain deposited in the DSM with the number DSM34011 or a live strain having all the identifying characteristics of the *Vibrio* species deposited in the DSM with the number DSM34011.

[0227] 60. The method as described in any one of Examples 45-59, wherein the *Cryptococcus* species comprises *Cryptococcus variabilis*.

[0228] 61. The method as described in Example 60, wherein the common marine cephalococcus comprises a strain deposited in the DSM, numbered DSM 34030, or a live strain having all the identifying characteristics of the marine cephalococcus species deposited in the DSM, numbered DSM 34030.

[0229] 62. The method as described in any one of Examples 43-61, wherein the one or more immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both.

[0230] 63. The method of any one of Examples 43-62, wherein the one or more immune system functional mediators comprise IL-10, IFN-γ, IL-1b, IL-6, IL-12, IL-23, TNF-α, TGF-β and / or any combination thereof.

[0231] 64. The method as described in any one of Examples 43-63, wherein one or more immune system functional mediators are increased or decreased by 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of immune system functional mediators in the subject before administration of the composition.

[0232] 65. The method as described in any one of Examples 43-64, wherein the one or more immune system functional mediators induce immune enhancement or immune stimulation.

[0233] 66. The method as described in any one of Examples 43-65, wherein the one or more immune system functional mediators induce an anti-inflammatory effect.

[0234] 67. The method as described in any one of Examples 43-66, wherein the one or more immune system functional mediators are associated with immune system-related disorders.

[0235] 68. The method as described in Example 67, wherein the immune system-related disorder includes non-inflammatory disorders, acute inflammatory disorders, and / or chronic inflammatory disorders.

[0236] 69. The method of any one of Examples 43-68, wherein the composition comprises probiotics, prebiotics, post-biotics, human milk oligosaccharides, xylitol, betaine and / or plant preparations.

[0237] 70. The method as described in any one of Examples 43-69, wherein the composition has been pasteurized or heat-treated.

[0238] 71. The method of any one of Examples 43-70, wherein the composition is lyophilized, freeze-dried, or spray-dried.

[0239] 72. The method as described in any one of Examples 43-71, wherein the composition is encapsulated or coated.

[0240] 73. The method of any one of Examples 43-72, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

[0241] 74. The method of any one of Examples 43-73, wherein the composition is formulated as tablets, lozenges, extended-release capsules, extended-release granules, powders, sachets, nasal sprays, ointments, serums, lotions, or adhesives.

[0242] 75. The method of any one of Examples 43-74, wherein the composition is formulated as a food product, food ingredient, dietary supplement or pharmaceutical agent.

[0243] 76. The method as described in any one of Examples 43-75, wherein the composition comprises at least about 1 x 10 4 CFU / g to at least approximately 1 x 10⁻⁶ 14 The CFU / g is essentially a pure bacterial species.

[0244] 77. The method as described in any one of Examples 43-76, wherein the treatment and / or prevention comprises administering the composition for at least 1 day.

[0245] 78. The method as described in any one of Examples 43-77, wherein the treatment and / or prevention comprises applying the composition for at least 1 week.

[0246] 79. The method as described in any one of Examples 43-78, wherein the treatment and / or prevention comprises applying the composition for at least one month. Example

[0247] Example 1: Identification of Akkermansia masais

[0248] This example describes the growth and isolation of a new species of Akkermania, previously identified by the authors as Akkermania species DSM 33459 and subsequently renamed Akkermania marsie (Ndongo et al., 2022. Nature Scientific Reports 12(21747), the contents of which are incorporated herein by reference in their entirety).

[0249] Based on the previously analyzed 16S community analysis, clinical samples were obtained and the abundance of their top-ranked candidate bacteria was analyzed. During a second round of isolation following the general first round, strain P122_6884 (DSM 33459) was selected from fecal sample F015V3 on YCFA medium containing 10 g / L mucin, with any top-ranked candidate bacteria as the target. Briefly, the samples used in this round of isolation were previously prepared by mixing fecal material from sample F015V3 with glycerol to achieve a final glycerol concentration of 25%. These samples were stored at -80°C until needed. One sample was removed from the freezer, placed in the anaerobic chamber, and thawed at room temperature for approximately 10 minutes. Unless otherwise specified, all work was performed in the anaerobic chamber using a gas mixture of N2 / CO2 / H2 (85% / 10% / 5%). The measured fractions were removed and serially diluted in mucin-free YCFA broth. -4 10 -5 and 10 -6 Take 100 µl of the diluted sample and spread it onto YCFA+ mucin agar in an Omni dish. Use approximately twelve sterile glass beads to spread the bacterial cells evenly on the agar surface. Incubate the plate at 37°C in an anaerobic environment created by an anaerobic chamber containing small packets of the solution for approximately 72 hours.

[0250] Dispense 350 µl of deoxygenated growth medium (YCFA + 10 g / L mucin) into 1 ml deep-well plates. Pick a single colony and inoculate it into a plate pre-spread medium, one colony per well. Cover the plate with a breathable cap to allow gas exchange. Incubate the plate at 37°C for approximately 216 hours. Gently mix the cultures using a 96-well Integra pipette. Use one culture sample for 16S PCR analysis, and mix the remaining culture with sterile, deoxygenated 50% glycerol + 1 g / L L-cysteine ​​to achieve a final glycerol concentration of 25%. Transfer these cultures to appropriate long-term storage vials and store at -80°C.

[0251] 16S Identification: The cell culture sample used for PCR was diluted approximately 1:100 with sterile water. This aqueous dilution was used as the template in the 16S PCR reaction. The PCR primers used to amplify the 16S gene were: 8F: AGA GTT TGA TYMTGG CTC and 1492R: CGG TTA CCT TGT TAC GAC TT. The PCR reaction conditions and thermal cycler settings for polymerase Q5 were standard. The 16S PCR sample was run on a gel to confirm the presence of the expected size of the 16S PCR product. The 16S PCR sample was enzymatically purified using the ExoSAP-IT Express PCR purification kit. The sample was then sent to an external third-party supplier for Sanger sequencing. The most commonly used 16S primer for 16S Sanger sequencing is 515F: GTG CCA GCM GCC GCG GTA A.

[0252] The 16S sequence was then compared with 16S amplicon sequences from the top-ranked candidate bacterial list. These results indicated that the closest matching candidate was *Akermansia myxophilus*. Vials corresponding to the wells containing the desired strain were removed from the freezer and placed in an anaerobic chamber so that a small fraction of the frozen culture could be taken from the vials and streaked onto a YCFA+ 10 g / L myxophilus agar plate. The plate was incubated at 37°C in an anaerobic chamber with pouch culture until sufficient growth was available for preparing the cryoprotectant and extracting DNA.

[0253] Genome extraction of strain P122_6884 (DSM 33459): The DNA extraction kit used was the Qiagen MagAttract® PowerSoil® DNA KF (King Fisher) kit. The most recently streaked growth was scraped from a YCFA + 10 g / L mucin agar plate, and the cells were resuspended in the first solution from the DNA extraction kit. Cell clumps were gently broken up by pipetting. This cell resuspension was evenly distributed into multiple wells of a PowerMag bead plate. The number of wells was determined based on the amount and density of cells in the cell suspension. DNA was then extracted following the manufacturer's protocol. After DNA extraction, similar wells were merged into a single DNA sample, and the DNA concentration was determined using the Ingenium Quant-iTPicoGreen dsDNA Quantification Kit. The DNA was then sent for whole genome sequencing.

[0254] Sequencing libraries were prepared using the Nextera Flex kit (Illumina) and sequenced in pairs of 2 x 150 nt reads on MiSeq (Illumina). To improve assembly quality, long reads were added using the Oxford Nanopore GridION system. Illumina reads were first quality-trimmed using pickle (v. 1.33) (Joshi NA2011 software) and de novo hybridization assembly was performed using the Unicycler (v0.4.7) workflow, which included read error correction steps using SPAdes (v.3.10.0) (Wick et al., 2017 PLoSComput Biol [PLOS Computational Biology], 13(6): p. e1005595). The integrity of genome assembly was quantitatively assessed using BUSCO (Seppey et al., 2019 Methods Mol Biol [Molecular Biology Methods], 1962: pp. 227-245), an assessment based on gene content from near-universal single-copy orthologs.

[0255] The draft genome of strain P122_6884 (DSM 33459) consists of 11 contigs with an N50 of 2,020,938 bp. The genome size is 3.21 Mb, larger than two other Akkermania species: *Ackermania myxophilus* Muc. T (2.66 Mbp) and Agrobacterium pyt. T The genome size of the type strain was 3.07 Mb. The G+C content of the genomic DNA was 57.7%.

[0256] Taxonomy of strain P122_6884 (DSM 33459) was established by comparing the full-length 16S rRNA sequence and genomic average nucleotide identity (ANI) with reference strains of the genus *Ackermania* (Table 1). The 16S rRNA sequence was 99.2% similar to the *Ackermania* type strain ATCC BAA-835; however, genomic comparison between the two strains showed an ANI of only 87.5%. This is below the representative threshold of 96% for speciation (Goris et al., 2007 Int J SystEvol Microbiol. [International Journal of Systematic and Evolutionary Microbiology] 57(Pt 1):81-91.). Phylogenetic trees were constructed for 1000 single-copy genes from selected types of Akkermansia strains, including the newly proposed but unverified species *Akkermansia masai* (Masai-P6666T) and *Akkermansia timonensis* (Akk0196T). Figure 1 (Ndongo et al., 2022). Both phylogenetic and genetic analyses showed that the Akkermania species P122_6884 (DSM 33459) was more similar to the proposed type strain Akkermania masai, a new species, Marsai-P6666 (16S rRNA similarity = 99.7%; ANI = 99.85%).

[0257] Table 1. Genomic mean nucleotide identity (ANI) between Akkermania species P122_6884 (DSM 33459) and Akkermania type strains.

[0258]

[0259] Fatty acid methyl ester (FAME) analysis of cellular fatty acids (Welch, 1991. Applications of cellular fatty-acid analysis. Clin. Microbiol. Rev. 4:422-438) was performed by Microbial ID Inc. (Delaware, USA), where standard samples of strain P122_6884 (DSM 33459) and Akkermansia myxophilus ATCC strain BAA835 were prepared by growing on BHIA to extract fatty acid methyl esters for identification. The samples were then loaded onto a gas chromatograph for analysis. FAME profiles of the samples were generated using Sherlock® pattern recognition software. The samples were then compared to determine similarity. As shown in Table 2, the FAME profiles revealed significant differences between strain P122_6884 (DSM 33459) and ATCC strain BAA835.

[0260] Table 2: Comparative FAME characteristics of strains BAA835 and P122_6884 (DSM 33459).

[0261]

[0262] Strain P122_6884 (DSM 33459) was subsequently deposited by DuPont NutritionBiosciences ApS (Langebrogade 1, DK-1411, Copenhagen K, Denmark) under the Budapest Treaty at the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) (Inhoffenstrasse 7B, 38124 Braunschweig, Germany), where it is recorded under the following accession number: strain P122_6884 (DGCC12918); and on March 4, 2020, under accession number DSM. 33459 is a collection.

[0263] Example 2: The activity of Akkermania strains in the production of immune cell cytokines.

[0264] This example illustrates the ability of *Akermansia masei* to modulate immune responses by influencing cytokine production in macrophages and dendritic cells.

[0265] The *Ackermania myxophilus* DSM22959 and *Ackermania masei* DSM33459 microbial strains were grown similarly to those described in Example 1. Briefly, the microbial strains were grown three times on YCFAC plates over a period of 3–4 days under anaerobic conditions (80% N2, 10% H2, 10% CO2). For cell experiments, the microbial strains were grown on YCFAC plates for 4 days and then resuspended in RPMI medium at a concentration of 10^8 bacteria / ml and a bacteria:host cell ratio of 10:1. BactoBox and / or flow cytometry were used to determine the bacterial concentration.

[0266] Peripheral blood mononuclear cells (PBMCs) were obtained from freshly collected leukocyte-rich white membrane layers from four healthy blood donors from the Finnish Red Cross Blood Service (License No. 46 / 2016). The use of human blood was approved by the Ethics Committee of the Helsinki and Neue Prairie Hospital District, Finland (License No. 216 / 13 / 03 / 00 / 2016). PBMCs were separated by density gradient centrifugation and the mononuclear cells were purified using CD14+ magnetic beads. For macrophage assays, the purified mononuclear cells were centrifuged at 3 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1000 IU / ml onto 24-well plates and differentiated for 7 days in macrophage-SFM containing 1000 IU / ml of recombinant human GM-CSF (Miltenyi Biotech) and 1% antibiotic-antifungal agent (Gibco, Life Technologies, Grand Arena, NY, USA). For dendritic cell assays, monocytes were seeded at 5 x 10⁻⁶ cells / well. 5 Cells / well were plated onto 12-well plates (Falcon, Corning, NY, USA) and kept for 7 days in RPMI-1640 (Sigma) supplemented with 1% antibiotic-antifungal agent, 10% fetal bovine serum, IL-4 (400 IU / ml) and GM-CSF (1000 IU / ml).

[0267] Macrophages or dendritic cells from the above-mentioned blood donors were stimulated with Akkermansia myxophila DSM22959 or Akkermansia marsei DSM33459, with ordinary culture medium used as a negative control. The stimulation time was 24 h for macrophages or 48 h for dendritic cells.

[0268] Cell culture supernatants were analyzed using Quanterix multiplex enzyme-linked immunosorbent assay (ELISA) (Quanterix, Billerica, Massachusetts, USA). For dendritic cells, IL-12, IL-10, IL-6, IL-1β, TNF-α, IFN-γ, TGF-β, and IL-23 were measured; for macrophages, IL-12, IL-10, IL-6, IL-1β, TNF-α, and IFN-γ were measured. Results were analyzed using CiraSoft software (Quanterix).

[0269] exist Figure 2 The table shows the cytokine production (in pg / ml) in the cell culture supernatant of macrophages after 24 hours, with standard error present. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, =p < 0.01, = p < 0.001, and = p < 0.0001. Table 3 shows the statistical analysis of cytokine production in macrophages, where for each combination of cytokines and cell types, a linear fixed-effects model (corresponding to one-way ANOVA) was used to model the data separately, and pairwise comparisons were performed using model contrast. Estimates of the comparison between group 1 and group 2 are presented as standard errors and p-values. A p-value < 0.05 was considered statistically significant.

[0270] Table 3. Statistical analysis of cytokine production in macrophages.

[0271]

[0272] exist Figure 3 The table shows the cytokine production (in pg / ml) in the cell culture supernatant of dendritic cells after 48 hours, with standard error present. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001. Table 4 shows the statistical analysis of cytokine production in dendritic cells. For each combination of cytokines and cell types, a linear fixed-effects model (corresponding to one-way ANOVA) was used to model the data, and pairwise comparisons were performed using model contrast. Estimates of the comparison between group 1 and group 2 are presented as standard errors and p-values. A p-value < 0.05 was considered statistically significant.

[0273] Table 4. Statistical analysis of cytokine production in dendritic cells.

[0274]

[0275] As in Figure 2 and Figure 3 As shown in the figures and summarized in Tables 3 and 4, *Ackermania masae* DSM33459 was able to induce anti-inflammatory interleukin-10 (IL-10) from human monocyte-derived dendritic cells and macrophages compared to control cells, and was able to induce higher levels of IL-10 from dendritic cells compared to the *Ackermania myxophilus* model strain. Furthermore, compared to control cells and *Ackermania myxophilus*, *Ackermania masae* DSM33459 was able to induce higher levels of IL-12, IL-6, IL-1β, TNF-α, IFN-γ, TGF-β, and IL-23 from dendritic cells, and was able to induce higher levels of IL-6, IL-1β, and TNF-α from macrophages compared to control cells.

[0276] Example 3: Identification of other symbiotic species

[0277] This example describes the growth and isolation of several species derived from fecal samples, similar to that described in Example 1. These species were identified by the authors, similar to those described in International Publication No. WO 2023 / 092141, the contents of which are incorporated herein by reference in their entirety. The growth and isolation included a new species of *Oscillatoria* identified by the authors as belonging to the genus *Oscillatoria*, which was subsequently deposited under the Budapest Treaty at the Leibniz-Institut Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ) , Leibniz Institute of Microbiology and Cell Culture (Inhoffenstrasse 7B, 38124, Braunschweig, Germany). Braunschweig, Germany), and recorded with the following accession number: strain P3_135 (DGCC13804); deposited on September 8, 2021 with accession number DSM34011.

[0278] Based on the previously analyzed 16S community analysis, clinical samples were obtained and the abundance of their top-ranking candidate bacteria was analyzed. Strains isolated from the fecal sample of Subject 1 were selected using M2GSC medium C and then maintained on YCFAC medium. Briefly, the samples used in this round of isolation were previously prepared by mixing fecal material from Subject 1 with glycerol to achieve a final glycerol concentration of 25%. These samples were stored at -80°C until needed. One sample was removed from the freezer, placed in the anaerobic chamber, and thawed at room temperature for approximately 10 minutes. Unless otherwise specified, all work was performed in the anaerobic chamber using a gas mixture of N2 / CO2 / H2 (85% / 10% / 5%). The measured fractions were removed and serially diluted in YCFA broth. -4 10 -5 and 10 -6 Take 100 µl of the diluted sample and spread it onto YCFA in an Omni dish. Use approximately twelve sterile glass beads to spread the bacterial cells evenly on the agar surface. Incubate the plate at 37°C in an anaerobic environment created by an anaerobic chamber containing small packets of the solution for approximately 72 hours.

[0279] Dispense 350 µl of deoxygenated growth medium (YCFA) into 1 ml deep-well plates. Pick a single colony and inoculate it into a plate pre-spread medium, one colony per well. Cover the plate with a breathable cap to allow gas exchange. Incubate the plate at 37°C for approximately 3 days. Gently mix the cultures using a 96-well Integra pipette. Use one culture sample for 16S PCR analysis, and mix the remaining culture with sterile, deoxygenated 50% glycerol + 1 g / L L-cysteine ​​to achieve a final glycerol concentration of 25%. Transfer these cultures to appropriate long-term storage vials and store at -80°C.

[0280] Genome of *Oscillatoria* strain DSM34011: The DNA extraction kit used was the Qiagen MagAttract® PowerSoil® DNA KF (King Fisher) kit. The most recently streaked growth was scraped from a YCFA agar plate, and the cells were resuspended in the first solution from the DNA extraction kit. Cell clumps were gently broken up by pipetting. This cell resuspension was evenly distributed into multiple wells of a PowerMag bead plate. The number of wells was determined based on the amount and density of cells in the cell suspension. DNA was then extracted following the manufacturer's protocol. After DNA extraction, similar wells were merged into a single DNA sample, and the DNA concentration was determined using the Ingenium Quant-iT PicoGreen dsDNA Quantification Kit. The DNA was then sent for whole-genome sequencing.

[0281] Sequencing libraries were prepared using the Nextera Flex kit (Illumina) and sequenced in pairs of 2 x 150 nt reads on MiSeq (Illumina). To improve assembly quality, long reads were added using the Oxford Nanopore GridION system. Illumina reads were first quality-trimmed using pickle (v. 1.33) (Joshi NA 2011 software) and de novo hybridization was performed using the Unicycler (v0.4.7) workflow, which included read error correction steps using SPAdes (v.3.10.0) (Wick et al., 2017 PLoS Comput Biol, 13(6): p. e1005595). The integrity of genome assembly was quantitatively assessed using BUSCO (Seppey et al., 2019 Methods Mol Biol [Molecular Biology Methods], 1962: pp. 227-245), an assessment based on gene content from near-universal single-copy orthologs.

[0282] Genome sequencing and assembly: The genomes of strains of the genus *Oscillatoria* were sequenced using paired-end 2 x 250 bp reads from an Illumina NovaSeq 6000. Reads were quality filtered, errors were corrected using BFC, and then assembled using SPAdes (Bankevich et al., 2012; Li, 2015). Assembly was further corrected using Pilon and annotated using Prokka (Seemann, 2014; Walker et al., 2014). The integrity of the genome assembly was evaluated using BUSCO based on gene content from near-universal single-copy orthologs (Simao et al., 2015). The resulting draft genome of the *Oscillatoria* strains consisted of 121 contigs, with a total genome size of 3.39 Mbp, an N50 of 127,403, and an average G+C content of 55.7%.

[0283] Taxonomyces species DSM34011 was determined by comparing the full-length 16S rRNA gene and genome-wide average nucleotide identity (ANI) with the type strains of closely related species. The 16S rRNA sequence similarity of the closely related *Dysosmobacter welbionis*, *Oscillibacter valericigenes*, and *Oscillibacter ruminantium* was greater than 98.6% of the recognized species boundary (Table 5) (Kim et al., 2014). The genome-wide ANI between strain DSM34011 and *Dysosmobacter welbionis* J115 was 81.4%, and the genome-wide ANI between strain DSM34011 and *Oscillibacter valericigenes* Sjm18-20 was 78.0%. The ANI of DSM34011 and its closely related species is greater than 95%, which is a representative threshold for speciation (Table 6) (Goris et al., 2007). A phylogenetic tree containing *Oscillatoria*-like species, including type strains and other publicly available genomes, was constructed. One hundred single-copy core genes were compared using RAxML at the bootstrap values ​​shown in 100 iterations. Figure 4 (Stamatakis, 2014). The *Oscillatoria* species DSM34011 does not clearly cluster with type strains from known species of the *Oscillatoria* or *Non-Penetrating Bacteria* genera. Based on taxonomic and phylogenetic analyses, *Oscillatoria* species DSM34011 is proposed to be a novel species.

[0284] Table 5: 16S rRNA similarity between strain DSM34011 and the closest type strain.

[0285]

[0286] Table 6: Genome-wide average nucleotide identity (ANI) between strain DSM34011 and the closest type strain.

[0287]

[0288] Example 4: The activity of other symbiotic strains in the production of immune cell cytokines.

[0289] This example describes the ability of the following strains to modulate immune responses by influencing cytokine production in macrophages and dendritic cells: *Ostomyces undulans* strain deposited in DSM 34031, *Ostomyces oncogenes* strain deposited in DSM 34033, *Bacteroides aureus* strain deposited in DSM 34013, *Barnesia esculenta* strain deposited in DSM 34012, *Barnesia esculenta* strain deposited in DSM 34032, *Enteromonas masei* strain deposited in DSM 33460, *Oscillatoria* strain deposited in DSM 34011, and *Cryptospira marinum* strain deposited in DSM 34030.

[0290] The following microbial strains were grown similarly to those described in Examples 1 and 3: *Ostomyces undulans* strain preserved in DSM 34031, *Ostomyces onychomycosis* strain preserved in DSM 34033, *Bacteroides micranthum* strain preserved in DSM 34013, *Barnesia esculenta* strain preserved in DSM 34012, *Barnesia esculenta* strain preserved in DSM 34032, *Enteromonas masei* strain preserved in DSM 33460, *Oscillatoria* strain preserved in DSM 34011, and *Cryptospira marinum* strain preserved in DSM 34030. In summary, the microbial strains were grown three times on YCFAC plates over a period of 3–4 days under anaerobic conditions (80% N2, 10% H2, 10% CO2). For cell experiments, the microbial strain was grown on YCFAC plates for 4 days and then resuspended in RPMI medium at a concentration of 10^8 bacteria / ml and a bacteria:host cell ratio of 10:1. BactoBox and / or flow cytometry were used to determine the bacterial concentration.

[0291] Peripheral blood mononuclear cells (PBMCs) were obtained from freshly collected leukocyte-rich white membrane layers from four healthy blood donors from the Finnish Red Cross Blood Service (License No. 46 / 2016). The use of human blood was approved by the Ethics Committee of the Helsinki and Neue Prairie Hospital District, Finland (License No. 216 / 13 / 03 / 00 / 2016). PBMCs were separated by density gradient centrifugation and the mononuclear cells were purified using CD14+ magnetic beads. For macrophage assays, the purified mononuclear cells were centrifuged at 3 x 10⁻⁶ cells / mL. 5 Cells were seeded at a density of 1000 IU / ml onto 24-well plates and differentiated for 7 days in macrophage-SFM containing 1000 IU / ml of recombinant human GM-CSF (Miltenyi Biotech) and 1% antibiotic-antifungal agent (Gibco, Life Technologies, Grand Arena, NY, USA). For dendritic cell assays, monocytes were seeded at 5 x 10⁻⁶ cells / well. 5 Cells / well were plated onto 12-well plates (Falcon, Corning, NY, USA) and kept for 7 days in RPMI-1640 (Sigma) supplemented with 1% antibiotic-antifungal agent, 10% fetal bovine serum, IL-4 (400 IU / ml) and GM-CSF (1000 IU / ml).

[0292] Macrophages or dendritic cells from the above blood donors were stimulated with the following strains of bacteria: *Ostomyces undulans* preserved in DSM 34031, *Ostomyces onychomycosis* preserved in DSM 34033, *Bacteroides aureus* preserved in DSM 34013, *Barnesia esculenta* preserved in DSM 34012, *Barnesia esculenta* preserved in DSM 34032, *Enteromonas masei* preserved in DSM 33460, *Oscillatoria* preserved in DSM 34011, and *Cryptospira marinum* preserved in DSM 34030. Ordinary culture medium was used as a negative control. Stimulation was performed on macrophages for 24 h or on dendritic cells for 48 h.

[0293] Cell culture supernatants were analyzed using Quanterix multiplex enzyme-linked immunosorbent assay (ELISA) (Quanterix, Billerica, Massachusetts, USA). For dendritic cells, IL-12, IL-10, IL-6, IL-1β, TNF-α, IFN-γ, TGF-β, and IL-23 were measured; for macrophages, IL-12, IL-10, IL-6, IL-1β, TNF-α, and IFN-γ were measured. Results were analyzed using CiraSoft software (Quanterix).

[0294] exist Figure 5 The figure shows the logarithmic levels of cytokine production in the cell culture supernatant of macrophages after 24 hours relative to the negative control. 10 The fold change, shown as a box plot, has the median of the four donors and whiskers at the minimum and maximum values. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, = p < 0.001, and = p < 0.0001. Table 7 shows the statistical analysis of cytokine production in macrophages, where for each combination of cytokines and cell types, a linear fixed-effects model (corresponding to one-way ANOVA) was used to model the data separately, and pairwise comparisons were performed using model contrast. Estimates of the comparison between Group 1 and Group 2 (control, "ctrl") are presented as standard errors and p-values. A p-value < 0.05 was considered statistically significant.

[0295] Table 7. Statistical analysis of cytokine production in macrophages.

[0296]

[0297] exist Figure 6 The figure shows the log10-fold change in cytokine production in the culture supernatant of dendritic cells at 48 hours relative to the negative control, presented as a box plot with the median and whisker lines at the minimum and maximum values ​​for the four donors. Statistical significance between different stimuli is expressed as ns = not significant. = p < 0.05, = p < 0.01, =p < 0.001, and = p < 0.0001. Table 8 shows the statistical analysis of cytokine production in dendritic cells, where for each combination of cytokines and cell types, a linear fixed-effects model (corresponding to one-way ANOVA) was used to model the data separately, and pairwise comparisons were performed using model contrast. Estimates of the comparison between Group 1 and Group 2 (control, "ctrl") are presented as standard errors and p-values. A p-value < 0.05 was considered statistically significant.

[0298] Table 8. Statistical analysis of cytokine production in dendritic cells.

[0299]

[0300] As in Figure 5 and Figure 6 As shown and summarized in Tables 7 and 8, compared with control cells, *Alternaria fusca* DSM34031, *Alternaria ongol* DSM 34033, *Bacteroides cylindrica* DSM 34013, *Barnesia pulmonata* DSM 34012, *Barnesia pulmonata* DSM 34032, *Enteromonas masei* DSM 33460, *Oscillatoria* species DSM 34011, and *Cryptospira marinum* DSM 34030 were able to induce anti-inflammatory interleukin-10 (IL-10) from human monocyte-derived dendritic cells and macrophages, indicating control of inflammatory and / or immune responses; or, in the case of dendritic cells, demonstrating the ability of dendritic cells to polarize toward immune regulation and induce regulatory T cells. Furthermore, compared with control cells, *Alternaria alternata* DSM 34031, *Alternaria ongol* DSM 34033, *Bacteroides aurea* DSM 34013, *Barnesia natans* DSM 34012, *Barnesia natans* DSM34032, *Enteromonas masei* DSM 33460, *Oscillatoria* species DSM 34011, and *Cryptospira marines* DSM34030 were able to induce higher levels of IL-12, IL-6, IL-1β, TNF-α, and IFN-γ from macrophages, indicating an immunostimulatory effect.

[0301] Furthermore, compared with control cells, the following bacteria (DSM 34031, DSM 34033, DSM 34013, DSM 34012, DSM 34032, DSM 33460, DSM 34011, and DSM 34030) were able to induce higher levels of IL-12 and IFN-γ from dendritic cells, indicating the polarization of dendritic cells towards type 1 immunity (or in other words, T helper cell type 1 immunity). Furthermore, compared with control cells, the following bacteria (DSM 34031, DSM 34033, DSM 34013, DSM 34012, DSM 34032, DSM 33460, DSM 34011, and DSM 34030) were able to induce higher levels of IL-6 and IL-23 from dendritic cells, indicating the polarization of dendritic cells towards type 3 immunity (or in other words, type 17 T helper cell immunity).

[0302] Example 5: Effects of Akkermania strains on the production of cytokines and chemokines under various conditions

[0303] This example describes the ability of various strains of Akkermansia masei to modulate immune responses by influencing cytokine production in THP-1 monocyte-derived macrophages.

[0304] Akkermansia masei DSM33459 (Amas DSM33459), the model strain of Akkermansia masei CECT 30548 (Amas(TS)), and Akkermansia masei DSM 22959 (Amuc) were grown under anaerobic conditions for 48 h on carbohydrate-containing yeast casein peptone fatty acid (YCFAC) agar plates (Anaerobe Systems, USA) in a Whitley A95 anaerobic chamber (Don Whitley Scientific Ltd, UK). The gas mixture in the chamber was (80% N2, 10% H2, and 10% CO2). All strains were refreshed twice on sterile YCFAC plates and grown under the same conditions as above. On the day of the experiment, the microbial biomass was suspended in RPMI sterile medium supplemented with 1% antibiotic-antifungal agent (AB, Gibco, Life Technologies) and maintained under anaerobic conditions. A final concentration of 10 was obtained using Bactobox (SBT Instruments, Denmark). 8 One bacteria per ml of live bacteria.

[0305] THP-1 cells (ECACC, Public Health England, 88081201 growth culture) were maintained at +37°C under a 5% CO2 atmosphere in Roswell Park Memorial Institute 1640 (RPMI with L-glutamine, Gibbec Life Sciences) medium supplemented with 10% fetal bovine serum (FBS, Gibbec Life Sciences) and 1% antibiotic-antifungal agent (AB, Gibbec Life Sciences). When the culture reached 8 x 10⁸ cells / year, the culture was maintained at 37°C. 5 -1 x 10 6 At a density of 100 cells / ml, passage the cells at least twice before using them in experiments. 7.5 x 10⁻⁶ cells / ml were cultured in 200 µl of complete RPMI medium supplemented with phorbol 12-tetradecanoate 13-acetate (PMA, Sigma-Aldrich). 4 Cells / well were added to 96-well plates to differentiate THP-1 cells into macrophage-like cells. Cell differentiation was allowed to occur for 3 days (approximately 72 hours) prior to the experiment. Differentiation was confirmed by microscopic examination to visualize morphological changes in the cells.

[0306] Macrophage-like cells were incubated in the presence of either *Akermansia masei* DSM33459 (Amas DSM33459), the *Akermansia masei* model strain CECT 30548 (Amas(TS)), or *Akermansia masei* DSM 22959 (Amuc) in isolation (without stimulation, ( Figure 7A Alternatively, incubation can be performed in the presence of two separate compounds that are stimulating to macrophages: a combination of 15 µg / ml polyinosinic acid:polycytidylic acid (PolyIC) and 5 µM retinomod (R848) (PolyIC, Figure 7C ); or incubate with TNFα stimulation at a final concentration of 1 ng / ml ( ); Figure 7B Immediately after adding bacteria, the irritant compound was added to the macrophages. Cells and bacteria were incubated under different conditions for 24 hours, after which the plates were centrifuged at 1000 rpm for 5 min, and the supernatant was collected for enzyme-linked immunosorbent assay (ELISA) or immediately frozen at -80°C until further use.

[0307] Following the manufacturer's instructions, multiplex ELISA was performed on the supernatant samples using the Luminex technology platform provided by Bio-Rad (Bio-Plex 200). Analytes were tested on a multiplex assay set (Bio-Plex Pro Human Cytokine Screening Set) that included interleukin (IL) 1β (IL-1β), IL-6, IL-10, tumor necrosis factor α (TNFα), interferon-γ-induced protein 10 (IP-10, also known as CXCL10), and monocyte chemoattractant protein 1 (MCP-1, also known as CC motif chemokine 2 or CCL2).

[0308] like Figures 7A-7C As shown, under no-excitation conditions ( Figure 7A Compared to the *Amuc* species, both *Akermansia masei* strains (Amas(TS) and Amas DSM33459) were able to induce the cytokine IL-6 and chemokines MCP-1 and IP-10 to higher levels, and also showed higher levels of TNF-α (… Figure 7B ) and PolyIC ( Figure 7C Under both conditions, the chemokine IP-10 was induced to a higher degree compared to the *Amuc* species. Furthermore, under TNF-α conditions (…), Figure 7B Under conditions of ), compared with the Akermansia muciniphila species (Amuc), the Akermansia masais strain DSM33459 was able to induce the chemokine MCP-1 and cytokines IL-6 and IL-10 to a greater extent.

[0309] Statistical analysis was performed using parametric one-way ANOVA (i.e., a linear fixed-effects model), followed by model comparisons, comparing results to a reference, or pairwise comparisons between Amas DSM33459 and Amas(TS), Amas DSM33459 and Amuc, or Amas(TS) and Amuc. Logarithmic transformation was performed on the data before statistical analysis to ensure a good fit between the model and the data. Two additional biological replicates (in triplicate) were used to test all strains. The obtained p-values ​​were adjusted using the Benjamini-Hochberg method. Statistical significance is expressed as... (p < 0.0001) (p < 0.001) (p < 0.01) (p < 0.05). sequence

[0310] Barnsley bacillus - DSM 34032 16S

[0311]

[0312] Barnsley bacillus - DSM 34012 16S

[0313]

[0314] *Ostomyces onychomycosis* - DSM 34033 16S

[0315]

[0316] Bacteroides aureus DSM 34013 16S

[0317]

[0318] Common marine coccidia DSM 34030 16S

[0319]

[0320] Species of the genus *Vibrio* - DSM 34011 16S

[0321]

[0322] Enteromonas masei DSM 33460 16S

[0323]

[0324] Useless Alternariae DSM 34031 16S

[0325]

Claims

1. A method for treating and / or preventing one or more immune system-related disorders in subjects in need, the method comprising administering an effective amount of a composition comprising one or more substantially pure bacterial species.

2. The method of claim 1, wherein the one or more substantially pure bacterial species are selected from Akkermansia species and Alistipes species, Bacteroides species, Barnesiella species, Intestinimonas species, Oscillibacter species, and Phocaeicola species.

3. The method of claim 1 or claim 2, wherein the Akkermania species comprises Akkermansia massiliensis.

4. The method of claim 2 or claim 3, wherein the Akkermania species comprises a strain deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of the Akkermania species deposited in the DSM, numbered DSM 33459.

5. The method according to any one of claims 2-4, wherein the *Alistipes* species comprises *Alistipes inops* and / or *Alistipes onderdonkii*, and optionally comprises a *Alistipes inops* strain deposited in the DSM, numbered DSM 34031, or a live strain having all the identification characteristics of the *Alistipes inops* strain deposited in the DSM, numbered DSM 34031; and / or a *Alistipes onderdonkii* strain deposited in the DSM, numbered DSM 34033, or a live strain having all the identification characteristics of the *Alistipes onderdonkii* strain deposited in the DSM, numbered DSM 34033.

6. The method of any one of claims 2-5, wherein the Bacteroides species comprises Bacteroides finegoldii., and optionally comprises a Bacteroides finegoldii strain deposited in DSM 34013 or a live strain having all the identifying characteristics of the Bacteroides finegoldii strain deposited in DSM 34013.

7. The method of any one of claims 2-6, wherein the *Barnesiella* species comprises *Barnesiella intestinihominis*, and optionally comprises a *Barnesiella intestinihominis* strain deposited in the DSM, designated DSM 34012, or a live strain having all the identification characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, designated DSM 34012; and / or a strain deposited in the DSM, designated DSM 34032, or a live strain having all the identification characteristics of the *Barnesiella intestinihominis* strain deposited in the DSM, designated DSM 34032.

8. The method of any one of claims 2-7, wherein the Enteromonas species comprises Enteromonas masei, and optionally comprises an Enteromonas masei strain deposited in the DSM, numbered DSM 33460, or a live strain having all the identifying characteristics of the Enteromonas masei strain deposited in the DSM, numbered DSM 33460.

9. The method according to any one of claims 2-8, wherein the *Oscillatoria* species and the *Oscillatoria* species deposited in DSM 34011 contain at least about 95% whole-genome average nucleotide identity (gANI).

10. The method of claim 9, wherein the *Vibrio* strain comprises a strain deposited in the DSM, numbered DSM34011, or a live strain having all the identifying characteristics of the *Vibrio* species deposited in the DSM, numbered DSM 34011.

11. The method according to any one of claims 2-10, wherein the *Phocaeicola vulgatus* species comprises *Phocaeicola vulgatus*, and optionally comprises a *Phocaeicola vulgatus* strain deposited in the DSM with the number DSM 34030 or a live strain having all the identifying characteristics of the *Phocaeicola vulgatus* species deposited in the DSM with the number DSM 34030.

12. The method of any one of claims 1-11, wherein the treatment and / or prevention comprises stimulating an immune response in the subject.

13. The method of any one of claims 1-11, wherein the treatment and / or prevention comprises downregulating the immune response in the subject.

14. The method of any one of claims 1-13, wherein the one or more immune system-related disorders are selected from viral infections, bacterial infections, yeast infections, parasitic infections, allergies, autoimmune diseases, tumors, cardiovascular diseases, respiratory diseases, metabolic disorders, gastrointestinal diseases, and age-related disorders.

15. The method of any one of claims 1-14, wherein the one or more immune system-related disorders are not metabolic disorders.

16. The method of any one of claims 1-15, wherein the one or more immune system-related disorders include acute inflammatory disorders or chronic inflammatory disorders.

17. The method of claim 16, wherein the acute or chronic inflammatory disorder is selected from irritable bowel syndrome, colitis, non-atopic eczema, Alzheimer's disease, Parkinson's disease, cancer, cancer treatment-associated mucositis, atopic dermatitis, food allergy, allergic rhinitis, sinusitis, asthma, Addison's disease, alopecia, ankylosing spondylitis, antiphospholipid syndrome, Behcet's disease, chronic fatigue syndrome, Crohn's disease, ulcerative colitis, fibromyalgia, pulmonary hemorrhage nephritis syndrome, Graves' disease, idiopathic thrombocytopenic purpura, lupus, Meniere's disease, multiple sclerosis, myasthenia gravis, pemphigus vulgaris, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, rheumatic fever, sarcoidosis, scleroderma, vasculitis, or vitiligo.

18. The method of any one of claims 1-17, wherein the treatment and / or prevention comprises modulating the production of one or more immune system functional mediators.

19. A method for regulating the production of one or more immune system functional mediators in a subject in need, the method comprising administering an effective amount of a composition comprising one or more substantially pure bacterial species.

20. The method of claim 19, wherein the one or more substantially pure bacterial species are selected from Akkermania species and Alternaria species, Bacteroides species, Barnes species, Enteromonas species, Vibrio parahaemolyticus species and Cyclosporium species.

21. The method of claim 19 or claim 20, wherein the Akkermania species comprises Akkermania marsiegei.

22. The method of claim 20 or claim 21, wherein the Akkermania species comprises a strain deposited in the DSM, numbered DSM 33459, or a live strain having all the identifying characteristics of the Akkermania species deposited in the DSM, numbered DSM 33459.

23. The method of any one of claims 20-22, wherein the *Alternaria* species comprises *Alternaria fusca* and / or *Alternaria onychomycosis*, and optionally comprises a strain of *Alternaria fusca* deposited in the DSM, numbered DSM 34031, or a live strain having all the identification characteristics of the *Alternaria fusca* strain deposited in the DSM, numbered DSM 34031; and / or a strain of *Alternaria onychomycosis* deposited in the DSM, numbered DSM 34033, or a live strain having all the identification characteristics of the *Alternaria onychomycosis* strain deposited in the DSM, numbered DSM 34033.

24. The method of any one of claims 20-23, wherein the Bacteroides species comprises Bacteroides aurea, and optionally comprises a Bacteroides aurea strain deposited in the DSM, numbered DSM 34013, or a live strain having all the identifying characteristics of the Bacteroides aurea strain deposited in the DSM, numbered DSM 34013.

25. The method of any one of claims 20-24, wherein the *Barnesia* species comprises *Barnesia enterica*, and optionally comprises a *Barnesia enterica* strain deposited in the DSM, designated DSM 34012, or a live strain having all the identification characteristics of the *Barnesia enterica* strain deposited in the DSM, designated DSM 34012; and / or a strain deposited in the DSM, designated DSM 34032, or a live strain having all the identification characteristics of the *Barnesia enterica* strain deposited in the DSM, designated DSM 34032.

26. The method of any one of claims 20-25, wherein the Enteromonas species comprises Enteromonas masei, and optionally comprises an Enteromonas masei strain deposited in the DSM, numbered DSM 33460, or a live strain having all the identifying characteristics of the Enteromonas masei strain deposited in the DSM, numbered DSM 33460.

27. The method of any one of claims 20-26, wherein the *Vibrio* species and the *Vibrio* species deposited in DSM34011 contain at least about 95% whole-genome average nucleotide identity (gANI).

28. The method of claim 27, wherein the *Vibrio* strain comprises a strain deposited in the DSM, numbered DSM34011, or a live strain having all the identifying characteristics of the *Vibrio* species deposited in the DSM, numbered DSM 34011.

29. The method of any one of claims 20-28, wherein the *C. marinei* species comprises *C. marinei*, and optionally comprises a *C. marinei* strain deposited in the DSM, numbered DSM 34030, or a live strain having all the identifying characteristics of the *C. marinei* species deposited in the DSM, numbered DSM 34030.

30. The method of any one of claims 18-29, wherein the one or more immune system functional mediators are produced by dendritic cells, macrophages, or a combination of both.

31. The method of any one of claims 18-30, wherein the one or more immune system functional mediators comprise IL-10, IFN-γ, IL-1β, IL-6, IL-12, IL-23, TNF-α, TGF-β, IP-10, MCP-1 and / or any combination thereof.

32. The method of any one of claims 18-31, wherein the one or more immune system functional mediators are increased or decreased by 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% compared to the level of immune system functional mediators in the subject prior to administration of the composition.

33. The method of any one of claims 18-32, wherein one or more immune system functional mediators induce immune enhancement or immune stimulation.

34. The method of any one of claims 18-33, wherein one or more immune system functional mediators induce anti-inflammatory effects.

35. The method of any one of claims 1-34, wherein the composition has been pasteurized or heat-treated.

36. The method of any one of claims 1-35, wherein the composition is lyophilized, freeze-dried, or spray-dried.

37. The method of any one of claims 1-36, wherein the composition is encapsulated or coated.

38. The method of any one of claims 1-37, wherein the composition is a pharmaceutical composition and further comprises at least one pharmaceutically acceptable carrier and / or excipient.

39. The method of any one of claims 1-38, wherein the composition is formulated as a tablet, lozenge, extended-release capsule, extended-release granule, powder, sachet, nasal spray, ointment, serum, lotion, or adhesive.

40. The method of any one of claims 1-39, wherein the composition is formulated as a food product, food ingredient, dietary supplement, or pharmaceutical agent.

41. The method of any one of claims 1-40, wherein the composition comprises probiotics, prebiotics, postbiotics, human milk oligosaccharides, xylitol, betaine and / or plant preparations.

42. The method of any one of claims 1-41, wherein the composition comprises at least about 1 x 10 4 CFU / g to at least approximately 1 x 10⁻⁶ 14 The CFU / g values ​​indicate essentially pure bacterial species.

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