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WO2022178209A1 - Compositions and methods for treating metabolic diseases and disorders using christensenellaceae bacteria - Google Patents

Compositions and methods for treating metabolic diseases and disorders using christensenellaceae bacteria Download PDF

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Publication number
WO2022178209A1
WO2022178209A1 PCT/US2022/016919 US2022016919W WO2022178209A1 WO 2022178209 A1 WO2022178209 A1 WO 2022178209A1 US 2022016919 W US2022016919 W US 2022016919W WO 2022178209 A1 WO2022178209 A1 WO 2022178209A1
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WIPO (PCT)
Prior art keywords
bacteria
composition
christensenella
mevs
antagonists
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PCT/US2022/016919
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French (fr)
Inventor
Alicia BALLOK
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Evelo Biosciences, Inc.
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Publication of WO2022178209A1 publication Critical patent/WO2022178209A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • a composition e.g., a bacterial and/or pharmaceutical composition
  • a composition comprising non-living Christensenellaceae bacteria and/or microbial extracellular vesicles (mEVs) derived from Christensenellaceae bacteria.
  • the Christensenellaceae bacteria can be, for example, bacteria of the genus Christensenella, such as Christensenella minula, including, for example, one or more species with a 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and/or mixtures thereof.
  • the Christensenellaceae is of a strain that is resistant to polymyxin.
  • the Christensenellaceae bacteria and/or mEVs in the composition are lyophilized.
  • a composition e.g., a bacterial and/or pharmaceutical composition
  • a composition comprising non-living Christensenellaceae bacteria and/or extracellular vesicles (mEVs) derived from Christensenellaceae bacteria.
  • the Christensenellaceae bacteria can be, for example, bacteria of the genus Christensenella, such as Christensenella minula, including, for example, one or more species with a 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and/or mixtures thereof.
  • the Christensenellaceae is of a strain that is resistant to polymyxin.
  • the Christensenellaceae bacteria and/or mEVs in the composition are lyophilized.
  • the composition comprises no more than 10 8 colony forming units (CPUs) of Christensenellaceae bacteria.
  • the composition comprises no more than 10 6 colony forming units (CPUs) (e.g., no more than 10 5 , 10 4 , 10 3 , 500, 100, 50, 10, or fewer CPUs) of Christensenella bacteria.
  • CPUs colony forming units
  • substantially all or all of the Christensenellaceae bacteria in the administered composition are non-living.
  • At least 4 x 10 10 cells of the Christensenellaceae bacteria are administered to the subject daily.
  • mEVs fiom Christensenellaceae bacteria can be administered at doses e.g., of about IxlO 7 to about 1x10 15 particles, e.g., as measured by NTA.
  • the dose of mEVs is about 1 x 10 5 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • the dose of mEVs fiom Christensenellaceae bacteria is about 1 x 10 10 to about 7 x 10 13 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)).
  • administration of the disclosed compositions does not increase the levels of Christensenellaceae bacteria, relative to the levels of other bacteria, in the gastrointestinal tract of the subject.
  • the composition comprises isolated Christensenellaceae bacteria microbial extracellular vesicles (mEVs) (e.g., secreted microbial extracellular vesicles (smEVs), processed microbial extracellular vesicles (pmEVs)).
  • mEVs microbial extracellular vesicles
  • pmEVs processed microbial extracellular vesicles
  • the composition comprises Christensenellaceae bacteria microbial extracellular vesicles (mEVs) and Christensenellaceae bacteria.
  • the composition can be formulated as a tablet, capsule, oral liquid preparation, reconstitutable powder or suspension.
  • the composition can comprise food or nutritional supplements containing Christensenellaceae bacteria and/or mEVs, e.g., a composition of substantially purified Christensenellaceae bacteria and/or mEVs.
  • the composition can be administered on a daily or weekly basis.
  • the subject to whom a composition of the invention is administered can have metabolic disease.
  • the composition is administered in two or more doses.
  • the administration to the subject of the two or more doses are separated by at least 1 day. In other embodiments, the administration of the two or more doses are separated by at least 1 week.
  • the composition comprises killed bacteria. In further embodiments, the composition comprises irradiated bacteria. In other embodiments, the composition comprises gamma irradiated bacteria.
  • the composition comprises lyophilized bacteria.
  • the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the metabolic disease does not include hypertriglyceridemia.
  • the metabolic disease comprises obesity. In some embodiments, the obesity does not include hypertriglyceridemia.
  • the metabolic disease comprises metabolic syndrome. In some embodiments, the metabolic syndrome does not include hypertriglyceridemia.
  • the metabolic disease comprises diabetes.
  • administration of the composition treats the metabolic disease.
  • the method further comprises administering to the subject an additional therapeutic.
  • the additional therapeutic is selected from the group consisting of an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), a cytokine antagonist, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprofen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, M
  • the additional therapeutic is an antibiotic.
  • the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, anti- mycobacterial compounds and combinations thereof.
  • the additional therapeutic is a metabolic disease therapeutic.
  • the metabolic disease therapeutic is metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3- fatty acids, statins, ezetimibe, or ursodeoxycholic acid.
  • the method further comprises administering to the subject a second therapeutic bacterial strain, e.g., a composition thereof.
  • the second therapeutic bacterial strain is of the Methanobacteriaceae family.
  • the second therapeutic bacterial strain is Methanobacterium aarhusense, Methanobacterium aggregans, Methanobacterium alcaliphilum, Methanobacterium arcticum, Methanobacterium beijingense, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium curvum, Methanobacterium espanolae, Methanobacterium ferruginis, Methanobacterium flexile, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium kanagiense, Methanobacterium lacus, Methanobacterium movens, Methanobacterium movilense, Methanobacterium oryzae, Methanobacterium
  • the method further comprises administering a prebiotic to the subject.
  • the prebiotic is a fructooligosaccharide, a galactooligosaccharide, a trans-galactooligosaccharide, a xylooligosaccharide, a chitooligosaccharide, a soy oligosaccharide, a gentiooligosaccharide, an isomaltooligosaccharide, a mannooligosaccharide, a maltooligosaccharide, a mannanoligosaccharide, lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, or a cyclodextrin.
  • the composition is formulated as a food or drink.
  • the subject is a human.
  • subject is a non-human mammal.
  • the mammal is selected from the group consisting of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • compositions comprising mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, obtained from Christensenellaceae bacteria.
  • mEVs such as smEVs and/or pmEVs
  • bacteria or any combination thereof, obtained from Christensenellaceae bacteria.
  • compositions comprising ChristenseneUaceae mEVs isolated from bacteria.
  • the disclosure provides a composition described herein for use in treating and/or preventing a metabolic disease in a subject.
  • the disclosure provides use of a composition described herein for the preparation of a medicament for treating and/or preventing a metabolic disease in a subject.
  • the disclosure provides a composition described herein for use in inhibiting weight gain, promoting weight loss, or reducing adiposity in a subject.
  • the disclosure provides use of a composition described herein for the preparation of a medicament for inhibiting weight gain, promoting weight loss, or reducing adiposity in a subject.
  • bioreactors comprising ChristenseneUaceae bacteria.
  • the bacteria are a strain comprising at least 90% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
  • the mEVs are from bacteria of a strain comprising at least 90% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
  • the bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
  • the mEVs are from bacteria of a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
  • the Christensenellaceae bacteria are Christensenella minuta. In some embodiments, the mEVs are from Christensenella minuta bacteria.
  • kits for growing bacteria in a bioreactor comprising providing a bioreactor as described herein; and fermenting the bacteria for a period of time.
  • compositions that have substantially purified bacteria and/or mEVs of the family Christensenellaceae and/or the genus Christensenella, and uses of these compositions.
  • the administration of Christensenellaceae (bacteria and/or mEVs) to an individual can treat or prevent metabolic diseases.
  • adjuvant or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a subject (e.g., human).
  • an adjuvant might help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines.
  • an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent.
  • an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
  • administering broadly refers to a route of administration of a composition to a subject.
  • routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection.
  • Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration.
  • compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial.
  • transdermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g., using any standard patch
  • intradermal e.g.
  • compositions described herein are administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In some embodiments, the compositions described herein are administered orally.
  • a “carbohydrate” refers to a sugar or polymer of sugars.
  • saccharide polysaccharide
  • carbohydrate oligosaccharide
  • Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnHinOn.
  • a carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide.
  • the most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose.
  • Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose.
  • an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units.
  • Exemplary polysaccharides include starch, glycogen, and cellulose.
  • Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’-fluororibose, deoxyribose, and hexose).
  • Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
  • Cellular augmentation broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself.
  • Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells.
  • a “combination” of bacteria from two or more strains includes the physical coexistence of the bacteria, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the bacteria from the two or more strains.
  • the term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state.
  • Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).
  • Dysbiosis refers to a state of the microbiota or microbiome of the gut or other body area, including, e.g., mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks “microbiome”) are disrupted.
  • a state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period.
  • Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents, host genotype, host diet and/or stress.
  • a dysbiosis may result in: a change (e.g., increase or decrease) in the prevalence of one or more bacteria types (e.g., anaerobic), species and/or strains, change (e.g., increase or decrease) in diversity of the host microbiome population composition; a change (e.g., increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (e.g., pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.
  • ecological consortium is a group of bacteria that trades metabolites and positively co-regulates one another, in contrast to two bacteria that induce host synergy through activating complementary host pathways for improved efficacy.
  • engineered bacteria are any bacteria that have been genetically altered from their natural state by human activities and the progeny of any such bacteria.
  • Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution.
  • the term “gene” is used broadly to refer to any nucleic acid associated with a biological function.
  • the term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
  • nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., etal., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo etal.
  • FASTA Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444
  • the term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10 ⁇ 3 fold, 10 ⁇ 4 fold, 10 ⁇ 5 fold, 10 ⁇ 6 fold, and/or 10 ⁇ 7 fold greater after treatment when compared to a pre-treatment state.
  • Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).
  • ITS is a piece of non-fimctional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi.
  • rRNA structural ribosomal RNAs
  • the rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively.
  • Insulin resistance has its common meaning in the art. Insulin resistance is a physiological condition where the natural hormone insulin, becomes less effective at lowering blood sugars. The resulting increase in blood glucose may raise levels outside the normal range and cause adverse health effects such as metabolic syndrome, dyslipidemia and subsequently type 2 diabetes mellitus.
  • insulin resistance- related complications and “insulin resistance-related conditions” as used herein encompass, without limitation, metabolic syndrome, dyslipidemia and type 2 diabetes mellitus, as well as insulin resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease and lipodystrophy syndromes).
  • isolated or “enriched” encompasses a microbe (such as a bacterium) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man.
  • isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pine.
  • a substance is “pure” if it is substantially free of other components.
  • purify refers to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production.
  • a microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.”
  • purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure.
  • the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type.
  • Microbial compositions are generally purified from residual habitat products.
  • Methodabolic disease refers a cluster of conditions that occur together, increasing the risk of heart disease, stroke and type 2 diabetes. These conditions include increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels.
  • the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • Metal refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.
  • Merobe refers to any natural or engineered organism characterized as a bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism.
  • gut microbes examples include: Actinomyces graevenitzii, Actinomyces odontofyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XTV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Cory
  • Microbial extracellular vesicles may be naturally-produced vesicles derived from bacteria, such as smEVs. mEVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations).
  • mEV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy).
  • purified mEV composition or “mEV composition” refers to a preparation of mEVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the mEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components.
  • Microbial extracellular vesicles may also be obtained from mammalian cells and from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites. Microbial extracellular vesicles from any of these sources can be prepared into a solution and/or dried form as described herein.
  • Microbial extracellular vesicles may be artificially-produced vesicles prepared from bacteria, such as pmEVs, for example, obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, and can also be prepared into a solution and/or dried form as described herein.
  • “Microbiome” broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses.
  • microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner.
  • the microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome.
  • the microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state or treatment conditions (e.g., antibiotic treatment, exposine to different microbes).
  • the microbiome occurs at a mucosal surface.
  • the microbiome is a gut microbiome.
  • a “microbiome profile” or a “microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome.
  • a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome.
  • “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form.
  • Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity.
  • Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that its increases or decreases virulence.
  • a gene is “overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
  • polynucleotide and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • a polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • nucleotide structure may be imparted before or after assembly of the polymer.
  • a polynucleotide may be further modified, such as by conjugation with a labeling component.
  • U nucleotides are interchangeable with T nucleotides.
  • “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species.
  • the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence.
  • the entire genomes of two entities are sequenced and compared.
  • select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared.
  • OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g. Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940.
  • OTUs For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU.
  • OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof.
  • Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein.
  • a substance is “pine” if it is substantially free of other components.
  • strain refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species.
  • the genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g.
  • strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
  • subject refers to any mammal.
  • a subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease.
  • Mammals i.e., mammalian animals
  • mammals include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents).
  • the subject may be a human.
  • the subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
  • the subject or patient may be healthy, or may be suffering from a metabolic disease at any developmental stage.
  • treating refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • a pharmaceutical treatment e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening.
  • “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
  • prevention of cancer includes, for example, reducing the number of detectable tumors in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable tumors in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
  • a value is “greater than” another value if it is higher by any amount (e.g., each of 100, 50, 20, 12, 11, 10.6, 10.1, 10.01, and 10.001 is at least 10). Similarly, as used herein, a value is ‘less than” another value if it is lower by any amount (e.g., each of 1, 2, 4, 6, 8, 9, 9.2, 9.4, 9.6, 9.8, 9.9, 9.99, 9.999 is no more than 10).
  • a test value “is” an anchor value when the test value rounds to the anchor value (e.g., if “an ingredient mass is 10% of a total mass,” in which case 10% is the anchor value, the test values of 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, and 10.4 would also meet the “ingredient mass is 10% of the total mass” feature).
  • ChristenseneUaceae are a family of anaerobic bacteria of the order Clostridiales. Multiple members of the ChristenseneUaceae, and multiple species of Christensenella, have been identified on the basis of homologous operational taxonomic unit (OTU) sequences in microbial phylogeny databases, such as Greengenes, a database of 16SRNA genetic sequences (DeSantis, T. Z., et al. Appl Environ Microbiol 72:5069-72 (2006)).
  • OFTU operational taxonomic unit
  • a bacterium can be defined as a member of the ChristenseneUaceae if ( 1 ) the 16S rRNA gene sequence of that bacterium shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, preferably 97% or greater identity, with any 16S rRNA gene sequence in a database of 16S rRNA gene sequences, such as the Greengenes database that has the taxonomic classification of ChristenseneUaceae, or (2) a phylogenetic analysis reveals that the 16S rRNA gene sequence of that bacterium is most closely related to the 16S rRNA gene sequence of a member of the ChristenseneUaceae, such that this sequence and a sequence from a member of the Christensenellaceae share a common ancestor sequence that is unique to the family Christensenellaceae (and members of other families do not share that ancestor).
  • Phylogenetic analysis approaches that are acceptable as general practice in the field of microbiology for 16S rRNA gene phylogenetics include: (a) a multiple sequence alignment of the 16S rRNA gene sequences (members of the Christensenellaceae-+the novel sequence+non- Christensenellaceae) using secondary' structure information, followed by (b) use of either the General-Time reversible model of evolution in a Maximum Likelihood (ML) analysis with bootstrapping (Harris, J. K., et al., Appl. Environ. Microbiol. 70:845-849 (2004)), or a Bayesian phylogenetics analysis (Huelsenbeck, J. P. and F.
  • ML Maximum Likelihood
  • Christensenella is a recently identified genus of the Christensenellaceae.
  • the first isolated species of Christensenella was Christensenella minute, which was isolated from a human fecal sample (Morotomi, et al ., Int. J. Syst. Evol. Microbiol. 62:144-149 (2012).
  • C. minuta has a 16S ribosomal RNA gene sequence of SEQ ID NO: 1.
  • the C. minuta 16S rRNA sequence is identified by Genbank Accession No. AB490809.
  • Bacteria belonging to Christensenellaceae and Christensenella can be identified by amplifying the 16S rRNA gene from a sample.
  • PCR primers that amplify universally conserved regions of the 16S rRNA gene are known in the art. For example, using the known 16S rRNA gene primers 515F (SEQ ID NO: 3) and 806R (SEQ ID NO: 4) to amplify the 16S rRNA gene sequence from Christensenella minuta amplifies a portion of SEQ ID NO: 1 disclosed herein as SEQ ID NO: 2.
  • a bacterium belonging to the Christensenella genus is identified as having a 16S RNA gene sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Additional 16S sequences for bacteria within the genus of Christensenella are provided as SEQ ID NOS: 5-8 (OTU numbers 701845, 177179,1146771, and 361793 in the Greengenes database, respectively).
  • each sequence relative to SEQ ID NO: 1 is as follows: SEQ ID NO: 5, 93% identity to SEQ ID NO: 1 ; SEQ ID NO: 6, 95% identity to SEQ ID NO: 1; SEQ ID NO: 7, 97% identity to SEQ ID NO: 1; SEQ ID NO: 8, 95% identity to SEQ ID NO: 1.
  • compositions of substantially purified bacteria of the family are disclosed herein.
  • the Christensenellaceae are bacteria of the genus
  • Christensenella In a further embodiment, the Christensenella bacteria are selected from
  • Christensenella minuta one or more Christensenella species with the 16S sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof.
  • the bacteria are C. minuta bacteria.
  • Christensenella in the administered composition are non-living.
  • a ‘Viable” or ‘living” bacterium is capable of forming a colony, for example, under conditions suitable for growth of said bacterium.
  • a viable or living bacterium can also be referred to as a colony forming unit (CFU).
  • Christensenellaceae and Christensenella can be cultured under anaerobic conditions using, for example, the methods disclosed in Morotomi, et al. (Int. J. Syst. Evol. Microbiol. 62:144-149 (2012)).
  • cultures may be inoculated into tubes or plates for growth in anaerobic conditions at 32-42°C, preferably 37°C, with a culture medium of modified Gifu anaerobic medium (GAM broth; Nissui Pharmaceutical) containing l%-2%, preferably 1.5% (w/v) agar supplemented with 2-8% bile (Bacto oxgall; Difco) and NaCl.
  • GAM broth modified Gifu anaerobic medium
  • l%-2% preferably 1.5% (w/v) agar supplemented with 2-8% bile (Bacto oxgall; Difco) and NaCl.
  • Christensenella can be cultured in brain heart infusion broth supplemented with yeast (2-8 g/1, preferably 5 g/1), menadione (0.5- 2.0 mg/1, preferably 1.0 g/1), hemin (5-15 mg/1, preferably 10 mg/1), and L-cysteine-HCL (0.2-0.8 g/1, preferably 0.5 g/1) at 32-42°C., preferably 37°C, under anaerobic conditions.
  • the composition includes lyophilized Christensenellaceae or Christensenella bacteria. Lyophilized compositions of the inventions contain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% not viable Christensenellaceae or Christensenella bacteria.
  • the composition contains less than 10* CFU of bacteria (e.g., less than 10 8 CFU of bacteria/g,) and more particularly less than 10 6 CFU of bacteria (e.g., less than 10 6 CFU of bacteria/g) of Christensenellaceae or Christensenella bacteria.
  • the composition contains less than 10* CFU of bacteria/g of support, and more particularly less than 10 6 CFU of bacteria/g of support of Christensenellaceae or Christensenella bacteria.
  • support is meant the food product or the pharmaceutically acceptable excipient.
  • the terms “about” and “approximately” indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. In one nonlimiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.
  • Bacteria and strains of the family Christensenellaceae, and bacteria and strains of the genus Christensenella are probiotic.
  • a probiotic bacterium or strain itis meant a non-pathogenic microorganism which, when ingested live, exercises a beneficial effect on the host's health.
  • These probiotic strains generally have the ability to survive the passage through the upper part of the digestive tract. Without being limited, it is thought that these bacteria can exercise their beneficial effect on health on the one hand via ecological interactions with the resident flora in the digestive tract (i.e., the “gut microbiome”), and on the other hand via their ability to influence various aspects of the host physiology.
  • compositions of substantially purified mEVs from bacteria of the family Christensenellaceae and the genus Christensenella are provided herein.
  • compositions that comprise mEVs (such as smEVs and/or pmEVs) from Christensenellaceae bacteria are provided herein.
  • the Christensenellaceae mEVs are mEVs of the genus Christensenella.
  • the mEVs are from Christensenella bacteria selected from Christensenella minuta, one or more Christensenella species with the 16S sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof.
  • the mEVs are from C. minuta bacteria.
  • the composition includes lyophilized mEVs from Christensenellaceae or Christensenella bacteria.
  • compositions disclosed herein can include substantially purified Christensenellaceae or Christensenella bacteria and one or more acceptable excipients.
  • acceptable excipients include: sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols; emulsifiers such as sucrose esters of fatty acids, glycerin esters of fatty acids and lecithin; thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum; acidifiers such as citric acid, lactic acid and malic acid; fruit juices such as lemon juice, orange juice and berry
  • compositions of the invention may be prepared by admixture, usually adapted for oral administration.
  • Such compositions may be in the form of tablets, capsules, oral liquid preparations, conventional food products, powders, granules, lozenges, reconstitutable powders or suspensions.
  • Tablets and capsules for oral administration can also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; and lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be in the form of a dryproduct for reconstitution with water or another suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and if desired, conventional flavorings or colorants.
  • compositions comprising Christensenellaceae bacteria and/or mEVs, and methods of using a composition comprising Christensenellaceae bacteria and/or mEVs derived from Christensenellaceae bacteria.
  • the Christensenellaceae bacteria are bacteria of genus Christensenella.
  • the Christensenellaceae bacteria of genus Christensenella are Christensenella minuta bacteria.
  • the bacteria is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic, 16S or CRISPR nucleotide sequence) of the bacterial strains listed in Table 1.
  • sequence identity e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. ⁇ 122.
  • the deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
  • the bacteria described herein are modified to enhance their immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent).
  • the bacteria described herein are modified to enhance immune activation (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins, vesicles).
  • the bacteria described herein are modified to improve bacterial manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
  • the Christensenellaceae bacteria e.g., Christensenella minuta
  • administration of the disclosed compositions increases the levels of Christensenella, relative to the levels of other bacteria, in the gastrointestinal tract of a subject.
  • the Christensenellaceae is resistant to polymyxin.
  • the Christensenellaceae bacteria are modified to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) of the mEVs (such as smEVs and/or pmEVs), bacteria for compositions, or any combination thereof, (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof (e.g., through
  • the engineered Christensenellaceae bacteria described herein are modified to improve manufacturing mEVs (such as smEVs and/or pmEVs), bacteria for compositions, or any combination thereof (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times).
  • the engineered Christensenellaceae bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes.
  • the engineered Christensenellaceae bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet tight mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
  • compositions of substantially purified bacteria and mEVs of the family Christensenellaceae and/or the genus Christensenella are disclosed herein.
  • compositions that comprise bacteria and mEVs (such as smEVs and/or pmEVs) fiom Christensenellaceae bacteria are provided herein.
  • the composition comprises bacteria of only one strain of bacteria. In some embodiments, the composition comprises bacteria of only one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises mEVs fiom only one strain of bacteria. In some embodiments, the composition comprises mEVs fiom only one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises bacteria and mEVs fiom only one strain of bacteria, e.g., one strain of Christensenellaceae bacteria.
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Christensenellaceae bacteria.
  • 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Christensenellaceae bacteria.
  • at least 99% of the bacteria in the composition are of the Christensenellaceae bacteria.
  • the bacteria in the composition are essentially (e.g., about 100%) of the Christensenellaceae bacteria.
  • At least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the mEVs in the composition are of the Christensenellaceae bacteria. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the mEVs in the composition are of the Christensenellaceae bacteria. In some embodiments, at least 99% of the mEVs in the composition are of the Christensenellaceae bacteria. In some embodiments, the mEVs in the composition are essentially (e.g., about 100%) of the Christensenellaceae bacteria.
  • the composition comprises bacteria from more than one strain of bacteria. In some embodiments, the composition comprises bacteria from more than one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises mEVs from more than one strain of bacteria. In some embodiments, the composition comprises mEVs from more than one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises bacteria and mEVs from more than one strain of bacteria. In some embodiments, the composition comprises bacteria and mEVs from more than one strain of Christensenellaceae bacteria.
  • the composition comprises only one strain of bacteria, e.g. , Christensenellaceae bacteria.
  • the composition comprises more than one strain of bacteria, e.g., Christensenellaceae bacteria, and the therapeutic effect caused by the composition is due to the presence of the Christensenellaceae bacteria present in the composition.
  • strain of bacteria e.g., Christensenellaceae bacteria
  • the composition comprises mEVs from only one strain of bacteria, e.g., Christensenellaceae bacteria.
  • the composition comprises mEVs from more than one strain of bacteria, e.g., Christensenellaceae bacteria, and the therapeutic effect caused by the composition is due to the presence of the mEVs from Christensenellaceae bacteria present in the composition.
  • the Christensenellaceae bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed in Table 1.
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, described herein are obtained from a strain of Christensenellaceae bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence as provided in Table 1.
  • sequence identity e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are lyophilized.
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are UV irradiated.
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are acid treated.
  • the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are oxygen sparged (e.g., at 0.1 wm for two hours).
  • the phase of growth can affect the amount or properties of bacteria and/or mEVs (such as smEVs and/or pmEVs) produced by bacteria.
  • bacteria can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • mEVs such as smEVs and/or pmEVs
  • mEVs can be prepared from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • Christensenellaceae bacteria are lyophilized. In some embodiments, Christensenellaceae bacteria are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, Christensenellaceae bacteria are UV irradiated. In some embodiments, Christensenellaceae bacteria are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours). In some embodiments, Christensenellaceae bacteria are acid treated. In some embodiments, Christensenellaceae bacteria are oxygen sparged (e.g., at 0.1 vvm for two hours).
  • the mEVs are lyophilized.
  • the mEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • the mEVs are UV irradiated.
  • the mEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns).
  • the mEVs are acid treated.
  • the mEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
  • the phase of growth can affect the amount or properties of Christensenellaceae bacteria and/or smEVs produced by Christensenellaceae bacteria.
  • smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • pmEVs can be prepared from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the Christensenellaceae bacteria and/or smEVs produced by Christensenellaceae bacteria are grown in growth media comprising yeast extract (e.g., Yeast Extract 19512), soy peptone (e.g., Soy Peptone El 10 19885, Soy Peptone A3 SC 19685), potato peptone (e.g., Potato Peptone E210 19425), tri-sodium citrate, K2HPO4, KH2PO4, magnesium chloride, manganese chloride, L-cysteine-HCl, FeSO4, NH4CI, Xylose, and vitamin B12.
  • yeast extract e.g., Yeast Extract 19512
  • soy peptone e.g., Soy Peptone El 10 19885, Soy Peptone A3 SC 19685
  • potato peptone e.g., Potato Peptone E210 19425
  • tri-sodium citrate K2HPO4, KH2PO4, magnesium chloride, manganese chloride, L-cystein
  • the yeast extract (e.g., Yeast Extract 19512) is prepared in the growth media at 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • Yeast Extract 19512 is prepared in the growth media at 0.001 g/L, 0.005 g/L, 0.01 g/L
  • the soy peptone (e.g., Soy Peptone El 10 19885, Soy Peptone A3 SC 19685) is prepared at 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • Soy peptone e.g., Soy Peptone El 10 19885, Soy Pe
  • the tri-sodium citrate is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the K2HPO4 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the KH2PO4 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L,
  • the magnesium chloride is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the manganese chloride is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the L-cysteine-HCl is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the FeSCh is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L,
  • the NH4CI is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the Xylose is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • the vitamin B 12 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
  • Enriched growth media is used to grow and prepare the bacterium for in vitro and in vivo use.
  • media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins.
  • Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.).
  • Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested.
  • media may be prepared and the selected bacterium grown as shown by Saarela et al., J. Applied Microbiology. 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze- drying survival, storage stability, and acid and bile exposine of the selected bacterium produced without milk-based ingredients.
  • the growth media is sterilized.
  • Sterilization may be by Ultra High Temperature (UHT) processing.
  • UHT processing is performed at very high temperature for short periods of time.
  • the UHT range may be from 135-180°C.
  • the medium may be sterilized from between 10 to 30 seconds at 135°C.
  • inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored.
  • the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume.
  • bioreactor volume can be at least 2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.
  • the bioreactor before inoculation, is prepared with medium at desired pH, temperature, and oxygen concentration.
  • the initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0.
  • the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide.
  • the temperature may be controlled from 25°C to 45°C, for example at 37°C. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8 mg/L to 0 mg/L.
  • nitrogen or gas mixtures may be used in order to establish anaerobic conditions.
  • no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium.
  • the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.
  • a frozen vial is diluted to 0.1% in a IL media at 37°C for 12-16 hours.
  • the media is PM11 with 1 g/1 L-sodium lactate (no FeSO4, no NH4C1, no malate).
  • the IL media is diluted to 1% in a 15 L bioreactor at 37°C, 150 rpm, gas of 5% CO2 and 95% Ni, uncontrolled pH for 16-18 hours.
  • the feed is 10X YEP, 33 g/1 L-sodium lactate (no G2) (Constant feed: 11 mL/Lh). It is then centrifuged at 10,000 x g, 10°C, for 10 minutes to collect 90 g pellet/15 L. Then placed in a new stabilizer: sucrose-dextran- cysteine 0.18 g stab/g pellet.
  • Reviving microbes from a frozen state may require special considerations.
  • Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material.
  • the kinetics of transfer or passage of seed material to fresh medium, for the pinposes of increasing the seed volume or maintaining the microbial growth state, may be influenced by the current state of the microbes (ex. exponential growth, stationary growth, unstressed, stressed).
  • the initial state of the bioreactor system must be optimized to facilitate successfill and consistent production.
  • the fraction of seed culture to total medium (e.g. a percentage) has a dramatic impact on growth kinetics.
  • the range may be 1-5% of the fermenter’s working volume.
  • the initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration.
  • Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc.
  • Optimal temperature range for growth may vary with strain. The range may be 20-40 °C.
  • Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Microbes may have alternate kinetics when excess nutrients are available.
  • microbes may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and downstream processing.
  • a change in temperature (often reducing to 20-5°C) may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter.
  • Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1-2 points can improve effectiveness of concentration but can also be detrimental to cells.
  • Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization dining downstream.
  • Separation methods and technology may impact how efficiently microbes are separated from the culture medium.
  • Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1-2 points may improve effectiveness of concentration but can also be detrimental to cells.
  • Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Microbes may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g-minutes to successfully centrifuge. Excipients can be added before after separation.
  • Excipients can be added for cryo protection or for protection during lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants.
  • Prior to lyophilization droplets of cell pellets mixed with excipients are submerged in Equid nitrogen.
  • Harvesting can be performed by continuous centrifugation. Product may be resuspended with various excipients to a desired final concentration.
  • Excipients can be added for cryo protection or for protection dining lyophilization.
  • Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants.
  • droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
  • Lyophilization of material begins with primary drying.
  • the ice is removed.
  • a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime.
  • product bound water molecules are removed.
  • the temperature is raised higher than in the primary drying phase to break any physicochemical interactions that have formed between the water molecules and the product material.
  • the pressure may also be lowered further to enhance desorption during this stage.
  • the chamber may be filled with an inert gas, such as nitrogen.
  • the product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants.
  • the mEVs (such as smEVs and/or pmEVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
  • the mEVs described herein are modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead).
  • the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria.
  • the magnetic and/or paramagnetic moiety is linked to and/or a part of an mEV-binding moiety that that binds to the mEV.
  • the mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP.
  • the mEV-binding moiety has binding specificity for the mEV (e.g., by having binding specificity for a bacterial antigen).
  • the mEV-binding moiety comprises an antibody or antigen binding fiagment thereof.
  • the mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR).
  • the mEV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fiagment thereof.
  • co-administration of the magnetic and/or paramagnetic moiety with the mEVs can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present.
  • mEVs Processed Microbial Extracellular Vesicles
  • the pmEVs described herein can be prepared using any method known in the art.
  • the pmEVs are prepared without a pmEV purification step.
  • Christensenellaceae bacteria ftom which the pmEVs described herein are released are killed using a method that leaves Christensenellaceae bacterial pmEVs intact, and the resulting Christensenellaceae bacterial components, including the pmEVs, are used in the methods and compositions described herein.
  • the Christensenellaceae bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the Christensenellaceae bacteria are killed using UV irradiation.
  • the pmEVs described herein are purified ftom one or more other Christensenellaceae bacterial components. Methods for purifying pmEVs ftom Christensenellaceae bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared ftom Christensenellaceae bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. (Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety.
  • the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C).
  • the supernatants are discarded and cell pellets are frozen at -80°C.
  • cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/mL DNase I.
  • cells are lysed using an Emulsiflex C-3 (A vestin, Inc.) under conditions recommended by the manufacturer.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hr at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C.
  • pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.
  • pmEVs are obtained by methods adapted from Sandrini et al, 2014.
  • ChristenseneUaceae bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C.
  • cell pellets are frozen at -80°C and supernatants are discarded.
  • cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme.
  • samples are incubated with mixing at room temp or at 37°C for 30 min.
  • samples are refrozen at -80°C and thawed again on ice.
  • DNase I is added to a final concentration of 1.6 mg/mL and MgC12 to a final concentration of 100 mM.
  • samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off.
  • debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C.
  • supernatants are then centrifuged at 110,000 x g for 15 min at 4°C.
  • pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at - 20°C.
  • a method of forming (e.g., preparing) isolated ChristenseneUaceae bacterial pmEVs comprises the steps of: (a) centrifuging a ChristenseneUaceae bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supematant;(c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated ChristenseneUaceae bacterial pmEVs.
  • the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution.
  • the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4°C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80 °C .
  • the solution in step (c) is 100 mM Tris-HCl, pH 7.5 supplemented with Img/ml DNasel. In some embodiments, the solution in step (c) is 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, supplemented with 0.1 mg/ml lysozyme.
  • step (c) further comprises incubating for 30 minutes at 37°C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at -80°C . In some embodiments, step (c) further comprises adding DNasel to a final concentration of 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgChto a final concentration of 100 mM.
  • the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication.
  • the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication.
  • the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4 °C or room temperature.
  • the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature.
  • the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10 mM Tris- HCl pH 8.0, 2% triton X-100.
  • step (g) further comprises incubating the solution for 1 hour at 4°C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at 110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4°C or room temperature. In some embodiments, the third solution in step (i) is 100 mM Tris-HCl, pH 7.5.
  • the third solution in step (i) is PBS.
  • the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4°C or room temperature.
  • the fourth solution in step (k) is 100 mM Tris- HCl, pH 7.5 or PBS.
  • pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfide precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column.
  • Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 horns at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfide precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3- 24 hours at 4°C.
  • pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.
  • the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.
  • select pmEVs are isolated and enriched by chromatography and binding surface moieties on pmEVs.
  • select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
  • pmEVs are lyophilized. In some embodiments, pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, pmEVs are UV irradiated. In some embodiments, pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns). In some embodiments, pmEVs are acid treated. In some embodiments, pmEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
  • pmEVs can be isolated , e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • the smEVs described herein can be prepared using any method known in the art.
  • the smEVs are prepared without an smEV purification step.
  • bacteria described herein are killed using a method that leaves the smEVs intact and the resulting Christensenellaceae bacterial components, including the smEVs, are used in the methods and compositions described herein.
  • the Christensenellaceae bacteria are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the Christensenellaceae bacteria are killed using UV irradiation.
  • the Christensenellaceae bacteria are heat-killed.
  • the smEVs described herein are purified from one or more other Christensenellaceae bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs are prepared from Christensenellaceae bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety.
  • the Christensenellaceae bacteria are cultured to high optical density and then centrifuged to pellet Christensenellaceae bacteria (e.g., at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C).
  • the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter).
  • the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS.
  • filtered supernatants are centrifuged to pellet bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C).
  • the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C).
  • Optiprep iodixanol gradient or gradient
  • centrifugation e.g., at 200,000 x g for 4-20 hours at 4°C.
  • smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C).
  • the purified smEVs can be stored, for example, at -80°C or -20°C until use.
  • the smEVs are further purified by treatment with DNase and/or proteinase K.
  • cultures of ChristenseneUaceae bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria.
  • Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells.
  • Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration.
  • ammonium sulfate precipitation 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C.
  • Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria smEVs and other debris.
  • filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes.
  • a filtration technique such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
  • smEVs can be obtained from ChristenseneUaceae bacteria cultures continuously dining growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen).
  • ATF alternating tangential flow
  • the ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection.
  • the system may be configured so that the ⁇ 0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor.
  • the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. smEVs collected by this method may be further purified and/or concentrated by ultracentrifiigation or filtration as described above for filtered supernatants.
  • smEVs obtained by methods provided herein may be fiirther purified by sizebased column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifiigation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifiigation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0.
  • the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfide precipitation or ultracentrifiigation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample.
  • the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep.
  • Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
  • smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
  • smEVs used for in vivo injections purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v).
  • smEVs in PBS are sterile-filtered to ⁇ 0.22 pm.
  • samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
  • filtration e.g., Amicon Ultra columns
  • dialysis e.g., dialysis
  • ultracentrifugation 200,000 x g, > 3 hours, 4°C
  • the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.
  • select smEVs are isolated and enriched by chromatography and binding surface moieties on smEVs.
  • select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
  • the smEV s can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
  • smEVs are lyophilized.
  • smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy).
  • smEVs are UV irradiated.
  • smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns).
  • smEVs s are acid treated.
  • smEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
  • the growth environment e.g., culture conditions
  • the yield of smEVs can be increased by an smEV inducer, as provided in Table 2.
  • the method can optionally include exposing a culture of Christensenellaceae bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture.
  • the culture of Christensenellaceae bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
  • compositions such as pharmaceutical compositions
  • mEVs such as smEVs and/or pmEVs
  • bacteria or any combination thereof, obtained from Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • compositions comprising Christensenellaceae bacteria (e.g., bacteria of the genus Christensenella) described herein and a pharmaceutically acceptable carrier.
  • Christensenellaceae bacteria e.g., bacteria of the genus Christensenella
  • the compositions comprises less than 10 3 , 500, 100, 50, 10, or fewer colony forming units (CFU) of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • CFU colony forming units
  • the compositions comprises about 1 x 10 s , 5 x 10 s , 1 x 10 6 , , , , , , , total cells of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • the Christensenellaceae bacteria are quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
  • compositions comprises at least , , total cells of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • the compositions comprises at most , , total cells of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • the composition comprises live, killed, attenuated, lyophilized, and/or irradiated (e.g., UV or gamma irradiated) bacteria.
  • Bacteria may be heat-killed by pasteurization, sterilization, high temperature treatment, spray cooking and/or spray drying (heat treatments can be performed at 50°C, 65°C, 85°C or a variety of other temperatures and/or a varied amount of time). Bacteria may also be killed or inactivated using y-irradiation (gamma irradiation), exposune to UV light, formalin- inactivation, and/or freezing methods, or a combination thereof.
  • irradiated e.g., UV or gamma irradiated
  • the bacteria may be exposed to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50kGy of radiation prior to administration.
  • bacteria are killed using gamma irradiation.
  • the bacteria are killed or inactivated using electron irradiation (e.g., beta radiation) or x-ray irradiation.
  • the bacteria in the composition described herein are killed using a method that leaves the disease modulating activity of the bacteria intact and the resulting bacterial components are used in the methods and compositions described herein.
  • the bacteria in the composition described herein are killed using an antibiotic (e.g., using an antibiotic described herein).
  • the bacteria in the composition described herein are killed using UV irradiation.
  • the bacteria in the composition described herein are killed using heat (temperature) sterilization, filtration, and radiation using methods known to those skilled in the art (Garg M., see the World Wide Web at biologydiscussion.com/microorganisms/sterilizatiion/top-3-physical-methods-used-to- kill-microorganisms/55243).
  • the bacteria may be killed via E-beam using methods known to those skilled in the art (StLiNDtR M. et al, FABAD J. Pham. Sci., 34, 43-53, 2009).
  • the bacteria in the composition described herein are killed and/or attenuated by a chemical agent, for example, aldehydes, e.g., formaldehyde, glutaraldehyde, and the like; food preservative agents such as SCh, sorbic acid, benzoic, acid, nitrate, and nitrite salts; gases such as ethylene oxide; halogens, such as iodine, chlorine, and the like; peroxygens, such as ozone, peroxide, peracetic acid; bisphenols; phenols; phenolics; biguanides, e.g., chlorhexidine; and the like.
  • aldehydes e.g., formaldehyde, glutaraldehyde, and the like
  • food preservative agents such as SCh, sorbic acid, benzoic, acid, nitrate, and nitrite salts
  • gases such as ethylene oxide
  • halogens such as iod
  • Bacteria may be grown to various growth phases and tested for efficacy at different dilutions and at different points during the growth phase. For example, bacteria may be tested for efficacy following administration at stationary phase (including early or late stationary phase), or at various time points during exponential phase. In addition to inactivation by various methods, bacteria may be tested for efficacy using different ratios of live versus inactivated cells, or different ratios of cells at various growth phases.
  • compositions comprising mEVs (such as smEVs and/or pmEVs) (e.g., an mEV composition (e.g., an smEV composition or a pmEV composition)) from Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
  • the mEV composition comprises mEVs (such as smEVs and/or pmEVs) and/or a combination of mEVs (such as smEVs and/or pmEVs) described herein and a pharmaceutically acceptable carrier.
  • the smEV composition comprises smEVs and/or a combination of smEVs described herein and a pharmaceutically acceptable carrier.
  • the pmEV composition comprises pmEVs and/or a combination of pmEVs described herein and a pharmaceutically acceptable carrier.
  • the compositions comprise mEVs (such as smEVs and/or pmEVs) substantially or entirely free of whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the compositions comprise both mEVs and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the composition comprises lyophilized mEVs (such as smEVs and/or pmEVs). In some embodiments, the composition comprises gamma irradiated mEVs (such as smEVs and/or pmEVs). The mEVs (such as smEVs and/or pmEVs) can be gamma irradiated after the mEVs are isolated (e.g., prepared).
  • mEVs such as smEVs and/or pmEVs
  • electron microscopy e.g., EM of ultrathin frozen sections
  • NTA nanoparticle tracking analysis
  • Coulter counting Coulter counting
  • DLS dynamic light scattering
  • Combined results from Coulter counting and NTA can reveal the numbers of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a given sample.
  • Coulter counting reveals the numbers of particles with diameters of 0.7-10 pm.
  • the Coulter counter alone can reveal the number of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a sample.
  • pmEVs are 20-600 nm in diameter.
  • a Nanosight instrument can be obtained from Malvern Pananlytical.
  • the NS300 can visualize and measure particles in suspension in the size range 10-2000 nm.
  • NTA allows fbr counting of the numbers of particles that are, fbr example, 50-1000 nm in diameter.
  • DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 pm.
  • mEVs can be characterized by analytical methods known in the art (e.g., Jeppesen, et al. Cell 177:428 (2019)).
  • the mEVs may be quantified based on particle count. For example, total protein content of an mEV preparation can be measured using NTA.
  • the mEVs may be quantified based on the amount of protein, lipid, or carbohydrate.
  • a dose of mEV can be determined by particle count of an mEV preparation can be measured using the Bradford assay or BCA.
  • the mEVs are isolated away from one or more other bacterial components of the source bacteria.
  • the composition further comprises other bacterial components.
  • the mEV preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (e.g., size, density, protein content, binding affinity) of the subpopulations.
  • One or more of the mEV subpopulations can then be incorporated into the compositions of the invention.
  • compositions comprising mEVs (such as smEVs and/or pmEVs) usefill for the treatment and/or prevention of disease (e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such mEVs, and methods of using such compositions (e.g., for the treatment and/or prevention of a disease or a health disorder (e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, and/or a metabolic disease), either alone or in combination with other therapeutics).
  • disease e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease
  • a health disorder e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, and/or a metabolic disease
  • the compositions comprise both mEVs (such as smEVs and/or pmEVs), and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria).
  • the compositions comprise mEVs (such as smEVs and/or pmEVs) in the absence of bacteria.
  • the compositions comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from the genus Christensenella.
  • the compositions comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from Christensenella minuta.
  • compositions comprising Christensenellaceae bacteria and/or Christensenellaceae bacteria mEVs described herein.
  • the bacteria are bacteria of the genus Christensenella, such as Christensenella minuta, including, for example, one or more species with the 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof.
  • the composition comprises at least 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
  • the composition comprises about 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
  • the composition comprises no more than 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1,
  • the composition comprises at least 1 Christensenellaceae bacterial mEV particle (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3,
  • the composition comprises about 1 Christensenellaceae bacterial mEV particle (e.g., fiom the genus Christensenella) for every 1, 1.1, 1.2, 1.3,
  • the composition comprises no more than 1 ChristenseneUaceae bacteria mEV particle (e.g., fiom the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1,
  • 1 ChristenseneUaceae bacteria mEV particle e.g., fiom the genus Christensenella
  • compositions fbr administration to a subject e.g., human subject.
  • the compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format.
  • the composition is combined with an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
  • an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
  • the composition comprises at least one carbohydrate.
  • the composition comprises at least one lipid.
  • the lipid comprises at least one fetty acid selected fiom lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), dococosanoic acid (22:
  • the composition comprises at least one supplemental mineral or mineral source.
  • supplemental mineral or mineral source examples include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium.
  • Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
  • the composition comprises at least one supplemental vitamin.
  • the at least one vitamin can be fat-soluble or water soluble vitamins.
  • Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B 12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin.
  • Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
  • the composition comprises an excipient.
  • suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
  • the excipient is a buffering agent.
  • suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
  • the excipient comprises a preservative.
  • suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
  • the composition comprises a binder as an excipient.
  • suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, Cn-Cis fiitty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
  • the composition comprises a lubricant as an excipient.
  • suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfiite, magnesium lauryl sulfiite, and light mineral oil.
  • the composition comprises a dispersion enhancer as an excipient.
  • suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
  • the composition comprises a disintegrant as an excipient.
  • the disintegrant is a non-eflfervescent disintegrant.
  • suitable non-effervescent disintegrants include starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth.
  • the disintegrant is an effervescent disintegrant.
  • suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
  • the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infonts, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed.
  • a food product e.g., a food or beverage
  • a food or beverage for infonts e.g., a health food or beverage
  • a food or beverage for infonts e.g., a food or beverage for infonts
  • a food or beverage for pregnant women e.g., athletes, senior citizens or other specified group
  • a functional food e.g., a beverage
  • a beverage e.g., a food or beverage for infonts
  • a food or beverage for pregnant women e.g., athletes, senior citizens or other specified group
  • a functional food
  • the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, carb
  • the composition is a food product for animals, including humans.
  • the animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like.
  • Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
  • Chnstensenellaceae bacteria and/or Christensenella bacteria can be measured by amplifying the 16S rRNA gene from bacteria in a sample.
  • the levels of Chnstensenellaceae bacteria and/or Christensenella bacteria are measured in a sample by performing quantitative PCR using primers that specifically amplify members of the family or of the genus, and these levels are normalized to the total 16S rRNA in a sample.
  • the prevalent strategy for amplifying the 16S rRNA gene from microorganisms in a sample involves utilizing probes and primers that anneal to conserved regions at the edges of known variable domains in the 16S rRNA gene sequence, for amplification and sequencing across the variable domains to identify species-specific variable sequences and thus identify species within the sample.
  • primer pairs have been designed to amplify a single variable (“ V 1 ') region, such as the 16S V3, V4, or V5 region.
  • V 1 ' a single variable
  • Common primers to amplify the V4 region include universal 16S primers 515F (SEQ ID NO: 3) and 806R (SEQ ID NO: 4).
  • sequencing technologies include, but are not limited to, sequencing-by-synthesis (e.g., Illumina dye sequencing; Single Molecule Real Time Sequencing platform by Pacific Biosciences); sequencing by ligation (Solid or Polony sequencing platform, Applied Biosystems); pyrosequencing (454 Sequencing, Roche Diagnostics); and ion semiconductor sequencing (Ton Torrent Sequencing, Life Technologies).
  • sequencing-by-synthesis e.g., Illumina dye sequencing; Single Molecule Real Time Sequencing platform by Pacific Biosciences
  • sequencing by ligation Solid or Polony sequencing platform, Applied Biosystems
  • pyrosequencing (454 Sequencing, Roche Diagnostics
  • ion semiconductor sequencing Teon Torrent Sequencing, Life Technologies
  • PCR amplicons can be purified, for example using a magnetic bead system, and quantified, using commercially available products for purification and quantification. Donavan be sequenced and analyzed using any one of several sequencing platforms, such as an Illumina MISEQ platform (Illumina Inc., San Diego, Calif, USA).
  • Illumina MISEQ platform Illumina Inc., San Diego, Calif, USA.
  • Quality filtering to remove “noise” generated by random sequencing errors
  • analysis of 16S rRNA gene sequence data can be performed using publicly available bioinformatic analytic tools for taxon identification from raw DNA sequencing data, such as the Quantitative Insights Into Microbial Ecology' (QIIME) analytic tool (Caporaso et al.. Nature Methods 7:335-336 (2010)).
  • QIIME Quantitative Insights Into Microbial Ecology'
  • Christensenellaceae bacteria are identified based on (1) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity, preferably 97% identity or greater, of the 16S rRNA gene sequence or sequences of the bacteria with Christensenellaceae 16S rRNA gene sequences, using an available database of 16S rRNA gene sequences, or (2) phylogenetic analysis of the 16S rRNA gene sequence of the bacteria identifying the bacteria as Christensenellaceae, using known methods for determining phylogeny.
  • the levels of Christensenellaceae in a sample can be determined by comparing the abundance or ratio of sequences identified as Christensenellaceae to the total bacterial abundance in a sample, or to the abundance or ratio of sequences identified as not belonging to Christensenellaceae.
  • the abundance or ratio of Christensenellaceae in a sample, relative to other bacterial species can be measured by use of analytic tools, such as QIIME. These tools can generate taxonomic distributions in a sample by comparing the bacterial 16S rRNA gene sequences from the sample to the 16SrRNA gene sequences in a database such as Greengenes, which have previously been assigned taxonomic classifications.
  • Christensenella bacteria are identified based on at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 (the portion of SEQ ID NO: 1 generated by amplification with the 515F and 806R universal 16S primers).
  • the levels of Christensenella in a sample can be measured by comparing the abundance or ratio of sequences having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 to the abundance or ratio of sequences falling outside of this range.
  • the abundance or ratio or Christensenella in a sample, relative to other bacterial species can be measured by use of analytic tools, such as QIIME, to generate taxonomic distributions in a sample by comparing the bacterial 16S rRNA gene sequences from the sample to the 16S rRNA gene sequences in a database such as Greengenes, which have previously been assigned taxonomic classifications.
  • QIIME analytic tools
  • the subject can benefit by administration of a composition as disclosed herein.
  • the subject can be treated by administration of a dosage of less than 10 x 8 CPU of Christensenellaceae or Christensenella bacteria/dose, and more particularly less than 10 x 6 CPU of Christensenellaceae.
  • the bacteria and/or mEVs described herein are administered in separate administrations to a subject.
  • Separate administrations can include any number of two or more administrations (e.g., doses), including two, three, four, five or six administrations.
  • doses e.g., doses
  • One skilled in the art can readily determine the number of administrations to perform, or the desirability of performing one or more additional administrations, according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein.
  • the doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks.
  • the methods provided herein include methods of providing to the subject one or more administrations of bacteria, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results, including, but not limited to, the overall health of the subject and/or the weight of the subject.
  • the time period between administrations of the bacteria described herein can be any of a variety of time periods.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the bacteria from normal tissue.
  • the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
  • provided herein is a method of delivering bacteria and/or mEVs and/or a composition described herein to a subject.
  • the bacteria, mEVs, and/or a composition thereof are administered in conjunction with the administration of an additional therapeutic.
  • the bacteria and/or mEVs are co-formulated in a composition with the additional therapeutic.
  • the bacteria and/or mEVs are co-administered with the additional therapeutic.
  • the additional therapeutic is administered to the subject before administration of the bacteria and/or mEVs (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before).
  • the bacteria and/or mEVs e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before.
  • the additional therapeutic is administered to the subject after administration of the bacteria and/or mEVs (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after).
  • the same mode of delivery is used to deliver both the bacteria and the additional therapeutic.
  • different modes of delivery are used to administer the bacteria and the additional therapeutic.
  • the bacteria and/or mEVs are administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, intramuscular and/or intratumoral injection).
  • the composition is administered orally, rectally, intravenously, intradermally, intraperitoneally, or subcutaneously. In some embodiments, the composition is administered orally.
  • the composition can be administered on a daily or weekly basis.
  • the subject to whom a composition of the invention is administered can have metabolic disease.
  • the subject to whom a composition of the invention is administered can have obesity, metabolic syndrome, and/or diabetes.
  • the composition can be for inhibiting weight gain, promoting weight loss, or reducing adiposity.
  • the composition is administered in two or more doses.
  • the administration to the subject of the two or more doses are separated by at least 1 day. In other embodiments, the administration of the two or more doses are separated by at least 1 week.
  • administration of the composition treats the metabolic disease. In some embodiments, administration of the composition treats the obesity, metabolic syndrome, and/or diabetes. In some embodiments, administration of the composition inhibits weight gain, promotes weight loss, or reduces adiposity.
  • compositions described herein can be administered in conjunction with any other conventional metabolic disease therapeutic agents. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the compositions described herein.
  • the dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art.
  • appropriate miniminn dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate.
  • the methods of treatment described herein may be suitable for the treatment of a metabolic disease (e.g., diabetes).
  • the dose of the pharmaceutical compositions described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like.
  • the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day.
  • the effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
  • the dose administered to a subject is sufficient to prevent the metabolic disease, delay its onset, or slow or stop its progression or prevent a relapse of the metabolic disease. In some embodiments, the dose administered to a subject is sufficient to prevent the obesity, metabolic syndrome, and/or diabetes, delay its onset, or slow or stop its progression or prevent a relapse of the obesity, metabolic syndrome, and/or diabetes. In some embodiments, the dose administered to a subject is sufficient to inhibit weight gain, promote weight loss, or reduce adiposity.
  • dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect.
  • Suitable doses and dosage regimens can be determined by conventional rangefinding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached.
  • An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD”) of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
  • MTD maximal tolerable dose
  • the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering therapy, among other factors affecting the selected dosage.
  • the dose should be sufficient to result in slowing, and preferably regressing, the advancement of an immune disorder.
  • the bacteria, mEVs, and/or composition and an additional therapeutic can be administered separately.
  • Separate administrations can include any number of two or more administrations (e.g. , doses), including two, three, four, five or six administrations.
  • doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks.
  • the methods provided herein include methods of providing to the subject one or more administrations of bacteria, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results, including, but not limited to, the overall health of the subject and/or the weight of the subject.
  • the time period between administrations can be any of a variety of time periods.
  • the time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the bacteria from normal tissue.
  • the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month.
  • the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
  • the delivery of the bacteria and/or mEVs described herein reduces the adverse effects and/or improves the efficacy of the metabolic disease therapeutic.
  • the administration of the composition treats the metabolic disease.
  • the administration of the composition treats the obesity, metabolic syndrome, and/or diabetes.
  • the administration of the composition inhibits weight gain, promotes weight loss, or reduces adiposity.
  • the methods provided herein include administering to a subject a composition described herein (e.g., a composition comprising a Christensenellaceae bacteria and/or mEVs) either alone or in combination with another therapeutic.
  • a composition described herein e.g., a composition comprising a Christensenellaceae bacteria and/or mEVs
  • the composition and the other therapy can be administered to the subject in any order.
  • the composition and the other therapy are administered conjointly.
  • composition is administered to the subject before the additional therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
  • composition is administered to the subject after the additional therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
  • the composition and the additional therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an horn* of each other).
  • the subject is administered an antibiotic before the composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).
  • the subject is administered an antibiotic after the composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after).
  • the composition and the antibiotic are
  • the subject may undergo surgery.
  • Types of surgery include but are not limited to preventative, diagnostic or staging, cinative and palliative surgery.
  • the additional therapeutic is an antibiotic.
  • antibiotics broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability, or their spectrum of target microbe (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs.
  • antibiotics can be used to selectively target bacteria of a specific niche.
  • antibiotics known to treat a particular infection that includes a metabolic disease niche may be used to target metabolic-disease-associated microbes, including metabolic-disease-associated bacteria in that niche.
  • antibiotics are administered after the bacterial treatment.
  • antibiotics are administered after the bacterial treatment to remove the engrafiment.
  • antibiotics can be selected based on their bactericidal or bacteriostatic properties.
  • Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., P-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones).
  • Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis.
  • some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties.
  • bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics.
  • bactericidal and bacteriostatic antibiotics are not combined.
  • Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti- mycobacterial compounds, and combinations thereof.
  • Aminoglycosides include, but are not limited to Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin.
  • Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia colt and Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or SOS ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin.
  • Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
  • Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
  • Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Grampositive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
  • Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole.
  • Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin-resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • MRSA methicillin-resistant Staphylococcus aureus
  • Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Grampositive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus and Streptococcus. Lincosamides are believed to bind to the bacterial SOS ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
  • Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g., against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or SOS ribosomal subunit, thereby inhibiting bacterial protein synthesis.
  • Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g., against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
  • Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
  • Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cioxacillin, Dicloxacillin, Flucioxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin.
  • Penicillins are effective, e.g., against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus, Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
  • Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
  • Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E.
  • Polypeptide Antibiotics are effective, e.g., against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.
  • Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin.
  • Quinolones/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria.
  • Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
  • Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide, Sulfodiazine, Silver sulfodiazine, Sulfodimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co- trimoxazole), and Sulfonamidochrysoidine. Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.
  • Tetracyclines include, but are not limited to, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
  • Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinaniide, Rifampicin, Rifabutin, Rifopentine, and Streptomycin.
  • Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleand
  • the additional therapeutic is metabolic disease therapeutic, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof.
  • Representative agents include, but are not limited to, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen,
  • metabolic disease therapeutics include, but are not limited to, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, and/or ursodeoxycholic acid.
  • the method further comprises administering to the subject a second therapeutic bacterial strain.
  • the second therapeutic bacterial strain is of the Methanobacteriaceae family.
  • the second therapeutic bacterial strain v& Methanobacterium aarhusense, Methanobacterium aggregans, Methanobacterium alcaliphilum, Methanobacterium arcticum, Methanobacterium beijingense, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium curvum, Methanobacterium espanolae, Methanobacterium ferruginis, Methanobacterium flexile, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium kanagiense, Methanobacterium lacus, Methanobacterium movens, Methanobacterium movilense, Methanobacterium oryzae, Methanobacterium paludis, Methanobacterium
  • the methods can optionally include measuring the levels of Christensenellaceae in a biological sample from a subject; determining the amount of a composition of substantially purified Christensenellaceae to administer to the subject, based on the levels of Christensenellaceae in said sample; and administering a composition to the subject in an amount effective to treat the condition.
  • Metabolic syndrome is characterized by the presence of three or more of these components: an elevated waist circumference in men of equal to or greater than 40 inches (102 cm); an elevated waist circumference in women of equal to or greater than 35 inches (88 cm); elevated triglycerides of equal to or greater than 150 mg/dL; reduced HDL (“good”) cholesterol in men of less than 40 mg/dL, or less than 50 mg/dL in women; elevated blood pressure of equal to or greater than 130/85 mm Hg; and elevated fasting glucose equal to or greater than 100 mg/dL.
  • the metabolic syndrome does not comprise elevated triglycerides of equal to or greater than 150 mg/dL, e.g., does not comprise hypertriglyceridemia.
  • Diabetes mellitus is a condition in which a person has a high blood sugar (glucose) level, either because the body doesn't produce enough insulin, or because body cells don't properly respond to the insulin that is produced. Diabetes can be diagnosed, for example, from an oral glucose tolerance test as a 2-hour plasma glucose concentration of greater than or equal to 200 mg/dl. Insulin is a hormone produced in the pancreas that enables body cells to absorb glucose, to turn into energy. If the body cells do not absorb the glucose, the glucose accumulates in the blood (hyperglycemia), leading to vascular, nerve, and other complications. Type 1 diabetes results from the body's failure to produce insulin, and presently requires the person to inject insulin. Type 2 diabetes results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency.
  • a therapeutically effective amount can be administered to a patient in one or more doses sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease, or reduce the symptoms of the disease.
  • the amelioration or reduction need not be permanent, but can be for a period of time ranging from at least one hour, at least one day, or at least one week or more.
  • the effective amount is generally determined by the physician on a case- by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition, as well as the route of administration, dosage form and regimen and the desired result.
  • the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • the methods and compositions described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fiitty liver disease (NAFLD), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • compositions described herein can be used, for example, for preventing or treating (reducing, partially or completely, the adverse effects of) a metabolic disease, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fiitty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease.
  • the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
  • Fractionated HSP Spin in ultracentrifuge at 200,000 x g, 1 hr, 4°C.
  • Christensenella are amended with non-living C. minuta bacteria or extracellular vesicles, and weight gain of recipient mice is monitored.
  • One obese human donor is selected from the 21 donors from the first transplant experiment based on its lack of detectable OTUs assigned to the genus Christensenella.
  • mice receiving the nonliving C. minuta bacteria or extracellular vesicles treatment are weighed and may weigh significantly less than those that receive unamended stool.
  • Adiposity is measured and may be significantly lower for mice receiving the non-living C. minuta bacteria or extracellular vesicles treatment. Energy content for stool collected at Day 21 is measured and may not differ between treatments.

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Abstract

Provided herein are methods and compositions comprising non-living Christensenellaceae bacteria and/or extracellular vesicles (EVs) from Christensenellaceae bacteria. In certain embodiments, the methods and compositions provided herein are useful for the treatment and/or prevention of metabolic diseases.

Description

Compositions and Methods for Treating Metabolic Diseases and Disorders Using Christensenellaceae Bacteria
CROSS-REFERENCE TO RELATED APPLICATION
[001] This application claims the benefit of U.S. Provisional Application serial number 63/151,088, filed February 19, 2021, the entire contents of which are incorporated herein by reference.
SUMMARY
[002] In certain aspects, provided herein are methods of treating and/or preventing a metabolic disease in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition (e.g., a bacterial and/or pharmaceutical composition) comprising non-living Christensenellaceae bacteria and/or microbial extracellular vesicles (mEVs) derived from Christensenellaceae bacteria. The Christensenellaceae bacteria can be, for example, bacteria of the genus Christensenella, such as Christensenella minula, including, for example, one or more species with a 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and/or mixtures thereof. In some embodiments, the Christensenellaceae is of a strain that is resistant to polymyxin. In some embodiments, the Christensenellaceae bacteria and/or mEVs in the composition are lyophilized.
[003] In certain aspects, provided herein are methods of inhibiting weight gain, promoting weight loss, or reducing adiposity in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition (e.g., a bacterial and/or pharmaceutical composition) comprising non-living Christensenellaceae bacteria and/or extracellular vesicles (mEVs) derived from Christensenellaceae bacteria. The Christensenellaceae bacteria can be, for example, bacteria of the genus Christensenella, such as Christensenella minula, including, for example, one or more species with a 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and/or mixtures thereof. In some embodiments, the Christensenellaceae is of a strain that is resistant to polymyxin. In some embodiments, the Christensenellaceae bacteria and/or mEVs in the composition are lyophilized.
[004] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% of the Christensenellaceae bacteria in the composition are non- living (e.g., non-live or not live or not viable). In further embodiments, the composition comprises no more than 108 colony forming units (CPUs) of Christensenellaceae bacteria. In further embodiments, the composition comprises no more than 106 colony forming units (CPUs) (e.g., no more than 105, 104, 103, 500, 100, 50, 10, or fewer CPUs) of Christensenella bacteria. In some embodiments, substantially all or all of the Christensenellaceae bacteria in the administered composition are non-living.
[005] In certain embodiments of the methods provided herein, at least 4 x 1010 cells of the Christensenellaceae bacteria are administered to the subject daily. In some embodiments, at least 4 x 1010 cells, 5 x 1010 cells, 6 x 1010 cells, 7 x 1010 cells, 8 x 1010 cells, 9 x 1010 cells, 1.0 x 1011 cells, 1.1 x 1011 cells, 1.2 x 1011 cells, 1.3 x 1011 cells, 1.4 x 1011 cells, 1.5 x 1011 cells, 1.6 x 1011 cells, 1.7 x 1011 cells, 1.8 x 1011 cells, 1.9 x 1011 cells, 2.0 x 1011 cells, 2.1 x 1011 cells, 2.2 x 1011 cells, 2.3 x 1011 cells, 2.4 x 1011 cells, 2.5 x 1011 cells, 2.6 x 1011 cells, 2.7 x 1011 cells, 2.8 x 1011 cells, 2.9 x 1011 cells, 3.0 x 1011 cells, 3.1 x 1011 cells, 3.2 x 1011 cells, 3.3 x 1011 cells, 3.4 x 1011 cells, 3.5 x 1011 cells, 3.6 x 1011 cells, 3.7 x 1011 cells, 3.8 x 1011 cells, 3.9 x 1011 cells, 4.0 x 1011 cells, 4.1 x 1011 cells, 4.2 x 1011 cells, 4.3 x 1011 cells, 4.4 x 1011 cells, 4.5 x 1011 cells, 4.6 x 1011 cells, 4.7 x 1011 cells, 4.8 x 1011 cells, 4.9 x 1011 cells, 5.0 x 1011 cells, 5.1 x 1011 cells, 5.2 x 1011 cells, 5.3 x 1011 cells, 5.4 x 1011 cells, 5.5 x 1011 cells, 5.6 x IO11 cells, 5.7 x IO11 cells, 5.8 x IO11 cells, 5.9 x IO11 cells, 6.0 x IO11 cells, 6.1 x IO11 cells, 6.2 x IO11 cells, 6.3 x IO11 cells, 6.4 x IO11 cells, 6.5 x IO11 cells, 6.6 x IO11 cells, 6.7 x IO11 cells, 6.8 x IO11 cells, 6.9 x IO11 cells, 7.0 x IO11 cells, 7.1 x IO11 cells, 7.2 x IO11 cells, 7.3 x IO11 cells, 7.4 x IO11 cells, 7.5 x IO11 cells, 7.6 x IO11 cells, 7.7 x IO11 cells, 7.8 x IO11 cells, 7.9 x IO11 cells, or 8.0 x IO11 cells of the Christensenellaceae bacteria are administered to the subject daily. In some embodiments, the Christensenellaceae bacteria are quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
[006] In certain embodiments of the methods provided herein, mEVs fiom Christensenellaceae bacteria can be administered at doses e.g., of about IxlO7 to about 1x1015 particles, e.g., as measured by NTA. In some embodiments, the dose of mEVs is about 1 x 105 to about 7 x 1013 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)). In some embodiments, the dose of mEVs fiom Christensenellaceae bacteria is about 1 x 1010 to about 7 x 1013 particles (e.g., wherein particle count is determined by NTA (nanoparticle tracking analysis)). [007] In some embodiments, administration of the disclosed compositions does not increase the levels of Christensenellaceae bacteria, relative to the levels of other bacteria, in the gastrointestinal tract of the subject.
[008] In some embodiments, the composition comprises isolated Christensenellaceae bacteria microbial extracellular vesicles (mEVs) (e.g., secreted microbial extracellular vesicles (smEVs), processed microbial extracellular vesicles (pmEVs)). In further embodiments, the composition comprises Christensenellaceae bacteria microbial extracellular vesicles (mEVs) and Christensenellaceae bacteria.
[009] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacterial mEVs.
[010] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacteria particles.
[OH] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacterial mEV proteins.
[012] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition are Christensenellaceae bacterial proteins.
[013] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacterial mEV lipids.
[014] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacterial lipids. [015] In some embodiments, the composition is administered orally, rectally, intravenously, intradermally, intraperitoneally, or subcutaneously. In some embodiments, the composition is administered orally.
[016] In some embodiments, the composition can be formulated as a tablet, capsule, oral liquid preparation, reconstitutable powder or suspension. [017] In some embodiments, the composition can comprise food or nutritional supplements containing Christensenellaceae bacteria and/or mEVs, e.g., a composition of substantially purified Christensenellaceae bacteria and/or mEVs.
[018] In some embodiments, the composition can be administered on a daily or weekly basis. The subject to whom a composition of the invention is administered can have metabolic disease.
[019] In some embodiments, the composition is administered in two or more doses. In further embodiments, the administration to the subject of the two or more doses are separated by at least 1 day. In other embodiments, the administration of the two or more doses are separated by at least 1 week.
[020] In some embodiments, the composition comprises killed bacteria. In further embodiments, the composition comprises irradiated bacteria. In other embodiments, the composition comprises gamma irradiated bacteria.
[021] In some embodiments, the composition comprises lyophilized bacteria.
[022] In some embodiments, the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease. In further embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema. In some embodiments, the metabolic disease does not include hypertriglyceridemia.
[023] In some embodiments, the metabolic disease comprises obesity. In some embodiments, the obesity does not include hypertriglyceridemia.
[024] In some embodiments, the metabolic disease comprises metabolic syndrome. In some embodiments, the metabolic syndrome does not include hypertriglyceridemia.
[025] In some embodiments, the metabolic disease comprises diabetes.
[026] In some embodiments, administration of the composition treats the metabolic disease.
[027] In some embodiments, the method further comprises administering to the subject an additional therapeutic. [028] In some embodiments, the additional therapeutic is selected from the group consisting of an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), a cytokine antagonist, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprofen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetaminophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs, hydroxychloroquine, chloroquine, sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists, TNF alpha antagonists, TNF alpha receptor antagonists, ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra ZActemra®), integrin antagonists, TYSABRI® (natalizumab), IL-1 antagonists, ACZ885 (Haris), Anakinra (Kineret®), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists, Atacicept, Benlysta®/ LymphoStat-B® (belimumab), p38 Inhibitors, CD20 antagonists, Ocrelizumab, Ofatumumab (Arzerra®), interferon gamma antagonists, Fontolizumab, prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura- 100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D - ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus, Prograf®, RADOO1, rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone, Curcumin, Longvida™, Rosuvastatin, Maraviroc, ramipnl, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists, Tysarbri® (natalizumab), VGEF antagnosits, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists, IL-1 beta antagonsits, IL-23 antagonists, receptor decoys, antagonistic antibodies, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, antileukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines, cytokine inhibitors, anti-IL-6 antibodies, TNF inhibitors, infliximab, adalimumab, certolizumab pegol, golimumab, and etanercept.
[029] In some embodiments, the additional therapeutic is an antibiotic. In some embodiments, the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, anti- mycobacterial compounds and combinations thereof.
[030] In some embodiments, the additional therapeutic is a metabolic disease therapeutic. In some embodiments, the metabolic disease therapeutic is metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3- fatty acids, statins, ezetimibe, or ursodeoxycholic acid.
[031] In some embodiments, the method further comprises administering to the subject a second therapeutic bacterial strain, e.g., a composition thereof. In some embodiments, the second therapeutic bacterial strain is of the Methanobacteriaceae family. In some embodiments, the second therapeutic bacterial strain is Methanobacterium aarhusense, Methanobacterium aggregans, Methanobacterium alcaliphilum, Methanobacterium arcticum, Methanobacterium beijingense, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium curvum, Methanobacterium espanolae, Methanobacterium ferruginis, Methanobacterium flexile, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium kanagiense, Methanobacterium lacus, Methanobacterium movens, Methanobacterium movilense, Methanobacterium oryzae, Methanobacterium paludis, Methanobacterium palustre, Methanobacterium petrolearium, Methanobacterium subterraneum, Methanobacterium thermaggregans, Methanobacterium uliginosum, Methanobacterium veterum, Methanobrevibacter acididurans, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanobrevibacter curvatus, Methanobrevibacter cuticularis, Methanobrevibacter filiformis, Methanobrevibacter gottschalkii, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanobrevibacter oralis, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter thaueri, Methanobrevibacter woesei, Methanobrevibacter wolinii, Methanosphaera cuniculi, Methanosphaera stadtmanae, Methanothermobacter crinale, Methanothermobacter defluvii, Methanothermobacter marburgensis, Methanothermobacter tenebrarum, Methanothermobacter thermautotrophicus, Methanothermobacter thermoflexus, Methanothermobacter thermophiles, or Methanothermobacter wolfeii. In preferred embodiments, the second therapeutic bacterial strain is Methanobrevibacter smithii. [032] In some embodiments, the second therapeutic bacterial strain is administered as part of an ecological consortium.
[033] In some embodiments, the method further comprises administering a prebiotic to the subject. In some embodiments, the prebiotic is a fructooligosaccharide, a galactooligosaccharide, a trans-galactooligosaccharide, a xylooligosaccharide, a chitooligosaccharide, a soy oligosaccharide, a gentiooligosaccharide, an isomaltooligosaccharide, a mannooligosaccharide, a maltooligosaccharide, a mannanoligosaccharide, lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, or a cyclodextrin.
[034] In some embodiments, the composition is formulated as a food or drink. [035] In some embodiments, the subject is a human.
[036] In some embodiments, subject is a non-human mammal. In some embodiments, the mammal is selected from the group consisting of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
[037] In certain aspects, provided herein are compositions comprising mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, obtained from Christensenellaceae bacteria. In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacterial mEVs.
[038] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacteria particles.
[039] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacterial mEV protein.
[040] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition are Christensenellaceae bacteria proteins. [041] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are ChristenseneUaceae bacterial mEV lipids.
[042] In some embodiments, at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are ChristenseneUaceae bacteria lipids. [043] In certain aspects, provided herein are compositions comprising ChristenseneUaceae bacteria isolated from mEVs.
[044] In certain aspects, provided herein are compositions comprising ChristenseneUaceae mEVs isolated from bacteria.
[045] In some aspects, the disclosure provides a composition described herein for use in treating and/or preventing a metabolic disease in a subject.
[046] In some aspects, the disclosure provides use of a composition described herein for the preparation of a medicament for treating and/or preventing a metabolic disease in a subject.
[047] In some aspects, the disclosure provides a composition described herein for use in inhibiting weight gain, promoting weight loss, or reducing adiposity in a subject.
[048] In some aspects, the disclosure provides use of a composition described herein for the preparation of a medicament for inhibiting weight gain, promoting weight loss, or reducing adiposity in a subject.
[049] In certain aspects, provided herein are bioreactors comprising ChristenseneUaceae bacteria. [050] In some embodiments, the bacteria are a strain comprising at least 90% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1. In some embodiments, the mEVs are from bacteria of a strain comprising at least 90% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
[051] In some embodiments, the bacteria are a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1. In some embodiments, the mEVs are from bacteria of a strain comprising at least 99% genomic, 16S and/or CRISPR sequence identity to the nucleotide sequence of a bacterial strain listed in Table 1.
[052] In some embodiments, the Christensenellaceae bacteria are Christensenella minuta. In some embodiments, the mEVs are from Christensenella minuta bacteria.
[053] In certain aspects, provided herein are methods of growing bacteria in a bioreactor comprising providing a bioreactor as described herein; and fermenting the bacteria for a period of time.
DETAILED DESCRIPTION
[054] Disclosed herein are compositions that have substantially purified bacteria and/or mEVs of the family Christensenellaceae and/or the genus Christensenella, and uses of these compositions. The administration of Christensenellaceae (bacteria and/or mEVs) to an individual can treat or prevent metabolic diseases.
Definitions
[055] “Adjuvant” or “Adjuvant therapy” broadly refers to an agent that affects an immunological or physiological response in a subject (e.g., human). For example, an adjuvant might help absorb an antigen presenting cell antigen, activate macrophages and lymphocytes and support the production of cytokines. By changing an immune response, an adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, an adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.
[056] “Administration” broadly refers to a route of administration of a composition to a subject. Examples of routes of administration include oral administration, rectal administration, topical administration, inhalation (nasal) or injection. Administration by injection includes intravenous (IV), intramuscular (IM), intratumoral (IT) and subcutaneous (SC) administration. The compositions described herein can be administered in any form by any effective route, including but not limited to intratumoral, oral, parenteral, enteral, intravenous, intraperitoneal, topical, transdermal (e.g., using any standard patch), intradermal, ophthalmic, (intra)nasally, local, non-oral, such as aerosol, inhalation, subcutaneous, intramuscular, buccal, sublingual, (trans)rectal, vaginal, intraarterial, and intrathecal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), intravesical, intrapulmonary, intraduodenal, intragastrical, and intrabronchial. In preferred embodiments, the compositions described herein are administered orally, rectally, topically, intravesically, by injection into or adjacent to a draining lymph node, intravenously, by inhalation or aerosol, or subcutaneously. In some embodiments, the compositions described herein are administered orally.
[057] A “carbohydrate” refers to a sugar or polymer of sugars. The terms “saccharide,” “polysaccharide,” “carbohydrate,” and “oligosaccharide” may be used interchangeably. Most carbohydrates are aldehydes or ketones with many hydroxyl groups, usually one on each carbon atom of the molecule. Carbohydrates generally have the molecular formula CnHinOn. A carbohydrate may be a monosaccharide, a disaccharide, trisaccharide, oligosaccharide, or polysaccharide. The most basic carbohydrate is a monosaccharide, such as glucose, sucrose, galactose, mannose, ribose, arabinose, xylose, and fructose. Disaccharides are two joined monosaccharides. Exemplary disaccharides include sucrose, maltose, cellobiose, and lactose. Typically, an oligosaccharide includes between three and six monosaccharide units (e.g., raffinose, stachyose), and polysaccharides include six or more monosaccharide units. Exemplary polysaccharides include starch, glycogen, and cellulose. Carbohydrates may contain modified saccharide units such as 2’-deoxyribose wherein a hydroxyl group is removed, 2’-fluororibose wherein a hydroxyl group is replaced with a fluorine, or N-acetylglucosamine, a nitrogen-containing form of glucose (e.g., 2’-fluororibose, deoxyribose, and hexose). Carbohydrates may exist in many different forms, for example, conformers, cyclic forms, acyclic forms, stereoisomers, tautomers, anomers, and isomers.
[058] “Cellular augmentation” broadly refers to the influx of cells or expansion of cells in an environment that are not substantially present in the environment prior to administration of a composition and not present in the composition itself. Cells that augment the environment include immune cells, stromal cells, bacterial and fungal cells. [059] A “combination” of bacteria from two or more strains includes the physical coexistence of the bacteria, either in the same material or product or in physically connected products, as well as the temporal co-administration or co-localization of the bacteria from the two or more strains.
[060] The term “decrease” or “deplete” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 1/100, 1/1000, 1/10,000, 1/100,000, 1/1,000,000 or undetectable after treatment when compared to a pre-treatment state. Properties that may be decreased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).
[061] “Dysbiosis” refers to a state of the microbiota or microbiome of the gut or other body area, including, e.g., mucosal or skin surfaces (or any other microbiome niche) in which the normal diversity and/or function of the host gut microbiome ecological networks “microbiome”) are disrupted. A state of dysbiosis may result in a diseased state, or it may be unhealthy under only certain conditions or only if present for a prolonged period. Dysbiosis may be due to a variety of factors, including, environmental factors, infectious agents, host genotype, host diet and/or stress. A dysbiosis may result in: a change (e.g., increase or decrease) in the prevalence of one or more bacteria types (e.g., anaerobic), species and/or strains, change (e.g., increase or decrease) in diversity of the host microbiome population composition; a change (e.g., increase or reduction) of one or more populations of symbiont organisms resulting in a reduction or loss of one or more beneficial effects; overgrowth of one or more populations of pathogens (e.g., pathogenic bacteria); and/or the presence of, and/or overgrowth of, symbiotic organisms that cause disease only when certain conditions are present.
[062] The term “ecological consortium” is a group of bacteria that trades metabolites and positively co-regulates one another, in contrast to two bacteria that induce host synergy through activating complementary host pathways for improved efficacy.
[063] As used herein, “engineered bacteria” are any bacteria that have been genetically altered from their natural state by human activities and the progeny of any such bacteria. Engineered bacteria include, for example, the products of targeted genetic modification, the products of random mutagenesis screens and the products of directed evolution. [064] The term “gene” is used broadly to refer to any nucleic acid associated with a biological function. The term “gene” applies to a specific genomic sequence, as well as to a cDNA or an mRNA encoded by that genomic sequence.
[065] ‘Identity” as between nucleic acid sequences of two nucleic acid molecules can be determined as a percentage of identity using known computer algorithms such as the “FASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCG program package (Devereux, J., etal., Nucleic Acids Research 12(I):387 (1984)), BLASTP, BLASTN, FASTA Atschul, S. F., et al., J Molec Biol 215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego, 1994, and Carillo etal. (1988) SIAM J Applied Math 48: 1073). For example, the BLAST function of the National Center for Biotechnology Information database can be used to determine identity. Other commercially or publicly available programs include DNAStar “MegAlign” program (Madison, Wis.) and the University of Wisconsin Genetics Computer Group (UWG) “Gap” program (Madison Wis.)).
[066] The term “increase” means a change, such that the difference is, depending on circumstances, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 2-fold, 4-fold, 10-fold, 100-fold, 10^3 fold, 10^4 fold, 10^5 fold, 10^6 fold, and/or 10^7 fold greater after treatment when compared to a pre-treatment state. Properties that may be increased include the number of immune cells, bacterial cells, stromal cells, myeloid derived suppressor cells, fibroblasts, metabolites; the level of a cytokine; or another physical parameter (such as ear thickness (e.g., in a DTH animal model) or tumor size).
[067] The “internal transcribed spacer” or ‘ITS” is a piece of non-fimctional RNA located between structural ribosomal RNAs (rRNA) on a common precursor transcript often used for identification of eukaryotic species in particular fungi. The rRNA of fungi that forms the core of the ribosome is transcribed as a signal gene and consists of the 8S, 5.8S and 28S regions with ITS4 and 5 between the 8S and 5.8S and 5.8S and 28S regions, respectively. These two intercistronic segments between the 18S and 5.8S and 5.8S and 28S regions are removed by splicing and contain significant variation between species for barcoding pinposes as previously described (Schoch et al Nuclear ribosomal internal transcribed spacer (ITS) region as a universal DNA barcode marker for Fungi. PNAS 109:6241-6246. 2012). 18S rDNA is traditionally used for phylogenetic reconstruction however the ITS can serve this function as it is generally highly conserved but contains hypervariable regions that harbor sufficient nucleotide diversity to differentiate genera and species of most fungus.
[068] The term "insulin resistance" has its common meaning in the art. Insulin resistance is a physiological condition where the natural hormone insulin, becomes less effective at lowering blood sugars. The resulting increase in blood glucose may raise levels outside the normal range and cause adverse health effects such as metabolic syndrome, dyslipidemia and subsequently type 2 diabetes mellitus. The term "insulin resistance- related complications" and "insulin resistance-related conditions" as used herein encompass, without limitation, metabolic syndrome, dyslipidemia and type 2 diabetes mellitus, as well as insulin resistance in endocrine diseases (e.g., obese subjects with type 1 diabetes mellitus, Cushing's disease and lipodystrophy syndromes).
[069] The term “isolated” or “enriched” encompasses a microbe (such as a bacterium) or other entity or substance that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, purified, and/or manufactured by the hand of man. In some embodiments, isolated microbes are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pine. As used herein, a substance is “pure” if it is substantially free of other components. The terms “purify,” “purifying” and “purified” refer to a microbe or other material that has been separated from at least some of the components with which it was associated either when initially produced or generated (e.g., whether in nature or in an experimental setting), or during any time after its initial production. A microbe or a microbial population may be considered purified if it is isolated at or after production, such as from a material or environment containing the microbe or microbial population, and a purified microbe or microbial population may contain other materials up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or above about 90% and still be considered “isolated.” In some embodiments, purified microbes or microbial population are more than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. In the instance of microbial compositions provided herein, the one or more microbial types present in the composition can be independently purified from one or more other microbes produced and/or present in the material or environment containing the microbial type. Microbial compositions are generally purified from residual habitat products.
[070] “Metabolic disease” as used herein refers a cluster of conditions that occur together, increasing the risk of heart disease, stroke and type 2 diabetes. These conditions include increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels. In some embodiments, the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglyceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
[071] “Metabolite” as used herein refers to any and all molecular compounds, compositions, molecules, ions, co-factors, catalysts or nutrients used as substrates in any cellular or microbial metabolic reaction or resulting as product compounds, compositions, molecules, ions, co-factors, catalysts or nutrients from any cellular or microbial metabolic reaction.
[072] “Microbe” refers to any natural or engineered organism characterized as a bacterium, fungus, microscopic alga, protozoan, and the stages of development or life cycle stages (e.g., vegetative, spore (including sporulation, dormancy, and germination), latent, biofilm) associated with the organism. Examples of gut microbes include: Actinomyces graevenitzii, Actinomyces odontofyticus, Akkermansia muciniphila, Bacteroides caccae, Bacteroides fragilis, Bacteroides putredinis, Bacteroides thetaiotaomicron, Bacteroides vultagus, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bilophila wadsworthia, Blautia, Butyrivibrio, Campylobacter gracilis, Clostridia cluster III, Clostridia cluster IV, Clostridia cluster IX (Acidaminococcaceae group), Clostridia cluster XI, Clostridia cluster XIII (Peptostreptococcus group), Clostridia cluster XTV, Clostridia cluster XV, Collinsella aerofaciens, Coprococcus, Corynebacterium sunsvallense, Desulfomonas pigra, Dorea formicigenerans, Dorea longicatena, Escherichia coli, Eubacterium hadrum, Eubacterium rectale, Faecalibacteria prausnitzii, Gemella, Lactococcus, Lanchnospira, Mollicutes cluster XVI, Mollicutes cluster XVIII, Prevotella, Rothia mucilaginosa, Ruminococcus callidus, Ruminococcus gnavus, Ruminococcus torques, and Streptococcus.
[073] “Microbial extracellular vesicles” (emEVs) may be naturally-produced vesicles derived from bacteria, such as smEVs. mEVs are comprised of bacterial lipids and/or bacterial proteins and/or bacterial nucleic acids and/or bacterial carbohydrate moieties, and are isolated from culture supernatant. The natural production of these vesicles can be artificially enhanced (for example, increased) or decreased through manipulation of the environment in which the bacterial cells are being cultured (for example, by media or temperature alterations). Further, mEV compositions may be modified to reduce, increase, add, or remove bacterial components or foreign substances to alter efficacy, immune stimulation, stability, immune stimulatory capacity, stability, organ targeting (for example, lymph node), absorption (for example, gastrointestinal), and/or yield (for example, thereby altering the efficacy). As used herein, the term “purified mEV composition” or “mEV composition” refers to a preparation of mEVs that have been separated from at least one associated substance found in a source material (for example, separated from at least one other bacterial component) or any material associated with the mEVs in any process used to produce the preparation. It can also refer to a composition that has been significantly enriched for specific components. Microbial extracellular vesicles may also be obtained from mammalian cells and from microbes such as archaea, fungi, microscopic algae, protozoans, and parasites. Microbial extracellular vesicles from any of these sources can be prepared into a solution and/or dried form as described herein. Microbial extracellular vesicles may be artificially-produced vesicles prepared from bacteria, such as pmEVs, for example, obtained by chemically disrupting (for example, by lysozyme and/or lysostaphin) and/or physically disrupting (for example, by mechanical force) bacterial cells and separating the bacterial membrane components from the intracellular components through centrifugation and/or ultracentrifugation, or other methods, and can also be prepared into a solution and/or dried form as described herein. [074] “Microbiome” broadly refers to the microbes residing on or in body site of a subject or patient. Microbes in a microbiome may include bacteria, viruses, eukaryotic microorganisms, and/or viruses. Individual microbes in a microbiome may be metabolically active, dormant, latent, or exist as spores, may exist planktonically or in biofilms, or may be present in the microbiome in sustainable or transient manner. The microbiome may be a commensal or healthy-state microbiome or a disease-state microbiome. The microbiome may be native to the subject or patient, or components of the microbiome may be modulated, introduced, or depleted due to changes in health state or treatment conditions (e.g., antibiotic treatment, exposine to different microbes). In some aspects, the microbiome occurs at a mucosal surface. In some aspects, the microbiome is a gut microbiome.
[075] A “microbiome profile” or a “microbiome signature” of a tissue or sample refers to an at least partial characterization of the bacterial makeup of a microbiome. In some embodiments, a microbiome profile indicates whether at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more bacterial strains are present or absent in a microbiome.
[076] “Modified” in reference to a bacteria broadly refers to a bacteria that has undergone a change from its wild-type form. Bacterial modification can result from engineering bacteria. Examples of bacterial modifications include genetic modification, gene expression modification, phenotype modification, formulation modification, chemical modification, and dose or concentration. Examples of improved properties are described throughout this specification and include, e.g., attenuation, auxotrophy, homing, or antigenicity. Phenotype modification might include, by way of example, bacteria growth in media that modify the phenotype of a bacterium such that its increases or decreases virulence.
[077] As used herein, a gene is “overexpressed” in a bacteria if it is expressed at a higher level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions. Similarly, a gene is “underexpressed” in a bacteria if it is expressed at a lower level in an engineered bacteria under at least some conditions than it is expressed by a wild-type bacteria of the same species under the same conditions.
[078] The terms “polynucleotide” and “nucleic acid” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A polynucleotide may comprise modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. A polynucleotide may be further modified, such as by conjugation with a labeling component. In all nucleic acid sequences provided herein, U nucleotides are interchangeable with T nucleotides.
[079] “Operational taxonomic units” and “OTU(s)” refer to a terminal leaf in a phylogenetic tree and is defined by a nucleic acid sequence, e.g., the entire genome, or a specific genetic sequence, and all sequences that share sequence identity to this nucleic acid sequence at the level of species. In some embodiments the specific genetic sequence may be the 16S sequence or a portion of the 16S sequence. In other embodiments, the entire genomes of two entities are sequenced and compared. In another embodiment, select regions such as multilocus sequence tags (MLST), specific genes, or sets of genes may be genetically compared. For 16S, OTUs that share > 97% average nucleotide identity across the entire 16S or some variable region of the 16S are considered the same OTU. See e.g. Claesson MJ, Wang Q, O’Sullivan O, Greene-Diniz R, Cole JR, Ross RP, and O’Toole PW. 2010. Comparison of two next-generation sequencing technologies for resolving highly complex microbiota composition using tandem variable 16S rRNA gene regions. Nucleic Acids Res 38: e200. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. For complete genomes, MLSTs, specific genes, other than 16S, or sets of genes OTUs that share > 95% average nucleotide identity are considered the same OTU. See e.g., Achtman M, and Wagner M. 2008. Microbial diversity and the genetic nature of microbial species. Nat. Rev. Microbiol. 6: 431-440. Konstantinidis KT, Ramette A, and Tiedje JM. 2006. The bacterial species definition in the genomic era. Philos Trans R Soc Lond B Biol Sci 361: 1929-1940. OTUs are frequently defined by comparing sequences between organisms. Generally, sequences with less than 95% sequence identity are not considered to form part of the same OTU. OTUs may also be characterized by any combination of nucleotide markers or genes, in particular highly conserved genes (e.g., “house-keeping” genes), or a combination thereof. Operational Taxonomic Units (OTUs) with taxonomic assignments made to, e.g., genus, species, and phylogenetic clade are provided herein. [080] As used herein, a substance is “pine” if it is substantially free of other components.
[081] “Strain” refers to a member of a bacterial species with a genetic signature such that it may be differentiated from closely-related members of the same bacterial species. The genetic signature may be the absence of all or part of at least one gene, the absence of all or part of at least on regulatory region (e.g., a promoter, a terminator, a riboswitch, a ribosome binding site), the absence (“curing”) of at least one native plasmid, the presence of at least one recombinant gene, the presence of at least one mutated gene, the presence of at least one foreign gene (a gene derived from another species), the presence at least one mutated regulatory region (e.g. , a promoter, a terminator, a riboswitch, a ribosome binding site), the presence of at least one non-native plasmid, the presence of at least one antibiotic resistance cassette, or a combination thereof. Genetic signatures between different strains may be identified by PCR amplification optionally followed by DNA sequencing of the genomic region(s) of interest or of the whole genome. In the case in which one strain (compared with another of the same species) has gained or lost antibiotic resistance or gained or lost a biosynthetic capability (such as an auxotrophic strain), strains may be differentiated by selection or counter-selection using an antibiotic or nutrient/metabolite, respectively.
[082] The terms “subject” or “patient” refers to any mammal. A subject or a patient described as “in need thereof’ refers to one in need of a treatment (or prevention) for a disease. Mammals (i.e., mammalian animals) include humans, laboratory animals (e.g., primates, rats, mice), livestock (e.g., cows, sheep, goats, pigs), and household pets (e.g., dogs, cats, rodents). The subject may be a human. The subject may be a non-human mammal including but not limited to of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee. The subject or patient may be healthy, or may be suffering from a metabolic disease at any developmental stage.
[083] As used herein, the term “treating” a disease in a subject or “treating” a subject having or suspected of having a disease refers to administering to the subject to a pharmaceutical treatment, e.g., the administration of one or more agents, such that at least one symptom of the disease is decreased or prevented from worsening. Thus, in one embodiment, “treating” refers inter alia to delaying progression, expediting remission, inducing remission, augmenting remission, speeding recovery, increasing efficacy of or decreasing resistance to alternative therapeutics, or a combination thereof.
[084] As used herein, the term “preventing” is art-recognized, and when used in relation to a condition, such as a local recurrence, is well understood in the art, and includes administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. Thus, prevention of cancer includes, for example, reducing the number of detectable tumors in a population of patients receiving a prophylactic treatment relative to an untreated control population, and/or delaying the appearance of detectable tumors in a treated population versus an untreated control population, e.g., by a statistically and/or clinically significant amount.
[085] As used herein, a value is “greater than” another value if it is higher by any amount (e.g., each of 100, 50, 20, 12, 11, 10.6, 10.1, 10.01, and 10.001 is at least 10). Similarly, as used herein, a value is ‘less than” another value if it is lower by any amount (e.g., each of 1, 2, 4, 6, 8, 9, 9.2, 9.4, 9.6, 9.8, 9.9, 9.99, 9.999 is no more than 10). In contrast, as used herein, a test value “is” an anchor value when the test value rounds to the anchor value (e.g., if “an ingredient mass is 10% of a total mass,” in which case 10% is the anchor value, the test values of 9.5, 9.6, 9.7, 9.8, 9.9, 10, 10.1, 10.2, 10.3, and 10.4 would also meet the “ingredient mass is 10% of the total mass” feature).
ChristenseneUaceae Bacteria
[086] ChristenseneUaceae are a family of anaerobic bacteria of the order Clostridiales. Multiple members of the ChristenseneUaceae, and multiple species of Christensenella, have been identified on the basis of homologous operational taxonomic unit (OTU) sequences in microbial phylogeny databases, such as Greengenes, a database of 16SRNA genetic sequences (DeSantis, T. Z., et al. Appl Environ Microbiol 72:5069-72 (2006)). [087] A bacterium can be defined as a member of the ChristenseneUaceae if ( 1 ) the 16S rRNA gene sequence of that bacterium shares at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity, preferably 97% or greater identity, with any 16S rRNA gene sequence in a database of 16S rRNA gene sequences, such as the Greengenes database that has the taxonomic classification of ChristenseneUaceae, or (2) a phylogenetic analysis reveals that the 16S rRNA gene sequence of that bacterium is most closely related to the 16S rRNA gene sequence of a member of the ChristenseneUaceae, such that this sequence and a sequence from a member of the Christensenellaceae share a common ancestor sequence that is unique to the family Christensenellaceae (and members of other families do not share that ancestor). Phylogenetic analysis approaches that are acceptable as general practice in the field of microbiology for 16S rRNA gene phylogenetics include: (a) a multiple sequence alignment of the 16S rRNA gene sequences (members of the Christensenellaceae-+the novel sequence+non- Christensenellaceae) using secondary' structure information, followed by (b) use of either the General-Time reversible model of evolution in a Maximum Likelihood (ML) analysis with bootstrapping (Harris, J. K., et al., Appl. Environ. Microbiol. 70:845-849 (2004)), or a Bayesian phylogenetics analysis (Huelsenbeck, J. P. and F. Ronquist, Bioinformatics 17:754-755 (2001). Ronquist, F. and. P. Huelsenbeck, Bioinformatics 19:1572-1574 (2003)). These tools are commonly available and implemented in many different standard sequence analysis software packages. Visualization of resulting phylogenetic trees can be performed software available in the art for phylogenetic tree visualization. For inclusion in the family Christensenellaceae, the bootstrap support in an ML analysis is 70%, or a 70% probability for a Bayesian analysis.
[088] Christensenella is a recently identified genus of the Christensenellaceae. The first isolated species of Christensenella was Christensenella minute, which was isolated from a human fecal sample (Morotomi, et al ., Int. J. Syst. Evol. Microbiol. 62:144-149 (2012). C. minuta has a 16S ribosomal RNA gene sequence of SEQ ID NO: 1. The C. minuta 16S rRNA sequence is identified by Genbank Accession No. AB490809.
[089] Bacteria belonging to Christensenellaceae and Christensenella can be identified by amplifying the 16S rRNA gene from a sample. PCR primers that amplify universally conserved regions of the 16S rRNA gene are known in the art. For example, using the known 16S rRNA gene primers 515F (SEQ ID NO: 3) and 806R (SEQ ID NO: 4) to amplify the 16S rRNA gene sequence from Christensenella minuta amplifies a portion of SEQ ID NO: 1 disclosed herein as SEQ ID NO: 2. A bacterium belonging to the Christensenella genus is identified as having a 16S RNA gene sequence with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Additional 16S sequences for bacteria within the genus of Christensenella are provided as SEQ ID NOS: 5-8 (OTU numbers 701845, 177179,1146771, and 361793 in the Greengenes database, respectively). The identity' of each sequence relative to SEQ ID NO: 1 is as follows: SEQ ID NO: 5, 93% identity to SEQ ID NO: 1 ; SEQ ID NO: 6, 95% identity to SEQ ID NO: 1; SEQ ID NO: 7, 97% identity to SEQ ID NO: 1; SEQ ID NO: 8, 95% identity to SEQ ID NO: 1.
Table 1: Christensenella mtnuta Sequences
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
Figure imgf000027_0001
Con itions of Purified Christensenellaceae and Christensenella Bacteria and/or mEVs
[090] Disclosed herein are compositions of substantially purified bacteria of the family
Christensenellaceae and the genus Christensenella.
[091] In one embodiment, the Christensenellaceae are bacteria of the genus
Christensenella. In a further embodiment, the Christensenella bacteria are selected from
Christensenella minuta, one or more Christensenella species with the 16S sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof. In a specific example, the bacteria are C. minuta bacteria.
[092] In one example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, or 99% of Christensenellaceae or Christensenella bacteria in the disclosed composition are non-living (e.g., not viable). In some embodiments, all of the
Christensenella in the administered composition are non-living. A ‘Viable” or ‘living” bacterium is capable of forming a colony, for example, under conditions suitable for growth of said bacterium. A viable or living bacterium can also be referred to as a colony forming unit (CFU).
[093] In one example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,
98%, or 99% of Christensenellaceae or Christensenella bacteria in the disclosed composition are attenuated bacteria. In some embodiments, all of the Christensenella in the administered composition are attenuated bacteria.
[094] Christensenellaceae and Christensenella can be cultured under anaerobic conditions using, for example, the methods disclosed in Morotomi, et al. (Int. J. Syst. Evol. Microbiol. 62:144-149 (2012)). For example, cultures may be inoculated into tubes or plates for growth in anaerobic conditions at 32-42°C, preferably 37°C, with a culture medium of modified Gifu anaerobic medium (GAM broth; Nissui Pharmaceutical) containing l%-2%, preferably 1.5% (w/v) agar supplemented with 2-8% bile (Bacto oxgall; Difco) and NaCl. As another example, Christensenella can be cultured in brain heart infusion broth supplemented with yeast (2-8 g/1, preferably 5 g/1), menadione (0.5- 2.0 mg/1, preferably 1.0 g/1), hemin (5-15 mg/1, preferably 10 mg/1), and L-cysteine-HCL (0.2-0.8 g/1, preferably 0.5 g/1) at 32-42°C., preferably 37°C, under anaerobic conditions. [095] In another example, the composition includes lyophilized Christensenellaceae or Christensenella bacteria. Lyophilized compositions of the inventions contain at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% not viable Christensenellaceae or Christensenella bacteria.
[096] In an example, the composition contains less than 10* CFU of bacteria (e.g., less than 108 CFU of bacteria/g,) and more particularly less than 106 CFU of bacteria (e.g., less than 106 CFU of bacteria/g) of Christensenellaceae or Christensenella bacteria. In a further example, the composition contains less than 10* CFU of bacteria/g of support, and more particularly less than 106 CFU of bacteria/g of support of Christensenellaceae or Christensenella bacteria. By support is meant the food product or the pharmaceutically acceptable excipient.
[097] Throughout this application, the terms “about” and “approximately” indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects. In one nonlimiting embodiment the terms are defined to be within 10%, preferably within 5%, more preferably within 1%, and most preferably within 0.5%.
[098] Bacteria and strains of the family Christensenellaceae, and bacteria and strains of the genus Christensenella, are probiotic. Generally, by a probiotic bacterium or strain itis meant a non-pathogenic microorganism which, when ingested live, exercises a beneficial effect on the host's health. These probiotic strains generally have the ability to survive the passage through the upper part of the digestive tract. Without being limited, it is thought that these bacteria can exercise their beneficial effect on health on the one hand via ecological interactions with the resident flora in the digestive tract (i.e., the “gut microbiome”), and on the other hand via their ability to influence various aspects of the host physiology. These bacteria, when given in a sufficient number, have the ability to progress live through the intestine, however they do not cross the intestinal barrier and their primary effects are therefore induced in the lumen and/or the wall of the gastrointestinal tract. They then form part of the resident flora during the administration period and ideally on an extended basis subsequent to administration. This colonization (or transient colonization) allows the probiotic bacteria to exercise a beneficial effect both through interactions with the gut microbiome and on the host itself.
[099] Disclosed herein are compositions of substantially purified mEVs from bacteria of the family Christensenellaceae and the genus Christensenella. In certain aspects, provided herein are compositions that comprise mEVs (such as smEVs and/or pmEVs) from Christensenellaceae bacteria.
[100] In one embodiment, the Christensenellaceae mEVs are mEVs of the genus Christensenella. In a further embodiment, the mEVs are from Christensenella bacteria selected from Christensenella minuta, one or more Christensenella species with the 16S sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof. In a specific example, the mEVs are from C. minuta bacteria.
[101] In another example, the composition includes lyophilized mEVs from Christensenellaceae or Christensenella bacteria.
[102] Compositions disclosed herein (e.g., of bacteria and/or mEVs) can include substantially purified Christensenellaceae or Christensenella bacteria and one or more acceptable excipients. Examples of acceptable excipients include: sugars such as sucrose, isomerized sugar, glucose, fructose, palatinose, trehalose, lactose and xylose; microcrystalline cellulose and other celluloses, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate, glycine, starch, milk sugar and high molecular weight polyethylene glycols; emulsifiers such as sucrose esters of fatty acids, glycerin esters of fatty acids and lecithin; thickeners (stabilizers) such as carrageenan, xanthan gum, guar gum, pectin and locust bean gum; acidifiers such as citric acid, lactic acid and malic acid; fruit juices such as lemon juice, orange juice and berry juice; vitamins such as vitamin A, vitamin B, vitamin C, vitamin D and vitamin E; and minerals such as calcium, iron, manganese and zinc. [103] Compositions of the invention (e.g., compositions comprising bacteria and/or mEVs) may be prepared by admixture, usually adapted for oral administration. Such compositions may be in the form of tablets, capsules, oral liquid preparations, conventional food products, powders, granules, lozenges, reconstitutable powders or suspensions.
[104] Tablets and capsules for oral administration can also contain one or more of: excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine; disintegrants such as starch (preferably com, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates; granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia; and lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc.
[105] Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be in the form of a dryproduct for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), preservatives, and if desired, conventional flavorings or colorants.
[106] In certain aspects, provided herein are compositions comprising Christensenellaceae bacteria and/or mEVs, and methods of using a composition comprising Christensenellaceae bacteria and/or mEVs derived from Christensenellaceae bacteria. In some embodiments, the Christensenellaceae bacteria are bacteria of genus Christensenella. In some embodiments, the Christensenellaceae bacteria of genus Christensenella are Christensenella minuta bacteria.
[107] In some embodiments, the bacteria is a strain comprising at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the nucleotide sequence (e.g., genomic, 16S or CRISPR nucleotide sequence) of the bacterial strains listed in Table 1.
[108] Applicant represents that the ATCC is a depository affording permanence of the deposit and ready accessibility thereto by the public if a patent is granted. All restrictions on the availability to the public of the material so deposited will be irrevocably removed upon the granting of a patent. The material will be available during the pendency of the patent application to one determined by the Commissioner to be entitled thereto under 37 CFR 1.14 and 35 U.S.C. § 122. The deposited material will be maintained with all the care necessary to keep it viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposited plasmid, and in any case, for a period of at least thirty (30) years after the date of deposit or for the enforceable life of the patent, whichever period is longer. Applicant acknowledges its duty to replace the deposit should the depository be unable to furnish a sample when requested due to the condition of the deposit.
[109] In some embodiments, the bacteria described herein are modified to enhance their immunomodulatory and/or therapeutic effect (e.g., either alone or in combination with another therapeutic agent). In some embodiments, the bacteria described herein are modified to enhance immune activation (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins, vesicles). In some embodiments, the bacteria described herein are modified to improve bacterial manufacturing (e.g., higher oxygen tolerance, improved freeze-thaw tolerance, shorter generation times).
[110] The Christensenellaceae bacteria (e.g., Christensenella minuta) can be cultured according to methods known in the art.
[111] In some embodiments, administration of the disclosed compositions increases the levels of Christensenella, relative to the levels of other bacteria, in the gastrointestinal tract of a subject.
[112] In some embodiments, the Christensenellaceae is resistant to polymyxin.
[113] In some embodiments, the Christensenellaceae bacteria are modified to reduce toxicity or other adverse effects, to enhance delivery) (e.g., oral delivery) of the mEVs (such as smEVs and/or pmEVs), bacteria for compositions, or any combination thereof, (e.g., by improving acid resistance, muco-adherence and/or penetration and/or resistance to bile acids, digestive enzymes, resistance to anti-microbial peptides and/or antibody neutralization), to target desired cell types (e.g., M-cells, goblet cells, enterocytes, dendritic cells, macrophages), to enhance their immunomodulatory and/or therapeutic effect of the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof (e.g., either alone or in combination with another therapeutic agent), and/or to enhance immune activation or suppression by the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof (e.g., through modified production of polysaccharides, pili, fimbriae, adhesins). In some embodiments, the engineered Christensenellaceae bacteria described herein are modified to improve manufacturing mEVs (such as smEVs and/or pmEVs), bacteria for compositions, or any combination thereof (e.g., higher oxygen tolerance, stability, improved freeze-thaw tolerance, shorter generation times). For example, in some embodiments, the engineered Christensenellaceae bacteria described include bacteria harboring one or more genetic changes, such change being an insertion, deletion, translocation, or substitution, or any combination thereof, of one or more nucleotides contained on the bacterial chromosome or endogenous plasmid and/or one or more foreign plasmids, wherein the genetic change may results in the overexpression and/or underexpression of one or more genes. The engineered Christensenellaceae bacteria may be produced using any technique known in the art, including but not limited to site-directed mutagenesis, transposon mutagenesis, knock-outs, knock-ins, polymerase chain reaction mutagenesis, chemical mutagenesis, ultraviolet tight mutagenesis, transformation (chemically or by electroporation), phage transduction, directed evolution, or any combination thereof.
[114] Disclosed herein are compositions of substantially purified bacteria and mEVs of the family Christensenellaceae and/or the genus Christensenella. In certain aspects, provided herein are compositions that comprise bacteria and mEVs (such as smEVs and/or pmEVs) fiom Christensenellaceae bacteria.
[115] In some embodiments, the composition comprises bacteria of only one strain of bacteria. In some embodiments, the composition comprises bacteria of only one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises mEVs fiom only one strain of bacteria. In some embodiments, the composition comprises mEVs fiom only one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises bacteria and mEVs fiom only one strain of bacteria, e.g., one strain of Christensenellaceae bacteria.
[116] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Christensenellaceae bacteria. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the bacteria in the composition are of the Christensenellaceae bacteria. In some embodiments, at least 99% of the bacteria in the composition are of the Christensenellaceae bacteria. In some embodiments, the bacteria in the composition are essentially (e.g., about 100%) of the Christensenellaceae bacteria.
[117] In some embodiments, at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the mEVs in the composition are of the Christensenellaceae bacteria. 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% of the mEVs in the composition are of the Christensenellaceae bacteria. In some embodiments, at least 99% of the mEVs in the composition are of the Christensenellaceae bacteria. In some embodiments, the mEVs in the composition are essentially (e.g., about 100%) of the Christensenellaceae bacteria.
[118] In some embodiments, the composition comprises bacteria from more than one strain of bacteria. In some embodiments, the composition comprises bacteria from more than one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises mEVs from more than one strain of bacteria. In some embodiments, the composition comprises mEVs from more than one strain of Christensenellaceae bacteria. In some embodiments, the composition comprises bacteria and mEVs from more than one strain of bacteria. In some embodiments, the composition comprises bacteria and mEVs from more than one strain of Christensenellaceae bacteria.
[119] In some embodiments, the composition comprises only one strain of bacteria, e.g. , Christensenellaceae bacteria.
[120] In some embodiments, the composition comprises more than one strain of bacteria, e.g., Christensenellaceae bacteria, and the therapeutic effect caused by the composition is due to the presence of the Christensenellaceae bacteria present in the composition.
[121] In some embodiments, the composition comprises mEVs from only one strain of bacteria, e.g., Christensenellaceae bacteria.
[122] In some embodiments, the composition comprises mEVs from more than one strain of bacteria, e.g., Christensenellaceae bacteria, and the therapeutic effect caused by the composition is due to the presence of the mEVs from Christensenellaceae bacteria present in the composition.
[123] In some embodiments, the Christensenellaceae bacterial strain is a bacterial strain having a genome that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% sequence identity to a strain listed in Table 1.
[124] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, described herein are obtained from a strain of Christensenellaceae bacteria comprising a 16S sequence that is at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity (e.g., at least 99.5% sequence identity, at least 99.6% sequence identity, at least 99.7% sequence identity, at least 99.8% sequence identity, at least 99.9% sequence identity) to the 16S sequence as provided in Table 1.
[125] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are lyophilized.
[126] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are gamma irradiated (e.g., at 17.5 or 25 kGy).
[127] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are UV irradiated.
[128] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours).
[129] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are acid treated.
[130] In some embodiments, the mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, of the compositions described herein are oxygen sparged (e.g., at 0.1 wm for two hours).
[131] The phase of growth can affect the amount or properties of bacteria and/or mEVs (such as smEVs and/or pmEVs) produced by bacteria. For example, in the methods of bacteria preparation provided herein, bacteria can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached. As another example, in the methods of preparing mEVs (such as smEVs and/or pmEVs) provided herein, mEVs (such as smEVs and/or pmEVs) can be prepared from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached. [132] In some embodiments, Christensenellaceae bacteria are lyophilized. In some embodiments, Christensenellaceae bacteria are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, Christensenellaceae bacteria are UV irradiated. In some embodiments, Christensenellaceae bacteria are heat inactivated (e.g., at 50°C for two hours or at 90°C for two hours). In some embodiments, Christensenellaceae bacteria are acid treated. In some embodiments, Christensenellaceae bacteria are oxygen sparged (e.g., at 0.1 vvm for two hours).
[133] In some embodiments, the mEVs are lyophilized. In some embodiments, the mEVs are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, the mEVs are UV irradiated. In some embodiments, the mEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns). In some embodiments, the mEVs are acid treated. In some embodiments, the mEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
[134] The phase of growth can affect the amount or properties of Christensenellaceae bacteria and/or smEVs produced by Christensenellaceae bacteria. For example, in the methods of smEVs preparation provided herein, smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached. As another example, in the methods of pmEV preparation provided herein, pmEVs can be prepared from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
[135] In some embodiments, the Christensenellaceae bacteria and/or smEVs produced by Christensenellaceae bacteria are grown in growth media comprising yeast extract (e.g., Yeast Extract 19512), soy peptone (e.g., Soy Peptone El 10 19885, Soy Peptone A3 SC 19685), potato peptone (e.g., Potato Peptone E210 19425), tri-sodium citrate, K2HPO4, KH2PO4, magnesium chloride, manganese chloride, L-cysteine-HCl, FeSO4, NH4CI, Xylose, and vitamin B12.
[136] In some embodiments, the yeast extract (e.g., Yeast Extract 19512) is prepared in the growth media at 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[137] In some embodiments, the soy peptone (e.g., Soy Peptone El 10 19885, Soy Peptone A3 SC 19685) is prepared at 0.001 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[138] In some embodiments, the tri-sodium citrate is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[139] In some embodiments, the K2HPO4 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[140] In some embodiments, the KH2PO4 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L,
19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[141] In some embodiments, the magnesium chloride is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[142] In some embodiments, the manganese chloride is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[143] In some embodiments, the L-cysteine-HCl is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, ll g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[144] In some embodiments, the FeSCh is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L,
20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[145] In some embodiments, the NH4CI is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[146] In some embodiments, the Xylose is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, 10 g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[147] In some embodiments, the vitamin B 12 is prepared in the growth media at 1 g/L, 0.005 g/L, 0.01 g/L, 0.05 g/L, 0.1 g/L, 0.5 g/L, 1 g/L, 2 g/L, 3 g/L, 4 g/L, 5 g/L, 6 g/L, 7 g/L, 8 g/L, 9 g/L, lO g/L, 11 g/L, 12 g/L, 13 g/L, 14 g/L, 15 g/L, 16 g/L, 17 g/L, 18 g/L, 19 g/L, 20 g/L, 21 g/L, 22 g/L, 23 g/L, 24 g/L, or 25 g/L.
[148] Enriched growth media is used to grow and prepare the bacterium for in vitro and in vivo use. For example, media may contain sugar, yeast extracts, plant based peptones, buffers, salts, trace elements, surfactants, anti-foaming agents, and vitamins. Composition of complex components such as yeast extracts and peptones may be undefined or partially defined (including approximate concentrations of amino acids, sugars etc.). Microbial metabolism may be dependent on the availability of resources such as carbon and nitrogen. Various sugars or other carbon sources may be tested. Alternatively, media may be prepared and the selected bacterium grown as shown by Saarela et al., J. Applied Microbiology. 2005. 99: 1330-1339, which is hereby incorporated by reference. Influence of fermentation time, cryoprotectant and neutralization of cell concentrate on freeze- drying survival, storage stability, and acid and bile exposine of the selected bacterium produced without milk-based ingredients.
[149] In some embodiments, the growth media is sterilized. Sterilization may be by Ultra High Temperature (UHT) processing. The UHT processing is performed at very high temperature for short periods of time. The UHT range may be from 135-180°C. For example, the medium may be sterilized from between 10 to 30 seconds at 135°C.
[150] In some embodiments, inoculum can be prepared in flasks or in smaller bioreactors and growth is monitored. For example, the inoculum size may be between approximately 0.5 and 3% of the total bioreactor volume. Depending on the application and need for material, bioreactor volume can be at least 2 L, 10 L, 80 L, 100 L, 250 L, 1000 L, 2500 L, 5000 L, 10,000 L.
[151] In some embodiments, before inoculation, the bioreactor is prepared with medium at desired pH, temperature, and oxygen concentration. The initial pH of the culture medium may be different that the process set-point. pH stress may be detrimental at low cell centration; the initial pH could be between pH 7.5 and the process set-point. For example, pH may be set between 4.5 and 8.0. During the fermentation, the pH can be controlled through the use of sodium hydroxide, potassium hydroxide, or ammonium hydroxide. The temperature may be controlled from 25°C to 45°C, for example at 37°C. Anaerobic conditions are created by reducing the level of oxygen in the culture broth from around 8 mg/L to 0 mg/L. For example, nitrogen or gas mixtures (Ni, CO2, and Hi) may be used in order to establish anaerobic conditions. Alternatively, no gases are used and anaerobic conditions are established by cells consuming remaining oxygen from the medium. Depending on strain and inoculum size, the bioreactor fermentation time can vary. For example, fermentation time can vary from approximately 5 hours to 48 hours.
[152] For example, a frozen vial is diluted to 0.1% in a IL media at 37°C for 12-16 hours. The media is PM11 with 1 g/1 L-sodium lactate (no FeSO4, no NH4C1, no malate). The IL media is diluted to 1% in a 15 L bioreactor at 37°C, 150 rpm, gas of 5% CO2 and 95% Ni, uncontrolled pH for 16-18 hours. The feed is 10X YEP, 33 g/1 L-sodium lactate (no G2) (Constant feed: 11 mL/Lh). It is then centrifuged at 10,000 x g, 10°C, for 10 minutes to collect 90 g pellet/15 L. Then placed in a new stabilizer: sucrose-dextran- cysteine 0.18 g stab/g pellet.
[153] Reviving microbes from a frozen state may require special considerations. Production medium may stress cells after a thaw; a specific thaw medium may be required to consistently start a seed train from thawed material. The kinetics of transfer or passage of seed material to fresh medium, for the pinposes of increasing the seed volume or maintaining the microbial growth state, may be influenced by the current state of the microbes (ex. exponential growth, stationary growth, unstressed, stressed).
[154] Inoculation of the production fermenter(s) can impact growth kinetics and cellular activity. The initial state of the bioreactor system must be optimized to facilitate successfill and consistent production. The fraction of seed culture to total medium (e.g. a percentage) has a dramatic impact on growth kinetics. The range may be 1-5% of the fermenter’s working volume. The initial pH of the culture medium may be different from the process set-point. pH stress may be detrimental at low cell concentration; the initial pH may be between pH 7.5 and the process set-point. Agitation and gas flow into the system during inoculation may be different from the process set-points. Physical and chemical stresses due to both conditions may be detrimental at low cell concentration. [155] Process conditions and control settings may influence the kinetics of microbial growth and cellular activity. Shifts in process conditions may change membrane composition, production of metabolites, growth rate, cellular stress, etc. Optimal temperature range for growth may vary with strain. The range may be 20-40 °C. Optimal pH for cell growth and performance of downstream activity may vary with strain. The range may be pH 5-8. Gasses dissolved in the medium may be used by cells for metabolism. Adjusting concentrations of O2, CO2, and N2 throughout the process may be required. Availability of nutrients may shift cellular growth. Microbes may have alternate kinetics when excess nutrients are available.
[156] The state of microbes at the end of a fermentation and during harvesting may impact cell survival and activity. Microbes may be preconditioned shortly before harvest to better prepare them for the physical and chemical stresses involved in separation and downstream processing. A change in temperature (often reducing to 20-5°C) may reduce cellular metabolism, slowing growth (and/or death) and physiological change when removed from the fermenter. Effectiveness of centrifugal concentration may be influenced by culture pH. Raising pH by 1-2 points can improve effectiveness of concentration but can also be detrimental to cells. Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization dining downstream.
[157] Separation methods and technology may impact how efficiently microbes are separated from the culture medium. Solids may be removed using centrifugation techniques. Effectiveness of centrifugal concentration can be influenced by culture pH or by the use of flocculating agents. Raising pH by 1-2 points may improve effectiveness of concentration but can also be detrimental to cells. Microbes may be stressed shortly before harvest by increasing the concentration of salts and/or sugars in the medium. Cells stressed in this way may better survive freezing and lyophilization during downstream. Additionally, Microbes may also be separated via filtration. Filtration is superior to centrifugation techniques for purification if the cells require excessive g-minutes to successfully centrifuge. Excipients can be added before after separation. Excipients can be added for cryo protection or for protection during lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants. Prior to lyophilization, droplets of cell pellets mixed with excipients are submerged in Equid nitrogen. [158] Harvesting can be performed by continuous centrifugation. Product may be resuspended with various excipients to a desired final concentration. Excipients can be added for cryo protection or for protection dining lyophilization. Excipients can include, but are not limited to, sucrose, trehalose, or lactose, and these may be alternatively mixed with buffer and anti-oxidants. Prior to lyophilization, droplets of cell pellets mixed with excipients are submerged in liquid nitrogen.
[159] Lyophilization of material, including live bacteria, begins with primary drying. During the primary drying phase, the ice is removed. Here, a vacuum is generated and an appropriate amount of heat is supplied to the material for the ice to sublime. During the secondary drying phase, product bound water molecules are removed. Here, the temperature is raised higher than in the primary drying phase to break any physicochemical interactions that have formed between the water molecules and the product material. The pressure may also be lowered further to enhance desorption during this stage. After the freeze-drying process is complete, the chamber may be filled with an inert gas, such as nitrogen. The product may be sealed within the freeze dryer under dry conditions, preventing exposure to atmospheric water and contaminants.
Modified mEVs
[160] In some aspects, the mEVs (such as smEVs and/or pmEVs) described herein are modified such that they comprise, are linked to, and/or are bound by a therapeutic moiety.
[161] In some embodiments, the mEVs described herein are modified such that they comprise, are linked to, and/or are bound by a magnetic and/or paramagnetic moiety (e.g., a magnetic bead). In some embodiments, the magnetic and/or paramagnetic moiety is comprised by and/or directly linked to the bacteria. In some embodiments, the magnetic and/or paramagnetic moiety is linked to and/or a part of an mEV-binding moiety that that binds to the mEV. In some embodiments, the mEV-binding moiety is a fragment of or a full-length peptidoglycan recognition protein, such as PGRP. In some embodiments the mEV-binding moiety has binding specificity for the mEV (e.g., by having binding specificity for a bacterial antigen). In some embodiments, the mEV-binding moiety comprises an antibody or antigen binding fiagment thereof. In some embodiments, the mEV-binding moiety comprises a T cell receptor or a chimeric antigen receptor (CAR). In some embodiments, the mEV-binding moiety comprises a ligand for a receptor expressed on the surface of a cancer cell or a receptor-binding fiagment thereof. In certain embodiments, co-administration of the magnetic and/or paramagnetic moiety with the mEVs (either together or in separate administrations) can be used to increase the targeting of the mEVs (e.g., to cancer cells and/or a part of a subject where cancer cells are present. production of Processed Microbial Extracellular Vesicles (pmEVs)
[162] In certain aspects, the pmEVs described herein can be prepared using any method known in the art.
[163] In some embodiments, the pmEVs are prepared without a pmEV purification step. For example, in some embodiments, Christensenellaceae bacteria ftom which the pmEVs described herein are released are killed using a method that leaves Christensenellaceae bacterial pmEVs intact, and the resulting Christensenellaceae bacterial components, including the pmEVs, are used in the methods and compositions described herein. In some embodiments, the Christensenellaceae bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the Christensenellaceae bacteria are killed using UV irradiation.
[164] In some embodiments, the pmEVs described herein are purified ftom one or more other Christensenellaceae bacterial components. Methods for purifying pmEVs ftom Christensenellaceae bacteria (and optionally, other bacterial components) are known in the art. In some embodiments, pmEVs are prepared ftom Christensenellaceae bacterial cultures using methods described in Thein, et al. (J. Proteome Res. 9(12):6135-6147 (2010)) or Sandrini, et al. (Bio-protocol 4(21): el287 (2014)), each of which is hereby incorporated by reference in its entirety. In some embodiments, the bacteria are cultured to high optical density and then centrifuged to pellet bacteria (e.g., at 10,000- 15,000 x g for 10- 15 min at room temperature or 4°C). In some embodiments, the supernatants are discarded and cell pellets are frozen at -80°C. In some embodiments, cell pellets are thawed on ice and resuspended in 100 mM Tris-HCl, pH 7.5 supplemented with 1 mg/mL DNase I. In some embodiments, cells are lysed using an Emulsiflex C-3 (A vestin, Inc.) under conditions recommended by the manufacturer. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min at 4°C. In some embodiments, supernatants are then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in ice-cold 100 mM sodium carbonate, pH 11, incubated with agitation for 1 hr at 4°C, and then centrifuged at 120,000 x g for 1 hour at 4°C. In some embodiments, pellets are resuspended in 100 mM Tris-HCl, pH 7.5, re centrifuged at 120,000 x g for 20 min at 4°C, and then resuspended in 0.1 M Tris-HCl, pH 7.5 or in PBS. In some embodiments, samples are stored at -20°C.
[165] In certain aspects, pmEVs are obtained by methods adapted from Sandrini et al, 2014. In some embodiments, ChristenseneUaceae bacterial cultures are centrifuged at 10,000-15,500 x g for 10-15 min at room temp or at 4°C. In some embodiments, cell pellets are frozen at -80°C and supernatants are discarded. In some embodiments, cell pellets are thawed on ice and resuspended in 10 mM Tris-HCl, pH 8.0, 1 mM EDTA supplemented with 0.1 mg/mL lysozyme. In some embodiments, samples are incubated with mixing at room temp or at 37°C for 30 min. In some embodiments, samples are refrozen at -80°C and thawed again on ice. In some embodiments, DNase I is added to a final concentration of 1.6 mg/mL and MgC12 to a final concentration of 100 mM. In some embodiments, samples are sonicated using a QSonica Q500 sonicator with 7 cycles of 30 sec on and 30 sec off. In some embodiments, debris and unlysed cells are pelleted by centrifugation at 10,000 x g for 15 min. at 4°C. In some embodiments, supernatants are then centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in 10 mM Tris-HCl, pH 8.0, 2% Triton X-100 and incubated 30-60 min with mixing at room temperature. In some embodiments, samples are centrifuged at 110,000 x g for 15 min at 4°C. In some embodiments, pellets are resuspended in PBS and stored at - 20°C.
[166] In certain aspects, a method of forming (e.g., preparing) isolated ChristenseneUaceae bacterial pmEVs, described herein, comprises the steps of: (a) centrifuging a ChristenseneUaceae bacterial culture, thereby forming a first pellet and a first supernatant, wherein the first pellet comprises cells; (b) discarding the first supematant;(c) resuspending the first pellet in a solution; (d) lysing the cells; (e) centrifuging the lysed cells, thereby forming a second pellet and a second supernatant; (f) discarding the second pellet and centrifuging the second supernatant, thereby forming a third pellet and a third supernatant; (g) discarding the third supernatant and resuspending the third pellet in a second solution, thereby forming the isolated ChristenseneUaceae bacterial pmEVs.
[167] In some embodiments, the method further comprises the steps of: (h) centrifuging the solution of step (g), thereby forming a fourth pellet and a fourth supernatant; (i) discarding the fourth supernatant and resuspending the fourth pellet in a third solution. In some embodiments, the method further comprises the steps of: (j) centrifuging the solution of step (i), thereby forming a fifth pellet and a fifth supernatant; and (k) discarding the fifth supernatant and resuspending the fifth pellet in a fourth solution.
[168] In some embodiments, the centrifugation of step (a) is at 10,000 x g. In some embodiments the centrifugation of step (a) is for 10-15 minutes. In some embodiments, the centrifugation of step (a) is at 4°C or room temperature. In some embodiments, step (b) further comprises freezing the first pellet at -80 °C . In some embodiments, the solution in step (c) is 100 mM Tris-HCl, pH 7.5 supplemented with Img/ml DNasel. In some embodiments, the solution in step (c) is 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, supplemented with 0.1 mg/ml lysozyme. In some embodiments, step (c) further comprises incubating for 30 minutes at 37°C or room temperature. In some embodiments, step (c) further comprises freezing the first pellet at -80°C . In some embodiments, step (c) further comprises adding DNasel to a final concentration of 1.6 mg/ml. In some embodiments, step (c) further comprises adding MgChto a final concentration of 100 mM. In some embodiments, the cells are lysed in step (d) via homogenization. In some embodiments, the cells are lysed in step (d) via emulsiflex C3. In some embodiments, the cells are lysed in step (d) via sonication. In some embodiments, the cells are sonicated in 7 cycles, wherein each cycle comprises 30 seconds of sonication and 30 seconds without sonication. In some embodiments, the centrifugation of step (e) is at 10,000 x g. In some embodiments, the centrifugation of step (e) is for 15 minutes. In some embodiments, the centrifugation of step (e) is at 4 °C or room temperature.
[169] In some embodiments, the centrifugation of step (f) is at 120,000 x g. In some embodiments, the centrifugation of step (f) is at 110,000 x g. In some embodiments, the centrifugation of step (f) is for 1 hour. In some embodiments, the centrifugation of step (f) is for 15 minutes. In some embodiments, the centrifugation of step (f) is at 4°C or room temperature. In some embodiments, the second solution in step (g) is 100 mM sodium carbonate, pH 11. In some embodiments, the second solution in step (g) is 10 mM Tris- HCl pH 8.0, 2% triton X-100. In some embodiments, step (g) further comprises incubating the solution for 1 hour at 4°C. In some embodiments, step (g) further comprises incubating the solution for 30-60 minutes at room temperature. In some embodiments, the centrifugation of step (h) is at 120,000 x g. In some embodiments, the centrifugation of step (h) is at 110,000 x g. In some embodiments, the centrifugation of step (h) is for 1 hour. In some embodiments, the centrifugation of step (h) is for 15 minutes. In some embodiments, the centrifugation of step (h) is at 4°C or room temperature. In some embodiments, the third solution in step (i) is 100 mM Tris-HCl, pH 7.5. In some embodiments, the third solution in step (i) is PBS. In some embodiments, the centrifugation of step (j) is at 120,000 x g. In some embodiments, the centrifugation of step (j) is for 20 minutes. In some embodiments, the centrifugation of step (j) is at 4°C or room temperature. In some embodiments, the fourth solution in step (k) is 100 mM Tris- HCl, pH 7.5 or PBS.
[170] pmEVs obtained by methods provided herein may be further purified by size based column chromatography, by affinity chromatography, and by gradient ultracentrifugation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfide precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 horns at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfide precipitation or ultracentrifugation were used to concentrate the filtered supernatants, pellets are resuspended in 35% Optiprep in PBS. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3- 24 hours at 4°C.
[171] In some embodiments, to confirm sterility and isolation of the pmEV preparations, pmEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated pmEVs may be DNase or proteinase K treated.
[172] In some embodiments, the sterility of the pmEV preparations can be confirmed by plating a portion of the pmEVs onto agar medium used for standard culture of the bacteria used in the generation of the pmEVs and incubating using standard conditions.
[173] In some embodiments select pmEVs are isolated and enriched by chromatography and binding surface moieties on pmEVs. In other embodiments, select pmEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
[174] The pmEVs can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
[175] In some embodiments, pmEVs are lyophilized. In some embodiments, pmEVs are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, pmEVs are UV irradiated. In some embodiments, pmEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns). In some embodiments, pmEVs are acid treated. In some embodiments, pmEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
[176] The phase of growth can affect the amount or properties of bacteria. In the methods of pmEV preparation provided herein, pmEVs can be isolated , e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
Production of Secreted Microbial Extracellular Vesicles (smEVs)
[177] In certain aspects, the smEVs described herein can be prepared using any method known in the art.
[178] In some embodiments, the smEVs are prepared without an smEV purification step. For example, in some embodiments, bacteria described herein are killed using a method that leaves the smEVs intact and the resulting Christensenellaceae bacterial components, including the smEVs, are used in the methods and compositions described herein. In some embodiments, the Christensenellaceae bacteria are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the Christensenellaceae bacteria are killed using UV irradiation. In some embodiments, the Christensenellaceae bacteria are heat-killed.
[179] In some embodiments, the smEVs described herein are purified from one or more other Christensenellaceae bacterial components. Methods for purifying smEVs from bacteria are known in the art. In some embodiments, smEVs are prepared from Christensenellaceae bacterial cultures using methods described in S. Bin Park, et al. PLoS ONE. 6(3):el7629 (2011) or G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015) or Jeppesen, et al. Cell 177:428 (2019), each of which is hereby incorporated by reference in its entirety. In some embodiments, the Christensenellaceae bacteria are cultured to high optical density and then centrifuged to pellet Christensenellaceae bacteria (e.g., at 10,000 x g for 30 min at 4°C, at 15,500 x g for 15 min at 4°C). In some embodiments, the culture supernatants are then passed through filters to exclude intact bacterial cells (e.g., a 0.22 pm filter). In some embodiments, the supernatants are then subjected to tangential flow filtration, during which the supernatant is concentrated, species smaller than 100 kDa are removed, and the media is partially exchanged with PBS. In some embodiments, filtered supernatants are centrifuged to pellet bacterial smEVs (e.g., at 100,000-150,000 x g for 1-3 hours at 4°C, at 200,000 x g for 1-3 hours at 4°C). In some embodiments, the smEVs are further purified by resuspending the resulting smEV pellets (e.g., in PBS), and applying the resuspended smEVs to an Optiprep (iodixanol) gradient or gradient (e.g., a 30-60% discontinuous gradient, a 0-45% discontinuous gradient), followed by centrifugation (e.g., at 200,000 x g for 4-20 hours at 4°C). smEV bands can be collected, diluted with PBS, and centrifuged to pellet the smEVs (e.g., at 150,000 x g for 3 hours at 4°C, at 200,000 x g for 1 hour at 4°C). The purified smEVs can be stored, for example, at -80°C or -20°C until use. In some embodiments, the smEVs are further purified by treatment with DNase and/or proteinase K.
[180] For example, in some embodiments, cultures of ChristenseneUaceae bacteria can be centrifuged at 11,000 x g for 20-40 min at 4°C to pellet bacteria. Culture supernatants may be passed through a 0.22 pm filter to exclude intact bacterial cells. Filtered supernatants may then be concentrated using methods that may include, but are not limited to, ammonium sulfate precipitation, ultracentrifugation, or filtration. For example, for ammonium sulfate precipitation, 1.5-3 M ammonium sulfate can be added to filtered supernatant slowly, while stirring at 4°C. Precipitations can be incubated at 4°C for 8-48 hours and then centrifuged at 11,000 x g for 20-40 min at 4°C. The resulting pellets contain bacteria smEVs and other debris. Using ultracentrifugation, filtered supernatants can be centrifuged at 100,000-200,000 x g for 1-16 hours at 4°C. The pellet of this centrifugation contains bacteria smEVs and other debris such as large protein complexes. In some embodiments, using a filtration technique, such as through the use of an Amicon Ultra spin filter or by tangential flow filtration, supernatants can be filtered so as to retain species of molecular weight > 50 or 100 kDa.
[181] Alternatively, smEVs can be obtained from ChristenseneUaceae bacteria cultures continuously dining growth, or at selected time points during growth, for example, by connecting a bioreactor to an alternating tangential flow (ATF) system (e.g., XCell ATF from Repligen). The ATF system retains intact cells (>0.22 pm) in the bioreactor, and allows smaller components (e.g., smEVs, free proteins) to pass through a filter for collection. For example, the system may be configured so that the <0.22 pm filtrate is then passed through a second filter of 100 kDa, allowing species such as smEVs between 0.22 pm and 100 kDa to be collected, and species smaller than 100 kDa to be pumped back into the bioreactor. Alternatively, the system may be configured to allow for medium in the bioreactor to be replenished and/or modified during growth of the culture. smEVs collected by this method may be further purified and/or concentrated by ultracentrifiigation or filtration as described above for filtered supernatants.
[182] smEVs obtained by methods provided herein may be fiirther purified by sizebased column chromatography, by affinity chromatography, by ion-exchange chromatography, and by gradient ultracentrifiigation, using methods that may include, but are not limited to, use of a sucrose gradient or Optiprep gradient. Briefly, using a sucrose gradient method, if ammonium sulfate precipitation or ultracentrifiigation were used to concentrate the filtered supernatants, pellets are resuspended in 60% sucrose, 30 mM Tris, pH 8.0. If filtration was used to concentrate the filtered supernatant, the concentrate is buffer exchanged into 60% sucrose, 30 mM Tris, pH 8.0, using an Amicon Ultra column. Samples are applied to a 35-60% discontinuous sucrose gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C. Briefly, using an Optiprep gradient method, if ammonium sulfide precipitation or ultracentrifiigation were used to concentrate the filtered supernatants, pellets are resuspended in PBS and 3 volumes of 60% Optiprep are added to the sample. In some embodiments, if filtration was used to concentrate the filtered supernatant, the concentrate is diluted using 60% Optiprep to a final concentration of 35% Optiprep. Samples are applied to a 0-45% discontinuous Optiprep gradient and centrifuged at 200,000 x g for 3-24 hours at 4°C, e.g., 4-24 hours at 4°C.
[183] In some embodiments, to confirm sterility and isolation of the smEV preparations, smEVs are serially diluted onto agar medium used for routine culture of the bacteria being tested, and incubated using routine conditions. Non-sterile preparations are passed through a 0.22 pm filter to exclude intact cells. To further increase purity, isolated smEVs may be DNase or proteinase K treated.
[184] In some embodiments, for preparation of smEVs used for in vivo injections, purified smEVs are processed as described previously (G. Norheim, et al. PLoS ONE. 10(9): e0134353 (2015)). Briefly, after sucrose gradient centrifugation, bands containing smEVs are resuspended to a final concentration of 50 pg/mL in a solution containing 3% sucrose or other solution suitable for in vivo injection known to one skilled in the art. This solution may also contain adjuvant, for example aluminum hydroxide at a concentration of 0-0.5% (w/v). In some embodiments, for preparation of smEVs used for in vivo injections, smEVs in PBS are sterile-filtered to < 0.22 pm.
[185] In certain embodiments, to make samples compatible with further testing (e.g., to remove sucrose prior to TEM imaging or in vitro assays), samples are buffer exchanged into PBS or 30 mM Tris, pH 8.0 using filtration (e.g., Amicon Ultra columns), dialysis, or ultracentrifugation (200,000 x g, > 3 hours, 4°C) and resuspension.
[186] In some embodiments, the sterility of the smEV preparations can be confirmed by plating a portion of the smEVs onto agar medium used for standard culture of the bacteria used in the generation of the smEVs and incubating using standard conditions.
[187] In some embodiments, select smEVs are isolated and enriched by chromatography and binding surface moieties on smEVs. In other embodiments, select smEVs are isolated and/or enriched by fluorescent cell sorting by methods using affinity reagents, chemical dyes, recombinant proteins or other methods known to one skilled in the art.
[188] The smEV s can be analyzed, e.g., as described in Jeppesen, et al. Cell 177:428 (2019).
[189] In some embodiments, smEVs are lyophilized. In some embodiments, smEVs are gamma irradiated (e.g., at 17.5 or 25 kGy). In some embodiments, smEVs are UV irradiated. In some embodiments, smEVs are heat inactivated (e.g., at 50°C for two hours or at 90°C for two horns). In some embodiments, smEVs s are acid treated. In some embodiments, smEVs are oxygen sparged (e.g., at 0.1 wm for two hours).
[190] The phase of growth can affect the amount or properties of Christensenellaceae bacteria and/or smEVs produced by Christensenellaceae bacteria. For example, in the methods of smEV preparation provided herein, smEVs can be isolated, e.g., from a culture, at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
[191] The growth environment (e.g., culture conditions) can affect the amount of smEVs produced by Christensenellaceae bacteria. For example, the yield of smEVs can be increased by an smEV inducer, as provided in Table 2.
Table 2: Culture Techniques to Increase smEV Production
Figure imgf000048_0001
Figure imgf000049_0001
[192] In the methods of smEVs preparation provided herein, the method can optionally include exposing a culture of Christensenellaceae bacteria to an smEV inducer prior to isolating smEVs from the bacterial culture. The culture of Christensenellaceae bacteria can be exposed to an smEV inducer at the start of the log phase of growth, midway through the log phase, and/or once stationary phase growth has been reached.
Compositions
[193] In certain aspects, provided herein are compositions (such as pharmaceutical compositions) that comprise mEVs (such as smEVs and/or pmEVs), bacteria, or any combination thereof, obtained from Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
[194] In certain aspects, provided herein are compositions comprising Christensenellaceae bacteria (e.g., bacteria of the genus Christensenella) described herein and a pharmaceutically acceptable carrier.
[195] In some embodiments, the compositions comprises less than
Figure imgf000050_0004
Figure imgf000050_0003
103, 500, 100, 50, 10, or fewer colony forming units (CFU) of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
[196] In some embodiments, the compositions comprises about 1 x 10s, 5 x 10s, 1 x 106,
Figure imgf000050_0001
, , , , , , , , total cells of Christensenellaceae
Figure imgf000050_0002
bacteria, e.g., bacteria of the genus Christensenella. [197] In some embodiments, the Christensenellaceae bacteria are quantified based on total cells, e.g., total cell count (TCC) (e.g., determined by Coulter counter).
[198] In some embodiments, the compositions comprises at least
Figure imgf000051_0004
, ,
Figure imgf000051_0001
Figure imgf000051_0005
total cells of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
[199] In some embodiments, the compositions comprises at most
Figure imgf000051_0006
, ,
Figure imgf000051_0002
Figure imgf000051_0003
total cells of Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella.
[200] In some embodiments, the composition comprises live, killed, attenuated, lyophilized, and/or irradiated (e.g., UV or gamma irradiated) bacteria. Bacteria may be heat-killed by pasteurization, sterilization, high temperature treatment, spray cooking and/or spray drying (heat treatments can be performed at 50°C, 65°C, 85°C or a variety of other temperatures and/or a varied amount of time). Bacteria may also be killed or inactivated using y-irradiation (gamma irradiation), exposune to UV light, formalin- inactivation, and/or freezing methods, or a combination thereof. For example, the bacteria may be exposed to 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, or 50kGy of radiation prior to administration. In some embodiments, bacteria are killed using gamma irradiation. In some embodiments, the bacteria are killed or inactivated using electron irradiation (e.g., beta radiation) or x-ray irradiation.
[201] In some embodiments, the bacteria in the composition described herein are killed using a method that leaves the disease modulating activity of the bacteria intact and the resulting bacterial components are used in the methods and compositions described herein. In some embodiments, the bacteria in the composition described herein are killed using an antibiotic (e.g., using an antibiotic described herein). In some embodiments, the bacteria in the composition described herein are killed using UV irradiation.
[202] In some embodiments, the bacteria in the composition described herein are killed using heat (temperature) sterilization, filtration, and radiation using methods known to those skilled in the art (Garg M., see the World Wide Web at biologydiscussion.com/microorganisms/sterilizatiion/top-3-physical-methods-used-to- kill-microorganisms/55243). The bacteria may be killed via E-beam using methods known to those skilled in the art (StLiNDtR M. et al, FABAD J. Pham. Sci., 34, 43-53, 2009). In some embodiments, the bacteria in the composition described herein are killed and/or attenuated by a chemical agent, for example, aldehydes, e.g., formaldehyde, glutaraldehyde, and the like; food preservative agents such as SCh, sorbic acid, benzoic, acid, nitrate, and nitrite salts; gases such as ethylene oxide; halogens, such as iodine, chlorine, and the like; peroxygens, such as ozone, peroxide, peracetic acid; bisphenols; phenols; phenolics; biguanides, e.g., chlorhexidine; and the like.
[203] Bacteria may be grown to various growth phases and tested for efficacy at different dilutions and at different points during the growth phase. For example, bacteria may be tested for efficacy following administration at stationary phase (including early or late stationary phase), or at various time points during exponential phase. In addition to inactivation by various methods, bacteria may be tested for efficacy using different ratios of live versus inactivated cells, or different ratios of cells at various growth phases.
[204] In certain embodiments, provided herein are compositions comprising mEVs (such as smEVs and/or pmEVs) (e.g., an mEV composition (e.g., an smEV composition or a pmEV composition)) from Christensenellaceae bacteria, e.g., bacteria of the genus Christensenella. In some embodiments, the mEV composition comprises mEVs (such as smEVs and/or pmEVs) and/or a combination of mEVs (such as smEVs and/or pmEVs) described herein and a pharmaceutically acceptable carrier. In some embodiments, the smEV composition comprises smEVs and/or a combination of smEVs described herein and a pharmaceutically acceptable carrier. In some embodiments, the pmEV composition comprises pmEVs and/or a combination of pmEVs described herein and a pharmaceutically acceptable carrier.
[205] In some embodiments, the compositions comprise mEVs (such as smEVs and/or pmEVs) substantially or entirely free of whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the compositions comprise both mEVs and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the composition comprises lyophilized mEVs (such as smEVs and/or pmEVs). In some embodiments, the composition comprises gamma irradiated mEVs (such as smEVs and/or pmEVs). The mEVs (such as smEVs and/or pmEVs) can be gamma irradiated after the mEVs are isolated (e.g., prepared).
[206] In some embodiments, to quantify the numbers of mEVs (such as smEVs and/or pmEVs) and/or bacteria present in a bacterial sample, electron microscopy (e.g., EM of ultrathin frozen sections) can be used to visualize the mEVs (such as smEVs and/or pmEVs) and/or bacteria and count their relative numbers. Alternatively, nanoparticle tracking analysis (NTA), Coulter counting, or dynamic light scattering (DLS) or a combination of these techniques can be used. NTA and the Coulter counter count particles and show their sizes. DLS gives the size distribution of particles, but not the concentration. Bacteria frequently have diameters of 1-2 um (microns). The full range is 0.2-20 um. Combined results from Coulter counting and NTA can reveal the numbers of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a given sample. Coulter counting reveals the numbers of particles with diameters of 0.7-10 pm. For most bacterial and/or mEV (such as smEV and/or pmEV) samples, the Coulter counter alone can reveal the number of bacteria and/or mEVs (such as smEVs and/or pmEVs) in a sample. pmEVs are 20-600 nm in diameter. For NTA, a Nanosight instrument can be obtained from Malvern Pananlytical. For example, the NS300 can visualize and measure particles in suspension in the size range 10-2000 nm. NTA allows fbr counting of the numbers of particles that are, fbr example, 50-1000 nm in diameter. DLS reveals the distribution of particles of different diameters within an approximate range of 1 nm - 3 pm. [207] mEVs can be characterized by analytical methods known in the art (e.g., Jeppesen, et al. Cell 177:428 (2019)).
[208] In some embodiments, the mEVs may be quantified based on particle count. For example, total protein content of an mEV preparation can be measured using NTA.
[209] In some embodiments, the mEVs may be quantified based on the amount of protein, lipid, or carbohydrate. For example, a dose of mEV can be determined by particle count of an mEV preparation can be measured using the Bradford assay or BCA.
[210] In some embodiments, the mEVs are isolated away from one or more other bacterial components of the source bacteria. In some embodiments, the composition further comprises other bacterial components.
[211] In certain embodiments, the mEV preparation obtained from the source bacteria may be fractionated into subpopulations based on the physical properties (e.g., size, density, protein content, binding affinity) of the subpopulations. One or more of the mEV subpopulations can then be incorporated into the compositions of the invention.
[212] In certain aspects, provided herein are compositions comprising mEVs (such as smEVs and/or pmEVs) usefill for the treatment and/or prevention of disease (e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, or a metabolic disease), as well as methods of making and/or identifying such mEVs, and methods of using such compositions (e.g., for the treatment and/or prevention of a disease or a health disorder (e.g., a cancer, an autoimmune disease, an inflammatory disease, a dysbiosis, and/or a metabolic disease), either alone or in combination with other therapeutics). In some embodiments, the compositions comprise both mEVs (such as smEVs and/or pmEVs), and whole bacteria (e.g., live bacteria, killed bacteria, attenuated bacteria). In some embodiments, the compositions comprise mEVs (such as smEVs and/or pmEVs) in the absence of bacteria. In some embodiments, the compositions comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from the genus Christensenella. In some embodiments, the compositions comprise mEVs (such as smEVs and/or pmEVs) and/or bacteria from Christensenella minuta.
[213] In certain aspects, provided herein are compositions comprising Christensenellaceae bacteria and/or Christensenellaceae bacteria mEVs described herein. In some embodiments, the bacteria are bacteria of the genus Christensenella, such as Christensenella minuta, including, for example, one or more species with the 16S rRNA gene sequence of any of SEQ ID NOS: 1 or 5-8, and mixtures thereof. [214] In some embodiments, the composition comprises at least 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7. 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.
5.9. 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 10s, 2 x 10s, 3 x 10s, 4 x 10s, 5 x 10s, 6 x 10s, 7 x 10s, 8 x 10s, 9 x 10s, 1 x 106, 2 x 106, 3 x IO6, 4 x 106, 5 x IO6, 6 x 106, 7 x IO6, 8 x IO6, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x IO8, 2 x IO8, 3 x 108, 4 x IO8, 5 x IO8, 6 x IO8, 7 x IO8, 8 x IO8, 9 x IO8 , 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x IO10, 2 x IO10, 3 x IO10, 4 x IO10, 5 x IO10, 6 x IO10, 7 x IO10, 8 x IO10, 9 x IO10, 1 x IO11, 2 x IO11, 3 x IO11, 4 x IO11, 5 x IO11, 6 x 10u, 7 x IO11, 8 x IO11, 9 x 10u, and/or 1 x 1012 Christensenellaceae bacteria mEV particles (e.g., from the genus Christensenella).
[215] In some embodiments, the composition comprises about 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6,
1.7. 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7,
3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8.
5.9. 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x IO4, 2 x IO4, 3 x IO4, 4 x IO4, 5 x IO4, 6 x IO4, 7 x 104, 8 x IO4, 9 x IO4, 1 x 10s, 2 x 10s, 3 x 10s, 4 x 10s, 5 x 10s, 6 x 10s, 7 x 10s, 8 x 105, 9 x 105, 1 x IO6, 2 x 106, 3 x IO6, 4 x 106, 5 x IO6, 6 x 106, 7 x IO6, 8 x IO6, 9 x
106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108, 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x IO10, 2 x IO10, 3 x IO10, 4 x IO10, 5 x IO10, 6 x IO10, 7 x IO10, 8 x IO10, 9 x IO10, 1 x IO11, 2 x IO11, 3 x IO11, 4 x IO11, 5 x IO11, 6 x 1OU, 7 x IO11, 8 x IO11, 9 x 1OU, and/or 1 x 1012 Christensenellaceae bacteria mEV particles (e.g., from the genus Christensenella).
[216] In some embodiments, the composition comprises no more than 1 Christensenellaceae bacterium (e.g., from the genus Christensenella) for every 1, 1.1,
1.2. 1.3. 1.4. 1.5. 1.6. 1.7. 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2,
3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3,
5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4,
7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5,
9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x 104, 2 x 104, 3 x 104, 4 x 104, 5 x 104, 6 x 104, 7 x 104, 8 x 104, 9 x 104, 1 x 105, 2 x 105, 3 x 105, 4 x 105, 5 x 103, 6 x 105, 7 x 105, 8 x 105, 9 x 105, 1 x 106, 2 x 106, 3 x 106, 4 x 106, 5 x 106, 6 x IO6, 7 x IO6, 8 x IO6, 9 x 106, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x
107, 8 x 107, 9 x 107, 1 x IO8, 2 x IO8, 3 x IO8, 4 x IO8, 5 x IO8, 6 x IO8, 7 x IO8, 8 x IO8, 9 x IO8 , 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x IO10, 2 x IO10, 3 x IO10, 4 x IO10, 5 x IO10, 6 x IO10, 7 x IO10, 8 x IO10, 9 x IO10, 1 x IO11, 2 x IO11, 3 x IO11, 4 x IO11, 5 x IO11, 6 x IO11, 7 x IO11, 8 x IO11, 9 x IO11, and/or 1 x 1012 Christensenellaceae bacteria mEV particles (e.g., from the genus Christensenella).
[217] In some embodiments, the composition comprises at least 1 Christensenellaceae bacterial mEV particle (e.g., from the genus Christensenella) for every 1, 1.1, 1.2, 1.3,
1.4. 1.5. 1.6. 1.7. 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,
5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,
31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,
55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78.
79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x IO4, 2 x IO4, 3 x 104, 4 x l04, 5 x l04, 6 x l04, 7 x l04, 8 x l04, 9 x l04, l x 10s, 2 x 10s, 3 x 10s, 4 x 10s, 5 x 10s, 6 x 10s, 7 x 10s, 8 x 10s, 9 x 10s, 1 x IO6, 2 x IO6, 3 x IO6, 4 x IO6, 5 x IO6, 6 x IO6, 7 x IO6, 8 x 106, 9 x IO6, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x l07, l x IO8, 2 x 10s, 3 x 10s, 4 x 10s, 5 x 10s, 6 x 10s, 7 x l08, 8 x 10s, 9 x 10s, l x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x IO10, 2 x IO10, 3 x IO10, 4 x IO10, 5 x IO10, 6 x IO10, 7 x IO10, 8 x IO10, 9 x IO10, 1 x IO11, 2 x IO11, 3 x IO11, 4 x IO11, 5 x IO11, 6 x IO11, 7 x IO11, 8 x IO11, 9 x IO11, and/or 1 x 1012 Christensenellaceae bacteria (e.g., fiom the genus Christensenella).
[218] In some embodiments, the composition comprises about 1 Christensenellaceae bacterial mEV particle (e.g., fiom the genus Christensenella) for every 1, 1.1, 1.2, 1.3,
1.4. 1.5. 1.6. 1.7. 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4,
3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5,
5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6,
7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7,
9.8, 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30,
31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54,
55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78.
79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150,
200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1 x 103, 2 x 103, 3 x 103, 4 x 103, 5 x 103, 6 x 103, 7 x 103, 8 x 103, 9 x 103, 1 x IO4, 2 x IO4, 3 x 104, 4 x l04, 5 x l04, 6 x l04, 7 x l04, 8 x l04, 9 x l04, l x 10s, 2 x 10s, 3 x 10s, 4 x 10s, 5 x 10s, 6 x 10s, 7 x 10s, 8 x 10s, 9 x 10s, 1 x IO6, 2 x IO6, 3 x IO6, 4 x IO6, 5 x IO6, 6 x IO6, 7 x IO6, 8 x 106, 9 x IO6, 1 x 107, 2 x 107, 3 x 107, 4 x 107, 5 x 107, 6 x 107, 7 x 107, 8 x 107, 9 x 107, 1 x 108, 2 x 108, 3 x 108, 4 x 108, 5 x 108, 6 x 108, 7 x 108, 8 x 108, 9 x 108 , 1 x 109, 2 x 109, 3 x 109, 4 x 109, 5 x 109, 6 x 109, 7 x 109, 8 x 109, 9 x 109, 1 x IO10, 2 x IO10, 3 x IO10, 4 x IO10, 5 x IO10, 6 x IO10, 7 x IO10, 8 x IO10, 9 x IO10, 1 x IO11, 2 x IO11, 3 x 1011, 4 x 1011, 5 x 1011, 6 x IO11, 7 x IO11, 8 x IO11, 9 x IO11, and/or 1 x 1012 ChristenseneUaceae bacteria (e.g., fiom the genus Christensenella).
[219] In some embodiments, the composition comprises no more than 1 ChristenseneUaceae bacteria mEV particle (e.g., fiom the genus Christensenella) for every 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1,
5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2,
7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3,
9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10, 11, 12, 13, 14, 15, 16, 17, 18. 19, 20, 21, 22, 23, 24, 25, 26, 27, 28. 29, 30, 31, 32, 33, 34, 35, 36, 37, 38. 39, 40, 41, 42, 43, 44, 45, 46, 47, 48. 49, 50, 51, 52, 53, 54, 55, 56, 57, 58. 59, 60, 61, 62, 63, 64, 65, 66, 67, 68. 69, 70, 71, 72, 73, 74, 75, 76, 77, 78. 79, 80, 81, 82, 83, 84, 85, 86, 87, 88. 89, 90, 91, 92, 93, 94, 95, 96, 97, 98. 99, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, ChristenseneUaceae bacteria (e.g., from the genus Christensenella).
[220] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are ChristenseneUaceae bacterial mEVs (e.g., from the genus Christensenella).
[221] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are ChristenseneUaceae bacteria (e.g., from the genus Christensenella). [222] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of total the particles in the composition are Christensenellaceae bacterial mEVs (e.g., from the genus Christensenella).
[223] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacteria (e.g., from the genus Christensenella).
[224] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacterial mEVs (e.g., from the genus Christensenella).
[225] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenellaceae bacteria (e.g., fiom the genus Christensenella).
[226] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacterial mEV protein (e.g., fiom the genus Christensenella).
[227] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacteria protein (e.g., fiom the genus Christensenella).
[228] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacterial mEV protein (e.g., fiom the genus Christensenella).
[229] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacteria protein (e.g., fiom the genus Christensenella).
[230] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacterial mEV protein (e.g., fiom the genus Christensenella).
[231] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total protein in the composition is Christensenellaceae bacteria protein (e.g., fiom the genus Christensenella).
[232] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacterial mEV lipids (e.g., fiom the genus Christensenella). [233] In some embodiments, at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacteria lipids (e.g., from the genus Christensenella).
[234] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacterial mEV lipids (e.g., fiom the genus Christensenella).
[235] In some embodiments, no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacteria lipids(e.g., from the genus Christensenella).
[236] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacterial mEV lipids (e.g., fiom the genus Chris t ens ene Ila).
[237] In some embodiments, about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenellaceae bacteria lipids (e.g., fiom the genus Christensenella).
[238] In certain aspects, provided are compositions fbr administration to a subject (e.g., human subject). In some embodiments, the compositions are combined with additional active and/or inactive materials in order to produce a final product, which may be in single dosage unit or in a multi-dose format. In some embodiments, the composition is combined with an adjuvant such as an immuno-adjuvant (e.g., a STING agonist, a TLR agonist, or a NOD agonist).
[239] In some embodiments, the composition comprises at least one carbohydrate.
[240] In some embodiments, the composition comprises at least one lipid. In some embodiments the lipid comprises at least one fetty acid selected fiom lauric acid (12:0), myristic acid (14:0), palmitic acid (16:0), palmitoleic acid (16:1), margaric acid (17:0), heptadecenoic acid (17:1), stearic acid (18:0), oleic acid (18:1), linoleic acid (18:2), linolenic acid (18:3), octadecatetraenoic acid (18:4), arachidic acid (20:0), eicosenoic acid (20: 1), eicosadienoic acid (20:2), eicosatetraenoic acid (20:4), eicosapentaenoic acid (20:5) (EPA), docosanoic acid (22:0), docosenoic acid (22: 1), docosapentaenoic acid (22:5), docosahexaenoic acid (22:6) (DHA), and tetracosanoic acid (24:0).
[241] In some embodiments, the composition comprises at least one supplemental mineral or mineral source. Examples of minerals include, without limitation: chloride, sodium, calcium, iron, chromium, copper, iodine, zinc, magnesium, manganese, molybdenum, phosphorus, potassium, and selenium. Suitable forms of any of the foregoing minerals include soluble mineral salts, slightly soluble mineral salts, insoluble mineral salts, chelated minerals, mineral complexes, non-reactive minerals such as carbonyl minerals, and reduced minerals, and combinations thereof.
[242] In some embodiments, the composition comprises at least one supplemental vitamin. The at least one vitamin can be fat-soluble or water soluble vitamins. Suitable vitamins include but are not limited to vitamin C, vitamin A, vitamin E, vitamin B 12, vitamin K, riboflavin, niacin, vitamin D, vitamin B6, folic acid, pyridoxine, thiamine, pantothenic acid, and biotin. Suitable forms of any of the foregoing are salts of the vitamin, derivatives of the vitamin, compounds having the same or similar activity of the vitamin, and metabolites of the vitamin.
[243] In some embodiments, the composition comprises an excipient. Non-limiting examples of suitable excipients include a buffering agent, a preservative, a stabilizer, a binder, a compaction agent, a lubricant, a dispersion enhancer, a disintegration agent, a flavoring agent, a sweetener, and a coloring agent.
[244] In some embodiments, the excipient is a buffering agent. Non-limiting examples of suitable buffering agents include sodium citrate, magnesium carbonate, magnesium bicarbonate, calcium carbonate, and calcium bicarbonate.
[245] In some embodiments, the excipient comprises a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as alpha-tocopherol and ascorbate, and antimicrobials, such as parabens, chlorobutanol, and phenol.
[246] In some embodiments, the composition comprises a binder as an excipient. Nonlimiting examples of suitable binders include starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, Cn-Cis fiitty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, and combinations thereof.
[247] In some embodiments, the composition comprises a lubricant as an excipient. Non-limiting examples of suitable lubricants include magnesium stearate, calcium stearate, zinc stearate, hydrogenated vegetable oils, sterotex, polyoxyethylene monostearate, talc, polyethyleneglycol, sodium benzoate, sodium lauryl sulfiite, magnesium lauryl sulfiite, and light mineral oil.
[248] In some embodiments, the composition comprises a dispersion enhancer as an excipient. Non-limiting examples of suitable dispersants include starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose as high HLB emulsifier surfactants.
[249] In some embodiments, the composition comprises a disintegrant as an excipient. In some embodiments the disintegrant is a non-eflfervescent disintegrant. Non-limiting examples of suitable non-effervescent disintegrants include starches such as com starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pectin, and tragacanth. In some embodiments the disintegrant is an effervescent disintegrant. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid, and sodium bicarbonate in combination with tartaric acid.
[250] In some embodiments, the composition is a food product (e.g., a food or beverage) such as a health food or beverage, a food or beverage for infonts, a food or beverage for pregnant women, athletes, senior citizens or other specified group, a functional food, a beverage, a food or beverage for specified health use, a dietary supplement, a food or beverage for patients, or an animal feed. Specific examples of the foods and beverages include various beverages such as juices, refreshing beverages, tea beverages, drink preparations, jelly beverages, and functional beverages; alcoholic beverages such as beers; carbohydrate-containing foods such as rice food products, noodles, breads, and pastas; paste products such as fish hams, sausages, paste products of seafood; retort pouch products such as curries, food dressed with a thick starchy sauces, and Chinese soups; soups; dairy products such as milk, dairy beverages, ice creams, cheeses, and yogurts; fermented products such as fermented soybean pastes, yogurts, fermented beverages, and pickles; bean products; various confectionery products, including biscuits, cookies, and the like, candies, chewing gums, gummies, cold desserts including jellies, cream caramels, and frozen desserts; instant foods such as instant soups and instant soy-bean soups; microwavable foods; and the like. Further, the examples also include health foods and beverages prepared in the forms of powders, granules, tablets, capsules, liquids, pastes, and jellies.
[251] In some embodiments, the composition is a food product for animals, including humans. The animals, other than humans, are not particularly limited, and the composition can be used for various livestock, poultry, pets, experimental animals, and the like. Specific examples of the animals include pigs, cattle, horses, sheep, goats, chickens, wild ducks, ostriches, domestic ducks, dogs, cats, rabbits, hamsters, mice, rats, monkeys, and the like, but the animals are not limited thereto.
Measuring Levels of Chnstensenellaceae in a Sample
[252] The levels of Chnstensenellaceae bacteria and/or Christensenella bacteria can be measured by amplifying the 16S rRNA gene from bacteria in a sample.
[253] In one example, the levels of Chnstensenellaceae bacteria and/or Christensenella bacteria are measured in a sample by performing quantitative PCR using primers that specifically amplify members of the family or of the genus, and these levels are normalized to the total 16S rRNA in a sample. The prevalent strategy for amplifying the 16S rRNA gene from microorganisms in a sample involves utilizing probes and primers that anneal to conserved regions at the edges of known variable domains in the 16S rRNA gene sequence, for amplification and sequencing across the variable domains to identify species-specific variable sequences and thus identify species within the sample.
Typically, primer pairs have been designed to amplify a single variable (“ V1') region, such as the 16S V3, V4, or V5 region. Common primers to amplify the V4 region include universal 16S primers 515F (SEQ ID NO: 3) and 806R (SEQ ID NO: 4).
[254] Traditional amplification methods such as Sanger sequencing, or next generation sequencing methods, are suitable for amplification and sequencing of microorganisms. However, given the potentially large number and variety of microorganisms which can be present in a given sample, high-throughput/next generation sequencing methods, which are capable of rapidly generating large amounts of sequence data, are particularly well suited for the rapid and accurate identification of microorganisms in a sample. “Next generation” sequencing technologies enable a large number of distinct nucleic acid sequences to be sequenced simultaneously and at a high density. Such sequencing technologies include, but are not limited to, sequencing-by-synthesis (e.g., Illumina dye sequencing; Single Molecule Real Time Sequencing platform by Pacific Biosciences); sequencing by ligation (Solid or Polony sequencing platform, Applied Biosystems); pyrosequencing (454 Sequencing, Roche Diagnostics); and ion semiconductor sequencing (Ton Torrent Sequencing, Life Technologies).
PCR amplicons can be purified, for example using a magnetic bead system, and quantified, using commercially available products for purification and quantification. Donavan be sequenced and analyzed using any one of several sequencing platforms, such as an Illumina MISEQ platform (Illumina Inc., San Diego, Calif, USA). [255] Once the 16S rRNA gene in a sample has been amplified and sequenced, microorganisms present in the sample are then identified based on identification of species-specific 16S DNA amplicon sequences generated from the sample. Quality filtering (to remove “noise” generated by random sequencing errors) and analysis of 16S rRNA gene sequence data can be performed using publicly available bioinformatic analytic tools for taxon identification from raw DNA sequencing data, such as the Quantitative Insights Into Microbial Ecology' (QIIME) analytic tool (Caporaso et al.. Nature Methods 7:335-336 (2010)).
[256] As disclosed elsewhere in this application, Christensenellaceae bacteria are identified based on (1) at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identity, preferably 97% identity or greater, of the 16S rRNA gene sequence or sequences of the bacteria with Christensenellaceae 16S rRNA gene sequences, using an available database of 16S rRNA gene sequences, or (2) phylogenetic analysis of the 16S rRNA gene sequence of the bacteria identifying the bacteria as Christensenellaceae, using known methods for determining phylogeny. The levels of Christensenellaceae in a sample can be determined by comparing the abundance or ratio of sequences identified as Christensenellaceae to the total bacterial abundance in a sample, or to the abundance or ratio of sequences identified as not belonging to Christensenellaceae. Alternatively, the abundance or ratio of Christensenellaceae in a sample, relative to other bacterial species, can be measured by use of analytic tools, such as QIIME. These tools can generate taxonomic distributions in a sample by comparing the bacterial 16S rRNA gene sequences from the sample to the 16SrRNA gene sequences in a database such as Greengenes, which have previously been assigned taxonomic classifications.
Christensenella bacteria are identified based on at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to the sequence of SEQ ID NO: 1 or SEQ ID NO: 2 (the portion of SEQ ID NO: 1 generated by amplification with the 515F and 806R universal 16S primers). Thus, the levels of Christensenella in a sample can be measured by comparing the abundance or ratio of sequences having at least 90% identity to SEQ ID NO: 1 or SEQ ID NO: 2 to the abundance or ratio of sequences falling outside of this range. Alternatively, the abundance or ratio or Christensenella in a sample, relative to other bacterial species, can be measured by use of analytic tools, such as QIIME, to generate taxonomic distributions in a sample by comparing the bacterial 16S rRNA gene sequences from the sample to the 16S rRNA gene sequences in a database such as Greengenes, which have previously been assigned taxonomic classifications.
[257] In one example, if the relative abundance of Christensenellaceae in a sample from a subject is less than 0.01 (out of a total bacterial abundance=l), or if the ratio or level of Christensenellaceae in the sample is below 1.0% of the total bacteria in the sample, the subject is diagnosed with low levels of Christensenellaceae. Accordingly, the subject can benefit by administration of a composition as disclosed herein. The subject can be treated by administration of a dosage of less than 10 x8 CPU of Christensenellaceae or Christensenella bacteria/dose, and more particularly less than 10 x6 CPU of Christensenellaceae.
Admini
Figure imgf000068_0001
iQn
[258] In certain aspects, the bacteria and/or mEVs described herein (e.g., a composition comprising the bacteria) are administered in separate administrations to a subject.
Separate administrations can include any number of two or more administrations (e.g., doses), including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform, or the desirability of performing one or more additional administrations, according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. In some embodiments, the doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of bacteria, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results, including, but not limited to, the overall health of the subject and/or the weight of the subject.
[259] The time period between administrations of the bacteria described herein (e.g., a composition comprising the bacteria) can be any of a variety of time periods. The time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the bacteria from normal tissue. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month. In another example, the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
[260] In certain aspects, provided herein is a method of delivering bacteria and/or mEVs and/or a composition described herein to a subject. In some embodiments of the methods provided herein, the bacteria, mEVs, and/or a composition thereof are administered in conjunction with the administration of an additional therapeutic. In some embodiments, the bacteria and/or mEVs are co-formulated in a composition with the additional therapeutic. In some embodiments, the bacteria and/or mEVs are co-administered with the additional therapeutic. In some embodiments, the additional therapeutic is administered to the subject before administration of the bacteria and/or mEVs (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes before, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours before, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days before). In some embodiments, the additional therapeutic is administered to the subject after administration of the bacteria and/or mEVs (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 or 55 minutes after, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours after, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 days after). In some embodiments, the same mode of delivery is used to deliver both the bacteria and the additional therapeutic. In some embodiments, different modes of delivery are used to administer the bacteria and the additional therapeutic. For example, in some embodiments, the bacteria and/or mEVs are administered orally while the additional therapeutic is administered via injection (e.g., an intravenous, intramuscular and/or intratumoral injection). [261] In some embodiments, the composition is administered orally, rectally, intravenously, intradermally, intraperitoneally, or subcutaneously. In some embodiments, the composition is administered orally.
[262] In some embodiments, the composition can be administered on a daily or weekly basis. The subject to whom a composition of the invention is administered can have metabolic disease. The subject to whom a composition of the invention is administered can have obesity, metabolic syndrome, and/or diabetes.
[263] The composition can be for inhibiting weight gain, promoting weight loss, or reducing adiposity.
[264] In some embodiments, the composition is administered in two or more doses. In further embodiments, the administration to the subject of the two or more doses are separated by at least 1 day. In other embodiments, the administration of the two or more doses are separated by at least 1 week.
[265] In some embodiments, administration of the composition treats the metabolic disease. In some embodiments, administration of the composition treats the obesity, metabolic syndrome, and/or diabetes. In some embodiments, administration of the composition inhibits weight gain, promotes weight loss, or reduces adiposity.
[266] In certain embodiments, the compositions described herein can be administered in conjunction with any other conventional metabolic disease therapeutic agents. These treatments may be applied as necessary and/or as indicated and may occur before, concurrent with or after administration of the compositions described herein.
[267] The dosage regimen can be any of a variety of methods and amounts, and can be determined by one skilled in the art according to known clinical factors. As is known in the medical arts, dosages for any one patient can depend on many factors, including the subject's species, size, body surface area, age, sex, immunocompetence, and general health, the particular microorganism to be administered, duration and route of administration, the kind and stage of the disease, for example, tumor size, and other compounds such as drugs being administered concurrently. In addition to the above factors, such levels can be affected by the infectivity of the microorganism, and the nature of the microorganism, as can be determined by one skilled in the art. In the present methods, appropriate miniminn dosage levels of microorganisms can be levels sufficient for the microorganism to survive, grow and replicate. The methods of treatment described herein may be suitable for the treatment of a metabolic disease (e.g., diabetes). The dose of the pharmaceutical compositions described herein may be appropriately set or adjusted in accordance with the dosage form, the route of administration, the degree or stage of a target disease, and the like. For example, the general effective dose of the agents may range between 0.01 mg/kg body weight/day and 1000 mg/kg body weight/day, between 0.1 mg/kg body weight/day and 1000 mg/kg body weight/day, 0.5 mg/kg body weight/day and 500 mg/kg body weight/day, 1 mg/kg body weight/day and 100 mg/kg body weight/day, or between 5 mg/kg body weight/day and 50 mg/kg body weight/day. The effective dose may be 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, or 1000 mg/kg body weight/day or more, but the dose is not limited thereto.
[268] In some embodiments, the dose administered to a subject is sufficient to prevent the metabolic disease, delay its onset, or slow or stop its progression or prevent a relapse of the metabolic disease. In some embodiments, the dose administered to a subject is sufficient to prevent the obesity, metabolic syndrome, and/or diabetes, delay its onset, or slow or stop its progression or prevent a relapse of the obesity, metabolic syndrome, and/or diabetes. In some embodiments, the dose administered to a subject is sufficient to inhibit weight gain, promote weight loss, or reduce adiposity. One skilled in the art will recognize that dosage will depend upon a variety of factors including the strength of the particular compound employed, as well as the age, species, condition, and body weight of the subject. The size of the dose will also be determined by the route, timing, and frequency of administration as well as the existence, nature, and extent of any adverse side-effects that might accompany the administration of a particular compound and the desired physiological effect.
[269] Suitable doses and dosage regimens can be determined by conventional rangefinding techniques known to those of ordinary skill in the art. Generally, treatment is initiated with smaller dosages, which are less than the optimum dose of the compound. Thereafter, the dosage is increased by small increments until the optimum effect under the circumstances is reached. An effective dosage and treatment protocol can be determined by routine and conventional means, starting e.g., with a low dose in laboratory animals and then increasing the dosage while monitoring the effects, and systematically varying the dosage regimen as well. Animal studies are commonly used to determine the maximal tolerable dose ("MTD") of bioactive agent per kilogram weight. Those skilled in the art regularly extrapolate doses for efficacy, while avoiding toxicity, in other species, including humans.
[270] In accordance with the above, in therapeutic applications, the dosages of the active agents used in accordance with the invention vary depending on the active agent, the age, weight, and clinical condition of the recipient patient, and the experience and judgment of the clinician or practitioner administering therapy, among other factors affecting the selected dosage. Generally, the dose should be sufficient to result in slowing, and preferably regressing, the advancement of an immune disorder.
[271] The bacteria, mEVs, and/or composition and an additional therapeutic (e.g., a conventional metabolic disease therapeutic agent) can be administered separately. Separate administrations can include any number of two or more administrations (e.g. , doses), including two, three, four, five or six administrations. One skilled in the art can readily determine the number of administrations to perform, or the desirability of performing one or more additional administrations, according to methods known in the art for monitoring therapeutic methods and other monitoring methods provided herein. In some embodiments, the doses may be separated by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days or 1, 2, 3, or 4 weeks. Accordingly, the methods provided herein include methods of providing to the subject one or more administrations of bacteria, where the number of administrations can be determined by monitoring the subject, and, based on the results of the monitoring, determining whether or not to provide one or more additional administrations. Deciding on whether or not to provide one or more additional administrations can be based on a variety of monitoring results, including, but not limited to, the overall health of the subject and/or the weight of the subject.
[272] The time period between administrations can be any of a variety of time periods. The time period between administrations can be a function of any of a variety of factors, including monitoring steps, as described in relation to the number of administrations, the time period for a subject to mount an immune response and/or the time period for a subject to clear the bacteria from normal tissue. In one example, the time period can be a function of the time period for a subject to mount an immune response; for example, the time period can be more than the time period for a subject to mount an immune response, such as more than about one week, more than about ten days, more than about two weeks, or more than about a month; in another example, the time period can be less than the time period for a subject to mount an immune response, such as less than about one week, less than about ten days, less than about two weeks, or less than about a month. In another example, the time period can be a function of the time period for a subject to clear the bacteria from normal tissue; for example, the time period can be more than the time period for a subject to clear the bacteria from normal tissue, such as more than about a day, more than about two days, more than about three days, more than about five days, or more than about a week.
[273] In some embodiments, the delivery of the bacteria and/or mEVs described herein (e.g., a composition comprising the bacteria and/or mEVs), or delivery of a metabolic disease therapeutic in combination with the bacteria and/or mEVs described herein, reduces the adverse effects and/or improves the efficacy of the metabolic disease therapeutic. In some embodiments, the administration of the composition treats the metabolic disease. In some embodiments, the administration of the composition treats the obesity, metabolic syndrome, and/or diabetes. In some embodiments, the administration of the composition inhibits weight gain, promotes weight loss, or reduces adiposity.
Therapeutic Agents
[274] In certain aspects, the methods provided herein include administering to a subject a composition described herein (e.g., a composition comprising a Christensenellaceae bacteria and/or mEVs) either alone or in combination with another therapeutic. In some embodiments, the composition and the other therapy can be administered to the subject in any order. In some embodiments, the composition and the other therapy are administered conjointly.
[275] In some embodiments the composition is administered to the subject before the additional therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before). In some embodiments, the composition is administered to the subject after the additional therapeutic is administered (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours after or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, the composition and the additional therapeutic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an horn* of each other). In some embodiments, the subject is administered an antibiotic before the composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days before).In some embodiments, the subject is administered an antibiotic after the composition is administered to the subject (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hours before or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 days after). In some embodiments, the composition and the antibiotic are administered to the subject simultaneously or nearly simultaneously (e.g., administrations occur within an hour of each other).
[276] In certain embodiments, the subject may undergo surgery. Types of surgery include but are not limited to preventative, diagnostic or staging, cinative and palliative surgery.
[277] In some embodiments, the additional therapeutic is an antibiotic. For example, if the presence of a metabolic-disease-associated bacteria and/or a metabolic-disease- associated microbiome profile is detected according to the methods provided herein, antibiotics can be administered to eliminate the metabolic-disease-associated bacteria from the subject. “Antibiotics” broadly refers to compounds capable of inhibiting or preventing a bacterial infection. Antibiotics can be classified in a number of ways, including their use for specific infections, their mechanism of action, their bioavailability, or their spectrum of target microbe (e.g., Gram-negative vs. Gram-positive bacteria, aerobic vs. anaerobic bacteria, etc.) and these may be used to kill specific bacteria in specific areas of the host (“niches”) (Leekha, et al 2011. General Principles of Antimicrobial Therapy. Mayo Clin Proc. 86(2): 156-167). In certain embodiments, antibiotics can be used to selectively target bacteria of a specific niche. In some embodiments, antibiotics known to treat a particular infection that includes a metabolic disease niche may be used to target metabolic-disease-associated microbes, including metabolic-disease-associated bacteria in that niche. In other embodiments, antibiotics are administered after the bacterial treatment. In some embodiments, antibiotics are administered after the bacterial treatment to remove the engrafiment.
[278] In some aspects, antibiotics can be selected based on their bactericidal or bacteriostatic properties. Bactericidal antibiotics include mechanisms of action that disrupt the cell wall (e.g., P-lactams), the cell membrane (e.g., daptomycin), or bacterial DNA (e.g., fluoroquinolones). Bacteriostatic agents inhibit bacterial replication and include sulfonamides, tetracyclines, and macrolides, and act by inhibiting protein synthesis. Furthermore, while some drugs can be bactericidal in certain organisms and bacteriostatic in others, knowing the target organism allows one skilled in the art to select an antibiotic with the appropriate properties. In certain treatment conditions, bacteriostatic antibiotics inhibit the activity of bactericidal antibiotics. Thus, in certain embodiments, bactericidal and bacteriostatic antibiotics are not combined.
[279] Antibiotics include, but are not limited to aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, and anti- mycobacterial compounds, and combinations thereof.
[280] Aminoglycosides include, but are not limited to Amikacin, Gentamicin, Kanamycin, Neomycin, Netilmicin, Tobramycin, Paromomycin, and Spectinomycin. Aminoglycosides are effective, e.g., against Gram-negative bacteria, such as Escherichia colt and Klebsiella, Pseudomonas aeruginosa, and Francisella tularensis, and against certain aerobic bacteria but less effective against obligate/facultative anaerobes. Aminoglycosides are believed to bind to the bacterial 30S or SOS ribosomal subunit thereby inhibiting bacterial protein synthesis.
[281] Ansamycins include, but are not limited to, Geldanamycin, Herbimycin, Rifamycin, and Streptovaricin. Geldanamycin and Herbimycin are believed to inhibit or alter the function of Heat Shock Protein 90.
[282] Carbacephems include, but are not limited to, Loracarbef. Carbacephems are believed to inhibit bacterial cell wall synthesis.
[283] Carbapenems include, but are not limited to, Ertapenem, Doripenem, Imipenem/Cilastatin, and Meropenem. Carbapenems are bactericidal for both Grampositive and Gram-negative bacteria as broad-spectrum antibiotics. Carbapenems are believed to inhibit bacterial cell wall synthesis.
[284] Cephalosporins include, but are not limited to, Cefadroxil, Cefazolin, Cefalotin, Cefalothin, Cefalexin, Cefaclor, Cefamandole, Cefoxitin, Cefprozil, Cefuroxime, Cefixime, Cefdinir, Cefditoren, Cefoperazone, Cefotaxime, Cefpodoxime, Ceftazidime, Ceftibuten, Ceftizoxime, Ceftriaxone, Cefepime, Ceftaroline fosamil, and Ceftobiprole. Selected Cephalosporins are effective, e.g., against Gram-negative bacteria and against Gram-positive bacteria, including Pseudomonas, certain Cephalosporins are effective against methicillin-resistant Staphylococcus aureus (MRSA). Cephalosporins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[285] Glycopeptides include, but are not limited to, Teicoplanin, Vancomycin, and Telavancin. Glycopeptides are effective, e.g., against aerobic and anaerobic Grampositive bacteria including MRSA and Clostridium difficile. Glycopeptides are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[286] Lincosamides include, but are not limited to, Clindamycin and Lincomycin. Lincosamides are effective, e.g., against anaerobic bacteria, as well as Staphylococcus and Streptococcus. Lincosamides are believed to bind to the bacterial SOS ribosomal subunit thereby inhibiting bacterial protein synthesis.
[287] Lipopeptides include, but are not limited to, Daptomycin. Lipopeptides are effective, e.g., against Gram-positive bacteria. Lipopeptides are believed to bind to the bacterial membrane and cause rapid depolarization.
[288] Macrolides include, but are not limited to, Azithromycin, Clarithromycin, Dirithromycin, Erythromycin, Roxithromycin, Troleandomycin, Telithromycin, and Spiramycin. Macrolides are effective, e.g., against Streptococcus and Mycoplasma. Macrolides are believed to bind to the bacterial or SOS ribosomal subunit, thereby inhibiting bacterial protein synthesis.
[289] Monobactams include, but are not limited to, Aztreonam. Monobactams are effective, e.g., against Gram-negative bacteria. Monobactams are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[290] Nitrofurans include, but are not limited to, Furazolidone and Nitrofurantoin.
[291] Oxazolidonones include, but are not limited to, Linezolid, Posizolid, Radezolid, and Torezolid. Oxazolidonones are believed to be protein synthesis inhibitors.
[292] Penicillins include, but are not limited to, Amoxicillin, Ampicillin, Azlocillin, Carbenicillin, Cioxacillin, Dicloxacillin, Flucioxacillin, Mezlocillin, Methicillin, Nafcillin, Oxacillin, Penicillin G, Penicillin V, Piperacillin, Temocillin and Ticarcillin. Penicillins are effective, e.g., against Gram-positive bacteria, facultative anaerobes, e.g., Streptococcus, Borrelia, and Treponema. Penicillins are believed to inhibit bacterial cell wall synthesis by disrupting synthesis of the peptidoglycan layer of bacterial cell walls.
[293] Penicillin combinations include, but are not limited to, Amoxicillin/clavulanate, Ampicillin/sulbactam, Piperacillin/tazobactam, and Ticarcillin/clavulanate.
[294] Polypeptide antibiotics include, but are not limited to, Bacitracin, Colistin, and Polymyxin B and E. Polypeptide Antibiotics are effective, e.g., against Gram-negative bacteria. Certain polypeptide antibiotics are believed to inhibit isoprenyl pyrophosphate involved in synthesis of the peptidoglycan layer of bacterial cell walls, while others destabilize the bacterial outer membrane by displacing bacterial counter-ions.
[295] Quinolones and Fluoroquinolone include, but are not limited to, Ciprofloxacin, Enoxacin, Gatifloxacin, Gemifloxacin, Levofloxacin, Lomefloxacin, Moxifloxacin, Nalidixic acid, Norfloxacin, Ofloxacin, Trovafloxacin, Grepafloxacin, Sparfloxacin, and Temafloxacin. Quinolones/Fluoroquinolone are effective, e.g., against Streptococcus and Neisseria. Quinolones/Fluoroquinolone are believed to inhibit the bacterial DNA gyrase or topoisomerase IV, thereby inhibiting DNA replication and transcription.
[296] Sulfonamides include, but are not limited to, Mafenide, Sulfacetamide, Sulfodiazine, Silver sulfodiazine, Sulfodimethoxine, Sulfamethizole, Sulfamethoxazole, Sulfanilimide, Sulfasalazine, Sulfisoxazole, Trimethoprim-Sulfamethoxazole (Co- trimoxazole), and Sulfonamidochrysoidine. Sulfonamides are believed to inhibit folate synthesis by competitive inhibition of dihydropteroate synthetase, thereby inhibiting nucleic acid synthesis.
[297] Tetracyclines include, but are not limited to, Demeclocycline, Doxycycline, Minocycline, Oxytetracycline, and Tetracycline. Tetracyclines are effective, e.g., against Gram-negative bacteria. Tetracyclines are believed to bind to the bacterial 30S ribosomal subunit thereby inhibiting bacterial protein synthesis.
[298] Anti-mycobacterial compounds include, but are not limited to, Clofazimine, Dapsone, Capreomycin, Cycloserine, Ethambutol, Ethionamide, Isoniazid, Pyrazinaniide, Rifampicin, Rifabutin, Rifopentine, and Streptomycin.
[299] Suitable antibiotics also include arsphenamine, chloramphenicol, fosfomycin, fusidic acid, metronidazole, mupirocin, platensimycin, quinupristin/dalfopristin, tigecycline, tinidazole, trimethoprim amoxicillin/clavulanate, ampicillin/sulbactam, amphomycin ristocetin, azithromycin, bacitracin, buforin II, carbomycin, cecropin Pl, clarithromycin, erythromycins, furazolidone, fusidic acid, Na fusidate, gramicidin, imipenem, indolicidin, josamycin, magainan II, metronidazole, nitroimidazoles, mikamycin, mutacin B-Ny266, mutacin B-JH1 140, mutacin J-T8, nisin, nisin A, novobiocin, oleandomycin, ostreogrycin, piperacillin/tazobactam, pristinamycin, ramoplanin, ranalexin, reuterin, rifaximin, rosamicin, rosaramicin, spectinomycin, spiramycin, staphylomycin, streptogramin, streptogramin A, synergistin, taurolidine, teicoplanin, telithromycin, ticarcillin/clavulanic acid, triacetyloleandomycin, tylosin, tyrocidin, tyrothricin, vancomycin, vemamycin, and virginiamycin.
[300] In some embodiments, the additional therapeutic is metabolic disease therapeutic, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof. Representative agents include, but are not limited to, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs (e g., hydroxychloroquine and chloroquine), sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNF alpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra /Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Haris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®/ LymphoStat-B® (belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists (Fontolizumab), prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura- 100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D - ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus (Prograf®), RADOO1, rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone, Curcumin (Longvida™), Rosuvastatin, Maraviroc, ramipnl, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonistis, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists (Tysarbri® (natalizumab)), VGEF antagnosits, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonsits), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.). Examples of metabolic disease therapeutics include, but are not limited to, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3-fatty acids, statins, ezetimibe, and/or ursodeoxycholic acid.
[301] In some embodiments, the method further comprises administering to the subject a second therapeutic bacterial strain. In some embodiments, the second therapeutic bacterial strain is of the Methanobacteriaceae family. In some embodiments, the second therapeutic bacterial strain v& Methanobacterium aarhusense, Methanobacterium aggregans, Methanobacterium alcaliphilum, Methanobacterium arcticum, Methanobacterium beijingense, Methanobacterium bryantii, Methanobacterium congolense, Methanobacterium curvum, Methanobacterium espanolae, Methanobacterium ferruginis, Methanobacterium flexile, Methanobacterium formicicum, Methanobacterium ivanovii, Methanobacterium kanagiense, Methanobacterium lacus, Methanobacterium movens, Methanobacterium movilense, Methanobacterium oryzae, Methanobacterium paludis, Methanobacterium palustre, Methanobacterium petrolearium, Methanobacterium subterraneum, Methanobacterium thermaggregans, Methanobacterium uliginosum, Methanobacterium veterum, Methanobrevibacter acididurans, Methanobrevibacter arboriphilus, Methanobrevibacter boviskoreani, Methanobrevibacter curvatus, Methanobrevibacter cuticularis, Methanobrevibacter filiformis, Methanobrevibacter gottschaUcii, Methanobrevibacter millerae, Methanobrevibacter olleyae, Methanobrevibacter oralis, Methanobrevibacter ruminantium, Methanobrevibacter smithii, Methanobrevibacter thaueri, Methanobrevibacter woesei, Methanobrevibacter wolimi, Methanosphaera cuniculi, Methanosphaera stadtmanae, Methanothermobacter crinale, Methanothermobacter defluvii, Methanothermobacter marburgensis, Methanothermobacter tenebrarum, Methanothermobacter thermautotrophicus, Methanothermobacter thermoflexus, Methanothermobacter thermophiles, or Methanothermobacter wolfeii. In preferred embodiments, the second therapeutic bacterial strain is Methanobrevibacter smithii.
Metabolic Disease
[302] Further disclosed are methods of treating a condition selected from overweight, obesity, metabolic syndrome, excess adiposity, or diabetes in a subject. The methods can optionally include measuring the levels of Christensenellaceae in a biological sample from a subject; determining the amount of a composition of substantially purified Christensenellaceae to administer to the subject, based on the levels of Christensenellaceae in said sample; and administering a composition to the subject in an amount effective to treat the condition.
[303] Overweight and obese individuals frequently suffer from weight-related disorders such as insulin resistance, diabetes, including type 2 diabetes, and dyslipidemia. These individuals also frequently suffer from hypertension, increased risk for cardiovascular diseases such as atherosclerosis and coronary heart disease. Other health conditions caused or exacerbated by overweight and obesity' include sleep apnea, obesity-related hypoventilation, back and joint problems, non-alcoholic fatty liver disease and gastroesophageal reflux disease. In some embodiments, the obesity does not comprise hypertriglyceridemia.
[304] Metabolic syndrome is characterized by the presence of three or more of these components: an elevated waist circumference in men of equal to or greater than 40 inches (102 cm); an elevated waist circumference in women of equal to or greater than 35 inches (88 cm); elevated triglycerides of equal to or greater than 150 mg/dL; reduced HDL (“good”) cholesterol in men of less than 40 mg/dL, or less than 50 mg/dL in women; elevated blood pressure of equal to or greater than 130/85 mm Hg; and elevated fasting glucose equal to or greater than 100 mg/dL. In some embodiments, the metabolic syndrome does not comprise elevated triglycerides of equal to or greater than 150 mg/dL, e.g., does not comprise hypertriglyceridemia. [305] Diabetes mellitus, often simply referred to as diabetes, is a condition in which a person has a high blood sugar (glucose) level, either because the body doesn't produce enough insulin, or because body cells don't properly respond to the insulin that is produced. Diabetes can be diagnosed, for example, from an oral glucose tolerance test as a 2-hour plasma glucose concentration of greater than or equal to 200 mg/dl. Insulin is a hormone produced in the pancreas that enables body cells to absorb glucose, to turn into energy. If the body cells do not absorb the glucose, the glucose accumulates in the blood (hyperglycemia), leading to vascular, nerve, and other complications. Type 1 diabetes results from the body's failure to produce insulin, and presently requires the person to inject insulin. Type 2 diabetes results from insulin resistance, a condition in which cells fail to use insulin properly, sometimes combined with an absolute insulin deficiency.
[306] The single greatest risk factor for developing type 2 diabetes is being overweight or obese. Almost 90% of people living with type 2 diabetes are overweight or obese. People who are overweight or obese have added pressure on their body's ability to use insulin to properly control blood sugar levels, and are therefore more likely to develop diabetes.
[307] A therapeutically effective amount can be administered to a patient in one or more doses sufficient to palliate, ameliorate, stabilize, reverse or slow the progression of the disease, or otherwise reduce the pathological consequences of the disease, or reduce the symptoms of the disease. The amelioration or reduction need not be permanent, but can be for a period of time ranging from at least one hour, at least one day, or at least one week or more. The effective amount is generally determined by the physician on a case- by-case basis and is within the skill of one in the art. Several factors are typically taken into account when determining an appropriate dosage to achieve an effective amount. These factors include age, sex and weight of the patient, the condition being treated, the severity of the condition, as well as the route of administration, dosage form and regimen and the desired result.
[308] In some embodiments, the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, nonalcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease. In further embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
[309] In some embodiments, the methods and compositions described herein relate to the treatment or prevention of a metabolic disease or disorder a, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fiitty liver disease (NAFLD), or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
[310] The methods described herein can be used to treat any subject in need thereof.
[311] The compositions described herein can be used, for example, for preventing or treating (reducing, partially or completely, the adverse effects of) a metabolic disease, such as type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fiitty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease. In some embodiments, the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
EXAMPLES
Example 1: Christensenella minuta Growth Media
[312] Christensenella minuta was grown in the following media:
Table 3: Christensenella minuta Growth Media
Figure imgf000082_0001
Figure imgf000083_0001
Example 2: Christensenella minuta smEV Preparation
[313] Christensenella minuta can be prepared as follows:
[314] 1. Clarification a. Centrifuge cells (15,500 x g, 15 min, 4°C) b. Filter to 0.2 μ
Figure imgf000083_0002
m. c. Aliquot 1.3 mL clarified supernatant. Freeze at -20°C.
[315] 2. Concentration a. Tangential flow filtration (TFF) (remove < 100 kDa species, concentrate supe, and buffer exchange with PBS). b. High-speed pellet (HSP):
1. Pellet mEVs by ultracentrifugation. Spin at 200,000 x g, 1 hr, 4°C.
2. Resuspend in a minimal volume of PBS. Raise volume to (2.5x + 0.3) mL. Set aside 0.3 mL for further analysis; store at 4°C for up to a week or - 20°C longer.
[316] 3. Purification: Optiprep gradients a. Dilute sample to 45% Optiprep by adding 3 volumes of 60% Optiprep and mixing thoroughly. Set up step gradient with steps of 35%, 25%, 15%, and no Optiprep, diluted with PBS. b. Spin in ultracentrifuge at 200,000 x g, 4-24 hr, 4°C. c. Remove and save fractions fiom the gradient. Store fractions at 4°C for up to a week or -20°C indefinitely. d. Quantify fractions, such as by protein concentration. e. Remove Optiprep from fractions of interest:
1. Dilute fraction at least 15-fold in PBS. Mix thoroughly.
2. Fractionated HSP (FHSP): Spin in ultracentrifuge at 200,000 x g, 1 hr, 4°C.
3. Resuspend pellet in minimal volume of PBS. Store at 4°C for up to a week or -20°C longer.
Example 3: Administration of Christensenella Inhibits Weight and Adiposity Gains
[317] Christensenella minuta is added to Donor Stool to Reduce Adiposity Gains in Recipient Mice. Experiments in which a donor stool lacking detectable
Christensenella are amended with non-living C. minuta bacteria or extracellular vesicles, and weight gain of recipient mice is monitored. One obese human donor is selected from the 21 donors from the first transplant experiment based on its lack of detectable OTUs assigned to the genus Christensenella. At Day 21 post-gavage, mice receiving the nonliving C. minuta bacteria or extracellular vesicles treatment are weighed and may weigh significantly less than those that receive unamended stool. Adiposity is measured and may be significantly lower for mice receiving the non-living C. minuta bacteria or extracellular vesicles treatment. Energy content for stool collected at Day 21 is measured and may not differ between treatments.
Incorporation bv Reference
[318] All publications patent applications mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. In case of conflict, the present application, including any definitions herein, will control.
Equivalents
[319] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

What is claimed is:
1. A method of treating and/or preventing a metabolic disease in a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising Christensenella minuta bacteria, wherein at least 50% of the Christensenella minuta in said composition are non-living.
2. The method of claim 1, wherein the metabolic disease comprises obesity.
3. The method of claim 1, wherein the metabolic disease comprises metabolic syndrome.
4. The method of claim 1, wherein the metabolic disease comprises diabetes.
5. A method of inhibiting weight gain, promoting weight loss, or reducing adiposityin a mammalian subject in need thereof, the method comprising administering to the subject an effective amount of a composition comprising Christensenella minuta bacteria, wherein at least 50% of the Christensenella minuta in said composition are non-living.
6. The method of any one of claims 1 to 5, wherein the composition comprises fewer than 108 colony forming units (CPUs) of the Christensenella minuta bacteria.
7. The method of claim 6, wherein the composition comprises fewer than 106 colony forming units (CPUs) of the Christensenella minuta bacteria.
8. The method of any one of claims 1 to 7, wherein at least 90% of the Christensenella minuta in said composition are non-living.
9. The method of any one of claims 1 to 7, wherein substantially all of the Christensenella minuta in said composition are non-living.
10. The method of claim 1 to 9, wherein said administration does not increase the level of living Christensenella minuta bacteria relative to the levels of other bacteria in the gastrointestinal tract of said subject.
11. The method of any one of claims Ito 10, wherein the Christensenella minuta is a strain comprising at least 90% 16S sequence identity to any one of SEQ ID NOS: 1 or 5- 8.
12. The method of any one of claims Ito 10, wherein the Christensenella minuta is a strain comprising at least 99% 16S sequence identity to SEQ ID NO: 1 or 5-8.
13. A method of treating and/or preventing a metabolic disease in a mammalian subject in need thereof, comprising administering to the subject of an effective amount of a composition comprising Christensenella minuta microbial extracellular vesicles (mEVs).
14. The method of claim 13, wherein the metabolic disease comprises obesity.
15. The method of claim 13, wherein the metabolic disease comprises metabolic syndrome.
16. The method of claim 13, wherein the metabolic disease comprises diabetes.
17. A method of inhibiting weight gain, promoting weight loss, or reducing adiposity in a mammalian subject in need thereof, comprising administering to the subject of an effective amount of a composition comprising Christensenella minuta microbial extracellular vesicles (mEVs).
18. The method of any one of claims 9 to 17, where the composition comprises isolated Christensenella minuta microbial extracellular vesicles (mEVs).
19. The method of claim 18, wherein the composition comprises Christensenella minuta microbial extracellular vesicles (mEVs) are substantially free from living Christensenella minuta bacteria.
20. The method of any one of claims 9-19, wherein at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% of the composition is mEVs.
21. The method of any one of claims 9-20, wherein the composition comprises secreted mEVs (smEVs).
22. The method of any one of claims 9-20, wherein the composition comprises processed mEVs (pmEVs).
23. The method of any one of claims 9-22, wherein the Christensenella minuta is a strain comprising at least 90% 16S sequence identity to any one of SEQ ID NOS: 1 or 5- 8.
24. The method of any one of claims 9-22, wherein the Christensenella minuta is a strain comprising at least 99% 16S sequence identity to SEQ ID NO: 1 or 5-8.
25. The method of any one of claims 9-24, wherein the mEVs comprise pmEVs and the pmEV s are produced from bacteria that have been gamma irradiated, UV irradiated, heat inactivated, acid treated or oxygen sparged.
26. The method of any one of claims 9-24, wherein the mEVs comprise pmEVs and the pmEVs are produced from live bacteria.
27. The method of any one of claims 9-24, wherein the mEVs are lyophilized (e.g., the lyophilized product further comprises a pharmaceutically acceptable excipient).
28. The method of any one of claims 9-23, wherein the dose of mEVs is about 5 mg to about 900 mg total protein (e.g., wherein total protein is determined by Bradford assay).
29. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenella mEVs.
30. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenella bacteria particles.
31. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total proteins in the composition are Christensenella mEVs protein.
32. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total proteins in the composition are Christensenella bacteria proteins.
33. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenella mEVs lipids.
34. The method of any one of claims 9-28, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenella bacteria lipids.
35. The method of claim 1, wherein the composition comprises non-living Christensenella minuta bacteria isolated from mEVs.
36. The method of any preceding claim, wherein the composition is administered orally, rectally, intravenously, intradermally, intraperitoneally, or subcutaneously.
37. The method of any one of claims 1 to 36, wherein said administration comprises administration on a daily or weekly basis.
38. The method of any one of claims 1 to 37, wherein the composition is administered in two or more doses.
39. The method of claim 38, wherein the administration to the subject of the two or more doses are separated by at least 1 day.
40. The method of claim 39, wherein the administration of the two or more doses are separated by at least 1 week.
41. The method of any one of claims 1 to 40, wherein the composition comprises attenuated bacteria.
42. The method of any one of claims 1 to 40, wherein the composition comprises killed bacteria.
43. The method of claim 42, wherein the composition comprises irradiated bacteria.
44. The method of claim 42, wherein the composition comprises gamma irradiated bacteria.
45. The method of any one of claims 1 to 44, wherein the composition comprises lyophilized bacteria.
46. The method of any one of claims 1 to 45, wherein the metabolic disease is type II diabetes, impaired glucose tolerance, insulin resistance, obesity, hyperglycemia, hyperinsulinemia, fatty liver, non-alcoholic steatohepatitis, hypercholesterolemia, hypertension, hyperlipoproteinemia, hyperlipidemia, hypertriglylceridemia, ketoacidosis, hypoglycemia, thrombotic disorders, dyslipidemia, non-alcoholic fatty liver disease (NAFLD), or a related disease.
47. The method of claim 46, wherein the related disease is cardiovascular disease, atherosclerosis, kidney disease, nephropathy, diabetic neuropathy, diabetic retinopathy, sexual dysfunction, dermatopathy, dyspepsia, or edema.
48. The method of any one of claims 1 to 47, wherein administration of the composition treats the metabolic disease.
49. The method of any one of claims 1 to 48, wherein the method further comprises administering to the subject an additional therapeutic.
50. The method of claim 49, wherein the additional therapeutic is selected from the group consisting of an immunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), a cytokine antagonist, cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprofen, cholin magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib, acetaminophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs, hydroxychloroquine, chloroquine, sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline, cyclophosphamide, D- penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists, TNF alpha antagonists, TNF alpha receptor antagonists, ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOUZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simpom®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra ZActemra®), integrin antagonists, TYSABRI® (natalizumab), IL-1 antagonists, ACZ885 (Haris), Anakinra (Kineret®), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists, Atacicept, Benlysta®/ LymphoStat-B® (belimumab), p38 Inhibitors, CD20 antagonists, Ocrelizumab, Ofatumumab (Arzerra®), interferon gamma antagonists, Fontolizumab, prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura- 100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D - ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus, Prograf®, RADOO1, rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium, rosightazone, Curcumin, Longvida™, Rosuvastatin, Maraviroc, ramipnl, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAK1 and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL-6 antagonists, CD20 antagonists, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist, integrin antagonists, Tysarbri® (natalizumab), VGEF antagnosits, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists, IL-1 beta antagonsits, IL-23 antagonists, receptor decoys, antagonistic antibodies, corticosteroids, mesalazine, mesalamine, sulfasalazine, sulfasalazine derivatives, immunosuppressive drugs, cyclosporin A, mercaptopurine, azathiopurine, prednisone, methotrexate, antihistamines, glucocorticoids, epinephrine, theophylline, cromolyn sodium, antileukotrienes, anti-cholinergic drugs for rhinitis, TLR antagonists, inflammasome inhibitors, anti-cholinergic decongestants, mast-cell stabilizers, monoclonal anti-IgE antibodies, vaccines, cytokine inhibitors, anti-IL-6 antibodies, TNF inhibitors, infliximab, adalimumab, certolizumab pegol, golimumab, and etanercept.
51. The method of claim 38 or 39, wherein the additional therapeutic is an antibiotic.
52. The method of claim 51, wherein the antibiotic is selected from the group consisting of aminoglycosides, ansamycins, carbacephems, carbapenems, cephalosporins, glycopeptides, lincosamides, lipopeptides, macrolides, monobactams, nitrofurans, oxazolidonones, penicillins, polypeptide antibiotics, quinolones, fluoroquinolone, sulfonamides, tetracyclines, anti-mycobacterial compounds and combinations thereof.
53. The method of claim 49, wherein the additional therapeutic is a metabolic disease therapeutic.
54. The method of claim 53, wherein the metabolic disease therapeutic is metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin, pioglitazone, rosiglitazone, pentoxifylline, omega-3- fetty acids, statins, ezetimibe, or ursodeoxycholic acid.
55. The method of any one of claims 1 to 54, wherein the method further comprises administering to the subject a second therapeutic bacterial strain.
56. The method of any one of claims 1 to 55, wherein the method further comprises administering a prebiotic to the subject.
57. The method of claim 56, wherein the prebiotic is a fructooligosaccharide, a galactooligosaccharide, a trans-galactooligosaccharide, a xylooligosaccharide, a chitooligosaccharide, a soy oligosaccharides, a gentiooligosaccharide, an isomaltooligosaccharide, a mannooligosaccharide, a maltooligosaccharide, a mannanoligosaccharide, lactulose, lactosucrose, palatinose, glycosyl sucrose, guar gum, gum Arabic, tagalose, amylose, amylopectin, pectin, xylan, or a cyclodextrin.
58. The method of any one of claims 1 to 57, wherein the composition is formulated as a food or drink.
59. The method of any one of claims 1 to 58, wherein the subject is a human.
60. The method of any one of claims 1 to 58, wherein the subject is a non-human mammal.
61. The method of claim 60, wherein the mammal is selected from the group consisting of a dog, a cat, a cow, a horse, a pig, a donkey, a goat, a camel, a mouse, a rat, a guinea pig, a sheep, a llama, a monkey, a gorilla or a chimpanzee.
62. A composition comprising isolated Christensenella microbial extracellular vesicles (mEVs).
63. A composition comprising Christensenella microbial extracellular vesicles (EVs) and non-living Christensenella bacteria.
64. The composition claim 62 or 63, wherein the Christensenella mtnuta is a strain comprising at least 90% 16S sequence identity to any one of SEQ ID NOS: 1 or 5-8.
65. The composition claim 62 or 63, wherein the Christensenella mtnuta is a strain comprising at least 99% 16S sequence identity to SEQ ID NO: 1 or 5-8.
66. The composition of any one of claims 62-65, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenella mEVs.
67. The composition of any one of claims 62-65, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total particles in the composition are Christensenella. bacteria particles.
68. The composition of any one of claims 62-65, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total proteins in the composition are Christensenella mEVs proteins.
69. The composition of any one of claims 62-65, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total proteins in the composition are Christensenella bacteria proteins.
70. The composition of any one of claims 62-65 composition of any one of claims 54- 57, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenella mEVs lipids.
71. The composition of any one of claims 62-65, wherein at least, about, or no more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the total lipids in the composition are Christensenella bacteria lipids.
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