Methods and compositions for treating infectious, autoimmune, and allergic diseases

Abstract

The present disclosure provides methods and compositions for treating food allergies, infections, autoimmune conditions, and other allergic conditions. Accordingly, the present disclosure relates to a method for treating an infectious disease, an autoimmune disease, or an allergic disease in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes caccae. A further aspect relates to a method for treating a food allergy, reducing an allergic response to an allergen, or treating or preventing an anaphylactic reaction in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes caccae and a prebiotic.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 755,945, filed November 5, 2018, which is incorporated herein by reference in its entirety.

[0002] 1. Field of the Invention The present invention relates to the fields of molecular biology and medicine. [Background technology]

[0003] 2.Background Food allergy has become a major public health problem due to the significant intergenerational increase in disease prevalence in Westernized societies (1, 2). One hypothesis for this rapid increase in prevalence proposes that recent lifestyle factors, including increased antibiotic use, dietary changes, and higher rates of cesarean section and artificial feeding, have altered the composition of the intestinal commensal microbiota, increasing susceptibility to allergic diseases. Host-microbiota interactions are essential for establishing proper immune homeostasis, and disruption of naturally selected bacterial populations, often referred to as dysbiosis, has been implicated in many different pathologies; some studies suggest that early-life dysbiosis may be particularly harmful (3, 4).

[0004] There is a need in the art for compositions that modify the microbiome for the effective treatment of allergies and other related conditions. Summary of the Invention

[0005] The present disclosure fulfills a need in the art by providing methods and compositions for treating food allergies, infections, autoimmune conditions, and other atopic conditions. Accordingly, the present disclosure relates to a method for treating an infectious disease, an autoimmune disease, or an allergic disease in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae. A further aspect relates to a method for treating a food allergy, reducing an allergic response to an allergen, or treating or preventing an anaphylactic reaction in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. In some aspects, the method relates to treating a subject with or at risk of developing a food allergy or an anaphylactic reaction, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. For example, a subject may be at risk because of a family history of food allergy or a genetic predisposition to food allergy, or may be at risk for an anaphylactic reaction due to accidental exposure to an allergen. In some aspects, the subject has an adverse microbial profile or signs that a food allergy may exist or may develop in the subject. In other aspects, a method relates to reducing an allergic response to an allergen in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. In a further aspect, a method relates to treating or preventing an anaphylactic reaction in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. A further aspect relates to a method for treating an atopic disease in a subject in need thereof, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic.In some aspects, the method is for treating atopy in a subject by administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic.

[0006] A further aspect relates to a method of diagnosing a subject as having a food allergy, comprising determining a protective / non-protective operational taxonomic unit (OTU) ratio, wherein the subject is diagnosed with a food allergy if the ratio is less than 3. A further aspect relates to a method of diagnosing a subject as having an allergic condition, infection, or autoimmune disorder, comprising determining a protective / non-protective operational taxonomic unit (OTU) ratio, wherein the subject is diagnosed with an allergic condition, infection, or autoimmune disorder if the ratio is less than 3. In some embodiments, the protective / non-protective operational taxonomic unit (OTU) ratio is less than, greater than, or about 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5 (or any range derivable therein).

[0007] A further aspect of the present disclosure relates to a composition comprising the bacterium Anaerostipes cacae and a prebiotic. A further aspect relates to a tablet, capsule, or powder comprising the composition of the present disclosure.

[0008] Listed below are specific embodiments that can be used in conjunction with any of the aspects described above and herein.

[0009] In some embodiments of the above aspects, the method relates to treating an atopic disease, hi some embodiments, the atopic disease includes eczema, atopic dermatitis, asthma, or allergic rhinitis.

[0010] In some embodiments, the method is for reducing an allergic response to an allergen in a subject. In some embodiments, the subject has been determined to have an allergic response to an allergen. Allergic responses can take multiple forms and have multiple levels of severity or intensity. These allergic responses can vary from person to person, and also vary over time for a given individual. Systems for scoring the severity or intensity of a response are known and described in the art. For example, a scoring system is described in Sampson et al., J. Allergy Clin. Immunol. Vol 130(6) p.1260 (2012), which is incorporated by reference for all purposes. The phrase "reduced allergic response" should be understood to mean a reduction in the severity or intensity of an allergic response as measured by one of the scoring systems known in the art. In some embodiments, the reduction of an allergic response relates to a statistically significant reduction in allergic response. In some embodiments, the average severity or intensity of allergic responses in a population of humans or animals is reduced. In some embodiments, allergic reaction may refer to a response to any atopic disease, including food allergies, eczema, asthma, and allergic rhinitis. In some embodiments, the allergic reaction is reduced by at least one grade as scored by the scoring system described in Sampson et al. For example, in certain embodiments, the allergic reaction is reduced from grade 3 to grade 2. In some embodiments, the allergic reaction is reduced from grade 2 to grade 1. In some embodiments, the allergic reaction is reduced from grade 1 to grade 0. In some embodiments, the allergic reaction is reduced from grade 3 to grade 1. In some embodiments, the allergic reaction is reduced from grade 3 to grade 0. In some embodiments, the allergic reaction is reduced from grade 3 to grade 0. In some embodiments, the allergic reaction is reduced from grade 2 to grade 0.

[0011] The term "prebiotic" refers to oligosaccharides or polysaccharides with a degree of polymerization of 2 or greater that are resistant to digestion or degradation before entering the upper gastrointestinal tract, such as the small intestine, and that are fermentable or digestible by microorganisms or other processes in the colon, where the fermented or digested oligosaccharides, or by-products of digestion, modify the microbiome or provide a benefit to humans or animals.

[0012] In some embodiments, the method is for treating an anaphylactic reaction. In some embodiments, the anaphylactic reaction is due to a food allergy. In some embodiments, the anaphylactic reaction is due to a drug allergy. In some embodiments, the anaphylactic reaction is due to an insect sting.

[0013] In some embodiments, the short chain oligosaccharides are 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, 30, 40, 50, 60, 65, 70, 75, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, The degree of polymerization is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 40, 50, 60, 65, 70, 75, or 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, 30, 40, 50, 60, 65, 70, 75. In some embodiments, the prebiotic comprises galactooligosaccharides (GOS). GOS can be found in large amounts in human breast milk, which often contains significant amounts of lactose, and can be degraded by β-galactosidase. Specific examples of GOS include stachyose, raffinose, verbascose, β(1-4)galactosyl lactose (4'-galactooligosaccharide) and β(1-6)galactosyl lactose (6'-galactooligosaccharide), and lactulose. In some embodiments, prebiotics include fructans (including short-chain fructooligosaccharides (scFOS), fructooligosaccharides (FOS), and inulin), galactans, glucans, and other oligosaccharides.Examples of such include short-chain FOS (shorter chains of fructose, described below, with a degree of polymerization of 2-4); fructooligosaccharides (with a degree of polymerization of 4-20); inulin or phlein (with a degree of polymerization of >20); soybean oligosaccharides (SOS); galactooligosaccharides (GOS); isomaltooligosaccharides (IMOS) (derived from wheat, barley, corn, oat, tapioca, rice, potato starch), such as isomaltose, isomaltotriose, and panose; soybean oligosaccharides (SOS) (derived from soybeans), such as raffinose and the tetrasaccharide stachyose; xylooligosaccharides (XOS), such as Examples include xylans, xylobioses, xylotrioses, and xylotetraoses (derived from starches found in bamboo shoots, fruits, vegetables, milk, and honey); pectic oligosaccharides (POS), such as pectin; chitooligosaccharides, such as chitin; lactulose; beta-glucans (derived from grains such as oats, barley, wheat, and rye); and type III resistant starches, including those formed when starch-containing foods (e.g., pasta, potatoes, and rice) are cooled. Further examples include polyols such as isomalt, maltitol, mannitol, sorbitol, xylitol, lactitol, erythritol, and polyglycitol. Sources of polyols include apples, apricots, avocados, blackberries, cherries, lychees, nectarines, peaches, pears, plums, prunes, watermelons, cauliflower, and mushrooms. Further examples include dextrins, such as maltodextrin, cyclodextrin, and pyrodextrin (derived from potato and corn starch), wheat dextrin, high amylose corn starch (and corn starch), amylose, and non-fructans such as amylopectin. In some embodiments, the prebiotic includes one or more of galactooligosaccharides, lactulose, lactitol, erythritol, isomalt, polyglycitol, acetic acid, and lactic acid.In some embodiments, the prebiotic includes one or more of galactooligosaccharides, lactulose, lactic acid, acetic acid, and lactitol.

[0014] Derivatives and processed forms of the compounds described herein are also included. Derivatives or processed forms may be modified to change the fermentation properties of prebiotics, for example, by making them more digestible or specific to certain types of bacteria, or to increase the yield of fermentation products such as short-chain fatty acids (SCFAs) and / or other metabolites. Also included are foods and food derivatives that are known to contain large amounts of these compounds and / or can have prebiotic effects. These foods may be processed to isolate starch, or may be administered for consumption without isolating starch, for example, by grinding the whole food. Examples of such foods include onions, artichokes, garlic, wheat, bananas, asparagus, chicory, leeks, tomatoes, bamboo shoots, fruits, vegetables, milk, honey, wheat, rye, barley, corn, oats, tapioca, rice, and potatoes.

[0015] The term "prebiotic derivative" refers to modified forms of prebiotics prepared prior to consumption to increase fermentation properties, digestibility, or to increase fermentation by products such as short-chain fatty acids and other metabolites.

[0016] In some embodiments, the prebiotics comprise one or both of digestible and non-digestible oligosaccharides. In some embodiments, the prebiotics comprise at least 6 grams of non-digestible oligosaccharides. In some embodiments, the prebiotics comprise 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, 30, or 40 grams, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, The plant material may contain 20, 21, 22, 23, 24, 25, 30, or 40 grams, or 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, 30, or 40 grams (or any range derivable therein) of digestible and / or non-digestible oligosaccharides. The term "non-digestible oligosaccharides" as used herein refers to parts of a plant that cannot be digested by animals. Non-digestible oligosaccharides may be water-soluble (those that can be dissolved in water) or water-insoluble (those that do not dissolve in water). Specific examples of suitable non-digestible oligosaccharides useful in the methods and compositions of the present disclosure include one or more of inulin (derived from chicory or agave), flax fiber, soy fiber, oat fiber, corn fiber, guar gum, gum Arabic, larch bark, bean gum, gum acacia, pumpkin fiber, chia fiber, and combinations thereof.

[0017] In some embodiments, the oligosaccharides include modified oligosaccharides. In some embodiments, the modified oligosaccharides include butyrate-releasing oligosaccharides that are fermentable by A. caccae. Butyrate-releasing refers to the production of the metabolic product butyrate as a by-product of fermentation. In some embodiments, the oligosaccharides increase the concentration, mass, or amount of lactic acid in the gastrointestinal tract after fermentation or digestion. Examples include wheat, rye, corn, oats, rice, and potato. In some embodiments, the butyrate-releasing oligosaccharides fermentable by A. caccae include compounds that can be fermented to butyrate by A. caccae.

[0018] In some embodiments, the method further comprises administering a butyrate transport compound. In some embodiments, the butyrate transport compound comprises pHPMA-b-pBMA, as further described in WO 2018 / 195067. In some embodiments, the butyrate transport compound comprises those disclosed in WO 2018 / 195067, which is incorporated by reference. In some embodiments, the butyrate transport compound is administered by gavage.

[0019] In some embodiments, 1 x 10 6 ~1×10 15 Cells or CFU of A. cacae are administered to the subject. In some embodiments, at least 1 x 10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , or 1×10 15 cells or CFU, or at most 1 x 10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×1013 , 1×10 14 , or 1×10 15 cells or CFU, or approximately 1 x 10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , or 1×10 15 Cells or CFU (or any range derivable therefrom) of A. cacae are administered.

[0020] In some embodiments, A. cacae, prebiotics, and / or butyrate transport compounds are administered simultaneously. In some embodiments, A. cacae is administered at least 1 hour before the prebiotics and / or butyrate transport compounds. In some embodiments, A. cacae and / or butyrate transport compounds are administered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, or 18 hours, or 1, 2, 3, 4, 5, or 6 days, or 1, 2, 3, 4, 5, 6, 7, or 8 weeks (or any range derivable therein) before or after the prebiotics and / or butyrate transport compounds. In some embodiments, at least 10 grams of prebiotics are administered to the subject. 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, 30, 40, or 50 grams, or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, In some embodiments, 21, 22, 23, 24, 25, 30, 40, or 50 grams of prebiotic is administered to a subject, or 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, 30, 40, or 50 grams (or any range derivable therein). In some embodiments, the ratio of colony forming units of A. cocae to grams of prebiotic and / or butyrate transport compound is 1000:1 to 10000:1. In some embodiments, the ratio is at least 1:1, 2:1, 4:1, 5:1, 8:1, 10:1, 20:1, 40:1, 50:1, 80:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1000:1, 1500:1, 2000:1, 2500:1, 3000:1, 40 ...0:1, 6000:1, 6000:1, 7000:1, 7000:1, 8000:1, 8000:1, 9000:1, 9000:1, 10000:1, 10000:1, 110000:1, 120000:1, 13000:1, 14000:1, 15000:1, 16000:1, 17000:1, 18000:1, 19000:1, 21000:1, 22000:1, 23000:1, 24000:1, 25000:1, 26000:1, 27000:1, 28000:1, 29000:1, 30000:1, 31000:1, 32000:1, 33000:1, 3400 000:1, 3500:1, 4000:1, 4500:1, 5000:1, 7500:1, 10000:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:50, 1:80, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500, 1:1000,1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:7500, or 1:10000, or at most 1:1, 2:1, 4:1, 5:1, 8:1, 10:1, 20:1, 40:1, 50:1, 80:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1000:1, 1500:1, 2000:1, 25 00:1, 3000:1, 3500:1, 4000:1, 4500:1, 5000:1, 7500:1, 10000:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:50, 1:80, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500 , 1:5000, 1:7500, or 1:10000, or about 1:1, 2:1, 4:1, 5:1, 8:1, 10:1, 20:1, 40:1, 50:1, 80:1, 100:1, 150:1, 200:1, 250:1, 300:1, 400:1, 500:1, 1000:1, 1500:1, 2000:1, 2500:1, 3000:1, 3500:1, 4000:1, 4500:1, 5000:1, 7500:1, 1000 The butyrate transport compound is 0:1, 1:2, 1:4, 1:5, 1:8, 1:10, 1:20, 1:40, 1:50, 1:80, 1:100, 1:150, 1:200, 1:250, 1:300, 1:400, 1:500, 1:1000, 1:1500, 1:2000, 1:2500, 1:3000, 1:3500, 1:4000, 1:4500, 1:5000, 1:7500, or 1:10000 (or any range derivable therein). In some embodiments, the butyrate transport compound is administered after the prebiotic. In some embodiments, the butyrate transport compound and the prebiotic are in the same composition. In some embodiments, the butyrate transport compound is administered immediately after administration of the prebiotic, or within 5, 10, 20, 30, or 60 minutes (or any range derivable therein) after administration of the prebiotic.

[0021] In some embodiments, the food allergy includes milk allergy. In some embodiments, the food allergy includes peanut allergy or egg allergy. In some embodiments, the subject has been diagnosed with a food allergy, milk allergy, peanut allergy, egg allergy, soy allergy, wheat / gluten allergy, shellfish allergy, sesame allergy, tree nut (pistachio, cashew, walnut, almond, hazelnut, macadamia) allergy, allergic condition, autoimmune disease, or infection. In some embodiments, the subject has previously received treatment for a food allergy, allergic condition, autoimmune disease, or infection. In some embodiments, the subject has been determined to be resistant to previous treatment. In some embodiments, the subject has not been diagnosed with a food allergy, allergic condition, autoimmune disease, or infection and / or does not exhibit symptoms of a food allergy, allergic condition, autoimmune disease, or infection.

[0022] In some embodiments, the subject is a human. In some embodiments, the subject is a newborn, under 1 year old, under 5 years old, under 12 years old, or under 18 years old. In some embodiments, the subject is under 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 year old, or under 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month old, or under 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 month old, or under 14 days old, 13 days old, 12 days old, 11 days old, 10 days old, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day old (or any range derived therein). In particular embodiments, the patient is a pediatric patient, i.e., under the age of 18. In other embodiments, the patient is an adult patient.

[0023] In some embodiments, the A. cacae includes a live bacterial product. In some embodiments, the bacteria are lyophilized or freeze-dried. In some embodiments, the A. cacae and / or prebiotics are administered orally. In some embodiments, the A. cacae and / or prebiotics are administered in a tablet or capsule. In some embodiments, the A. cacae and / or prebiotics are administered by a route of administration described herein.

[0024] The term "food" or "food derivative" refers to cooked or uncooked foods that contain prebiotics found in the raw product or that have been processed to further isolate the prebiotics.

[0025] In some embodiments, the method further comprises administering a formula or food containing lactic acid. In some embodiments, the food is apple, apricot, avocado, blackberry, cherry, lychee, nectarine, peach, pear, plum, prune, watermelon, cauliflower, and mushroom, onion, artichoke, garlic, wheat, banana, asparagus, chicory, leek, tomato, bamboo shoot, fruit, vegetable, milk, honey, wheat, rye, barley, corn or maize, oat, tapioca, rice, and potato.

[0026] In some embodiments, the subject is determined to have a protective / non-protective operational taxonomic unit (OTU) ratio of less than 3. In some embodiments, the protective / non-protective operational taxonomic unit (OTU) ratio is less than, greater than, or about 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5 (or any range derivable therein).

[0027] In some embodiments, the protective OTUs include 1111294, 360015, New Reference OTU147, 628226, 349024, 712677, 780650, 843459, 262095, New Reference OTU166, 579851, 551822, 298247, 345540, 582691, 259772, 325419, 797229, 557627, 828483, 299267, 4448, as listed in Supplementary Table 3. 331, 3376513, 813217, New.CleanUp.Reference OTU56927, 335701, 191999, 183865, 231787, 342397, 581782, 304641, 4389289, or 541119. In some embodiments, the non-protective OTUs include one or more OTUs selected from 195258, 315846, 551902, 318190, 591635, 585227, 370225, 199354, 198866, 583398, 583656, 535375, 1111191, 580629, 365181, 359809, 585914, 365385, 583117, 180082, 589071, 514272, 484304, or 589277, as listed in Supplementary Table 3.

[0028] In some embodiments, the composition further comprises a pharmaceutical excipient. In some embodiments, the composition is formulated for oral administration.

[0029] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the method of measurement or quantification.

[0030] The use of the words "a" or "an," when used with the term "comprises," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0031] The term "and / or" means "and" or "or." By way of example, A, B, and / or C includes A alone, B alone, C alone, A and B in combination, A and C in combination, B and C in combination, or A, B and C in combination. In other words, "and / or" functions as an inclusive "or."

[0032] The words "comprising" (and any form of "comprising", such as "comprise" and "comprises"), "having" (and any form of "having", such as "have" and "has"), "including" (and any form of "including", such as "includes" and "include"), or "containing" (and any form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0033] Compositions and methods of their use can "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the scope of the claims to specified materials or steps that do not materially affect the basic and novel characteristics of the claimed invention.

[0034] It is contemplated that any embodiment described herein can be implemented with respect to any method or composition of the invention, and vice versa. Further, a composition of the invention can be used to achieve a method of the invention.

[0035] [The present invention 1001] A method for treating a food allergy, reducing an allergic reaction to an allergen, or treating or preventing an anaphylactic reaction in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes caccae and a prebiotic. [The present invention 1002] A method for treating a patient at risk of a food allergy or anaphylactic reaction, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. [The present invention 1003] A method for treating an atopic disease in a subject in need thereof, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae and a prebiotic. [The present invention 1004] 1004. The method of claim 1003, wherein said atopic disease comprises eczema, atopic dermatitis, asthma, or allergic rhinitis. [The present invention 1005] The method of any of claims 1001 to 1004, wherein the prebiotic comprises one or more of galactooligosaccharides, lactulose, lactitol, erythritol, isomalt, polyglycitol, acetic acid, and lactic acid. [The present invention 1006] 1005. The method of claim 10, wherein said prebiotic comprises one or more of galactooligosaccharides, lactulose, lactic acid, acetic acid, and lactitol. [The present invention 1007] The method of any of claims 1001 to 1006, wherein said prebiotics comprise one or more of digestible and non-digestible oligosaccharides. [The present invention 1008] 8. The method of any of claims 1001 to 1007, wherein said prebiotic comprises at least 6 grams of non-digestible oligosaccharides. [The present invention 1009] 1007. The method of any one of claims 1007 to 1008, wherein the oligosaccharide comprises a modified oligosaccharide. [The present invention 1010] 1009. The method of claim 10, wherein the modified oligosaccharides comprise butyrate-releasing oligosaccharides fermentable by A. caccae. [The present invention 1011] 1×10 6 ~1×10 15 The method of any of claims 1001 to 1010, wherein CFU of A. coccae are administered to said subject. [The present invention 1012] The method of any of claims 1001 to 1011, further comprising administration of a butyrate transport compound. [The present invention 1013] 1012. The method of claim 1012, wherein said butyrate transport compound comprises pHPMA-b-pBMA. [The present invention 1014] 1014. The method of any one of claims 1012 to 1013, wherein said butyrate transport compound is administered orally. [The present invention 1015] The method of any one of claims 1001 to 1014, wherein said A. coccae, said prebiotic and / or said butyrate transport compound are administered simultaneously. [The present invention 1016] 1012. The method of any one of claims 1001 to 1011, wherein said A. coccae is administered prior to said prebiotic and / or butyrate transport compound. [The present invention 1017] 1012. The method of any one of claims 1001 to 1011, wherein said A. coccae is administered after said prebiotic and / or butyrate transport compound. [The present invention 1018] 10. The method of any of claims 1001 to 1017, wherein said A. coccae is administered at least 1 hour before said prebiotic and / or butyrate transport compound. [The present invention 1019] The method of any of claims 1001 to 1017, wherein said butyrate transport compound is administered after said prebiotics. [The present invention 1020] Any of the methods of claims 1001 to 1019, wherein at least 10 grams of prebiotics are administered to said subject. [The present invention 1021] 10. The method of any one of claims 1001 to 1020, wherein the ratio of colony forming units of A. coccae to grams of prebiotic is 1000:1 to 10000:1. [The present invention 1022] 1022. The method of any one of claims 1001 to 1021, wherein said food allergy comprises milk allergy, egg allergy, peanut allergy, soybean allergy, wheat / gluten allergy, shellfish allergy, sesame allergy, or nut allergy. [The present invention 1023] The method of any of claims 1001 to 1022, wherein said subject has been diagnosed with a food allergy or an atopic disease. [The present invention 1024] The method of any of claims 1001 to 1023, wherein said subject has previously been treated for a food allergy or an atopic disease. [The present invention 1025] The method of claim 1024, wherein said subject has been determined to be refractory to a previous treatment. [The present invention 1026] The method of any one of claims 1001 to 1025, wherein the subject is a human. [The present invention 1027] The method of claim 1026, wherein the subject is under 1 year old, under 5 years old, under 12 years old, or under 18 years old. [The present invention 1028] 1028. The method of any one of claims 1001 to 1027, wherein said A. coccae comprises a live bacterial product. [The present invention 1029] The method according to any one of claims 1001 to 1028, wherein the bacterium is lyophilized or freeze-dried. [The present invention 1030] 1029. The method of any one of claims 1001 to 1029, wherein said A. cacae and / or said prebiotics are administered orally. [The present invention 1031] The method of claim 1030, wherein said A. cacae and / or said prebiotics are administered in a tablet or capsule. [The present invention 1032] 1032. The method of any one of claims 1001 to 1031, further comprising administering a formula or food containing lactic acid. [The present invention 1033] The method of any of claims 1001 to 1032, wherein the subject is determined to have a protective / non-protective operational taxonomic unit (OTU) ratio of less than 3. [The present invention 1034] A method for diagnosing a subject as having a food allergy, comprising determining a protective / non-protective operational taxonomic unit (OTU) ratio, wherein the subject is diagnosed as having a food allergy if the ratio is less than 3. [This invention 1035] The method of claim 1034, further comprising treating a subject diagnosed with a food allergy with a composition comprising the bacterium Anaerostipes cacae and a prebiotic. [The present invention 1036] 1035. The method of claim 1035, wherein said prebiotic comprises one or more of galactooligosaccharides, lactulose, lactitol, erythritol, isomalt, polyglycitol, lactic acid, and acetic acid. [This invention 1037] 1036. The method of claim 1036, wherein said prebiotic comprises one or more of galactooligosaccharides, lactulose, lactic acid, acetic acid, and lactitol. [The present invention 1038] 1038. The method of any of claims 1035 to 1037, wherein the prebiotic comprises one or more of digestible and non-digestible oligosaccharides. [This invention 1039] 1039. The method of any of claims 1035 to 1038, wherein the prebiotic comprises at least 6 grams of non-digestible oligosaccharides. [The present invention 1040] 1039. The method of any one of claims 1038 to 1039, wherein the oligosaccharide comprises a modified oligosaccharide. [The present invention 1041] 1040. The method of claim 1040, wherein the modified oligosaccharides comprise butyrate-releasing oligosaccharides fermentable by A. cocoa. [The present invention 1042] 1×10 6 ~1×10 15 1042. The method of any one of claims 1035 to 1041, wherein CFU of A. cocae are administered to said subject. [This invention 1043] The method of any of claims 1035 to 1042, further comprising administration of a butyrate transport compound. [This invention 1044] 1043. The method of claim 1043, wherein said butyrate transport compound comprises pHPMA-b-pBMA. [This invention 1045] 1045. The method of any one of claims 1043 to 1044, wherein said butyrate transport compound is administered orally. [The present invention 1046] 1046. The method of any one of claims 1035 to 1045, wherein said A. coccae, said prebiotic and / or said butyrate transport compound are administered simultaneously. [This invention 1047] 1043. The method of any of claims 1035 to 1042, wherein said A. coccae is administered prior to said prebiotic and / or butyrate transport compound. [This invention 1048] 1043. The method of any one of claims 1035 to 1042, wherein said A. coccae is administered after said prebiotic and / or butyrate transport compound. [This invention 1049] 1049. The method of any of claims 1035 to 1048, wherein said A. coccae is administered at least 1 hour before said prebiotic and / or butyrate transport compound. [The present invention 1050] 1049. The method of any of claims 1043 to 1049, wherein said butyrate transport compound is administered after said prebiotics. [This invention 1051] Any of the methods of claims 1035 to 1050, wherein at least 10 grams of prebiotics are administered to the subject. [This invention 1052] 1035-1051. The method of any one of claims 1035-1051, wherein the ratio of colony forming units of A. coccae to grams of prebiotic is 1000:1 to 10000:1. [This invention 1053] The method of any one of claims 1034 to 1052, wherein the food allergy includes a milk allergy. [This invention 1054] The method of any of claims 1034 to 1053, wherein said subject has not been diagnosed with a food allergy and / or does not exhibit symptoms of a food allergy. [This invention 1055] The method of any one of claims 1034 to 1054, wherein the subject is a human. [This invention 1056] The method of claim 1055, wherein the subject is under 1 year old, under 5 years old, under 12 years old, or under 18 years old. [This invention 1057] 1056. The method of any one of claims 1035 to 1056, wherein the A. cacae comprises a probiotic. [This invention 1058] The method according to any one of claims 1035 to 1057, wherein the bacterium is lyophilized or freeze-dried. [This invention 1059] 1058. The method of any one of claims 1035 to 1058, wherein the A. cacae and / or prebiotics are administered orally. [The present invention 1060] 1059. The method of claim 1059, wherein said A. cacae and / or prebiotics are administered in a tablet or capsule. [This invention 1061] The method of any of claims 1035 to 1060, further comprising administering a formula or food containing lactic acid. [This invention 1062] A composition comprising the bacterium Anaerostipes cacae and a prebiotic. [The present invention 1063] 1062. The composition of claim 1062, wherein the prebiotic comprises one or more of galactooligosaccharides, lactulose, lactitol, erythritol, isomalt, and polyglycitol. [This invention 1064] The composition of claim 1063, wherein the prebiotic comprises one or more of galactooligosaccharides, lactulose, and lactitol. [This invention 1065] The composition of any one of claims 1062 to 1064, further comprising a butyrate transport compound. [The present invention 1066] 1065. The composition of claim 1065, wherein the butyrate transport compound comprises pHPMA-b-pBMA. [This invention 1067] The composition of any one of claims 1062 to 1064, wherein the prebiotic comprises one or both of digestible oligosaccharides and non-digestible oligosaccharides. [The present invention 1068] 8. The composition of any of claims 1062 to 1067, wherein the prebiotic comprises at least 6 grams of non-digestible oligosaccharides. [The present invention 1069] The composition of any one of claims 1067 to 1068, wherein the oligosaccharide comprises a modified oligosaccharide. [The present invention 1070] 1069. The composition of claim 1069, wherein the modified oligosaccharides comprise butyrate-releasing oligosaccharides fermentable by A. cocoa. [This invention 1071] The dosage of A. cacae is 1 x 10 6 ~1×10 15 Any of the compositions of 1062 to 1070 of the present invention, which is CFU. [This invention 1072] Any of the compositions of inventions 1062 to 1071, wherein the ratio of colony forming units of A. coccae to grams of prebiotic is 1000:1 to 10000:1. [This invention 1073] The composition of any one of claims 1062 to 1072, further comprising a pharmaceutical excipient. [This invention 1074] The composition of any one of claims 1062 to 1073, which is formulated for oral administration. [This invention 1075] A tablet, capsule, or powder containing any one of the compositions of present inventions 1062 to 1074. [This invention 1076] A method for treating an infectious disease, autoimmune disease, or allergic disease in a subject, comprising administering to the subject a composition comprising the bacterium Anaerostipes cacae. [This invention 1077] 1076. The method of claim 1076, wherein the disease comprises an atopic disease. [This invention 1078] 1078. The method of claim 1077, wherein said atopic disease comprises eczema, atopic dermatitis, asthma, or allergic rhinitis. [This invention 1079] 1×10 6 ~1×10 15 The method of any of claims 1076 to 1078, wherein CFU of A. cacae are administered to said subject. [The present invention 1080] The method of any of claims 1076 to 1079, wherein said subject has been diagnosed with an infectious disease, an autoimmune disease, or an allergic disease. [This invention 1081] The method of any of claims 1001 to 1023, wherein said subject has previously been treated for an infectious disease, an autoimmune disease, or an allergic disease. [This invention 1082] The method of claim 1081, wherein the subject has been determined to be resistant to a previous treatment. [This invention 1083] The method of any one of claims 1076 to 1082, wherein the subject is a human. [This invention 1084] The method of claim 1083, wherein the subject is under 1 year old, under 5 years old, under 12 years old, or under 18 years old. [This invention 1085] The method of any one of claims 1076 to 1084, wherein the A. cacae comprises a probiotic. [The present invention 1086] The method of any one of claims 1076 to 1085, wherein the bacterium is lyophilized or freeze-dried. [This invention 1087] The method of any one of claims 1076 to 1086, wherein the A. cacae is administered orally. [This invention 1088] 1087. The method of claim 1087, wherein said A. cacae is administered in a tablet or capsule. [This invention 1089] 108. The method of any of claims 1076 to 1088, further comprising administering a formula or food containing lactic acid. [The present invention 1090] 1089. The method of any of claims 1076 to 1089, wherein the subject is determined to have a protective / non-protective operational taxonomic unit (OTU) ratio of less than 3. Other objects, features, and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. [Brief explanation of the drawings]

[0036] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0037] [Figure 1] Figure 1A-D: Transfer of healthy infant microbiota prevents allergic reactions to food, but not CMA infant microbiota. (a) Changes in core body temperature at the indicated time points after initial challenge with beta-lactoglobulin (BLG) in germ-free mice sensitized with BLG + cholera toxin (CT), and in mice transplanted with feces from each of eight donors (four healthy, four CMA; see Supplementary Table 1), collected from two independent experiments (n = 42 CMA mice, n = 31 healthy mice, and n = 24 GF mice; 4-12 mice for each of the eight donors). (b) BLG-specific IgE, (c) BLG-specific IgG1, and (d) mMCPT-1 in serum from mice in (a). For (a), circles represent the mean, and error bars represent the s.e.m. For (b)-(d), circles represent individual mice, and bars represent the mean + s.e.m. A linear mixed-effects model was used to compare groups in (a)–(d) with the Benjamini-Hochberg FDR (BH-FDR) method to correct for multiple testing. *P<0.05, **P<0.01, ***P<0.001. [Figure 2-1]Figure 2A-F: Analysis of fecal samples from eight human infant donors reveals taxonomic signatures correlated with allergic phenotypes. (a) Heatmap of OTUs differentially present between CMA and healthy donors. Horizontal columns represent 58 OTUs identified as differing at a false discovery rate (FDR) controlled at 0.10 and present in at least four human fecal samples and at least two transplanted mouse groups (see Supplementary Table 3). Vertical columns represent each donor (D) or transplanted mouse group (M). For donors, n = 2-3 technical replicates; for transplanted mouse groups, n = 1-4 mice, based on feces obtained 2 and 3 weeks after transplantation (see Methods). Bar graphs above the heatmap represent the abundance scores of potentially protective (orange) or non-protective (blue) OTUs calculated for each donor or mouse group. (b-d) Ratio of protective to non-protective OTUs from the transplanted mice in (a) plotted against the levels of BLG-specific IgE (b), BLG-specific IgG1 (c), and mMCPT-1 (d) from all mice in Figure 1 (see Figure 10B). Each circle represents the average result from all mice transplanted with feces from each of four healthy (orange) or CMA (blue) donors. (e) LEfSe analysis of taxa significantly enriched in healthy (orange) or CMA (blue) transplanted mice from the samples in (a) (n = 8 mice in the healthy group, n = 9 mice in the CMA group, from fecal samples collected 2 and 3 weeks after transplantation). (f) Cladogram showing the microbiota composition of the transplanted mouse samples from (a). Taxa detected as differentially present by LEfSe analysis are colored by group (healthy = orange and CMA = blue). In (a), the discrete false discovery rate (DS-FDR) method was used to compare groups, and in (e), the Kruskal-Wallis test was performed (see Methods). [Figure 2-2] See description of Figure 2-1. [Figure 3]Figure 3A-B: A unique ileal transcriptome signature distinguishes healthy from CMA-transplanted mice. (a) Heatmap of 32 differentially expressed genes (DEGs) in ileal IECs isolated from GF mice (n = 3), healthy mice (n = 18), or CMA-transplanted mice (n = 18) pooled from at least two independent experiments 7 days after transplantation (see Supplementary Table 4). Each column represents an individual mouse transplanted with the indicated donor feces. Four types of gene expression changes are shown: (1) Up in Healthy: genes upregulated in healthy mice compared to both CMA and GF; (2) Up in CMA: genes upregulated in CMA mice compared to both healthy and GF; (3) Down in Healthy: genes downregulated in healthy mice compared to both CMA and GF; and (4) Down in CMA: genes downregulated in CMA mice compared to healthy and GF. (b) Gene Ontology (GO) terms and KEGG pathways (bold) significantly enriched in DEGs from (a) associated with healthy transplanted mice (orange) or CMA-transplanted mice (blue). In (b), the hypergeometric distribution test was used with the Benjamini-Hochberg FDR (BH-FDR) method for multiple testing correction (see Methods). [Figure 4-1]Figure 4A-L: Correlation between ileal OTUs and DEGs in the ileum of healthy transplanted mice identifies a Clostridium species, A. cacae, that prevents allergic reactions to food. (a) Heatmap showing Spearman's rank correlation coefficients between the relative abundance of ileal OTUs (horizontal rows) and the expression of DEGs (columns) from ileal IEC samples of CMA mice versus healthy mice (see Figure 3A and Methods). (b) Spearman's correlation between the abundance of OTU259772 (Lachnospiraceae) from the ileal 16S dataset (see Supplementary Table 5) and the RNA-Seq expression of Ror2, Fbp1, Tgfbr3, Acot12, and Me1 in ileal IECs. Circles represent individual mice, and shaded bands indicate 95% confidence intervals fitted by linear regression. (c, d) Abundance of OTU259772 (Lachnospiraceae) by 16S sequencing (c) and Anaerostipes cacae by qPCR (d) in ileum samples from healthy and CMA-implanted mice. LD indicates samples that were below the detection limit of the assay. (e) Spearman correlation between abundance of OTU259772 (Lachnospiraceae; 16S sequencing) and abundance of Anaerostipes cacae (qPCR) in ileum samples from healthy and CMA-implanted mice. Circles represent individual mice, and shaded bands indicate 95% confidence intervals fitted by linear regression. Ileum samples that were above LD in both the 16S and qPCR experiments are shown (n = 19). (f) Gene expression of Ror2, Fbp1, Tgfbr3, Acot12, and Me1 in ileal IECs isolated from GF mice, healthy mice, CMA-implanted mice, or mice implanted with A. cacae alone by qPCR. Data are normalized to Hprt as a housekeeping gene and shown as fold change in expression relative to GF, which was set at 1. (g) Changes in core body temperature at the indicated time points after the initial challenge with BLG in CMA-implanted mice sensitized with BLG+CT and A. cacae-implanted mice alone. (h-j) BLG-specific IgE (h), BLG-specific IgG1 (i), and mMCPT-1 (j) in serum from mice in (g).(k, l) IL-13 (k) and IL-4 (l) in the culture supernatant of splenocytes from CMA- or A. cacae-implanted mice that were stimulated with BLG for 72 hours and sacrificed 24 hours after challenge. For (c), (d), (f), and (h)-(l), circles represent individual mice, and bars represent the mean + s.e.m. For (g), circles represent the mean, and bars represent the s.e.m. For each group in (a)-(b), n = 18 healthy or 18 CMA-implanted mice. For each group in (c) and (d), n = 19 healthy or 21 CMA-implanted mice. For each group in (f), n = 14 GF mice, n = 20 A. cacae-implanted mice, n = 18 healthy or 23 CMA-implanted mice. For (g)–(j), n = 16 CMA-implanted mice and n = 16 A. cacae-implanted mice were collected from three independent experiments with two different CMA donors (5 and 6). Bars represent the mean + s.e.m. For (k) and (l), n = 6 CMA-implanted mice and n = 9 A. cacae-implanted mice were collected from a single experiment. Circles represent individual mice, and bars represent the mean + see. For comparison of groups, in (c), the DS-FDR method was used; in (d), a two-tailed Student's t-test was used; in (f), one-way analysis of variance (ANOVA) with Bonferroni's multiple testing correction was used; or in (g), a linear mixed-effects model was used; and in (h)–(l), a two-tailed t-test after logarithmic transformation was used. *P<0.05, **P<0.01, ***P<0.001. [Figure 4-2] See description of Figure 4-1. [Figure 4-3] See description of Figure 4-1. [Figure 5] Sensitization of healthy or CMA-implanted mice with BLG + cholera toxin does not result in intestinal pathology. Representative images of histological samples from healthy or CMA-implanted mice sensitized with BLG + cholera toxin 24 hours after challenge for donors 1 (healthy) and 5 (CMA, see Supplementary Table 1). All sections were stained with H&E or PAS, as indicated. Scale bar = 100 μm. [Figure 6] Long-term transplantation of feces from healthy or CMA infants into GF mice does not result in intestinal pathology. Representative images of histological samples from naive, healthy, or CMA-transplanted mice collected 5–6 months after transplantation for the donors listed in Supplementary Table 1. All sections stained with H&E or PAS, as indicated. Scale bar = 100 µm. [Figure 7] Figure 7A-B: Diversity analysis of fecal samples from healthy or CMA-transplanted mice. (a) Shannon diversity index and (b) Pielou evenness index in feces from healthy (orange) and CMA-transplanted mice (blue) from Figure 2A. For each transplanted mouse group, n = 1–4 mice (based on feces collected 2 and 3 weeks after transplantation) (see Methods). Each circle represents one fecal sample, and the bars represent the mean ± SEM. The eight human formula-fed fecal donors are listed in Supplementary Table 1. [Figure 8] Figure 8A-D: Transfer of healthy, exclusively breastfed infant microbiota prevents anaphylactic reactions to sensitization with BLG + cholera toxin. (a) Changes in core body temperature at the indicated time points after initial challenge with BLG in mice transplanted with feces from breastfed healthy or CMA infant donors (n = 13 mice per group, collected from at least two independent experiments). (b-d) BLG-specific IgE (b), BLG-specific IgG1 (c), and mMCPT-1 (d) in serum from mice in (a). Four of the CMA-transplanted mice sensitized with BLG + CT died of anaphylaxis after challenge. For (a), symbols represent the mean, and bars represent the s.e.m. For (b)-(d), symbols represent individual mice, and bars represent the mean + s.e.m. In (a), a linear mixed-effects model was used to compare groups, and in (b), a two-tailed Student's t-test was used after logarithmic transformation. The two human breast-fed fecal donors are listed in Supplementary Table 2. *P<0.05. [Figure 9]Figure 9A-D: Continuous milk exposure does not induce tolerance to BLG in germ-free mice fed water or Enfamil and sensitized with BLG + cholera toxin. (a) Changes in core body temperature at the indicated time points after initial challenge with BLG in mice fed water (n = 12) or Enfamil (n = 10), collected from three independent experiments. (b-d) BLG-specific IgE (b), BLG-specific IgG1 (c), and mMCPT-1 (d) in serum from mice in (a). For (a), circles represent the mean, and error bars represent s.e.m. For (b)-(d), circles represent individual mice, and bars represent the mean + s.e.m. To compare groups in (a), a linear mixed-effects model was used; for (b)-(d), a two-tailed Student's t-test was used after logarithmic transformation. **P<0.01. ns = not significant (P=0.36). [Figure 10A] Figure 10A-B: Binary representation of protective and non-protective OTUs in CMA donors, healthy donors, and transplanted mouse groups. (a) Binary map of the protective / non-protective OTU presence / absence ratio in CMA donors and healthy donors with the same layout as Figure 2a. Columns represent each donor (D) or transplanted mouse group (M). For donors, n = 2–3 technical replicates; for each transplanted mouse group, n = 1–4 mice (from feces collected 2 and 3 weeks after transplantation) (see Methods). Rows represent 58 OTUs present in at least four human fecal samples and at least two transplanted mouse groups, FDR-controlled at 0.10 (see Methods), in the comparison of human CMA donors versus healthy donors (see Supplementary Table 3). The bar graph above the grid map represents the total number of potentially protective (more abundant in healthy donors; orange) and potentially non-protective OTUs (more abundant in CMA donors; blue) in each individual donor or group of mice. The grid map represents the presence (green) or absence (white) of protective and non-protective OTUs in each sample. [Figure 10B](b) The protective / non-protective OTU ratio was calculated for each donor or mouse group in (a), taking into account the presence or absence of 58 OTUs. Donors and their mouse transfer recipients are indicated by squares and circles, respectively. The vertical dashed line represents a ratio of 2.6. [Figure 11] Validation of protective / non-protective OTU ratios using a larger, independent cohort of healthy and CMA infant donors. Analysis of protective / non-protective OTU ratios in fecal samples from healthy infants (n = 19) and CMA infants (n = 19), as previously isolated and described from reference 5 (see Figures 2 and 10). The horizontal center line indicates the median, boxes represent the 25th and 75th percentiles, and whiskers extend to the furthest data point within a maximum of 1.5 times the interquartile range (IQR). All individual points are shown, and each circle represents a subject. Of the 58 OTUs shown in Figure 2A, 55 OTUs were assigned known reference IDs and 3 OTUs were assigned novel reference IDs. Novel reference OTU IDs were not comparable between different analysis cohorts; therefore, we focused on OTUs with known reference IDs. Fifty-two of the 55 known OTUs (29 protective and 23 non-protective) were detected in this cohort and used for ratio calculations (see Methods). The other three were not detected. A two-tailed Wilcoxon rank sum test was used. *P<0.05. [Figure 12]Figure 12A-C: Healthy to CMA OTU abundance ratios are significantly correlated between fecal and ileal samples from mice. (a) Bubble plots show similar patterns in fecal samples (n = 8 mice in the healthy group, n = 9 mice in the CMA group, with fecal samples collected 2 and 3 weeks after transplantation, identical to Figure 2A) and ileal samples (n = 22 mice in the healthy group, n = 25 mice in the CMA group) from healthy and CMA-transplanted mice. The 58 OTUs significantly differentially present between CMA and healthy donors are shown in the same order as in Figure 2A. Circle size indicates the magnitude of relative abundance enrichment toward either CMA or healthy. Color intensity indicates statistical significance calculated using the DS-FDR permutation test (see Methods). (b and c) Healthy to CMA OTU abundance ratios are significantly correlated between fecal and ileal samples from mice. Each dot represents one individual OTU. For (b), the mean abundance for each OTU was calculated at the group level for 8 healthy and 9 CMA-transplanted mice for fecal samples, and for 22 healthy and 25 CMA-transplanted mice for ileal samples. The ratio of OTU abundance in feces is plotted on the x-axis, and the ratio of OTU abundance in the ileum is plotted on the y-axis. For (c), n = 35 mice (n = 15 healthy and n = 20 CMA-transplanted mice) pooled from at least two independent experiments were used to calculate the OTU abundance ratios for both feces and ileum. Here, fecal and ileal samples were collected from the same individual mice. For further details, see "Methods." [Figure 13]Figure 13A-C: The abundance of OTU259772 (Lachnospiraceae) and Anaerostipes cacae correlates in fecal samples from healthy and CMA-transplanted mice. Abundance of OTU259772 (Lachnospiraceae) from the 16S dataset (a) and abundance of Anaerostipes cacae by qPCR (b) in fecal samples from healthy (n = 7) and CMA-transplanted mice (n = 8) from Figure 2. For each individual mouse, one to two fecal samples were collected 2 and 3 weeks after transplantation. LD indicates samples that were below the detection limit of the assay. (c) Spearman correlation between abundance of OTU259772 (Lachnospiraceae; 16S sequencing) and abundance of Anaerostipes cacae (qPCR) in fecal samples from healthy and CMA-transplanted mice from Figure 2. Fecal samples that were above LD in both the 16S and qPCR experiments are shown (n=13). Each circle represents one fecal sample. For (a) and (b), the bars indicate the mean + sem. For (c), the shaded bands indicate the 95% confidence intervals fitted by linear regression. In (a), the DS-FDR method was used to compare groups, and in (b), a two-tailed Student's t-test was used. ***P<0.001. [Figure 14] The abundance of Anaerostipes cacae in ileal samples correlates with gene expression in ileal IECs. Spearman correlation between the abundance of Anaerostipes cacae by qPCR and the RNA-Seq expression of Ror2, Fbp1, Tgfbr3, Acot12, and Me1 in ileal IECs (see Figure 3A). Circles represent individual mice, and shaded bands represent 95% confidence intervals fitted by linear regression. n = 36 mice (n = 18 healthy transplanted mice and n = 18 CMA transplanted mice) collected from at least two independent experiments. Samples with values ​​above the detection limit are indicated (A. cacae abundance > 0). [Figure 15] A schematic diagram of the isolation and characterization of A. cacae is shown. [Figure 16]A. cacae lah is highly sensitive to ampicillin and somewhat sensitive to tetracycline. [Figure 17] A. cacae_lah cannot ferment complex carbohydrates in monoculture, but it can ferment simple sugars such as those found in infant formula, e.g., lactose. A. cacae_lah was grown from frozen stocks in CMG broth for 24 hours, then 10 μl was transferred to minimal peptone yeast (PY) broth supplemented with 10 mg / ml carbohydrate. Growth and butyrate production were measured after 48 hours. All experiments were performed in duplicate, and groups were analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. PY alone. [Figure 18] A. cacae_lah can use both lactate and acetate to produce butyrate. A. cacae_lah was grown from a frozen glycerol stock in CMG broth for 24 hours, then 10 μl was transferred to minimal PY broth supplemented with 33 mM acetate and / or 40 mM lactate. Growth and butyrate production were measured after 48 hours. All experiments were performed in duplicate, and groups were analyzed by one-way ANOVA. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 vs. PY alone. [Figure 19] A. cacae_lah produces substantially more butyrate from fiber in co-culture with a complex bacterial mixture derived from an allergic (CMA) infant donor. A. cacae_lah or human CMA fecal samples were grown separately from frozen glycerol stocks in CMG broth for 24 hours, and then 10 μl total was transferred to minimal PY broth supplemented with 10 mg / ml potato starch or cellobiose. Growth and butyrate production were measured after 48 hours. All experiments were performed in duplicate. [Figure 20A]Figure 20A-B: CMA repository. While bacterial mixtures derived from mouse feces produce more butyrate at baseline than human CMA feces derived from frozen glycerol stocks (A), the addition of lactate and acetate to A. cacae_lah further results in significant differences in butyrate levels (B). A. cacae_lah or CMA mixtures derived from human or mouse feces were grown separately from frozen glycerol stocks in CMG broth for 24 hours, and then 10 μl total was transferred to minimal PY broth supplemented with 10 mg / ml carbohydrate. In the 24-hour group, CMA was transferred at time 0, and A. cacae_lah and carbohydrate were transferred at time 24 hours. Growth and butyrate production were measured after 48 or 72 hours. All experiments were performed in duplicate. [Figure 20B] See legend to Figure 20A. [Figure 21] 1 shows the experimental design for transplantation of A. cacae_lah into CMA-implanted mice and determining their dependence on prebiotic supplements. DETAILED DESCRIPTION OF THE INVENTION

[0038] Description of exemplary embodiments To better understand the role of the microbiota in controlling allergic diseases in humans, we transplanted bacteria derived from the feces of healthy or cow's milk-allergic (CMA) infants into germ-free (GF) mice. Here, we show that transplantation of bacteria from healthy infants into germ-free mice prevented sensitization to the cow's milk allergen β-lactoglobulin. Mice transplanted with bacteria from CMA infants exhibited anaphylactic responses to BLG challenge and significantly increased serum BLG-specific IgE. Differences in bacterial composition separated healthy and CMA populations in both human donors and transplanted mice. RNA-Seq analysis of ileal epithelial cells revealed differentially expressed genes (DEGs) that distinguished healthy and CMA-transplanted mice across all donors. Correlation of ileal OTUs with DEGs in the ileum of healthy transplanted mice identified a Clostridium species, Anaerostipes cacae, that prevented allergic reactions to food. The findings demonstrate that the composition of the gut microbiota is important for the control of allergic responses to dietary antigens and suggest that interventions modulating the bacterial community may be therapeutically relevant in the context of food allergies.

[0039] I. Definition The term "unit dose" or "dosage" refers to physically discrete units suitable for use in a subject, each containing a predetermined amount of a therapeutic composition calculated to produce the desired response described herein in conjunction with its administration, i.e., an appropriate route and treatment regimen. The amount administered, both in terms of number of treatments and unit dose, will depend on the desired effect. The actual dosage of a composition of embodiments of the present invention administered to a patient or subject can be determined by physical and physiological factors, such as the subject's weight, age, health, and sex, the type of disease being treated, the depth of the disease, previous or concurrent therapeutic interventions, the patient's idiopathic nature, the route of administration, and the efficacy, stability, and toxicity of the specific therapeutic agent. For example, dosages can include from about 1 μg / kg / body weight to about 1000 mg / kg / body weight per administration (including such ranges), or more, and any specific dosage derivable therefrom. In non-limiting examples of ranges derivable from the numbers listed herein, ranges from about 5 μg / kg / body weight to about 100 mg / kg / body weight, from about 5 μg / kg / body weight to about 500 mg / kg / body weight, etc. may be administered. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose for the individual subject.

[0040] "Subject" and "patient" refer to either humans or non-humans such as primates, mammals, and vertebrates. In a specific embodiment, the subject is a human.

[0041] As used herein, the terms "treat," "treatment," "treating," or "amelioration," when used in reference to a disease, disorder, or medical condition, refer to therapeutic treatment of a condition with the goal of reversing, alleviating, ameliorating, inhibiting, slowing, or halting the progression or severity of the symptoms or condition. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of the condition. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, treatment is "effective" if the progression of the condition is reduced or halted. That is, "treatment" includes not only the improvement of symptoms or markers, but also the halting or at least slowing of the progression or worsening of symptoms that would be expected in the absence of treatment. Beneficial or desired clinical results include, but are not limited to, alleviation of one or more symptoms, a reduction in the degree of disability, and an increase in lifespan compared to that expected in the absence of treatment.

[0042] The term "isolated" encompasses bacteria or other objects or substances that are (1) separated from at least some of the components with which they were originally associated (whether in nature or in an experimental setting) and / or (2) artificially created, prepared, purified, and / or manufactured. Isolated bacteria may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were originally associated. In some embodiments, isolated bacteria have a purity of greater 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 greater than about 99%. As used herein, a substance is "pure" if it is substantially free of other components.

[0043] The terms "purify," "purifying," and "purified" refer to bacteria or other materials that have been separated from at least some of the components with which they were associated when originally produced or generated (e.g., in nature or in an experimental setting), or any time period after initial production. Bacteria or bacterial populations can be considered purified, for example, if they are isolated from the material or environment containing the bacteria or bacterial population at the time of or after production; purified bacteria or bacterial populations can be considered "isolated" even if they contain up to about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than about 90% other materials. In some embodiments, purified bacteria and bacterial populations have a purity of greater 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 greater than about 99%. In the case of the bacterial compositions provided herein, one or more bacterial types present in the composition may be purified independently from one or more other bacteria produced and / or present in the material or environment containing the bacterial type. Bacterial compositions and their bacterial components are generally purified from residual habitat products.

[0044] As used herein, the terms "comprising" or "comprises" are used in reference to compositions, methods, and their respective components that are essential to the invention, but may also include non-specified elements, whether essential or not.

[0045] As used herein, the term "consisting essentially of" refers to elements required for a given embodiment. The term allows for the presence of additional elements that do not materially affect the basic novel or functional characteristics of that embodiment of the invention. With respect to pharmaceutical compositions, the term "consisting essentially of" includes the recited active ingredients and excludes other active ingredients, but does not exclude pharmaceutical excipients or other ingredients that have no therapeutic activity.

[0046] The term "consisting of" refers to compositions, methods, and their respective components described herein, excluding elements not recited in that description of an embodiment.

[0047] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "the method" includes one or more methods, and / or steps described herein and / or that will become apparent to those skilled in the art upon review of this disclosure, and so forth.

[0048] As used herein, "essentially free of," with respect to a named component, means that none of the named components are intentionally formulated in the composition and / or are present only as contaminants or in trace amounts. Thus, the total amount of the named component resulting from unintentional contamination of the composition is well below 0.01%. Most preferred are compositions in which the amount of the named component cannot be detected by standard analytical methods.

[0049] Although the use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or that the alternatives are mutually exclusive, the present disclosure supports the definition referring to alternatives only and "and / or." As used herein, the term "other" can mean at least a second or more.

[0050] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the device, the method being utilized to determine the value, or the variation that exists among study subjects.

[0051] The phrases "effective amount" or "therapeutically effective amount" or "sufficient amount" refer to a dosage of a drug or agent sufficient to produce a desired result.

[0052] II. Microbial composition Aspects of the present disclosure relate to microbial compositions for treating infectious, autoimmune, or allergic diseases.

[0053] The present disclosure also provides pharmaceutical compositions comprising one or more of the above-mentioned microbial cultures.Therefore, the bacterial species is present in the dosage form as a live bacterium, whether in a dried form or a lyophilized form.It can preferably be suitable for suitable administration, for example, in the form of a tablet or powder, which may have an enteric coating for oral treatment.

[0054] In a specific aspect, the composition is formulated for oral administration.Oral administration can be achieved by using chewable formulations, dissolving formulations, encapsulated / coated formulations, multi-layered lozenges (to separate active ingredients and / or to separate active ingredients and excipients), sustained / sustained release formulations, or other suitable formulations known to those skilled in the art.Although the word "tablet" is used herein, the formulation can take various physical forms, which can be generally referred to by other terms, such as lozenges, pills, capsules, etc.

[0055] The compositions of the present disclosure are preferably formulated for oral administration, although other routes of administration may be utilized, including, but not limited to, subcutaneous, intramuscular, intradermal, transdermal, intraocular, intraperitoneal, mucosal, vaginal, rectal, and intravenous.

[0056] The desired dose of the compositions of the present disclosure may be presented in multiple (e.g., 2, 3, 4, 5, 6, or more) sub-doses administered at appropriate intervals throughout the day, week, month, or year.

[0057] In one aspect, the disclosed compositions can be prepared as a capsule. The capsule (i.e., carrier) can be a hollow, generally cylindrical capsule formed from a variety of materials such as gelatin, cellulose, carbohydrates, and the like.

[0058] In another aspect, the disclosed compositions can be prepared as suppositories.Suppositories can contain one or more carriers, such as, but not limited to, bacteria and polyethylene glycol, gum arabic, acetylated monoglyceride, carnauba wax, cellulose acetate phthalate, corn starch, dibutyl phthalate, sodium docusate, gelatin, glycerin, iron oxide, kaolin, lactose, magnesium stearate, methylparaben, pharmaceutical glaze, povidone, propylparaben, sodium benzoate, sorbitan monooleate, sucrose, talc, titanium dioxide, white wax, and coloring agents.

[0059] In some aspects, the disclosed microbial compositions can be prepared as tablets. Tablets can include bacteria and one or more tableting agents (i.e., carriers) such as calcium hydrogen phosphate, stearic acid, croscarmellose, silica, cellulose, and cellulose coatings. Tablets can be formed using direct compression, although those skilled in the art will recognize that various techniques can be used to form tablets.

[0060] In other aspects, the disclosed microbial compositions may be formulated as a food or beverage, or as an additive to a food or beverage, in which case an appropriate amount of bacteria is added to the food or beverage to make the food or beverage the carrier.

[0061] In some embodiments, the microbial composition can further comprise a food or nutritional supplement effective to stimulate the growth of A. coccae present in the gastrointestinal tract of the subject. In some embodiments, the nutritional supplement is produced by another bacterium associated with the healthy human gut microbiome.

[0062] III. Administration of Therapeutic Compositions The treatments provided herein include the administration of a combination of a microbial composition and a therapeutic agent, such as a prebiotic. The treatment can be administered in any suitable manner known in the art. For example, the microbial composition and the prebiotic can be administered sequentially (at different times) or simultaneously (at the same time). In some embodiments, the microbial composition and the prebiotic are in separate compositions. In some embodiments, the microbial composition and the prebiotic are in the same composition.

[0063] Embodiments of the present disclosure relate to compositions and methods comprising bacteria and one or more prebiotics. The bacteria and / or prebiotics may be administered in one composition, or in multiple compositions, such as two, three, or four compositions. For example, various combinations of agents may be utilized, such as the following (where bacteria (or a composition comprising bacteria) is "A" and prebiotic is "B"): TIFF0007737098000001.tif22128

[0064] In some embodiments, the microbial composition is administered prior to the prebiotic. In some embodiments, the microbial composition is administered at least 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or at most 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks (or any range derivable therein) prior to the prebiotic. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any range derivable therein) of the microbial composition are administered at least 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or at most 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks (or any range derivable therein) before the prebiotic. In some embodiments, the microbial composition is administered after the prebiotic. In some embodiments, the microbial composition is administered at least 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or at most 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks (or any range derivable therein) after the prebiotic, or at least one of the prebiotics, or at least two of the prebiotics.In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 doses (or any range derivable therein) of the microbial composition are administered at least 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or at most 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks, or about 1, 2, 3, 5, 6, 12, 24 hours, or 2, 3, 4, 6, 8, 10 days, or 2, 3, 4, 5, 6, 7, or 8 weeks (or any range derivable therein) after the prebiotic, or at least one of the prebiotics, or at least two of the prebiotics.

[0065] In some embodiments, the microbial modulator composition is formulated for oral administration. A variety of formulations are known to those skilled in the art that can include live or killed microorganisms and can be presented as a dietary supplement (e.g., pills, tablets, etc.) or as a functional food such as a beverage or fermented yogurt.

[0066] The agents of the present disclosure may be administered by the same administration route or by different administration routes. In some embodiments, the prebiotics are administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. In some embodiments, the microbial composition is administered intravenously, intramuscularly, subcutaneously, topically, orally, transdermally, intraperitoneally, intraorbitally, by implantation, by inhalation, intrathecally, intracerebroventricularly, or intranasally. The appropriate dosage can be determined based on the type of disease being treated, the severity and course of the disease, the individual's clinical condition, the individual's medical history and response to treatment, and the discretion of the attending physician.

[0067] For example, a therapeutically effective or sufficient amount of each of at least one isolated or purified bacterial population, or each of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 isolated or purified bacterial populations of a microbial modulator composition of the present embodiments administered to a human may be at least about 1 x 10 3 colony-forming units (CFU) of bacteria, or at least approximately 1 x 10 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 In some embodiments, a single dose will be at least 1 x 10 CFU (or any range derivable therefrom). 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 CFU or more, or at most 1 x 10 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 1015 CFU or more, or approximately 1 x 10 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 In some embodiments, a single dose will contain bacteria (such as a specific bacteria, or species, genus, or family described herein) present in an amount of at least 1 x 10 CFU, or more (or any range derivable therein). 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 CFU or more, or at most 1 x 10 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 10 7 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 CFU or more, or approximately 1 x 10 4 CFU, 1 × 10 5 CFU, 1 × 10 6 CFU, 1 × 107 CFU, 1 × 10 8 CFU, 1 × 10 9 CFU, 1 × 10 10 CFU, 1 × 10 11 CFU, 1 × 10 12 CFU, 1 × 10 13 CFU, 1 × 10 14 CFU, 1 × 10 15 It will contain CFU or more (or any range derivable therefrom) of total bacteria.

[0068] In some embodiments, the therapeutically effective or sufficient amount of each of the at least one isolated or purified bacterial population of the microbial composition of the present embodiments administered to a human is at least 1 x 10 3 Bacteria, or at least about 1 x 10 cells 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 In some embodiments, a single dose will be at least 1 x 10 cells (or any range derivable therefrom). 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 cells, or more, or at most 1 × 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10, 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 cells, or more, or approximately 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 In some embodiments, a single dose will contain bacteria (such as a specific bacterium, or species, genus, or family described herein) present in an amount of at least 1 x 10 cells, or more (or any range derivable therefrom). In some embodiments, a single dose will contain at least 1 x 10 cells, or more (or any range derivable therefrom). 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 cells, or more, or at most 1 × 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×10 9 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 cells, or more, or about 1 x 10 4 , 1×10 5 , 1×10 6 , 1×10 7 , 1×10 8 , 1×109 , 1×10 10 , 1×10 11 , 1×10 12 , 1×10 13 , 1×10 14 , 1×10 15 It may contain whole bacteria, or more (or any range derivable therefrom).

[0069] The treatment may comprise various "unit doses." A unit dose is defined as containing a predetermined amount of a therapeutic composition. The amount to be administered, as well as the specific route and formulation, are within the skill of those skilled in the clinical arts to determine. A unit dose need not be administered as a single injection, but may comprise continuous infusion over a set period of time. In some embodiments, a unit dose comprises a single administrable dose.

[0070] The amount to be administered, both in terms of the number of treatments and the unit dose, depends on the desired therapeutic effect. An effective dose is understood to refer to the amount necessary to achieve a specific effect. Indeed, in certain embodiments, it is contemplated that a dose ranging from 10 mg / kg to 200 mg / kg can affect the protective capabilities of these agents. Thus, it is contemplated that doses include about 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, and 200, 300, 400, 500, 1000 μg / kg, mg / kg, μg / day, or mg / day, or any range derivable therein. Furthermore, such doses may be administered at multiple time points during a day and / or multiple days, weeks, or months.

[0071] In certain embodiments, an effective dose of the pharmaceutical composition is one that can provide blood levels of about 1 μM to 150 μM. In other embodiments, an effective dose provides blood levels of about 4 μM to 100 μM; or about 1 μM to 100 μM; or about 1 μM to 50 μM; or about 1 μM to 40 μM; or about 1 μM to 30 μM; or about 1 μM to 20 μM; or about 1 μM to 10 μM; or about 10 μM to 150 μM; or about 10 μM to 100 μM; or about 10 μM to 50 μM; or about 25 μM to 150 μM; or about 25 μM to 100 μM; or about 25 μM to 50 μM; or about 50 μM to 150 μM; or about 50 μM to 100 μM (or any range derivable therein). In other embodiments, the dose can provide the following blood levels of drug resulting from the therapeutic agent administered to a subject: 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, 99, or is 100 μM or mM, or at least 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, 99, or 100 μM or mM, or at most 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, 99, or 100 μM or mM, or any range derivable therein. In certain embodiments, a therapeutic agent administered to a subject is metabolized in the body to a metabolized therapeutic agent, in which case blood levels can refer to the amount of that agent. Alternatively, unless the therapeutic agent is metabolized by the subject, blood levels described herein can refer to the unmetabolized therapeutic agent.

[0072] Precise amounts of the therapeutic composition depend on the judgment of the practitioner and are peculiar to each individual. Factors affecting dosage include the physical and clinical condition of the patient, the route of administration, the intended goal of treatment (whether symptomatic relief or cure), and the efficacy, stability, and toxicity of the particular therapeutic agent or other treatment to which the subject may be subjected.

[0073] It will be understood and appreciated by those skilled in the art that dosage units of μg / kg or mg / kg of body weight can be converted and expressed in comparable concentration units of μg / ml or μM or mM (blood levels), such as 4 μM to 100 μM. It is also understood that uptake is species and organ / tissue dependent. Applicable conversion factors, as well as physiological assumptions made regarding uptake and concentration measurements, are well known and will allow one skilled in the art to convert one concentration measurement to another and make reasonable comparisons and conclusions regarding the doses, efficacies, and results described herein.

[0074] Prebiotics can be formulated using pharmaceutical formulation techniques known in the art. They can also be formulated using known special techniques for delivery to specific regions of the gastrointestinal tract. Two examples known in the art are described in published PCT application WO 2018 / 195067 A1 and U.S. patent application US15 / 257,673, each of which is incorporated by reference. Other formulations for prebiotics or combinations of prebiotics and A. cacae are known in the art. In some embodiments, the compositions of the present disclosure include a butyrate transport compound, such as that described in WO 2018 / 195067.

[0075] IV. Treatment method The disclosed methods relate to the treatment of infectious, autoimmune, or allergic diseases. In some embodiments, the methods are for treating food allergies. In some embodiments, the food allergy includes milk allergy. In some embodiments, the milk allergy includes cow's milk allergy. In some embodiments, the milk allergy includes cow's milk allergy, goat's milk allergy, or sheep's milk allergy. In some embodiments, the methods are for treating allergies, asthma, diabetes (e.g., type 1 diabetes), transplant rejection, arthritis (rheumatoid arthritis, e.g., acute arthritis, chronic rheumatoid arthritis, gout or gouty arthritis, acute gouty arthritis, acute immunological arthritis, chronic inflammatory arthritis, degenerative arthritis, type II collagen arthritis, infectious arthritis, Lyme arthritis, proliferative arthritis, psoriatic arthritis, Still's disease, spondyloarthritis, and systemic juvenile-onset rheumatoid arthritis, osteoarthritis, chronic progressive arthritis, osteoarthritis deformans), chronic primary polyarthritis, reactive arthritis, and ankylosing spondylitis), inflammatory hyperproliferative skin diseases, psoriasis, e.g., plaque psoriasis, guttate psoriasis, pustular psoriasis, and psoriasis of the nails, atopy, e.g., atopic diseases, e.g., hay fever and Job's syndrome, dermatitis, e.g., contact dermatitis, chronic contact dermatitis, exfoliative dermatitis, allergic dermatitis, allergic contact dermatitis, dermatitis herpetiformis, nummular eczema, seborrheic dermatitis, nonspecific dermatitis, primary irritant contact dermatitis, and atopic dermatitis, x-linked hyper IgM syndrome, allergic intraocular inflammatory disease, urticaria, e.g., chronic allergic rhinitis, allergic urticaria and chronic idiopathic urticaria, e.g., chronic autoimmune urticaria, myositis, polymyositis / dermatomyositis, juvenile dermatomyositis, toxic epidermal necrolysis, scleroderma (e.g., systemic sclerosis), sclerosis, e.g., systemic sclerosis, multiple sclerosis (MS), e.g., optic-spinal MS, primary progressive MS (PPMS), and relapsing-remitting MS (RRMS), progressive systemic sclerosis, atherosclerosis, arteriosclerosis, disseminated sclerosis, ataxic sclerosis, neuromyelitis optica (NMO), inflammatory bowel disease (IBD) (e.g., Crohn's disease, autoimmune gastrointestinal diseases), colitis, e.g., ulcerative colitis, colitis ulcerosa,Microscopic colitis, collagenous colitis, polypoid colitis (colitis polyposa), necrotizing colitis, and transmural colitis, and autoimmune inflammatory bowel disease), intestinal inflammation, pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, respiratory distress syndromes, such as adult or acute respiratory distress syndrome (ARDS), meningitis, inflammation of all or part of the uvea, iritis, choroiditis, autoimmune hematological disorders, rheumatoid spondylitis, rheumatoid synovitis, hereditary angioedema, cranial nerve damage such as in the case of meningitis, pregnancy Herpes gestationis, pemphigoid of pregnancy, scrotal pruritus, autoimmune premature ovarian failure, sudden hearing loss due to autoimmune conditions, IgE-mediated diseases such as anaphylaxis and allergic rhinitis and atopic rhinitis, encephalitis such as Rasmussen's encephalitis and limbic encephalitis and / or brainstem encephalitis, uveitis such as anterior uveitis, acute anterior uveitis, granulomatous uveitis, non-granulomatous uveitis, lens-induced uveitis, posterior uveitis Uveitis, or autoimmune uveitis, glomerulonephritis (GN) with or without nephrotic syndrome, e.g., chronic or acute glomerulonephritis, e.g., primary GN, immune-mediated GN, membranous GN (membranous nephropathy), idiopathic membranous GN, or idiopathic membranous nephropathy, membranoproliferative GN (MPGN), e.g., types I and II, and rapidly progressive GN, proliferative nephritis, autoimmune polyendocrinopathy, balanitis, e.g., plasma cell balanitis, balanitis capitis, erythema annulare centrifugally, erythema dyschromatosis perstans, erythema multiforme, granuloma annulare, lichen nitidus, lichen sclerosus atrophicus, localized neurodermatitis, lichen spinous, lichen planus, ichthyosis lamellar, epidermolytic hyperkeratosis, premalignant keratosis, pyoderma gangrenosum, allergic conditions and responses, allergic reactions, eczema, e.g., allergic or atopic eczema, asteatotic eczema, dyshidrotic eczema, and bullous palmoplantar eczema, asthma, e.g., bronchial asthma bronchiale, bronchial asthma, and autoimmune asthma, conditions involving T cell infiltration and chronic inflammatory responses, immune responses to foreign antigens such as fetal ABO blood group during pregnancy, chronic pulmonary inflammatory diseases, autoimmune myocarditis, leukocyte adhesion deficiency, lupus, e.g., lupus nephritis, lupus encephalitis, pediatric lupus, non-renal lupus, extrarenal lupus, discoid lupus, and discoid lupus erythematosus, alopecia lupus,for systemic lupus erythematosus (SLE), such as cutaneous SLE or subacute cutaneous SLE, neonatal lupus syndrome (NLE), and disseminated lupus erythematosus, for juvenile-onset (type I) diabetes, such as insulin-dependent diabetes mellitus (IDDM) of childhood, and adult-onset diabetes (type II diabetes), and for autoimmune diabetes. Immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, sarcoidosis, granulomatous diseases, e.g., lymphomatoid granulomatosis, Wegener's granulomatosis, agranulocytosis, vasculitic syndromes, e.g., vasculitis, e.g., large vasculitis (e.g., polymyalgia rheumatica and giant cell (Takayasu) arteritis), medium vasculitis (e.g., Kawasaki disease and polyarteritis nodosa / periarteritis nodosa), microscopic polyarteritis, immune vasculitis, CNS vasculitis, cutaneous vasculitis, hypersensitivity vasculitis, necrotizing vasculitis, e.g., systemic necrotizing vasculitis, and ANCA-associated vasculitis, e.g., Churg-Strauss vasculitis or syndrome (CSS), and ANCA-associated small vasculitis, temporal arteritis, aplastic anemia, autoimmune aplastic anemia, Coombs' positive anemia, Diamond-Blackfan anemia, hemolytic anemia, or immune-mediated hemolytic anemia, e.g., autoimmune hemolytic anemia (AIHA), Addison's disease, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte extravasation, CNS inflammatory disorders, Alzheimer's disease, Parkinson's disease, multiple organ injury syndromes, e.g., sepsis, trauma, or hemorrhage sequelae, antigen-antibody complex-mediated diseases, antiglomerular basement membrane disease, antiphospholipid syndrome, allergic neuritis, Behcet's disease / syndrome, Castleman syndrome, Goodpasture's syndrome, Raynaud's syndrome, Sjögren's syndrome, Stevens-Johnson syndrome, pemphigoid, e.g., bullous pemphigoid and cutaneous pemphigoid, pemphigus (e.g., pemphigus vulgaris, pemphigus foliaceus, pemphigus mucous membrane pemphigoid) mucusmembrane pemphigoid), and pemphigus erythematosus), autoimmune polyendocrinopathy, Reiter's disease or syndrome, burns, preeclampsia, immune complex disorders such as immune complex nephritis, antibody-mediated nephritis, polyneuropathy, chronic neuropathy such as IgM polyneuropathy or IgM-mediated neuropathy,autoimmune or immune-mediated thrombocytopenia, for example idiopathic thrombocytopenic purpura (ITP), for example chronic or acute ITP; scleritis, for example idiopathic keratoscleritis, episcleritis; autoimmune diseases of the testes and ovaries, for example autoimmune orchitis and oophoritis; primary hypothyroidism, hypoparathyroidism; autoimmune endocrine diseases, for example thyroiditis, for example autoimmune thyroiditis, Hashimoto's disease, chronic thyroiditis (Hashimoto's thyroiditis), or subacute thyroiditis; autoimmune thyroid diseases, idiopathic hypothyroidism, Graves' disease; polyglandular syndromes, for example autoimmune polyglandular syndrome (or polyendocrinopathy syndrome); paraneoplastic syndromes, for example neurological paraneoplastic syndromes, for example Lambert-Eaton myasthenic syndrome or Eaton-Lambert syndrome, stiff-man syndrome or stiff-person syndrome; encephalomyelitis, for example allergic encephalomyelitis; encephalomyelitis or allergic encephalomyelitis (encephalomyelitis allergica), and experimental allergic encephalomyelitis (EAE), experimental autoimmune encephalomyelitis, myasthenia gravis, e.g., thymoma-associated myasthenia gravis, cerebellar degeneration, neuromyotonia, opsoclonus or opsoclonus-myoclonus syndrome (OMS), and sensory neuropathy, multifocal motor neuropathy, Sheehan's syndrome, autoimmune hepatitis, chronic hepatitis, lupoid hepatitis, giant cell hepatitis, chronic active hepatitis or autoimmune chronic active hepatitis, lymphocytic interstitial pneumonia (LIP), bronchiolitis obliterans (non-transplant) vs. NSIP, Guillain-Barré syndrome, Burger's disease (IgA nephropathy), idiopathic IgA nephropathy, linear IgA dermatosis, acute febrile neutrophilic dermatosis, subcorneal pustular dermatosis, transient acantholytic dermatosis, cirrhosis, e.g., primary biliary cirrhosis and pulmonary cirrhosis, autoimmune enteropathy syndrome, celiac disease, celiac sprue (gluten enteropathy), refractory sprue, idiopathic sprue, cryoglobulinemia, amyotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune ear diseases, e.g., autoimmune inner ear disease (AIED), autoimmune hearing loss, polychondritis, e.g., refractory or relapsing or relapsing polychondritis, pulmonary alveolar proteinosis, Cogan's syndrome / non-syphilitic interstitial keratitis, Bell's palsy, Sweet's disease / syndrome, autoimmune rosacea, shingles-associated pain, amyloidosis,Non-cancerous lymphocytosis, primary lymphocytosis including monoclonal B-cell lymphocytosis (e.g., benign monoclonal gammopathy and monoclonal gammopathy of undetermined significance, MGUS), peripheral neuropathies, paraneoplastic syndromes, channelopathies such as epilepsy, migraine, cardiac arrhythmias, muscle disorders, hearing loss, blindness, periodic paralysis, and channelopathies of the CNS, autism, inflammatory myopathies, focal or segmental glomerulosclerosis (FSGS), endocrine eye diseases, retinal uveitis , chorioretinitis, autoimmune hepatic pathological disorders, fibromyalgia, polyendocrinopathy, Schmidt's syndrome, adrenalitis, gastric atrophy, presenile dementia, demyelinating diseases such as autoimmune demyelinating diseases and chronic inflammatory demyelinating polyneuropathy, Dressler's syndrome, alopecia areata, alopecia totalis, crest syndrome (calcinosis, Raynaud's phenomenon, esophageal hypoperistalsis, sclerodactyly, and telangiectasia), male and female autoimmune infertility due to, for example, antisperm antibodies, mixed connective tissue disease, Chagas' disease, rheumatic fever fever), recurrent abortion, farmer's lung, erythema multiforme, postcardiotomy syndrome, Cushing's syndrome, bird breeder's disease, allergic granulomatous vasculitis, benign lymphocytic vasculitis, Alport syndrome, alveolitis, e.g. allergic alveolitis and fibrosing alveolitis, interstitial lung disease, transfusion reactions, leprosy, malaria, parasitic diseases, e.g. leishmaniasis, trypanosomiasis, schistosomiasis, ascariasis, aspergillosis, Samter's syndrome, Kaplan's syndrome, dengue fever, endocarditis, endomyocardial fibrosis, diffuse interstitial pulmonary fibrosis, interstitial pulmonary fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, cystic fibrosis, endophthalmitis, erythema elevatum, erythroblastosis fetalis, eosinophilic fasciitis, Shulman's syndrome syndrome, Felty's syndrome, filariasis, cyclitis, e.g., chronic cyclitis, metachronous cyclitis, iridocyclitis (acute or chronic), or Fuchs' cyclitis, Henoch-Schönlein purpura, human immunodeficiency virus (HIV) infection, SCID, acquired immune deficiency syndrome (AIDS), echovirus infection, sepsis, endotoxemia, pancreatitis, thyrotoxicosis, parvovirus infection, rubella virus infection, post-vaccination syndrome, congenital rubella infection, Epstein-Barr virus infection, parotitis, Evans' syndrome, autoimmune dysgonadism, Sydenham's chorea, post-streptococcal nephritis, thromboangiitis obliterans, thyrotoxicosis, tabes dorsalis, choroiditis,Giant cell polymyalgia, chronic hypersensitivity pneumonitis, keratoconjunctivitis sicca, epidemic keratoconjunctivitis, idiopathic nephritic syndrome, minimal change nephrosis, benign familial, and ischemia-reperfusion injury, transplanted organ reperfusion, retinal autoimmunity, joint inflammation, bronchitis, chronic obstructive airway / pulmonary disease, silicosis, aphthous stomatitis, arteriosclerotic disorders, asperniogeneic disorders, autoimmune hemolysis, Beck's disease, cryoglobulinemia, Dupuytren's contracture, lenticular hypersensitivity endophthalmitis, allergic enteritis, erythema nodosum leprosum, idiopathic facial nerve palsy, chronic fatigue syndrome, rheumatic fever, Hammann-Rich disease, sensorineural hearing loss, paroxysmal hemoglobinuria, hypogonadism, and regional ileitis. regionalis), leukopenia, infectious mononucleosis (mononucleosis infectiosa), transverse myelitis, primary idiopathic myelitis, Edema, nephrosis, ophthalmia symphatica, granulomatous orchitis, pancreatitis, acute polyradiculitis, pyoderma gangrenosum, Quervain's thyroiditis, acquired splenic atrophy, nonmalignant thymoma, vitiligo, toxic shock syndrome, food poisoning, conditions involving T-cell infiltration, leukocyte adhesion deficiency, immune responses related to acute and delayed hypersensitivity mediated by cytokines and T lymphocytes, diseases involving leukocyte extravasation, multiple organ injury syndrome, antigen-antibody complex-mediated diseases, anti-glomerular basement membrane disease, allergic neuritis, autoimmune polyendocrinopathy, oophoritis, primary myxedema, autoimmune atrophic gastritis, sympathetic ophthalmiaophthalmia), rheumatic diseases, mixed connective tissue disease, nephrotic syndrome, insulitis, polyendocrine glandular dysfunction, autoimmune polyglandular syndrome type I, adult-onset idiopathic hypoparathyroidism (AOIH), cardiomyopathies such as dilated cardiomyopathy, epidermolysis bullosa acquisita (EBA), hemochromatosis, myocarditis, nephrotic syndrome, primary sclerosing cholangitis, suppurative or non-suppurative sinusitis, acute or chronic sinusitis, ethmoid sinusitis, frontal sinusitis, upper respiratory tract infections, maxillary sinusitis or sphenoid sinusitis, eosinophil-related diseases such as eosinophilia, pulmonary infiltrate eosinophilia, eosinophilic myalgia syndrome, Löffler's syndrome, chronic eosinophilic pneumonia, tropical pulmonary eosinophilia, bronchopneumonic aspergillosis, aspergilloma, or eosinophil-containing granuloma, anaphylaxis, seronegative spondyloarthritis, polyendocrine autoimmune disease, sclerosing cholangitis, scleral, episcleral, chronic mucocutaneous candidiasis, Bruton's syndrome, transient infantile hypogamma Globulinemia, Wiskott-Aldrich syndrome, ataxia-telangiectasia syndrome, vascular ectasia, autoimmune disorders associated with connective tissue diseases, rheumatism, neurological disorders, lymphadenitis, decreased blood pressure response, vascular insufficiency, tissue damage, cardiovascular ischemia, hyperalgesia, renal ischemia, cerebral ischemia, and diseases involving angiogenesis, allergic hypersensitivity disorders, glomerulonephritis, reperfusion injury, ischemia-reperfusion injury, reperfusion injury of myocardium or other tissues, lymphomatous tracheobronchitis Also contemplated are inflammatory skin diseases, skin diseases with an acute inflammatory component, multiple organ failure, bullous disease, renal cortical necrosis, acute purulent meningitis or other central nervous system inflammatory disorders, ocular and orbital inflammatory disorders, granulocyte transfusion-associated syndrome, cytokine-induced toxicity, narcolepsy, acute severe inflammation, chronic refractory inflammation, pyelitis, intimal hyperplasia, peptic ulcer, valvular inflammation, graft-versus-host disease, contact hypersensitivity, asthmatic airway hyperresponsiveness, and endometriosis.

[0076] V. Kit Certain aspects of the present disclosure also include kits for practicing the methods of the present disclosure, such as kits containing the compositions described herein. The kits may include a container with a label. Suitable containers include, for example, bottles, vials, and test tubes. The containers may be formed from a variety of materials, such as glass or plastic. The containers may hold compositions containing probes useful for prognostic or non-prognostic applications, such as those described above. The label on the container may indicate that the composition is to be used for a specific prognostic or non-prognostic application and may also indicate instructions for either in vivo or in vitro use, such as those described above. The kits may include the containers described above and one or more other containers containing materials desirable from a commercial and user standpoint, such as buffers, diluents, filters, needles, syringes, and package inserts containing instructions for use. [Example]

[0077] VI. Working Examples The following examples are included to demonstrate preferred embodiments of the invention. It will be recognized by those of skill in the art that the techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the invention, and therefore can be considered to constitute preferred modes for its practice. However, in light of the present disclosure, those of skill in the art will recognize that many changes can be made in the specific embodiments disclosed and still obtain like or similar results without departing from the spirit and scope of the invention.

[0078] Example 1 - Healthy infants harbor gut bacteria that protect against food allergies B. Results Studies from our laboratory and others have demonstrated that the fecal microbiota of infants with CMA differs significantly from that of their healthy counterparts (5, 6). Based on these results and evidence that members of the microbiota can be allergy-protective (7), we used a gnotobiotic mouse model to investigate whether commensal bacteria play a necessary role in preventing allergic responses to the cow's milk allergen β-lactoglobulin (BLG). Human feces from four age-, sex-, and birth-matched healthy infant donors and four infant donors with IgE-mediated cow's milk allergy (CMA) were transplanted into germ-free (GF) mice (8, 9) (Supplementary Table 1). It has previously been reported that diet is important for stable transplantation of human feces into germ-free mice (10). To support the growth of human bacteria in mouse hosts, mice received feces from formula-fed healthy or CMA infants and were fed a plant-based mouse diet plus the same formula consumed by their human infant donors. CMA infant donors received extensively hydrolyzed casein formula (EHCF) to manage ongoing allergic symptoms, while healthy donors received standard cow's milk-based formula (5). The initial transfer recipients were used as survival repositories for subsequent experiments (see Methods).

[0079] Groups of GF mice and mice transplanted with either healthy or CMA infant bacterial flora were sensitized with BLG and the mucosal adjuvant cholera toxin (CT). GF mice not receiving bacterial transplants were highly susceptible to anaphylactic reactions to food, as evidenced by a decrease in core body temperature (Fig. 1A) and the production of BLG-specific IgE and IgG1 (Fig. 1B, C) (7, 11). We also measured a substantial decrease in core body temperature in response to BLG challenge in mice transplanted with fecal samples from each of the four CMA donors (Fig. 1A). Sensitized CMA-transplanted mice produced significantly higher serum concentrations of BLG-specific IgE (Fig. 1B), IgG1 (Fig. 1C), and mMCPT-1 (Fig. 1D) compared with healthy-transplanted mice. Strikingly, all mice receiving the microbiota from four healthy infants were protected from anaphylactic reactions to BLG challenge; postchallenge core body temperatures were significantly different from those measured in GF mice or CMA-transplanted mice (Figure 1A). Histological analysis revealed no evidence of pathology or inflammation in ileal or colonic tissue samples collected after challenge (Figure 5) or long-term transplantation (Figure 6). Microbial analysis revealed that the diversity and evenness of the microbiota were similar between the healthy-transplanted and CMA-transplanted mouse groups (Figure 7). To investigate whether milk-containing formula contributed to the microbiota-independent protection from anaphylaxis in the healthy-transplanted mice, we performed additional fecal transfers from breast-fed healthy and CMA-transplanted donors (Supplementary Table 2). Recipient mice received only a plant-based mouse diet. Mice transplanted with feces from breast-fed healthy donors were protected from anaphylactic reactions to BLG sensitization and challenge. However, mice transplanted with feces from breast-fed CMA donors exhibited significantly greater core body temperature reductions (Figure 8A) and higher levels of BLG-specific IgE (Figure 8B) compared with healthy donor mice. Sensitization to BLG was also compared in GF mice receiving water or Enfamil. Both groups of mice responded robustly to BLG sensitization (Figure 9).There were no significant differences in the decrease in core body temperature or serum concentrations of BLG-specific IgE or IgG1 after challenge; however, serum mMCPT-1 was suppressed in mice fed the cow's milk-containing formula.

[0080] Analysis of fecal samples from eight formula-fed human infant donors (Supplementary Table 1) identified 58 operational taxonomic units (OTUs) differentially present between healthy and CMA infants (Figure 2A; Supplementary Table 3). Because variation exists between each donor and mouse transfer recipient at the single OTU level, we investigated whether differences in the composition of the donor-derived microbiome could distinguish between transplanted mouse groups. As an aggregate measure for presenting the data, we calculated the number of potentially "protective" (more abundant in healthy donors, n = 34) and potentially "non-protective" (more abundant in CMA donors, n = 24) OTUs to obtain a presence / absence ratio for each donor (Figure 10A; see "Methods"). Additionally, we calculated a weighted score (hereafter referred to as the abundance score) for each OTU based on its relative abundance in the sample (Figure 2A; see "Methods"). When OTU abundance scores were plotted against the presence / absence ratio, the ratio separated donors into healthy and CMA groups. This threshold also separated CMA- and healthy-transplanted mice by biological phenotype (Fig. 10B, squares), demonstrating that the donor-derived collective microbiota signature was validated in mouse transfer recipients. The significantly higher protective / non-protective OTU ratio in healthy infants compared with those with CMA was independently confirmed in an unrelated sample set from the same Naples cohort by reanalysis of 16S fecal sample data collected in a previously published study (5) (Fig. 11). Donor-derived OTU ratios also separated healthy and CMA-transplanted mice when plotted against biomarkers of allergic disease, including BLG-specific IgE (Fig. 2B), BLG-specific IgG1 (Fig. 2C), and mMCPT-1 (Fig. 2D). Interestingly, linear discriminant effect size (LEfSe) analysis (Fig. 2E,F) showed that the family Lachnospiraceae of the class Clostridia was enriched in healthy transplanted mice (7).

[0081] Tolerance to dietary antigens begins with absorption in the small intestine (4, 12). Most commensal bacteria reside in the colon; within the small intestine, bacteria are most abundant in the ileum (13). The interaction of these bacteria with IECs is central to immune regulation at the host-microbe interface (13, 14). Ileal IECs were isolated from groups of mice transplanted with each of eight infant donors, and gene expression was quantified by RNA-Seq (Figure 3A). Healthy transplanted mice upregulated a unique set of ileal genes compared with CMA-transplanted mice (Figure 3A; Supplementary Table 4). For example, Fbp1 (15), which encodes a key gluconeogenic enzyme abundantly expressed in small intestinal epithelial cells, was significantly upregulated in all healthy transplanted mice (Figure 3A). Decreased expression of Fbp1 alters epithelial oxygenation and contributes to dysbiosis (18), and is associated with a metabolic switch from oxidative phosphorylation to aerobic glycolysis (16, 17). Tgfbr3 and Ror2 were downregulated in the ileum of CMA-implanted mice compared with healthy control mice (Figure 3A). Tgfbr3 encodes a receptor for the growth factor TGFβ and is highly expressed in the small intestine of lactating rats (19). Soluble TGFβRIII and TGFβ2 are present in high concentrations in breast milk; activation of TGFβ signaling by Wnt5a is mediated by Ror2 and is important for epithelial repair (20). In contrast, Acot12 and Me1, genes involved in pyruvate metabolism, were upregulated in the ileum of CMA-implanted mice compared with healthy control mice. These metabolic and molecular processes are reflected in the gene ontology pathways that were significantly altered in CMA- and healthy-transplanted mice, shown in Figure 3B.

[0082] To determine whether the fecal OTU signature identified in Figure 2 also reflects the ileal bacterial population, we investigated the correlation between ileal OTUs and fecal signatures in healthy and CMA-transplanted mice (Figure 12A). The majority of taxa were found to change in the same direction (increased or decreased abundance) between fecal and ileal samples in healthy compared with CMA-transplanted mice (Figure 12B, C). The identification of differential gene expression in ileal IECs from healthy and CMA-transplanted mice (Figure 3A) suggested that ileal bacteria regulate host immunity, contributing to allergic sensitization. Integrated analysis of ileal bacteria and differentially expressed genes (DEGs) in the ileum revealed that nine OTUs were significantly and consistently correlated with genes upregulated in the ileum of healthy or CMA-transplanted mice (Figure 4A). Interestingly, three-fifths of the protective OTUs associated with DEGs up-regulated in the ileum of healthy transplanted mice are members of the Lachnospiraceae family. BLAST searches of assembled 16S sequences against the NCBI database (16S ribosomal RNA, Bacteria, and Archaea) revealed that three protective Lachnospiraceae OTUs (259772, New18, and 177986) up-regulated in healthy transplanted mice all had Anaerostipes cacae as their most closely matching species. Notably, OTU259772 was annotated with A. cacae in a previous study of human infant feces and diet (21). A. cacae is non-sporeforming, utilizes lactate and acetate, and produces butyrate (22, 23). Spearman correlations between Lachnospiraceae OTU259772 and several highly correlated ileal DEGs of interest from Figure 3A (Ror2, Fbp1, Tgfbr3, Acot12, and Me1) are shown in Figure 4B. Analysis of ileal and fecal samples using quantitative PCR (qPCR) with previously validated species-specific primers (24) provided independent confirmation of A. cacae enrichment in healthy transplanted mice (Figures 4C-E and Figures S13A-C). The abundance of A. cacae in ileal samples also correlated with DEGs derived from ileal IECs (Figure S14).Notably, two of the highly correlated DEGs (Acot12 and Me1) are involved in pyruvate metabolism. Butyrate is an important energy source for colonic epithelial cells (25). Butyrate drives oxygen consumption by colonocytes through β-oxidation, thereby maintaining a locally hypoxic niche for butyrate-producing obligate anaerobes (26). Under dysbiotic conditions, colonocytes generate energy through glycolysis, a process that involves the production of pyruvate as a key intermediate (27). It is tempting to speculate that the negative correlation between the abundance of butyrate-producing A. cacae and pyruvate metabolism-related genes in IECs from CMA-implanted mice reflects a metabolic shift in ileal epithelial function under dysbiotic conditions.

[0083] Next, we investigated whether A. cacae monotransplantation into GF mice could mimic the gene expression changes associated with healthy microbiota and protection from anaphylaxis (see "Methods"). Several of the genes significantly upregulated in healthy transplanted mice (Fbp1, Tgfbr3) were also significantly upregulated in A. cacae monotransplanted mice compared with GF or CMA transplanted mice (Figure 4F). Acot12 expression was significantly upregulated in CMA transplanted mice, but not in healthy or A. cacae monotransplanted mice (Figure 4F). A. cacae monotransplanted mice sensitized with BLG + CT were protected from anaphylactic reactions to BLG challenge. Similar to Figure 1, CMA transplanted mice showed a significant decrease in core body temperature, indicative of anaphylaxis (Figure 4G). Both changes in core body temperature and serum concentrations of mMCPT-1 were significantly reduced in mice transplanted with A. coli alone compared with mice transplanted with CMA (Fig. 4G, J). Antigen-specific Th2-dependent antibodies (BLG-specific IgE and IgG1 in serum) (Fig. 4H, I) and cytokine responses, IL-13 and IL-4 (Fig. 4K, L), were all reduced in mice transplanted with A. coli alone.

[0084] We have shown that anaerobic mucosal-associated bacteria of the Clostridia class have attracted considerable interest because of their reported roles in maintaining intestinal homeostasis through the induction of regulatory T cells (28, 29), the production of immunomodulatory metabolites (30, 31), and the control of colonization resistance (32). We have demonstrated that such immunomodulatory bacteria are present in the ileum, the site of food absorption, and demonstrated their role in protecting against anaphylactic reactions to food. The mechanistic analysis of the Clostridium-associated changes in ileal gene expression described herein is likely to reveal additional pathways important for maintaining tolerance to dietary antigens. The model described in this report does not resolve whether the allergic state drives dysbiosis (33) or whether dysbiosis precedes allergy. Indeed, many factors likely contribute to the development of food allergy. These data demonstrate that commensal bacteria play an important role in preventing allergic reactions to food and provide proof-of-concept for the development of microbiome-modulating strategies to prevent or treat this disease.

[0085] C. Method Gnotobiotic Mouse Husbandry. All mice were bred and housed at the Gnotobiotic Research Animal Facility (GRAF). Mice were maintained in Trexler flexible film isolator housing units (Class Biologically Clean) containing Ancare polycarbonate mouse cages (catalog no. N10HT) and Teklad Pine Shavings (7088; autoclaved) at a room temperature of 20–24°C with a 12-hour light / dark cycle. Mice received USP-grade autoclaved sterile water at pH 5.2 ad libitum. Bedding was changed weekly; formula-fed mouse cages required nearly daily bedding changes due to formula leakage from the bottles. All mice were fed Purina Lab Diet® 5K67, stored in a temperature-controlled environment in accordance with the Guide for the Care and Use of Laboratory Animals (8th Edition, 2013). Diets were sterilized by autoclaving at 121°C for 30 minutes. Sterility of the isolators was checked weekly by both fecal culture and 16S rRNA analysis by qPCR. Aerobic and anaerobic incubations at 37°C and aerobic incubations at 42°C were performed for 96 hours in BHI broth, nutrient broth, and Sabbaroud broth. All mice were initially screened at rederivation or receipt for all internal and external parasites, complete serological profiles and / or PCR, bacteriology, and gross and histological analysis of major organs by either IDEXX Radil or Charles River Labs using the Axenic Profile Screen. Germ-free (GF) C3H / HeN mice were transferred to the facility from T. Golovkina (University of Chicago).

[0086] Preparation of human fecal samples. Healthy (non-allergic) fecal samples were obtained from participants in a vaccination program. These subjects were not at risk for atopic disorders, and their medical history was negative for allergic conditions. Infants with CMA were diagnosed at a tertiary pediatric allergy center (Pediatric Allergy Program at the Department of Translational Medical Science of the University of Naples 'Federico II'); for complete patient information, see Supplementary Tables 1 and 2. All aspects of this study were conducted in accordance with the Declaration of Helsinki and approved by the Ethics Committee of the University of Naples 'Federico II'. Written informed consent was obtained from the parents / guardians of all children participating in the study. Fresh fecal samples were collected in the clinic into sterile tubes, weighed, and mixed with 2 mL of LB broth + 30% glycerol per 100–500 mg. They were then aliquoted into sterile cryovials and immediately stored at -80°C. Samples were shipped on dry ice to the University of Chicago, where they were stored at -80°C until homogenization. For transplantation into mice, frozen fecal samples were introduced into an anaerobic chamber and thawed. The thawed feces were mixed with 3 mm borosilicate glass beads in a sterile 50 mL tube containing 2.5 mL of pre-reduced PBS + 0.05% cysteine ​​and gently vortexed to dissociate. The resulting homogenate was filtered through a 100 μm filter. This homogenization and filtration process was repeated three more times, and the final filtrate was mixed with an equal volume of 30% glycerol + 0.05% cysteine. This solution was aliquoted into Balch tubes with rubber stoppers for transport and introduction into gnotobiotic isolators. The remaining fecal solution was aliquoted and frozen at -80°C.

[0087] Transplantation of Germ-Free Mice. All mice were weaned onto a plant-based mouse diet (Purina Lab Diet® 5K67) at 3 weeks of age and transplanted at weaning. GF mice received autoclaved sterile water. Both male and female mice were used for all experiments. Each experiment was a littermate control. All mice were identified by a unique 5-digit ear tag. All studies were conducted in accordance with Institutional Biosafety and Animal Care and Use Committees. Each human infant donor transfer was maintained in its own flexible film isolator to avoid cross-contamination. In all experiments, repository mice were generated from human fecal donors by gavaging 500 μL of freshly prepared infant fecal homogenate into GF mice. These repositories were then used to transplant subsequent experimental mice via mouse-to-mouse transfer via gavage of mouse feces. Fecal samples from both repository and experimental mice were routinely examined by 16S rRNA analysis, demonstrating that gavage-mediated transfer of fecal matter from repository mice to experimental mice was highly reproducible and stable over time. For experimental mouse transplants, freshly excreted fecal pellets from repository mice were homogenized in 1 mL of sterile PBS, and 250 μL of this homogenate was used to gavage one recipient mouse. For formula-fed mice, drinking water was replaced with formula 4 hours before transplantation. Mice transplanted with healthy infant feces received Enfamil Infant (Mead Johnson Nutrition, Evansville, IN) ad libitum, while CMA-transplanted mice received extensively hydrolyzed casein formula (EHCF), Nutramigen I (Mead Johnson Nutrition). Both dry and liquid forms of formula were utilized. Dry formula was mixed with USP grade autoclaved sterile water according to the manufacturer's instructions. All formula was changed daily.

[0088] For mice transplanted with Anaerostipes cacae alone, A. cacae (DSM-14662, DSMZ) was cultured in an anaerobic chamber (Coy, Model B) in reduced Schaedler broth (Remel) overnight at 37 °C until an optical density (OD600) of 1.08 was reached. 250 μL (approximately 2.5 × 10 8 CFU) were gavaged into GF mice. These mice were monitored for transplantation by qPCR with species-specific primers (Supplementary Table 6) and maintained as viable repositories. For transplantation of experimental mice, Enfamil infant formula (liquid) was added to the drinking water 4 hours before transplantation. Freshly excreted fecal pellets from repository mice were then homogenized in 1 mL of sterile PBS, and 250 μL of this homogenate was used to gavage one recipient mouse. Single transplantation of A. cacae was confirmed for all experimental mice by 16S rRNA-targeted sequencing of fecal samples collected at the time of sacrifice.

[0089] 16S rRNA-targeted sequencing. Bacterial DNA was extracted using the Power Soil DNA Isolation Kit (MoBio). 16S rRNA gene amplicon sequencing was performed on an Illumina MiSeq at the Environmental Sample Preparation and Sequencing Facility at Argonne National Laboratory. The method described in reference 34 was used to generate 151-bp paired-end reads from fecal samples with 12-bp barcodes. The V4 region of the 16S rRNA gene was PCR amplified with region-specific primers (515F-806R) containing sequencer adapter sequences used in an Illumina flow cell. A signature cohort of microbiota (n = 99) consisting of fecal samples from infant donors and fecal and ileal samples from gnotobiotic mice was analyzed using Quantitative Insights into Microbial Ecology (QIIME) (version 1.9) (35). Raw reads were trimmed to remove low-quality bases; overlapping paired-end 3' sequences were merged using SeqPrep (found on the World Wide Web at github.com / jstjohn / SeqPrep). An open-reference OTU picking protocol was used against the Greengenes database (08 / 2013 release) at 97% sequence identity (36). Sequences were aligned using PyNAST (37). Taxonomic assignment was performed using the uclust consensus taxonomy assigner (38); predicted chimeric sequences were removed using ChimeraSlayer (v20110519) (found on the World Wide Web at microbiomeutil.sourceforge.net).Data were rarefied to a mean depth of 3,160 reads for the donor and transplant mouse cohort (n = 99, consisting of donor fecal samples, mouse fecal samples 2 and 3 weeks after transplantation, and mouse ileum samples) and 10,050 reads for the mouse cohort shown in Figure 12C (n = 70, consisting of paired fecal and ileum samples from 35 mice 1 week after transplantation). α (Shannon index) and β diversity metrics were compared between the CMA and healthy groups using a two-tailed Mann-Whitney-Wilcoxon test (nonparametric) and PERMANOVA (39) with weighted UniFrac distances in the R package vegan (v2.4.5), respectively. Peel's evenness index, J', is The variance was calculated using the Shannon exponent (H') and S as the maximum number of OTUs. The discrete false discovery rate (DS-FDR) (40) was used to identify differentially abundant bacterial taxa between the fecal biota of the CMA group and the healthy control group with the parameters "transform_type=normdata, method=meandiff, alpha=0.10, numperm=1000, fdr_method=dsfdr" (accessed 02262018) (https: / / github.com / biocore / dsFDR). Compared to the Benjamini-Hochberg FDR (BH-FDR) method, the DS-FDR method has increased power due to limited sample size and is robust to sparse data structures (low proportion of non-zero values ​​in the microbial abundance table), making it uniquely suited for microbiome data analysis (40). The DS-FDR algorithm does not calculate adjusted P values; instead, it estimates the false discovery rate from a permutation test (default 1,000 permutations) that controls the FDR at the desired level (0.10). Therefore, it calculates raw P values, test statistics, and rejected hypotheses in its output (Supplementary Tables 3 and 5). OTUs present in fewer than four samples in each comparison were removed before applying the DS-FDR test. Linear discriminant analysis effect size (LEfSe) was used to identify genera significantly enriched in the CMA or healthy control groups compared with the other, using a per-sample normalization value of 1,000,000 and default values ​​for other parameters (41). In the LEfSe analysis, linear discriminant analysis (LDA) scores were calculated for taxa differentially present between the two groups. Taxa with P < 0.05 (Kruskal-Wallis test) and log(LDA) ≥ 2.0 (or ≤ -2.0) were considered significant.For Figure 2A, after testing for differential abundance in donor CMA vs. healthy comparisons using DS-FDR, we further filtered significant OTUs by requiring their presence in at least two mouse groups, leaving a total of 58 OTUs for further analysis. The OTU ratio was calculated for each sample by dividing the total number of potentially protective (more abundant in healthy) OTUs by the total number of potentially non-protective (more abundant in CMA) OTUs. Furthermore, an OTU abundance score was calculated taking into account the abundance of the 58 OTUs identified in CMA compared to healthy donor fecal samples, as shown in Figure 2A. First, a data transformation was applied to the relative abundance to approximate a Gaussian distribution. A constant (1 x 10) was applied to the relative abundance of each OTU so that all values ​​were greater than 1. 6 ), which was log10 transformed and scaled by dividing the value by the root mean square of the sample. The abundance of potentially non-protective OTUs was multiplied by (-1). The sum of the transformed abundances of the 58 OTUs was then calculated to generate an aggregate score. To validate the differences in OTU ratios in an independent cohort, we reanalyzed the 16S sequencing data of fecal samples (n = 38) collected from healthy and CMA infants in reference 5 using the same analytical protocol as above, with data rarefied to an average depth of 6,424 reads. Of the 58 OTUs shown in Figure 2A, 55 OTUs were assigned known reference IDs and three were assigned novel reference IDs (Supplementary Table 3). Novel reference OTU IDs were not comparable between different analysis cohorts; therefore, we focused on OTUs with known reference IDs. Fifty-two of the 55 known OTUs were concordant in the reanalyzed independent cohort and were used to calculate the protective / non-protective OTU ratios shown in FIG.

[0090] Sensitization and challenge with food allergens. The protocol was adapted from Reference 7. All mice were weaned to a plant-based mouse diet (Purina Lab Diet® 5K67) at 3 weeks of age. GF mice received autoclaved sterile water. For mice transplanted with feces from infant donors or mice transplanted with A. cacae alone, drinking water was replaced with formula 4 hours before transplantation. Mice transplanted with healthy feces or A. cacae received Enfamil; CMA-transplanted mice received Nutramigen (both from Mead Johnson). On day 0, 1 week after weaning (GF), or at the time of transplantation (healthy / A. cacae / CMA), all mice were fasted for 4 hours and then gavaged with 200 mM sodium bicarbonate. After 30 minutes, mice were given 20 mg BLG (Sigma) + 10 μg CT (List Biologicals). This protocol was repeated weekly for 5 weeks. For formula-fed mice, the formula was replaced with sterile water one week after the final sensitization. Before the challenge on day 42, mice were fasted for 4 hours and given sodium bicarbonate by gavage. Then, 100 mg of BLG was administered twice by gavage, 30 minutes apart. Core body temperature was measured blindly using a rectal probe (PhysiTemp) before allergen challenge and every 5 minutes after the first challenge, until at least 30 minutes after the second challenge. Serum was collected 1 hour after the second challenge to measure mMCPT-1 levels. Serum was collected 24 hours after challenge to measure antibodies.

[0091] ELISA. mMCPT-1 was quantified in serum collected 1 h after the second challenge according to the manufacturer's protocol (eBioscience). BLG-specific ELISA was performed using a protocol modified from reference 7. Briefly, plates were coated with 100 μg / mL BLG in 100 mM carbonate-bicarbonate buffer (pH 9.6) overnight at 4°C. Plates were blocked with 3% BSA for 2 h at room temperature. Samples were added to 1% BSA and incubated overnight at 4°C. Assays were standardized using BLG-specific antibodies (IgE or IgG1) purified on a CNBr-Sepharose affinity column from mice immunized with BLG plus alum (42). BLG-specific antibodies were detected with goat anti-mouse IgE-UNLB (Southern Biotech) and rabbit anti-goat IgG-AP (ThermoFisher), followed by development with p-NPP (KPL Labs) or IgG1-HRP (Southern Biotech) and TMB (Sigma).

[0092] For cytokine analysis, spleens were collected 24 hours after challenge from mice sensitized with BLG+CT for 5 weeks and transplanted with A. cacae or CMA. Splenocytes were cultured at 2 × 10 in 10 mg / ml BLG (Sigma) in cDMEM containing 4% FCS (HyClone), 10 mM HEPES (Gibco), 100 U / ml penicillin / streptomycin (Gibco), and 55 μM 2-mercaptoethanol (Gibco) at 37°C and 10% CO. 6 The cells were stimulated at a concentration of 1000 cells / ml. Cytokine concentrations in the 72-hour culture supernatant were determined by ELISA for IL-13 and IL-4 (both manufactured by Invitrogen).

[0093] Epithelial cell isolation. As in the sensitization experiments, mice were weaned at 3 weeks of age and maintained on formula prior to transplantation. Seven days after transplantation, mice were euthanized and the ileum was removed. For IEC isolation, the tissue was cleaned and inverted as described in reference (43). IECs were collected by distending the inverted tissue in Cell Recovery Solution (Corning) every 5 minutes for 30 minutes. IEC samples were lysed with TRIZol (ThermoFisher), and RNA was extracted using the PureLink RNA Mini Kit (Ambion) with on-column DNAse treatment (PureLink DNAse Set, Ambion).

[0094] RNASeq. RNA libraries were prepared using the TruSeq Stranded Total Library Preparation Kit with Ribo-Zero human / mouse / rat (Illumina). Samples were sequenced at the University of Chicago Functional Genomics Core using 50-bp single-read chemistry on a HiSeq2500 machine with two sequencing lanes. Raw read quality was assessed by FastQC (v0.11.5) (44). The QC30 score, which represents the percentage of bases with a quality score ≥ Q30, was 96.81% ± 0.06% (mean ± sem) across 39 RNAseq samples. Alignment to the mouse reference transcriptome was performed using Gencode gene annotation (vM16, GRCm38) with Kallisto (v0.43.1) in strand-specific mode (45). This mode implements a kmer-based pseudoalignment algorithm to accurately quantify transcripts from RNASeq data while robustly detecting errors in the reads. The average mapping rate was 62.77% ± 1.10% (mean ± SEM) based on Kallisto pseudoalignment with the reference transcriptome. On average, 35 million raw sequencing reads were generated for each sample, and 22 million were mapped to the transcriptome using Kallisto. Transcript abundance was quantified using a strand-specific protocol, summarized to the gene level using tximport (v1.4.0) (46), normalized by the trimmed mean M-value (TMM) method, and log2-transformed. Genes expressed in at least three samples (counts per million reads (CPM) > 3) were retained for further analysis. Differentially expressed genes between groups were identified using the limma voom algorithm with precision weights (v3.34.5) ( 47 ).The duplicateCorrelation function was used to estimate correlations between mouse samples, using donor (1–8) as a blocking factor. The lmFit function was used to fit all mouse samples (n = 39, 18 CMA transplants, 18 healthy transplants, and 3 GF transplants) to a single linear model incorporating the correlation structure calculated above. Contrasts were set as CMA vs. healthy, CMA vs. GF, and healthy vs. GF ​​to identify DEGs in each comparison. Genes significantly differentially expressed between CMA and healthy mice and also different from GF mice were identified using a two-step approach: (1) genes were detected as different in the CMA vs. healthy comparison with a fold change of ≥ 1.5 or ≤ -1.5 at a false discovery rate (FDR)-corrected p-value of < 0.10; (2) genes from step (1) were further filtered by a fold change of ≥ 1.5 or ≤ -1.5 in the CMA vs. GF ​​or healthy vs. GF ​​comparison at an FDR of 0.05. A more stringent FDR threshold (0.05) was applied in step (2) to prioritize potential true positives when compared to the negative control (GF). Multiple testing correction was performed using the Benjamini-Hochberg FDR (BH-FDR) method (48). A total of 32 DEGs, representing four types of gene expression changes in transplanted mice, passed these thresholds: (1) Up in Healthy: genes upregulated in healthy mice compared to both CMA and GF; (2) Up in CMA: genes upregulated in CMA mice compared to both healthy and GF; (3) Down in Healthy: genes downregulated in healthy mice compared to both CMA and GF; and (4) Down in CMA: genes downregulated in CMA mice compared to healthy and GF. The four groups of DEGs are shown in Figures 3A and 4A. Significantly enriched gene ontology and KEGG pathways in the 32 DEGs of interest were identified using clusterprofiler (v3.6.0) ( 49 ) with a false discovery rate (FDR)-corrected p -value (BH-FDR method, hypergeometric distribution test) of less than 0.10.For this analysis, the DEGs were divided into two groups: (1) healthy, which included all genes that were up in healthy and down in CMA; and (2) CMA, which included all genes that were up in CMA and down in healthy. Correlations between DEGs and ileal OTUs that were significantly differentially present between CMA and healthy samples were calculated using Spearman's rank correlation method, followed by the following filters: (1) retain OTUs from the designated group that showed significant correlation with at least one DEG at P<0.05. For potentially protective (more abundant in healthy) OTUs, they correlated with genes from the "up in healthy" or "down in healthy" group; for potentially non-protective (more abundant in CMA) OTUs, they correlated with genes from the "up in CMA" or "down in CMA" group; and (2) retain OTUs from the designated group that showed a relatively consistent trend of positive correlation (Spearman's ρ>0.20) in at least 60% of the DEGs. For potentially protective OTUs, they were correlated with the combined genes from the "up in healthy" or "down in CMA" groups; for potentially non-protective OTUs, they were correlated with the combined genes from the "up in CMA" or "down in healthy" groups; (3) OTUs present in at least three CMA mice and three healthy mice were retained. Nine ileal OTUs passed these correlation filters and are shown in Figure 4A. Correlations between relative abundance of OTUs and gene expression were calculated using Spearman's correlation method with samples exceeding the detection limit of the assay.

[0095] qPCR. Gene expression was measured by qPCR as described in reference 7. Briefly, cDNA was prepared from RNA using the iScript cDNA synthesis kit (BioRad). Gene expression was measured using PowerUp SYBR Green Master Mix (Applied Biosystems) according to the manufacturer's instructions. Primers are listed in Supplementary Table 7 (20, 50-54). Expression of genes of interest was normalized to Hprt. Relative expression was measured using ΔΔCt centered at the geometric mean; GF mice were used as the reference.

[0096] The presence of A. cacae in fecal and ileal samples was confirmed using qPCR as described in reference (25). Bacterial DNA was extracted using the Power Soil DNA Isolation Kit (MoBio), and qPCR was performed using 4 μl of each primer with a 10 μM working dilution and 2 μl of bacterial DNA using PowerUp SYBR Green Master Mix (Applied Biosystems). Primers are listed in Supplementary Table 6. The cycling conditions for the reaction consisted of an activation cycle at 50°C for 2 min, followed by one cycle at 95°C for 10 min, and 40 cycles of 94°C for 20 s, 55°C for 20 s, and 72°C for 50 s. The fluorescent probe was detected during the final step of this cycle. A melting curve was performed at the end of PCR to confirm the specificity of the PCR products. Relative abundance was measured using the PowerUp SYBR Green Master Mix (Applied Biosystems). -Ct was normalized to the total 16S rRNA copy number per gram of fecal material, and a constant (1 × 10 25 )

[0097] Histopathological Analysis. For histological analysis, 3 mm tissue sections of the mid-colon and mid-ileum were fixed in either 10% formalin for H&E staining or Carnoy's fixative for periodic acid-Schiff (PAS) staining. Sectioning and staining were performed by the Human Tissue Resource Center at the University of Chicago. All sections were examined blindly by a GI pathologist.

[0098] Statistical analysis. Prism 7.0 (GraphPad) was used to perform one-way ANOVA (Figure 4F) with Bonferroni correction for multiple comparisons and two-tailed Student's t-tests (Figure 4D and Figure 13B), as indicated in the figure legends. The DS-FDR method was used to identify significant OTUs comparing the CMA group with the healthy group (Figure 2A, Figure 4C, and Figure 13A). The BH-FDR method was used to correct for multiple testing in the RNA-Seq analysis (Figure 3A) and GO enrichment analysis (Figure 3B). Shannon diversity and Pirou evenness were compared using a two-tailed nonparametric Mann-Whitney-Wilcoxon test (Figure 7A, B). Analysis of protective / non-protective OTU ratios in a larger, independent cohort of infants was performed using a two-tailed Mann-Whitney-Wilcoxon test (Figure 11). The biological responses of mice transplanted with different donors to BLG sensitization (Figures 1A–D, 4G, 8A, and 9A) were explored using linear mixed-effects models (55) based on restricted maximum likelihood (REML) in R (ImerTest v3.0.1) (56). Temperature changes (both linear and quadratic) over time for groups (GF, healthy, and CMA in Figure 1A; A. cacae and CMA in Figure 4G; healthy BFD and CMA BFD in Figure 8A; HO and Enfamil in Figure 9A) were modeled as Temperature = Group + Time*Group + Time*Time*Group, with random intercepts and slopes estimated for individual mice. Contrasts between group temperature trends were performed using t-tests with Benjamini-Hochberg FDR (BH-FDR) correction for multiple comparisons. To control for cases where a group contained multiple donors (Figure 1A), the previous model was revised to include mice nested within each donor as a random effect, and contrasts were repeated. Results from the two models were consistent, so for consistency of methodology, results from the first model are reported. For Figures 1B–D, antibody concentrations were log-transformed and modeled as log(Concentration) = Group, with donor as a random effect.Contrasts of group differences were performed using previously mentioned methods. For Figures 4H-I, 8B-D, and 9B-D, antibody and cytokine concentrations were log-transformed and compared using t-tests. Data analysis commands (including data files and R markdown files for reproducibility) are available from the authors upon request.

[0099] D.Table Supplementary Table 1. Patient data for the formula-fed infant donors used in this study TIFF0007737098000003.tif243142

[0100] Supplementary Table 2. Patient data for breast-fed infant donors in Supplementary Figure 4. TIFF0007737098000004.tif122167

[0101] Supplementary Table 3. 58 OTUs differentially present between CMA and healthy donor fecal samples shown in Figure 2a TIFF0007737098000005.tif231163TIFF0007737098000006.tif245163TIFF0007737098000007.tif243163 TIFF0007737098000008.tif245163TIFF0007737098000009.tif243163TIFF0007737098000010.tif224163

[0102] Supplementary Table 4. 32 genes differentially expressed in ileum RNAseq samples from CMA-transplanted and healthy-transplanted mice shown in Figure 3a and different from the negative control (GF mice). TIFF0007737098000011.tif246141NE: Not assessable; If a DEG does not meet the filtering criteria in either the "CMA vs. GF" or "healthy vs. GF" comparison, the directional change is not assessable. ND: Undetermined; if a DEG meets the filtering criteria in both the "CMA vs. GF" or "healthy vs. GF" comparisons, the one with the higher P value is reset to undetermined to resolve the discrepancy.

[0103] Supplementary Table 5. 108 OTUs differentially present between CMA and healthy mouse ileum samples TIFF0007737098000012.tif226160TIFF0007737098000013.tif240160TIFF000 7737098000014.tif240160TIFF0007737098000015.tif240160TIFF0007737098 000016.tif244160TIFF0007737098000017.tif245160TIFF0007737098000018. tif241160TIFF0007737098000019.tif240160TIFF0007737098000020.tif95160

[0104] Supplementary Table 6. Primer sequences used for qPCR analysis of Anaerostipes cacae TIFF0007737098000021.tif12159

[0105] Supplementary Table 7. Primer sequences used for qPCR analysis in Figure 4f TIFF0007737098000022.tif72160

[0106] Example 2: Isolation and characterization, in vitro fermentation, initial in vivo experimental design Isolation and characterization of AA cacae A. cacae was isolated from healthy infant feces frozen in 30% glycerol stocks. First, undiluted fecal glycerol stocks from healthy donor #2 were plated onto brain heart infusion (BHI) agar supplemented with vitamin K, hemin, and antibiotics. The antibiotics used were 16 μg / ml ciprofloxacin or a mixture of 16 μg / ml ciprofloxacin, 6 μg / ml gentamicin, 5 μg / ml aztreonam, and 10 μg / ml colistin (58, 60). This donor was chosen because previous sequencing data indicated that he had the highest relative abundance of A. cacae among four healthy donors. Plates were incubated in an anaerobic chamber at 37°C for 6 days. Plates were then scraped and suspended in 50% glycerol solution. The suspension is then aliquoted, and one aliquot is used for qPCR, while the other is frozen at -80°C for future cultivation. A. coccae abundance is quantified using qPCR with species-specific primers to the 16S rRNA gene (24). Frozen aliquots of the suspension from the plate shown to have the highest abundance of A. coccae are diluted, plated on BHI agar, and incubated in an anaerobic chamber at 37°C for 6 days. Single colonies are then isolated from 10 -5A selection from the dilution was inoculated into pre-reduced chopped meat and glucose broth (CMG) and incubated overnight in an anaerobic chamber. An aliquot of this broth culture was then taken for A. cacae-specific PCR and universal PCR 16S rRNA amplification for Sanger sequencing. Another aliquot was taken, diluted 1:1 with 50% glycerol, and frozen in a cryovial at -80°C. Colonies that were A. cacae-positive by both PCR and Sanger sequencing were then streaked from the frozen cryovial onto BHI agar to increase purity. A single colony from this plate was then inoculated into chopped meat and glucose broth and grown overnight at 37°C in an anaerobic chamber. An aliquot of this stock culture was diluted 1:1 with 50% glycerol and stored in a cryovial at -80°C for future studies. This isolation is shown in Figure 15.

[0107] Further confirmation of the A. cacae isolates was performed using CosmosID® whole genome analysis. The method described in Figure 15 generated seven A. cacae isolates (all from healthy donor #2). The three colonies deemed most distinct from each other by Sanger sequencing (designated 66a_Rep_1_1_IonXpress_011_trimmed, D24_colony_4_2_IonXpress_015, and D24_colony_5_IonXpress_016) were subjected to whole genome sequencing. All three analyzed colonies are highly similar to A. cacae 3 2 56FAA and A. cacae DSM 14662, but are distinct strains from these known reference strains. Although they fall within the cutoff homology range for classification as A. cacae (>98.5% homology), the SNP distance (>100 SNPs) identifies this isolate as a distinct strain from reference strains 3_2_56FAA and DSM 14662. However, the isolates are all identical to each other (0 SNPs); therefore, this strain is referred to herein as A. cacae_lah. Whole genome analysis by CosmosID® also confirmed the absence of virulence genes in this isolate; the only antibiotic resistance gene recorded was for the tetracycline class. This is further illustrated in the table below.

[0108] TIFF0007737098000023.tif23488

[0109] The antibiotic susceptibility of A. cacae_lah was characterized by growth inhibition around antibiotic disks. Ice chips of frozen A. cacae_lah stock were grown overnight in CMG broth, and 100 μl of the broth was spread on BHI agar. Antibiotic disks (10 μg streptomycin, 30 μg kanamycin, 30 μg tetracycline, and 10 μg ampicillin) were then placed on the agar to measure the zone of clearance, which indicates antibiotic susceptibility. The plates were incubated at 37°C under anaerobic conditions for 6 days. A. cacae_lah is highly sensitive to ampicillin, as indicated by the large clearance diameter (Figure 16). It is also somewhat sensitive to tetracycline, despite carrying the tetracycline resistance gene (Figure 16).

[0110] B. In vitro fermentation A. cacae_lah cannot ferment complex carbohydrates in monoculture, but it can ferment simple sugars, such as those found in infant formula, such as lactose. A. cacae_lah stock ice chips were grown in CMG broth at 37°C under anaerobic conditions for 24 hours. This preculture method will be used for all future in vitro fermentation experiments. Ten microliters of the preculture was then transferred to 7 mL of minimal peptone yeast (PY) broth alone or supplemented with 10 mg / mL of glucose, sucrose, lactose, cellobiose, or potato starch. All of these PY variations were pre-reduced and prepared anaerobically by Anaerobe Systems. Growth and butyrate production were measured after 48 hours. As measured by OD600, A. cacae_lah grew abundantly when PY broth was supplemented with sucrose, glucose, and lactose compared to cellobiose or potato starch. Butyrate in solution was quantified by HPLC-UV-Vis as described in references 61 and 62. Similarly, A. cacae_lah produced the most butyrate when grown in sucrose, glucose, or lactose. These simple sugars are similar to those found in infant formula. To test the abundance and butyrate production of A. cacae_lah in infant formula, A. cacae_lah was grown in PY broth supplemented with Nutramigen® (10 mg / ml carbohydrate), an iron-fortified hypoallergenic infant formula designed for infants with CMA, or Enfamil® (10 mg / ml carbohydrate), a standard infant formula containing cow's milk. These are the same formulas consumed by healthy infant donors (Enfamil) and CMA infant donors (Nutramigen), and by all mice transplanted with these respective microbiomes. A. cacae_lah abundance was similar when grown in Enfamil or Nutramigen as PY broth, demonstrating that A. cacae_lah does not depend on milk consumption for growth.However, butyrate production was highest in Nutramigen compared to PY broth alone. The majority of sugars in Nutramigen are corn syrup containing fructose and sucrose, which reflects the high butyrate production by A. cacae_lah when supplemented with sucrose alone. All groups were analyzed by one-way ANOVA. PY vs. Nutramigen. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. The results are shown in FIG.

[0111] A. cacae_lah can use both lactate and acetate to produce butyrate. Although A. cacae and other Clostridia are generally considered fiber-fermenting species, previous data suggest that A. cacae_lah alone cannot consume complex fibers for growth or butyrate production. Other groups have shown that the dominant degrading bacteria, i.e., Bacteroides species, can degrade complex fibers, producing the metabolites lactate and butyrate, which Clostridium species can then consume to produce acetate (59). To investigate whether this complex cross-feeding can be mimicked in vitro, we supplemented PY broth with only these metabolites to stimulate butyrate production by A. cacae_lah. A. cacae_lah preculture was grown from a frozen glycerol stock as described above, and 10 μl was then transferred to minimal PY broth supplemented with 33 mM acetate and / or 40 mM lactate (57). Growth and butyrate production were measured after 48 hours. As shown in Figure 18, A. cacae_lah had significant growth when supplemented with one or both metabolites. Interestingly, the strain produced significantly higher levels of butyrate only when supplemented with both lactate and acetate compared to the single supplementation. This data supports the cross-feeding mechanism and metabolic cycle described by other publications for bacteria of this family. All groups were analyzed by one-way ANOVA. PY alone vs. * P<0.05, ** P<0.01,*** P<0.001、 **** P<0.0001。

[0112] As shown in Figure 19, A. cacae_lah produces substantially more butyrate from complex carbohydrates in coculture with a complex bacterial mixture derived from an allergic (CMA) infant donor. Precultures of A. cacae_lah or human CMA fecal samples (frozen glycerol stocks from human donor 6) were grown as described above. A total of 10 μl (10 μl A. cacae_lah, 10 μl CMA, or 5 μl A. cacae_lah + 5 μl CMA) was then transferred to minimal PY broth alone or supplemented with 10 mg / ml potato starch or cellobiose (Anaerobe Systems). Growth and butyrate production were measured after 48 hours. A. cacae_lah grew similarly in all three media in monoculture and was below the limit of detection in CMA culture. When A. cacae_lah was co-cultured with the CMA bacterial mixture, abundance measured by qPCR was similar to or slightly higher than that observed in monoculture growth. This demonstrates that A. cacae_lah can compete for substrates and establish a niche in complex co-cultures. As previously shown, A. cacae_lah failed to produce significant butyrate in monoculture when grown in media supplemented with any carbohydrate. Butyrate production by CMA-only cultures in PY and PY + starch was also minimal. However, when supplemented with any carbohydrate, especially starch, the co-culture produced significantly higher levels of butyrate compared to A. cacae_lah alone or CMA alone. There is some evidence that potato starch may be an excellent prebiotic supplement to support A. cacae_lah growth and butyrate production in vivo. When supplemented with cellobiose, the CMA and co-culture produced more butyrate than A. cocae_lah alone, suggesting that the CMA bacterial mixture contains several species capable of producing butyrate from fiber degradation and that the addition of A. cocae_lah did not contribute additional butyrate to the system. Therefore, cellobiose may not be a suitable prebiotic supplement.

[0113] Because feces from CMA-transplanted repository mice will be used to transplant all future CMA mice, we determined whether the bacterial mixture derived from these repository mice's feces behaves similarly to the human CMA mixture. Fecal pellets from previously transplanted repository mice from CMA donor 6 were collected, homogenized in sterile PBS, diluted 1:1 with 50% glycerol, and frozen in cryovials at -80°C. Precultures of A. cacae_lah, human CMA flora (hCMA), and mouse CMA flora (msCMA) were prepared as described above. The precultures were then inoculated into PY broth alone (10 μl) or together (5 μl A. cacae + 5 μl CMA), with or without supplements, or A. cacae_lah and supplements were added 24 hours later. Supplements (Nutramigen, lactate / acetate) were added at the same concentrations as previously described, and approximately 10 mg / ml wheat bran was used. In the groups supplemented at later time points, growth and butyrate production were measured at t = 48 h or 72 h to allow A. cacae growth for a full 48 h. As expected, neither CMA mixture had measurable A. cacae. While bacterial mixtures derived from CMA repository mouse feces produce more butyrate than freshly thawed human CMA feces at baseline, the addition of A. cacae_lah with lactate and acetate further resulted in a significant increase in butyrate concentrations (Figure 20). Many factors could contribute to the differences between the two CMA sources, including species loss during transfer into mice, increased abundance of butyrate-producing bacteria in repository mice over time, or differences in the time elapsed during frozen culture.

[0114] A. cacae_lah can grow in vitro when inoculated at lower abundances. Because A. cacae reaches much lower relative abundances when introduced into CMA-implanted mice, in this series of experiments we determined whether A. cacae_lah can grow in coculture even when inoculated at a lower abundance than the CMA mixture. This experiment was performed to more accurately reflect the feasibility of introducing A. cacae_lah into CMA-implanted mice than simultaneous 1:1 inoculation. Precultures of A. cacae_lah or CMA were performed as described herein, and then 10 μl was transferred to minimal PY broth supplemented with 10 mg / ml carbohydrate (10 mg / ml carbohydrate Nutramigen, Nutramigen + 10 mg / ml scFOS (short-chain fructooligosaccharides, a clinical prebiotic), or Nutramigen + LA (40 mM lactate + 33 mM acetate)). Precultures of A. cacae_lah and CMA were transferred to supplemented PY broth or CMG broth in various ratios: 10 μl A. cacae_lah: 0 μl CMA; 5 μl A. cacae_lah: 5 μl CMA; 3 μl A. cacae_lah: 7 μl CMA; 1 μl A. cacae_lah: 9 μl CMA. As shown by species-specific qPCR, the volume of A. cacae_lah did not significantly affect its growth or butyrate production. Addition of scFOS reduced the total butyrate concentration compared to medium containing Nutramigen alone. Addition of lactate and acetate resulted in the greatest butyrate concentration. The amount of butyrate in this medium was higher in the co-culture than in A. cacae_lah alone; in A. cacae_lah monocultures, no detectable lactate or acetate was present in the medium at 48 hours. This suggests that the CMA species in the culture may contribute additional lactate and acetate to the system, which may allow continued butyrate production by A. cacae_lah after the supplemented metabolites are depleted.

[0115] Prior to initiating the in vivo transplantation experiments, the presence of A. cacae in healthy repository mice and its absence in CMA repository mice were confirmed and measured by qPCR. Repository mice transplanted with feces from healthy infant donor 2 exhibited measurable A. cacae abundance by qPCR, whereas repository mice transplanted with feces from CMA donor 6 did not. We predict that providing A. cacae_lah to CMA-transplanted mice along with a prebiotic supplement will enable the species to grow and establish a niche in the host. Ideally, we would be able to administer A. cacae_lah so that the abundance of A. cacae_lah in CMA-transplanted mice reached levels similar to those detected in healthy-transplanted mice.

[0116] C. Experimental Design for Initial In Vivo Studies (Figure 21) In this experiment, we aim to validate the transplantation of A. cacae_lah into CMA-transplanted mice and its dependence on prebiotic supplements. Germ-free (GF) C3H / HeN germ-free mice are weaned and fed Nutramigen. Simultaneously, to establish a CMA microenvironment in the mouse model, the mice are given fecal slurry from CMA repository mice (human donor 6) via gavage (IG). CMA microbes are given for 7 days for transplantation. The mice receive a continuous supply of Nutramigen throughout the experiment. By providing the mice with ad libitum access to Nutramigen along with additional lactate / acetate or carbohydrate supplements, sufficient continuous substrate can be provided to support the transplantation of A. cacae. Starting 7 days after weaning, the mice receive IG gavage of both A. cacae_lah and prebiotics or control. Cohorts of mice receive 100 ul of PBS (control) or one prebiotic supplement (10 mg / ml lactic acid + 10 mg / ml acetic acid or 10 mg / ml potato starch) immediately after fecal collection. Thirty minutes later, mice receive 250 ul of live biotherapeutic product (LBP), i.e., approximately 1 x 106 Mice received 100 CFU of A. cacae_lah or an equal volume of sterile CMG broth in glycerol as a negative control. After the first week, gavage of LBP was discontinued, but prebiotics (or control) continued to be administered for another week. This was to determine whether prebiotic administration was sufficient to maintain the LBP (A. cacae_lah) population in mice without introducing additional bacteria. Feces were collected daily for these two weeks to analyze A. cacae_lah abundance by qPCR and fecal butyrate concentration by HPLC UV-Vis. Mice were sacrificed on day 42, or the first time point at which A. cacae abundance was no longer detectable in the feces. After sacrifice, cecal butyrate was measured, and expression of the genes Ror2, Fbp1, Tgfbr3, Acot1, and Me1 in ileal epithelial cells (iIECs) was analyzed by qPCR. Because most butyrate produced in the colon is immediately consumed by colonocytes, cecal butyrate is generally a more sensitive measure than fecal butyrate and may be a better measure of butyrate production by A. cacae_lah in vivo. These specific genes were chosen for analysis in iIEC because they were immunologically relevant and shown to be differentially expressed between healthy, CMA-, and A. cacae-implanted mice (see Figure 4f).

[0117] In additional experiments, we aim to enhance A. cacae_lah transplantation by additionally delivering a butyrate transport compound to mice via IG gavage. Examples of butyrate transport compounds are described in Hubbell et al.'s published PCT application WO 2018 / 195067 A1. As described in Hubbell et al., an 80 mg / mL solution of the butyrate delivery polymer pHPMA-b-pBMA is prepared and diluted to 53.3 mg / mL. In the experiment described immediately above [paragraph 0116], a cohort of mice receiving a prebiotic supplement also receives 125 μL of this diluted butyrate delivery polymer solution via IG gavage immediately after receiving the prebiotic supplement. The remainder of the experiment is performed as described above.

[0118] All of the methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of the present invention have been described with reference to preferred embodiments, it will be apparent to those skilled in the art that variations may be made in the methods, steps, or the order of method steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically related agents may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutions and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0119] References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference. TIFF0007737098000024.tif187160TIFF0007737098000025.tif238160TIFF00077370980 00026.tif231160TIFF0007737098000027.tif245160TIFF0007737098000028.tif107160

Claims

1. A composition comprising the bacterium Anaerostipes cacae and a prebiotic for use in a method for treating a patient having or at risk of having a food allergy, the method comprising administering the composition to a subject, wherein the prebiotic comprises potato starch.

2. (a) the prebiotic comprises one or more of potato starch, a combination of lactic acid and acetic acid, galactooligosaccharides, lactulose, lactitol, erythritol, isomalt, polyglycitol, acetic acid, short-chain fructooligosaccharides (scFOS), guar gum, bean gum, amylose, mannitol, sorbitol, polyglycitol, raffinose, isomaltose, isomaltotriose, pectin, beta-glucan, and lactic acid; and / or (b) the prebiotic comprises one or more of digestible and non-digestible oligosaccharides, optionally the oligosaccharides comprising modified oligosaccharides, and further optionally the modified oligosaccharides comprising butyrate-releasing oligosaccharides fermentable by A. caccae; and / or (c) the prebiotic comprises at least 6 grams of non-digestible oligosaccharides, optionally the oligosaccharides comprising modified oligosaccharides, and further optionally the modified oligosaccharides comprising butyrate-releasing oligosaccharides fermentable by A. cacae; and / or (d) 1×10 6 ~1×10 15 CFU of A. cacae are administered to the subject, optionally before or after the prebiotic and / or pHPMA-b-pBMA. and / or (e) the method further comprises administering pHPMA-b-pBMA, optionally wherein the pHPMA-b-pBMA is administered orally; and / or (f) the A. cacae, the prebiotic, and / or pHPMA-b-pBMA are administered simultaneously; 10. The composition of claim 1.

3. The composition of claim 1 or 2, wherein the A. cacae is administered before or after the prebiotic and / or pHPMA-b-pBMA.

4. (a) the A. cacae is administered at least one hour before the prebiotic and / or pHPMA-b-pBMA, or the pHPMA-b-pBMA is administered after the prebiotic; and / or (b) at least 10 grams of a prebiotic is administered to the subject; and / or (c) the ratio of colony-forming units of A. cacae to grams of prebiotic is between 1000:1 and 10,000:1; and / or (d) The food allergy includes a milk allergy, egg allergy, peanut allergy, soy allergy, wheat / gluten allergy, shellfish allergy, sesame allergy, or tree nut allergy; and / or (e) the subject has been diagnosed with a food allergy; and / or (f) the subject has previously been treated for a food allergy, and optionally the subject has been determined to be refractory to the previous treatment; and / or (g) the subject is a human, and optionally the subject is under 1 year of age, under 5 years of age, under 12 years of age, or under 18 years of age; and / or (h) the A. coccae comprises a live bacterial product; and / or (i) the bacteria are lyophilized or freeze-dried; and / or (j) the A. cacae and / or the prebiotic are administered orally, and optionally the A. cacae and / or the prebiotic are administered in a tablet or capsule; and / or (k) the method further comprises administering a formula or food containing lactic acid; and / or (l) the subject is determined to have a protective / non-protective operational taxonomic unit (OTU) ratio of less than 3, wherein the protective / non-protective operational taxonomic unit (OTU) ratio is the total number of protective OTUs divided by the total number of non-protective OTUs identified in a fecal sample from the subject, wherein protective OTUs are selected from the OTUs shown to be more abundant in individual healthy samples as set forth in Supplementary Table 3, and non-protective OTUs are selected from the OTUs shown to be more abundant in individual cow's milk allergy (CMA) samples as set forth in Supplementary Table 3. The composition of any one of claims 1 to 3.

5. 5. The composition of any one of claims 1 to 4, wherein the prebiotic consists essentially of potato starch.

6. 6. The composition of any one of claims 1 to 5, wherein the bacteria in the composition consists essentially of Anaerostipes caccae.

7. 7. The composition of any one of claims 1 to 6, wherein the Anaerostipes caccae comprises Anaerostipes caccae_lah.

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