A bacteroides thetaiotaomicron strain and its use in reducing inflammation

Description

Field of the Invention

The present invention relates to microorganisms that can positively modulate inflammatory diseases and that can be used in therapy or in preventive medicine. Background of the invention

[0002] Bacteroides thetaiotaomicron has potent anti-inflammatory effects in vitro and in vivo (Kelly et al. Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclearcytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol. 2004 Jan; 5 (1): 104-12 ). It modulates the molecular pathways of NF-kB signaling (Kelly et al., Commensal anaerobic gut bacteria attenuate inflammation by regulating nuclear-cytoplasmic shuttling of PPAR-gamma and RelA. Nat Immunol. 2004 Jan; 5 (1): 104-12). In particular, it stops the binding of the active ingredient (RelA) of NF-kB to key genes in the nucleus, thereby preventing the activation of pro-inflammatory pathways (Kelly et al., Supra 2004). The complete genome of B. thetaiotaomicron was sequenced and annotated by the Gordon Group (Washington University School of Medicine, USA) in 2003 [Xu et al., A genomic view of the humanBacteroides thetaiotaomicron symbiosis. Science. 2003 Mar 28; 299 (5615): 2074-6]. Moran, 2014 (Microbial Wealth, Innovation Chemistry and Industry, vol. 78, no. 6, pages 20-23, June 1, 2014) discusses the commercial exploitation of the microbiome and in this context refers to a single-species live biotherapy, Bacteroides thetaiotaomicron as a treatment for pediatric Crohn's disease. WO 03/046580 discloses the use of Bacteroides thetaiotaomicron in general as a medicament for the treatment of an inflammatory disease.

Summary of the invention

[0003] The present invention is based on the discovery of the strain Bacteroides thetaiotaomicron (BT) which has surprising efficacy against inflammatory disorders. Accordingly, the BT strain is useful as a therapeutic or prophylactic drug against inflammatory and / or autoimmune disorders and / or allergic disorders.

[0004] According to a first aspect of the invention, there is a Bacteroides thetaiotaomicron deposited as NCIMB accession number 42341.

[0005] According to a second aspect of the invention, there is a composition comprising Bacteriodes thetaiotaomicron as defined in aspect 1.

[0006] According to a third aspect of the invention, there is a pharmaceutical composition comprising Bacteriodes thetaiotaomicron as defined in aspect 1.

[0007] According to a fourth aspect of the invention, there is a nutritional supplement containing

Bacteroides thetaiotaomicron as defined in aspect 1 and a nutritionally acceptable excipient, carrier or diluent.

[0008] According to a fifth aspect of the invention, there is a feed, food product, dietary supplement or food supplement comprising a Bacteroides thetaiotaomicron as defined in aspect 1. According to a sixth aspect of the invention, Bacteroides thetaiotaomicron as defined in aspect 1, composition as defined in aspect 2, pharmaceutical composition as defined in aspect 3, nutritional supplement as defined in aspect 4 or feed, food product, dietary supplement or food supplement as defined in aspect in aspect 5, for use in reducing inflammation of a tissue or organ in a subject.

According to a seventh aspect of the invention, Bacteroides thetaiotaomicron as defined in aspect 1, composition as defined in aspect 2, pharmaceutical composition as defined in aspect 3, dietary supplement as defined in aspect 4, or feed, food product, dietary supplement or food additive. as defined in aspect 5, for use in treating and / or preventing a Condition in a subject; wherein said condition is an inflammatory disorder and / or an autoimmune disorder.

[0010] According to the eighth aspect of the invention, Bacteroides thetaiotaomicron as defined in aspect 1, composition as defined in aspect 2, pharmaceutical composition as defined in aspect 3, food supplement as defined in aspect 4 or feed, food product, dietary supplement or a food additive as defined in aspect 5 for use in reducing a disorder in the colon of a subject, preferably said subject has IBD.

According to a ninth aspect of the invention, Bacteroides thetaiotaomicron as defined in aspect 1, composition as defined in aspect 2, pharmaceutical composition as defined in aspect 3, food supplement as defined in aspect 4 or feed, food product, dietary supplement or a food additive as defined in aspect 5, for use in reducing the expression of one or more pro-inflammatory genes in a cell or cells of a subject.

[0012] According to a tenth aspect of the invention, Bacteroides thetaiotaomicron as defined in aspect 1, composition as defined in aspect 2, pharmaceutical composition as defined in aspect 3, dietary supplement as defined in aspect 4 or feed, food product, dietary supplement or a food additive as defined in aspect 5 for use in increasing the percentage of regulatory T cells (Treg) in the gastrointestinal tract or part of the gastrointestinal tract.

According to an eleventh aspect of the invention, a method for preparing a pharmaceutical composition according to aspect 3, said method comprising admixing said Bacteroides thetaiotaomicron with a pharmaceutically acceptable excipient, carrier or diluent, said Bacteroides thetaiotaomicron being optionally encapsulated in said method.

Brief description of the drawings

[0014] The invention has been described with reference to the accompanying drawings, wherein:

Figure 1 illustrates the attenuation of colitis by expansion of Treg cells with B. thetaiotaomicron strain BT2013 in a DSS-induced colitis model. Figure 2 illustrates that B. thetaiotaomicron strain BT2013 does not affect Treg cells but affects Teff cells in conventional mice;

Figure 3: illustrates the myeloperoxidase (MPO) activity in the ileum (a) and caecum (b) of mice dosed with DSS with or without a daily dose of B. thetaiotaomicron

Figure 4: illustrates the histopathology of the ascending colon of C57BI / 6 female mice treated with DSS (a) or DSS and B. thetaiotaomicron (b)

Figure 5: illustrates the mean histopathological evaluation of colon ascending tissue from mice colonized with B. thetaiotaomicron strains E1 and BT2013 during DSS-induced colitis;

Figure 6: illustrates the expression of pro-inflammatory genes (IL-1β and IL-6) and anti-inflammatory gene (IL10) in the ascending colon of mice treated with B. thetaiotaomicron strains E1, E2 and BT2013; Figure 7: illustrates IL-8 expression in Caco-2 cells incubated with PMA and E1, E2 and BT2013 bacterial medium or cells.

Detailed Description of the Invention

[0015] The present invention is based on the discovery that the BT301 strain has a stronger anti-inflammatory effect compared to the BT control strains.

[0016] The BT2013 BT strain was deposited under accession number 42341 on December 3, 2014. At the National Collections of Industrial, Food and Marine Bacteria (NCIMB) at NCIMB Ltd, Ferguson Building, Craibstone Estate, Bucksburn, Aberdeen, UK, AB21 9YA . The deposit was made in accordance with the terms of the Budapest Treaty. The deposit was made by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland). GT Biologics Ltd. then changed its name to 4D Pharma Research Limited.

Derivative

[0017] The present disclosure includes derivatives of the deposited strain. The term "derivative" includes progeny (progeny) or strains bred (subcloned) from the original but modified in some way (including at the genetic level) without negatively altering biological activity, i.e. the derived strain will have at least the same immunomodulatory activity as the original strain BT2013.

Biotypes

[0018] The bacterial strains which are biotypes of the bacteria deposited under accession number NCIMB 42341 are also expected to be effective in the treatment or prevention of inflammatory and / or autoimmune disorders and / or allergic disorders. A biotype is a closely related strain that has the same or very similar physiological and biochemical properties.

[0019] In some embodiments disclosed herein, the bacterial strain for use in the disclosure has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99, 9% identical to the 16s rRNA sequence of the bacterium as deposited under accession number NCIMB 42341.

[0020] Alternatively, strains which are biotypes of the bacteria deposited under accession number NCIMB 42341 and which are suitable for use in the disclosure may be identified by sequencing other nucleotide sequences for the bacteria deposited under accession number NCIMB 42341. For example, substantially the entire genome may be sequenced and the biotype strain for use in the disclosure may have at least 95%, 96%, 97%, 98%, 99%, 99.5%, or 99.9% sequence identity of at least 80%. the entire genome (e.g., at least 85%, 90%, 95%, or 99%, or the entire genome). Other suitable sequences for use in identifying biotype strains may include hsp60 or repeat sequences such as BOX, ERIC, (GTG) .5 or REP (Masco et al. (2003) Systematic and Applied Microbiology, 26: 557-563). Biotypic strains may have sequences of at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacteria deposited under accession number NCIMB 42341.

Alternatively, strains that are biotypes of the bacteria deposited under accession number NCIMB 42341 and which are suitable for use in the disclosure can be identified by the deposit accession number NCIMB 42341 and restriction fragment analysis and / or PCR analysis. for example, using fluorescently amplified fragment length polymorphism (FAFLP) and "fingerprint" PCR analysis of the repeat DNA element (rep) or protein profiling or partial 16S or 23s rDNA sequencing.

[0022] In some embodiments, the strains that are biotypes of the bacteria deposited under accession number NCIMB 42341 and suitable for use in the disclosure are strains that provide the same pattern as the bacteria deposited under accession number NCIMB 42341 when analyzed. by restriction analysis of amplified ribosome DNA (ARDRA), for example when using the restriction enzyme Sau3AI (for exemplary methods and guidelines see, for example, SrQtkova et al. (2011) J. Microbiol. Methods, 87 (1): 10-6). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacteria deposited under accession number NCIMB 42341.

[0023] Bacterial strains that are biotypes of the bacteria deposited under accession number NCIMB 42341 and that are useful in the compositions and methods of the disclosure may be identified by any suitable method or strategy. For example, bacterial strains that have similar growth patterns, metabolic type, and / or surface antigens to the bacteria deposited under accession number NCIMB 42341 may be useful in the disclosure. The biotype strain will have a similar modulatory immune activity to the NCIMB 42341 strain. For example, a biotype strain will produce comparable effects in DSS-induced colitis models and a comparable effect on Treg levels, MPO enzymatic activity, inflammation-related gene expression, and colon histopathology versus the effects shown in Functional Tests that can be identified by using protocols described in Functional Tests.

Ailments

[0024] The strain BT2013 Bacteroides thetaiotaomicron can be used to treat and / or prevent a condition in a subject, the condition being an inflammatory condition and / or an autoimmune condition.

[0025] In one embodiment, the disorder affects the gastrointestinal tract, digestive tract, liver, liver cells, immune cells, epithelial cells, epidermal cells, neuronal cells, endothelial cells, fibroblasts, pancreas, and / or pancreatic cells (such as islets of Langerhans). ).

Examples of sections (i.e. portions) of the gastrointestinal tract include the esophagus, stomach and intestine (such as the small intestine (e.g. duodenum, jejunum, and ileum) and / or large intestine (e.g., cecum, ascending colon, transverse colon) , descending colon and sigmoid colon)).

[0027] Examples of epithelial cells include intestinal epithelial cells. Examples of immune cells include dendritic cells, monocytes / macrophages, T cells, and neutrophils.

[0028] In one embodiment, the condition is selected from the group consisting of:

1. Organ related conditions such as irritable bowel syndrome (IBS), inflammatory bowel disease including Crohn's disease and ulcerative colitis, necrotizing enteritis, pocketitis, celiac disease, multiple sclerosis (brain), type I diabetes, Goodpasture syndrome 'a, Hashimoto's thyroiditis, chronic active hepatitis, cardiomyopathy, uveitis, and rhinitis.

2. Systemic conditions such as rheumatoid arthritis, systemic lupus erythematosus, scleroderma, psoriasis, atopic dermatitis, vitiligo, multiple sclerosis, alopecia areata, sarcoidosis, polymyositis, and combinations thereof.

[0029] In one aspect, the condition affects the gut.

[0030] In one aspect, the disease is an inflammatory disorder. For example, the condition is an inflammatory bowel disease (IBD) such as Crohn's disease.

[0031] In one aspect, the disease is an autoimmune disease. For example, the autoimmune disease is selected from the group consisting of ulcerative colitis, bursitis, rheumatoid arthritis, psoriasis, multiple sclerosis, type I diabetes, allergies (including celiac disease), atopic dermatitis, and rhinitis.

Subject

[0032] In one embodiment, the subject is a monogastric animal.

[0033] Examples of monogastric animals include poultry, humans, rats, pigs, dogs, cats, horses, and rabbits.

[0034] In another embodiment, the subject is a mammal, such as a monogastric mammal. [0035] Examples of monogastric mammals include omnivores (such as humans, rats, and pigs), carnivores (such as dogs and cats), and herbivores (such as horses and rabbits).

[0036] Preferably, the subject is a human.

[0037] In one aspect, In one aspect, the subject has a condition selected from the group consisting of enteritis (IBD), colitis, rheumatoid arthritis, psoriasis, multiple sclerosis, type I diabetes, celiac disease, atopic dermatitis, and mucositis. rhinitis, irritable bowel syndrome (IBS), ulcerative colitis, bursitis, Crohn's disease, functional dyspepsia, atopic diseases, necrotizing enterocolitis, nonalcoholic fatty liver disease, gastrointestinal infection, and combinations thereof. For example, the subject has an IBD.

Modulation / regulation

[0038] The terms "modulation" and "regulation" may be used interchangeably herein.

[0039] In one embodiment, B. thetaiotaomicron strain BT2013 is used to modulate inflammation of a cell, tissue or organ in a subject.

[0040] In one embodiment, the term "modulation" refers to an increase and / or induction and / or excitation and / or activation. In an alternative embodiment, the term "modulation" refers to reduction and / or reduction and / or inhibition.

[0041] In one embodiment, the term "adjusting" refers to an increase. In an alternative embodiment, the term "regulating" refers to a reduction.

[0042] In one embodiment, the B. thetaiotaomicron strain BT2013 as described herein reduces inflammation of a cell, tissue or organ. For example, inflammation of the gastrointestinal tract, a section (i.e. part) of the gastrointestinal tract (such as the intestine), liver, liver cells, epithelial cells, epidermal cells, nerve cells, endothelial cells, fibroblasts, pancreas, and / or pancreatic cells (such as like the islets of Langerhans).

[0043] In one example, inflammation in the gastrointestinal tract or a portion thereof (such as the intestine) is reduced.

[0044] In another example, inflammation caused by immune cells of the tissue or organ is reduced.

[0045] In another example, inflammation caused by epithelial cells of a tissue or organ is reduced.

[0046] As used herein, the term "inflammation" refers to one or more of the following: redness, swelling, pain, tenderness, heat and disruption of a cell, tissue or organ due to an inflammatory process caused by an overactive immune system.

[0047] In one embodiment, the number of cells that are inflamed in a subject is at least 10%, 20%, 30%, 40%, or 50% lower following administration of a polypeptide or polynucleotide or a host cell as described herein. compared with the numbers of cells that are inflamed in the subject before the strain BT2013 as described herein is administered to the subject.

[0048] In one embodiment of dying, the amount of tissue or organ that is inflamed in the subject is at least 10%, 20%, 30%, 40% or 50% lower after administration of the BT2013 strain, as compared to the amount of tissue or an organ that is inflamed in a subject prior to administration of the BT2013 strain to the subject.

[0049] In one embodiment, strain BT2013 reduces inflammation caused by tissue or organ epithelial cells.

[0050] For example, epithelial cells are epithelial cells of the gastrointestinal tract or a portion thereof (such as the intestine).

[0051] Without wishing to be bound by theory, strain BT2013 increases the production of T cells (such as regulatory T cells, which may also be referred to as Treg) in a subject. This increase in Tregs can counteract the effects of other T effector cells (also referred to as Teff) such as Th1, Th17 and Th2 which drive inflammation, autoimmunity and allergic / atopic conditions. In Crohn's disease and ulcerative colitis, there is a loss of the balance of Teff / Treg lymphocytes.

[0052] In one embodiment, the subject's T cell production is increased such that there is at least 10%, 20%, 30%, 40%, or 50% more T cells or more than 100% more T cells following administration of the polypeptide or of a polynucleotide or host cell as described herein compared to the number of T cells in the subject before the BT2013 strain is administered to the subject.

The integrity of the intestinal barrier

[0053] In one embodiment, strain BT2013 is used to improve the intestinal barrier integrity of a subject.

[0054] The term "improvement of intestinal barrier integrity" as used herein refers to the reduction of the number and / or types of microorganisms that spread from the gut to other cells in a subject after administration of the BT2013 strain, as compared to numbers and / or types. microorganisms that spread from the intestine to other cells of the subject prior to administration of the BT2013 strain, as described herein.

[0055] In one embodiment, the number of microorganisms that spread from the gut to other cells in the subject is at least 10%, 20%, 30%, 40%, or 50% lower after administration of the BT2013 strain as compared to the number of microorganisms that spread from the gut to other cells. spread from the gut to other cells after administration to the subject.

[0056] In one embodiment, at least 5%, 10%, 15% or 20% fewer types of microorganisms spread from the gut to other cells in the subject following administration of the BT2013 strain as compared to the types of microorganisms that have spread from the gut to other cells. cells in the subject prior to administration.

Disorder in the intestine

[0057] In one embodiment, strain BT2013 is used to reduce a disorder in the gut (e.g., colon) of a subject (such as an IBD subject).

[0058] The term "subject gut disorder" as used herein refers to an effect on the integrity of the mucosal epithelium and / or an effect on the number of goblet cells in the epithelium and / or an effect on the number of immune cells invading the lamina propria.

[0059] In one embodiment, strain BT2013 reduces or prevents the impairment of mucosal epithelial integrity and / or reduces or prevents a reduction in the number of goblet cells in the epithelium and / or reduces or prevents immune cell infiltration into the lamina propria.

[0060] In one embodiment, a reduction in mucosal epithelial dysfunction is a reduction of at least 5%, 10%, 15% or 20% in the number of bacteria transferred from the intestinal lumen to the intestinal cells after administration of the BT2013 strain, as compared to the number of bacteria transferred from the intestinal tract. intestinal lumen into the intestinal cells of the subject prior to administration.

[0061] In one embodiment, the reduction in the number of goblet cells in the epithelium is a reduction of at least 2%, 5%, 10%, 15% or 20% in the number of goblet cells in the epithelium of the subject after administration of the BT2013 strain as compared to the number of goblet cells in the subject. subject epithelium prior to administration.

[0062] In one embodiment, the reduction of immune cell infiltration into the lamina propria is such that over a predetermined period of time (such as 24 hours) there is a reduction of at least 5%, 10%, 15%, 20%, or 30% in the number of cells. immune cells (e.g., T lymphocytes) transferred to lamina propria cells after administration of strain BT2013, compared to the number of immune cells (e.g., T lymphocytes) transferred to lamina propria in the subject prior to administration.

Pro-inflammatory genes and barrier integrity genes

[0063] In one embodiment, strain BT2013 is used to regulate the expression of one or more proinflammatory genes and / or one or more barrier integrity genes in a cell or cells of a subject.

[0064] In one embodiment, the term "regulate" refers to increasing the expression of one or more pro-inflammatory genes. In an alternative embodiment, the term "regulate" refers to reducing the expression of one or more pro-inflammatory genes.

[0065] In one embodiment, the strain BT2013 reduces the expression of one or more pro-inflammatory genes in a cell or cells of a subject.

[0066] The term "pro-inflammatory gene" as used herein refers to a gene that promotes inflammation when expressed. Examples of proinflammatory genes include genes encoding, but not limited to, IL1-β, IL4, IL5, IL6, IL8, IL12, IL13, IL17, IL21, IL22, IL23, IL27, IFN, CCL2, CCL3, CCL5, CCL20, CXCL5, CXCL10, CXCL12, CXCL13 and TNF-α.

[0067] In one embodiment, the pro-inflammatory gene is selected from the group consisting of IL1-p, IL6 and IL8.

[0068] In one embodiment, the expression level (e.g., mRNA level) of one or more of the pro-inflammatory genes is reduced (i.e. lowered) such that the level is at least 10%, 20%, 30%, 40%, or 50% lower. after administration of strain BT2013, compared to the level in the subject prior to administration.

[0069] The term "barrier integrity genes" as used herein refers to a gene which, when expressed, plays a role in such a barrier function as barrier repair and prevention of microbial passage through the barrier. Examples of barrier integrity genes include the genes encoding RetnIg | RetnIb, Si, Defa24, Hsd11b2, Hsd17b2, and Nr1d1 | Thra.

[0070] In one embodiment, the term "regulate" refers to increasing the expression of one or more barrier integrity genes. In an alternative embodiment, the term "regulate" refers to reducing the expression of one or more barrier integrity genes.

[0071] In one embodiment, strain BT2013 increases the expression of barrier integrity genes in a cell or cells of a subject.

[0072] In one embodiment, the barrier integrity gene is selected from the group consisting of RetnIg | RetnIb, Si, Defa24, Hsdllb2, Hsd17b2, and Nr1d1 | Thra.

[0073] In one embodiment, the expression level (e.g., mRNA level) of one or more barrier integrity genes is increased (i.e., increased) such that the level is at least 10%, 20%, 30%, 40%, or 50%. higher after administration of strain BT2013 as compared to the level in the subject before administration.

Digestive tract

[0074] Parts of the digestive tract include the esophagus, stomach and intestine (such as the small intestine (e.g. duodenum, jejunum, and ileum) and / or large intestine (e.g., cecum, ascending colon, transverse colon, descending colon, and sigmoid colon) ).

[0075] As used herein, the term "large intestine" may be used interchangeably with the term "colon".

[0076] In one embodiment, strain BT2013 is used to improve gastrointestinal health in a subject.

[0077] As used herein, the term "improvement in gastrointestinal health" refers to the reduction of the level of inflammation in the gastrointestinal tract or parts thereof and / or the improvement of the intestinal microflora.

[0078] In one embodiment, the level of inflammation in the gastrointestinal tract is at least 10%, 20%, 30%, 40% or 50% lower after administration of the BT2013 strain as compared to the level of inflammation in the patient's gastrointestinal tract before administration.

[0079] In one embodiment, strain BT2013 is used to improve the intestinal microflora in a subject.

[0080] As used herein, the term "intestinal microflora" refers to microorganisms that live in the digestive tract of host animals. These microorganisms perform a wide variety of metabolic, structural, protective, and other beneficial functions.

[0081] As used herein, the term "improvement of the intestinal microflora" refers to increasing the number and / or type of desired microorganisms present in the gut of a subject (e.g., host) and / or increasing the activity of said desired microorganisms with respect to their metabolic, structural functions. , protective and other beneficial functions. The term "improving the gut microflora" may also refer to reducing the number and / or type of undesirable microorganisms present in the gut of a subject (eg, host) and / or reducing the activity of these undesirable microorganisms with respect to their metabolic, structural, protective, and other beneficial functions.

[0082] The microorganisms desired in the host gut are those microorganisms that exert a protective and beneficial function. Firmicutes and bacteroidetes are examples of microorganisms desired in the host gut.

[0083] Microorganisms that are undesirable in the host gut are those microorganisms that can interfere with the metabolic, structural, protective, and other beneficial functions of the desired microbes that have a protective and beneficial function in the gut. Additionally or alternatively, undesirable microorganisms are those which cause, for example, inflammation and / or diarrhea. E coli is an example of an undesirable microorganism in the gut of the host.

[0084] For example, a change in the microflora balance between a desired microorganism (such as bacteria firmicutes and bacteroidetes) and unwanted microorganisms (such as E coli: ETEC, EPEC, EIEC, EHEC, and EAEC) in the gut can occur in individuals with inflammatory bowel disease (IBD) (IBD). IBD) after administration of strain BT2013 to the subject.

[0085] In one embodiment, the number of desired microorganisms (such as bacteria firmicutes and bacteroidetes) present in the gut of a subject (e.g., host) is increased such that the number of microorganisms is at least 10%, 20%, 30%, 40% or 50% greater or greater than 100% greater after administration of the BT2013 strain as compared to the subject prior to administration. Additionally or alternatively, the number of types of desired microorganisms (such as firmicutes and bacteroidetes) present in the gut of the subject (e.g., host) is so increased that there is at least 2%, 5%, 10%, or 15% more of these types of microorganisms after administration. strain BT2013, compared to the types in the subject prior to administration.

[0086] In one embodiment, in one embodiment, the number of unwanted microorganisms (such as E. coliETEC, EPEC, EIEC, EHEC, and EAEC) present in the gut of the subject (e.g., host) is reduced such that the number of microorganisms is at least 10%, 20%, 30%, 40% or 50% less after administration of the BT2013 strain as compared to the subject's pre-administration level. Additionally or alternatively, a number of types of undesirable microorganisms (such as E. coli ETEC, EPEC, EIEC, EHEC, and EAEC) present in the gut of the subject (e.g., host), is so reduced that there is at least 1%, 2%, 5%, or 10% less types of microorganisms after administration of strain BT2013. compared to the types in the subject prior to administration.

Encapsulation

[0087] In one embodiment, B. thetaiotaomicron strain BT2013 is encapsulated.

[0088] In an additional embodiment, the pharmaceutical composition containing the BT2013 strain is encapsulated.

[0089] In one embodiment, the nutritional supplement containing the BT2013 strain is encapsulated.

[0090] In an additional embodiment, the feed, food product, dietary supplement or food supplement as described herein is encapsulated.

[0091] The term "encapsulated as used herein refers to means of protecting the BT2013 strain from an incompatible environment by physical separation such that it can be delivered to a target site (e.g., the intestine) without degradation or significant degradation so that the BT2013 strain can have impact on the target site. An example is an enteric-coated capsule or a gastric juice resistant capsule.

[0092] Even if the purpose of encapsulation is to isolate the strain from its surroundings, the protective coating or shell must be broken at the time of the desired action. Breakage of the protective shell or shell is usually caused by the application of chemical and physical stimuli, such as pressure, enzymatic attack, chemical reaction, and physical disintegration.

[0093] For example, encapsulation ensures that the strain can be ingested such that the microorganisms can be delivered to the target site (e.g., the intestine) in an amount that is effective to produce an effect at the target site.

Pharmaceutical composition

[0094] In one embodiment, the pharmaceutical composition comprises microorganisms from the BT2013 strain and optionally a pharmaceutically acceptable excipient, carrier or diluent.

[0095] The pharmaceutical composition can be any pharmaceutical composition. In one aspect, the pharmaceutical composition is to be administered orally, enterally, or rectally. For example, the composition may be an edible composition. "Edible" means material that is permitted for human or animal consumption.

[0096] The pharmaceutical compositions can be used in humans or animals in human and veterinary medicine.

[0097] Examples of such suitable excipients for the various different forms of the pharmaceutical compositions described herein can be found in the "Handbook of Pharmaceutical Excipients, 2nd edition, (1994), edited by A Wade and PJ Weller.

[0098] Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in Remington's Pharmaceutical Sciences, Mack Publishing Co. (AR Gennaro, ed. 1985).

[0099] Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnesium stearate, mannitol, sorbitol and the like.

[0100] Examples of suitable diluents include one or more of: water, ethanol, glycerol, propylene glycol, and glycerin, and combinations thereof.

[0101] The choice of the pharmaceutical carrier, excipient or diluent can be made with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may contain as a carrier, excipient or diluent, or in addition, any suitable binder (s), lubricant (s), suspending agent (s), coating agent (s), solubilizing agent (s).

[0102] Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flowing lactose, beta-lactose, corn sweeteners, natural and synthetic gums such as acacia, tragacanth or sodium alginate, carboxymethylcellulose and glycol. polyethylene.

[0103] Examples of suitable lubricants include sodium oleate, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride and the like.

[0104] Preservatives, stabilizers, colorants, and even flavoring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid, and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents can also be used.

[0105] In one aspect, the microorganisms of the pharmaceutical composition of the BT2013 strain are encapsulated.

[0106] The pharmaceutical may be in the form of a solution or a solid - depending on the use and / or the mode of application and / or the mode of administration.

[0107] As used herein, the term "medicament" includes medicaments for human and animal use in human and veterinary medicine. Furthermore, as used herein, the term "drug" means any substance that provides a therapeutic and / or beneficial effect. As used herein, the term "drug" is not necessarily limited to substances that require authorization, but may include substances that may be used in cosmetics, nutraceuticals, food (including food and drink), probiotic cultures, supplements. and natural remedies. In addition, as used herein, the term "medicament" includes a product intended to be incorporated into animal feed, for example, livestock and / or pet food.

Nutritional supplements

[0108] Nutritionally acceptable carriers, diluents and excipients include those suitable for human or animal consumption and which are used as standard in the food industry. Conventional nutritionally acceptable carriers, diluents and excipients will be known to those skilled in the art.

[0109] In one embodiment, the dietary supplement comprises microorganisms of the BT2013 strain or host cells containing an expression vector containing said polynucleotide sequence and a nutritionally acceptable excipient, carrier or diluent.

[0110] In one example, strain BT2013 microorganisms are encapsulated.

Feed / products

[0111] A further aspect of the invention relates to feed, food products, dietary supplements and food additives containing the microorganisms of the BT2013 strain.

[0112] The terms "feed", "food product", "food additive" and "dietary supplement" as used herein are meant to include all consumer products that may be solid, jelly-like or liquid.

[0113] The term "food product" is used in a broad sense - and includes food for humans as well as food for animals (ie, food). In one aspect, the food product is for human consumption. Examples of food products include dairy products (such as milk, cheese, whey protein drinks, milk drinks, lactic acid bacteria drinks, yogurt, drinking yoghurt), bakery products, beverages and powdered beverages.

[0114] The "feed", "food product", "food additive" and "dietary supplement" can be in the form of a solution or in a solid form - depending on the use and / or the mode of application and / or the mode of administration.

[0115] As used herein, the term "dietary supplement" includes a formulation that is or can be added to a food or feed product as a nutritional supplement. As used herein, the term "dietary supplement" also refers to formulations that can be used at low levels in a wide variety of products that require gelation, texturing, stabilization, suspension, film formation and structure, keeping juiciness and improving mouthfeel, without adding viscosity.

[0116] Suitable food products may include, for example, functional food products, food compositions, pet food, livestock feed, health food, pet food, and the like. In one aspect, the food product is healthy food.

[0117] As used herein, the term "functional food product" means a food that is capable of providing not only a nutritional effect, but is also capable of providing the consumer with additional beneficial effects. Accordingly, a functional food is an ordinary food that contains ingredients or ingredients (such as those described herein) that give it a specific functional - e.g. a medical or physiological benefit other than a purely nutritional effect.

[0118] Examples of specific food products that apply to the present invention include milk-based products, ready-to-eat desserts, powders for reconstitution with e.g. milk or water, chocolate milk drinks, malt beverages, ready-to-eat meals, instant meals. or human beverages or food compositions representing a complete or partial diet for pets or livestock.

[0119] In one aspect, the feed, food product, dietary supplement or food additive in accordance with the present invention is for human, domestic or farm animals, such as monogastric animals. The feed, food product, dietary supplement or food additive may be for animals selected from the group consisting of dogs, cats, pigs, horses or poultry. In a further embodiment, the food product, dietary supplement or food additive is intended for an adult species, particularly adult humans.

[0120] The term "milk-based product" as used herein means any liquid or semi-solid milk-based or whey-based product having a different fat content. The milk-based product can be, for example, cow's milk, goat's milk, sheep's milk, skim milk, whole milk, recombined milk made from powdered milk and whey without any processing or a processed product such as yogurt, curds, cottage cheese, sour milk. , sour whole milk, buttermilk, and other sour milk products. Another important group includes milk beverages such as whey beverages, fermented beverages, condensed milk, infant or toddler milk; flavored milks, ice cream; products containing milk, such as sweets.

[0121] The feeds, food products, dietary supplements or food additives of the present invention may be - or may be added to - food supplements, also referred to herein as dietary or nutritional supplements or food additives.

[0122] The feed products, food products, dietary supplements or food additives of the invention can also be used in animal nutrition (for example in pig nutrition), particularly in the early weaning and fattening periods. Feed, food products, dietary supplements or food additives are expected to improve immune function and prevent infectious diseases, favorably change the composition of the microflora, and improve animal growth and performance, for example by increasing feed efficiency.

[0123] In one embodiment, the feed, food product, dietary supplement or food supplement is encapsulated.

Live biotherapeutic product

[0124] The microorganisms of the BT2013 strain can be used in or as a live biotherapeutic product (LBP).

[0125] In one aspect, the LBP is a composition for the oral administration of metabolically active bacteria, ie, live and / or lyophilized or non-viable, heat killed, irradiated or lysed. The LBP may contain other ingredients. LBP can be administered orally, i.e. in the form of a tablet, capsule or powder. The LBP may additionally contain other bacterial species, for example R. hominis bacterial species. Encapsulated products are preferred for R. hominis because it is an anaerobic. Other ingredients (such as, for example, vitamin C) can be introduced as oxygen scavengers and substrates (such as those to improve colonization and survival in vivo). Alternatively, the LBP of the invention may be administered orally as a food or nutritional product, such as a fermented milk or whey based milk product, or as a pharmaceutical product.

[0126] A suitable daily dose of bacteria in an LBP is about 1 x 103 to about 1 x 10¹²colony forming units (CFU); for example, from about 1 x 107 to about 1 x 10¹⁰ CFU; in another example, from about 1 x 106 to about 1 x 10¹⁰ CFU.

[0127] In one aspect, the LBP comprises bacterial species and / or their cellular components as active ingredients in an amount of from about 1x106 to about 1x106.¹² CFU / g, based on the weight of the composition; for example, from about 1x108 to about 1x10¹⁰ CFU / g. Typically, the LBP is optionally combined with at least one suitable prebiotic compound. The prebiotic is usually a non-digestible carbohydrate, such as an oligo- or polysaccharide, or a sugar alcohol that does not degrade or be absorbed in the upper gastrointestinal tract. Known prebiotics include commercial products such as inulin and transgalacto-oligosaccharides.

[0128] In one aspect, the LBP as used herein comprises the prebiotic in an amount of from about 1 to about 30% by weight, based on the total weight of the composition (eg, from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo-oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalto-oligosaccharides, pectins, xyl-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS-modified and COS) starches (or COS) for resin, polydextrose, D-tagatose, acacia, carob, oats and citrus. In one aspect, the prebiotics are short chain fructooligosaccharides (for simplicity set forth below as FOSs-cc); these FOSs-cc are non-digestible carbohydrates, usually obtained by converting beet sugar and containing a sucrose molecule to which three glucose molecules are bound.

Administration

The pharmaceutical compositions, nutritional supplements, feeds, food products, dietary supplements, or food additives of the present invention can be adapted to the oral, rectal, vaginal, parenteral, intramuscular, intraperitoneal, intraarterial, intrathecal, intrabronchial, subcutaneous, intradermal, administration routes. , intravenous, intranasal, buccal or sublingual.

[0130] In one aspect, the pharmaceutical compositions, nutritional supplements, feeds, food products, dietary supplements or food additives of the present invention are adapted for oral, rectal, vaginal, parenteral, intranasal, buccal or sublingual administration.

[0131] In a further aspect, the pharmaceutical compositions, nutritional supplements, feeds, food products, dietary supplements or food additives of the present invention are adapted for oral administration.

[0132] For oral administration, especially compressed tablets, pills, tablets, gellules, drops and capsules are used.

[0133] Other forms of administration include solutions or emulsions that can be injected intravenously, intra-arterially, intrathecally, subcutaneously, intradermally, intraperitoneally, or intramuscularly, and which are prepared from sterile or sterilized solutions. The pharmaceutical compositions of the present invention may also be in the form of suppositories, pessaries and suspensions. The pharmaceutical compositions, nutritional supplements, feeds, food products, dietary supplements, or food additives can be formulated in unit dosage form, i.e., as separate portions containing a unit dose or a multiple or sub-unit of the unit dose.

Dosage

[0134] One skilled in the art can easily determine the appropriate dosage of the BT2013 strain to be administered to a subject without undue experimentation. Typically, a physician will determine the actual dosage which will be most appropriate for an individual patient and will depend on many factors including the activity of the strain used, the metabolic stability and duration of action of that strain, age, body weight, general health, sex, diet, method and the time of administration, rate of excretion, combination of drugs, the severity of the particular disease state, and the subject being treated. The dosages disclosed herein are exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are required, and such are within the scope of this invention.

Associations

[0135] In one aspect, microorganisms of the BT2013 strain are administered in combination with one or more other active agents. In such cases, microorganisms of the BT2013 strain may be administered sequentially, simultaneously or sequentially with one or more other active agents.

Functional tests:

In vivo model

[0136] C57BL / 6 mice (6 weeks) were used to evaluate the therapeutic effect of B. thetaiotaomicron strains E1, E2 and BT2013 on DSS-induced colitis. Mice were colonized with one of the B. thetaiotaomicron strains prior to DSS treatment. The animals were euthanized and intestinal tissue sampling was performed. The small intestine was collected for immunological analysis by flow cytometry and measurements of the enzymatic activity of the myeloperoxidase enzyme (MPO). The ascending colon was divided into equal parts and transferred to neutral buffered formalin (NBF, Sigma-Aldrich) for histological analysis or RNAlater (Ambion) for molecular analysis.

[0137] Flow cytometry analysis of the T cell population in the lamina propria of the small intestine was performed (Fig. 1 and Fig. 2). Treatment with DSS and B. thetaiotaomicron alone had no effect on the overall percentage of the CD3 + CD4 + CD8- population. Populations affected by DSS alone and B. thetaiotaomicron were Treg lymphocytes (CD25 + FoxP3 + * and FR4^hiCD25 + *) and Teff (FR4^loCD25 + *) (Figures 1 and 2). The percentage of Treg was increased in mice treated with B. thetaiotaomicron strain BT2013 compared to DSS alone. The E1W strain did not seem to have any effect on Treg. (fig. 1). The effects of BT2013 on Treg were only seen in mice when treated with DSS. The strain had no effect on the Treg in untreated mice, but it did affect the Teff lymphocyte population (Fig. 2).

[0138] The enzymatic activity of MPO in the ileum and cecum was determined (Figures 3a and 3b). MPO is a pro-inflammatory enzyme stored in the azurophilic granules of neutrophils. MPO is used as an indicator of inflammation, in particular neutrophil recruitment and accumulation. Lower levels of MPO activity detected in ileal or cecum tissue samples from mice treated with B. thetaiotaomicron / DSS, compared to the DSS alone, indicates a reduction in neutrophil recruitment and thus a reduction in inflammation.

[0139] Histological analysis of the ascending colon was performed (Figures 4 and 5 and Table 1). The pattern of the histopathological evaluation was based on the criteria of Berg et al 1996, as summarized:

= Shallow crypts, no or few infiltrating inflammatory cells, intact epithelium, goblet cells are full of mucin. ie no pathology = Crypts may show slight epithelial cell proliferation, little diffuse infiltrating inflammatory cells may be seen between the crypts, lumen epithelium appears intact, goblet cells may appear slightly mucin depleted.

= Crypts appear deeper with clear evidence of epithelial hyperplasia, depletion of goblet cell mucin, inflammatory infiltration evident and may be multifocal, although infiltrates are not visible in the submucosa.

= Changes affected a larger area of ​​the mucosa and / or were more frequent than those seen in Grade 2. The lesions did not extend to the submucosa. The lumen epithelial cells showed small erosions. The lesions are not transmural.

= The crypt epithelium seems to be destroyed. There may be abscesses.

[0140] The lumen epithelial cells appear irregular, sometimes with a complete loss.

[0141] Transmural infiltration is observed - this has often been associated with a complete loss of epithelial cells to the lumen.

Colon disruption from DSS-induced colitis was significantly reduced by treating mice with B. thetaiotaomicron strains E1, E2 and BT2013. The expression of inflammation related genes in the ascending colon was reduced in B. thetaiotaomicron-colonized mice compared to mice treated with DSS alone. Strains El and BT2013 significantly reduced the expression of inflammatory IL1B and IL6 genes compared to strain E2. (Fig. 6)

Table 1

<td>1 1 LSI</td><td>CONTROL</td><td>BT El</td><td>BT 2013</td><td>BT E2</td>

<td>DSS</td><td> 0,000</td><td> 0,032</td><td> 0,041</td><td> 0,089</td>

In vitro model

[0143] Inflammatory gene expression interleukin-8 induced in intestinal epithelial cells after PMA exposure was modulated in the presence of B. thetaiotaomicron strains E1, E2 and BT2013 (Figure 7).

Sequencing of the genome of strain BT2013

[0144] A DNA sample from strain BT2013 was sequenced on MiSeq (v2 nano 2x250 bp) using the Nextera XT library for rapid fragmentation and labeling with sequencing adapters for a total of 4605120 readings (1115615927 bases).

[0145] The data analysis is summarized below:

a. Mapping to the reference sequence (NC_004663 and NC_004703) using bowtie2 (2.2.2)

b. Calling SNV and small InDels using VarScan (2.3.7) and SNVer (0.5.3) by making consensus call to avoid false positives

c. Annotate the varieties with reference gff

d. Invoking large InDeli with the pindel tool (0.2.5a3)

e. De-novo Compilation of Unmapped Readings Using SOAPdenovo (2.04)

f. Blast of assembled contigs with the NCBI database on

g. Sub-sampling of all sample readings up to 50%

h. De-novo submission on subsamples of readings using SOAPdenovo (2.04)

The sequences were mapped to a reference sequence (NC_004663 and NC_004703) using bowtie2 (2.2.2). Nucleotide variations and small insertions and / or deletions were identified using VarScan (2.3.7) and SNVer (0.5.3) to avoid false positives during sequencing , and the variants were described using a reference sequence. Large insertions and deletions were identified using the pindel tool (0.2.5a3). Unmapped readings were assembled de novo using SOAPdenovo (2.04). The sequencing fragments were reassembled into contigs which were blasted against the NCBI nucleotide database. For all readings, samples up to 50% were subsampled and then assembled de novo using SOAPdenovo (2.04) to provide a combined version of the de novo assembled BT2013 sequence.

4D Pharma Research Limited, United Kingdom Agents

1760

EP 3 065 748 B1

Z-16691/17