OA18687A - Compositions comprising bacterial strains - Google Patents

Compositions comprising bacterial strains Download PDF

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OA18687A
OA18687A OA1201700486 OA18687A OA 18687 A OA18687 A OA 18687A OA 1201700486 OA1201700486 OA 1201700486 OA 18687 A OA18687 A OA 18687A
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composition
asthma
compositions
disease
cancer
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OA1201700486
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Angela Margaret PATTERSON
George Grant
Imke MULDER
Seanin MCCLUSKEY
Emma RAFTIS
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4D Pharma Research Limited
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Abstract

The invention provides compositions comprising bacterial strains for treating and preventing inflammatory and autoimmune diseases.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS
TECHNICAL FIELD
This invention is in the field of compositions comprising bacterial strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease.
BACKGROUND TO THE INVENTION
The human intestine is thought to be stérile in utero, but it is exposed to a large variety of maternai and environmental microbes immediately after birth. Thereafter, a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host génotype, ail of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1]. The human gut microbiota contains more than 500-1000 different phylotypes belonging essentially to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2]. The successful symbiotic relationships arising from bacterial colonization of the human gut hâve yielded a wide variety of metabolic, structural, protective and other bénéficiai fonctions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplifïed in germfree animais which hâve an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5].
Dramatic changes in microbiota composition hâve been documented in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, the levels of Clostridium cluster XlVa bacteria are reduced in IBD patients whilst numbers of E. coli are increased, suggesting a shift in the balance of symbionts and pathobionts within the gut [6-9]. Interestingly, this microbial dysbiosis is also associated with imbalances in T effector cell populations.
In récognition of the potential positive effect that certain bacterial strains may hâve on the animal gut, various strains hâve been proposed for use in the treatment of various diseases (see, for example, [10-13]). Also, certain strains, including mostly Lactobacillus and Bifldobacterium strains, hâve been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [14] and [15] for reviews). However, the relationship between different diseases and different bacterial strains, and the précisé effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases, are poorly characterised.
There is a requirement in the art for new methods of treating inflammatory and autoimmune diseases. There is also a requirement for the potential effects of gut bacteria to be characterised so that new thérapies using gut bacteria can be developed.
SUMMARY OF THE INVENTION
The inventors hâve developed new thérapies for treating and preventing inflammatory and autoimmune diseases. In particular, the inventors hâve developed new thérapies for treating and preventing diseases and conditions mediated by IL-17 or the Th 17 pathway. In particular, the inventors hâve identified that bacterial strains from the genus Bacteroides can be effective for reducing the Thl7 inflammatory response. As described in the examples, oral administration of compositions comprising Bacteroides coprocola may reduce the severity of the inflammatory response, including the Th 17 inflammatory response, in mouse models of asthma, rheumatoid arthritis and multiple sclerosis.
Therefore, in a first embodiment, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a disease or condition mediated by IL-17 or the Thl7 pathway. The inventors hâve identified that treatment with bacterial strains from this genus can reduce levels of cytokines that are part of the Thl 7 pathway, including IL-17, can alleviate the Thl 7 inflammatory response and can provide clinical benefits in mouse models of inflammatory and autoimmune diseases mediated by IL-17 and the Th 17 pathway.
In particular embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing a disease or condition selected from the group consisting of: multiple sclerosis; arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvénile idiopathic arthritis; neuromyelitis optica (Devic's disease); ankylosing spondylitis; spondyloarthritis; psoriasis; systemic lupus erythematosus; inflammatory bowel disease, such as Crohn’s disease or ulcerative colitis; celiac disease; asthma, such as allergie asthma or neutrophilie asthma; chronic obstructive pulmonary disease (COPD); cancer, such as breast cancer, colon cancer, lung cancer or ovarian cancer; uveitis; scleritis; vasculitis; Behcet's disease; atherosclerosis; atopie dermatitis; emphysema; periodontitis; allergie rhinitis; and allograft rejection. The effect shown for the bacterial strains from the genus Bacteroides on the Thl 7 inflammatory response may provide therapeutic benefits for diseases and conditions mediated by IL-17 and the Thl7 pathway, such as those listed above.
In preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing asthma, such as neutrophilie asthma or allergie asthma. The inventors hâve identified that treatment with Bacteroides strains can reduce recruitment of neutrophils and eosinophils into the lungs, which can help treat or prevent asthma. Furthermore, the inventors hâve tested and demonstrated the efficacy of Bacteroides strains in mouse models of asthma. In certain embodiments, the composition is for use in a method of treating or preventing neutrophilie asthma or éosinophilie asthma. The effect shown for the compositions of the invention on neutrophils and eosinophils mean that they may be particularly effective for treating or preventing neutrophilie asthma and éosinophilie asthma. Indeed, in certain embodiments, the composition is for use in a method of reducing a neutrophilie inflammatory response in the treatment or prévention of asthma, or the composition is for use in a method of reducing an éosinophilie inflammatory response in the treatment or prévention of asthma. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides coprocola, for use in the treatment of asthma, and in particular neutrophilie asthma. Bacteroides coprocola is shown to hâve a particularly pronounced effect on neutrophils in asthma models and treatment with Bacteroides coprocola may be particularly effective for treating neutrophilie asthma. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides thetaiotaomicron for use in the treatment of asthma, and in particular éosinophilie or allergie asthma. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides fragilis for use in the treatment of asthma, and in particular éosinophilie or allergie asthma.
In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing rheumatoid arthritis. The inventors hâve identified that treatment with Bacteroides strains can provide clinical benefits in a mouse model of rheumatoid arthritis and can reduce joint swelling. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides coprocola, for use în the treatment of rheumatoid arthritis. Compositions using Bacteroides coprocola may be particularly effective for treating rheumatoid arthritis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides thetaiotaomicron, for use in the treatment of rheumatoid arthritis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides fragilis, for use in the treatment of rheumatoid arthritis.
In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing multiple sclerosis. The inventors hâve identified that treatment with Bacteroides strains can reduce disease incidence and disease severity in a mouse model of multiple sclerosis. In preferred embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides coprocola, for use in the treatment of multiple sclerosis. Compositions using Bacteroides coprocola may be particularly effective for treating multiple sclerosis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides thetaiotaomicron, for use in the treatment of multiple sclerosis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides fragilis, for use in the treatment of multiple sclerosis.
In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing cancer, such as breast, lung or liver cancer. Compositions comprising a bacterial strain of the genus Bacteroides may reduce tumour growth in mouse models of breast, lung and liver cancer. In certain embodiments, the composition is for use in a method of reducing tumour size or preventing tumour growth in the treatment of cancer. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides coprocola, for use in the treatment of cancer. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides thetaiotaomicron, for use in the treatment of cancer. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides fragilis, for use in the treatment of cancer.
In further preferred embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing uveitis, such as posterior uveitis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides coprocola, for use in the treatment of uveitis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides thetaiotaomicron, for use in the treatment of uveitis. In certain embodiments, the invention provides a composition comprising a bacterial strain of the species Bacteroides fragilis, for use in the treatment of uveitis.
In certain embodiments, the compositions of the invention are for use in a method of reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of a disease or condition mediated by IL-17 or the Th 17 pathway. In particular, the compositions of the invention may be used in reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of asthma, rheumatoid arthritis or multiple sclerosis. Preferably, the invention provides compositions comprising a bacterial strain of the species Bacteroides coprocola, for use in reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of asthma, rheumatoid arthritis or multiple sclerosis, or of asthma, rheumatoid arthritis, multiple sclerosis, uveitis or cancer. In certain embodiments, the invention provides compositions comprising a bacterial strain of the species Bacteroides thetaiotaomicron, for use in reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of asthma, rheumatoid arthritis, multiple sclerosis, uveitis or cancer. In certain embodiments, the invention provides compositions comprising a bacterial strain of the species Bacteroides fragilis, for use in reducing IL-17 production or reducing Thl7 cell différentiation in the treatment or prévention of asthma, rheumatoid arthritis, multiple sclerosis, uveitis or cancer.
In certain embodiments, the composition is for use in a patient with elevated IL-17 levels or Thl7 cells. The effect on the Thl7 inflammatory response shown for Bacteroides strains may be particularly bénéficiai for such patients.
In preferred embodiments of the invention, the bacterial strain in the composition is of Bacteroides coprocola. Closely related strains may also be used, such as bacterial strains that hâve 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 a bacterial strain of Bacteroides coprocola. Preferably, the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1, 2, 3 or 4. Preferably, the sequence identity is to SEQ ID NO:4. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:4.
In further preferred embodiments of the invention, the bacterial strain in the composition is of Bacteroides thetaiotaomicron. Closely related strains may also be used, such as bacterial strains that hâve 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 a bacterial strain of Bacteroides thetaiotaomicron. Preferably, the bacterial strain has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:5.
In further preferred embodiments of the invention, the bacterial strain in the composition is of Bacteroides fragilis. Closely related strains may also be used, such as bacterial strains that hâve a genome with sequence identity to CR626927.1.
In certain embodiments, the composition of the invention is for oral administration. Oral administration of the strains of the invention can be effective for treating IL-17- or Thl 7 pathwaymediated diseases and conditions. Also, oral administration is convenient for patients and practitioners and allows delivery to and / or partial or total colonisation of the intestine.
In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers.
In certain embodiments, the composition of the invention comprises a bacterial strain that has been lyophilised. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.
In certain embodiments, the invention provides a food product comprising the composition as described above.
In certain embodiments, the invention provides a vaccine composition comprising the composition as described above.
Additionally, the invention provides a method of treating or preventing a disease or condition mediated by IL-17 or the Thl7 pathway, comprising administering a composition comprising a bacterial strain of the genus Bacteroides.
In developing the above invention, the inventors hâve identified and characterised a bacterial strain that is particularly useful for therapy. The Bacteroides coprocola strain of the invention is shown to be effective for treating the diseases described herein, such as arthritis, asthma and multiple sclerosis. Therefore, in another aspect, the invention provides a cell of the Bacteroides coprocola strain deposited under accession number NCIMB 42408, or a dérivative thereof. The invention also provides compositions comprising such cells, or biologically pure cultures of such cells. The invention also provides a cell of the Bacteroides coprocola strain deposited under accession number NCIMB 42408, or a dérivative thereof, for use in therapy, in particular for the diseases described herein.
BRIEF DESCRIPTION OF DRAWINGS
Figure 1: Mouse model of house dust mite-induced asthma - Total BAL fluid cell counts.
Figure 2: Mouse model of house dust mite-induced asthma - Total eosinophil count in BALF.
Figure 3: Mouse model of house dust mite-induced asthma - Proportion of eosinophils in BALF.
Figure 4: Mouse model of house dust mite-induced asthma - Total macrophage count in BALF.
Figure 5: Mouse model of house dust mite-induced asthma - Proportion of macrophages in BALF.
Figure 6: Mouse model of house dust mite-induced asthma - Total neutrophil count in BALF.
Figure 7: Mouse model of house dust mite-induced asthma - Proportion of neutrophils in BALF.
Figure 8: Mouse model of house dust mite-induced asthma - Total lymphocyte count in BALF.
Figure 9: Mouse model of house dust mite-induced asthma - Proportion of lymphocytes in BALF;
Figure 10: Mouse model of severe neutrophilie asthma - Total BAL fluid cell counts.
Figure 11: Mouse model of severe neutrophilie asthma - Total eosinophil count in BALF.
Figure 12: Mouse model of severe neutrophilie asthma - Proportion of eosinophils in BALF.
Figure 13: Mouse model of severe neutrophilie asthma - Total macrophage count in BALF.
Figure 14: Mouse model of severe neutrophilie asthma - Proportion of macrophages in BALF.
Figure 15: Mouse model of severe neutrophilie asthma - Total neutrophil count in BALF.
Figure 16: Mouse model of severe neutrophilie asthma - Proportion of neutrophils in BALF.
Figure 17: Mouse model of severe neutrophilie asthma - Total lymphocyte count in BALF.
Figure 18: Mouse model of severe neutrophilie asthma - Proportion of lymphocytes in BALF.
Figure 19: Mouse model of rheumatoid arthritis - Bodyweights, days -14 to 0. Data are presented as Mean ± SEM percentages of the initial (Day -14) bodyweights. Statistical significance: ▲ p < 0.05 and A AÀ A p <0.0001 whencomparedto the vehicle-treated group.
Figure 20: Mouse model of rheumatoid arthritis - Bodyweights, days 0 to 42. Data are presented as Mean ± SEM percentages of the initial (Day 0) bodyweights. ▲ p < 0.05, ♦ p < 0.05, ▲ ▲ ▲ p < 0.001, ···· p < 0.0001 when compared to the vehicle-treated group.
Figure 21: Mouse model of rheumatoid arthritis - Clinical Scores. Data are presented as Mean ± SEM. **** p < 0.0001 when compared to Day 21 in the vehicle-treated group. Ι,θρ< 0.05 when compared to the vehicle-treated group on a given day.
Figure 22 : Mouse model of rheumatoid arthritis - Splénocyte proliférative response to Collagen IL Media background subtracted [ClI-stimulated - media background] counts per minute based on 3HTdR incorporation. Ail data are presented as Mean ± SEM.
Figure 23: Mouse model of rheumatoid arthritis - Levels of IFNy in tissue culture supematants from Vehicle-treated group. Lines represent group médian values.
Figure 24: Mouse model of rheumatoid arthritis - Levels of IL-17A in tissue culture supematants from Vehicle-treated group. Lines represent group médian values.
Figure 25: Mouse model of rheumatoid arthritis - Levels of IL-10 in tissue culture supematants from Vehicle-treated group. Lines represent group médian values.
Figure 26: Mouse model of rheumatoid arthritis - Levels of IL-6 in tissue culture supematants from Vehicle-treated group. Lines represent group médian values.
Figure 27 : Mouse model of rheumatoid arthritis - Levels of cytokine in tissue culture supematants from biotherapeutic #675-treated group (Group 4). Lines represent group médian values.
Figure 28: Mouse model of house dust mite-induced asthma - Total IgE in Sérum
Figure 29: Mouse model of house dust mite-induced asthma — HDM spécifie IgGl in Sérum
Figure 30: Mouse model of house dust mite-induced asthma - Total IgE in BALF
Figure 31: Mouse model of house dust mite-induced asthma - HDM spécifie IgGl in BALF
Figure 32: Mouse model of house dust mite-induced asthma - Histological Analysis - Mean Peribronchiolar Infiltration Score
Figure 33: Mouse model of house dust mite-induced asthma - Hîstological Analysis - Mean Perivascular Infiltration Score
Figure 34: Mouse model of house dust mite-induced asthma - Hîstological Analysis - Mean Inflammatory Score (Average of both Peribronchiolar and Perivascular Infiltration Score)
Figure 35: Mouse model of house dust mite-induced asthma — Hîstological Analysis - Mucus Score
Figure 36: Mouse model of house dust mite-induced asthma — IL-9 level in lung tissue
Figure 37: Mouse model of house dust mite-induced asthma- IL-la level in lung tissue
Figure 38: Mouse model of house dust mite-induced asthma - IFNy level in lung tissue
Figure 39: Mouse model of house dust mite-induced asthma - IL-17A level in lung tissue
Figure 40: Mouse model of house dust mite-induced asthma IL-4 level in lung tissue
Figure 41: Mouse model of house dust mite-induced asthma — IL-5 level in lung tissue
Figure 42: Mouse model of house dust mite-induced asthma - IL-lb level in lung tissue
Figure 43: Mouse model of house dust mite-induced asthma - RANTES level in lung tissue
Figure 44: Mouse model of house dust mite-induced asthma - ΜΙΡ-la level in lung tissue
Figure 45: Mouse model of house dust mite-induced asthma - KC level in lung tissue
Figure 46: Mouse model of house dust mite-induced asthma - MIP-2 level in lung tissue
Figure 47: Mouse model of severe neutrophilie asthma - HDM spécifie IgGl in Sérum
Figure 48: Mouse model of severe neutrophilie asthma — HDM spécifie IgG2a in Sérum
Figure 49: Mouse model of severe neutrophilie asthma — HDM spécifie IgGl in BALF
Figure 50: Mouse model of severe neutrophilie asthma - HDM spécifie IgG2a in BALF
Figure 51: Mouse model of severe neutrophilie asthma - Hîstological Analysis — Mean Peribronchiolar Infiltration Score
Figure 52: Mouse model of severe neutrophilie asthma - Hîstological Analysis - Mean Perivascular Infiltration Score
Figure 53: Mouse model of severe neutrophilie asthma — Hîstological Analysis - Mean Inflammatory Score (Average of both Peribronchiolar and Perivascular Infiltration Score)
Figure 54: Mouse model of severe neutrophilie asthma — TNFa level in lung tissue
Figure 55: Mouse model of severe neutrophilie asthma — IL-la level in lung tissue
Figure 56: Mouse model of severe neutrophilie asthma — IFNy level in lung tissue
Figure 57: Mouse model of severe neutrophilie asthma - IL-17F level in lung tissue
Figure 58: Mouse model of severe neutrophilie asthma - IL-lb level in lung tissue
Figure 59: Mouse model of severe neutrophilie asthma - RANTES level in lung tissue
Figure 60: Mouse model of severe neutrophilie asthma - MIP-2 level in lung tissue
Figure 61: Mouse model of severe neutrophilie asthma - KC level in lung tissue
Figure 62: Mouse model of severe neutrophilie asthma — IL-17A level in lung tissue
Figure 63: Mouse model of severe neutrophilie asthma -ΜΙΡ-la level in lung tissue
Figure 64: Mouse model of severe neutrophilie asthma - IL-33 level in lung tissue
Figure 65: Mouse model of rheumatoid arthritis - Visual Template for Histopathology Scoring. Représentative images showing composite scores from mouse tarsal joints in a collagen-induced arthritis study.
Figure 66: Mouse model of rheumatoid arthritis - Histopathology: Inflammation Scores. Data are presented as Mean ± SEM. ** p < 0.01 when compared to the vehicle-treated group.
Figure 67: Mouse model of rheumatoid arthritis - Histopathology: Vehicle-treated group Cartilage Scores. Data are presented as Mean ± SEM.
Figure 68: Mouse model of rheumatoid arthritis - Histopathology: Bone Scores. Data are presented as Mean ± SEM.
Figure 69: Mouse model of rheumatoid arthritis - Histopathology: Total Scores. Data are presented as Mean ± SEM.
Figure 70: Mouse model of rheumatoid arthritis - Histopathology: Strain #675. Data are presented as Mean ± SEM.
Figure 71: Mouse model of rheumatoid arthritis - Histopathology: Représentative Pictures. Animal ID (#n.n) and limb (R for right, L for left) are indicated between brackets. Top left image (vehicle): extensive joint and bone destruction with inflammation and fibrosis extending to the peri-articular soft tissues. Lower image (strain #675): synovitis and bursitis extending focally to peri-articular tissues, mild articular cartilage damage and intra-articular débris, bone structure unaffected.
Figure 72: Mouse model of multiple sclerosis - clinical score.
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Figure 73: Mouse model of multiple sclerosis - disease incidence.
DISCLOSURE OF THE INVENTION
Bacterial strains
The compositions of the invention comprise a bacterial strain of the genus Bacteroides. The examples demonstrate that bacteria of this genus are useful for treating or preventing diseases and conditions mediated by IL-17 or the Thl7 pathway. The preferred bacterial strains are of the species Bacteroides coprocola. Further preferred bacterial strains are of the species Bacteroides thetaiotaomicron or Bacteroides fragilis.
Examples of Bacteroides species for use in the invention include Bacteroides massiliensis, Bacteroides coprocola, Bacteroides thetaiotaomicron and Bacteroides caccae. A further example of a Bacteroides species for use in the invention is Bacteroides fragilis. Bacteroides is a genus of Gramnegative, obligately anaérobie bacteria. Bacteroides species are non-endospore-forming bacilli, and may be either motile or non-motile, depending on the species. Bacteroides species make up a substantial portion of the mammalian gastrointestinal flora and are essential for processing complex molécules.
Bacteroides coprocola cells cultivated on EG blood agar plates are strictly anaérobie, non-sporeforming, non-motile and Gram-negative. The short rods or rod-shaped cells are about 0 8pm in width and variable in length, generally in the range 1—4pm. Example strains of species Bacteroides coprocola are described in [16]. The type strain, M16T (=JCM 12979T=DSM 17136T), was isolated from faeces of a healthy human. Two additional strains [MH (=JCM 12980) and Ml56 (=JCM 12981)] are included in this species. GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene sequence of these Bacteroides coprocola strains are AB200223, AB200224 and AB200225 (disclosed herein as SEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3).
The Bacteroides coprocola bacterium deposited under accession number NCIMB 42408 was tested in the Examples and is also referred to herein as strain 675. A 16S rRNA sequence for the 675 strain that was tested is provided in SEQ ID NO:4. Strain 675 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 13th May 2015 as “Bacteroidales 675” and was assigned accession number NCIMB 42408.
The genome of strain 675 comprises a chromosome and plasmid. A chromosome sequence for strain 675 is provided in SEQ ID NO:6 of WO2016203217. A plasmid sequence for strain 675 is provided in SEQ ID NO:7. These sequences were generated using the PacBio RS II platform.
Bacterial strains closely related to the strain tested in the examples are also expected to be effective for treating or preventing diseases and conditions mediated by IL-17 or the Th.17 pathway. In certain embodiments, the bacterial strain for use in the invention 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 a bacterial strain of Bacteroides coprocola. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1, 2, 3 or 4. Preferably, the sequence identity is to SEQ ID NO:4. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO:4.
A further preferred bacterial strain for use in the invention is the Bacteroides thetaiotaomicron strain deposited under accession number NCIMB 42341. This strain was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) on 3rd December 2014.
Further preferred Bacteroides thetaiotaomicron strains for use in the invention are the type strain ATCC 29148 = CCUG 10774 = CIP 104206 = DSM 2079 = JCM 5827 = NCTC 10582 = VPI 5482 and strain WAL 2926 = ATCC 29741. A further preferred Bacteroides thetaiotaomicron strain for use in the invention is the strain described in EP1448995. The accession number for the 16S rRNA gene sequence of Bacteroides thetaiotaomicron strain WAL 2926 is M58763 (disclosed herein as SEQIDNO:5).
In certain embodiments, the bacterial strain for use in the invention 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 a bacterial strain of Bacteroides thetaiotaomicron. Preferably, the bacterial strain for use in the invention has a 16s rRNA sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:5. Preferably, the bacterial strain for use in the invention has the 16s rRNA sequence represented by SEQ ID NO: 5.
A preferred Bacteroides fragilis strain for use in the invention is the type strain ATCC 25285 = CCUG 4856 = CIP 77.16 = DSM 2151 = JCM 11019 = LMG 10263 = NCTC 9343. The accession number for the Bacteroides fragilis NCTC 9343 strain complété genome is CR626927.1 (version: CR626927.1 GF60491031).
In certain embodiments, the bacterial strain for use in the invention has a genome with sequence identity to CR626927.1. In preferred embodiments, the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to CR626927.1 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of CR626927.1. For example, the bacterial strain for use in the invention may hâve a genome with at least 90% sequence identity to CR626927.1 across 70% of CR626927.1, or at least 90% sequence identity to CR626927.1 across 80% of CR626927.1, or at least 90% sequence identity to CR626927.1 across 90% of CR626927.1, or at least 90% sequence identity to CR626927.1 across 100% of CR626927.1, or at least 95% sequence identity to
CR626927.1across 70% of CR626927.1, or at least 95% sequence identity to CR626927.1 across 80% of CR626927.1, or at least 95% sequence identity to CR626927.1 across 90% of CR626927.1, or at least 95% sequence identity to CR626927.1 across 100% of CR626927.1, or at least 98% sequence identity to CR626927.1 across 70% of CR626927.1, or at least 98% sequence identity to CR626927.1 across 80% of CR626927.1, or at least 98% sequence identity to CR626927.1 across 90% of CR626927.1, or at least 98% sequence identity to CR626927.1 across 100% of CR626927.1.
In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:6 of WO2016203217. In preferred embodiments, the bacterial strain for use in the invention has a chromosome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:6 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:6. For example, the bacterial strain for use in the invention may hâve a chromosome with at least 90% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 90% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 95% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 70% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 80% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 90% of SEQ ID NO:6, or at least 98% sequence identity to SEQ ID NO:6 across 100% of SEQ ID NO:6.
In certain embodiments, the bacterial strain for use in the invention has a plasmid with sequence identity to SEQ ID NO:7. In preferred embodiments, the bacterial strain for use in the invention has a plasmid with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:7 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:7. For example, the bacterial strain for use in the invention may hâve a plasmid with at least 90% sequence identity to SEQ ID NO:7 across 70% of SEQ ID NO:7, or at least 90% sequence identity to SEQ ID NO:7 across 80% of SEQ ID NO: 7, or at least 90% sequence identity to SEQ ID NO: 7 across 90% of SEQ ID NO: 7, or at least 90% sequence identity to SEQ ID NO:7 across 100% of SEQ ID NO:7, or at least 95% sequence identity to SEQ ID NO:7 across 70% of SEQ ID NO:7, or at least 95% sequence identity to SEQ ID NO:7 across 80% of SEQ ID NO:7, or at least 95% sequence identity to SEQ ID NO:7 across 90% of SEQ ID NO:7, or at least 95% sequence identity to SEQ ID NO:7 across 100% of SEQ ID NO:7, or at least 98% sequence identity to SEQ ID NO:7 across 70% of SEQ ID NO:7, or at least 98% sequence identity to SEQ ID NO:7 across 80% of SEQ ID NO:7, or at least 98% sequence identity to
SEQ ID NO:7 across 90% of SEQ ID NO:7, or at least 98% sequence identity to SEQ ID NO:7 across 100% of SEQ ID NO:7.
In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:6 of WO2016203217 and aplasmid with sequence identity to SEQ ID NO:7.
Bacterial strains that are biotypes of the bacterium deposited under accession number 42408 are also expected to be effective for treating or preventing diseases and conditions mediated by IL-17 or the Thl7 pathway. Bacterial strains that are biotypes of a bacterium deposited under accession number NCIMB 42341, ATCC 29148 or ATCC 29741 are also expected to be effective for treating or preventing diseases and conditions mediated by IL-17 or the Th 17 pathway. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.
Strains that are biotypes of a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 and that are suitable for use in the invention may be identified by sequencing other nucléotide sequences for a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may hâve at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). Other suitable sequences for use in idcntilying biotype strains may include hsp60 or répétitive sequences such as BOX, ERIC, (GTG)5, or REP or [17]. Biotype strains may hâve sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741.
Altematively, strains that are biotypes of a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42408 deposit or the accession number NCIMB deposit 42341 or the accession number ATCC 29148 deposit or the accession number ATCC 29741 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and répétitive DNA element (rep)PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Bacteroides coprocola or Bacteroides thetaiotaomicron strains.
In certain embodiments, strains that are biotypes of a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 and that are suitable for use in the invention are strains that provide the same pattern as a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example, [18]). Alternatively, biotype strains are identified as strains that hâve the same carbohydrate fermentation patterns as a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741.
Other Bacteroides strains that are useful in the compositions and methods of the invention, such as biotypes of a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741, may be identified using any appropriate method or strategy, including the assays described in the examples. For instance, strains for use in the invention may be identified by culturing in anaérobie YCFA and/or administering the bacteria to the type II collagen-induced arthritis mouse model and then assessing cytokine levels. In particular, bacterial strains that hâve similar growth patterns, metabolic type and/or surface antigens to a bacterium deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 may be useful in the invention. A useful strain will hâve comparable immune modulatory activity to the NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 strain. In particular, a biotype strain will elicit comparable effects on the asthma, arthritis and multiple sclerosis disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples.
A particularly preferred strain of the invention is the Bacteroides coprocola strain deposited under accession number NCIMB 42408. This is the exemplaiy 675 strain tested in the examples and shown to be effective for treating disease. Therefore, the invention provides a cell, such as an isolated cell, of the Bacteroides coprocola strain deposited under accession number NCIMB 42408, or a dérivative thereof. The invention also provides a composition comprising a cell of the Bacteroides coprocola strain deposited under accession number NCIMB 42408, or a dérivative thereof. The invention also provides a biologically pure culture of the Bacteroides coprocola strain deposited under accession number NCIMB 42408. The invention also provides a cell of the Bacteroides coprocola strain deposited under accession number NCIMB 42408, or a dérivative thereof, for use in therapy, in particular for the diseases described herein.
A dérivative of the strain deposited under accession number NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A dérivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeutically active. A dérivative strain will hâve comparable immune modulatory activity to the original NCIMB 42408, NCIMB 42341, ATCC 29148 or ATCC 29741 strain. In particular, a dérivative strain will elicit comparable effects on the asthma, arthritis and multiple sclerosis disease models and comparable effects on cytokine levels to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. A dérivative of the NCIMB 42408 strain will generally be a biotype of the NCIMB 42408 strain. A dérivative of the NCIMB 42341, ATCC 29148 or ATCC 29741 strain will generally be a biotype of the NCIMB 42341, ATCC 29148 or ATCC 29741 strain.
References to cells of the Bacteroides coprocola strain deposited under accession number NCIMB 42408 encotnpass any cells that hâve the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42408, and such cells are encompassed by the invention. References to cells of the Bacteroides thetaiotaomicron strain deposited under accession numbers NCIMB 42341, ATCC 29148 or ATCC 29741 encompass any cells that hâve the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42341, ATCC 29148 or ATCC 29741, and such cells are encompassed by the invention.
Tn preferred embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.
Therapeutic uses
As demonstrated in the examples, the bacterial compositions of the invention are effective for reducing the Thl7 inflammatory response. In particular, treatment with compositions of the invention achieves a réduction in IL-17A levels and other Thl7 pathway cytokines, and clinical improvements in animal models of conditions mediated by IL-17 and the Thl7 pathway. Therefore, the compositions of the invention may be useful for treating or preventing inflammatory and autoimmune diseases, and in particular diseases or conditions mediated by IL-17. In particular, the compositions of the invention may be useful for reducing or preventing élévation of the IL-17 inflammatory response.
Thl7 cells are a subset of T helper cells that produce, for example, IL-17A, IL17-F, IL-21 and IL-22. Thl7 cell différentiation and IL-17 expression may be driven by IL-23. These cytokines and others form important parts of the Th 17 pathway, which is a well-established inflammatory signalling pathway that contributes to and underlies a number of inflammatory and autoimmune diseases (as described in, for example, [19-24]). Diseases wherein the Thl7 pathway is activated are Thl7 pathway-mediated diseases. Thl7 pathway-mediated diseases can be ameliorated or alleviated by repressing the Thl7 pathway, which may be through a réduction in the différentiation of Thl7 cells or a réduction in their activity or a réduction in the level of Thl7 pathway cytokines. Diseases mediated by the Th 17 pathway may be characterised by increased levels of cytokines produced by Thl7 cells, such as IL-17A, IL-17F, IL-21, IL-22, IL-26, IL-9 (reviewed in [25]). Diseases mediated by the Thl7 pathway may be characterised by increased expression of Th-17-related genes, such as Stat3 or IL-23R. Diseases mediated by the Thl7 pathway may be associated with increased levels of Thl7 cells.
IL-17 is a pro-inflammatory cytokine that contributes to the pathogenesis of several inflaimnatory and autoimmune diseases and conditions. IL-17 as used herein may refer to any member of the IL-17 family, including IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F. IL-17-mediated diseases and conditions are characterised by high expression of IL-17 and/or the accumulation or presence of IL-17-positive cells in a tissue affected by the disease or condition. Similarly, IL-17-mediated diseases and conditions are diseases and conditions that are exacerbated by high IL-17 levels or an increase in IL-17 levels, and. that are alleviated by low IL-17 levels or a réduction in IL-17 levels. The IL-17 inflammatory response may be local or systemic.
Examples of diseases and conditions that may be mediated by IL-17 or the Thl7 pathway include multiple sclerosis; arthritis, such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvénile idiopathic arthritis; neuromyelitis optica (Devic's disease); ankylosîng spondylitis; spondyloarthritis; psoriasis; systemic lupus erythematosus; inflammatory bowel disease, such as Crohn’s disease or ulcerative colitis; celiac disease; asthma, such as allergie asthma or neutrophilie asthma; chronic obstructive pulmonaiy disease (COPD); cancer, such as breast cancer, colon cancer, lung cancer or ovarian cancer; uveitis; scleritis; vasculitis; Behcet's disease; atherosclerosis; atopie dermatitis; emphysema; periodontitis; allergie rhinitis; and allograft féjection. In preferred embodiments, the compositions of the invention are used for treating or preventing one or more of these conditions or diseases. In further preferred embodiments, these conditions or diseases are mediated by IL-17 or the Thl7 pathway.
In certain embodiments, the compositions of the invention are for use in a method of reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of a disease or condition mediated by IL-17 or the Thl7 pathway. In certain embodiments, the compositions of the invention are for use in treating or preventing an inflammatory or autoimmune disease, wherein said treatment or prévention is achieved by reducing or preventing élévation of the Thl7 inflammatory response. In certain embodiments, the compositions of the invention are for use in treating a patient with an inflammatory or autoimmune disease, wherein the patient has elevated IL-17 levels or elevated Th 17 cells or is exhibiting a Thl7 inflammatory response. In certain embodiments, the patient may hâve been diagnosed with a chronic inflammatory or autoimmune disease or condition, or the composition of the invention may be for use in preventing an inflammatory or autoimmune disease or condition developing into a chronic inflammatory or autoimmune disease or condition. In certain embodiments, the disease or condition may not be responsive to treatment with TNF-a inhibitors. These uses of the invention may be applied to any of the spécifie disease or conditions listed in the preceding paragraph.
IL-17 and the Thl7 pathway are often associated with chronic inflammatory and autoimmune diseases, so the compositions of the invention may be particularly useful for treating or preventing chronic diseases or conditions as listed above. In certain embodiments, the compositions are for use in patients with chronic disease. In certain embodiments, the compositions are for use in preventing the development of chronic disease.
The compositions of the invention may be useful for treating diseases and conditions mediated by IL17 or the Thl7 pathway and for addressing the Thl7 inflammatory response, so the compositions of the invention may be particularly useful for treating or preventing chronic disease, treating or preventing disease in patients that hâve not responded to other thérapies (such as treatment with TNF-α inhibitors), and/or treating or preventing the tissue damage and symptoms associated with IL17 and Thl7 cells. For example, IL-17 is known to activate matrix destruction in cartilage and bone tissue and IL-17 has an inhibitory effect on matrix production in chondrocytes and osteoblasts, so the compositions of the invention may be useful for treating or preventing bone érosion or cartilage damage.
In certain embodiments, treatment with compositions of the invention provides a réduction or prevents an élévation in IL-17 levels, in particular IL-17A levels. In certain embodiments, treatment with compositions of the invention provides a réduction or prevents an élévation in IFN-γ, IL-Ιβ, RANTES, ΜΙΡ-Ια, IL-8 or IL-6 levels. Such réduction or prévention of elevated levels of these cytokines may be useful for treating or preventing inflammatory and autoimmune diseases and conditions, in particular those mediated by IL-17 or the Th 17 pathway.
Asthma
In preferred embodiments, the compositions of the invention are for use in treating or preventing asthma. The examples demonstrate that the compositions of the invention achieve a réduction in the recruitment of neutrophils and/or eosinophils into the airways following sensitisation and challenge with house dust mite extract and so they may be usefùl in the treatment or prévention of asthma. Asthma is a chronic disease characterised by inflammation and restriction of the airways. The inflammation in asthma may be mediated by IL-17 and/or Thl7 cells, and so the compositions of the invention may be particularly effective for preventing or treating asthma. The inflammation in asthma may be mediated by eosinophils and/or neutrophils.
In certain embodiments, the asthma is éosinophilie or allergie asthma. Eosinophilie and allergie asthma are characterised by increased numbers of eosinophils in peripheral blood and in airway sécrétions and is associated pathologically with thickening of the basement membrane zone and pharmacologîcally by corticosteroid responsiveness [26]. Compositions that reduce or inhibit eosinophil recruitment or activation may be useful for treating or preventing éosinophilie and allergie asthma.
In additional embodiments, the compositions of the invention are for use in treating or preventing neutrophilie asthma (or non-eosinophilic asthma). High neutrophil numbers are associated with severe asthma that may be insensitive to corticosteroid treatment. Compositions that reduce or inhibit neutrophil recruitment or activation may be useful for treating or preventing neutrophilie asthma.
Eosinophilie and neutrophilie asthma are not mutually exclusive conditions and treatments that help address either the eosinophil and neutrophil responses may be useful for treating asthma in general.
Increased IL-17 levels and activation of the Th 17 pathway are associated with severe asthma, so the compositions of the invention may be useful for preventing the development of severe asthma or for treating severe asthma.
In certain embodiments, the compositions of the invention are for use in methods reducing an éosinophilie inflammatory response in the treatment or prévention of asthma, or for use in methods of reducing a neutrophilie inflammatory response in the treatment or prévention of asthma. As noted above, high levels of eosinophils in asthma is associated pathologically with thickening of the basement membrane zone, so reducing éosinophilie inflammatory response in the treatment or prévention of asthma may be able to specifically address this feature of the disease. Also, elevated neutrophile, either in combination with elevated eosinophils or in their absence, is associated with severe asthma and chronic airway narrowing. Therefore, reducing the neutrophilie inflammatory response may be particularly useful for addressing severe asthma.
In certain embodiments, the compositions reduce peribronchiolar infiltration in allergie asthma, or are for use in reducing peribronchiolar infiltration in the treatment of allergie asthma. In certain embodiments, the compositions reduce peribronchiolar and/or perivascular infiltration in neutrophilie asthma, or are for use in reducing peribronchiolar and/or perivascular infiltration in the treatment of allergie neutrophilie asthma.
In certain embodiments, treatment with compositions of the invention provides a réduction or prevents an élévation in IL-Ιβ, IFNy, RANTES, ΜΙΡ-Ια or IL-8 levels.
In certain embodiments, the compositions of the invention are for use in a method of treating asthma that results in a réduction of the éosinophilie and/or neutrophilie inflammatory response. In certain embodiments, the patient to be treated has, or has previously been identified as having, elevated neutrophil or eosinophil levels, for example as identified through blood sampling or sputum analysis.
The compositions of the invention may be useful for preventing the development of asthma in a newbom when administered to the new-bom, or to a prégnant woman. The compositions may be useful for preventing the development of asthma in children. The compositions of the invention may be useful for treating or preventing adult-onset asthma. The compositions of the invention may be useful for managing or alleviating asthma. The compositions of the invention may be particularly usefiil for reducing symptoms associated with asthma that is aggravaied by allergens, such as house dust mites.
Treatment or prévention of asthma may refer to, for example, an alleviation of the severity of symptoms or a réduction in the frequency of exacerbations or the range of triggers that are a problem for the patient.
Arthritis
In preferred embodiments, the compositions of the invention are for use in treating or preventing rheumatoid arthritis (RA). The examples demonstrate that the compositions of the invention achieve a réduction in the clinical signs of RA in a mouse model, reduce cartilage and bone damage, and reduce the IL-17 inflammatory response, and so they may be useful in the treatment or prévention of RA. RA is a systemic inflammatory disorder that primarily affects joints. RA is associated with an inflammatory response that results in swelling of joints, synovial hyperplasia, and destruction of cartilage and bone. IL-17 and Thl7 cells may hâve a key rôle in RA, for example because IL-17 inhibîts matrix production in chondrocytes and osteoblasts and activâtes the production and function of matrix metalloproteinases and because RA disease activity is correlated to IL-17 levels and Th-17 cell numbers [27,28], so the compositions of the invention may be particularly effective for preventing or treating RA.
In certain embodiments, the compositions of the invention are for use in lowering IL-17 levels or preventing élévation of IL-17 levels in the treatment or prévention of RA. In certain embodiments, treatment with compositions of the invention provides a réduction or prevents an élévation in IL-17 levels, in particular IL-17A levels. In certain embodiments, treatment with compositions of the invention provides a réduction or prevents an élévation in IFN-γ or IL-6 levels.
In certain embodiments, treatment with the compositions of the invention results in a réduction in the swelling of joints. In certain embodiments, the compositions of the invention are for use in patients with swollen joints or patients identified as at risk of having swollen joints. In certain embodiments, the compositions of the invention are for use in a method of reducing joint swelling in RA.
In certain embodiments, treatment with the compositions of the invention results in a réduction in cartilage damage or bone damage. In certain embodiments, the compositions of the invention are for use in reducing or preventing cartilage or bone damage in the treatment of RA. In certain embodiments, the compositions are for use in treating patient with severe RA that are at risk of cartilage or bone damage.
Increased IL-17 levels and Thl7 cell numbers are associated with cartilage and bone destruction in RA [27,28]. IL-17 is known to activate matrix destruction in cartilage and bone tissue and IL-17 has an inhibitory effect on matrix production in chondrocytes and osteoblasts. Therefore, in certain embodiments, the compositions of the invention are for use in preventing bone érosion or cartilage damage in the treatment of RA. In certain embodiments, the compositions are for use in treating patients that exhibit bone érosion or cartilage damage or patients identified as at risk of bone érosion or cartilage damage.
TNF-α is also associated with RA, but TNF-α is not involved in the pathogenesis of the later stages of the disease. In contrast, IL-17 has a rôle throughout ail stages of chronic disease [29]. Therefore, in certain embodiments the compositions of the invention are for use in treating chronic RA or latestage RA, such as disease that includes joint destruction and loss of cartilage. In certain embodiments, the compositions of the invention are for treating patients that hâve previously received anti-TNF-α therapy. In certain embodiments, the patients to be treated do not respond or no longer respond to anti-TNF-α therapy.
The compositions of the invention may be usefül for modulating a patient’s immune System, so in certain embodiments the compositions of the invention are for use in preventing RA in a patient that has been identified as at risk of RA, or that has been diagnosed with early-stage RA. The compositions of the invention may be useful for preventing the development of RA.
The compositions of the invention may be useful for managing or allevîating RA. The compositions of the invention may be particularly usefül for reducing symptoms associated with joint swelling or bone destruction. Treatment or prévention of RA may refer to, for example, an alleviation of the severity of symptoms or a réduction in the frequency of exacerbations or the range of triggers that are a problem for the patient.
Multiple sclerosis
In preferred embodiments, the compositions of the invention are for use in treating or preventing multiple sclerosis. The examples demonstrate that the compositions of the invention achieve a réduction in the disease incidence and disease severity in a mouse model of multiple sclerosis (the EAE model), and so they may be useful in the treatment or prévention of multiple sclerosis. Multiple sclerosis is an inflammatory disorder associated with damage to the myelin sheaths of neurons, particularly in the brain and spinal column. Multiple sclerosis is a chronic disease, which is progressively incapacitating and which evolves in épisodes. IL-17 and Thl7 cells may hâve a key rôle in multiple sclerosis, for example because IL-17 levels may correlate with multiple sclerosis lésions, IL-17 can disrupt blood brain barrier endothélial cell tight jonctions, and Thl7 cells can migrate into the central nervous System and cause neuronal loss [30,31], Therefore, the compositions of the invention may be particularly effective for preventing or treating multiple sclerosis.
In certain embodiments, treatment with the compositions of the invention results in a réduction in disease incidence or disease severity. In certain embodiments, the compositions of the invention are for use in reducing disease incidence or disease severity. In certain embodiments, treatment with the compositions of the invention prevents a décliné in motor function or results in improved motor function. In certain embodiments, the compositions of the invention are for use in preventing a décliné in motor function or for use in improving motor function. In certain embodiments, treatment with the compositions of the invention prevents the development of paralysis. In certain embodiments, the compositions of the invention are for use in preventing paralysis in the treatment of multiple sclerosis.
The compositions of the invention may be useful for modulating a patient’s immune System, so in certain embodiments the compositions of the invention are for use in preventing multiple sclerosis in a patient that has been identified as at risk of multiple sclerosis, or that has been diagnosed with early-stage multiple sclerosis or “relapsing-remitting” multiple sclerosis. The compositions of the invention may be useful for preventing the development of sclerosis. Indeed, the examples show that administration of compositions of the invention prevented the development of disease in many mice.
The compositions of the invention may be useful for managing or alleviating multiple sclerosis. The compositions of the invention may be particularly useful for reducing symptoms associated with multiple sclerosis. Treatment or prévention of multiple sclerosis may refer to, for example, an alleviation of the severity of symptoms or a réduction in the frequency of exacerbations or the range of triggers that are a problem for the patient.
Cancer
In preferred embodiments, the compositions of the invention are for use in treating or preventing cancer. IL-17 and the Th 17 pathway hâve central rôles in cancer development and progression, and so the compositions of the invention may be useful for treating or preventing cancer.
Although the rôles of IL-17 and Th 17 cells in cancer are not fully understood, numerous pro-tumour effects of ÎL-17 and Thl7 cells are known. For example, Thl7 cells and IL-17 can promote angiogenesis, increase prolifération and survival of tumor cells and activate tumour-promoting transcription factors [32-34].
In certain embodiments, treatment with the compositions of the invention results in a réduction in tumour size or a réduction in tumour growth. In certain embodiments, the compositions of the invention are for use in reducing tumour size or reducing tumour growth. The compositions of the invention may be effective for reducing tumour size or growth. In certain embodiments, the compositions of the invention are for use in patients with solid tumours. In certain embodiments, the compositions of the invention are for use in reducing or preventing angiogenesis in the treatment of cancer. IL-17 and Th 17 cells hâve central rôles in angiogenesis. In certain embodiments, the compositions of the invention are for use in preventing metastasis.
In certain embodiments, the compositions of the invention are for use in treating or preventing breast cancer. The compositions of the invention may be effective for treating breast cancer, and IL-17 and Thl7 cells hâve important rôles in breast cancer [35]. In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of breast cancer. In preferred embodiments the cancer is mammary carcinoma. In preferred embodiments the cancer is stage IV breast cancer.
In certain embodiments, the compositions of the invention are for use in treating or preventing lung cancer. The compositions of the invention may be effective for treating lung cancer, and IL-17 and Thl7 cells hâve important rôles in lung cancer [36], In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of lung cancer. In preferred embodiments the cancer is lung carcinoma.
In certain embodiments, the compositions of the invention are for use in treating or preventing liver cancer. The compositions of the invention may be effective for treating liver cancer, and IL-17 and Th 17 cells hâve important rôles in liver cancer [37], In certain embodiments, the compositions of the invention are for use in reducing tumour size, reducing tumour growth, or reducing angiogenesis in the treatment of liver cancer. In preferred embodiments the cancer is hepatoma (hepatocellular carcinoma).
In certain embodiments, the compositions of the invention are for use in treating or preventing carcinoma. The compositions of the invention may be particularly effective for treating carcinoma. In certain embodiments, the compositions of the invention are for use in treating or preventing nonimmunogenic cancer. The compositions of the invention may be effective for treating nonimmunogenic cancers.
In further embodiments, the compositions of the invention are for use in treating or preventing acute lymphoblastic leukemia (ALL), acute myeloid leukemia, adrenocortical carcinoma, basal-cell carcinoma, bile duct cancer, bladder cancer, bone tumor, osteosarcoma/malignant fibrous histiocytoma, brainstem glioma, brain tumor, cerebellar astrocytoma, cérébral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, breast cancer, bronchial adenomas/carcinoids, Burkitt's lymphoma, carcinoid tumor, cervical cancer, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, endométrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, intraocular melanoma, retinoblastoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, glioma, childhood visual pathway and hypothalamic, Hodgkin lymphoma, melanoma, islet cell carcinoma, Kaposi sarcoma, rénal cell cancer, laryngeal cancer, leukaemias, lymphomas, mesothelioma, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, parathyroid cancer, pharyngeal cancer, pituitary adenoma, plasma cell neoplasia, prostate cancer, rénal cell carcinoma, retinoblastoma, sarcoma, testicular cancer, thyroid cancer, or uterine cancer.
The compositions of the invention may be particularly effective when used in combination with further therapeutic agents. The immune-modulatory effects of the compositions of the invention may be effective when combined with more direct anti-cancer agents. Therefore, in certain embodiments, the invention provides a composition comprising a bacterial strain of the genus Bacteroides and an anticancer agent. In preferred embodiments the anticancer agent is an immune checkpoint inhibitor, a targeted antibody immunotherapy, a CAR-T cell therapy, an oncolytic virus, or a cytostatic drug. In preferred embodiments, the composition comprises an anti-cancer agent selected from the group consisting of: Yervoy (ipilimumab, BMS); Keytruda (pembrolizumab, Merck); Opdivo (nivolumab, BMS); MEDI4736 (AZ/Medlmmune); MPDL3280A (Roche/Genentech); Tremelimumab (AZ/Medlmmune); CT-011 (pidilizumab, CureTech); BMS-986015 (lirilumab, BMS); MEDI0680 (AZ/Medlmmune); MSB-0010718C (Merck); PF-05082566 (Pfizer); MEDI6469 (AZ/Medlmmune); BMS-986016 (BMS); BMS-663513 (urelumab, BMS); IMP321 (Prima Biomed); LAG525 (Novartis); ARGX-110 (arGEN-X); PF-05082466 (Pfizer); CDX-1127 (varlilumab; CellDex Therapeutics); TRX-518 (GITR Inc.); MK-4166 (Merck); JTX-2011 (Jounce Therapeutics); ARGX115 (arGEN-X); NLG-9189 (indoximod, NewLink Genetics); INCB024360 (Incyte); IPH2201 (Innate Immotherapeutics/AZ); NLG-919 (NewLink Genetics); anti-VISTA (JnJ); Epacadostat (INCB24360, Incyte); F001287 (Flexus/BMS); CP 870893 (University of Pennsylvania); MGA271 (Macrogenix); Emactuzumab (Roche/Genentech); Galunisertib (Eli Lilly); Ulocuplumab (BMS); BKT140/BL8040 (Biokine Therapeutics); Bavituximab (Peregrine Pharmaceuticals); CC 90002 (Celgene); 852A (Pfizer); VTX-2337 (VentiRx Pharmaceuticals); IMO-2055 (Hybridon, Idera Pharmaceuticals); LY2157299 (Eli Lilly); EW-7197 (Ewha Women’s University, Korea); Vemurafenib (Plexxikon); Dabrafenib (Genentech/GSK); BMS-777607 (BMS); BLZ945 (Memorial Sloan-Kettering Cancer Centre); Unituxin (dinutuximab, United Therapeutics Corporation); Blincyto (blinatumomab, Amgen); Cyramza (ramucirumab, Eli Lilly); Gazyva (obinutuzumab, Roche/Biogen); Kadcyla (ado-trastuzumab emtansine, Roche/Genentech); Perjeta (pertuzumab, Roche/Genentech); Adcetris (brentuximab vedotin, Takeda/Millennium); Arzerra (ofatumumab, GSK); Vectibix (panitumumab, Amgen); Avastin (bevacizumab, Roche/Genentech); Erbitux (cetuximab, BMS/Merck); Bexxar (tositumomab-1131, GSK); Zevalin (ibritumomab tiuxetan, Biogen); Campath (alemtuzumab, Bayer); Mylotarg (gemtuzumab ozogamicin, Pfizer); Herceptin (trastuzumab, Roche/Genentech); Rituxan (rituximab, Genentech/Biogen); volociximab (Abbvie); Enavatuzumab (Abbvie); ABT-414 (Abbvie); Efotuzumab (Abbvie/BMS); ALX-0141 (Ablynx); Ozaralizumab (Ablynx); Actimab-C (Actinium); Actimab-P (Actinium); Milatuzumab-dox (Actinium); Emab-SN-38 (Actinium); Naptumonmab estafenatox (Active Biotech); AFM13 (Affîmed); AFM11 (Affimed); AGS-16C3F (Agensys); AGS-16M8F (Agensys); AGS-22ME (Agensys); AGS-15ME (Agensys); GS-67E (Agensys); ALXN6000 (samalizumab, Alexion); ALT836 (Altor Bioscience); ALT-801 (Altor Bioscience); ALT-803 (Altor Bioscience); AMG780 (Amgen); AMG 228 (Amgen); AMG820 (Amgen); AMG172 (Amgen); AMG595 (Amgen); AMG110 (Amgen); AMG232 (adecatumumab, Amgen); AMG211 (Amgen/Medlmmune); BAY2010112 (Amgen/Bayer); Rilotumumab (Amgen); Denosumab (Amgen); AMP-514 (Amgen); MEDI575 (AZ/Medlmmune); MEDI3617 (AZ/Medlmmune); MEDI6383 (AZ/Medlmmune); MEDI551 (AZ/Medlmmune); Moxetumomab pasudotox (AZ/Medlmmune); MEDI565 (AZ/Medlmmune); MEDI0639 (AZ/Medlmmune); MEDI0680 (AZ/Medlmmune); MEDI562 (AZ/Medlmmune); AV-380 (AVEO); AV203 (AVEO); AV299 (AVEO); BAY79-4620 (Bayer); Anetumab ravtansine (Bayer); vantictumab (Bayer); BAY94-9343 (Bayer); Sibrotuzumab (Boehringer Ingleheim); BI-836845 (Boehringer Ingleheim); B-701 (BioClin); ΒΠΒ015 (Biogen); Obinutuzumab (Biogen/Genentech); BI-505 (Bioinvent); BI-1206 (Bioinvent); TB-403 (Bioinvent); BT-062 (Biotest) BIL-OlOt (Biosceptre); MDX-1203 (BMS); MDX-1204 (BMS); Necitumumab (BMS); CAN-4 (Cantargia AB); CDX-011 (Celldex); CDX1401 (Celldex); CDX301 (Celldex); U31565 (Daiichî Sankyo); patritumab (Daiîchi Sankyo); tigatuzumab (Daiichi Sankyo); nimotuzumab (Daiichi Sankyo); DS-8895 (Daiichi Sankyo); DS-8873 (Daiichi Sankyo); DS-5573 (Daiichi Sankyo); MORab-004 (Eisai); MORab-009 (Eisai); MORab-003 (Eisai); MORab-066 (Eisai); LY3012207 (Eli Lilly); LY2875358 (Eli Lilly); LY2812176 (Eli Lilly); LY3012217(Eli Lilly); LY2495655 (Eli Lilly); LY3012212 (Eli Lilly); LY3012211 (Eli Lilly); LY3009806 (Eli Lilly); cixutumumab (Eli Lilly); Flanvotumab (Eli Lilly); IMC-TR1 (Eli Lilly); Ramucirumab (Eli Lilly); Tabalumab (Eli Lilly); Zanolimumab (Emergent Biosolution); FG-3019 (FibroGen); FPA008 (Five Prime Therapeutics); FP-1039 (Five Prime Therapeutics); FPA144 (Five Prime Therapeutics); catumaxomab (Fresenius Biotech); IMAB362 (Ganymed); IMAB027 (Ganymed); HuMax-CD74 (Genmab); HuMax-TFADC (Genmab); GS-5745 (Gilead); GS-6624 (Gilead); OMP-21M18 (demcizumab, GSK); mapatumumab (GSK); IMGN289 (ImmunoGen); IMGN901 (ImmunoGen); IMGN853 (ImmunoGen); IMGN529 (ImmunoGen); IMMU-130 (Immunomedics); milatuzumabdox (Immunomedics); IMMU-115 (Immunomedics); IMMU-132 (Immunomedics); IMMU-106 (Immunomedics); IMMU-102 (Immunomedics); Epratuzumab (Immunomedics); Clivatuzumab (Immunomedics); IPH41 (Innate Immunotherapeutics); Daratumumab (Janssen/Genmab); CNTO-95 (Intetumumab, Janssen); CNTO-328 (siltuximab, Janssen); KB004 (KaloBios); mogamulizumab (Kyowa Hakko Kirrin); KW-2871 (ecromeximab, Life Science); Sonepcizumab (Lpath); Margetuximab (Macrogenics); Enoblituzumab (Macrogenics); MGD006 (Macrogenics); MGF007 (Macrogenics); MK-0646 (dalotuzumab, Merck); MK-3475 (Merck); Sym004 (Symphogen/Merck Serono); DI17E6 (Merck Serono); MOR208 (Morphosys); MOR202 (Morphosys); Xmab5574 (Morphosys); BPC-1C (ensituximab, Précision Biologics); TAS266 (Novartis); LFA102 (Novartis); BHQ880 (Novartis/Morphosys); QGE031 (Novartis); HCD122 (lucatumumab, Novartis); LJM716 (Novartis); AT355 (Novartis); OMP-21M18 (Demcizumab, OncoMed); OMP52M51 (Oncomed/GSK); OMP-59R5 (Oncomed/GSK); vantictumab (Oncomed/Bayer); CMC-544 (inotuzumab ozogamicîn, Pfizer); PF-03446962 (Pfizer); PF-04856884 (Pfizer); PSMA-ADC (Progenics); REGN1400 (Regeneron); REGN910 (nesvacutnab, Regeneron/Sanofî); REGN421 (enoticumab, Regeneron/Sanofi); RG7221, RG7356, RG7155, RG7444, RG7116, RG7458, RG7598, RG7599, RG7600, RG7636, RG7450, RG7593, RG7596, DCDS3410A, RG7414 (parsatuzumab), RG7160 (imgatuzumab), RG7159 (obintuzumab), RG7686, RG3638 (onartuzumab), RG7597 (Roche/Genentech); SAR307746 (Sanofi); SAR566658 (Sanofi); SAR650984 (Sanofi); SAR153192 (Sanofî); SAR3419 (Sanofi); SAR256212 (Sanofi), SGN-LIV1A (lintuzumab, Seattle Genetics); SGN-CD33A (Seattle Genetics); SGN-75 (vorsetuzumab mafodotin, Seattle Genetics); SGN-19A (Seattle Genetics) SGN-CD70A (Seattle Genetics); SEA-CD40 (Seattle Genetics); ibritumomab tiuxetan (Spectrnm); MLN0264 (Takeda); ganitumab (Takeda/Amgen); CEP-37250 (Teva); TB-403 (Thrombogenic); VB4-845 (Viventia); Xmab2512 (Xencor); Xmab5574 (Xencor); nimotuzumab (YM Biosciences); Carlumab (Janssen); NY-ESO TCR (Adaptimmune); MAGE-A-10 TCR (Adaptimmune); CTL019 (Novartis); JCAR015 (Juno Therapeutics); KTE-C19 CAR (Kite Pharma); UCART19 (Cellectis); BPX-401 (Bellicum Pharmaceuticals); BPX-601 (Bellicum Pharmaceuticals); ATTCK20 (Unum Therapeutics); CAR-NKG2D (Celyad); Onyx-015 (Onyx Pharmaceuticals); H101 (Shanghai Sunwaybio); DNX-2401 (DNAtrix); VCN-01 (VCN Biosciences); Colo-Adl (PsiOxus Therapeutics); ProstAtak (Advantagene); Oncos-102 (Oncos Therapeutics); CG0070 (Cold Genesys); Pexa-vac (JX-594, Jennerex Biotherapeutics); GL-ONC1 (Genelux); T-VEC (Amgen); G207 (Medigene); HF10 (Takara Bio); SEPREHVIR (HSV1716, Virttu Biologics); OrienXOlO (OrienGene Biotechnology); Reolysin (Oncolytics Biotech); SW-001 (Neotropix); Cacatak (CVA21, Viralytics); Alimta (Eli Lilly), cisplatin, oxaliplatin, irinotecan, folinic acid, methotrexate, cyclophosphamide, 5-fluorouracil, Zykadia (Novartis), Tafinlar (GSK), Xaikori (Pfizer), Tressa (AZ), Gilotrif (Boehringer Ingelheim), Tarceva (Astellas Pharma), Halaven (Eisai Pharma), Veliparib (Abbvie), AZD9291 (AZ), Alectinib (Chugai), LDK378 (Novartis), Genetespib (Synta Pharma), Tergenpumatucel-L (NewLink Genetics), GV1001 (Kael-GemVax), Tivantinib (ArQule); Cytoxan (BMS); Oncovin (Eli Lilly); Adriamycin (Pfizer); Gemzar (Eli Lilly); Xeloda (Roche); Ixempra (BMS); Abraxane (Celgene); Trelstar (Debiopharm); Taxotere (Sanofi); Nexavar (Bayer); IMMU-132 (Immunomedics); E7449 (Eisai); Thermodox (Celsion); Cometriq (Exellxis); Lonsurf (Taiho Pharmaceuticals); Camptosar (Pfizer); UFT (Taiho Pharmaceuticals); and TS-1 (Taiho Pharmaceuticals).
Uveitis
In further preferred embodiments, the compositions of the invention are for use in treating or preventing uveitis. The compositions of the invention may achieve a réduction in disease incidence and disease severity in an animal model of uveitis and so they may be useful in the treatment or prévention of uveitis. Uveitis is inflammation of the uvea and can resuit in retinal tissue destruction. It can présent in different anatomical forms (anterior, intermediate, posterior or diffuse) and resuit from different, but related, causes, including systemic autoimmune disorders. IL-17 and the Thl7 pathway are centrally involved in uveitis, so the compositions of the invention may be particularly effective for preventing or treating uveitis. References [38-45] describe elevated sérum levels of interleukin-17A in uveitis patients, spécifie association of IL17A genetic variants with panuveitis, the rôle of Thl7-associated cytokines in the pathogenesis of experimental autoimmune uveitis, the imbalance between Th 17 Cells and regulatory T Cells during monophasic experimental autoimmune uveitis, the up-regulation of IL-17A in patients with uveitis and active Adamantiades-Behçet and Vogt-Koyanagi-Harada (VKH) diseases, the treatment of non-infectious uveitis with seeukinumab (anti-IL-17A antibody), and Th 17 inuveitic eyes.
In certain embodiments, the uveitis is posterior uveitis. Posterior uveitis présents primarily with inflammation of the retina and choroid and the compositions of the invention may be effective for reducing retinal inflammation and damage.
In certain embodiments, treatment with the compositions of the invention results in a réduction in retinal damage, hi certain embodiments, the compositions of the invention are for use in reducing or preventing retinal damage in the treatment of uveitis. In certain embodiments, the compositions are for use in treating patients with severe uveitis that are at risk of retinal damage. In certain embodiments, treatment with the compositions of the invention results in a réduction in optic dise inflammation. In certain embodiments, the compositions of the invention are for use in reducing or preventing optic dise inflammation. In certain embodiments, treatment with the compositions of the invention results in a réduction in retinal tissue infiltration by inflammatory cells. In certain embodiments, the compositions of the invention are for use in reducing retinal tissue infiltration by inflammatory cells. In certain embodiments, treatment with the compositions of the invention results in vision being maintained or improved. In certain embodiments, the compositions of the invention are for use in maintaining or improving vision.
In certain embodiments, the compositions are for use in treating or preventing uveitis associated with a non-infectious or autoimmune disease, such as Behçet disease, Crohn's disease, Fuchs hétérochromie iridocyclitis, granulomatosis with polyangiitis, HLA-B27 related uveitis, juvénile idiopathic arthritis, sarcoidosis, spondyloarthritis, sympathetic ophthalmia, tubulointerstitial nephritis and uveitis syndrome or Vogt-Koyanagi-Harada syndrome. IL-17A has been shown to be involved in, for example, Behçet and Vogt-Koyanagi-Harada diseases.
Treatment or prévention of uveitis may refer to, for example, an alleviation of the severity of symptoms or a prévention of relapse.
Modes of administration
Preferably, the compositions of the invention are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the intestine with the bacterial strain of the invention. Generally, the compositions of the invention are administered orally, but they may be administered rectally, intranasally, or via buccal or sublingual routes.
In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a gel.
In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for example in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.
In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopie gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jéjunum and other suitable access ports.
The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily.
In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be repeated if delivery of and / or partial or total colonisation with the strain of the invention is not achieved such that efficacy is not observed, or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed.
In certain embodiments, the composition of the invention may be administered to a prégnant animal, for example a mammal such as a human in order to prevent an inflammatory or autoimmune disease developing in her child in utero and / or after it is bom.
The compositions of the invention may be administered to a patient that has been diagnosed with a disease or condition mediated by IL-17 or the Th 17 pathway, or that has been identified as being at risk of a disease or condition mediated by IL-17 or the Th 17 pathway. The compositions may also be administered as a prophylactic measure to prevent the development of diseases or conditions mediated by IL-17 or the Thl7 pathway in a healthy patient.
The compositions of the invention may be administered to a patient that has been identified as having an abnormal gut microbiota. For example, the patient may hâve reduced or absent colonisation by Bacteroides, and in particular Bacteroides coprocola.
The compositions of the invention may be administered as a food product, such as a nutritional supplément.
Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animais including monogastrîc mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animais. If administered to animais, oral gavage may be used.
Compositions
Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. For example, the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial sixain of the invention.
Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [46-48],
Altematîvely, the composition of the invention may comprise a live, active bacterial culture.
In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from dégradation until delivery at the target location through, for example, rupturing with Chemical or physical stimuli such as pressure, enzymatic activity, or physical disintegration, which may be triggered by changes in pH. Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachaient or adsorption on solid carrier surfaces, selfaggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [49] and [50].
The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Bacteroides are anaerobes. Other ingrédients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo. Altematîvely, the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product.
The composition may be formulated as a probiotic.
A composition of the invention includes a therapeutically effective amount of a bacterial strain of the invention. A therapeutically effective amount of a bacterial strain is sufficient to exert a bénéficiai effect upon a patient. A therapeutically effective amount of a bacterial strain may be suffîcient to resuit in delivery to and / or partial or total colonisation of the patient’s intestine.
A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 103 to about 1 x 1011 colony forming units (CFU); for example, from about 1 x 107 to about 1 x 1O10 CFU; in another example from about 1 x 106 to about 1 x 1O10 CFU.
In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 106 to about 1 x 1011 CFU/g, respect to the weight of the composition; for example, from about 1 x 108 to about 1 x 1O10 CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.
Typîcally, a probiotic, such as the composition of the invention, is optionaily combined with at least one suitable prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and transgalactooligosaccharides.
In certain embodiments, the probiotic composition of the présent invention includes a prebiotic compound in an amount of from about 1 to about 30% by weight, respect to the total weight composition, (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomaltoligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), betaglucans, arable gum modified and résistant starches, polydextrose, D-tagatose, acacia fîbers, carob, oats, and citrus fîbers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molécule to which three glucose molécules are bonded.
The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [51]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [52], Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol and the like. Examples of suitable diluents include éthanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, freeflow lactose, beta-lactose, corn sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilîzers, dyes and even flavouring 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 may be also used.
The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefît in addition to the therapeutîc effect of the invention, such as in a nutritional supplément. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, the composition of the invention is formulated as a milk-based product. The term milk-based product means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milk-based product can be, e.g., cow's milk, goat's milk, sheep's milk, skimmed milk, whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.
In certain embodiments, the compositions of the invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially free from other species of organism.
The compositions for use in accordance with the invention may or may not require marketing approval.
In some cases, the lyophilised bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.
The compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.
In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat a disorder when administered to a subject in need thereof; and wherein the disorder is selected from the group consisting of asthma, allergie asthma, neutrophilie asthma, osteoarthritis, psoriatic arthritis, juvénile idiopathic arthritis, neuromyelitis optica (Devic's disease), ankylosing spondylitis, spondyloarthritis, systemic lupus erythematosus, celiac disease, chronic obstructive pulmonary disease (COPD), cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, uveitis, scleritis, vasculitis, Behcet's disease, atherosclerosis, atopie dermatitis, emphysema, periodontitis, allergie rhinitis, and allograft rejection.
In certain embodiments, the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition mediated by IL-17 or the Th 17 pathway. In preferred embodiments, said disease or condition is selected from the group consisting of rheumatoid arthritis, multiple sclerosis, psoriasis, inflammatory bowel disease, Crohn’s disease, ulcerative colitis, celiac disease, asthma, allergie asthma, neutrophilie asthma, osteoarthritis, psoriatic arthritis, juvénile idiopathic arthritis, neuromyelitis optica (Devic's disease), ankylosing spondylitis, spondyloarthritis, systemic lupus eiythematosus, chronic obstructive pulmonary disease (COPD), cancer, breast cancer, colon cancer, lung cancer, ovarian cancer, uveitis, scleritis, vasculitis, Behcet's disease, atherosclerosis, atopie dermatitis, emphysema, periodontitis, allergie rhinitis, and allograft rejection.
In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 * 103 to about 1 χ 1011 colony forming units per gram with respect to a weight of the composition.
In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.
In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subeutaneous, nasal, buccal, and sublingual.
In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol and sorbitol.
In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of éthanol, glycerol and water.
In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, freeflow lactose, beta-lactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate and sodium chloride.
In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.
In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid and esters of phydroxybenzoic acid.
In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.
In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25°C and the container is placed in an atmosphère having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.
Culturing methods
The bacterial strains for use in the présent invention can be cultured using standard microbiology techniques as detailed in, for example, references [53-55],
Bacterial strains of the genus Bacteroides may be cultured using a method such as that outlined below, which may provide good growth and reliability. This method is particularly useful for culturing strains of the species Bacteroides coprocola.
Bacterial strains of the genus Bacteroides, and in particular of the species Bacteroides coprocola, may be cultured by using a liquid stock to inoculate the plate (or a larger liquid culture), rather than a scrape of frozen stock as may generally be used (see, for example, reference [56]). The establishment of mature colonies is reliable and quick if frozen stocks of Bacteroides strains are thawed and used as a liquid culture.
A method suitable for culturing a bacterial strain of the genus Bacteroides, may comprise:
(a) providing a frozen stock of the bacterial strain;
(b) thawing the frozen stock to provide a liquid stock;
(c) adding the liquid stock to a solid or liquid medium; and (d) incubating the solid or liquid media to provide a culture of the bacterial strain.
This method is particularly useful for culturing bacterial strains of Bacteroides coprocola.
The frozen stock may be a frozen glycerol stock. The solid or liquid medium may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCOs (0.4 g), cysteine (0.1 g), K2HPO4 (0.045 g), KH2PO4 (0.045 g), NaCl (0.09 g), (NH4)2SO4 (0.09 g), MgSO4 · 7H2O (0.009 g), CaCl2 (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), /i-aminobenzoic acid (3 pg), folie acid (5 pg), and pyridoxamine (15 pg).
The incubating in step (d) may be performed for at least 36 hours, such as 48 or 72 hours. The incubating in step (d) may be performed in an anaérobie environment, such as an anaérobie hood. The culture provided in step (d) may be used to subculture a larger culture of the bacterial strain. Such subculturing allows greater amounts of bacteria to be prepared and may be useful for providing compositions of the invention at a commercial scale.
The thawing in step (b) may be performed at room température, or by hand warming.
The amount of liquid stock added in step (c) may be between 300pI and 5ml, such 500pl. The stock may be 20% glycerol stock.
The above method may be used for preparing a pharmaceutical composition or a food product, in which case the method may comprise additional steps of isolating the bacterial strain, optionally lyophilising the bacterial strain, and combining the bacterial strain with one or more pharmaceutically acceptable excipients or carriers, or one or more food substances. This pharmaceutical composition or food product prepared by the method may be used in a method of treating or preventing a disease or condition mediated by IL-17 or the Thl 7 pathway.
An exemplary method of culturing bacterial strains of the genus Bacteroides, and in particular of the species Bacteroides coprocola, may comprise:
1. 500pl of 20% glycerol stock is plated onto YCFA agar.
2. Plates tire left in the anaérobie hood for 48/72 hours to generate mature colonies.
3. The bacteria are cultured in 10ml volumes, whether this be from a single colony or a 1% liquid subculture.
4. Plates with colonies are only used for 2/3 days after mature colony morphology is achieved.
Bacterial strains for use in vaccine compositions
The inventors hâve identified that the bacterial strains of the invention are useful for treating or preventing diseases or conditions mediated by IL-17 or the Thl 7 pathway. This is likely to be a resuit of the effect that the bacterial strains of the invention hâve on the host immune system. Therefore, the compositions of the invention may also be useful for preventing diseases or conditions mediated by IL-17 or the Th 17 pathway, when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subcutaneous injection.
General
The practice of the présent invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistiy, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fùlly in the literature. See, e.g., references [57] and [58-64], etc.
The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.
The term “about” in relation to a numerical value x is optional and means, for example, x+10%.
The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the définition ofthe invention.
References to a percentage sequence identity between two nucléotide sequences means that, when aligned, that percentage of nucléotides are the same in comparing the two sequences. This alignaient and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [65], A preferred alignment is determined by the Smith-Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The SmithWaterman homology search algorithm is disclosed in ref. [66].
Unless specifically stated, a process or method comprising numerous steps may comprise additional steps at the beginning or end of the method, or may comprise additional intervening steps. Also, steps may be combîned, omitted or performed in an alternative order, if appropriate.
Various embodiments of the invention are described herein. It will be appreciated that the features specified in each embodiment may be combîned with other specified features, to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combîned with each other (except when they are mutually exclusive).
MODES FOR CA RR Y ING OUT THE INVENTION
Example 1 — Efficacy of bacterial inocula in a mouse model of house dust mite-induced asthma
Summary
Mice were administered with compositions comprising bacterial strains according to the invention and were subsequently challenged with house dust mite (HDM) extract to elicit an allergie inflammatory response. The inflammatory response to HDM includes éosinophilie and neutrophilie components, is mediated by IL-17 and the Th 17 pathway, and is a model for asthma. The magnitude and characteristics of the inflammatory response exhibited by mice treated with compositions of the invention were compared to control groups. The compositions of the invention were found to alleviate the inflammatory response, and to reduce recruitment of eosinophils and neutrophils, indicating that they may be useful for treating IL-17- and Thl7-mediated conditions such as eosinophilia, neutrophilia and asthma.
Strain
675: Bacteroides coprocola
Study design
Groups:
1. Négative control group. Treatment with vehicle control (per oral).
3. Treatment with therapeutic bacteria inoculum strain 675 (per oral).
7. Positive control group. Treatment with Dexamethasone (i.p.).
8. Untreated Control Group.
Number of mice per group - 5
Day -14 to day 13: Daily administration of vehicle control per oral (Group 1).
Day -14 to day 13: Daily administration of therapeutic bacteria inoculum per oral (Group 2-6).
Day 0, 2, 4, 7, 9, 11 Administration of 15ug HDM (house dust mite extract Catalogue number: XPB70D3A25, Lot number: 231897, Greer Laboratories, Lenoir, NC, USA) in a volume of 30ul PBS per nasal (Group 1-8).
Day 0, 2, 4, 7, 9, 11 Administration of Dexamethasone (i.p., 3mg/kg, Sigma-Aldrich, Catalogue number DI 159) (Group 7).
Day 14 Sacrifice of ail animais for analysis.
Total number of mice = 40.
Endpoints and analysis
On day 14 animais were sacrificed by léthal intraperitoneal injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A) immediately followed by a bronchoalveolar lavage (BAL).
Cells were isolated from the BAL (bronchoalveolar lavage) fluid and differential cell counts performed (200 cell counts/ samples).
Material and Methods
Mice. Female 7 week old BALB/c mice were purchased from Charles River Laboratories and randomly allocated to cages totally 5 mice per cage (Ventilated cages sourced from Indulab AG, Gams, Switzerland Cage type: “The SealsafeTM - IVC cage. Product number 1248L). Cages were labeled with study number, group number and experimental starting date. Mice were monitored weekly and acclimatized to facility for 7 days prior to initiation of study (Study Day -14). Animais were 8 weeks old on Study Day -14. Potable water and food were available ad libitum. Cage enrichment was present. Daily care of the animais was performed according to local authorization license number 2283.1 (issued and approved by: Service de la consommation et des affaires vétérinaires du Canton de Vaud). Potable water and food were available ad libitum and refreshed once daily. Cage enrichment was present. Animal welfare régulations were observed as given by official authorities of Switzerland under ordinance 455.163 of the FVO (Fédéral Veterinary Office) on laboratory animal husbandry, production of genetically modified animais, and methods of animal expérimentation.
Culturing of bacteria inoculum. Within a stérile workstation, a cryo-vial of bacteria was thawed by warming in gloved hand and ~0.7 ml of contents injected into a Hungate tube (Cat Number, 1020471, Glasgerâtebau Ochs, Bovenden-Lenglem, Germany), containing 8 ml of anaérobie YCFA. Two tubes per strain were usually prepared. The Hungate tubes were then incubated (static) at 37°C for up to 24-26 hours (for strain 675).
Since the bacterial ODs of inoculum strain 675 was found to be variable, 3 different culturing approaches were performed each day. 2 vials were cultured as described above and a third sample was cultured utilizing a 400ul aliquot from the prior day’s culture for seeding. On 4 treatment days the latter approach was utilized, because of poor growth from the frozen stock. Of note, this approach resulted in robust growth of bacterial strain 675 on ail occasions.
Culturing of vehicle control. A Hungate tube containing 8 ml of anaérobie YCFA was incubated (static) at 37°C for 16h.
Administration of bacteria inoculum or vehicle control. 400ul of cultured bacteria inoculum or vehicle control were administered per day per oral gavage.
Intranasal sensitization. Mice were anesthetized by i.p. injection with 9.75 mg xylasol and 48.75 mg ketasol per kg (Dr. E. Graeub AG, Bem, Switzerland) and administered with 15ug of HDM (Catalogue number: XPB70D3A25, Lot number: 231897, Greer Laboratories, Lenoir, NC, USA) in a volume of 30ul PBS per nasal.
Préparation and administration of positive control compound Déxaméthasone. Déxaméthasone 21-phosphate disodium sait (Sigma-Aldrich, Catalogue number DI 159, Lot N° SLBD.1030V) was solved in H2O and administered to the animais in a dose of 3mg/kg in a volume of 200ul per oral at days indicated in study protocol above.
Terminal procedure. On day 14 animais were sacrifîced by léthal i.p. injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A) immediately followed by bronchoalveolar lavage (BAL) in 500 ul of saline.
Measurement of cellular infiltrâtes into BAL. Cells were isolated from the BAL fluid and differential cell counts were performed based upon standard morphological and cytochemical criteria.
Graphs and statistical analysis. Ail graphe were generated with Graphpad Prism Version 6 and a one-way ANOVA was applied. Results from the statistical analysis were provided with the individual data tables. Error bars represent Standard Error of the Mean (SEM).
Results and analysis
The results of the experiments are shown in Figures 1-9.
No morbidity or mortality was noted in the mice treated with the bacteria or the vehicle. The two Controls, vehicle treatment (négative control) and the dexamethasone treatment (positive control) behaved as expected, with impaired eosinophilia and neutrophilia noted following dexamethasone treatment.
The most important results of this experiment are displayed in Figures 6 and 7, which report on the total number and percentage of neutrophils detected in bronchiolar lavage following challenge with HDM. Strain 675 reduced total neutrophils and the proportion of neutrophils in BAL relative to the vehicle-only control.
Example 2 — Efficacy of bacterial inocula in a mouse model of severe neutrophilie asthma
Summary
Mice were administered with compositions comprising bacterial strains according to the invention and were subsequently sensitised with subeutaneous administrations of house dust mite (HDM) extract and challenged with an intranasal administration of HDM in order to model the inflammatory response of severe neutrophilie asthma. The magnitude and characteristics of the inflammatory response exhibited by mice treated with compositions of the invention were compared to control groups. The compositions of the invention were found to alleviate the inflammatory response, and in particular to reduce recruitment of neutrophils, in a manner comparable to the positive control comprising administrations of anti-IL-17 antibodies. The data therefore indicate that the compositions of the invention may be useful for treating IL-17- and Thl7-mediated conditions such as neutrophilia and asthma.
Strain
675: Bacteroides coprocola
Study design
Groups:
1. Négative control group. Treatment with vehicle control (per oral).
3. Treatment with therapeutic bacteria inoculum strain 675 (per oral).
7. Positive control group. Treatment anti-IL-17 (i.p.).
8. Untreated Control Group.
9: Healthy mice (baseline).
Number of mice per group (Group 1-8) = 5
Day -14 to day 17: Daily administration of vehicle control per oral (Group 1).
Day -14 to day 17: Daily administration of therapeutic bacteria inoculum per oral (Group 2-6).
Day 0: Sensitization with HDM in CFA (s.c.) (Group 1-8).
Day 7: Sensitization with HDM in CFA (s.c.) (Group 1-8).
Day 13, 15, 17: Administration of anti IL-17 neutralizing antibody per i.p. (Group 7).
Day 14, 15, 16, 17: Challenge with HDM in 30ul PBS per nasal (Group 1-8).
Day 18: Sacrifice of ail animais for analysis.
Endpoints and analysis:
On day 14 animais were sacrifîced by léthal intraperitoneal injection with pentabarbîtol (Streuli Pharma AG, Uznach, Cat: 1170139A) immediately followed by a bronchoalveolar lavage (BAL). Cells were isolated from the BAL fluid and differential cell counts performed (200 cell counts/ samples).
Material and Methods.
Mice. Female 7 week old C57BL/6 mice were purchased from Charles River Laboratories and randomly allocated to cages totally 5 mice per cage (Ventilated cages sourced from Indulab AG, Gains, Switzerland Cage type: “The SealsafeTM - IVC cage. Product number 1248L). Cages were labelled with study number, group number and experimental starting date. Mice were monitored weekly and acclimatized to facility for 7 days prior to initiation of study (Study Day -14). Animais were 8 weeks old on Study Day -14. Potable water and food were available ad libitum. Cage enrichment was présent. Daily care of the animais was performed according to local authorization license number 2283.1 (issued and approved by: Service de la consommation et des affaires vétérinaires du Canton de Vaud). Potable water and food were available ad libitum and refreshed once daily. Cage enrichment was présent. Animal welfare régulations were observed as given by official authorities of Switzerland under ordinance 455.163 of the FVO (Fédéral Veterinary Office) on laboratory animal husbandry, production of genetically modified animais, and methods of animal expérimentation.
Culturing of bacteria inoculum. Within a stérile workstation, a cryo-vial of bacteria was thawed by warming in gloved hand and ~0.7 ml of contents injected into a Hungate tube (Cat Number, 1020471, Glasgerâtebau Ochs, Bovenden-Lenglem, Germany), containing 8 ml of anaérobie YCFA. Two tubes per strain were usually prepared. The Hungate tubes were then incubated (static) at 37°C for up to 24-26 hours (for strain 675).
Since the bacterial ODs of inoculum strain 675 were variable, 3 different culturing approaches were performed each day. 2 vials were cultured as described above and a third sample was cultured utilizing a 400ul aliquot from the prior day’s culture for seeding. On 4 treatment days the latter approach was utilized, because of poor growth from the frozen stock. Of note, this approach resulted in robust growth of bacterial strain 675 on ail occasions.
Culturing of vehicle control. A Hungate tube containing 8 ml of anaérobie YCFA was incubated (static) at 37°C for 16h.
Administration of bacteria inoculum or vehicle control. 400ul of cultured bacteria inoculum or vehicle control were administered per day per oral gavage.
HDM sensitization. 50 pg of HDM (Catalogue number: XPB70D3A25, Lot number: 231897, Greer Laboratories, Lenoir, NC, USA) in PBS was emulsified in equal volume of complété Freund’s adjuvant (CFA Chondrex Inc. Washington, USA) and administered subcutaneously in a volume of 200 μΐ, twîce over two weeks on opposite flanks. A week after the second immunization, mice were anesthetized by i.p. injection with 9.75 mg xylasol and 48.75 mg ketasol per kg (Dr. E. Graeub AG, Bem, Switzerland) and then given intranasal challenges of 15 pg of HDM in a volume of 30ul PBS on 4 consecutive days. Analysis was performed one day after the final challenge.
Préparation and administration of positive control compound anti mouse IL-17 antibody. Anti-IL-17 neutralizing antibody was sourced from Bio X Cell and was stored at 4°C (Clone 17F3, Cat. Number BE0173, Bio X Cell) and administered per i.p. at a dose of 12.5 mg/kg at days indicated in study protocol above.
Terminal procedure. On day 18 animais were sacrifîced by léthal i.p. injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat: 1170139A) immediately followed by bronchoalveolar lavage (BAL) in 500 ul of saline.
Measurement of cellular infiltrâtes into BAL. Cells were isolated from the BAL fluid and differential cell counts were performed based upon standard morphological and cytochemical criteria.
Graphe and statistical analysis. Ail graphs were generated with Graphpad Prism Version 6 and a one-way ANOVA was applied. Results from the statistical analysis are provided with the individual data tables. Error bars represent Standard Error of the Mean (SEM).
Results and analysis
The results ofthe experiment are shown in Figures 10-18.
No morbidity or mortality was noted in the mice treated with the bacteria or the vehicle. As shown in Figures 15 and 16, strain 675 exhibited a strong effect and reduced total neutrophil numbers relative to the négative Controls. In addition, strain 675 reduced eosinophil numbers relative to the Controls, as shown in Figures 11 and 12.
Example 3 — Efficacy of bacterial inocula to treat arthritis in a type II collagen-induced arthritis mouse model
Materials and methods
Strain
675: Bacteroides coprocola
Bacterial cultures
Bacterial cultures were grown up for administration in an anaérobie workstation (Don Whitley Scientifîc) according to the growth scheme below.
- Mon/Weds/Fri: Transfer glycerol stock to ice and streak a YCFA plate from glycerol stock. Use plate as follows for a maximum of 3 days.
- Daily PM: Pick single colony of each strain from plate cultures, transfer to 8 ml hungate tube containing YCFA ovemight (ON1)
- AM: Subculture 80 ul (1%) ON1 into fresh 8 ml tube (DC1). Use ON1 culture for AM oral gavage.
- PM: Use DC1 culture for PM oral gavage.
Bacterial strain #675 was grown using glycerol stocks.
Glycerol stocks were stored at -80°C. Three times per week, glycerol stocks were thawed at room température and streaked on YCFA plates. A new glycerol aliquot was used on each occasion. Bacteria were allowed to grow on a given plate for up to 72 hours.
Solutions to be administered to the animais were prepared twice daily with an eight hour interval for moming (AM) and aftemoon (PM) treatments. A bacterial colony was picked from the streaked plate and transferred into a tube containing YCFA media. Bacterial strain #675 was allowed to grow for 16 hours before AM administrations. Bacteria were sub-cultured at 1% into YCFA media for PM administrations. OD values were recorded for each strain after moming and aftemoon treatment préparations.
Type II collagen-induced arthritis mouse model
Adult male DBA/1 mice were randomly allocated to experimental groups and allowed to acclimatise for two weeks. On Day 0, animais were administered by subcutaneous injection with 100 microliters of an émulsion containing 100 micrograms of type II collagen (Cil) in incomplète’s Freund’s adjuvant supplemented with 4 mg/ml Mycobacterium tuberculosis H37Ra. On Day 21, animais were administered by subcutaneous injection with a booster émulsion containing 100 pg of type II collagen in incomplète Freund’s adjuvant.
Treatments were given according to the administration schedule below. From Day -14 until the end of the experiment on Day 45, animais were weighed three times per week. From Day 21 until the end of the experiment, animais were scored three times per week for clinical signs of arthritis to include swelling of the hind- and front paws, radio-carpal (wrist) joints and tibio-tarsal (ankle) joints.
On Day 45 mice were culled and terminal blood samples were taken for cytokine analysis.
On Day -14, Day 0 and Day 45, faecal samples were collected for microbiological analysis, immediately snap-frozen and stored at -80°C.
The collagen-induced arthritis (CIA) mouse model is a well-established mouse model for rheumatoid arthritis [67]. Immunisation with CII causes a pathogenesis that includes several important pathological features of rheumatoid arthritis, including synovial hyperplasia, mononuclear cell infiltration and cartilage dégradation. Signifîcantly, the development of CIA is mediated by Thl7 cells through sécrétion of IL-17A [68]. The immune response underlying the arthritis model is enhanced by the use of Freund’s adjuvant supplemented with Mycobacterium tuberculosis.
On Day 21, spleens were collected from three satellite animais in each group. Cells were cultured for 72 hours in the presence or absence of type II collagen. Cytokines, including TNF-α, IL-6, IFN-γ, IL4, IL-10 and IL-17, were quantified in the culture supematants and in terminal sérum by Luminex. Cell prolifération was quantified using a tritiated thymidine incorporation method.
Treatment Groups and Dosages
Ail Groups were n=15 (n=12 for the main study group and n=3 for satellite groups)
The vehicle used for the biotherapeutics was Yeast extract-Casitone-Fatty Acids (YCFA) medium.
Group Dose Administration Disease Induction
Route Regimen
1 Vehicle 5 ml/kg PO BID: Day -I4*-End Day 0: Collagen/CFA, once, SC Day 21: Collagen/IFA, once, SC
4 Biotherapeutic #675 5 ml/kg
PO: oral gavage, SC: subcutaneous injection, BID: twice a day, CFA: complété Freund’s adjuvant. * Excep t Group 4 treated from Day 0.
Bodyweights
From Day -14 until the end of the experiment, animais were weighed three times per week. Data were graphed (Mean ± SEM).
Non-specifîc clinical observations
From Day -14 until the end of the experiment, animais were checked daily for non-specific clinical signs to include abnormal posture (hunched), abnormal coat condition (piloerection) and abnormal activity levels (reduced or increased activity).
Clinical Observations
From Day 21 until the end of the experiment on Day 45, animais were scored three times per week for clinical signs of arthritis to include swelling of the hind- and front paws, radio-carpal (wrist) joints and tibio-tarsal (ankle) joints. Each limb was scored using the following scale: (0) normal, (1) slight swelling, (2) mild swelling, (3) moderate swelling and (4) severe swelling. A clinical score was calculated by adding each limb score. The maximum possible clinical score for an animal was (16). Animais with a score equal to (12) on two consecutive occasions and animais with a score greater than (12) on any one occasion were culled. Data were graphed (Mean ± SEM).
Cell prolifération analysis
On Day 21, three satellite animais per group were culled and spleens were dissected out. Spleen cells were cultured for 72 hours in presence or absence of type II Collagen. After 72 hours, cells were pulsed ovemight in the presence of tritiated thymidine. Cell prolifération was quantifïed by measuring thymidine incorporation. Data were graphed (Mean ± SEM). Supernatants were taken and tested for the presence of key cytokines.
Cytokine analysis
Terminal supernatants from the spleen cell cultures were tested in order to quantitate TNF-α, IL-6,
IFN-γ, IL-4, IL-10 and IL-17 by Luminex. Data were graphed (Mean ± SEM).
Microbiological analysis
On Day -14, Day 0 and Day 45, faecal samples were collected from each animal, immediately snapfrozen, and stored at -80°C. Caeca (including content) were immediately snap-frozen and stored 5 at -80°C. A bacterial identification test was performed daily by plating the bacteria.
Histopathology
At the end of the experiment, hind paws were stored in tissue fixative. Samples were transferred into décalcification solution. Tissue samples were processed, sectioned and stained with Haematoxylin & Eosin. Sections were scored by a qualified histopathologist, blind to the experimental design, for 10 signs of arthritis to include inflammation, articular cartilage damage and damage to the underlying metaphyseal bone. A detailed scoring System was used (see below). Data were graphed (Mean ± SEM). Raw and analysed data were provided as well as représentative pictures.
Table 1: Histopathology Scoring System
Grade Description
Inflammation
Normal joint
Mild synovial hyperplasia with inflammation dominated by neutrophils. Low numbers of neutrophils and macrophages in joint space.
Synovial hyperplasia with moderate to marked inflammation involving both neutrophils and macrophages. Neutrophils and macrophages in joint space; may be some necrotic tissue débris.
Synovial hyperplasia with marked inflammation involving both neutrophils and macrophages. Loss of synoviocyte lining. Inflammation may extend from synovium to surrounding tissue including muscle. Numerous neutrophils and macrophages in joint space, together with significant necrotic tissue débris.
Articular cartilage damage
Normal joint
Articular cartilage shows only mild degenerative change. Early pannus formation may be présent peripherally.
Articular cartilage shows moderate degenerative change and focal loss.
Pannus formation is présent focally.
Significant disruption and loss of articular cartilage with extensive pannus formation.
Damage to the underlying metaphyseal bone
Normal joint
No change to underlying metaphyseal bone.
May be focal necrosis or fibrosis of metaphyseal bone.
Disruption or collapse of metaphyseal bone. Extensive inflammation, necrosis or fibrosis extending to medullary space of the metaphysis.
Results and analysis
Survival and Non-specific Clinical Observations
Some animais were culled prior to the scheduled end of the study due to the severity of the clinical signs of arthritis or due to the severity of the non-specific clinical observations.
One animal in Group 1 was culled (vehicle-treated, animal arrived from the supplier with broken leg).
Eleven animais were culled due to the severity of the clinical signs of arthritis: five animais in Group 1 (vehicle-treated), and six animais in Group 4 (biotherapeutic #675-treated).
Five animais were culled due to the severity of the non-specific clinical signs including abnormal posture (hunched), abnormal coat condition (piloerection), abnormal activity levels (reduced activity): three animais in Group 1 (vehicle-treated) and two animais in Group 4 (biotherapeutic #675-treated).
Bodyweights
Bodyweight data recorded from Day -14 until Day 0 and expressed as a percentage of the initial (Day -14) bodyweights were analysed by two-way ANOVA followed by Dunnett’s post-test for multiple comparisons with Day -14 then for multiple comparison with the vehicle-treated group. The data are presented in Figure 19. Data from animais culled prior to the scheduled end of the experiment were excluded from the analyses.
When compared to Day -14, twice daily administrations by oral gavage induced a significant bodyweight loss in the vehicle-treated group on Day -9 and Day -7.
Group 4 (untreated until Day 0 then biotherapeutic #675-treated) bodyweights were significantly higher than in the vehicle-treated group from Day -11 until Day -1 (p < 0.0001 except Day -4 where p < 0.05).
The bodyweights measured between Day -14 and Day -1 in the biotherapeutic-treated groups did not differ from the bodyweights measured in the vehicle-treated group on any given day.
Bodyweight data recorded from Day 0 until Day 28 and expressed as a percentage of the initial (Day
0) bodyweights were analysed by two-way ANOVA followed by Dunnett’s post-test for multiple comparisons with Day 0 in the Vehicle group then for multiple comparison with the vehicle-treated group. The data are presented in Figure 20. Data from animais culled prior to the scheduled end of the experiment and from Satellite animais were excluded from the analyses. Day 28, Day 35 and Day 42 data were further analysed by one-way ANOVA followed by Dunnett’s post-test for multiple comparisons to the vehicle-treated group.
The onset of clinical signs of arthritis was associated with a signifîcant bodyweight loss on Day 26 and Day 28 (p < 0.0001) when compared to Day 0 in the vehicle-treated group.
When compared to the vehicle-treated group, the bodyweights were signifïcantly higher in Group 4 (biotherapeutic #675-treated) on Day 3, Day 5, Day 10 (p < 0.05) and on Day 26 (p < 0.001).
When analysing the data by one-way ANOVA, the bodyweights were signifïcantly higher in Group 4 (biotherapeutic #675-treated) when compared to the vehicle-treated group on Day 28 (p < 0.01). There was no signifîcant différence between experimental groups on Day 35 or Day 42.
Clinical Observations
Clinical score data were analysed by two-way ANOVA followed by Dunnett’s post-test for multiple comparisons between days in the vehicle-treated group then for multiple comparisons between experimental groups and the vehicle-treated group each day. The data are presented in Figure 21. Data recorded from animais culled prior to the end of the experiment were excluded from the analysis. When animais were culled due to the severity of the clinical signs of arthritis, the last recorded score was reported for the following days and used in the statistical analyses.
A signifîcant increase of the clinical scores was observed in the vehicle-treated group from Day 28 until Day 45 (p < 0.0001) when compared to Day 21.
Biotherapeutic #675 did not reduce the clinical scores when compared to the vehicle-treated group. Animais in this group were immunised at a different time when compared to other experimental groups, which may explain the higher clinical scores observed.
Cell prolifération analysis
To validate the assay, splénocytes were cultured in the presence of soluble anti-CD3 and anti-CD28 (anti-CD3/CD28) as positive control stimuli to confirm the proliférative potential of the cells.
Strong proliférative responses to anti-CD3/CD28 were seen in ail experimental groups, showing cells were healthy, viable and able to respond to activation signais.
To test the proliférative response in presence of Collagen II (CII), splénocytes were cultured in the presence of Cil at 50 pg/ml. Splénocyte proliférative response to Cil were analysed by two-way ANOVA followed by Sydak’s post-test for multiple comparisons between unstimulated and CIIstimulated splénocytes and one-way ANOVA followed by Dunnett’s post-test for comparison of CIIstimulated response in different experimental groups with the vehicle-treated group. The data are presented in Figure 22.
Cil induced a highly significant increase of 3H-thymidine incorporation (cpm) when compared to the unstimulated splénocytes in the vehicle-treated group (p < 0.0001).
Splénocyte prolifération for group treated with biotherapeutic #675 was set up on a different day, therefore comparison with the vehicle-treated group was not preformed, although a notable réduction was observed.
Cytokine levels in tissue culture supematants
Levels of each cytokine were measured in tissue culture supematants derived from anti-CD3/CD28 stimulated cultures by luminex analysis. These showed robust responses for ail cytokines measured (mean levels in vehicle group were as follows: IL-4 = 6,406 pg/ml; IL-6 = 306 pg/ml; IL-10 = 10,987 pg/ml; IL-17A = 11,447 pg/ml; IFN-γ = 15,581 pg/ml; TNF-α = 76 pg/ml).
The following sections summarise the data obtained from the Collagen II-stimulated cultures. Where applicable, statistical analyses of the différences between cytokine levels in supematants of unstimulated and ClI-stimulated splénocytes were conducted using two-way ANOVA followed by Sidak’s post-test for multiple comparisons, while one-way ANOVA followed by Dunnett’s post-test was used for comparison of ClI-stimulated response in biotherapeutic-treated groups with the vehicle-treated group. There was no significant différence in cytokine levels between the groups in both cases. This is likely due to the small sample size used (n=3).
In order to more accurately présent the distribution of the data for the cytokines with substantial spread of the data, these are presented as scatter plots.
The group means of IL-4 in tissue culture supematants after stimulation with CH were <5pg/ml. These are not considered biologically significant and not included here. The group means of TNF-a in tissue culture supematants after stimulation with collagen were below limit of quantitation.
Supernatant levels of IFN-γ (Figure 23)
Along with IL-17, IFN-γ is the major cytokine driving disease in the CIA model. The scatter plot in Figure 23 demonstrates IFN-γ levels after CII stimulation, with group médian being higher for the Vehicle-treated group compared to the biotherapeutic (see Figure 27).
Supernatant levels of IL-17A (Figure 24)
Levels of IL-17A were 50pg/ml in ClI-stimulated cultures for the Vehicle-treated group. The levels of this cytokine appeared to be lower in the biotherapeutic group compared to the Vehicle-treated group (see Figure 27).
Supernatant levels of IL-10 (Figure 25)
Levels of IL-10 in Vehicle-treated group were 13 pg/ml and 2.1 pg/ml for ClI-stimulated, and media control cultures, respectively. Higher levels of IL-10 (which is an anti-inflammatory cytokine) for the vehicle-treated group may be expected because inflammation and pro-inflammatory cytokine induction could be accompanied by an anti-inflammatory feedback mechanism.
Supernatant levels of IL-6 (Figure 26)
Inflammatory cytokines such as IL-6 and TNF-α are not typically produced at high levels in anti-CII cultures. However, their levels may be altered as a resuit of immune modulation. Levels of IL-6 in ClI-stimulated cultures were modest, reaching lOpg/ml. Although higher than in media control cultures, these différences were too small to provide rationale for performing statistical analyses.
Tissue culture supernatants in Group 4 - Biotherapeutic #675 (Figure 27)
Splénocyte cultures for this group were set up on a different day and are therefore separate from the Vehicle-treated group. Although direct comparisons may not be appropriate, it appears that treatment with Biotherapeutic #675 was effective at lowering IFN-γ, IL-17A and IL-6 levels.
Microbiological analysis
Bacterial growth was confirmed by measuring the optical density at 600 nm using a spectrophotometer. Bacterial identity was confirmed by comparing streaked plate pictures to reference pictures.
Following the improved bacterial préparation method, consistently high doses of bacterial strain were administered from Day -2 and Day -3 (except for strain #675 from Day 0) as indicated by the high OD values measured.
Faecal samples were collected and snap-frozen on Day -14, Day 0 and at termination.
Histopathology
The histopathology results are shown in Figures 66-71. As expected for this model, intra-individual and inter-individual variability was observed in terms of the presence/absence of arthritis or the severity of change present. '
The nature of the pathology was as expected for this model, with extensive mixed chronic-active inflammation of the synovium and bursa extending to involve the peri-articular soft tissues (muscle, adipose tissue, dermal collagen). In the most severely affected joints there was articular cartilage degeneration and loss with intra-articular débris and inflammation and disruption of the joint and bone structure by fîbrosis and inflammation.
The incidence of histopathological changes was: vehicle - 80% (16/20); Biotherapeutic #675 83% (20/24). Biotherapeutic #675 was effective for reducing histopathological damage observed in hind limb joints and reducing joint inflammation scores, cartilage damage scores, bone damage scores and total histopathology scores (see Figure 70), although statistical comparisons with the vehicle group could not be performed.
Summary
Increased clinical scores were observed from Day 28 after the first administration of type II collagen, as expected in this model of arthritis in DBA/1 mice. Biotherapeutic #675 was shown to be effective at treating arthritis in this model. Animais treated with biotherapeutic #675 were immunised at a different time when compared to the vehicle-treated group, which may explain the higher clinical scores observed. However, biotherapeutic #675 was effective for reducing pathological disease in the joints, as demonstrated in the histopathological analysis.
Proliférative recall responses to Collagen II were seen in splénocyte cultures from ail experimental groups. Statistics were not performed for cultures of biotherapeutic #675, as these were established at a different time, but a réduction in collagen-specific response relative to the control was observed.
Most of the T cell cytokines tested showed détectable increases between Collagen II-stimulated and media Controls in the Vehicle-treated group. These increases were not as obvions in the biotherapeutic-treated group. This broadly supports the proliférative recall responses to Collagen II described above.
There was evidence of suppression of the Thl/Thl 7 axis, which is the pathogenic response in this model and in human RA. Corrélation of reduced levels of cytokines with reduced prolifération is suggestive of immune modulation. There was no evidence that this modulation resulted either from enhanced levels of Th2 associated IL-4 or with increases in the immune modulating cytokine, IL-10.
Example 4 — Further analysis of the effect of bacterial inocula in the mouse model of house dust mite-induced asthma
The mice tested in Example 1 were subjected to further analyses to further characterise the effect of the compositions of the invention on the allergie asthma inflammatory response.
Materials and methods
Blood withdrawal and sérum préparation on day 14. Blood samples of animais were collected via cardiac puncture. Sérum was isolated from the blood sample by centrifugation for 5 min at 14000g and stored at -20 °C.
Organ removal on day 14. Collection of the left lung lobe in formalin for follow-on histological analysis. Collection of the right lung lobes (ail remaining lobes) and removal of sérum for snap freezing and follow-on analysis. Remaining BAL fluid was snap frozen for follow-on analysis.
Measurement of antibody levels in sérum and BAL fluid
Total IgE and house-dust-mite (HDM) spécifie IgGl antibody production were measured in the BAL and sérum by ELISA assay.
Isolation of lung and histological analysis
Left lung lobes were fixed in formalin followed by embedment in paraffin, sectioning, and staining with hematoxylin and eosin and PAS. Subséquent histological scoring was performed blinded as followed: Five random fields of view per sample were scored for inflammation (peribronchial infiltration and perivascular infiltration) and mucus production. Inflammatory infiltration was scored with the following grading system:
- normal
- mild inflammatory infiltrâtes
- moderate inflammatory infiltrâtes
- marked inflammatory infiltrâtes
- severe inflammatory infiltrâtes
- very severe inflammatory infiltrâtes
In each field of view, airways were measured in size and mucus cell numbers were quantified/ um.
Measurement of inflammatory mediators in lung tissue
Right lung lobes (ail remaining lobes) isolated for quantification of inflammatory mediators were snap frozen for subséquent measurement of CCL11, IFN-gamma, IL-1 alpha, IL-1 beta, IL-4, IL-5, IL-9, IL-17A, CXCL1, CCL3, CXCL2 and CCL5 by commercially available multiplex assay (Merck-Millipore). Analysis was performed according to the manufacturer’s instructions.
Results and analysis
The results of the experiments are shown in Figures 28-46.
In support of the findings described in Example 1, analysis of the cellular infiltrâtes in the lung tissue of mice treated with strain 675 showed a notable and statistically signifîcant réduction in mean inflammation score (see Figures 32 and 34).
Antibody levels in the BAL fluid and sérum were analysed (see Figures 28-31). No clear effect of the bacterial treatment on sérum antibody levels was observed. This may reflect a failure in the experiment, because the spread of data and the error bars for each treatment are large, and the positive and négative Controls do not appear to hâve behaved as would be expected. Also, the baseline sérum antibody levels could hâve masked any changes.
Similarly, no clear effect of the bacterial treatment on cytokine levels in lung tissue was observed (see Figures 36-46). Again, this may reflect a failure in the experiment, because the spread of data and the error bars for each treatment are large, and the positive and négative Controls do not appear to hâve behaved as would be expected. It is also possible that the mechanism of action involved influences earlier cytokine responses that were no longer détectable on day 4 post the final HDM airway challenge. Some care should be taken when interpreting the cytokine data in the current study, due to the variability in the levels detected. This variability could in part be explained by the fact that the lung tissue was separated for the different analyses, and thus one lung lobe might not hâve been fully représentative or comparable to the same lobe in other mice due to patchy distribution of the inflammation.
Example 5 — Further analysis of the effect of bacterial inocula in the mouse model of severe neutrophilie asthma
The mice tested in Example 2 were subjected to further analyses to further characterise the effect of the compositions of the invention on the neutrophilie response associated with severe asthma.
Materials and methods
Organ removal on day 18. Collection of the left lung lobe in formalin for follow-on histological analysis. Collection of the right lung lobes (ail remaining lobes) and removal of sérum for snap freezing and follow-on analysis. Remaining BAL fluid was snap frozen for follow-on analysis.
Measurement of inflammatory mediators in lung tissue (follow-on analysis). Right lung lobes (ail remaining lobes) isolated for quantification of inflammatory mediators were snap frozen for subséquent measurement of IFN-gamma, IL-1 alpha, IL-1 beta, CXCL1, CCL3, CXCL2, CCL5, IL17A, TNF-alpha, IL-17F, IL-23 and IL-33 by commercially available multiplex assay (MerckMillipore). Analysis was performed according to the manufacturer’s instructions.
Measurement of antibody levels in sérum and BAL fluid (follow-on analysis). House-dust-mite (HDM) spécifie IgGl and IgG2a antibody production were measured in the BAL and sérum by ELISA assay.
Isolation of lung and histological analysis (follow-on analysis). Left lung lobes were fixed in formalin followed by embedment in paraffin, sectioning, and staining with hematoxylin and eosin and PAS. Subséquent histological scoring was performed blinded as followed: Five random fields of view per sample were scored for inflammation (peribronchial infiltration and perivascular infiltration) and mucus production. Inflammatory infiltration was scored with the following grading System:
- normal
- mild inflammatory infiltrâtes
- moderate inflammatory infiltrâtes
- marked inflammatory infiltrâtes
- severe inflammatory infiltrâtes
- very severe inflammatory infiltrâtes
Results and analysis
The results of the experiments are shown in Figures 47-64.
Further analysis of antibody levels revealed that the efficacy of bacterial strain 675 was also reflected in reduced HDM-specific IgGl levels in the BAL fluid and sérum (see Figures 47 and 49). Firm conclusions regarding an effect on IgG2a levels cannot be drawn. Overall, the data from the antibody analysis is suggestive of a réduction related to an overall reduced inflammatory response, as opposed to a sélective effect on antibody isotype switching.
Histological analysis supported the differential cell counts from the BAL fluid, showing a reduced cellular infiltrate in mice treated with Strain 675 (see Figures 51-53).
In relation to cytokine levels, as for Example 4, the spread of data and the error bars for each treatment are large, and the positive and négative Controls do not appear to hâve behaved as necessarily would be expected. It is also possible that the mechanism of action involves influencing earlier cytokine responses that were no longer détectable on day 4 post the final HDM airway challenge. Some care should be taken when interpreting the cytokine data in the current study, due to the variability in the levels detected. This variability could in part be explained by the fact that the lung tissue was separated for the different anidyses, and thus one lung lobe might not hâve been fully représentative or comparable to the same lobe in other mice due to patchy distribution of the inflammation. Despite this variability, a clear anti-inflammatory effect on cytokine levels for strain 675 was shown, and the positive control anti-IL-17 Ab generally behaved as expected.
With the above caveats, the data in Figures 56, 58, 59, 61 and 63 suggest that treatment with the bacterial strains of the invention may achieve a réduction in the levels of IL-lb, IFNy, RANTES, ΜΙΡ-la and KC (the mouse orthologue of human IL-8), which may be indicative of a mechanism of action related to influences on chemokine release (and thus recruitment of cells) by stromal or innate immune cells. These cytokines are part of the Thl7 pathway. Taking this dataset together, a clear conclusion can be drawn that Strain 675 was highly effective at protecting mice against inflammation in this mouse model of severe neutrophilie asthma.
Example 6 — Efficacy of bacterial inocula in a mouse model of multiple sclerosis
Summary
Mice were administered with compositions comprising bacterial strains according to the invention and the mice were subsequently immunised with myelin oligodendrocyte glycoprotein to induce experimental autoimmune encephalomyelitis (EAE). EAE is the most commonly used experimental model for human multiple sclerosis. The compositions of the invention were found to hâve a striking effect on disease incidence and disease severity.
Strain
675: bacteria deposited under accession number NCIMB 42408
Study design
Groups:
1. Négative control group. Treatment with vehicle control (per oral).
3. Treatment with therapeutic bacteria inoculum strain 675 (per oral).
9. Positive control group. Treatment with Dexamethasone (i.p.).
10. Untreated Control Group.
Number of mice per group =10
Days -14 to day 27: Daily administration of vehicle control per oral (Group 1).
Days -14 to day 27: Daily administration of therapeutic bacteria inoculum per oral (Group 4).
Days 0-28: administration of Dexamethasone (i.p.) three times a week (Group 9)
Day 0: MOG35-55 (myelin oligodendrocyte glycoprotein - 2mg/ml) and CFA (2mg/ml MTB) were mixed 1:1 resulting in Img/ml solutions. 100μΙ of the peptide-CFA mixture was injected subcutaneously into each hind leg. Administration of pertussis toxin intraperitoneally (300ng).
Day 1 : Administration of pertussis toxin intraperitoneally (300ng).
Days 7-onwards: Measurement of disease incidence and weight three times a week.
Endpoints and analysis
Mice were analysed for disease incidence and disease severity three times a week. Scoring was performed blind. Disease severity was assessed using a clinical score ranging from 0 to 5, with 5 indicating a dead mouse (see clinical scoring System below).
Monitoring
On the indicated days mice were weighed and observed for disease activity score and disease incidence.
Disease activity score observations:
- No obvious changes in motor function compared to non-immunized mice.
0.5 - Tip of tail is limp.
1.0- Limp tail.
1.5- Limp tail and hind leg inhibition.
2.0- Limp tail and weakness of hind legs.
OR - There are obvious signs of head tilting when the walk is observed. The balance is poor.
2.5 - Limp tail and dragging of hind legs.
- OR - There is a strong head tilt that causes the mouse to occasionally fall over.
3.0- Limp tail and complété paralysis of hind legs.
3.5 - Limp tail and complété paralysis of hind legs.
In addition to: Mouse is moving around the cage, but when placed on its side, is unable to right itself.
Hind legs are together on one side of body.
4.0 - Limp tail, complété hind leg and partial front leg paralysis.
- Mouse is minimally moving around the cage but appears alert and feeding
4.5 - Complété hind and partial front leg paralysis, no moventent around the cage.
Mouse is immediately euthanized and removed from cage.
5.0 Mouse is euthanized due to severe paralysis.
When an animal has equal or greater disease activity score of 1, it is considered to hâve a positive disease incidence score.
Results
The results of the study are shown in Figures 72 and 73.
Disease induction in the négative control groups was successiul with high scores shown by the vehicle control and the untreated cnnimi + c control group. These data tndleate the strain 675 may le ZZZ ‘° sclerosis. Y ™ f trcat]në or preventing multiple
Example 7—Stability testing
A composition described herem contain.ng m sealed container at 7ST nr λ'γ^ * - » * u red in a
Sequences
SEQ ID NO:1 (Bacteroides coprocola gene for 16S rRNA, partial sequence, strain: Mil AB200223)
* agagttcgat cctggcticag gai gaacgct agctacaggc ttaacacatg caagtcgagg
61 ggcagcatga àcttagcttg c t a a g t t τ g a tggcgaccgg cgcacgggrg agtaacacgt
121 atccaacctt ccgtttactc agggatagcc tttcgaaaga aagattaata cctgatagta
1S1 tggtgagatt gcatgatggc accatcaaag atttattggt aaacgatggg gatgcgttcc
241 attaggtagt aggcggggta acggcccacc tagcctgcga tggatagggg ttctgagagg
4 : a g 2 O C C C C O atëtTggaac rt fri il> il' I) Iri Ci 0 “ccaaactcc tâcgggaggc agcagtgagg
3 61 ΐ = t ê 1122LC aa „gggcgag ~ — * ? a s-- agccaagcag cg^gaaggat g a a g gtecta
421 cggattgtaa actcctttta tacgggaata aagtttccta cgtgtaggat tttgtatgta
4 ? Σ fil in <Q ύ >1' r 1 Q <2 O 0’ 1 J 0' u 0 m u i0 ιϋ i » ccagcagccg cggtaa~acg gaggatgega
- * -
- - — ------. _j«j = g^ _ a a g w = 3_> L· ·_ g g ô
621 aagtttgcgg ctcaaccgta il’ il) il) rt O CU i î Qi LT |J u IU u IU ty jj rl O O rt rt tQ Pi tQ rl gcagttgagg
€ € 1 c a□c c ce a a t g c g g _g a e_ 2 C ~ L g 3 z _ Ξ Σ cacgaagaac cccgattgcg
- ê 5 cctaaactcit aac:aac27 aacgcczcgaa agigrgggta tcaaacagga
r 2. TT ë g ë t âCCC ri aj il) ri Cri UJ O Ω Cl» cacggtaaac gatggatact cgctgttggc garataetgt
841 cagcggccaa gcgaaagcat taagtatccc acctggggag tacgccggca acggtgaaac
901 tcaaaggaat tgacgggggc ccgcacaagc ggaggaacat gtggtttaat tcgatgatac
961 gcgaggaacc ttacccgggc ttaaattgca gacgaattac gaggaaactt gtaagccgca
1021 aggcgtctgc gaaggtgctg cacggttgtc gtcagctcgt gccgtgaggt gtcggcttaa
1081 gtgccataac gagcgcaacc ctcgtggtca gttactaaca ggttaagctg agggctctgg
1141 ccagactgcc atcgtaagat gtgaggaagg tggggatgac gtcaaatcag cacggccctt
1201 acgtccgggg ctacacacgt gttacaatgg gaggtacaga aggccgctac ccggcaacgg
1261 gatgccaatc cccaaaacct ctctcagttc ggactggagt ctgcaacccg actccacgaa
1321 gctggattcg ctagtaatcg cgcatcagcc acggcgcggt gaatacgttc ccgggccttg
1381 tacacaccgc ccgtcaagcc atgaaagccg ggggtacctg aagtgcgtaa ccgcaaggag
1441 cgccctaggg taaaaccggt aattggggct aagtctaaca aggtaaccaa g SEQ ID NO:2 (Bacteroides coprocola gene for 16S rRNA, partial sequence, strain: Ml6 AB200224) agagtttgat cctggctcag gatgaacgct agctacaggc ttaacacatg caagtcgagg ggcagcatga acttagcttg ctaagtttga tggcgaccgg cgcacgggtg agtaacacgt
121 atccaacctt ccgtttactc agggatagcc tttcgaaaga aagattaata cctgatagta
181 tggtgagatt gcatgatggc accattaaag atttattggt aaacgatggg gatgcgttcc
241 attaggtagt aggcggggta acggcccacc tagcctgcga tggatagggg ttctgagagg
301 aaggtccccc acattggaac tgagacacgg tccaaactcc tacgggaggc agcagtgagg
361 aatattggtc aatgggcgag agcctgaacc agccaagtag cgtgaaggat gaaggtccta
421 cggattgtaa acttctttta tacgggaata aagtttccta cgtgtaggat tttgtatgta
481 ccgtatgaat aagcatcggc taactccgtg ccagcagccg cggtaatacg gaggatgcga
541 gcgttatccg gatttattgg gtttaaaggg agcgcagacg ggagattaag tcagttgtga
601 aagtttgcgg ctcaaccgta aaattgcagt tgatactggt ttccttgagt gcagttgagg
661 caggcggaat tcgtggtgta gcggtgaaat gcttagatat cacgaagaac cccgattgcg
721 aaggcagctt gctaaactgt aactgacgtt catgctcgaa agtgtgggta tcaaacagga
781 ttagataccc tggtagtcca cacggtaaac gatggatact cgctgttggc gatatactgt
841 cagcggccaa gcgaaagcat taagtatccc acctggggag tacgccggca acggtgaaac
901 tcaaaggaat tgacgggggc ccgcacaagc ggaggaacat gtggtttaat tcgatgatac
961 gcgaggaacc ttacccgggc ttaaattgca gacgaattac gaggaaactt gtaagccgca
1021 aggcgtctgt gaaggtgctg catggttgtc gtcagctcgt gccgtgaggt gtcggcttaa
1081 gtgccataac gagcgcaacc ctcgtggtca gttactaaca ggttaagctg agggctctgg
1141 ccagactgcc atcgtaagat gtgaggaagg tggggatgac gtcaaatcag cacggccctt
1201 acgtccgggg ctacacacgt gttacaatgg gaggtacaga aggccgctac ccggcaacgg
1261 gatgccaatc cccaaaacct ctctcagttc ggactggagt ctgcaacccg actccacgaa
1321 gctggattcg ctagtaatcg cgcatcagcc acggcgcggt gaatacgttc ccgggccttg
1381 tacacaccgc ccgtcaagcc atgaaagccg ggggtacctg aagtgcgtaa ccgcaaggag
1441 cgccctaggg taaaaccggt aattggggct aagtctaaca aggtaaccaa
SEQ ID NO:3 (Bacteroides coprocola gene for 16S rRNA, partial sequence, strain: Ml58 AB200225) agagtttgat cctggctcag gatgaacgct agctacaggc ttaacacatg caagtcgagg ggcagcatga acttagcttg ctaagtttga tggcgaccgg cgcacgggtg agtaacacgt 121 atccaacctt ccgtttactc agggatagcc tttcgaaaga aagattaata cctgatagta 181 tggtgagatt gcatgatagc accattaaag atttattggt aaacgatggg gatgcgttcc 241 attaggtagt aggcggggta acggcccacc tagcctncga tggatagggg ttctgagagg 301 aaggtccccc acattggaac tgagacacgg tccaaactcc tacgggaggc agcagtgagg 361 aatattggtc aatgggcgag agcctgaacc agccaagtag cgtgaaggat gaaggtccta 421 cggattgtaa acttctttta tacgggaata aagtatccta cgtgtaggat tttgtatgta 481 ccgtatgaat aagcatcggc taactccgtg ccagcagccg cggtaatacg gaggatgcga 541 gcgttatccg gatttattgg gtttaaaggg agcgcagacg ggagattaag tcagttgtga
601 aagtttgcgg ctcaaccgta aaattgcagt tgatactggt ttccttgagt gcagttgagg
661 caggcggaat tcgtggtgta gcggtgaaat gcttagatat cacgaagaac cccgattgcg
721 aaggcagctt gctaaactgt aactgacgtt catgctcgaa agtgtgggta tcaaacagga
781 ttagataccc tggtagtcca cacggtaaac gatggatact cgctgttggc gatatactgt
841 cagcggccaa gcgaaagcat taagtatccc acctggggag tacgccggca acggtgaaac
901 tcaaaggaat tgacgggggc ccgcacaagc ggaggaacat gtggtttaat tcgatgatac
961 gcgaggaacc ttacccgggc ttaaattgca gacgaattac gaggaaactt gtaagccgca
1021 aggcgtctgt gaaggtgctg catggttgtc gtcagctcgt gccgtgaggt gtcggcttaa
1081 gtgccataac gagcgcaacc ctcgtggtca gttactaaca ggttaagctg aggactctgg
1141 ccagactgcc atcgtaagat gtgaggaagg tggggatgac gtcaaatcag cacggccctt
1201 acgtccgggg ctacacacgt gttacaatgg gaggtacaga aggcagctac ccggcgacgg
1261 gatgccaatc cccaaaacct ctctcagttc ggactggagt ctgcaacccg actccacgaa
1321 gctggattcg ctagtaatcg cgcatcagcc acggcgcggt gaatacgttc ccgggccttg
1381 tacacaccgc ccgtcaagcc atgaaagccg ggggtacctg aagtgcgtaa ccgcaaggag
1441 cgccctaggg taaaaccggt aattggggct aagtcgtaac aaggtaacca a
SEQ ID NO:4 (consensus 16S rRNA sequence for Bacteroides coprocola strain 675)
GTCTGGCTCAKGATGAACGCTAGCTACAGGCTTAACACATGCAAGTCGAGGGGCAGCATGAACTTAGCTTGCTAAGT TTGATGGCGACCGGCGCACGGGTGAGTAACACGTATCCAACCTCCCGCTTACTCAGGAATAGCCTTTCGAAAGAAAG ATTAATGCCTGATGGTATCTTAAGCACACATGTAATTAAGATTAAAGATTTATCGGTAAGCGATGGGGATGCGTTCC ATTAGGTAGTAGGCGGGGTAACGGCCCACCTAGCCGACGATGGATAGGGGTTCTGAGAGGAAGGTCCCCCACATTGG AACTGAGACACGGTCCAAACTCCTACGGGAGGCAGCAGTGAGGAATATTGGTCAATGGGCGCGAGCCTGAACCAGCC AAGTAGCGTGAAGGATGAAGGTCCTATGGATTGTAAACTTCTTTTATACGGGAATAAAGTGGTCCACGTGTGGGCCT TTGCATGTACCGTATGAATAAGCATCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGATGCGAGCGTTATC CGGATTTATTGGGTTTAAAGGGAGCGCAGACGGGGGATTAAGTCAGTTGTGAAAGTTTGCGGCTCAACCGTAAAATT GCAGTTGATACTGGTTCCCTTGAGTGCAGTTGAGGCAGGCGGAATTCGTGGTGTAGCGGTGAAATGCATAGATATCA CGAAGAACCCCGATTGCGAAGGCAGCCTGCTAAGCTGTAACTGACGTTGAGGCTCGAAAGTGTGGGTATCAAACAGG ATTAGATACCCTGGTAGTCCACACGGTAAACGATGGATACTCGCTGTTGGCGATATACTGTCAGCGGCCAAGCGAAA GCATTAAGTATCCCACCTGGGGAGTACGCCGGCAACGGTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGA GGAACATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCCGGGCTTAAATTGCAGACGAATTACTTGGAAACAG GTAAGCCGCAAGGCGTCTGTGAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAGGTGTCGGCTTAAGTGCCAT AACGAGCGCAACCCTCGTGGCCAGTTACTAGCAGGTAACGCTGAGGACTCTGGCCAGACTGCCATCGTAAGATGCGA GGAAGGTGGGGATGACGTCAAATCAGCACGGCCCTTACGTCCGGGGCTACACACGTGTTACAATGGGAGGTACAGAA GGCAGCTACCCGGCGACGGGATGCCAATCTCCAAAGCCTCTCTCAGTTCGGACTGGAGTCTGCAACCCGACTCCACG AAGCTGGATTCGCTAGTAATCGCGCATCAGCCACGGCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC AAGCCATGAAAGCCGGGAGTACCTGAAGTGCGTAACCGCAAGGAGCGCCCTAGGGTAAAACCGGTAATTGGGGCTAA GTCNTACGGGG
SEQ ID NO:5 (Bacteroides thetaiotaomicron gene for 16S rRNA, partial sequence - M58763) cttntacaat gaagagtttg atcctggctc aggatnaacg ctagctacag gcttaacaca tgcaagtcna ggggcagcat ttcagtttgc ttgcaaactg gagatggcga ccggcgcacg 121 ggtgagtaac acgtatccaa cctgccgata actcggggat agcctttcga aagaaagatt 181 aatacçcnat ggtataatca gaccgcatng tcttrttatt aaagaatttc ggttatcgat 241 ggggatgcgt tccattaggc agttggtgag gtaacggctc acnnaacctt cgatggatag 301 gggttctgag aggaaggtcc cccacattgg aactgagaca cggtccaaac tcctacggga 361 ggcagcagtg aggaatattg gtcaatgggc gcaggcctga accagccaag tagcgtgaag 421 gatgactgcc ctatgggttg taaacttctt ttatatggga ataaagtttt ccacgtgtgg
-$8
481 aattttgtat gtaccatatg aataaggatc ggctaactcc gtgccagcag 541 acggagnatc cgagcgttat ccggatttat tgggtttaaa gggagcgtag 601 aagtcagttg tgaaagtttg cggctcaacc gtaaaattgc agttgàtact 661 agtacagtag aggtgggcgg aattcgtggt gtagcggtga aatgcttaga 721 aactccgatt gcgaaggcag ctcactggac tgcaactgac actgatgctc 781 gtatcaaaca ggattagatà ccctggtagt ccacacagta aacgatgaat 841 tgcgatatac agtaagcggc caagcgaaag cattaagtat tccacctggg 901 gcaacggtga aactcaaagg aattgacggg ggccngcaca agcggaggaa 961 aattcgatga tacgcgagga accttacccg ggcttaaatt gcatttgaat 1021 cagtatagcc gyaaggcaaa tgtgaaggtg ctgcatggtt gtcgtcagct 1081 ggtgtcggct taagtgccat aacgagcgca acccttatct ttagttacta 1141 ctgaggactc tagagagact gccgtcgtaa gatgtgagga aggtggggat 1201 cagcacngcc cntacgtccg gggctacaca cgtgttacaa tggggggtac 1261 tacctggtga caggatgcta atcccaaaag cctctctcag ttcggatcga 1321 ccgacttcgt gaagctggat tcgctagtaa tcgcgcatca gccatggcgc 1381 ttcccgggcn ttgtacacac cgcccgtcaa gccatgaaag ccgggggtac 1441 taaccgcaag gagcgtccta gggtaaaact ggtaattggg gc ccncgntnat gtggacagtt ggctgtcttg tatcacgaag gaaagtgtgg actcgctgtt gagtacgccg catgtggttt atattggaaa cgtgccgtga acaggtcatg gacgtcaaat agaaggcagc agtctgcaac ggtgaatacg ctgaagtacg
SEQ ID NO:6 (strain 675 chromosome sequence) - see WO2016203217.
SEQ ID NO:7 (strain 675 plasmid sequence) - see electronic sequence listing.
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Claims (22)

1. A composition comprising a bacterial strain of the genus Bacteroides, for use in a method of treating or preventing an inflammatory or autoimmune disease, or cancer, wherein the bacterial strain has a 16s rRNA sequence that is at least 95% identical to SEQ ID NO:4.
2. The composition of claim 1, wherein the composition is for use in a method of treating or preventing a disease or condition selected from the group consisting of asthma, including allergie asthma or neutrophilie asthma; arthritis, including rheumatoid arthritis, osteoarthritis, psoriatic arthritis, or juvénile idiopathic arthritis; multiple sclerosis; neuromyelitis optica (Devic's disease); ankylosing spondylitis; spondyloarthritis; psoriasis; systemic lupus erythematosus; inflammatory bowel disease, including Crohn’s disease or ulcerative colitis; celiac disease; chronic obstructive pulmonary disease (COPD); cancer, including breast cancer, colon cancer, lung cancer or ovarian cancer; uveitis; scleritis; vasculitîs; Behcet’s disease; atherosclerosis; atopie dermatitis; emphysema; periodontitis; allergie rhinitis; and allograft rejection.
3. The composition of claim 2, wherein the composition is for use in a method of treating or preventing asthma, including neutrophilie asthma or allergie asthma.
4. The composition of claim 3, wherein the composition is for use in a method of reducing neutrophilia or eosinophilia in the treatment of asthma.
5. The composition of claim 2, wherein the composition is for use in a method of treating or preventing rheumatoid arthritis.
6. The composition of claim 5, wherein the composition is for use in a method of reducing joint swelling in rheumatoid arthritis.
7. The composition of claim 2, wherein the composition is for use in a method of treating or preventing multiple sclerosis.
8. The composition of claim 7, wherein the composition is for use in a method of reducing disease incidence or disease severity.
9. The composition of claim 2, wherein the composition is for use in a method of treating or preventing cancer, including lung cancer, breast cancer or liver cancer.
10. The composition of claim 9, wherein the composition is for use in a method of reducing tumour size, reducing tumour growth, preventing metastasis or preventing angiogenesis.
11. The composition of claim 2, wherein the composition is for use in a method of treating or preventing uveitis.
b'i .
12. The composition of claim 11, wherein the composition is for use in a method of reducing or preventing retinal damage in uveitis.
13. The composition of any preceding claim, wherein the composition is for use in a method of reducing IL-17 production or reducing Th 17 cell différentiation in the treatment or prévention of the inflammatory or autoimmune disease, or cancer.
14. The composition of any preceding claim, wherein the composition is for use in a patient with elevated IL-17 levels or Thl7 cells.
15. The composition of any preceding claim, wherein the bacterial strain is of Bacteroides coprocola.
16. The composition of any preceding claim, wherein the bacterial strain has a 16s rRNA sequence that is at least 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:4, or wherein the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO:4.
17. The composition of any preceding claim, wherein the bacterial strain is lyophilised.
18. A food product or vaccine composition comprising the composition of any preceding claim, for the use of any preceding claim.
19. A cell of the strain deposited under accession number NCIMB 42408, or a dérivative thereof.
20. A biologically pure culture of the strain deposited under accession number NCIMB 42408, or a dérivative thereof.
21. A cell of the strain deposited under accession number NCIMB 42408, or a dérivative thereof, for use in therapy.
22. The cell of claim 21, wherein the cell is for use in a method defined in any of daims 1-14.
OA1201700486 2015-06-15 2016-06-15 Compositions comprising bacterial strains OA18687A (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
GB1510470.6 2015-06-15
GB1520510.7 2015-11-20
GB1603786.3 2016-03-04

Publications (1)

Publication Number Publication Date
OA18687A true OA18687A (en) 2019-05-17

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