U.S. patent application number 17/586968 was filed with the patent office on 2022-08-18 for compositions comprising bacterial strains.
The applicant listed for this patent is 4D Pharma Research Limited. Invention is credited to Alex STEVENSON.
Application Number | 20220257668 17/586968 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220257668 |
Kind Code |
A1 |
STEVENSON; Alex |
August 18, 2022 |
COMPOSITIONS COMPRISING BACTERIAL STRAINS
Abstract
The invention provides compositions comprising bacterial strains
for treating and preventing inflammatory and autoimmune
diseases.
Inventors: |
STEVENSON; Alex; (Leeds,
GB) |
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Applicant: |
Name |
City |
State |
Country |
Type |
4D Pharma Research Limited |
Aberdeen |
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GB |
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Appl. No.: |
17/586968 |
Filed: |
January 28, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16597673 |
Oct 9, 2019 |
11273185 |
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17586968 |
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15842635 |
Dec 14, 2017 |
10500237 |
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16597673 |
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PCT/GB2016/051774 |
Jun 15, 2016 |
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15842635 |
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International
Class: |
A61K 35/74 20060101
A61K035/74; C12N 1/20 20060101 C12N001/20; A61P 37/00 20060101
A61P037/00; A61P 11/06 20060101 A61P011/06; A61P 19/02 20060101
A61P019/02; A61K 9/19 20060101 A61K009/19 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 15, 2015 |
GB |
1510468.0 |
Apr 19, 2016 |
GB |
1606807.4 |
Claims
1. A composition comprising a bacterial strain of the genus
Roseburia, for use in a method of treating or preventing a disease
or condition mediated by IL-17 or the Th17 pathway.
2.-29. (canceled)
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 16/597,673, filed Oct. 9, 2019, which is a divisional of U.S.
application Ser. No. 15/842,635, filed Dec. 14, 2017, now U.S. Pat.
No. 10,500,237, issued Dec. 10, 2019, which is a continuation of
International Application No. PCT/GB2016/051774, filed Jun. 15,
2016; which claims the benefit of Great Britain Patent Application
No. 1510468.0, filed Jun. 15, 2015, and Great Britain Patent
Application No. 1606807.4, filed Apr. 19, 2016, all of which are
incorporated by reference herein in their entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 16, 2021, is named 56708_715_303_Sequence_Listing.TXT and
is 4,676,553 bytes in size.
TECHNICAL FIELD
[0003] 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
[0004] The human intestine is thought to be sterile in utero, but
it is exposed to a large variety of maternal 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
genotype, all 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
have yielded a wide variety of metabolic, structural, protective
and other beneficial functions. 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 exemplified in germfree animals which have an impaired
immune system that is functionally reconstituted following the
introduction of commensal bacteria [3-5].
[0005] Dramatic changes in microbiota composition have been
documented in gastrointestinal disorders such as inflammatory bowel
disease (IBD). For example, the levels of Clostridium cluster XIVa
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.
[0006] In recognition of the potential positive effect that certain
bacterial strains may have on the animal gut, various strains have
been proposed for use in the treatment of various diseases (see,
for example, [10-13]). Also, certain strains, including mostly
Lactobacillus and Bifidobacterium strains, have 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 precise effects of particular
bacterial strains on the gut and at a systemic level and on any
particular types of diseases, are poorly characterised.
[0007] 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 therapies using gut bacteria can be
developed.
SUMMARY OF THE INVENTION
[0008] The inventors have developed new therapies for treating and
preventing inflammatory and autoimmune diseases. In particular, the
inventors have developed new therapies for treating and preventing
diseases and conditions mediated by IL-17 or the Th17 pathway. In
particular, the inventors have identified that bacterial strains
from the genus Roseburia can be effective for reducing the Th17
inflammatory response. As described in the examples, oral
administration of compositions comprising Roseburia hominis may
reduce the severity of the inflammatory response, including the
Th17 inflammatory response, in mouse models of asthma, rheumatoid
arthritis and multiple sclerosis.
[0009] Therefore, in a first embodiment, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing a disease or
condition mediated by IL-17 or the Th17 pathway. The inventors have
identified that treatment with bacterial strains from this genus
can reduce levels of cytokines that are part of the Th17 pathway,
including IL-17, can alleviate the Th17 inflammatory response and
can provide clinical benefits in mouse models of inflammatory and
autoimmune diseases mediated by IL-17 and the Th17 pathway.
[0010] In particular embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
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 juvenile 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 allergic asthma or
neutrophilic 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; atopic dermatitis; emphysema; periodontitis;
allergic rhinitis; and allograft rejection. The effect shown for
the bacterial strains from the genus Roseburia on the Th17
inflammatory response may provide therapeutic benefits for diseases
and conditions mediated by IL-17 and the Th17 pathway, such as
those listed above.
[0011] In preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing asthma, such as
neutrophilic asthma or allergic asthma. The inventors have
identified that treatment with Roseburia strains can reduce
recruitment of neutrophils and eosinophils into the lungs, which
can help treat or prevent asthma. Furthermore, the inventors have
tested and demonstrated the efficacy of Roseburia strains in mouse
models of asthma. In certain embodiments, the composition is for
use in a method of treating or preventing neutrophilic asthma or
eosinophilic 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 neutrophilic
asthma and eosinophilic asthma. Indeed, in certain embodiments, the
composition is for use in a method of reducing a neutrophilic
inflammatory response in the treatment or prevention of asthma, or
the composition is for use in a method of reducing an eosinophilic
inflammatory response in the treatment or prevention of asthma. In
preferred embodiments, the invention provides a composition
comprising a bacterial strain of the species Roseburia hominis for
use in the treatment of asthma, and in particular eosinophilic or
allergic asthma. Roseburia hominis is shown to have a particularly
pronounced effect on eosinophils in asthma models and treatment
with Roseburia hominis may be particularly effective for treating
eosinophilic or allergic asthma.
[0012] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing rheumatoid arthritis.
The inventors have identified that treatment with Roseburia 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 Roseburia hominis, for use in the treatment of
rheumatoid arthritis. Compositions using Roseburia hominis may be
particularly effective for treating rheumatoid arthritis.
[0013] In further preferred embodiments, the invention provides a
composition comprising a bacterial strain of the genus Roseburia,
for use in a method of treating or preventing multiple sclerosis.
The inventors have identified that treatment with Roseburia 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
Roseburia hominis, for use in the treatment of multiple sclerosis.
Compositions using Roseburia hominis may be particularly effective
for treating multiple sclerosis.
[0014] In certain embodiments, the compositions of the invention
are for use in a method of reducing IL-17 production or reducing
Th17 cell differentiation in the treatment or prevention of a
disease or condition mediated by IL-17 or the Th17 pathway. In
particular, the compositions of the invention may be used in
reducing IL-17 production or reducing Th17 cell differentiation in
the treatment or prevention of asthma, rheumatoid arthritis or
multiple sclerosis. Preferably, the invention provides compositions
comprising a bacterial strain of the species Roseburia hominis, for
use in reducing IL-17 production or reducing Th17 cell
differentiation in the treatment or prevention of asthma,
rheumatoid arthritis or multiple sclerosis.
[0015] In certain embodiments, the composition is for use in a
patient with elevated IL-17 levels or Th17 cells. The effect on the
Th17 inflammatory response shown for Roseburia strains may be
particularly beneficial for such patients.
[0016] In preferred embodiments of the invention, the bacterial
strain in the composition is of Roseburia hominis. Closely related
strains may also be used, such as bacterial strains that have 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
Roseburia hominis. 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 or 3. Preferably, the sequence identity
is to SEQ ID NO:3. Preferably, the bacterial strain for use in the
invention has the 16s rRNA sequence represented by SEQ ID NO:3.
[0017] 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 Th17
pathway-mediated 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.
[0018] In certain embodiments, the composition of the invention
comprises one or more pharmaceutically acceptable excipients or
carriers.
[0019] 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.
[0020] In certain embodiments, the invention provides a food
product comprising the composition as described above.
[0021] In certain embodiments, the invention provides a vaccine
composition comprising the composition as described above.
[0022] Additionally, the invention provides a method of treating or
preventing a disease or condition mediated by IL-17 or the Th17
pathway, comprising administering a composition comprising a
bacterial strain of the genus Roseburia.
[0023] In developing the above invention, the inventors have
identified and characterised a bacterial strain that is
particularly useful for therapy. The Roseburia hominis 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
Roseburia hominis strain deposited under accession number NCIMB
42383, or a derivative thereof. The invention also provides
compositions comprising such cells, or biologically pure cultures
of such cells. The invention also provides a cell of the Roseburia
hominis strain deposited under accession number NCIMB 42383, or a
derivative thereof, for use in therapy, in particular for the
diseases described herein.
BRIEF DESCRIPTION OF DRAWINGS
[0024] FIG. 1: Mouse model of house dust mite-induced asthma--Total
BAL fluid cell counts.
[0025] FIG. 2: Mouse model of house dust mite-induced asthma--Total
eosinophil count in BALF.
[0026] FIG. 3: Mouse model of house dust mite-induced
asthma--Proportion of eosinophils in BALF.
[0027] FIG. 4: Mouse model of house dust mite-induced asthma--Total
macrophage count in BALF.
[0028] FIG. 5: Mouse model of house dust mite-induced
asthma--Proportion of macrophages in BALF.
[0029] FIG. 6: Mouse model of house dust mite-induced asthma--Total
neutrophil count in BALF.
[0030] FIG. 7: Mouse model of house dust mite-induced
asthma--Proportion of neutrophils in BALF.
[0031] FIG. 8: Mouse model of house dust mite-induced asthma--Total
lymphocyte count in BALF.
[0032] FIG. 9: Mouse model of house dust mite-induced
asthma--Proportion of lymphocytes in BALF.
[0033] FIG. 10: Mouse model of severe neutrophilic asthma--Total
BAL fluid cell counts.
[0034] FIG. 11: Mouse model of severe neutrophilic asthma--Total
eosinophil count in BALF.
[0035] FIG. 12: Mouse model of severe neutrophilic
asthma--Proportion of eosinophils in BALF.
[0036] FIG. 13: Mouse model of severe neutrophilic asthma--Total
macrophage count in BALF.
[0037] FIG. 14: Mouse model of severe neutrophilic
asthma--Proportion of macrophages in BALF.
[0038] FIG. 15: Mouse model of severe neutrophilic asthma--Total
neutrophil count in BALF.
[0039] FIG. 16: Mouse model of severe neutrophilic
asthma--Proportion of neutrophils in BALF.
[0040] FIG. 17: Mouse model of severe neutrophilic asthma--Total
lymphocyte count in BALF.
[0041] FIG. 18: Mouse model of severe neutrophilic
asthma--Proportion of lymphocytes in BALF.
[0042] FIG. 19: Mouse model of rheumatoid arthritis--Bodyweights,
days -14 to 0. Data are presented as Mean.+-.SEM percentages of the
initial (Day -14) bodyweights.
[0043] FIG. 20: Mouse model of rheumatoid arthritis--Bodyweights,
days 0 to 42. Data are presented as Mean.+-.SEM percentages of the
initial (Day 0) bodyweights.
[0044] FIG. 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.
[0045] FIG. 22: Mouse model of rheumatoid arthritis--Splenocyte
proliferative response to Collagen II. Media background subtracted
[CII-stimulated--media background] counts per minute based on
3H-TdR incorporation. All data are presented as Mean.+-.SEM. ***
p<0.001 compared to Vehicle group.
[0046] FIG. 23: Mouse model of rheumatoid arthritis--Levels of
IFN.gamma. in tissue culture supernatants. Lines represent group
median values.
[0047] FIG. 24: Mouse model of rheumatoid arthritis--Levels of
IL-17A in tissue culture supernatants. Lines represent group median
values.
[0048] FIG. 25: Mouse model of rheumatoid arthritis--Levels of
IL-10 in tissue culture supernatants. Lines represent group median
values.
[0049] FIG. 26: Mouse model of rheumatoid arthritis--Levels of IL-6
in tissue culture supernatants. Lines represent group median
values.
[0050] FIG. 27: Mouse model of house dust mite-induced
asthma--Total IgE in Serum
[0051] FIG. 28: Mouse model of house dust mite-induced asthma--HDM
specific IgG1 in Serum
[0052] FIG. 29: Mouse model of house dust mite-induced
asthma--Total IgE in BALF
[0053] FIG. 30: Mouse model of house dust mite-induced asthma--HDM
specific IgG1 in BALF
[0054] FIG. 31: Mouse model of house dust mite-induced
asthma--Histological Analysis--Mean Peribronchiolar Infiltration
Score
[0055] FIG. 32: Mouse model of house dust mite-induced
asthma--Histological Analysis--Mean Perivascular Infiltration
Score
[0056] FIG. 33: Mouse model of house dust mite-induced
asthma--Histological Analysis--Mean Inflammatory Score (Average of
both Peribronchiolar and Perivascular Infiltration Score)
[0057] FIG. 34: Mouse model of house dust mite-induced
asthma--Histological Analysis--Mucus Score
[0058] FIG. 35: Mouse model of house dust mite-induced asthma--IL-9
level in lung tissue
[0059] FIG. 36: Mouse model of house dust mite-induced
asthma--IL-1alevel in lung tissue
[0060] FIG. 37: Mouse model of house dust mite-induced
asthma--IFN.gamma. level in lung tissue
[0061] FIG. 38: Mouse model of house dust mite-induced
asthma--IL-17A level in lung tissue
[0062] FIG. 39: Mouse model of house dust mite-induced asthma--IL-4
level in lung tissue
[0063] FIG. 40: Mouse model of house dust mite-induced asthma--IL-5
level in lung tissue
[0064] FIG. 41: Mouse model of house dust mite-induced
asthma--IL-1blevel in lung tissue
[0065] FIG. 42: Mouse model of house dust mite-induced
asthma--RANTES level in lung tissue
[0066] FIG. 43: Mouse model of house dust mite-induced
asthma--MIP-1alevel in lung tissue
[0067] FIG. 44: Mouse model of house dust mite-induced asthma--KC
level in lung tissue
[0068] FIG. 45: Mouse model of house dust mite-induced
asthma--MIP-2 level in lung tissue
[0069] FIG. 46: Mouse model of severe neutrophilic asthma--HDM
specific IgG1 in Serum
[0070] FIG. 47: Mouse model of severe neutrophilic asthma--HDM
specific IgG2a in Serum
[0071] FIG. 48: Mouse model of severe neutrophilic asthma--HDM
specific IgG1 in BALF
[0072] FIG. 49: Mouse model of severe neutrophilic asthma--HDM
specific IgG2a in BALF
[0073] FIG. 50: Mouse model of severe neutrophilic
asthma--Histological Analysis--Mean Peribronchiolar Infiltration
Score
[0074] FIG. 51: Mouse model of severe neutrophilic
asthma--Histological Analysis--Mean Perivascular Infiltration
Score
[0075] FIG. 52: Mouse model of severe neutrophilic
asthma--Histological Analysis--Mean Inflammatory Score (Average of
both Peribronchiolar and Perivascular Infiltration Score)
[0076] FIG. 53: Mouse model of severe neutrophilic
asthma--TNF.alpha. level in lung tissue
[0077] FIG. 54: Mouse model of severe neutrophilic
asthma--IL-1alevel in lung tissue
[0078] FIG. 55: Mouse model of severe neutrophilic
asthma--IFN.gamma. level in lung tissue
[0079] FIG. 56: Mouse model of severe neutrophilic asthma--IL-17F
level in lung tissue
[0080] FIG. 57: Mouse model of severe neutrophilic
asthma--IL-1blevel in lung tissue
[0081] FIG. 58: Mouse model of severe neutrophilic asthma--RANTES
level in lung tissue
[0082] FIG. 59: Mouse model of severe neutrophilic asthma--MIP-2
level in lung tissue
[0083] FIG. 60: Mouse model of severe neutrophilic asthma--KC level
in lung tissue
[0084] FIG. 61: Mouse model of severe neutrophilic asthma--IL-17A
level in lung tissue
[0085] FIG. 62: Mouse model of severe neutrophilic asthma--MIP-1a
level in lung tissue
[0086] FIG. 63: Mouse model of severe neutrophilic asthma--IL-33
level in lung tissue
[0087] FIG. 64: Mouse model of rheumatoid arthritis--Visual
Template for Histopathology Scoring. Representative images showing
composite scores from mouse tarsal joints in a collagen-induced
arthritis study.
[0088] FIG. 65: Mouse model of rheumatoid
arthritis--Histopathology: Inflammation Scores. Data are presented
as Mean.+-.SEM.
[0089] FIG. 66: Mouse model of rheumatoid
arthritis--Histopathology: Cartilage Scores. Data are presented as
Mean.+-.SEM.
[0090] FIG. 67: Mouse model of rheumatoid
arthritis--Histopathology: Bone Scores. Data are presented as
Mean.+-.SEM.
[0091] FIG. 68: Mouse model of rheumatoid
arthritis--Histopathology: Total Scores. Data are presented as
Mean.+-.SEM.
[0092] FIG. 69: Mouse model of rheumatoid
arthritis--Histopathology: Representative Pictures. Animal ID
(#n.n) and limb (R for right, L for left) are indicated between
brackets. Left image (vehicle): extensive joint and bone
destruction with inflammation and fibrosis extending to the
peri-articular soft tissues.
[0093] FIG. 70: Mouse model of multiple sclerosis--clinical
score.
[0094] FIG. 71: Mouse model of multiple sclerosis--disease
incidence.
DISCLOSURE OF THE INVENTION
[0095] Bacterial Strains
[0096] The compositions of the invention comprise a bacterial
strain of the genus Roseburia. The examples demonstrate that
bacteria of this genus are useful for treating or preventing
diseases and conditions mediated by IL-17 or the Th17 pathway. The
preferred bacterial strains are of the species Roseburia
hominis.
[0097] Examples of Roseburia species for use in the invention
include Roseburia hominis, Roseburia cecicola, Roseburia faecis,
Roseburia intestinalis, and Roseburia inulinivorans. Roseburia
bacteria are slightly curved rod-shaped cells that are strictly
anaerobic and indigenous to the mammalian intestine. They are of
the phylogenetic cluster XIVa within the Firmicutes phylum. The
bacteria are butyrate-producing and are actively motile through
multiple flagella present along the concave side and in a cluster
at one end [16]. Roseburia hominis and Roseburia intestinalis are
recently described examples.
[0098] An example of Roseburia hominis is the strain deposited
under the terms of the Budapest Treaty at National Collections of
Industrial, Food and Marine Bacteria (NCIMB) at NCIMB Ltd, Ferguson
Building, Craibstone Estate, Bucksburn, Aberdeen, UK, AB21 9YA, on
21 October 2004 by the Rowett Research Institute under the
accession number NCIMB 14029.sup.T Roseburia hominis
A2-183.sup.T(DSM=16839.sup.T). Other exemplary Roseburia hominis
strains are described in [17]. GenBank/EMBL/DDBJ accession numbers
for the 16S rRNA gene sequence of strains of Roseburia hominis are
AY804148 and AJ270482 (disclosed herein as SEQ ID NO:1 and SEQ ID
NO:2). The NCIMB deposit was made under the terms of the Budapest
Treaty. Maintenance of a viable culture is assured for 30 years
from the date of deposit. All restrictions on the availability to
the public of the deposited microorganism will be irrevocably
removed upon the granting of a patent for this application.
[0099] An example of Roseburia intestinalis is the strain deposited
under the accession number NCIMB 13810 Roseburia intestinalis
L1-82.sup.T(DSM=14610.sup.T). Another example is the Roseburia
intestinalis strain as described in [17]. Reference [17] also
describes exemplary Roseburia faecis and Roseburia inulinivorans
strains.
[0100] The Roseburia hominis bacterium deposited under accession
number NCIMB 42383 was tested in the Examples and is also referred
to herein as strain 433. A 16S rRNA sequence for the 433 strain
that was tested is provided in SEQ ID NO:3. Strain 433 was
deposited with the international depositary authority NCIMB, Ltd.
(Ferguson Building, Aberdeen, AB21 9YA, Scotland) by GT Biologics
Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS,
Scotland) on 12th March 2015 as "Roseburia hominis 433" and was
assigned accession number NCIMB 42383. GT Biologics Ltd.
subsequently changed its name to 4D Pharma Research Limited. The
NCIMB deposit was made under the terms of the Budapest Treaty.
Maintenance of a viable culture is assured for 30 years from the
date of deposit. All restrictions on the availability to the public
of the deposited microorganism will be irrevocably removed upon the
granting of a patent for this application.
[0101] A genome sequence for strain 433 is provided in SEQ ID NO:4.
This sequence was generated using the PacBio RS II platform.
[0102] 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 Th17
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 Roseburia intestinalis. 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 or 3. Preferably, the sequence identity is to SEQ
ID NO:3. Preferably, the bacterial strain for use in the invention
has the 16s rRNA sequence represented by SEQ ID NO:3.
[0103] Bacterial strains that are biotypes of the bacterium
deposited under accession number 42383 are also expected to be
effective for treating or preventing diseases and conditions
mediated by IL-17 or the Th17 pathway. A biotype is a closely
related strain that has the same or very similar physiological and
biochemical characteristics.
[0104] Strains that are biotypes of the bacterium deposited under
accession number NCIMB 42383 and that are suitable for use in the
invention may be identified by sequencing other nucleotide
sequences for the bacterium deposited under accession number NCIMB
42383. For example, substantially the whole genome may be sequenced
and a biotype strain for use in the invention may have 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 identifying biotype strains may include hsp60 or repetitive
sequences such as BOX, ERIC, (GTG)5, or REP or [18]. Biotype
strains may have sequences with at least 95%, 96%, 97%, 98%, 99%,
99.5% or 99.9% sequence identity to the corresponding sequence of
the bacterium deposited under accession number NCIMB 42383.
[0105] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:4. 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 SEQ ID NO:4 across at least 60% (e.g. at least 65%, 70%, 75%,
80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO:4. For
example, the bacterial strain for use in the invention may have a
genome with at least 90% sequence identity to SEQ ID NO:4 across
70% of SEQ ID NO:4, or at least 90% sequence identity to SEQ ID
NO:4 across 80% of SEQ ID NO:4, or at least 90% sequence identity
to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at least 90% sequence
identity to SEQ ID NO:4 across 100% of SEQ ID NO:4, or at least 95%
sequence identity to SEQ ID NO:4 across 70% of SEQ ID NO:4, or at
least 95% sequence identity to SEQ ID NO:4 across 80% of SEQ ID
NO:4, or at least 95% sequence identity to SEQ ID NO:4 across 90%
of SEQ ID NO:4, or at least 95% sequence identity to SEQ ID NO:4
across 100% of SEQ ID NO:4, or at least 98% sequence identity to
SEQ ID NO:4 across 70% of SEQ ID NO:4, or at least 98% sequence
identity to SEQ ID NO:4 across 80% of SEQ ID NO:4, or at least 98%
sequence identity to SEQ ID NO:4 across 90% of SEQ ID NO:4, or at
least 98% sequence identity to SEQ ID NO:4 across 100% of SEQ ID
NO:4.
[0106] Alternatively, strains that are biotypes of the bacterium
deposited under accession number NCIMB 42383 and that are suitable
for use in the invention may be identified by using the accession
number NCIMB 42383 deposit and restriction fragment analysis and/or
PCR analysis, for example by using fluorescent amplified fragment
length polymorphism (FAFLP) and repetitive 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 Roseburia hominis strains. vIn certain
embodiments, strains that are biotypes of the bacterium deposited
under accession number NCIMB 42383 and that are suitable for use in
the invention are strains that provide the same pattern as the
bacterium deposited under accession number NCIMB 42383 when
analysed by amplified ribosomal DNA restriction analysis (ARDRA),
for example when using Sau3AI restriction enzyme (for exemplary
methods and guidance see, for example, [19]). Alternatively,
biotype strains are identified as strains that have the same
carbohydrate fermentation patterns as the bacterium deposited under
accession number NCIMB 42383.
[0107] Other Roseburia strains that are useful in the compositions
and methods of the invention, such as biotypes of the bacteria
deposited under accession number NCIMB 42383, 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 anaerobic 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 have similar growth patterns,
metabolic type and/or surface antigens to the bacterium deposited
under accession number NCIMB 42383 may be useful in the invention.
A useful strain will have comparable immune modulatory activity to
the NCIMB 42383 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.
[0108] A particularly preferred strain of the invention is the
Roseburia hominis strain deposited under accession number NCIMB
42383. This is the exemplary 433 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
Roseburia hominis strain deposited under accession number NCIMB
42383, or a derivative thereof. The invention also provides a
composition comprising a cell of the Roseburia hominis strain
deposited under accession number NCIMB 42383, or a derivative
thereof. The invention also provides a biologically pure culture of
the Roseburia hominis strain deposited under accession number NCIMB
42383. The invention also provides a cell of the Roseburia hominis
strain deposited under accession number NCIMB 42383, or a
derivative thereof, for use in therapy, in particular for the
diseases described herein.
[0109] A derivative of the strain deposited under accession number
NCIMB 42383 may be a daughter strain (progeny) or a strain cultured
(subcloned) from the original. A derivative of a strain of the
invention may be modified, for example at the genetic level,
without ablating the biological activity. In particular, a
derivative strain of the invention is therapeutically active. A
derivative strain will have comparable immune modulatory activity
to the original NCIMB 42383 strain. In particular, a derivative
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 derivative of the NCIMB 42383 strain
will generally be a biotype of the NCIMB 42383 strain.
[0110] References to cells of the Roseburia hominis strain
deposited under accession number NCIMB 42383 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 42383, and
such cells are encompassed by the invention.
[0111] In preferred embodiments, the bacterial strains in the
compositions of the invention are viable and capable of partially
or totally colonising the intestine.
[0112] Therapeutic Uses
[0113] As demonstrated in the examples, the bacterial compositions
of the invention are effective for reducing the Th17 inflammatory
response. In particular, treatment with compositions of the
invention achieves a reduction in IL-17A levels and other Th17
pathway cytokines, and clinical improvements in animal models of
conditions mediated by IL-17 and the Th17 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 elevation of the IL-17 inflammatory response.
[0114] Th17 cells are a subset of T helper cells that produce, for
example, IL-17A, IL17-F, IL-21 and IL-22. Th17 cell differentiation
and IL-17 expression may be driven by IL-23. These cytokines and
others form important parts of the Th17 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, [20-25]). Diseases wherein the Th17
pathway is activated are Th17 pathway-mediated diseases. Th17
pathway-mediated diseases can be ameliorated or alleviated by
repressing the Th17 pathway, which may be through a reduction in
the differentiation of Th17 cells or a reduction in their activity
or a reduction in the level of Th17 pathway cytokines. Diseases
mediated by the Th17 pathway may be characterised by increased
levels of cytokines produced by Th17 cells, such as IL-17A, IL-17F,
IL-21, IL-22, IL-26, IL-9 (reviewed in [26]). Diseases mediated by
the Th17 pathway may be characterised by increased expression of
Th-17-related genes, such as Stat3 or IL-23R. Diseases mediated by
the Th17 pathway may be associated with increased levels of Th17
cells.
[0115] IL-17 is a pro-inflammatory cytokine that contributes to the
pathogenesis of several inflammatory 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 reduction in IL-17 levels. The IL-17 inflammatory
response may be local or systemic.
[0116] Examples of diseases and conditions that may be mediated by
IL-17 or the Th17 pathway include multiple sclerosis; arthritis,
such as rheumatoid arthritis, osteoarthritis, psoriatic arthritis,
or juvenile 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 allergic asthma or neutrophilic 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; atopic dermatitis;
emphysema; periodontitis; allergic rhinitis; and allograft
rejection. 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 Th17
pathway.
[0117] In certain embodiments, the compositions of the invention
are for use in a method of reducing IL-17 production or reducing
Th17 cell differentiation in the treatment or prevention of a
disease or condition mediated by IL-17 or the Th17 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 prevention is achieved by reducing or
preventing elevation of the Th17 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 Th17
cells or is exhibiting a Th17 inflammatory response. In certain
embodiments, the patient may have 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-.alpha. inhibitors. These uses of the invention
may be applied to any of the specific disease or conditions listed
in the preceding paragraph.
[0118] IL-17 and the Th17 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.
[0119] The compositions of the invention may be useful for treating
diseases and conditions mediated by IL-17 or the Th17 pathway and
for addressing the Th17 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 have not responded to other therapies (such as
treatment with TNF-.alpha. inhibitors), and/or treating or
preventing the tissue damage and symptoms associated with IL-17 and
Th17 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 erosion or cartilage damage.
[0120] In certain embodiments, treatment with compositions of the
invention provides a reduction or prevents an elevation in IL-17
levels, in particular IL-17A levels. In certain embodiments,
treatment with compositions of the invention provides a reduction
or prevents an elevation in IFN-.gamma. or IL-6 levels. Such
reduction or prevention 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 Th17 pathway.
[0121] Asthma
[0122] 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
reduction 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 useful in the treatment or
prevention 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 Th17 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.
[0123] In certain embodiments, the asthma is eosinophilic or
allergic asthma. Eosinophilic and allergic asthma are characterised
by increased numbers of eosinophils in peripheral blood and in
airway secretions and is associated pathologically with thickening
of the basement membrane zone and pharmacologically by
corticosteroid responsiveness [27]. Compositions that reduce or
inhibit eosinophil recruitment or activation may be useful for
treating or preventing eosinophilic and allergic asthma.
[0124] In additional embodiments, the compositions of the invention
are for use in treating or preventing neutrophilic 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
neutrophilic asthma.
[0125] Eosinophilic and neutrophilic 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.
[0126] Increased IL-17 levels and activation of the Th17 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.
[0127] In certain embodiments, the compositions of the invention
are for use in methods reducing an eosinophilic inflammatory
response in the treatment or prevention of asthma, or for use in
methods of reducing a neutrophilic inflammatory response in the
treatment or prevention of asthma. As noted above, high levels of
eosinophils in asthma is associated pathologically with thickening
of the basement membrane zone, so reducing eosinophilic
inflammatory response in the treatment or prevention of asthma may
be able to specifically address this feature of the disease. Also,
elevated neutrophils, either in combination with elevated
eosinophils or in their absence, is associated with severe asthma
and chronic airway narrowing. Therefore, reducing the neutrophilic
inflammatory response may be particularly useful for addressing
severe asthma.
[0128] In certain embodiments, the compositions reduce
peribronchiolar infiltration in allergic asthma, or are for use in
reducing peribronchiolar infiltration in the treatment of allergic
asthma. In certain embodiments, the compositions reduce
peribronchiolar and/or perivascular infiltration in neutrophilic
asthma, or are for use in reducing peribronchiolar and/or
perivascular infiltration in the treatment of allergic neutrophilic
asthma.
[0129] In certain embodiments, treatment with compositions of the
invention provides a reduction or prevents an elevation in
IFN.gamma. levels.
[0130] In certain embodiments, the compositions of the invention
are for use in a method of treating asthma that results in a
reduction of the eosinophilic and/or neutrophilic 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.
[0131] The compositions of the invention may be useful for
preventing the development of asthma in a new-born when
administered to the new-born, or to a pregnant 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 useful for
reducing symptoms associated with asthma that is aggravated by
allergens, such as house dust mites.
[0132] Treatment or prevention of asthma may refer to, for example,
an alleviation of the severity of symptoms or a reduction in the
frequency of exacerbations or the range of triggers that are a
problem for the patient.
[0133] Arthritis
[0134] 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 reduction 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 prevention
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 Th17 cells may have a
key role in RA, for example because IL-17 inhibits matrix
production in chondrocytes and osteoblasts and activates the
production and function of matrix metalloproteinases and because RA
disease activity is correlated to IL-17 levels and Th-17 cell
numbers [28,29], so the compositions of the invention may be
particularly effective for preventing or treating RA.
[0135] In certain embodiments, the compositions of the invention
are for use in lowering IL-17 levels or preventing elevation of
IL-17 levels in the treatment or prevention of RA. In certain
embodiments, treatment with compositions of the invention provides
a reduction or prevents an elevation in IL-17 levels, in particular
IL-17A levels. In certain embodiments, treatment with compositions
of the invention provides a reduction or prevents an elevation in
IFN-.gamma. or IL-6 levels. In certain embodiments, treatment with
the compositions of the invention results in a reduction 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.
[0136] In certain embodiments, treatment with the compositions of
the invention results in a reduction 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.
[0137] Increased IL-17 levels and Th17 cell numbers are associated
with cartilage and bone destruction in RA [28,29]. 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
erosion or cartilage damage in the treatment of RA. In certain
embodiments, the compositions are for use in treating patients that
exhibit bone erosion or cartilage damage or patients identified as
at risk of bone erosion or cartilage damage.
[0138] TNF-.alpha. is also associated with RA, but TNF-.alpha. is
not involved in the pathogenesis of the later stages of the
disease. In contrast, IL-17 has a role throughout all stages of
chronic disease [30]. Therefore, in certain embodiments the
compositions of the invention are for use in treating chronic RA or
late-stage 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 have previously received
anti-TNF-.alpha. therapy. In certain embodiments, the patients to
be treated do not respond or no longer respond to anti-TNF-.alpha.
therapy.
[0139] 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 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
alleviating RA. The compositions of the invention may be
particularly useful for reducing symptoms associated with joint
swelling or bone destruction. Treatment or prevention of RA may
refer to, for example, an alleviation of the severity of symptoms
or a reduction in the frequency of exacerbations or the range of
triggers that are a problem for the patient.
[0140] Multiple Sclerosis
[0141] 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 reduction 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 prevention 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 episodes. IL-17
and Th17 cells may have a key role in multiple sclerosis, for
example because IL-17 levels may correlate with multiple sclerosis
lesions, IL-17 can disrupt blood brain barrier endothelial cell
tight junctions, and Th17 cells can migrate into the central
nervous system and cause neuronal loss [31,32]. Therefore, the
compositions of the invention may be particularly effective for
preventing or treating multiple sclerosis.
[0142] In certain embodiments, treatment with the compositions of
the invention results in a reduction 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 decline in motor function or results in
improved motor function. In certain embodiments, the compositions
of the invention are for use in preventing a decline 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.
[0143] 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.
[0144] 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 prevention of
multiple sclerosis may refer to, for example, an alleviation of the
severity of symptoms or a reduction in the frequency of
exacerbations or the range of triggers that are a problem for the
patient.
[0145] Modes of Administration
[0146] 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.
[0147] In certain embodiments, the compositions of the invention
may be administered as a foam, as a spray or a gel.
[0148] 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.
[0149] 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 endoscopic gastrostomy (PEG), or a port,
such as a chest wall port that provides access to the stomach,
jejunum and other suitable access ports.
[0150] 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.
[0151] 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.
[0152] In certain embodiments, the composition of the invention may
be administered to a pregnant 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 born.
[0153] 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 Th17 pathway, or that has been identified
as being at risk of a disease or condition mediated by IL-17 or the
Th17 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 Th17 pathway in a healthy
patient.
[0154] 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 have reduced or absent
colonisation by Roseburia, and in particular Roseburia hominis.
[0155] The compositions of the invention may be administered as a
food product, such as a nutritional supplement.
[0156] Generally, the compositions of the invention are for the
treatment of humans, although they may be used to treat animals
including monogastric 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 animals. If
administered to animals, oral gavage may be used.
[0157] Compositions
[0158] 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
strain of the invention.
[0159] 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 [33-35].
[0160] Alternatively, the composition of the invention may comprise
a live, active bacterial culture.
[0161] 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 degradation
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, attachment or adsorption on solid carrier
surfaces, self-aggregation 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 [36] and [37].
[0162] The composition may be administered orally and may be in the
form of a tablet, capsule or powder. Encapsulated products are
preferred because Roseburia are anaerobes. Other ingredients (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. Alternatively, 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.
[0163] The composition may be formulated as a probiotic.
[0164] 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 beneficial effect upon a patient. A
therapeutically effective amount of a bacterial strain may be
sufficient to result in delivery to and/or partial or total
colonisation of the patient's intestine.
[0165] A suitable daily dose of the bacteria, for example for an
adult human, may be from about 1.times.10.sup.3 to about
1.times.10.sup.11 colony forming units (CFU); for example, from
about 1.times.10.sup.7 to about 1.times.10.sup.10 CFU; in another
example from about 1.times.10.sup.6 to about 1.times.10.sup.10
CFU.
[0166] In certain embodiments, the composition contains the
bacterial strain in an amount of from about 1.times.10.sup.6 to
about 1.times.10.sup.11 CFU/g, respect to the weight of the
composition; for example, from about 1.times.10.sup.8 to about
1.times.10.sup.10 CFU/g. The dose may be, for example, 1 g, 3g, 5g,
and 10g.
[0167] Typically, a probiotic, such as the composition of the
invention, is optionally 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 transgalacto-oligosaccharides.
[0168] In certain embodiments, the probiotic composition of the
present 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,
isomalt-oligosaccharides, pectins, xylo-oligosaccharides (or XOS),
chitosan-oligosaccharides (or COS), beta-glucans, arable gum
modified and resistant starches, polydextrose, D-tagatose, acacia
fibers, carob, oats, and citrus fibers. 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 molecule to which three
glucose molecules are bonded.
[0169] The compositions of the invention may comprise
pharmaceutically acceptable excipients or carriers. Examples of
such suitable excipients may be found in the reference [38].
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art and are described, for example, in
reference [39]. Examples of suitable carriers include lactose,
starch, glucose, methyl cellulose, magnesium stearate, mannitol,
sorbitol and the like. Examples of suitable diluents include
ethanol, 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, free-flow 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 stearate, magnesium stearate, sodium benzoate,
sodium acetate, sodium chloride and the like. Preservatives,
stabilizers, 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.
[0170] The compositions of the invention may be formulated as a
food product. For example, a food product may provide nutritional
benefit in addition to the therapeutic effect of the invention,
such as in a nutritional supplement. 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.
[0171] 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.
[0172] The compositions for use in accordance with the invention
may or may not require marketing approval.
[0173] 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.
[0174] The compositions of the invention can comprise
pharmaceutically acceptable excipients, diluents or carriers.
[0175] 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, allergic asthma, neutrophilic asthma, osteoarthritis,
psoriatic arthritis, juvenile 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, atopic
dermatitis, emphysema, periodontitis, allergic rhinitis, and
allograft rejection.
[0176] 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
Th17 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, allergic
asthma, neutrophilic asthma, osteoarthritis, psoriatic arthritis,
juvenile idiopathic arthritis, neuromyelitis optica (Devic's
disease), ankylosing spondylitis, spondyloarthritis, systemic lupus
erythematosus, chronic obstructive pulmonary disease (COPD),
cancer, breast cancer, colon cancer, lung cancer, ovarian cancer,
uveitis, scleritis, vasculitis, Behcet's disease, atherosclerosis,
atopic dermatitis, emphysema, periodontitis, allergic rhinitis, and
allograft rejection.
[0177] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein the amount of the bacterial
strain is from about 1.times.10.sup.3 to about 1.times.10.sup.11
colony forming units per gram with respect to a weight of the
composition.
[0178] 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.
[0179] 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,
subcutaneous, nasal, buccal, and sublingual.
[0180] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising a carrier selected from the
group consisting of lactose, starch, glucose, methyl cellulose,
magnesium stearate, mannitol and sorbitol.
[0181] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising a diluent selected from the
group consisting of ethanol, glycerol and water.
[0182] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising an excipient selected from
the group consisting of starch, gelatin, glucose, anhydrous
lactose, free-flow lactose, beta-lactose, corn sweetener, acacia,
tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene
glycol, sodium oleate, sodium stearate, magnesium stearate, sodium
benzoate, sodium acetate and sodium chloride.
[0183] In certain embodiments, the invention provides the above
pharmaceutical composition, further comprising at least one of a
preservative, an antioxidant and a stabilizer.
[0184] 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
p-hydroxybenzoic acid.
[0185] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein said bacterial strain is
lyophilised.
[0186] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein when the composition is stored
in a sealed container at about 4.0 or about 25.0 and the container
is placed in an atmosphere 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.
[0187] Culturing Methods
[0188] The bacterial strains for use in the present invention can
be cultured using standard microbiology techniques as detailed in,
for example, references [40-42].
[0189] The solid or liquid medium used for culture may be YCFA agar
or YCFA medium. YCFA medium may include (per 100 ml, approximate
values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO.sub.3 (0.4
g), cysteine (0.1 g), K2HPO.sub.4 (0.045 g), KH.sub.2PO.sub.4
(0.045 g), NaCl (0.09 g), (NH.sub.4).sub.2SO.sub.4 (0.09 g),
MgSO.sub.4.7H.sub.2O (0.009 g), CaCl.sub.2 (0.009 g), resazurin
(0.1 mg), hemin (1 mg), biotin (1 .mu.g), cobalamin (1 .mu.g),
p-aminobenzoic acid (3 .mu.g), folic acid (5 .mu.g), and
pyridoxamine (15 .mu.g).
[0190] Bacterial Strains for use in Vaccine Compositions
[0191] The inventors have identified that the bacterial strains of
the invention are useful for treating or preventing diseases or
conditions mediated by IL-17 or the Th17 pathway. This is likely to
be a result of the effect that the bacterial strains of the
invention have 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 Th17 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.
[0192] General
[0193] The practice of the present invention will employ, unless
otherwise indicated, conventional methods of chemistry,
biochemistry, molecular biology, immunology and pharmacology,
within the skill of the art. Such techniques are explained fully in
the literature. See, e.g., references [43] and [44-50], etc.
[0194] 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.
[0195] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0196] 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 definition of the invention.
[0197] References to a percentage sequence identity between two
nucleotide sequences means that, when aligned, that percentage of
nucleotides are the same in comparing the two sequences. This
alignment 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 [51]. 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
Smith-Waterman homology search algorithm is disclosed in ref
[52].
[0198] 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 combined, omitted or performed in an alternative
order, if appropriate.
[0199] Various embodiments of the invention are described herein.
It will be appreciated that the features specified in each
embodiment may be combined with other specified features, to
provide further embodiments. In particular, embodiments highlighted
herein as being suitable, typical or preferred may be combined with
each other (except when they are mutually exclusive).
MODES FOR CARRYING OUT THE INVENTION
Example 1
Efficacy of Bacterial Inocula in a Mouse Model of House Dust
Mite-Induced Asthma
[0200] Summary
[0201] 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 allergic
inflammatory response. The inflammatory response to HDM includes
eosinophilic and neutrophilic components, is mediated by IL-17 and
the Th17 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 Th17-mediated conditions such as eosinophilia,
neutrophilia and asthma.
[0202] Strain
[0203] 433: Roseburia hominis
[0204] Study Design
[0205] Groups:
[0206] 1. Negative control group. Treatment with vehicle control
(per oral).
[0207] 2. Treatment with therapeutic bacteria inoculum strain 433
(per oral).
[0208] 7. Positive control group. Treatment with Dexamethasone
(i.p.).
[0209] 8. Untreated Control Group.
[0210] Number of mice per group=5
[0211] Day -14 to day 13: Daily administration of vehicle control
per oral (Group 1).
[0212] Day -14 to day 13: Daily administration of therapeutic
bacteria inoculum per oral (Group 2-6).
[0213] Day 0, 2, 4, 7, 9, 11 Administration of 15 ug HDM (house
dust mite extract--Catalogue number: XPB70D3A25, Lot number:
231897, Greer Laboratories, Lenoir, N.C., USA) in a volume of 30 ul
PBS per nasal (Group 1-8).
[0214] Day 0, 2, 4, 7, 9, 11 Administration of Dexamethasone (i.p.,
3 mg/kg, Sigma-Aldrich, Catalogue number D1159) (Group 7).
[0215] Day 14 Sacrifice of all animals for analysis.
[0216] Total number of mice=40.
[0217] Endpoints and Analysis
[0218] On day 14 animals were sacrificed by lethal intraperitoneal
injection with pentabarbitol (Streuli Pharma AG, Uznach, Cat:
1170139A) immediately followed by a bronchoalveolar lavage
(BAL).
[0219] Cells were isolated from the BAL (bronchoalveolar lavage)
fluid and differential cell counts performed (200 cell counts/
samples).
[0220] Material and Methods
[0221] 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 Sealsafe.TM.--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). Animals were 8 weeks old on Study Day -14. Potable
water and food were available ad libitum. Cage enrichment was
present. Daily care of the animals was performed according to local
authorization license number 2283.1 (issued and approved by:
Service de la consommation et des affaires veterinaires du Canton
de Vaud). Potable water and food were available ad libitum and
refreshed once daily. Cage enrichment was present. Animal welfare
regulations were observed as given by official authorities of
Switzerland under ordinance 455.163 of the FVO (Federal Veterinary
Office) on laboratory animal husbandry, production of genetically
modified animals, and methods of animal experimentation.
[0222] Culturing of bacteria inoculum. Within a sterile
workstation, a cryo-vial of bacteria was thawed by warming in
gloved hand and .about.0.7 ml of contents injected into a Hungate
tube (Cat Number, 1020471, Glasgeratebau Ochs, Bovenden-Lenglern,
Germany), containing 8 ml of anaerobic YCFA. Two tubes per strain
were usually prepared. The Hungate tubes were then incubated
(static) at 37.degree. C. for up to 24-26 hours (for strain
433).
[0223] Culturing of vehicle control. A Hungate tube containing 8 ml
of anaerobic YCFA was incubated (static) at 37.degree. C. for 16
h.
[0224] Administration of bacteria inoculum or vehicle control. 400
ul of cultured bacteria inoculum or vehicle control were
administered per day per oral gavage.
[0225] 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, Bern, Switzerland) and administered with 15 ug of HDM
(Catalogue number: XPB70D3A25, Lot number: 231897, Greer
Laboratories, Lenoir, N.C., USA) in a volume of 30 ul PBS per
nasal.
[0226] Preparation and administration of positive control compound
Dexamethasone. Dexamethasone 21-phosphate disodium salt
(Sigma-Aldrich, Catalogue number D1159, Lot N.degree. SLBD.1030V)
was solved in H.sub.2O and administered to the animals in a dose of
3 mg/kg in a volume of 200 ul per oral at days indicated in study
protocol above.
[0227] Terminal procedure. On day 14 animals were sacrificed by
lethal i.p. injection with pentabarbitol (Streuli Pharma AG,
Uznach, Cat: 1170139A) immediately followed by bronchoalveolar
lavage (BAL) in 500 ul of saline.
[0228] Measurement of cellular infiltrates into BAL. Cells were
isolated from the BAL fluid and differential cell counts were
performed based upon standard morphological and cytochemical
criteria.
[0229] Graphs and statistical analysis. All graphs 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).
[0230] Results and Analysis
[0231] The results of the experiments are shown in FIGS. 1-9.
[0232] No morbidity or mortality was noted in the mice treated with
the bacteria or the vehicle. The two controls, vehicle treatment
(negative control) and the dexamethasone treatment (positive
control) behaved as expected, with impaired eosinophilia and
neutrophilia noted following dexamethasone treatment.
[0233] Strain 433 was efficacious in alleviating the magnitude of
the allergic inflammatory response. As shown in FIGS. 2 and 3,
administration of strain 433 reduced total eosinophils and the
proportion of eosinophils in BAL, which indicates reduced
eosinophilia. In addition, administration of strain 433 resulted in
a statistically significant reduction in total neutrophils and the
proportion of neutrophils in BAL relative to the vehicle-only
control, as shown in FIGS. 6 and 7.
Example 2
Efficacy of Bacterial Inocula in a Mouse Model of Severe
Neutrophilic Asthma
[0234] Summary
[0235] Mice were administered with compositions comprising
bacterial strains according to the invention and were subsequently
sensitised with subcutaneous administrations of house dust mite
(HDM) extract and challenged with an intranasal administration of
HDM in order to model the inflammatory response of severe
neutrophilic 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
Th17-mediated conditions such as neutrophilia and asthma.
[0236] Strain
[0237] 433: Roseburia hominis
[0238] Study Design
[0239] Groups:
[0240] 1. Negative control group. Treatment with vehicle control
(per oral).
[0241] 2. Treatment with therapeutic bacteria inoculum strain 433
(per oral).
[0242] 7. Positive control group. Treatment anti-IL-17 (i.p.).
[0243] 8. Untreated Control Group.
[0244] 9: Healthy mice (baseline).
[0245] Number of mice per group (Group 1-8)=5
[0246] Day -14 to day 17: Daily administration of vehicle control
per oral (Group 1).
[0247] Day -14 to day 17: Daily administration of therapeutic
bacteria inoculum per oral (Group 2-6).
[0248] Day 0: Sensitization with HDM in CFA (s.c.) (Group 1-8).
[0249] Day 7: Sensitization with HDM in CFA (s.c.) (Group 1-8).
[0250] Day 13, 15, 17: Administration of anti IL-17 neutralizing
antibody per i.p. (Group 7).
[0251] Day 14, 15, 16, 17: Challenge with HDM in 30 ul PBS per
nasal (Group 1-8).
[0252] Day 18: Sacrifice of all animals for analysis.
[0253] Endpoints and Analysis:
[0254] On day 14 animals were sacrificed by lethal intraperitoneal
injection with pentabarbitol (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).
[0255] Material and Methods.
[0256] 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, Gams,
Switzerland Cage type: "The Sealsafe.TM.--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). Animals were 8 weeks old on Study Day -14. Potable
water and food were available ad libitum. Cage enrichment was
present. Daily care of the animals was performed according to local
authorization license number 2283.1 (issued and approved by:
Service de la consommation et des affaires veterinaires du Canton
de Vaud). Potable water and food were available ad libitum and
refreshed once daily. Cage enrichment was present. Animal welfare
regulations were observed as given by official authorities of
Switzerland under ordinance 455.163 of the FVO (Federal Veterinary
Office) on laboratory animal husbandry, production of genetically
modified animals, and methods of animal experimentation.
[0257] Culturing of bacteria inoculum. Within a sterile
workstation, a cryo-vial of bacteria was thawed by warming in
gloved hand and .about.0.7 ml of contents injected into a Hungate
tube (Cat Number,
[0258] 1020471, Glasgeratebau Ochs, Bovenden-Lenglern, Germany),
containing 8 ml of anaerobic YCFA. Two tubes per strain were
usually prepared. The Hungate tubes were then incubated (static) at
37.degree. C. for up to 24-26 hours (for strain 433).
[0259] Culturing of vehicle control. A Hungate tube containing 8 ml
of anaerobic YCFA was incubated (static) at 37.degree. C. for 16
h.
[0260] Administration of bacteria inoculum or vehicle control. 400
ul of cultured bacteria inoculum or vehicle control were
administered per day per oral gavage.
[0261] HDM sensitization. 50 .mu.g of HDM (Catalogue number:
XPB70D3A25, Lot number: 231897, Greer Laboratories, Lenoir, N.C.,
USA) in PBS was emulsified in equal volume of complete Freund's
adjuvant (CFA Chondrex Inc. Washington, USA) and administered
subcutaneously in a volume of 200 .mu.l, twice 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, Bern, Switzerland) and then given
intranasal challenges of 15 .mu.g of HDM in a volume of 30 ul PBS
on 4 consecutive days. Analysis was performed one day after the
final challenge.
[0262] Preparation 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.degree. 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.
[0263] Terminal procedure. On day 18 animals were sacrificed by
lethal i.p. injection with pentabarbitol (Streuli Pharma AG,
Uznach, Cat: 1170139A) immediately followed by bronchoalveolar
lavage (BAL) in 500 ul of saline.
[0264] Measurement of cellular infiltrates into BAL. Cells were
isolated from the BAL fluid and differential cell counts were
performed based upon standard morphological and cytochemical
criteria.
[0265] Graphs and statistical analysis. All 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).
[0266] Results and Analysis
[0267] The results of the experiment are shown in FIGS. 10-18.
[0268] No morbidity or mortality was noted in the mice treated with
the bacteria or the vehicle. As shown in FIGS. 11, 12, 15 and 16,
certain mice treated with strain 433 exhibited reduced eosinophilia
and neutrophilia.
Example 3
Efficacy of Bacterial Inocula to Treat Arthritis in a Type II
Collagen-Induced Arthritis Mouse Model
[0269] Materials and Methods
[0270] Strain
[0271] 433: Roseburia hominis
[0272] Bacterial Cultures
[0273] Bacterial cultures were grown up for administration in an
anaerobic workstation (Don Whitley Scientific).
[0274] Bacterial strain #433 was grown using glycerol stocks. The
glycerol stocks were stored at -80.degree. C. Three times per week,
glycerol stocks were thawed at room temperature 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.
[0275] Solutions to be administered to the animals were prepared
twice daily with an eight hour interval for morning (AM) and
afternoon (PM) treatments. A bacterial colony was picked from the
streaked plate and transferred into a tube containing YCFA media.
Bacterial strain #433 was allowed to grow for 24 hours before AM
administrations. Bacteria were sub-cultured at 1% into YCFA media
for PM administrations. OD values were recorded for each strain
after morning and afternoon treatment preparations.
[0276] Type II Collagen-Induced Arthritis Mouse Model
[0277] Adult male DBA/1 mice were randomly allocated to
experimental groups and allowed to acclimatise for two weeks. On
Day 0, animals were administered by subcutaneous injection with 100
microliters of an emulsion containing 100 micrograms of type II
collagen (CII) in incomplete's Freund's adjuvant supplemented with
4 mg/ml Mycobacterium tuberculosis H37Ra. On Day 21, animals were
administered by subcutaneous injection with a booster emulsion
containing 100 .mu.g of type II collagen in incomplete Freund's
adjuvant.
[0278] Treatments were given according to the administration
schedule below. From Day -14 until the end of the experiment on Day
45, animals were weighed three times per week. From Day 21 until
the end of the experiment, animals 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.
[0279] On Day 45 mice were culled and terminal blood samples were
taken for cytokine analysis.
[0280] On Day -14, Day 0 and Day 45, faecal samples were collected
for microbiological analysis, immediately snap-frozen and stored at
-80.degree. C.
[0281] The collagen-induced arthritis (CIA) mouse model is a
well-established mouse model for rheumatoid arthritis [53]
Immunisation with CII causes a pathogenesis that includes several
important pathological features of rheumatoid arthritis, including
synovial hyperplasia, mononuclear cell infiltration and cartilage
degradation. Significantly, the development of CIA is mediated by
Th17 cells through secretion of IL-17A [54]. The immune response
underlying the arthritis model is enhanced by the use of Freund's
adjuvant supplemented with Mycobacterium tuberculosis.
[0282] On Day 21, spleens were collected from three satellite
animals in each group. Cells were cultured for 72 hours in the
presence or absence of type II collagen. Cytokines, including
TNF-.alpha., IL-6, IFN-.gamma., IL-4, IL-10 and IL-17, were
quantified in the culture supernatants and in terminal serum by
Luminex. Cell proliferation was quantified using a tritiated
thymidine incorporation method.
[0283] Treatment Groups and Dosages
[0284] All 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.
TABLE-US-00001 Administration Disease Group Dose Route Regimen
Induction 1 Vehicle 5 ml/kg PO BID: Day 0: Day -14-End
Collagen/CFA, once, SC 6 Biotherapeutic #433 5 ml/kg Day 21:
Collagen/IFA, once, SC PO: oral gavage, SC: subcutaneous injection,
BID: twice a day, CFA: complete Freund's adjuvant.
[0285] Bodyweights
[0286] From Day -14 until the end of the experiment, animals were
weighed three times per week. Data were graphed (Mean.+-.SEM).
[0287] Non-specific clinical observations
[0288] From Day -14 until the end of the experiment, animals 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).
[0289] Clinical Observations
[0290] From Day 21 until the end of the experiment on Day 45,
animals 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).
Animals with a score equal to (12) on two consecutive occasions and
animals with a score greater than (12) on any one occasion were
culled. Data were graphed (Mean.+-.SEM).
[0291] Cell Proliferation Analysis
[0292] On Day 21, three satellite animals 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 overnight in the presence of tritiated thymidine. Cell
proliferation was quantified by measuring thymidine incorporation.
Data were graphed (Mean.+-.SEM). Supernatants were taken and tested
for the presence of key cytokines.
[0293] Cytokine Analysis
[0294] Terminal supernatants from the spleen cell cultures were
tested in order to quantitate TNF-.alpha., IL-6, IFN-.gamma., IL-4,
IL-10 and IL-17 by Luminex Data were graphed (Mean.+-.SEM).
[0295] Microbiological Analysis
[0296] On Day -14, Day 0 and Day 45, faecal samples were collected
from each animal, immediately snap-frozen, and stored at
-80.degree. C. Caeca (including content) were immediately
snap-frozen and stored at -80.degree. C. A bacterial identification
test was performed daily by plating the bacteria.
[0297] Histopathology
[0298] At the end of the experiment, hind paws were stored in
tissue fixative. Samples were transferred into decalcification
solution. Tissue samples were processed, sectioned and stained with
Haematoxylin & Eosin. Sections were scored by a qualified
histopathologist, blind to the experimental design, for 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 representative
pictures.
TABLE-US-00002 TABLE 1 Histopathology Scoring System Grade
Description Inflammation 0 Normal joint 1 Mild synovial hyperplasia
with inflammation dominated by neutrophils. Low numbers of
neutrophils and macrophages in joint space. 2 Synovial hyperplasia
with moderate to marked inflammation involving both neutrophils and
macrophages. Neutrophils and macrophages in joint space; may be
some necrotic tissue debris. 3 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 debris. Articular cartilage damage 0 Normal joint 1
Articular cartilage shows only mild degenerative change. Early
pannus formation may be present peripherally. 2 Articular cartilage
shows moderate degenerative change and focal loss. Pannus formation
is present focally. 3 Significant disruption and loss of articular
cartilage with extensive pannus formation. Damage to the underlying
metaphyseal bone 0 Normal joint 1 No change to underlying
metaphyseal bone. 2 May be focal necrosis or fibrosis of
metaphyseal bone. 3 Disruption or collapse of metaphyseal bone.
Extensive inflammation, necrosis or fibrosis extending to medullary
space of the metaphysis.
[0299] Results and Analysis
[0300] Survival and Non-specific Clinical Observations
[0301] Some animals 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.
[0302] Two animals were culled or found dead during the
pre-treatment period (Day -14 to Day 0): one animal in Group 1
(vehicle-treated, animal arrived from supplier with broken leg and
was culled) and one animal in Group 6 (biotherapeutic
#433-treated).
[0303] Ten animals were culled due to the severity of the clinical
signs of arthritis: five animals in Group 1 (vehicle-treated) and
five animals in Group 6 (biotherapeutic #433-treated).
[0304] Four animals 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 animals in Group 1 (vehicle-treated) and
one animal in Group 6 (biotherapeutic #433-treated).
[0305] Bodyweights
[0306] 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 FIG. 19. Data
from animals culled prior to the scheduled end of the experiment
were excluded from the analyses.
[0307] 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. 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 FIG. 20. Data from
animals culled prior to the scheduled end of the experiment and
from Satellite animals 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.
[0308] The onset of clinical signs of arthritis was associated with
a significant bodyweight loss on Day 26 and Day 28 (p<0.0001)
when compared to Day 0 in the vehicle-treated group.
[0309] Clinical Observations
[0310] 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 FIG. 21. Data recorded from animals culled
prior to the end of the experiment were excluded from the analysis.
When animals 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.
[0311] A significant 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.
[0312] Biotherapeutic #433 induced a reduction of the clinical
scores when compared to the vehicle-treated group from Day 28 until
Day 45, although the difference was non-significant.
[0313] Cell Proliferation Analysis
[0314] To validate the assay, splenocytes were cultured in the
presence of soluble anti-CD3 and anti-CD28 (anti-CD3/CD28) as
positive control stimuli to confirm the proliferative potential of
the cells. Strong proliferative responses to anti-CD3/CD28 were
seen in all experimental groups, showing cells were healthy, viable
and able to respond to activation signals.
[0315] To test the proliferative response in presence of Collagen
II (CII), splenocytes were cultured in the presence of CII at 50
.mu.g/ml. Splenocyte proliferative response to CII were analysed by
two-way ANOVA followed by Sydak's post-test for multiple
comparisons between unstimulated and CII-stimulated splenocytes and
one-way ANOVA followed by Dunnett's post-test for comparison of
CII-stimulated response in different experimental groups with the
vehicle-treated group. The data are presented in FIG. 22.
[0316] CII induced a highly significant increase of
.sup.3H-thymidine incorporation (cpm) when compared to the
unstimulated splenocytes in the vehicle-treated group
(p<0.0001).
[0317] The groups treated with biotherapeutic #433 demonstrated
significantly lower levels of CII-induced splenocyte proliferation
than the vehicle-treated group.
[0318] Cytokine Levels in Tissue Culture Supernatants
[0319] Levels of each cytokine were measured in tissue culture
supernatants derived from anti-CD3/CD28 stimulated cultures by
luminex analysis. These showed robust responses for all 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-.gamma.=15,581 pg/ml; TNF-.alpha.=76 pg/ml).
[0320] The following sections summarise the data obtained from the
Collagen II-stimulated cultures. Where applicable, statistical
analyses of the differences between cytokine levels in supernatants
of unstimulated and CII-stimulated splenocytes 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 CII-stimulated response in
biotherapeutic-treated groups with the vehicle-treated group. There
was no significant difference in cytokine levels between the groups
in both cases. This is likely due to the small sample size used
(n=3).
[0321] In order to more accurately present the distribution of the
data for the cytokines with substantial spread of the data, these
are presented as scatter plots.
[0322] The group means of IL-4 in tissue culture supernatants after
stimulation with CII were <5 pg/ml. These are not considered
biologically significant and not included here. The group means of
TNF-.alpha. in tissue culture supernatants after stimulation with
collagen were below limit of quantitation.
[0323] Supernatant Levels of IFN-.gamma. (FIG. 23)
[0324] Along with IL-17, IFN-.gamma. is the major cytokine driving
disease in the CIA model. The scatter plot in FIG. 23 demonstrates
IFN-.gamma. levels after CII stimulation, with group median being
higher for the Vehicle-treated group compared to the
biotherapeutic.
[0325] Supernatant Levels of IL-17A (FIG. 24)
[0326] Levels of IL-17A were 50 pg/ml in CII-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.
[0327] Supernatant Levels of IL-10 (FIG. 25)
[0328] Levels of IL-10 in Vehicle-treated group were 13 pg/ml and
2.1 pg/ml for CII-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.
[0329] Supernatant Levels of IL-6 (FIG. 26)
[0330] Inflammatory cytokines such as IL-6 and TNF-.alpha. are not
typically produced at high levels in anti-CII cultures. However,
their levels may be altered as a result of immune modulation.
Levels of IL-6 in CII-stimulated cultures were modest, reaching 10
pg/ml. Although higher than in media control cultures, these
differences were too small to provide rationale for performing
statistical analyses.
[0331] Microbiological Analysis
[0332] 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.
[0333] Following the improved bacterial preparation method,
consistently high doses of bacterial strain were administered from
Day -2 and Day -3 as indicated by the high OD values measured.
[0334] Faecal samples were collected and snap-frozen on Day -14,
Day 0 and at termination.
[0335] Histopathology
[0336] The histopathology results are shown in FIGS. 65-69. 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.
[0337] 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 debris and inflammation and disruption of the
joint and bone structure by fibrosis and inflammation.
[0338] The incidence of histopathological changes was: vehicle--80%
(16/20); Biotherapeutic #433--55% (12/22). Treatment with
Biotherapeutic #433 reduced the incidence of histopathological
scores in mouse hind limbs when compared to the vehicle-treated
group (see FIGS. 65-68). Histopathology scores were analysed by
one-way ANOVA for non-parametric data (Kruskal-Wallis test)
followed by Dunn's post-test for multiple comparisons to the
vehicle-treated group, although the reduction achieved by
Biotherapeutic #433 was not statistically significant in this
analysis. Biotherapeutic #433 induced a reduction of the joint
inflammation scores observed in histopathology when compared to the
vehicle-treated group. Biotherapeutic #433 induced a reduction of
the cartilage damage scores observed in histopathology when
compared to the vehicle-treated group. Biotherapeutic #433 induced
a reduction of the bone damage scores observed in histopathology
when compared to the vehicle-treated group. Biotherapeutic #433
induced a reduction of the total histopathology scores when
compared to the vehicle-treated group.
[0339] Summary
[0340] 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 #433 was shown to
be effective at treating arthritis in this model. Biotherapeutic
#433 was effective for reducing the severity of the clinical scores
and for reducing pathological disease in the joints, as
demonstrated in the histopathological analysis.
[0341] Proliferative recall responses to Collagen II were seen in
splenocyte cultures from all experimental groups. The
collagen-specific response was significantly reduced following
treatment with biotherapeutic #433 (Group 5).
[0342] Most of the T cell cytokines tested showed detectable
increases between Collagen II-stimulated and media controls in the
Vehicle-treated group. These increases were not as obvious in the
biotherapeutic-treated group. This broadly supports the
proliferative recall responses to Collagen II described above.
[0343] There was evidence of suppression of the Th1/Th17 axis,
which is the pathogenic response in this model and in human RA.
Correlation of reduced levels of cytokines with reduced
proliferation 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
[0344] The mice tested in Example 1 were subjected to further
analyses to further characterise the effect of the compositions of
the invention on the allergic asthma inflammatory response.
[0345] Materials and Methods
[0346] Blood withdrawal and serum preparation on day 14. Blood
samples of animals were collected via cardiac puncture. Serum was
isolated from the blood sample by centrifugation for 5 min at
14000g and stored at -20 .degree. C.
[0347] Organ removal on day 14. Collection of the left lung lobe in
formalin for follow-on histological analysis. Collection of the
right lung lobes (all remaining lobes) and removal of serum for
snap freezing and follow-on analysis. Remaining BAL fluid was snap
frozen for follow-on analysis.
[0348] Measurement of Antibody Levels in Serum and BAL Fluid
[0349] Total IgE and house-dust-mite (HDM) specific IgG1 antibody
production were measured in the BAL and serum by ELISA assay.
[0350] Isolation of Lung and Histological Analysis
[0351] Left lung lobes were fixed in formalin followed by embedment
in paraffin, sectioning, and staining with hematoxylin and eosin
and PAS. Subsequent 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:
[0352] 0--normal
[0353] 1--mild inflammatory infiltrates
[0354] 2--moderate inflammatory infiltrates
[0355] 3--marked inflammatory infiltrates
[0356] 4--severe inflammatory infiltrates
[0357] 5--very severe inflammatory infiltrates
[0358] In each field of view, airways were measured in size and
mucus cell numbers were quantified/um.
[0359] Measurement of Inflammatory Mediators in Lung Tissue
[0360] Right lung lobes (all remaining lobes) isolated for
quantification of inflammatory mediators were snap frozen for
subsequent measurement of CCL11, IFN-gamma, IL-1 alpha, IL-1 beta,
IL-4, IL-5, IL-9, IL-17A, CXCL1, CCL3, CXCL2 and CCLS by
commercially available multiplex assay (Merck-Millipore). Analysis
was performed according to the manufacturer's instructions.
[0361] Results and Analysis
[0362] The results of the experiments are shown in FIGS. 27-45.
[0363] In support of the findings described in Example 1, analysis
of the cellular infiltrates in the lung tissue of mice treated with
strain 433 showed a notable and statistically significant reduction
in mean inflammation score (see FIGS. 31 and 33).
[0364] Antibody levels in the BAL fluid and serum were analysed
(see FIGS. 27-30). No clear effect of the bacterial treatment on
serum 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 negative controls do
not appear to have behaved as would be expected. Also, the baseline
serum antibody levels could have masked any changes.
[0365] Similarly, no clear effect of the bacterial treatment on
cytokine levels in lung tissue was observed (see FIGS. 35-45).
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 negative controls do not appear to have behaved as
would be expected. It is also possible that the mechanism of action
involved influences earlier cytokine responses that were no longer
detectable 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 have been fully representative 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 Neutrophilic Asthma
[0366] The mice tested in Example 2 were subjected to further
analyses to further characterise the effect of the compositions of
the invention on the neutrophilic response associated with severe
asthma.
[0367] Materials and Methods
[0368] Organ removal on day 18. Collection of the left lung lobe in
formalin for follow-on histological analysis. Collection of the
right lung lobes (all remaining lobes) and removal of serum for
snap freezing and follow-on analysis. Remaining BAL fluid was snap
frozen for follow-on analysis.
[0369] Measurement of inflammatory mediators in lung tissue
(follow-on analysis). Right lung lobes (all remaining lobes)
isolated for quantification of inflammatory mediators were snap
frozen for subsequent measurement of IFN-gamma, IL-1 alpha, IL-1
beta, CXCL1, CCL3, CXCL2, CCLS, IL-17A, TNF-alpha, IL-17F, IL-23
and IL-33 by commercially available multiplex assay
(Merck-Millipore). Analysis was performed according to the
manufacturer's instructions.
[0370] Measurement of antibody levels in serum and BAL fluid
(follow-on analysis). House-dust-mite (HDM) specific IgG1 and IgG2a
antibody production were measured in the BAL and serum by ELISA
assay.
[0371] 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. Subsequent 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:
[0372] 0--normal
[0373] 1--mild inflammatory infiltrates
[0374] 2--moderate inflammatory infiltrates
[0375] 3--marked inflammatory infiltrates
[0376] 4--severe inflammatory infiltrates
[0377] 5--very severe inflammatory infiltrates
[0378] Results and Analysis
[0379] The results of the experiments are shown in FIGS. 46-63.
[0380] Further analysis of antibody levels revealed that the
efficacy of bacterial strain 433 was also reflected in reduced
HDM-specific IgG1 levels in the BAL fluid and serum (see FIGS. 46
and 48). Firm conclusions regarding an effect on IgG2a levels
cannot be drawn. Overall, the data from the antibody analysis is
suggestive of a reduction related to an overall reduced
inflammatory response, as opposed to a selective effect on antibody
isotype switching.
[0381] 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 negative controls do not appear to have behaved as
necessarily would be expected. It is also possible that the
mechanism of action involves influencing earlier cytokine responses
that were no longer detectable 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 have been fully representative 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 433 was
shown, and the positive control anti-IL-17 Ab generally behaved as
expected.
[0382] With the above caveats, the data in FIG. 55 suggests that
treatment with strain 433 may achieve a reduction in the levels of
IFN.gamma., 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. IFN.gamma. is involved in
the Th17 pathway. Taking this dataset together, a clear conclusion
can be drawn that strains 433 was effective at protecting mice
against inflammation in this mouse model of severe neutrophilic
asthma.
Example 6
Efficacy of Bacterial Inocula in a Mouse Model of Multiple
Sclerosis
[0383] Summary
[0384] 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 have a striking
effect on disease incidence and disease severity.
[0385] Strain
[0386] 433: bacteria deposited under accession number NCIMB
43283
[0387] Study Design
[0388] Groups:
[0389] 1. Negative control group. Treatment with vehicle control
(per oral).
[0390] 5. Treatment with therapeutic bacteria inoculum strain 433
(per oral).
[0391] 9. Positive control group. Treatment with Dexamethasone
(i.p.).
[0392] 10. Untreated Control Group.
[0393] Number of mice per group=10
[0394] Days -14 to day 27: Daily administration of vehicle control
per oral (Group 1).
[0395] Days -14 to day 27: Daily administration of therapeutic
bacteria inoculum per oral (Group 5).
[0396] Days 0-28: administration of Dexamethasone (i.p.) three
times a week (Group 9)
[0397] Day 0: MOG35-55 (myelin oligodendrocyte glycoprotein--2
mg/ml) and CFA (2 mg/ml MTB) were mixed 1:1 resulting in 1 mg/ml
solutions. 100 .mu.1 of the peptide-CFA mixture was injected
subcutaneously into each hind leg. Administration of pertussis
toxin intraperitoneally (300 ng).
[0398] Day 1: Administration of pertussis toxin intraperitoneally
(300 ng).
[0399] Days 7-onwards: Measurement of disease incidence and weight
three times a week.
[0400] Endpoints and Analysis
[0401] 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).
[0402] Monitoring
[0403] On the indicated days mice were weighed and observed for
disease activity score and disease incidence. [0404] Disease
activity score observations: [0405] 0--No obvious changes in motor
function compared to non-immunized mice. [0406] 0.5--Tip of tail is
limp. [0407] 1.0--Limp tail. [0408] 1.5--Limp tail and hind leg
inhibition. [0409] 2.0--Limp tail and weakness of hind legs. [0410]
OR--There are obvious signs of head tilting when the walk is
observed. The balance is poor. [0411] 2.5--Limp tail and dragging
of hind legs. [0412] OR--There is a strong head tilt that causes
the mouse to occasionally fall over. [0413] 3.0--Limp tail and
complete paralysis of hind legs. [0414] 3.5--Limp tail and complete
paralysis of hind legs. [0415] In addition to: Mouse is moving
around the cage, but when placed on its side, is unable to right
itself. [0416] Hind legs are together on one side of body. [0417]
4.0--Limp tail, complete hind leg and partial front leg paralysis.
[0418] Mouse is minimally moving around the cage but appears alert
and feeding [0419] 4.5--Complete hind and partial front leg
paralysis, no movement around the cage. [0420] Mouse is immediately
euthanized and removed from cage. [0421] 5.0 Mouse is euthanized
due to severe paralysis.
[0422] When an animal has equal or greater disease activity score
of 1, it is considered to have a positive disease incidence
score.
[0423] Results
[0424] The results of the study are shown in FIGS. 70 and 71.
[0425] Disease induction in the negative control groups was
successful with high scores shown by the vehicle control and the
untreated control. The effect of treatment with strain 433 was
striking and the mice treated with strain 433 exhibited notably
reduced disease incidence and disease severity. These data indicate
the strain 433 may be useful for treating or preventing multiple
sclerosis.
Example 7
Stability Testing
[0426] A composition described herein containing at least one
bacterial strain described herein is stored in a sealed container
at 25.degree. C. or 4.degree. C. and the container is placed in an
atmosphere having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95%
relative humidity. After 1 month, 2 months, 3 months, 6 months, 1
year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%,
70%, 80% or 90% of the bacterial strain shall remain as measured in
colony forming units determined by standard protocols.
TABLE-US-00003 Sequences (Roseburia hominis strain A2-181 16S
ribosomal RNA gene, partial sequence-AY804148) SEQ ID NO: 1 1
taaaggttga tcctggctca ggatgaacgc tggaggcgtg cttaacacat gcaagtcgaa
61 cgaagcactt taattgattt cttcggaatg aagtttttgt gactgagtgg
cggacgggtg 121 agtaacgcgt gggtaacctc gctcatacag ggggataaca
gttggaaacg actgctaata 181 ccgcataagc gcacaggatt gcatgatcca
gtgtgaaaaa ctccggtggt atgagatgga 241 cccgcgtctg attagccagt
tggcggggta acggcccacc aaagcgacga tcagtagccg 301 acctgagagg
gtgaccggcc acattgggac tgagacacgg cccaaactcc tacgggaggc 361
agcagtgggt aatattgcac aatgggggaa accctgatgc agcgacgccg agtgagcgaa
421 gaagtatttc ggtatgtaaa gctctatcag caggaagaag aatgacggta
cctgactaaa 481 aagcaccggc taaatacgtg ccagcagccg cggtaatacg
tatggtgcaa gcgttatccg 541 gatttactgg gtgtaaaggg agcgcaggcg
gtacggcaag tctgatgtga aatcccgggg 601 ctcaaccccg gtactgcatt
ggaaactgtc ggactagggt gtctgagggg taagtggaat 661 tcctagtgta
gcggtgaaat gcgtagatat taggaggaac accagtggcg aaggcggctt 721
actggacgat tactgacgct gaggctcgaa agcgtgggga gcaaacagga ttagataccc
781 tggtagtcca cgccgtaaac gatgaatact aggtgtcggg gagcattgct
cttcggtgcc 841 gcagcaaacg caataagtat tccacctggg gagtacgttc
gcaagaatga aactcaaagg 901 aattgacggg gacccgcaca agcggtggag
catgtggttt aattcgaagc aacgcgaaga 961 accttaccaa gtcttgacat
cccactgaca aagtatgtaa tgtactttct cttcggagca 1021 gtggtgacag
gtggtgcatg gttgtcgtca gctcgtgtcg tgagatgttg ggttaagtcc 1081
cgcaacgagc gcaaccccta ttcttagtag ccagcggttt ggccgggcac tctagggaga
1141 ctgccaggga taacctggag gaaggtgggg atgacgtcaa atcatcatgc
cccttatgac 1201 ttgggctaca cacgtgctac aatggcgtaa acaaagggaa
gcaatcccgc gagggggagc 1261 aaatctcaaa aataacgtct cagttcggac
tgtagtctgc aactcgacta cacgaagctg 1321 gaatcgctag taatcgcgaa
tcagaatgtc gcggtgaata cgttcccggg tcttgtacac 1381 accgcccgtc
acaccatggg agttggtaat gcccgaagtc agtgacccaa ccgcaaggag 1441
ggagctgccg aagcaggact gataactggg gtgaagtcgt aacaagt (Roseburia
hominis A2-183 16S rRNA gene, type strain A2-183T-AJ270482) SEQ ID
NO: 2 1 gatcctggct caggatgaac gctggcggcg tgcttaacac atgcaagtcg
aacgaagcac 61 tttaattgat ttcttcggaa tgaagttttt gtgactgagt
ggcggacggg tgagtaacgc 121 gtgggtaacc tgcctcatac agggggataa
cagttggaaa cgactgctaa taccgcataa 181 gcgcacagga ttgcatgatc
cagtgtgaaa aactccggtg gtatgagatg gacccgcgtc 241 tgattagcca
gttggcgggg taacggccca ccaaagcgac gatcagtagc cgacctgaga 301
gggtgaccgg ccacattggg actgagacac ggcccaaact cctacgggag gcagcagtgg
361 ggaatattgc acaatggggg aaaccctgat gcagcgacgc cgcgtgagcg
aagaagtatt 421 tcggtatgta aagctctatc agcagggaag aagaatgcgg
tacctgacta agaagcaccg 481 gctaaatacg tgccagcagc cgcggtaata
cgtatggtgc aagcgttatc cggatttact 541 gggtgtaaag ggagcgcagg
cggtacggca agtctgatgt gaaatcccgg ggctcaaccc 601 cggtactgca
ttggaaactg tcggactaga gtgtcggagg ggtaagtgga attcctagtg 661
tagcggtgaa atgcgtagat attaggagga acaccagtgg cgaaggcggc ttactggacg
721 attactgacg ctgaggctcg aaagcgtggg gagcaaacag gattagatac
cctggtagtc 781 cacgccgtaa acgatgaata ctaggtgtcg gggagcattg
ctcttcggtg ccgcagcaaa 841 cgcaataagt attccacctg gggagtacgt
tcgcaagaat gaaactcaaa ggaattgacg 901 gggacccgca caagcggtgg
agcatgtggt ttaattcgaa gcaacgcgaa gaaccttacc 961 aagtcttgac
atcccactga cagagtatgt aatgtacttt ctcttcggag cagtggtgac 1021
aggtggtgca tggttgtcgt cagctcgtgt cgtgagatgt tgggttaagt cccgcaacga
1081 gcgcaacccc tattcttagt agccagcggt tcggccgggc actctaggga
gactgccagg 1141 gataacctgg aggaaggtgg ggatgacgtc aaatcatcat
gccccttatg acttgggcta 1201 cacacgtgct acaatggcgt aaacaaaggg
aagcaatccc gcgaggggga gcaaatctca 1261 aaaataacgt ctcagttcgg
actgtagtct gcaactcgac tacacgaagc tggaatcgct 1321 agtaatcgcg
aatcagaatg tcgcggtgaa tacgttcccg ggtcttgtac acaccgcccg 1381
tcacaccatg ggagttggta atgcccgaag tcagtgaccc aaccgcaagg agggagctgc
1441 cgaaggcagg actgataact ggggtgaagt cgtaacaagg gtacg (consensus
16S rRNA sequence for Roseburia hominis strain 433) SEQ ID NO: 3
AAGAGTTTGGGHCAGGCTCAGGATGAACGCTGGCGGCGTGCTTAACACATGCAAGTCGAACGAAGCACTTTAAT-
TGA
TTTCTTCGGAATGAAGTTTTTGTGACTGAGTGGCGGACGGGTGAGTAACGCGTGGGTAACCTGCCTCATACAGG-
GGG
ATAACAGTTGGAAACGACTGCTAATACCGCATAAGCGCACAGGATTGCATGATCCAGTGTGAAAAACTCCGGTG-
GTA
TGAGATGGACCCGCGTCTGATTAGCCAGTTGGCGGGGTAACGGCCCACCAAAGCGACGATCAGTAGCCGACCTG-
AGA
GGGTGACCGGCCACATTGGGACTGAGACACGGCCCAAACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAA-
TGG
GGGAAACCCTGATGCAGCGACGCCGCGTGAGCGAAGAAGTATTTCGGTATGTAAAGCTCTATCAGCAGGGAAGA-
AGA
ATGACGGTACCTGACTAAGAAGCACCGGCTAAATACGTGCCAGCAGCCGCGGTAATACGTATGGTGCAAGCGTT-
ATC
CGGATTTACTGGGTGTAAAGGGAGCGCAGGCGGTACGGCAAGTCTGATGTGAAATCCCGGGGCTCAACCCCGGT-
ACT
GCATTGGAAACTGTCGGACTAGAGTGTCGGAGGGGTAAGTGGAATTCCTAGTGTAGCGGTGAAATGCGTAGATA-
TTA
GGAGGAACACCAGTGGCGAAGGCGGCTTACTGGACGATTACTGACGCTGAGGCTCGAAAGCGTGGGGAGCAAAC-
AGG
ATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAATACTAGGTGTCGGGGAGCATTGCTCTTCGGTGCCGCA-
GCA
AACGCAATAAGTATNCCACCTGGGGAGTACGTTCGCAAGAATGAAACTCAAAGGAATTGACGGGGACCCGCACA-
AGC
GGTGGAGCNTGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAAGTCTTGACATCCCACTGACAGAGTATG-
TAA
TGTACTTTCTCTTCGGAGCAGTGGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT-
AAG
TCCCGCAACGAGCGCAACCCCTATTCTTAGTAGCCAGCGGTTTGGCCGGGCACTCTAGGGAGACTGCCAGGGAT-
AAC
CTGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACTTGGGCTACACACGTGCTACAATGGCGT-
AAA
CAAAGGGAAGCAATCCCGCGAGGGGGAGCAAATCTCAAAAATAACGTCTCAGTTCGGACTGTAGTCTGCAACTC-
GAC
TACACGAAGCTGGAATCGCTAGTAATCGCGAATCAGAATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACAC-
CGC
CCGTCACACCATGGGAGTTGGTAATGCCCGAAGTCAGTGACCCAACCGCAAGGAGGGAGCTGCCGAAGGCAGGA-
CTG ATAACTGGGGTGAAGTCTACRSAGGGTAGCCGTRMMC SEQ ID NO: 4 (strain 433
genome sequence)-see electronic sequence listing.
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Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220257668A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20220257668A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References