U.S. patent application number 17/679243 was filed with the patent office on 2022-08-18 for compositions comprising of bacterial strains.
The applicant listed for this patent is 4D Pharma Research Limited. Invention is credited to Marsilio ADRIANI, Suaad AHMED, Maria CHRISTOFI, Aurelie Pascale Patricia COUTURIER-MAILLARD, Philip COWIE, Margaret Inkster DELDAY, Anna ETTORRE, Emma Elizabeth Clare HENNESSY, Amy Beth HOLT, Delphine Louise Claudette LAUTE-CALY, Imke Elisabeth MULDER, DOMENICO PANZICA, Emma RAFTIS.
Application Number | 20220257745 17/679243 |
Document ID | / |
Family ID | |
Filed Date | 2022-08-18 |
United States Patent
Application |
20220257745 |
Kind Code |
A1 |
PANZICA; DOMENICO ; et
al. |
August 18, 2022 |
COMPOSITIONS COMPRISING OF BACTERIAL STRAINS
Abstract
The invention provides compositions comprising bacterial strains
for stimulating the immune system and treating and preventing
diseases.
Inventors: |
PANZICA; DOMENICO;
(Aberdeen, GB) ; HOLT; Amy Beth; (Aberdeen,
GB) ; AHMED; Suaad; (Aberdeen, GB) ; ETTORRE;
Anna; (Aberdeen, GB) ; MULDER; Imke Elisabeth;
(Aberdeen, GB) ; COWIE; Philip; (Aberdeen, GB)
; RAFTIS; Emma; (Aberdeen, GB) ; HENNESSY; Emma
Elizabeth Clare; (Aberdeen, GB) ; LAUTE-CALY;
Delphine Louise Claudette; (Aberdeen, GB) ;
COUTURIER-MAILLARD; Aurelie Pascale Patricia; (Aberdeen,
GB) ; DELDAY; Margaret Inkster; (Kemnay, GB) ;
ADRIANI; Marsilio; (Aberdeen, GB) ; CHRISTOFI;
Maria; (Aberdeen, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
4D Pharma Research Limited |
Aberdeen |
|
GB |
|
|
Appl. No.: |
17/679243 |
Filed: |
February 24, 2022 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17024628 |
Sep 17, 2020 |
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17679243 |
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PCT/EP2019/056894 |
Mar 19, 2019 |
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17024628 |
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International
Class: |
A61K 39/09 20060101
A61K039/09; A61K 35/744 20060101 A61K035/744 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 19, 2018 |
GB |
1804384.4 |
Jun 18, 2018 |
EP |
18178350.7 |
Jun 18, 2018 |
GB |
1809953.1 |
Jul 20, 2018 |
GB |
1811900.8 |
Jul 30, 2018 |
GB |
1812378.6 |
Aug 17, 2018 |
GB |
1813423.9 |
Aug 17, 2018 |
GB |
1813444.5 |
Oct 16, 2018 |
GB |
1816834.4 |
Oct 29, 2018 |
GB |
1817641.2 |
Jan 29, 2019 |
GB |
1901199.8 |
Jan 29, 2019 |
GB |
1901218.6 |
Feb 13, 2019 |
GB |
1901992.6 |
Feb 13, 2019 |
GB |
1901993.4 |
Claims
1. A composition comprising a bacterial strain of the species
Enterococcus gallinarum, for use in stimulating the immune system
in a subject.
2.-21. (canceled)
Description
CROSS-REFERENCE
[0001] This application is a continuation of U.S. application Ser.
No. 17/024,628, filed Sep. 17, 2020, which is a continuation of
International Application No. PCT/EP2019/056894, filed Mar. 19,
2019, which claims the benefit of Great Britain Application No.
1804384.4, filed Mar. 19, 2018, Great Britain Application No.
1809953.1, filed Jun. 18, 2018; Great Britain Application No.
1811900.8, filed Jul. 20, 2018; Great Britain Application No.
1812378.6, filed Jul. 30, 2018; Great Britain Application No.
1813423.9, filed Aug. 17, 2018; Great Britain Application No.
1813444.5, filed Aug. 17, 2018; Great Britain Application No.
1816834.4, filed Oct. 16, 2018; Great Britain Application No.
1817641.2, filed Oct. 29, 2018; European Application No.
18178350.7, filed Jun. 18, 2018; Great Britain Application No.
1901199.8, filed Jan. 29, 2019; Great Britain Application No.
1901218.6, filed Jan. 29, 2019; Great Britain Application No.
1901992.6, filed Feb. 13, 2019; Great Britain Application No.
1901993.4, filed Feb. 13, 2019, all of which are hereby
incorporated by reference 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 Oct. 6, 2020, is named 56708_740_302 Sequence Listing.txt and is
8,192 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, in
particular in stimulating the immune system 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 Finnicutes [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 [3-4].
[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 [5-6]. 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, [7-8]). 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 [9] and [10] for
reviews). Certain Streptococcus and Veillonella strains, and to a
lesser extent, Enterococcus and Lactobaccillus strains have been
suggested to have immunomodulatory effects, with varying effects on
different cytokines in vitro, suggesting that data obtained in
vitro with individual strains are unlikely to adequately represent
immune responses to mixtures of gut microbiota communities in vivo
[88]. 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 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 compositions comprising a
bacterial strain of the species Enterococcus gallinarum that can be
used in stimulating the immune system and treating and preventing
disease. The inventors have identified that strains of the species
Enterococcus gallinarum can potently activate the immune system and
can treat cancer, which indicates that they may able to also treat
other diseases where activation of the immune system may be useful.
The invention therefore provides a composition comprising a
bacterial strain of the species Enterococcus gallinarum, for use in
stimulating the immune system in subject.
[0009] In further aspects, the invention provides a composition
comprising a bacterial strain of the species Enterococcus
gallinarum, for use in treating, preventing or delaying
immunosenescence. In further aspects, the invention provides a
composition comprising a bacterial strain of the species
Enterococcus gallinarum, for use as a vaccine adjuvant.
[0010] In further aspects, the invention provides a composition
comprising a bacterial strain of the species Enterococcus
gallinarum, for use in enhancing a cell therapy, such as CAR-T.
[0011] Preferably, the bacteria used in the invention is the strain
deposited under accession number 42488 at NCIMB.
[0012] In further preferred embodiments, the bacteria used in the
invention is the strain deposited under accession number 42761 at
NCIMB.
BRIEF DESCRIPTION OF DRAWINGS
[0013] FIG. 1A: Mouse model of breast cancer--changes in tumour
volume post tumour induction and a table indicating the statistical
significance between each two treatments at each time point.
[0014] FIG. 1B: Upper panel: Area of necrosis in EMT6 tumours
(Untreated n=6, Vehicle n=6, MRx0518 n=8). Lower panel: Percentage
of dividing cells in EMT6 tumours. P=0.019 (Untreated n=4, total
number cells counted=37201, Vehicle n=6, total number of cells
counted=64297, MRx0518 n=6, total number cells counted=33539).
[0015] FIG. 1C: Mouse model of breast cancer--infiltrating immune
cells. Scatter plots represent cell counts of different immune
markers from individual animals from each treatment group.
[0016] FIG. 1D: Mouse model of breast cancer--Cytokine production
in tumour lysates. Columns represent the mean pg/mL of total
protein from each treatment group. *p<0.05 between groups using
one-way ANOVA followed by Dunnett's multiple comparisons test.
[0017] FIG. 1E: Mouse model of breast cancer --Cytokine production
in blood plasma. Columns represent the mean pg/mL from each
treatment group (+/-SEM).
[0018] FIG. 1F: Representative images of ileum cryosections from
vehicle, MRx0518 and anti-CTLA-4-treated mice immuno-labelled with
antibodies against CD8.alpha. (lower panels) and counter-stained
with DAPI (upper panels).
[0019] FIG. 1G: Plot quantifying animal study subsets with more
than 3 CD8.alpha.+ cells per field taken from the ileum crypt
region of mice treated with vehicle, MRx0518 or anti-CTLA-4.
[0020] FIG. 2: Mouse model of lung cancer--changes in tumour volume
post tumour induction and a table indicating the statistical
significance between each two treatments at each time point.
[0021] FIG. 3A: Mouse model of liver cancer--liver weight.
[0022] FIG. 3B: Mouse model of kidney cancer--changes in tumour
volume post tumour induction and a table indicating the statistical
significance between each two treatments at each time point.
[0023] FIG. 4A: Cytokine levels (pg/mLmL) in immature dendritic
cells (No bacteria).
[0024] FIG. 4B: Cytokine levels (pg/mLmL) in immature dendritic
cells after the addition of LPS.
[0025] FIG. 4C: Cytokine levels (pg/mLmL) in immature dendritic
cells after the addition of MRx0518.
[0026] FIG. 4D: Cytokine levels (pg/mLmL) in immature dendritic
cells after the addition of MRx0518 and LPS.
[0027] FIG. 5A: Cytokine levels in THP-1 cells (No bacteria).
[0028] FIG. 5B: Cytokine levels in THP-1 cells after addition of
bacterial sediment.
[0029] FIG. 5C: Cytokine levels in THP-1 cells after the addition
of MRx0518MRx0518 alone or in combination with LPS.
[0030] FIG. 6: Immunostimulatory response--TNF.alpha.
[0031] FIG. 7: Immunostimulatory response--TNF.alpha.
[0032] FIG. 8: Immunostimulatory response--IL-12p70
[0033] FIG. 9: Immunomodulatory response--IL-10
[0034] FIG. 10: Immunostimulatory response--IL-8
[0035] FIG. 11: Immunostimulatory response--IL-23
[0036] FIG. 12: Immunostimulatory response--IL-1.beta.
[0037] FIG. 13: Immunostimulatory response--IL-6
[0038] FIG. 14: Mechanism of action--activation of NF.kappa.B
[0039] FIG. 15: Mechanism of action--activation of TLR5
[0040] FIG. 16A: A schematic representation of the treatment
schedule of the different groups used in Example 8 described herein
below.
[0041] FIG. 16B: Mean tumour volume in mice bearing a tumour formed
by EMT-6 cells. The mice were either untreated or treated with a
YCFA vehicle (Vehicle), MRx0518 bacteria in YCFA medium (MRx0518),
an anti-CTLA-4 antibody and YCFA medium (Anti-CTLA-4) or a
combination of MRx0518 and the anti-CTLA-4 antibody. The provided
table indicates the statistical significance between each two
treatments at each time point.
[0042] FIG. 17: Mouse model of breast cancer--tumour volume.
[0043] FIG. 18: API 50 CHL profile of MRx0554.
[0044] FIG. 19: Mechanism of action--TLR9 activation by MRx0518
(MRx0518.sub.LV), heat-killed MRx0518 (MRx0518.sub.HK) and MRx0518
culture supernatant (MRx0518.sub.SN) in HEK-Blue.TM. hTLR9 reporter
cell lines. ODN2006 was used as a positive control and YCFA medium
was included as a negative control for MRx0518.sub.SN. The bar
graph represents an average of at least three biological
replicates. Statistical analysis was performed using GraphPad Prism
(ordinary one-way ANOVA analysis followed by Tukey's Multiple
comparison test). Statistically significant differences with the
relevant control are shown on the graphs as **** (p<0.0001).
[0045] FIGS. 20A-20B: Induction of T-cell differentiation in a
population of (FIG. 20A) T-helper cells and (FIG. 20B) Cytotoxic T
Lymphocytes (CTL), using heat-killed MRx0518 (HK 518), Supernatant
from MRx0518 culture or RPMI medium, without addition of cytokines
(no cyto). *=p.ltoreq.0.05; **=p.ltoreq.0.01; ***=p.ltoreq.0.001;
****=p.ltoreq.0.0001.
[0046] FIGS. 21A-21D: In-vitro cytokine production by (FIG. 21A)
PBMC cells; (FIG. 21B) Splenocytes; or (FIG. 21C) THP-1 cells;
which were treated with YCFA+medium ("Vehicle") or cell-free
bacterial supernatant of MRx0518 ("MRx0518"). FIG. 21D shows fold
change in cytokine expression following treatment of CaCo-2 cells
with live bacteria ("MRx0518") relative to untreated cells.
[0047] FIG. 21E: In-vitro cytokine production by splenocytes (N=3),
from cells that were either untreated ("Untreated"), treated with
YCFA blank media ("10% YCFA") or treated with MRx0518 cell-free
bacterial supernatant ("10% MRx0518").
[0048] FIG. 21F: Viability of splenocytes extracted from mice (N=4)
as measure by an MTT assay. Cells were either untreated
("Untreated"), treated with YCFA blank media ("10% YCFA") or
treated with MRx0518 cell-free bacterial supernatant ("10%
MRx0518").
[0049] FIGS. 22A-22D: NF-.kappa.B promoter activation in (FIG. 22A)
HEK-Blue.TM.-hNOD2 cells; (FIG. 22B) HEK-Blue.TM.-hTLR4 cells;
(FIG. 22C) HEK-Blue.TM.-hTLR9 cells or (FIG. 22D)
HEK-Blue.TM.-hTLR5 cells. Cells were either untreated, treated with
YCFA medium ("YCFA"), treated with MRx0518 ("MRx0518") or treated
with positive controls.
[0050] FIG. 23: Heat map representing NanoString analysis of EMT6
tumour microenvironment following treatment with YCFA vehicle
("Vehicle") or MRx0518 ("MRx0518").
DISCLOSURE OF THE INVENTION
Bacterial Strains
[0051] The compositions of the invention comprise a bacterial
strain of the species Enterococcus gallinarum. The examples
demonstrate that bacteria of this genus are useful for stimulating
the immune system and for treating disease.
[0052] Enterococcus gallinarum forms coccoid cells, mostly in pairs
or short chains. It is motile and colonies on blood agar or
nutrient agar are circular and smooth. Enterococcus gallinarum
reacts with Lancefield group D antisera. The type strain of
Enterococcus gallinarum is F87/276=PB21=ATCC 49573=CCUG 18658 =CIP
103013=JCM 8728=LMG 13129 32 NBRC 100675 32 NCIMB 702313 (formerly
NCDO 2313)=NCTC 12359 [11]. The GenBank accession number for a 16S
rRNA gene sequence of Enterococcus gallinarum is AF039900
(disclosed herein as SEQ ID NO:1). An exemplary Enterococcus
gallinarum strain is described in [11].
[0053] All microorganism deposits were made under the terms of the
Budapest Treaty and thus viability of the deposit is assured.
Maintenance of a viable culture is assured for 30 years from the
date of deposit. During the pendency of the application, access to
the deposit will be afforded to one determined by the Commissioner
of the United States Patent and Trademark Office to be entitled
thereto. All restrictions on the availability to the public of the
deposited microorganisms will be irrevocably removed upon the
granting of a patent for this application. The deposit will be
maintained for a term of at least thirty (30) years from the date
of the deposit or for the enforceable life of the patent or for a
period of at least five (5) years after the most recent request for
the furnishing of a sample of the deposited material, whichever is
longest. The deposit will be replaced should it become necessary
due to inviability, contamination or loss of capability to function
in the manner described in the specification.
[0054] The Enterococcus gallinarum bacterium deposited under
accession number NCIMB 42488 was tested in the Examples and is also
referred to herein as strain MRx0518. References to MRx0518 and
MRx0518 are used interchangeably. A 16S rRNA sequence for the
MRx0518 strain that was tested is provided in SEQ ID NO:2. Strain
MRx0518 was deposited with the international depositary authority
NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9YA, Scotland) by 4D
Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen,
AB25 2ZS, Scotland) on 16 Nov. 2015 as "Enterococcus sp" and was
assigned accession number NCIMB 42488.
[0055] The genome of strain MRx0518 comprises a chromosome and
plasmid. A chromosome sequence for strain MRx0518 is provided in
SEQ ID NO:3 OF WP2017/085520. A plasmid sequence for strain MRx0518
is provided in SEQ ID NO:4 OF WO2017/085520. These sequences were
generated using the PacBio RS II platform.
[0056] The Enterococcus gallinarum bacterium deposited under
accession number NCIMB 42761 was also tested in the Examples and is
also referred to herein as strain MRx0554. References to MRx0554
and MRx0554 are used interchangeably. Strain MRx0554 was deposited
with the international depositary authority NCIMB, Ltd. (Ferguson
Building, Aberdeen, AB21 9YA, Scotland) by 4D Pharma Research Ltd.
(Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland)
on 22 May 2017 as "Enterococcus gallinarum MRx0554" and was
assigned accession number NCIMB 42761. The genome sequence of this
bacterium is disclosed herein as SEQ ID NO:2 OF W02018/215782. The
genome sequence was assembled from multiple contigs. Ns in the
sequence represent gaps between the contigs. "N" may represent an
A, G, C or T nucleotide. A 16S rRNA gene sequence for the MRx0554
strain is provided in SEQ ID NO:3. SEQ ID NO:3 represents the full
length sequence present in the assembly, rather than a consensus of
the five 16S genes present in MRx0554.
[0057] Bacterial strains closely related to the strains tested in
the examples are also expected to be effective for simulating the
immune system. In certain embodiments, the bacterial strain for use
in the invention has a 16s rRNA gene sequence that is at least 95%,
96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or 2.
Preferably, the sequence identity is to SEQ ID NO:2. Preferably,
the bacterial strain for use in the invention has the 16s rRNA gene
sequence represented by SEQ ID NO:2. In certain embodiments, the
bacterial strain for use in the invention has a 16s rRNA gene
sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9%
identical to SEQ ID NO:3.
[0058] Bacterial strains that are biotypes of the bacterium
deposited under accession number 42488 are also expected to be
effective for stimulating the immune system. A biotype is a closely
related strain that has the same or very similar physiological and
biochemical characteristics.
[0059] Strains that are biotypes of the bacterium deposited under
accession number NCIMB 42488 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
42488. 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). For example, in some
embodiments, a biotype strain has at least 98% sequence identity
across at least 98% of its genome or at least 99% sequence identity
across 99% of its genome. Other suitable sequences for use in
identifying biotype strains may include hsp60 or repetitive
sequences such as BOX, ERIC, (GTG)5 (SEQ ID NO: 4), or REP or [12].
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
42488. In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of strain MRx0518 deposited as NCIMB
42488 and comprises a 16S rRNA gene sequence that is at least 99%
identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ
ID NO:2. In some embodiments, a biotype strain has a sequence with
at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity
to the corresponding sequence of strain MRx0518 deposited as NCIMB
42488 and has the 16S rRNA sequence of SEQ ID NO:2.
[0060] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:3 OF
W02017/085520. In preferred embodiments, the bacterial strain for
use in the invention has a chromosome with at least 90% sequence
identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity) to SEQ ID NO:3 OF W02017/085520 across at least
60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99%
or 100%) of SEQ ID NO:3 OF W02017/085520. For example, the
bacterial strain for use in the invention may have a chromosome
with at least 90% sequence identity to SEQ ID NO:3 OF W02017/085520
across 70% of SEQ ID NO:3 OF WO2017/085520, or at least 90%
sequence identity to SEQ ID NO:3 OF WO2017/085520 across 80% of SEQ
ID NO:3 OF WO2017/085520, or at least 90% sequence identity to SEQ
ID NO:3 OF WO2017/085520 across 90% of SEQ ID NO:3 OF
WO2017/085520, or at least 90% sequence identity to SEQ ID NO:3 OF
WO2017/085520 across 100% of SEQ ID NO:3 OF WO2017/085520, or at
least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across
70% of SEQ ID NO:3 OF WO2017/085520, or at least 95% sequence
identity to SEQ ID NO:3 OF WO2017/085520 across 80% of SEQ ID NO:3
OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:3
OF WO2017/085520 across 90% of SEQ ID NO:3 OF WO2017/085520, or at
least 95% sequence identity to SEQ ID NO:3 OF WO2017/085520 across
100% of SEQ ID NO:3 OF WO2017/085520, or at least 98% sequence
identity to SEQ ID NO:3 OF WO2017/085520 across 70% of SEQ ID NO:3
OF WO2017/085520, or at least 98% sequence identity to SEQ ID NO:3
OF WO2017/085520 across 80% of SEQ ID NO:3 OF WO2017/085520, or at
least 98% sequence identity to SEQ ID NO:3 OF WO2017/085520 across
90% of SEQ ID NO:3 OF WO2017/085520, or at least 98% identity to
SEQ ID NO:3 OF WO2017/085520 across 95% of SEQ ID NO:3 OF
WO2017/085520, or at least 98% sequence identity to SEQ ID NO:3 OF
WO02017/085520 across 100% of SEQ ID NO:3 OF WO2017/085520, or at
least 99.5% sequence identity to SEQ ID NO:3 OF WO2017/085520
across 90% of SEQ ID NO:3 OF WO2017/085520, or at least 99.5%
identity to SEQ ID NO:3 OF WO2017/085520 across 95% of SEQ ID NO:3
OF WO2017/085520, or at least 99.5% identity to SEQ ID NO:3 OF
WO2017/085520 across 98% of SEQ ID NO:3 OF WO2017/085520, or at
least 99.5% sequence identity to SEQ ID NO:3 OF WO02017/085520
across 100% of SEQ ID NO:3 OF WO02017/085520.
[0061] In certain embodiments, the bacterial strain for use in the
invention has a plasmid with sequence identity to SEQ ID NO:4 OF
WO2017/085520. In preferred embodiments, the bacterial strain for
use in the invention has a plasmid with at least 90% sequence
identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%
sequence identity) to SEQ ID NO:4 OF WO2017/085520 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 OF WO2017/085520. For example, the
bacterial strain for use in the invention may have a plasmid with
at least 90% sequence identity to SEQ ID NO:4 OF WO2017/085520
across 70% of SEQ ID NO:4 OF WO2017/085520, or at least 90%
sequence identity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ
ID NO:4 OF WO2017/085520, or at least 90% sequence identity to SEQ
ID NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF
WO2017/085520, or at least 90% sequence identity to SEQ ID NO:4 OF
WO2017/085520 across 100% of SEQ ID NO:4 OF WO2017/085520, or at
least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across
70% of SEQ ID NO:4 OF WO2017/085520, or at least 95% sequence
identity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4
OF WO2017/085520, or at least 95% sequence identity to SEQ ID NO:4
OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO2017/085520, or at
least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across
100% of SEQ ID NO:4 OF WO02017/085520, or at least 98% sequence
identity to SEQ ID NO:4 OF WO2017/085520 across 70% of SEQ ID NO:4
OF WO2017/085520, or at least 98% sequence identity to SEQ ID NO:4
OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520, or at
least 98% sequence identity to SEQ ID NO:4 OF WO2017/085520 across
90% of SEQ ID NO:4 OF W02017/085520, or at least 98% sequence
identity to SEQ ID NO:4 OF W02017/085520 across 100% of SEQ ID NO:4
OF WO2017/085520.
[0062] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:3 OF
WO2017/085520 and a plasmid with sequence identity to SEQ ID NO:4
OF WO2017/085520.
[0063] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:3 OF
WO2017/085520, for example as described above, and a 16S rRNA
sequence with sequence identity to any of SEQ ID NO:1 or 2, for
example as described above, preferably with a 16s rRNA sequence
that is at least 99% identical to SEQ ID NO: 2, more preferably
which comprises the 16S rRNA sequence of SEQ ID NO:2, and
optionally comprises a plasmid with sequence identity to SEQ ID
[0064] NO:4 OF WO2017/085520, as described above.
[0065] In certain embodiments, the bacterial strain for use in
invention has a chromosome with sequence identity to SEQ ID NO:3 OF
WO2017/085520, for example as described above, and optionally
comprises a plasmid with sequence identity to SEQ ID NO:4 OF
WO2017/085520, as described above, and is effective for stimulating
the immune system.
[0066] In certain embodiments, the bacterial strain for use in the
invention has a chromosome with sequence identity to SEQ ID NO:3 OF
WO2017/085520, for example as described above, and a 16S rRNA
sequence with sequence identity to any of SEQ ID NOs: 1 or 2, for
example as described above, and optionally comprises a plasmid with
sequence identity to SEQ ID NO:4 OF WO2017/085520, as described
above, and is effective for stimulating the immune system.
[0067] In certain embodiments, the bacterial strain for use in the
invention has a 16s rRNA sequence that is at least 99%, 99.5% or
99.9% identical to the 16s rRNA sequence represented by SEQ ID NO:
2 (for example, which comprises the 16S rRNA sequence of SEQ ID
NO:2) and a chromosome with at least 95% sequence identity to SEQ
ID NO:3 OF W02017/085520 across at least 90% of SEQ ID NO:3 OF
WO2017/085520, and optionally comprises a plasmid with sequence
identity to SEQ ID NO:4 OF WO2017/085520, as described above, and
which is effective for stimulating the immune system.
[0068] In certain embodiments, the bacterial strain for use in the
invention has a 16s rRNA sequence that is at least 99%, 99.5% or
99.9% identical to the 16s rRNA gene sequence represented by SEQ ID
NO: 2 (for example, which comprises the 16S rRNA sequence of SEQ ID
NO:2) and a chromosome with at least 98% sequence identity (e.g. at
least 99% or at least 99.5% sequence identity) to SEQ ID NO:3 OF
WO2017/085520 across at least 98% (e.g. across at least 99% or at
least 99.5%) of SEQ ID NO:3 OF WO02017/085520, and optionally
comprises a plasmid with sequence identity to SEQ ID NO:4 OF
WO2017/085520, as described above, and which is effective for
stimulating the immune system.
[0069] In certain embodiments, the bacterial strain for use in the
invention is a Enterococcus gallinarum and has a 16s rRNA sequence
that is at least 99%, 99.5% or 99.9% identical to the 16s rRNA
sequence represented by SEQ ID NO: 2 (for example, which comprises
the 16S rRNA sequence of SEQ ID NO:2) and a chromosome with at
least 98% sequence identity (e.g. at least 99% or at least 99.5%
sequence identity) to SEQ ID NO:3 OF WO2017/085520 across at least
98% (e.g. across at least 99% or at least 99.5%) of SEQ ID NO:3 OF
WO2017/085520, and optionally comprises a plasmid with sequence
identity to SEQ ID NO:4 OF WO2017/085520, as described above, and
which is effective for stimulating the immune system.
[0070] Alternatively, strains that are biotypes of the bacterium
deposited under accession number NCIMB 42488 and that are suitable
for use in the invention may be identified by using the accession
number NCIMB 42488 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 Enterococcus gallinarum strains.
[0071] In certain embodiments, strains that are biotypes of the
bacterium deposited under accession number NCIMB 42488 and that are
suitable for use in the invention are strains that provide the same
pattern as the bacterium deposited under accession number NCIMB
42488 when analysed by amplified ribosomal DNA restriction analysis
(ARDRA), for example when using Sau3AI restriction enzyme (for
exemplary methods and guidance see, for example,[13]).
Alternatively, biotype strains are identified as strains that have
the same carbohydrate fermentation patterns as the bacterium
deposited under accession number NCIMB 42488. In some embodiments,
the carbohydrate fermentation pattern is determined using the API
50 CHL panel (bioMerieux). In some embodiments, the bacterial
strain used in the invention is: [0072] (i) positive for
fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17 or all of): L-arabinose,
D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose,
N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose,
D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and
potassium gluconate; and/or [0073] (ii) intermediate for
fermentation of at least one of (e.g. at least 2, 3, 4 or all of):
D-mannitol, Methyl-.alpha.D-glycopyranoside, D-lactose, starch, and
L-fucose; preferably as determined by API 50 CHL analysis
(preferably using the API 50 CHL panel from bioMerieux).
[0074] Other Enterococcus gallinarum strains that are useful in the
compositions and methods of the invention, such as biotypes of the
bacterium deposited under accession number NCIMB 42488, 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 assessing their effects on
cytokine levels, as performed in the examples. In particular,
bacterial strains that have similar growth patterns, metabolic type
and/or surface antigens to the bacterium deposited under accession
number NCIMB 42488 may be useful in the invention. A useful strain
will have comparable immune modulatory activity to the NCIMB 42488
strain. In particular, a biotype strain will elicit comparable
effects on the cancer disease models to the effects shown in the
Examples, which may be identified by using the culturing and
administration protocols described in the Examples. According to
some embodiments, a biotype strain that may be used in the
invention is a strain which is able to elicit comparable effects on
the cancer disease models shown in the Examples when administered
in the method of the invention.
[0075] In some embodiments, the bacterial strain used in the
invention is: [0076] (i) Positive for at least one of (e.g. at
least 2, 3, 4, 5, 6, 7 or all of): mannose fermentation, glutamic
acid decarboxylase, arginine arylamidase, phenylalanine
arylamidase, pyroglutamic acid arylamidase, tyrosine arylamidase,
histidine arylamidase and serine arylamidase; and/or [0077] (ii)
Intermediate for at least one of (e.g. at least 2 or all of):
.beta.-galactosidase-6-phosphate, .beta.-glucosidase and
N-acetyl-.beta.-glucosaminidase; and/or [0078] (iii) Negative for
at least one of (e.g. at least 2, 3, 4, 5, 6 or all of): Raffinose
fermentation,
[0079] Proline arylamidase, Leucyl glycine arylamidase, Leucine
arylamidase, Alanine arylamidase, Glycine arylamidase and Glutamyl
glutamic acid arylamidase, preferably as determined by an assay of
carbohydrate, amino acid and nitrate metabolism, and optionally an
assay of alkaline phosphatase activity, more preferably as
determined by Rapid ID 32A analysis (preferably using the Rapid ID
32A system from bioMerieux).
[0080] In some embodiments, the bacterial strain used in the
invention is: [0081] (i) Negative for at least one of (e.g. at
least 2, 3, or all 4 of) glycine arylamidase, raffinose
fermentation, proline arylamidase, and leucine arylamidase, for
example, as determined by an assay of carbohydrate, amino acid and
nitrate metabolism, preferably as determined by Rapid ID 32A
analysis (preferably using the Rapid ID 32A system from
bioMerieux); and/or [0082] (ii) Intermediate positive for
fermentation of L-fucose, preferably as determined by API 50 CHL
analysis (preferably using the API 50 CHL panel from
bioMerieux).
[0083] In some embodiments, the bacterial strain used in the
invention is an extracellular ATP producer, for example one which
produces 6-6.7 ng/.mu.l (for example, 6.1-6.6 ng/.mu.l or 6.2-6.5
ng/.mu.l or 6.33.+-.0.10 ng/.mu.l ) of ATP as measured using the
ATP Assay Kit (Sigma-Aldrich, MAK190). Bacterial extracellular ATP
can have pleiotropic effects including activation of T
cell-receptor mediated signalling (Schenk et al., 2011), promotion
of intestinal Th17 cell differentiation (Atarashi et al., 2008) and
induction of secretion of the pro-inflammatory mediator IL-1.beta.
by activating the NLRP3 inflammasome (Karmarkar et al., 2016).
Accordingly, a bacterial strain which is an extracellular ATP
producer is useful for stimulating the immune system in the context
of the method of the invention.
[0084] In some embodiments, the bacterial strain for use in the
invention comprises one or more of the following three genes:
Mobile element protein; Xylose ABC transporter, permease component;
and FIG00632333: hypothetical protein. For example, in certain
embodiments, the bacterial strain for use in the invention
comprises genes encoding Mobile element protein and Xylose ABC
transporter, permease component; Mobile element protein and
FIG00632333: hypothetical protein; Xylose ABC transporter, permease
component and FIG00632333: hypothetical protein; or Mobile element
protein, Xylose ABC transporter, permease component, and
FIG00632333: hypothetical protein.
[0085] A particularly preferred strain of the invention is the
Enterococcus gallinarum strain deposited under accession number
NCIMB 42488. This is the exemplary MRx0518 strain tested in the
examples and shown to be effective for treating disease. The
invention provides, according to some embodiments, a bacterial
composition as part of the invention, comprising a cell of the
Enterococcus gallinarum strain deposited under accession number
NCIMB 42488, or a derivative thereof. A derivative of the strain
deposited under accession number NCIMB 42488 may be a daughter
strain (progeny) or a strain cultured (subcloned) from the
original.
[0086] A derivative of a strain of the composition comprised in 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 42488 strain. In particular, a derivative
strain will elicit comparable effects on the cancer disease models
when which may be identified by using the culturing and
administration protocols described in the Examples. A derivative of
the NCIMB 42488 strain will generally be a biotype of the NCIMB
42488 strain.
[0087] References to cells of the Enterococcus gallinarum strain
deposited under accession number NCIMB 42488 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 42488, and
such cells are encompassed by the the invention. Thus, in some
embodiments, reference to cells of the Enterococcus gallinarum
strain deposited under accession number NCIMB 42488 refers only to
the MRx0518 strain deposited under NCIMB 42488 and does not refer
to a bacterial strain that was not deposited under NCIMB 42488. In
some embodiments, reference to cells of the Enterococcus gallinarum
strain deposited under accession number NCIMB 42488 refers to cells
that have the same safety and therapeutic efficacy characteristics
as the strains deposited under accession number NCIMB 42488, but
which are not the strain deposited under NCIMB 42488.
[0088] Bacterial strains that are biotypes of the bacterium
deposited under accession number 42761 are also expected to be
effective for stimulating the immune system. A biotype is a closely
related strain that has the same or very similar physiological and
biochemical characteristics.
[0089] Strains that are biotypes of the bacterium deposited under
accession number NCIMB 42761 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
42761. 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). For example, in some
embodiments, a biotype strain has at least 98% sequence identity
across at least 98% of its genome or at least 99% sequence identity
across 99% of its genome. Other suitable sequences for use in
identifying biotype strains may include hsp60 or repetitive
sequences such as BOX, ERIC, (GTG)5 (SEQ ID NO: 4), or REP or [14].
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
42761. In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of strain MRx0554 deposited as NCIMB
42761. In some embodiments, a biotype strain has a sequence with at
least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to
the corresponding sequence of strain MRx0554 deposited as NCIMB
42761 and has a 16S rRNA gene sequence that is at least 99%
identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ
ID NO:3. In some embodiments, a biotype strain has a sequence with
at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity
to the corresponding sequence of strain MRx0554 deposited as NCIMB
42761 and has the 16S rRNA gene sequence of SEQ ID NO:3.
[0090] Alternatively, strains that are biotypes of the bacterium
deposited under accession number NCIMB 42761 and that are suitable
for use in the invention may be identified by using the accession
number NCIMB 42761 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.
[0091] In certain embodiments, strains that are biotypes of the
bacterium deposited under accession number NCIMB 42761 and that are
suitable for use in the invention are strains that provide the same
pattern as the bacterium deposited under accession number NCIMB
42761 when analysed by amplified ribosomal DNA restriction analysis
(ARDRA), for example when using Sau3AI restriction enzyme (for
exemplary methods and guidance see, for example, [15]).
Alternatively, biotype strains are identified as strains that have
the same carbohydrate fermentation patterns as the bacterium
deposited under accession number NCIMB 42761. In some embodiments,
the carbohydrate fermentation pattern is determined using the API
50 CHL panel (bioMerieux). In some embodiments, the bacterial
strain used in the invention is: [0092] (iii) positive for
fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17 or all of): L-arabinose,
D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose,
N-acetylglucosamine, amygdalin, arbutin, salicin, D-cellobiose,
D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and
potassium gluconate; and/or [0093] (iv) intermediate for
fermentation of at least one of (e.g. at least 2, 3, 4 or all of):
D-mannitol, Methyl-.alpha.D-glycopyranoside, D-lactose, starch, and
L-fucose; preferably as determined by API 50 CHL analysis
(preferably using the API 50 CHL panel from bioMerieux).
[0094] In some embodiments, the bacterial strain used in the
invention is: [0095] (i) positive for fermentation of at least one
of (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,
16, 17, 18 or all of): L-arabinose, D-ribose, D-xylose,
D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine,
amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose,
D-saccharose (sucrose), D-trehalose, gentiobiose, D-tagatose and
potassium gluconate; [0096] (ii) intermediate for fermentation of
at least one of (e.g. at least 2, 3, 4, 5 or all of): D-mannitol,
Methyl-.alpha.D-glycopyranoside, D-lactose, D-raffinose, amidon
(starch), and D-turanose; and/or [0097] (iii) negative for
fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or all
of): glycerol, erythritol, D-arabinose, L-xylose, D-adonitol,
methyl-.alpha.D-xylopryranoside, L-sorbose, L-rhamnose, dulcitol,
inositol, D-sorbitol, Methyl-.alpha.D-mannopyranoside, D-melibiose,
inulin, D-melezitose, glycogen, xylitol, D-lyxose, D-fucose,
L-fucose, D-arabitol, L-arabitol, potassium 2-ketogluconate and
potassium 5-ketogluconate; preferably as determined by API 50 CHL
analysis (preferably using the API 50 CHL panel from bioMerieux,
and preferably using the conditions described in Example 10).
[0098] Other Enterococcus gallinarum strains that are useful in the
compositions and methods of the invention, such as biotypes of the
bacterium deposited under accession number NCIMB 42761, may be
identified using any appropriate method or strategy, including the
assays described in the examples. For instance, strain 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 42761 may be useful in the invention.
A useful strain will have comparable immune modulatory activity to
the NCIMB 42761 strain. In particular, a biotype strain will elicit
comparable effects on the cancer disease models to the effects
shown in the Examples, which may be identified by using the
culturing and administration protocols described in the
Examples.
[0099] 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 42761 strain. In
particular, a derivative strain will elicit comparable effects on
the cancer disease models 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
42761 strain will generally be a biotype of the NCIMB 42761
strain.
[0100] References to cells of the Enterococcus gallinarum strain
deposited under accession number NCIMB 42761 encompass any cells
that have the same safety and therapeutic efficacy characteristics
as the strain deposited under accession number NCIMB 42761, and
such cells are encompassed by the invention. Thus, in some
embodiments, reference to cells of the Enterococcus gallinarum
strain deposited under accession number NCIMB 42761 refers only to
the MRx0554 strain deposited under NCIMB 42761 and does not refer
to a bacterial strain that was not deposited under NCIMB 42761.
[0101] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:2 OF
WO2018/215782. In some 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:2 OF WO2018/215782 across at least 60% (e.g.
across at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or
100%) of SEQ ID NO:2 OF W02018/215782.
[0102] For example, the bacterial strain for use in the invention
may have a genome with at least 90% sequence identity to SEQ ID
NO:2 OF WO02018/215782 across 70% of SEQ ID NO:2 OF WO2018/215782,
or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782
across 80% of SEQ ID NO:2 OF WO2018/215782, or at least 90%
sequence identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ
ID NO:2 OF WO2018/215782, or at least 90% sequence identity to SEQ
ID NO:2 OF WO2018/215782 across 100% of SEQ ID NO:2 OF
WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF
WO2018/215782 across 70% of SEQ ID NO:2 OF WO2018/215782, or at
least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across
80% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence
identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ ID NO:2
OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2
OF WO2018/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at
least 98% sequence identity to SEQ ID NO:2 OF WO2018/215782 across
70% of SEQ ID NO:2 OF WO2018/215782, or at least 98% sequence
identity to SEQ ID NO:2 OF WO2018/215782 across 80% of SEQ ID NO:2
OF WO2018/215782, or at least 98% sequence identity to SEQ ID NO:2
OF WO2018/215782 across 90% of SEQ ID NO:2 OF WO2018/215782, or at
least 98% identity across 95% of SEQ ID NO:2 OF WO2018/215782, or
at least 98% sequence identity to SEQ ID NO:2 OF WO2018/215782
across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 99.5%
sequence identity to SEQ ID NO:2 OF
[0103] Attorney Docket No. 56708-740.302 WO2018/215782 across 90%
of SEQ ID NO:2 OF WO2018/215782, or at least 99.5% identity across
95% of SEQ ID NO:2 OF WO2018/215782, or at least 99.5% identity
across 98% of SEQ ID NO:2 OF WO2018/215782, or at least 99.5%
sequence identity to SEQ ID NO:2 OF WO2018/215782 across 100% of
SEQ ID NO:2 OF WO2018/215782.
[0104] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:2 OF
WO2018/215782, for example as described above, and a 16S rRNA gene
sequence with sequence identity to SEQ ID NO:1 or 3, for example as
described above, preferably with a 16S rRNA gene sequence that is
at least 99% identical to SEQ ID NO:3, more preferably which
comprises the 16S rRNA gene sequence of SEQ ID NO:3.
[0105] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:2 OF
WO2018/215782, for example as described above, and is effective for
stimulating the immune system.
[0106] In certain embodiments, the bacterial strain for use in the
invention has a genome with sequence identity to SEQ ID NO:2 OF
WO2018/215782, for example as described above, and a 16S rRNA gene
sequence with sequence identity to SEQ ID NO: 1 or 3, for example
as described above, and is effective for stimulating the immune
system.
[0107] In certain embodiments, the bacterial strain for use in the
invention has a 16S rRNA gene sequence that is at least 99%, 99.5%
or 99.9% identical to the 16S rRNA gene sequence represented by SEQ
ID NO: 3 (for example, which comprises the 16S gene rRNA sequence
of SEQ ID NO:3) and a genome with at least 95% sequence identity to
SEQ ID NO:2 OF WO2018/215782 across at least 90% of SEQ ID NO:2 OF
WO2018/215782, and which is effective for stimulating the immune
system.
[0108] In certain embodiments, the bacterial strain for use in the
invention is a Enterococcus gallinarum and has a 16S rRNA gene
sequence that is at least 99%, 99.5% or 99.9% identical to the 16S
rRNA gene sequence represented by SEQ ID NO:3 (for example, which
comprises the 16S rRNA gene sequence of SEQ ID NO:3) and a genome
with at least 98% sequence identity (e.g. at least 99% or at least
99.5% sequence identity) to SEQ ID NO:2 OF WO2018/215782 across at
least 98% (e.g. across at least 99% or at least 99.5%) of SEQ ID
NO:2 OF WO2018/215782, and which is effective for stimulating the
immune system.
[0109] In preferred embodiments, the bacterial strains in the
compositions of the invention are viable and capable of partially
or totally colonising the intestine.
[0110] In alternative aspects of every embodiment of the invention,
the bacterial strain in the composition of the invention is of the
species Enterococcus caselliflavus. Enterococcus caselliflavus is
highly similar to Enterococcus gallinarum and is also
flagellated.
Therapeutic Uses
Stimulating the Immune System
[0111] The examples show that administration of the compositions of
the invention can lead to immune stimulation. Since administration
of the compositions of the invention were shown to have an
immunostimulatory effect, compositions of the invention may be
useful in the treatment of disease, in particular diseases
characterised by reduced immune activation and diseases treatable
by an increased immune response. In certain embodiments, the
compositions of the invention are for use in stimulating the immune
system. In certain embodiments, the compositions of the invention
are for use in treating disease by stimulating the immune system.
In certain embodiments, the compositions of the invention are for
use in promoting an immune response.
[0112] Compositions of the invention may be useful in the treatment
of diseases characterised by an increase in the percentage of Tregs
in a cell population. In one embodiment, the compositions of the
invention may be useful for treating or preventing diseases
characterised by an increase in the percentage of Tregs in a cell
population. In one embodiment, the compositions of the invention
may be useful for treating or preventing diseases characterised by
an increase in the percentage of CD4+CD25+CD127- cells in a cell
population. In one embodiment, the compositions of the invention
are for use in treating or preventing diseases by decreasing the
percentage of Tregs in cell populations. In one embodiment,
compositions of the invention are for use in reducing suppression
of the immune response by Tregs. In one embodiment, compositions of
the invention are for use in stimulating the immune response by the
selective reduction of Tregs. In one embodiment, compositions of
the invention are for use in immunostimulation, wherein the
compositions of the invention reduce the number or percentage of
Tregs.
[0113] Compositions of the invention may be useful in the treatment
of diseases characterised by a decrease in the ratio of CD8/Treg
and/or activated CD8/Treg cells. In one embodiment, the
compositions of the invention are for use in treating or preventing
diseases characterised by decrease in the ratio of CD8/Treg cells.
In one embodiment, the compositions of the invention are for use in
treating or preventing diseases characterised by decrease in the
ratio of activated CD8/Treg cells. In one embodiment, compositions
of the invention are for use in stimulating the immune response by
increasing the ratio of CD8/Treg cells. In one embodiment,
compositions of the invention are for use in stimulating the immune
response by increasing the ratio of activated CD8/Treg cells.
[0114] Compositions of the invention may be useful in the treatment
of diseases characterised by a decrease in the number or percentage
of B cells. In one embodiment, the compositions of the invention
are for use in treating or preventing diseases characterised by
decrease in the number or percentage of B cells. In one embodiment,
the compositions of the invention are for use in treating or
preventing diseases characterised by decrease in the number or
percentage of CD19.sup.+CD3- cells. In one embodiment, the
compositions of the invention are for use in treating or preventing
diseases by increasing the number or percentage of B cells in cell
populations, wherein the increase in number or percentage of B
cells results in immune stimulation. In one embodiment,
compositions of the invention are for use in stimulating the immune
response by increasing the number or percentage of B cells.
[0115] Compositions of the invention may be useful in the treatment
of diseases characterised by a decrease in the number or percentage
of CD8 T-cytotoxic cells. In one embodiment, the compositions of
the invention are for use in treating or preventing diseases
characterised by decrease in the number or percentage of CD8
T-cytotoxic cells. In one embodiment, the compositions of the
invention are for use in treating or preventing diseases by
increasing the number or percentage of CD8 T-cytotoxic cells in
cell populations, wherein the increase in number or percentage of
CD8 T-cytotoxic cells results in immune stimulation. In one
embodiment, compositions of the invention are for use in
stimulating the immune response by increasing the number or
percentage of CD8 T-cytotoxic cells.
[0116] Compositions of the invention may be useful in the treatment
of diseases characterised by a decrease in the number or percentage
of CD8.sup.+ activated cells. In one embodiment, the compositions
of the invention are for use in treating or preventing diseases
characterised by decrease in the number or percentage of CD8.sup.+
activated cells. In one embodiment, the compositions of the
invention are for use in treating or preventing diseases by
increasing the number or percentage of CD8.sup.+ activated cells in
cell populations, wherein the increase in number or percentage of
CD8.sup.+ activated cells results in immune stimulation. In one
embodiment, compositions of the invention are for use in
stimulating the immune response by increasing the number or
percentage of CD8.sup.+activated cells.
[0117] The examples show that administration of the compositions of
the invention can lead to an increase in expression of
pro-inflammatory molecules, such as pro-inflammatory cytokines.
Examples of pro-inflammatory molecules that showed an increase in
expression levels upon administration of compositions of the
invention include IL-8, IL-12p70, IL-23, TNF-.alpha., IL-1.beta.
(3, and IL-6. Since administration of the compositions of the
invention were shown to increase the expression of pro-inflammatory
molecules, compositions of the invention may be useful in the
treatment of diseases characterised by a decrease in expression of
pro-inflammatory molecules, such as pro-inflammatory cytokines. In
one embodiment, the compositions of the invention are for use in
treating or preventing diseases characterised by a decrease in the
expression and/or activity of pro-inflammatory molecules, in
particular diseases characterised by a decrease in the expression
and/or activity of pro-inflammatory cytokines. In a particular
embodiment, the compositions of the invention are for use in
treating or preventing diseases characterised by a decrease in the
expression and/or activity of IL-8, IL-12p70, IL-23, TNF-.alpha.,
IL-1.beta.- and/or IL-6. In one embodiment, the compositions of the
invention are for use in treating or preventing diseases by
increasing the expression and/or activity of IL-23, TNF-.alpha.,
IL-1.beta., and/or IL-6. In one embodiment, compositions of the
invention are for use in promoting the immune response by
increasing the expression and/or activity of IL-8, IL-12p70, IL-23,
TNF-.alpha., IL-1.beta. and/or IL-6.
[0118] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-1(3. IL-1(3 is a pro-inflammatory cytokine [16]. The
production and secretion of IL-1.beta. is regulated by the
inflammasome, a protein complex which is associated with activation
of the inflammatory response [17]. Since administration of the
compositions of the invention were shown to increase the expression
of IL-1.beta., compositions of the invention may be useful in the
treatment of diseases characterised by a decrease in expression of
IL-1.beta.. In a particular embodiment, the compositions of the
invention are for use in treating or preventing diseases
characterised by a decrease in the expression and/or activity of
IL-1.beta.. In one embodiment, the compositions of the invention
are for use in treating or preventing diseases by increasing the
expression and/or activity of IL-1.beta..
[0119] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-23. IL-23 has been linked to inflammation [18,19]. The
proposed functions of IL-23 in the immune response include
promoting the proliferation of CD4.sup.+ memory T cells and
promoting the secretion of IFN-.gamma. by dendritic cells (DCs)
[20]. Since administration of the compositions of the invention
were shown to increase the expression of IL-23, compositions of the
invention may be useful in the treatment of diseases characterised
by a decrease in expression of IL-23. In a particular embodiment,
the compositions of the invention are for use in treating or
preventing diseases characterised by a decrease in the expression
and/or activity of IL-23. In one embodiment, the compositions of
the invention are for use in treating or preventing diseases by
increasing the expression and/or activity of IL-23. In one
embodiment, compositions of the invention are for use in promoting
the immune response by increasing the expression and/or activity of
IL-23.
[0120] The examples show that administration of the compositions of
the invention can lead to an increase in expression of Tumour
Necrosis Factor alpha (TNF-.alpha.). TNF-.alpha. is a
pro-inflammatory cytokine which is known to be involved in various
signalling pathways to promote cell death. TNF-.alpha. initiates
apoptosis by binding to its cognate receptor, TNFR-1, which leads
to a cascade of cleavage events in the apoptotic pathway [21].
TNF-.alpha. can also trigger necrosis via a RIP kinase-dependent
mechanism [22]. Since administration of the compositions of the
invention show an increase in TNF-a expression, compositions of the
invention may be useful in the treatment of diseases, in particular
for use in treating or preventing diseases characterised by a
decrease in expression of by TNF-.alpha.. In one embodiment, the
compositions of the invention are for use in treating diseases
characterised by decreased TNF-a expression. In a particular
embodiment, the compositions of the invention are for use in
treating or preventing diseases characterised by a decrease in the
expression and/or activity of TNF-a. In one embodiment, the
compositions of the invention may be useful for treating or
preventing diseases by increasing the expression and/or activity of
TNF-a. In one embodiment, compositions of the invention are for use
in promoting the immune response by increasing the expression
and/or activity of TNF-.alpha..
[0121] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-6. IL-6 a pro-inflammatory cytokine that is produced during
inflammation, and promotes the differentiation of naive CD4.sup.+ T
cells and the differentiation of CD8.sup.+ T cells into cytotoxic T
cells [23]. Since administration of the compositions of the
invention were shown to increase the expression of IL-6,
compositions of the invention may be useful in the treatment of
diseases characterised by a decrease in expression of IL-6. In a
particular embodiment, the compositions of the invention are for
use in treating or preventing diseases characterised by a decrease
in the expression and/or activity of IL-6. In one embodiment, the
compositions of the invention are for use in treating or preventing
diseases by increasing the expression and/or activity of IL-6. In
one embodiment, compositions of the invention are for use in
promoting the immune response by increasing the expression and/or
activity of IL-6.
[0122] Bettelli et al. [24] reported that IL-6 inhibits the
expansion of Tregs. Since the examples show that compositions of
the invention increase the expression of IL-6, compositions of the
invention may selectively decrease the number or percentage of
Tregs by increasing the expression of IL-6. In one embodiment,
compositions of the invention are for use in immunostimulation by
increasing the expression of IL-6. In another embodiment,
compositions of the invention are for use in immunostimulation by
decreasing the number or percentage of Tregs.
[0123] In some embodiments, stimulating the immune system according
to the present invention comprises TLR5 activation or upregulation
of TLR5 activation. In some embodiments, stimulating the immune
system according to the present invention comprises TLR9 activation
or upregulation of TLR9 activation. In some embodiments,
stimulating the immune system according to the present invention
comprises activation of TLR5 and TLR9 or upregulation of TLR9 and
TLR5 activation. In some embodiments, stimulating the immune system
according to the present invention comprises inducing and/or
upregulating differentiation of T cells such as, but not limited
to, T helper cells and T cytotoxic cells.
[0124] TLR signalling pathways culminate in the activation of the
transcription factor nuclear factor-kappaB (NF-.kappa.KB).
NF-.kappa.B controls the expression of an array of inflammatory
cytokine genes, including TNF-.alpha.. Immune stimulation causes,
for example, the dimerization of TLR5, which subsequently recruits
MyD88 and activates protein kinases, including IRAK1, IRAK2, IRAK4
and IRAK-M. The activation of these kinases leads to the nuclear
localization of NF-.kappa.B, which is a proinflammatory cytokine
[25].
[0125] As demonstrated in the examples, compositions of the
invention lead to an increase in expression of NF-.kappa.B. Since
administration of the compositions of the invention increase the
expression of the proinflammatory cytokine NF-.kappa.B,
compositions of the invention may be useful in stimulating the
immune response. In addition, compositions of the invention may be
useful in the treatment of disease, in particular diseases
characterised by reduced immune activation and/or diseases
treatable by an increased immune response. In one embodiment, the
compositions of the invention are for use as an immune stimulant by
increasing the level and/or activity of NF-.kappa.B. In one
embodiment, the compositions of the invention are for use in
treating diseases characterised by reduced immune activation by
increasing the level and/or activity of NF-.kappa.B. In one
embodiment, the compositions of the invention are for use in
treating diseases treatable by an increased immune response by
increasing the level and/or activity of NF-.kappa.B.
[0126] In particular, compositions of the invention may be useful
in the treatment of diseases characterised by a decrease in
expression and/or activation of NF-.kappa.B. In one embodiment, the
compositions of the invention are for use in treating diseases
characterised by a decrease in expression and/or activation of
NF-.kappa.B
[0127] The activation of NF-.kappa.B is important for eliciting
innate immune responses and the subsequent development of adaptive
immune responses. Thus, agonists of TLR5, such as compositions of
the invention, are likely to be useful as adjuvants to treat
infectious diseases, allergies and tumours by promoting both innate
and adaptive immune responses [25]. In one embodiment, the
compositions of the invention are for use in treating infectious
diseases, allergies and/or tumours. In one embodiment, the
compositions of the invention are for use in treating infectious
diseases, allergies and/or tumours by increasing the level and/or
activity of NF-.kappa.B.
[0128] The examples also demonstrate that the compositions of the
invention promote the differentiation of T-helper cells and
cytotoxic T lymphocytes. Therefore, in certain embodiments, the
compositions of the invention are for use in stimulating the
differentiation of T-helper cells and/or cytotoxic T
lymphocytes.
[0129] In certain embodiments, the disease to be treated by the
compositions of the invention is not cancer.
Use as a Vaccine Adjuvant
[0130] The examples show that administration of the compositions of
the invention can lead to an increase in expression of Tumour
Necrosis Factor alpha (TNF-.alpha.). TNF-.alpha. is known to be
important for vaccine responses. For example, TNF-a has been shown
to be required for an efficient vaccine response in a flu
vaccination of the elderly population [26]. Since administration of
the compositions of the invention were shown to increase
TNF-.alpha.expression, compositions of the invention may be useful
as a vaccine adjuvant. In one embodiment, the compositions of the
invention are for use as a vaccine adjuvant by increasing the level
and/or activity of TNF-.alpha.. In one embodiment, the compositions
of the invention are for use as a vaccine adjuvant. In one
embodiment, the compositions of the invention are for use as a
vaccine adjuvant in influenza therapy. In certain embodiments, the
compositions of the invention are for use in enhancing an immune
response against an antigen. In certain embodiments, the invention
provides a composition to be administered in combination with an
antigen. In certain embodiments, the compositions of the invention
are for administration to a patient shortly prior to or after
vaccination.
[0131] Enterococcus gallinarum and in particular strain MRx0518 is
flagellated and flagellins can be TLR5 agonists. TLR agonists are
in development as vaccine adjuvants across a range of antigen
types, particularly in the elderly population [27]. Also, the data
in the examples confirm that MRx0518 flagellin is a TLR5 agonists.
Therefore, the compositions of the invention may be useful as
vaccine adjuvants, in particular for vaccine administered to
elderly patients (e.g. over 40, 50, 60, 70 or 80 years of age), who
may have reduced immune system activity. TLR5 signalling also plays
a key role in age-associated innate immune responses [28]. In
certain embodiments, the compositions are for use in enhancing an
innate immune response. Although TLR5 agonists are in development
as vaccine adjuvants, these are all from known pathogens and/or
synthetic. In contrast, the compositions of the invention comprise
commensal bacteria.
[0132] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-6. Increased IL-6 expression has been associated with vaccine
responses for many diseases. For example, IL-6 was produced by
CD14+CD16- inflammatory monocytes after adults were administered an
influenza vaccine [29], and higher levels of IL-6 were associated
with achieving a vaccine response to an influenza vaccine [30].
Furthermore, IL-6 was produced after injection of the AS03 adjuvant
system [31] and downregulation of IL-6 in mice was shown to reduce
the helper T cell response after administration of a tuberculosis
vaccine [32]. Since administration of the compositions of the
invention were shown to increase IL-6 expression, compositions of
the invention may be useful as a vaccine adjuvant. In one
embodiment, the compositions of the invention are for use as a
vaccine adjuvant by increasing the level and/or activity of IL-6.
In one embodiment, the compositions of the invention are for use as
a vaccine adjuvant. In one embodiment, the compositions of the
invention are for use as a vaccine adjuvant in tuberculosis
therapy.
[0133] Furthermore, IL-6 and TNF-a expression have been shown to be
correlated with the efficacy of a therapeutic HIV vaccine [Huang et
al] a tuberculosis vaccine and a chlamydia vaccine [33]. Su et al.
[34] showed that co-inoculation of IL-6 or TNF-.alpha. with the
FMDV DNA vaccine resulted in increased IFN-.gamma.expression by
CD4.sup.+ and CD8.sup.+ T cells, higher expression of IL-4 in
CD4.sup.+ T cells and a higher antigen-specific cytotoxic response.
Since administration of the compositions of the invention were
shown to increase IL-6 and TNF-.alpha. expression, compositions of
the invention may be useful as a vaccine adjuvant.
[0134] In one embodiment, the compositions of the invention may be
useful as a vaccine adjuvant by increasing the level and/or
activity of TNF-.alpha.. In one embodiment, the compositions of the
invention may be useful as a vaccine adjuvant by increasing the
level and/or activity of IL-6. In a particular embodiment, the
compositions of the invention may be useful as a vaccine adjuvant
by increasing the level and/or activity of TNF-.alpha. and IL-6. In
one embodiment, the compositions of the invention are for use as a
vaccine adjuvant in HIV therapy. In one embodiment, the
compositions of the invention are for use as a vaccine adjuvant in
chlamydia therapy.
[0135] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-1.beta.. Li et al. [35] showed that the adjuvant aluminium
hydroxide activated the secretion of IL-1.beta., and suggested that
IL-1.beta. itself can act as an adjuvant. Since administration of
the compositions of the invention were shown to increase IL-1.beta.
expression, compositions of the invention may be useful as a
vaccine adjuvant. The examples show that administration of the
compositions of the invention can T cells [36] and since
administration of the compositions of the invention were shown to
increase the ratio of CD8.sup.+ T cells to Tregs, compositions of
the invention may be useful as a vaccine adjuvant. In one
embodiment, compositions of the invention are for use as a vaccine
adjuvant. In one embodiment, the compositions of the invention are
for use as a vaccine adjuvant by increasing the ratio of CD8.sup.+
T cells to Tregs.
[0136] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
or levels of CXCR3 ligands CXCL9 and CXCL10. Known adjuvants such
as ASO3, CpG, GLA-SE, .alpha.GalCer all increase CXCL9 and 10
[37,38], which suggests the compositions of the invention will be
effective as adjuvants. Also, CXCL9 and 10 are associated with
IFN.gamma./Th1 responses and promote antibody responses [39].In
certain embodiments, the compositions of the invention are for use
in promoting an antibody response against an antigen, in particular
a pathogenic or cancer antigen. Also, CXCL9 is a more sensitive
measure than IFN-.gamma. of vaccine induced T-cell responses in
volunteers receiving investigated malaria vaccines [40].In certain
embodiments, the compositions of the invention are for use in
promoting an T-cell response against an antigen, in particular a
pathogenic or cancer antigen. In one embodiment, the compositions
of the invention are for use as a vaccine adjuvant by increasing
the level and/or activity of CXCL9 and CXCL10. In certain
embodiments, the compositions are for use in protecting against
malaria.
[0137] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
or levels of IL-12p70. This effect has been associated with vaccine
adjuvant efficiency and IL-12 has been proposed as an adjuvant
itself [41], which suggests the compositions of the invention will
be effective as adjuvants. In one embodiment, the compositions of
the invention are for use as a vaccine adjuvant by increasing the
level and/or activity of IL-12p70.
[0138] In some embodiments, when used as a vaccine adjuvant, the
compositions of the invention will be administered on their own to
provide an adjuvant effect for an antigen that has been separately
administered to the patient. In certain embodiments, the
composition of the invention is administered orally, whilst the
antigen is injected parenterally.
[0139] The compositions of the invention may be used for enhancing
an immune response to any useful antigen. Exemplary antigens for
use with the invention include: viral antigens, such as viral
surface proteins; bacterial antigens, such as protein and/or
saccharide antigens; fungal antigens; parasite antigens; and tumour
antigens. The invention is particularly useful for vaccines against
influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex
virus, rabies, respiratory syncytial virus, cytomegalovirus,
Staphylococcus aureus, chlamydia, SARS coronavirus, varicella
zoster virus, Streptococcus pneumoniae, Neisseria meningitidis,
Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus,
human papillomavirus, etc. Further antigens for use with the
invention include glycoprotein and lipoglycan antigens, archaea
antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen
(CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse
transcriptase (hTERT), Wilms tumour gene (WT1), CA-125,
prostate-specific antigen (PSA), Epstein-Barr virus antigens,
neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit
forming substances, for example nicotine, alcohol or opiates.
[0140] Preferred antigens for use with the invention include
pathogen antigens and tumour antigens. An antigen will elicit an
immune response specific for the antigen that will be effective for
protecting against infection with the pathogen or attacking the
tumour. Antigens may be, for example, peptides or
polysaccharides.
[0141] The invention also provides the use of: (i) an aqueous
preparation of an antigen; and (ii) a composition comprising a
bacterial strain of the species Enterococcus gallinarum, in the
manufacture of a medicament for raising an immune response in a
patient.
[0142] The immune response raised by these methods and uses will
generally include an antibody response, preferably a protective
antibody response.
[0143] In some embodiments, a bacterial strain of the species
Enterococcus gallinarum is engineered to present an antigen.
Presenting an antigen on the bacterial strain of the invention may
maximise the immunostimulatory activities and further enhance the
protective immune response generated against the antigen. In
addition, manufacturing and delivering therapeutics comprising an
antigen and a bacteria of the invention may be more efficient and
effective this way than when each of the antigen and the
composition comprising the bacterial strain are manufactured and
administered separately. Therefore, in some embodiments, the
invention provides a composition comprising a bacterial strain of
the species Enterococcus gallinarum that presents an antigen, for
example on its cell surface. In some embodiments, the composition
comprising the bacterial strain that presents an antigen is for use
as a vaccine antigen. In some embodiments, the antigen is derived
from HIV, hookworm, hepatitis B virus, herpes simplex virus,
rabies, respiratory syncytial virus, cytomegalovirus,
Staphylococcus aureus, chlamydia, SARS coronavirus, varicella
zoster virus, Streptococcus pneumoniae, Neisseria meningitidis,
Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus
or human papillomavirus. In some embodiments, the antigen is a
glycoprotein antigen, lipoglycan antigen, archaea antigen, melanoma
antigen E (MAGE),
[0144] Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn
(STn), human telomerase reverse transcriptase (hTERT), Wilms tumour
gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr
virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9
or a habit forming substance, such as, alcohol, opiates and the
like.
[0145] In some embodiments, the bacteria of the invention express
one or more antigens. Generally the antigen will be expressed
recombinantly and will be heterologous to the bacteria of the
invention. Therefore, the invention provides a bacterial strain of
the species Enterococcus gallinarum that expresses a heterologous
antigen. The antigen may be part of a fusion polypeptide expressed
with one or more polypeptides homologous to the bacteria. In some
embodiments, the bacteria express the antigen as a non-fusion
polypeptide. In some embodiments, the invention provides a
composition comprising a cell of a bacterial strain of the species
Enterococcus gallinarum, wherein the cell expresses a heterologous
antigen. In some embodiments, the composition is for use as a
vaccine. In some embodiments, the invention provides a cell of a
bacterial strain of the species Enterococcus gallinarum, wherein
the cell expresses a heterologous antigen. In some embodiments, the
cell is for use as a vaccine.
[0146] Exemplary antigens for use with the invention include: viral
antigens, such as viral surface proteins; bacterial antigens, such
as protein and/or saccharide antigens; fungal antigens; parasite
antigens; and tumor antigens. Further antigens for expressing in a
bacterial strain of the species Enterococcus gallinarum include
glycoprotein and lipoglycan antigens, archaea antigens, melanoma
antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2,
sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT),
Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA),
Epstein-Barr virus antigens, neoantigens, oncoproteins,
amyloid-beta, Tau, PCSK9 and habit forming substances, for example
nicotine, alcohol, opiates, or the like.
[0147] The invention may also be useful for enhancing the response
to vaccines against non-communicable diseases such as elevated
cholesterol (e.g. via the PCSK9 antigen).
[0148] The invention may also be useful for enhancing the response
to vaccines against habit forming substances, for example nicotine,
alcohol or opiates.
Cell Therapies
Chimeric Antigen Receptor T Cell (CAR-T) Therapy
[0149] The examples also show that administration of the
compositions of the invention can lead to an increase in expression
of IL-6. Increased IL-6 expression has been correlated with
response to CD19 CAR-T therapy of chronic lymphocyte leukaemia. An
increase in serum IL-6 was associated with CAR-T cell expansion,
whereas inhibition of IL-6 was associated with inhibition of CAR-T
cell proliferation [42]. Since administration of the compositions
of the invention were shown to increase IL-6 expression,
compositions of the invention may be useful in cell therapy, in
particular CAR-T cell therapy. In one embodiment, the compositions
of the invention are for use in cell therapy. In one embodiment,
the compositions of the invention are for use in CAR-T cell
therapy. In one embodiment, compositions of the invention are for
use in the treatment of chronic lymphocyte leukaemia.
[0150] Selective depletion of Tregs has been shown to enhance the
efficacy of cytotoxic lymphocytes [43]. CAR-T cells are a subset of
cytotoxic lymphocytes, and therefore it is thought that selective
depletion of Tregs is effective in CAR-T cell therapy. Since
administration of the compositions of the invention were shown to
deplete Tregs, compositions of the invention may be useful in cell
therapy, in particular CAR-T cell therapy.
[0151] Therefore, the compositions of the invention may be useful
in cell therapy, in particular in enhancing the response to a cell
therapy.
Mesenchymalstem Cell (MSC) Therapy
[0152] Mesenchymal stem cell (MSC) therapy has been reported to
have immunostimulatory properties. When MSCs are treated with LPS,
they upregulate pro-inflammatory cytokines IL-6 and IL-8 which
causes increased B cell proliferation [44]. Therefore, since
compositions of the invention were shown to increase the expression
of IL-6, they may be useful in combination with MSC cell
therapy.
Stem Cell Transplantation Therapy
[0153] It has been reported that, instead of using undifferentiated
stem cells in stem cell transplantation therapy, it may be
beneficial to differentiate stem cells to some extent prior to
transplantation. For example, Heng et al. [45] reported that
cardiomyogenic differentiation of stem cells may be beneficial by
having a higher engraftment efficiency, enhanced regeneration of
myocytes and increased restoration of heart function. Since
administration of the compositions of the invention initiated
neuronal differentiation in undifferentiated neuroblastoma cells,
compositions of the invention may be useful for stem cell
differentiation in stem cell transplantation therapy.
Hematopoietic Stem Cell Transplantation
[0154] Hematopoietic stem cell transplantation is the
transplantation of multipotent hematopoietic stem cells, usually
derived from bone marrow, peripheral blood, or umbilical cord
blood. Colonisation of the gut with Enterococci (Enterococcus
gallinarum and Enterococcus casseliflavus) prior to allogenic
hematopoietic stem cell transplantation has been shown to lead to a
significantly improved the 2-year survival of patients after due to
decreased nonrelapse mortality [46]. Therefore, the
immunomodulatory effect shown in the examples may be useful in
hematopoietic stem cell transplantation therapy. In certain
embodiments, the compositions of the invention may be useful in
improving survival after hematopoietic stem cell transplantation
and in particular after allogenic hematopoietic stem cell
transplantation.
[0155] The compositions of the invention may be useful in
combination with allogenic hematopoietic stem cell transplantation.
The compositions of the invention may be effective in boosting
successful patient response to allogenic hematopoietic stem cell
transplantation. In certain embodiments, the compositions of the
invention are administered prior to hematopoietic stem cell
transplantation. In certain embodiments, the compositions of the
invention are for administration to a patient scheduled to receive
hematopoietic stem cell transplantation. In certain embodiments,
the compositions of the invention are administered following
hematopoietic stem cell transplantation. In certain embodiments,
the compositions of the invention are for administration to a
patient that has received hematopoietic stem cell
transplantation.
Immunosenescence
[0156] Fulop et al [47] identified that an increase in Treg cell
number and a decrease in B cell number are associated with aging in
the adaptive immune system. Therefore, compositions of the
invention may be used to prevent or delay immunosenescence. In one
embodiment, compositions of the invention are for use in preventing
immunosenescence. In another embodiment, compositions of the
invention are for use in delaying immunosenescence characterised by
an increase in Treg cell number. In another embodiment,
compositions of the invention are for use in delaying
immunosenescence characterised by a decrease in B cell number. In
another embodiment, compositions of the invention are for use in
delaying immunosenescence characterised by an increase in Treg cell
number and a decrease in B cell number. In one embodiment,
compositions of the invention are for use in delaying
immunosenescence by decreasing Treg cell number. In one embodiment,
compositions of the invention are for use in delaying
immunosenescence by increasing B cell number. In another
embodiment, compositions of the invention are for use in delaying
immunosenescence by decreasing Treg cell number and increasing B
cell number. In one embodiment, compositions of the invention are
for use in treating diseases caused by immunosenescence. In one
embodiment, compositions of the invention are for use in treating
aging-related diseases by delaying and/or preventing
immunosenescence.
[0157] Furthermore, it has been proposed that vaccine adjuvants may
overcome immunosenescence [48]. Since the compositions of the
invention are suitable for use as a vaccine adjuvant, compositions
of the invention may be useful for preventing or delaying
immunosenescence. In another embodiment, compositions of the
invention are for use in delaying and/or preventing
immunosenescence as a vaccine adjuvant. In another embodiment,
compositions of the invention are for use as a vaccine adjuvant,
wherein the compositions delay and/or prevent immunosenescence.
[0158] Diseases that are associated with immunosenescence include
cardiovascular disease, cancer, diabetes mellitus type 2 [49] and
autoimmune disorders [50].
Modes of Administration
[0159] 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 (including
sublingual), but they may be administered rectally or
intranasally.
[0160] In certain embodiments, the compositions of the invention
may be administered as a foam, as a spray or a gel.
[0161] 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.
[0162] 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.
[0163] 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.
[0164] 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.
[0165] 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 reduce the likelihood of disease developing in
her child in utero and/or after it is born.
[0166] The compositions of the invention may be administered to a
patient that has been diagnosed with a disease or condition
mediated reduced immune activity, or that has been identified as
being at risk of a disease or condition mediated by reduced immune
activity. The compositions may also be administered as a
prophylactic measure to prevent the development of diseases or
conditions mediated by reduced immune activity in a healthy
patient.
[0167] The compositions of the invention may be administered to a
patient that has been diagnosed with deficient immune activity, or
that has been identified as being at risk of deficient immune
activity. For example, the patient may have reduced or absent
colonisation by Enterococcus, and in particular Enterococcus
gallinarum.
[0168] The compositions of the invention may be administered as a
food product, such as a nutritional supplement.
[0169] 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.
Compositions
[0170] 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.
[0171] 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 [51,52].
[0172] Alternatively, the composition of the invention may comprise
a live, active bacterial culture.
[0173] 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.
[0174] Guidance on encapsulation that may be useful for preparing
compositions of the invention is available in, for example,
references [53] and [54].
[0175] The composition may be administered orally and may be in the
form of a tablet, capsule or powder. Encapsulated products are
preferred because Enterococcus 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.
[0176] The composition may be formulated as a probiotic.
[0177] 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.
[0178] 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 x 10.sup.6 to about 1.times.10.sup.10 CFU; in
another example from about 1.times.10.sup.7 to about
1.times.10.sup.11 CFU; in another example from about
1.times.10.sup.8 to about 1.times.10.sup.10 CFU; in another example
from about 1.times.10.sup.8 to about 1.times.10.sup.11 CFU.
[0179] In certain embodiments, the dose of the bacteria is at least
10.sup.9 cells per day, such as at least 10.sup.10, at least
10.sup.11, or at least 10.sup.12 cells per day.
[0180] 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, 3 g, 5
g, and 10 g.
[0181] 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.
[0182] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein the composition is administered
at a dose of between 500 mg and 1000 mg, between 600 mg and 900 mg,
between 700 mg and 800 mg, between 500 mg and 750 mg or between 750
mg and 1000mg. In certain embodiments, the invention provides the
above pharmaceutical composition, wherein the lyophilised bacteria
in the pharmaceutical composition is administered at a dose of
between 500 mg and 1000 mg, between 600 mg and 900 mg, between 700
mg and 800 mg, between 500 mg and 750 mg or between 750 mg and 1000
mg.
[0183] 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.
[0184] 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.
[0185] The compositions of the invention may comprise
pharmaceutically acceptable excipients or carriers. Examples of
such suitable excipients may be found in the reference [55].
Acceptable carriers or diluents for therapeutic use are well known
in the pharmaceutical art and are described, for example, in
reference [56]. 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.
[0186] 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.
[0187] 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.
[0188] The compositions for use in accordance with the invention
may or may not require marketing approval.
[0189] 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.
[0190] The compositions of the invention can comprise
pharmaceutically acceptable excipients, diluents or carriers.
[0191] 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.
[0192] 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.
[0193] 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.
[0194] 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.
[0195] 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
pro-inflammatory cytokines, such as IL-1(3, TNF-.alpha.,
MIP-3.alpha., IL-23 or IL-6. In a preferred embodiment, 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 TNF-.alpha..
[0196] 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.
[0197] 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.
[0198] 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.
[0199] 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.
[0200] In certain embodiments, the invention provides the above
pharmaceutical composition, comprising a diluent selected from the
group consisting of ethanol, glycerol and water.
[0201] 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.
[0202] In certain embodiments, the invention provides the above
pharmaceutical composition, further comprising at least one of a
preservative, an antioxidant and a stabilizer.
[0203] 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.
[0204] In certain embodiments, the invention provides the above
pharmaceutical composition, wherein said bacterial strain is
lyophilised.
[0205] 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.degree. C. 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.
Culturing Methods
[0206] The bacterial strains for use in the present invention can
be cultured using standard microbiology techniques as detailed in,
for example, references [57,58].
[0207] The solid or liquid medium used for culture may be YCFA agar
or YCFA medium. YCFA medium may include (per 100mL, approximate
values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO3 (0.4 g),
cysteine (0.1 g), K.sub.2HPO.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).
Bacterial Strains for Use in Vaccine Compositions
[0208] The inventors have identified that the bacterial strains of
the invention are useful for treating or preventing diseases or
conditions associated with reduce immune activity. 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, when administered as vaccine compositions.
In certain such embodiments, the bacterial strains of the invention
may be killed, inactivated or attenuated. In certain such
embodiments, the compositions may comprise a vaccine adjuvant. In
certain embodiments, the compositions are for administration via
injection, such as via subcutaneous injection.
General
[0209] 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 [59] and [60,61], etc.
[0210] 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.
[0211] The term "about" in relation to a numerical value x is
optional and means, for example, x.+-.10%.
[0212] 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.
[0213] 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. [62]. 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.
[63].
[0214] 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.
[0215] 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 Mouse Models of
Cancer
Summary
[0216] This study tested the efficacy of compositions comprising
bacterial strains according to the invention in four tumor
models.
Materials
[0217] Test substance--Bacterial strain #MRx0518.
[0218] Reference substance--Anti-CTLA-4 antibody (clone: 9H10,
catalog: BE0131, isotype: Syrian Hamster IgG1, Bioxcell).
[0219] Test and reference substances vehicles--Bacterial culture
medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each
day of injection to mice, antibody was diluted with PBS (ref:
BE14-516F, Lonza, France).
[0220] Treatment doses--Bacteria: 2.times.10.sup.8 in 200 .mu.L.
The anti-CTLA-4 was injected at 10 mg/kg/inj. Anti-CTLA-4 was
administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse
weighing 20 g, 200 .mu.L of test substance will be administered)
according to the most recent body weight of mice.
[0221] Routes of administration--Bacterial inoculum was
administered by oral gavage (per os, PO) via a cannula. Cannulas
were decontaminated every day. Anti-CTLA-4 was injected into the
peritoneal cavity of mice (Intraperitoneally, IP).
[0222] Culture conditions of bacterial strain--The culture
conditions for the bacterial strain were as follows: [0223] Pipette
10 mL of YCFA (from the prepared 10 mL E&O lab bottles) into
Hungate tubes [0224] Seal the tubes and flush with CO.sub.2 using a
syringe input and exhaust system [0225] Autoclave the Hungate tubes
[0226] When cooled, inoculate the Hungate tubes with 1 mL of the
glycerol stocks [0227] Place the tubes in a static 37.degree. C.
incubator for about 16 hours. [0228] The following day, take 1 mL
of this subculture and inoculate 10 mL of YCFA (pre-warmed flushed
Hungate tubes again, all in duplicate) [0229] Place them in a
static 37.degree. C. incubator for 5 to 6 h
[0230] Cancer Cell Line and Culture Conditions--
[0231] The cell lines that were used are detailed in the table
below:
TABLE-US-00001 Cell line Type Mouse strain Origin EMT-6 Breast
carcinoma BALB/c ATCC LL/2 (LLC1) Lung carcinoma C57BL/6 ATCC
CRL1642 Hepa1-6 Hepatocellular C57BL/6 IPSEN carcinoma INNOVATION
RENCA Renal adenocarcinoma BALB/c ATCC
[0232] The EMT-6 cell line was established from a transplantable
murine mammary carcinoma that arose in a BALB/cCRGL mouse after
implantation of a hyperplastic mammary alveolar nodule [64].
[0233] The LL/2 (LLC1) cell line was established from the lung of a
C57BL/6 mouse bearing a tumour resulting from an implantation of
primary Lewis lung carcinoma [65].
[0234] The Hepa 1-6 cell line is a derivative of the BW7756 mouse
hepatoma that arose in a C57/L mouse [66].
[0235] Cell culture conditions--All cell lines were grown as
monolayer at 37.degree. C. in a humidified atmosphere (5% CO.sub.2,
95% air). The culture medium and supplement are indicated in the
table below:
TABLE-US-00002 Cell line Culture medium Supplement EMT6 RPMI 1640
containing 2 mM L-glutamine (ref: 10% foetal bovine serum (ref:
#3302, BE12-702F, Lonza) Lonza) LL/2 RPMI 1640 containing 2 mM
L-glutamine (ref: 10% foetal bovine serum (ref: #3302, (LLC1)
BE12-702F, Lonza) Lonza) Hepa1-6 DMEM (ref:11960-044, Gibco) 10%
foetal bovine serum (ref: #3302, Lonza) 2 mM L-Glutamine
penicillin-streptomycin (Sigma G-6784) RENCA DMEM 10% fetal bovine
serum, 2mM L-glutamine, 1 ug/mL puromycin
[0236] For experimental use, adherent tumour cells were detached
from the culture flask by a 5 minute treatment with trypsin-versene
(ref: BE17-161E, Lonza), in Hanks' medium without calcium or
magnesium (ref: BE10-543F, Lonza) and neutralized by addition of
complete culture medium. The cells were counted in a hemocytometer
and their viability will be assessed by 0.25% trypan blue exclusion
assay.
[0237] Use of Animals--
[0238] Healthy female BALB/C (BALB/cByJ) mice, of matching weight
and age, were obtained from CHARLES RIVER for the EMT6 and RENCA
model experiments.
[0239] Healthy female C57BL/6 (C57BL16J) mice, of matching weight
and age, were obtained from CHARLES RIVER (L'Arbresles) for the
LL/2(LLC1) and the Hepal-6 model experiments.
[0240] Animals were maintained in SPF health status according to
the FELASA guidelines, and animal housing and experimental
procedures according to the French and European Regulations and NRC
Guide for the Care and Use of Laboratory Animals were followed
[67,68]. Animals were maintained in housing rooms under controlled
environmental conditions: Temperature: 22.+-.2.degree. C., Humidity
55.+-.10%, Photoperiod (12 h light/12 h dark), HEPA filtered air,
15 air exchanges per hour with no recirculation. Animal enclosures
were provided with sterile and adequate space with bedding
material, food and water, environmental and social enrichment
(group housing) as described: 900 cm.sup.2 cages (ref: green,
Tecniplast) in ventilated racks, Epicea bedding (SAFE),10 kGy
Irradiated diet (A04-10, SAFE), Complete food for immuno-competent
rodents--R/M-H Extrudate, water from water bottles.
Experimental Design and Treatments
Antitumor Activity, EMT6 Model
[0241] Treatment schedule--The start of first dosing was considered
as DO. On DO, non-engrafted mice were randomized according to their
individual body weight into groups of 9/8 using Vivo manager.RTM.
software (Biosystemes, Couternon, France). On D0, the mice received
vehicle (culture medium) or bacterial strain. On D14, all mice were
engrafted with EMT-6 tumor cells as described below. On D24, mice
from the positive control group received anti-CTLA-4 antibody
treatments.
[0242] The treatment schedule is summarized in the table below:
TABLE-US-00003 No. Treatment Group Animals Treatment Dose Route
Schedule 1 8 Untreated -- -- -- 2 8 Vehicle (media) -- PO Q1Dx42 3
9 Bacterial strain #1 2x108 bacteria PO Q1Dx42 (MRx0518) 4 8
Anti-CTLA4 10 mg/kg IP TWx2
[0243] The monitoring of animals was performed as described
below.
[0244] Induction of EMT6 tumours in animals--On D14, tumours were
induced by subcutaneous injection of 1.times.10.sup.6 EMT-6 cells
in 200 .mu.L RPMI 1640 into the right flank of mice.
[0245] Euthanasia--Each mouse was euthanized when it reached a
humane endpoint as described below, or after a maximum of 6 weeks
post start of dosing.
Antitumor Activity, LL/2 (LLC1) Model
[0246] Treatment schedule--The start of first dosing was considered
as DO. On DO, non-engrafted mice were randomized according to their
individual body weight into 7 groups of 9/8 using Vivo manager.RTM.
software (Biosystemes, Couternon, France). On DO, the mice received
vehicle (culture medium) or bacterial strain. On D14, all mice were
engrafted with LL/2 tumor cells as described below. On D27, mice
from the positive control group received anti-CTLA-4 antibody
treatments.
[0247] The treatment schedule is summarized in the table below:
TABLE-US-00004 No. Treatment Group Animals Treatment Dose Route
Schedule 1 8 Untreated -- -- -- 2 9 Vehicle (media) -- PO Q1Dx42 3
9 Bacterial strain #1 2x10.sup.8 bacteria PO Q1Dx42 (MRx0518) 4 8
Anti-CTLA4 10 mg/kg IP TWx2
[0248] The monitoring of animals was performed as described
below.
[0249] Induction of LL/2 (LLC1) tumors in animals--On D14, tumors
were induced by subcutaneous injection of 1.times.10.sup.6 LL/2
(LLC1) cells in 200 .mu.L RPMI 1640 into the right flank of
mice.
[0250] Euthanasia--Each mouse was euthanized when it reached a
humane endpoint as described below, or after a maximum of 6 weeks
post start of dosing.
Antitumor activity, Hepal-6 Model
[0251] Treatment schedule--The start of first dosing was considered
as D0. On DO, non-engrafted mice were randomized according to their
individual body weight into 7 groups of 9 using Vivo manager.RTM.
software (Biosystemes, Couternon, France). On D0, the mice received
vehicle (culture medium) or bacterial strain. On D14, all mice were
engrafted with Hepa 1-6 tumor cells as described below. On D16,
mice from the positive control group received anti-CTLA-4 antibody
treatments.
[0252] The treatment schedule is summarized in the table below:
TABLE-US-00005 No. Treatment Group Animals Treatment Dose Route
Schedule 1 9 Untreated -- -- -- 2 9 Vehicle (media) -- PO Q1Dx42 6
9 Bacterial strain #4 2x10.sup.8 bacteria PO Q1Dx42 (MRx0518) 7 9
Anti-CTLA4 10 mg/kg IP TWx2
[0253] The monitoring of animals was performed as described
below.
[0254] Orthotopic induction of Hepa 1-6 tumor cells in animals by
intrasplenic injection--On D0, one million (1.times.10.sup.6) Hepa
1-6 tumor cells in 50 .mu.L RPMI 1640 medium were transplanted via
intra-splenic injection into mice. Briefly, a small left subcostal
flank incision was made and the spleen was exteriorized. The spleen
was exposed on a sterile gauze pad, and injected under visual
control with the cell suspension with a 27-gauge needle. After the
cell inoculation, the spleen was excised.
[0255] Euthanasia--Each mouse was euthanized when it reached a
humane endpoint as described in section below, or after a maximum
of 6 weeks post start of dosing.
[0256] Evaluation of tumour burden at euthanasia--At the time of
termination, livers were collected and weighed.
Antitumor Activity, RENCA Model
[0257] Treatment schedule--The start of first dosing was considered
as D0. On D0, non-engrafted mice were randomized according to their
individual body weight into groups of 12 mice. On D0, the mice
received vehicle (culture medium) or bacterial strain
(2.times.10.sup.8 in 100 .mu.L, PO). On D14, all mice were
engrafted with RENCA tumour cells injected SC into the left hind
flank as described below. Treatment with anti-CTLA-4 (clone 9D9, 10
mg/kg, IP) and anti-PDL1 (clone 10F.9G2, 10 mg/kg, IP) was
initiated from D17.
[0258] The treatment schedule is summarized in the table below:
TABLE-US-00006 No. Treatment Group Animals Treatment Dose Route
Schedule 1 12 Untreated -- -- -- 2 12 Vehicle (media) -- PO QD 3 12
Bacterial strain 2x10.sup.8 PO QD (MRx0518) bacteria 4 12
Paclitaxel 15 mg/kg IP Q4D (every four days) 5 12 Anti-CTLA4 + 10
mg/kg + IP BIW (twice Anti-PDL1 10 mg/kg weekly) From day 3
[0259] The monitoring of animals was performed as described
below.
[0260] On D14 (following 2 weeks of bacterial
dosing/pre-treatment), 5.times.10.sup.5 viable cells in 100 .mu.L
of PBS were injected subcutaneously into the left hind flank of
each mouse (which was sterilised with surgical spirit), 1 syringe
and needle per mouse. The implantation sites were shaved the day
prior to cell implantation.
[0261] Euthanasia--Each mouse was euthanized when it reached a
humane endpoint as described in section below, or after a maximum
of 6 weeks post start of dosing.
[0262] Evaluation of tumour burden at euthanasia--At the time of
termination, tumours were collected and their volume evaluated.
Animal Monitoring
[0263] Clinical monitoring--The length and width of the tumour was
measured 2-3 times a week with callipers and the volume of the
tumour was estimated by this formula [69]:
Tumor .times. .times. volume = width 2 .times. length 2
##EQU00001##
[0264] Humane endpoints [70]: Signs of pain, suffering or distress:
pain posture, pain face mask, behaviour; Tumor exceeding 10% of
normal body weight, but non-exceeding 2000 mm.sup.3; Tumors
interfering with ambulation or nutrition; Ulcerated tumour or
tissue erosion; 20% body weight loss remaining for 3 consecutive
days; Poor body condition, emaciation, cachexia, dehydration;
Prolonged absence of voluntary responses to external stimuli; Rapid
laboured breathing, anaemia, significant bleeding; Neurologic
signs: circling, convulsion, paralysis; Sustained decrease in body
temperature; Abdominal distension.
[0265] Anaesthesia--Isoflurane gas anesthesia was used for all
procedures: surgery or tumour inoculation, i.v. injections, blood
collection. Ketamine and Xylazine anesthesia was used for
stereotaxia surgical procedure.
[0266] Analgesia--Carprofen or multimodal carprofen/buprenorphine
analgesia protocol were adapted to the severity of surgical
procedure. Non-pharmacological care was provided for all painful
procedures. Additionally, pharmacological care not interfering with
studies (topic treatment) were provided at the recommendation of
the attending veterinarian.
[0267] Euthanasia--Euthanasia of animals was performed by gas
anesthesia over-dosage (Isoflurane) followed by cervical
dislocation or exsanguination.
Results
Antitumor Activity, EMT6 Model
[0268] The results are shown in FIG. 1A. Treatment with the
bacterial strain of the invention led to a clear reduction in
tumour volume relative to both the negative controls. The positive
control also led to a reduction in tumour volume, as would be
expected.
[0269] To further elucidate the mechanisms through which MRx0518
conveys its therapeutic effects in syngeneic tumour models, ex vivo
analysis was performed on the syngeneic EMT6 tumour model studies.
While tumour volume is the primary measurement in preclinical
oncology studies, tumours often consist of actively dividing tumour
cells along with a necrotic core. To investigate whether MRx0518
treatment had influence on the degree of necrosis found within EMT6
tumours, paraffin sections from the mid-belly region of the tumours
were stained with Haematoxylin and Eosin. MRx0518 treatment of a
murine EMT6 breast carcinoma model showed a tendency towards
increasing the cross-sectional area of necrosis within the tumour
(FIG. 1B, upper panel). To investigate whether MRx0518 treatment
had influence on dividing cells within the tumour, paraffin
sections from the mid-belly region of the tumours were stained with
the proliferation protein Ki67, along with DAPI counter stain, to
estimate the percentage of cells dividing within the EMT6 tumour.
MRx0518 treatment of a murine EMT6 breast carcinoma model
significantly decreased the percentage of dividing cells seen
within the tumour (FIG. 1B, lower panel, P=0.019).
Immune Cell Populations
[0270] Further investigation of the tumour microenvironment was
performed through flow cytometry of the tumour, to investigate the
hypothesis that the MRx0518 bacterial strain has the ability to
regulate the immune system into inducing an anti-tumour effect.
Tumours excised from the different treatment groups were cut into
pieces. One piece was subjected to flow cytometry analysis. To
assess the relative percentage of T lymphocytes, present within the
tumours, the following markers were used: CD45, CD3, CD4, CD8, CD25
and FoxP3.
[0271] The preliminary flow cytometry data presented in FIG. 1C
(and further supported by the below described data, presented in
FIG. 23) shows that the relative percentage of lymphocytes in
tumours was slightly decreased in both the MRx0518 and anti-CTLA-4
treated groups, when compared respectively to vehicle or control
animals. Likewise, the relative percentage of CD4.sup.+ cells
appeared to be decreased in MRx0518 and anti-CTLA-4 treated
animals, whilst the relative percentage of CD8.sup.+ cells followed
an opposite trend in both groups, albeit with different magnitude.
The relative percentage of CD4.sup.+ cells was lower in the
anti-CTLA-4 treated group when compared to the slight decrease in
MRx0518 treated animals; however, the reduction in the relative
percentage of CD4.sup.+CD25.sup.+ cells was noticeable only in the
anti-CTLA-4 treated group. The CD8+/FoxP3+ ratio showed a greater
increase in the anti-CTLA-4 treated group than in the MRx0518
animals. These data presented here supports the hypothesis that
anti-CTLA-4 antibody targets regulatory T cells (Tregs) by reducing
their cell numbers or attenuating their suppressive activity in
tumour tissue, whilst suggesting a different mode of action for
MRx0518.
[0272] Additional investigation of the tumour microenvironment was
performed using NanoString analysis of the tumour tissues, to
investigate whether the MRx0518 bacterial strain has the ability to
regulate the immune system into inducing an anti-tumour effect in
the EMT6 model. Tumours excised from vehicle and MRx0518-treated
groups were collected. RNA was extracted from tumour tissue using
TRIzol reagent (ThermoFisher) followed by a clean-up using the
RNeasy Mini kit (Qiagen) including a DNase I treatment (Qiagen).
RNA was then used for Nanostring analysis using the PanCancer Mouse
IO 360 panel. Genes previously shown to be characteristic of
various cell populations were used to measure these populations'
abundance:
TABLE-US-00007 Cell type Marker genes NK CD56dim cells Il21r,
Kir3dl1, Kir3dl2 Exhausted CD8 Cd244, Eomes, Lag3, Ptger4 DC Ccl2,
Cd209e, Hsd11b1 Cytotoxic cells Ctsw, Gzma, Gzmb, Klrb1, Klrd1,
Kirk1, Nkg7, Prf1 Macrophages Cd163, Cd68, Cd84, Ms4a4a T-cells
Cd3d, Cd3e, Cd3g, Cd6, Sh2d1a, Trat1 Mast cells Cpa3, Hdc, Ms4a2
Neutrophils Ceacam3, Csf3r, Fcgr4, Fpr1 B-cells Blk, Cd19, Fcrlb,
Ms4a1, Pnoc, Spib, Tcl1, Tnfrsf17 NK cells Ncr1, Xcl1 CD45
Ptprc
[0273] Z-scores for each cell population were calculated using the
linear cell type scores provided by the NanoString analysis (FIG.
23, heat map).
[0274] The NanoString data shows that the abundance of B cells,
CD45, T cells, cytotoxic and NK cells were increased in the tumour
tissue of MRx0518-treated group when compared to vehicle-treated
animals (FIG. 23). The data presented here supports the hypothesis
that MRx0518 has an immunostimulatory effect by increasing
leukocytes, in particular NK cells, T cells and cytotoxic cells in
the tumour microenvironment.
Cytokine Production
[0275] An additional tumour piece was used for total protein
extraction and subsequent cytokine analysis, together with plasma
samples. Protein levels of IL-10, CXCL1, CXCL2, CXCL10, IL-1 ,
IL-17A, GM-CSF, TNF-.alpha. a, IL-12p70 and IFN-.gamma. in the
tumour microenvironment were analysed by MagPix technology. While
IL-17A and GM-CSF were below levels of detection, all the other
markers were expressed at reasonable levels (FIG. 1D). A
significance difference was observed between the vehicle and
anti-CTLA-4 group for IFN-.gamma.. The production of the IL-10 and
IL-12p70 immune markers seemed reduced following MRx0518 treatment
compared to the control treatments.
[0276] Cytokine levels were also assessed in blood plasma of the
same animals. Protein levels of IL-23, IL-6, IL-10, VEGF, CXCL1,
CXCL2, CXCL10, IL-2, IL-1 , IL-17A, GM-CSF, TNF-.alpha., IL-12p70
and IFN-y were analysed by MagPix technology. Overall, little
cytokine production was detected in the blood plasma of animals
either before tumour induction or at the end of the study (FIG.
1E). VEGF and CXCL10 were detected at substantial levels, while
IL-23, IL-6, IL-10, CXCL1 and CXCL2 were detected at low levels.
IL-2, IL-1b, IL-17A, GM-CSF, TNF-.alpha., IL-12p70 and IFN-.gamma.
were not detected in the samples. MRx0518 significantly increased
production of IL-6 at Day 0. MRx0518 also seemed to increase IL-23
production. VEGF and CXCL10 were significantly downregulated in the
anti-CLTA-4 group at Day 22. Similarly to the results shown for the
immune cell populations, the differences in cytokine production in
the tumour and plasma, between MRx0518 and ant-CTLA-4 suggests that
each of them acts on a distinct and potentially complementary
mechanism.
Localisation of CD8.alpha. Positive Cells in the Ileum
[0277] 10 .mu.m cryo-sections of ileum were cut in cryostat (CM
1950 Leica), picked up onto poly-L Lysine slides. The sections were
then air-dried for 1 hour, fixed for 10 minutes in ice-cold
methanol, washed in PBS, blocked in 10% BSA in PBS pH 7.2 before
being incubated overnight with the primary antibody
(rat-anti-mouse-CD8.alpha. antibody, Sigma-Aldrich, Millipore).
[0278] The next morning the slides were washed in PBS and stained
with a secondary antibody: goat-anti-rat-antibody-Alexa488
(Molecular Probe, Invitrogen) for 1 hour at room temperature. After
another washing step, the slides were counterstained with
4',6-diamidino-2-phenylindole dihydrochloride (DAPI)
(Sigma-Aldrich, Millipore) and mounted in Vectashield (Vector
Laboratories). The slides were viewed and imaged using a Zeiss
Axioscope Microscope equipped with a mercury vapour lamp,
appropriate filters and a x20 apochromatic objective. Examples of
images obtained from slides from the vehicle, MRx0518, and
anti-CTLA4 animals are shown (FIG. 1F--upper panels: DAPI staining,
lower panels: CD8.alpha. staining).
[0279] Fields of view were examined from 20 animals and imaged
using manual exposure time. The number of animals and fields
analysed are shown in the following table:
TABLE-US-00008 Number of fields Number of Group analysed mice
Vehicle 53 5 MRx0518 70 7 Anti-CTLA4 71 8
[0280] The images were scored as follow: fields with .ltoreq.3
positive cells were scored as 0, whilst fields with more .gtoreq.3
cells were scored as 1. The results of this analysis are shown
(FIG. 1G).
[0281] Ileum cryosections stained with anti-CD8a showed a higher
number of CD8a positive cells localized in the crypt region tissues
from animals treated with MRx0518 and anti-CTLA-4 compared to the
vehicle group.
[0282] This observation is in line with CD8.sup.+ T cells being
present in the intestine in case of infection or inflammatory
microenvironment, as part of the immune response.
Antitumor Activity, LL/2 (LLC1) Model
[0283] The results are shown in FIG. 2. Treatment with the
bacterial strain of the invention led to a clear reduction in
tumour volume relative to both the negative controls.
Antitumor Activity, Hepal-6 Model
[0284] The results are shown in FIG. 3A. The untreated negative
control does not appear as would be expected, because liver weight
was lower in this group than the other groups. However, the vehicle
negative control and the positive control groups both appear as
would be expected, because mice treated with vehicle alone had
larger livers than mice treated with anti-CTLA4 antibodies,
reflecting a greater tumour burden in the vehicle negative control
group. Treatment with the bacterial strain of the invention led to
a clear reduction in liver weight (and therefore tumour burden)
relative to the mice in the vehicle negative control group.
Antitumor Activity, RENCA Model
[0285] The results are shown in FIG. 3B. Treatment with MRx0518
monotherapy reduced tumour volume with Test/Control of 51% (day 18)
compared with the vehicle-treated groups. Paclitaxel and
anti-CTLA-4+anti-PDL-1 showed an (almost) complete reduction in
tumour size at D18 and D22 compared to both the untreated and
vehicle groups.
[0286] These data indicate that strain MRx0518 may be useful for
treating or preventing other diseases associated with reduced
immune system activity.
Example 2--PCR Gene Analysis
[0287] A pure culture of bacteria MRx0518 was studied in a PCR gene
analysis. There were two arms to the experiment: 1) MRx0518 was
co-cultured with human colonic cells (CaCo2) to investigate the
effects of the bacteria on the host, and 2) MRx0518 was co-cultured
on CaCo2 cells that were stimulated with IL1 to mimic the effect of
the bacteria in an inflammatory environment. The effects in both
scenarios were evaluated through gene expression analysis. The
results are shown below:
TABLE-US-00009 Gene Fold change Function CXCL3 28412.73 CXCR2
ligand, CXCL2 135.42 CXCR2 ligand, 90% homology with CXCL1. CXCL9
34.76 CXCR3 ligand, primarily thought of as Th1 cell
chemoattractant (inducible by IFN-g) IL8 31.81 Cytokine,
chemoattractant (especially neutrophils), many receptors including
CXCR1 and CXCR2/ CXCL1 16.48 CXCR2 ligand, stimulates cell
proliferation as well as migration, overexpression is
neuroprotective in EAE. CD40 14.33 Co-stimulatory molecule, route
of T cell dependent DC activation. TNF 13.50 Major proinflammatory
cytokine IL17C 12.18 Promotes antibacterial response from
epthielium, synergistic with IL-22, CXCL10 10.66 Close homology
with CXCL9, think also CXCR3 ligand? HSPA1B 10.19 Heat shock
protein NFKBIA 8.87 NE.kappa.B signalling; PI3K JUN 7.61
Antibacterial response; GPCR signalling. TNFAIP3 6.63 TNF
signalling DUSP1 6.36 Anti-inflammatory phosphatase, inactivates
MAPKs JUNB 5.36 Transcription factor, JAK-STAT signalling BIRC3
4.86 Adherens junctions, tight junctions DUSP2 4.59
Anti-inflammatory, inactivates MAPK. IL32 4.29 Proinflammatory
cytokine, induced by IFN-g, IL-18 DUSP5 3.12 Anti-inflammatory,
inactivates MAPK FOS 3.03 Transcription factors, TLR signalling,
forms part of AP-1 GADD45B 2.89 Cell growth and proliferation CLDN4
2.61 Tight junctions ADM 2.57 NE.kappa.B signalling KLF10 2.49 Cell
arrest, TGF-b signaling. DEFB4A -2.34 Antimicrobial peptide APBA1
-2.53 Signalling IGFBP1 -2.72 Signalling pathway IL28B -2.73
IFN-lambda, antiviral immune defence, IL10 -3.38 Anti-inflammatory
cytokine NR4A1 -5.57 Nuclear receptor, anti-inflammatory, regulator
of T cell proliferation. T helper cell differentiation NOD2 -14.98
PRR, inflammasome activator, promotes autophagy INOS -26.88
Proinflammatory, generator of nitric oxide
[0288] These data appear to show two gene expression
signatures--CXCR1/2 ligands (CXCL3, CXCL2, CXCL1, IL-8), which is
associated with pro-inflammatory cell migration, and CXCR3 ligands
(CXCL9, CXCL10), which is more specifically indicative of
IFN-.gamma.-type responses, also supported by IL-32, which is
IFN-.gamma.-inducible. These data suggest that the compositions of
the invention are useful for stimulating the immune system.
Example 3--Stability Testing
[0289] 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.
Example 4--Cytokine Production in Immature Dendritic Cells Induced
by MRx0518 Compared to MRx0518 +LPS
Summary
[0290] This study tested the effect of the bacterial strain MRx0518
alone and in combination with lipopolysaccharide (LPS) on cytokine
production in immature dendritic cells.
[0291] A monocyte population was isolated from peripheral blood
mononuclear cells (PBMCs). The monocyte cells were subsequently
differentiated into immature dendritic cells. The immature
dendritic cells were plated out at 200,000 cells/well and incubated
with MRx0518 at a final concentration of 10.sup.7/mL, with the
optional addition of LPS at a final concentration of 100 ng/mL. The
negative control involved incubating the cells with RPMI media
alone and positive controls incubated the cells with LPS at a final
concentration of 100 ng/mL. The cytokine content of the cells was
then analysed.
Results
[0292] The results of these experiments can be seen in FIGS. 4a-d.
The addition of MRx0518 alone leads to a substantial increase in
the level of cytokines IL-6 and TNF-.alpha. compared to the
negative control (FIG. 4a and c). The addition of LPS (positive
control) leads to an increase in the level of IL-6 and TNF-a
compared to the negative control but not IL-1(3 (FIG. 4b). A
combination of MRx0518 and LPS led to a synergistic increase in the
level of IL-1(3 produced (FIG. 4d).
Conclusion
[0293] MRx0518 has the ability to induce higher IL-6 and
TNF-.alpha. cytokine production in immature dendritic cells. The
combination LPS and MRx0518 can increase the levels of cytokines
IL-1.beta. in immature dendritic cells. These data indicate that
MRx0518 alone or in combination with LPS can increase inflammatory
cytokines IL-1.beta., IL-6 and TNF-.alpha., which promotes
inflammation.
Example 5--Cytokine Production in THP-1 Cells Induced by MRx0518
Compared to MRx0518 +LPS
Summary
[0294] This study tested the effect of bacterial strain MRx0518
alone and in combination with LPS on cytokine production in THP-1
cells, a model cell line for monocytes and macrophages.
[0295] THF-1 cells were differentiated into MO medium for 48 h with
5 ng/mL phorbol-12-myristate-13-acetate (PMA). These cells were
subsequently incubated with MRx0518 at a final concentration of
10.sup.8/mL, with or without the addition of LPS at a final
concentration of 100 ng/mL. The bacteria were then washed off and
the cells allowed to incubate under normal growing conditions for
24 h. The cells were then spun down and the resulting supernatant
was analysed for cytokine content.
Results
[0296] The results of these experiments can be seen in FIGS. 5a-c.
The addition of MRx0518 without LPS leads to an increase in the
cytokine levels of IL-1(3, IL-6 and TNF-.alpha. compared to the no
bacterial and the bacterial sediment controls. The addition of LPS
and MRx0518 leads to a synergistic increase in the production of
cytokines.
Conclusion
[0297] MRx0518 has the ability to induce cytokine production in
THP-1 cells, which can be synergistically increased with the
addition of LPS. These data indicate that MRx0518 alone or in
combination with LPS can increase inflammatory cytokines
IL-1.beta., IL-6 and TNF-.alpha., which promotes inflammation.
Example 6--Cytokine Analysis
Introduction
[0298] The inventors sought to further analyse the
immunostimulatory effect of compositions of the invention. The
inventors analysed the expression of particular cytokines from
THP-1 macrophages and dendritic cells derived from monocytes upon
treatment with MRx0518. Macrophages and dendritic cells are key
components of the innate immune system, act as messengers between
the innate and adaptive immune systems and are resent in the gut
where they release a variety of cytokines to modulate the immune
response.
[0299] Cytokines involved in the innate immune response
(TNF-.alpha., IL-12 and IL-10) were analysed and also cytokines
involved in the recruitment and activation of adaptive immune cells
(IL-8, IL-23, IL-1.beta. and IL-6).
Method
Bacterial Strains
MRx0518
[0300] LPS used as positive control
Results
[0301] The results are shown in FIGS. 6-13. MRx0518 induces a
strong and characteristic immuno-stimulatory profile in
THP-1-derived macrophages and DCs derived from monocytes. Cytokines
involved in the innate immune response (TNF-.alpha., IL-12 and
IL-10) are significantly induced by MRx0518 in both DCs and
macrophages. MRx0518 induces a very strong and significant
induction of IL-8 in both macrophages and DCs. MRX0581 induces a
strong and significant induction of IL-23 and IL-6. MRx0518 also
induced IL-1.beta..
Discussion
[0302] These data shows that MRx0518 has immunostimulatory
properties, and may be an effective composition for
immunostimulation.
Example 7--Mechanism of Action
[0303] Further experiments were performed to characterise the
mechanism of action by which MRx0518 stimulates the immune system.
A TLR5 signalling reporter assay was selected and the data are
presented in FIGS. 14 and 15. MRx0518 supernatant was the most
potent activator of TLR5 and NF-.kappa.B. Also, the supernatant was
treated with various lytic enzymes and trypsin was found to
abrogate the majority of activity.
Example 8--Adjuvant Immunostimulatory Activity of MRx0518 in a
Therapeutic Combination with a CTLA-4 Inhibitor
Summary
[0304] This study compared the anti-tumour activity of MRx0518, a
CTLA-4 inhibitor and therapeutic combinations of MRx0518 with the
CTLA-4 inhibitor in mice bearing EMT-6 tumour cells.
Materials
[0305] Test and Reference Substances--Bacterial strain #MRx0518;
Anti-CTLA4 antibody (ref: BE0131, Bioxcell; clone: 9H10;
reactivity: mouse; isotype: Hamster IgG1; storage conditions:
+4.degree. C.).
[0306] Test and reference substances vehicles--The MRx0518 bacteria
were grown in a bacterial culture medium (Yeast extract, Casitone,
Fatty Acid medium (YCFA)) and kept as a glycerol stock at
-80.degree. C. The animals were dosed with the bacteria according
to the study protocol. The anti-CTLA-4 antibodies were diluted with
PBS (ref: BE14-516F, Lonza, France) on each day of injection to
mice.
[0307] Treatment doses--Bacteria: 2.times.10.sup.8 in 200 .mu.L.
The anti CTLA4 antibodies were administered at 10 mg/kg body weight
according to the most recent body weight of mice.
[0308] Routes of administration--The bacterial composition was
administered by oral gavage (per os, PO) via a gavage tube at a
volume of 200 .mu.L/inj. The anti CTLA-4 antibodies were injected
into the peritoneal cavity of mice (Intraperitoneally, IP) at a
volume of 10 mL/kg adjusted to the most recent individual body
weight of mice.
[0309] Cancer cell line and culture conditions--The cell line that
was used in this study is the EMT-6 cell line that was obtained
from the ATCC (American Type Culture Collection, Manassas,
Virginia, USA). The EMT-6 cell line was established from a
transplantable murine mammary carcinoma that arose in a BALB/cCRGL
mouse after implantation of a hyperplastic mammary alveolar
nodule.
[0310] Tumor cells were grown as monolayer at 37.degree. C. in a
humidified atmosphere (5% CO2, 95% air). The culture medium was
RPMI 1640 containing 2 mM L-glutamine (ref: BE12- 702F, Lonza,
Verviers, Belgium) supplemented with 10% fetal bovine serum (ref:
3302, Lonza). EMT-6 tumor cells are adherent to plastic flasks. For
experimental use, tumor cells were detached from the culture flask
by a 5-minute treatment with trypsin-versene (ref: BE02- 007E,
Lonza), in Hanks' medium without calcium or magnesium (ref:
BE10-543F, Lonza) and neutralized by addition of complete culture
medium. The cells were counted and their viability was assessed by
0.25% trypan blue exclusion assay.
[0311] Use of animals--Healthy female BALB/C (BALB/cByJ) mice, 5-7
weeks old, were obtained from CHARLES RIVER (L'Arbresles) and
maintained in SPF health status according to the FELASA guidelines.
Animal housing and experimental procedures were realized according
to the French and
[0312] European Regulations and NRC Guide for the Care and Use of
Laboratory Animals. Animals were maintained 3-4 per cage in housing
rooms under controlled environmental conditions: Temperature:
22.+-.2.degree. C., Humidity 55.+-.10%, Photoperiod (12 h light/12
h dark), HEPA filtered air, 15 air exchanges per hour with no
recirculation. Animal enclosures were provided with sterile and
adequate space with bedding material, food and water, environmental
and social enrichment (group housing) as described: Top filter
polycarbonate Eurostandard Type III or IV cages, Corn cob bedding
(ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet
(Ssniff.RTM. Soest, Germany), Complete food for immunocompetent
rodents--R/M-H Extrudate, Sterile, filtrated at 0.2 .mu.m water and
Environmental enrichment (SIZZLE-dri kraft--D20004 SERLAB, France).
Animals are individually identified with RFID transponder and each
cage was ladled with a specific code. Treatment of the animals
started after one week of acclimation for batches 2 and 3, or after
three weeks of acclimation for batch 1.
Experimental Design and Treatments
[0313] On day-14 (D-14), non-engrafted mice were randomized
according to their individual body weight into 3 groups of 30
animals and 2 groups of 10 animals using Vivo Manager.RTM. software
(Biosystemes, Couternon, France). The mice were separated into 3
batches of 10 animals per treatment group (batch 1: 10 animals of
groups 1, 2 and 3; batch 2: 10 animals of groups 1, 2 and 3 and
batch 3: 10 animals of groups 1 to 5) with different termination
points from the start of the study: D-14 or DO.
[0314] At termination, batch 3 was split into 2 cohorts, due to
termination and FACS analyses schedules; these were staggered over
1 day: D24/D25. Therefore, every cohort of animals had 5 animals
per treatment group (4 animals from cage one and one animal from
cage 2). Based on the ethical criteria, if the tumor volume were
higher than 1500mm.sup.3, the selection of the animals to be
sacrifice on D24 and D25 is based on tumor volume instead of the
cage. The experimental design is depicted in FIG. 16A and
summarized below:
[0315] 1) Batch 1 (groups 1, 2 and 3) started treatment on DO and
was culled at D14 (10 animals form groups 1 to 3). These did not
receive tumor cells and constituted the baseline group.
[0316] 2) Batch 2 (group 1, 2 and 3) started treatment on D-14 and
was culled at D7 (10 animals form groups 1 to 3).
[0317] 3) Batch 3 (groups 1 to 5) started treatment on D-14 and was
culled at D24/25 (10 animals form groups 1 to 5). The treatment of
Anti CTLA-4 started on D10.
[0318] On day 0 (D0) all mice of batches 2 and 3 (termination at
day 7 and 24/25, respectively) were engrafted with EMT-6 tumour
cells by a subcutaneous injection of 1.times.10.sup.6 EMT-6 cells
in 200 .mu.L RPMI 1640 into the right flank (the 30 mice from batch
1, that were sacrificed on D14, did not receive the tumour
injection). The mice were treated according to the following
treatment schedule groups (TWx2=twice a week):
TABLE-US-00010 Group No. Animals Treatment Dose Route Treatment
Schedule 1 30 = Untreated (+Tumour) -- -- -- 10 batch 1 10 batch 2
10 batch 3 2 30 = Vehicle (YCFA) -- PO Daily -14 to D0 10 batch 1
Daily -14 to D7 10 batch 2 Daily -14 to D24/25 10 batch 3 3 30 =
MRx0518 2x10.sup.8 PO Daily -14 to D0 10 batch 1 Daily -14 to D7 10
batch 2 Daily -14 to D24/25 10 batch 3 4 10 batch 3 Anti-CTLA-4 +
10 mg/kg IP + PO TWx2 from D10 YCFA YCFA Daily -14 to D24/25 5 10
batch 3 Anti- CTLA-4 + 10 mg/kg + IP + PO TWx2 from D10 MRx0518
2x10.sup.8 bacteria Bacteria Daily -14 to D24/25
Animal Monitoring
[0319] The viability and behaviour of the animals was recorded
every day. Body weights were measured twice a week. The length and
width of the tumour was measured twice a week with callipers and
the volume of the tumour was estimated by the following
formula:
Tumor .times. .times. volume = Width 2 .times. Length 2
##EQU00002##
[0320] The treatment efficacy was assessed in terms of the effects
of the test substance on the tumour volumes of treated animals
relative to control animals. The following evaluation criteria of
antitumor efficacy were determined using Vivo Manager.RTM. software
(Biosystemes, Couternon, France). Mean tumour volumes of groups 1
to 5 are depicted in FIG. 16B. Throughout the course of the study,
a progression in tumour growth was observed in all groups, with the
exception of the MRx0518+Anti-CTLA-4-treated group where a
regression of tumour growth occurred from Day 14 post tumour
induction. MRx0518+Anti-CTLA-4 treatment significantly reduced
tumour growth compared to the Vehicle-treated group on Day 21 and
Day 24 post tumour induction. The combination treatment of MRx0518
with Anti-CTLA-4 was the most efficacious for reducing tumour
growth in BALB/c mice bearing subcutaneously grafted EMT6 tumours.
These data demonstrate that MRx0518 has an immunostimulatory
effect.
Example 9--Efficacy of Bacterial Inocula in Mouse Models of
Cancer
Summary
[0321] This study tested the efficacy of compositions comprising
bacterial strain according to the invention in a tumor model.
Materials
[0322] Test substance--Bacterial strain #MRx0554.
[0323] Reference substance--Anti-CTLA-4 antibody (clone: 9H10,
catalog: BE0131, isotype: Syrian Hamster IgG1, Bioxcell).
[0324] Test and reference substances vehicles--Bacterial culture
medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each
day of injection to mice, antibody was diluted with PBS (ref:
BE14-516F, Lonza, France).
[0325] Treatment doses--Bacteria: 2.times.10.sup.8 in 200 .mu.L
YCFA. The anti-CTLA-4 was injected at 10 mg/kg/inj. Anti-CTLA-4 was
administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse
weighing 20 g, 200 .mu.L of test substance will be administered)
according to the most recent body weight of mice.
[0326] Routes of administration--Bacterial inoculum was
administered by oral gavage (per os, PO) via a cannula. Cannulas
were decontaminated every day. Anti-CTLA-4 was injected into the
peritoneal cavity of mice (Intraperitoneally, IP).
[0327] Culture conditions of bacterial strain--The culture
conditions for the bacterial strain were as follows: [0328] Pipette
10 mL of YCFA (from the prepared 10 mL E&O lab bottles) into
Hungate tubes [0329] Seal the tubes and flush with CO.sub.2 using a
syringe input and exhaust system [0330] Autoclave the Hungate tubes
[0331] When cooled, inoculate the Hungate tubes with 1 mL of the
glycerol stocks [0332] Place the tubes in a static 37.degree. C.
incubator for about 16 hours. [0333] The following day, take 1 mL
of this subculture and inoculate 10 mL of YCFA (pre-warmed flushed
Hungate tubes again, all in duplicate) [0334] Place them in a
static 37.degree. C. incubator for 5 to 6 h
Cancer Cell Line and Culture Conditions--
[0335] The cell lines that were used are detailed in the table
below:
TABLE-US-00011 Cell line Type Mouse strain Origin EMT-6 Breast
carcinoma BALB/c ATCC
[0336] The EMT-6 cell line was established from a transplantable
murine mammary carcinoma that arose in a BALB/cCRGL mouse after
implantation of a hyperplastic mammary alveolar nodule [71].
[0337] Cell culture conditions--All cell lines were grown as
monolayer at 37.degree. C. in a humidified atmosphere (5% CO.sub.2,
95% air). The culture medium and supplement are indicated in the
table below:
TABLE-US-00012 Cell line Culture medium Supplement EMT6 RPMI 1640
containing 2 mM 10% fetal bovine serum L-glutamine (ref: BE12-702F,
(ref: #3302, Lonza) Lonza)
[0338] For experimental use, adherent tumor cells were detached
from the culture flask by a 5 minute treatment with trypsin-versene
(ref: BE17-161E, Lonza), in Hanks' medium without calcium or
magnesium (ref: BE10-543F, Lonza) and neutralized by addition of
complete culture medium. The cells were counted in a hemocytometer
and their viability will be assessed by 0.25% trypan blue exclusion
assay.
Use of Animals--
[0339] Healthy female BALB/C (BALB/cByJ) mice, of matching weight
and age, were obtained from CHARLES RIVER (L'Arbresles) for the
EMT6 model experiments.
[0340] Animals were maintained in SPF health status according to
the FELASA guidelines, and animal housing and experimental
procedures according to the French and European Regulations and NRC
Guide for the Care and Use of Laboratory Animals were followed
[72,73]. Animals were maintained in housing rooms under controlled
environmental conditions: Temperature: 22.+-.2.degree. C., Humidity
55.+-.10%, Photoperiod (12 h light/12 h dark), HEPA filtered air,
15 air exchanges per hour with no recirculation. Animal enclosures
were provided with sterile and adequate space with bedding
material, food and water, environmental and social enrichment
(group housing) as described: 900 cm.sup.2 cages (ref: green,
Tecniplast) in ventilated racks, Epicea bedding (SAFE),10 kGy
Irradiated diet (A04-10, SAFE), Complete food for immuno-competent
rodents--R/M-H Extrudate, water from water bottles.
Experimental Design and Treatments
Antitumor Activity, EMT6 Model
[0341] Treatment schedule--The start of first dosing was considered
as DO. On DO, non-engrafted mice were randomized according to their
individual body weight into groups of 8-9 using Vivo manager.RTM.
software (Biosystemes, Couternon, France). On D0, the mice received
vehicle (culture medium) or bacterial strain. On D14, all mice were
engrafted with EMT-6 tumor cells as described below. On D24, mice
from the positive control group received anti-CTLA-4 antibody
treatments.
[0342] The treatment schedule is summarized in the table below:
TABLE-US-00013 No. Treatment Group Animals Treatment Dose Route
Schedule 1 8 Untreated -- -- -- 2 8 Vehicle -- PO 38 days EMT6
(media) 3 8 MRx0554 2x10.sup.8 PO 38 days EMT6 bacteria 4 8
Anti-CTLA4 10 mg/kg IP TWx2, D10, D13, D17 and D20 for EMT6
[0343] The monitoring of animals was performed as described
below.
[0344] Induction of EMT6 tumours in animals--On D14, tumors were
induced by subcutaneous injection of 1x10.sup.6 EMT-6 cells in 200
.mu.L RPMI 1640 into the right flank of mice.
[0345] Euthanasia--Each mouse was euthanized when it reached a
humane endpoint as described below, or after a maximum of 6 weeks
post start of dosing.
Animal Monitoring
[0346] Clinical monitoring--The length and width of the tumor was
measured twice a week with callipers and the volume of the tumor
was estimated by this formula [74]:
Tumor .times. .times. volume = width 2 .times. length 2
##EQU00003##
[0347] Humane endpoints [75]: Signs of pain, suffering or distress:
pain posture, pain face mask, behaviour;
[0348] Tumor exceeding 10% of normal body weight, but non-exceeding
2000 mm.sup.3; Tumors interfering with ambulation or nutrition;
Ulcerated tumor or tissue erosion; 20% body weight loss remaining
for 3 consecutive days; Poor body condition, emaciation, cachexia,
dehydration; Prolonged absence of voluntary responses to external
stimuli; Rapid laboured breathing, anaemia, significant bleeding;
Neurologic signs: circling, convulsion, paralysis; Sustained
decrease in body temperature; Abdominal distension.
[0349] Anaesthesia--Isoflurane gas anesthesia was used for all
procedures: surgery or tumor inoculation, i.v. injections, blood
collection. Ketamine and Xylazine anesthesia was used for
stereotaxia surgical procedure.
[0350] Analgesia--Carprofen or multimodal carprofen/buprenorphine
analgesia protocol were adapted to the severity of surgical
procedure. Non-pharmacological care was provided for all painful
procedures.
[0351] Additionally, pharmacological care not interfering with
studies (topic treatment) were provided at the recommendation of
the attending veterinarian.
[0352] Euthanasia--Euthanasia of animals was performed by gas
anesthesia over-dosage (Isoflurane) followed by cervical
dislocation or exsanguination.
Results
Antitumor Activity, EMT6 Model
[0353] The results are shown in FIG. 17. Treatment with the
bacterial strain of the invention led to a clear reduction in
tumour volume relative to both the negative controls. The positive
control also led to a reduction in tumour volume, as would be
expected.
[0354] These data indicate that strain MRx0554 may be useful for
treating or preventing other diseases associated with reduced
immune system activity.
Example 10--Analysis of Carbohydrate Metabolism--API 50 CHL
Analysis of MRx0554
[0355] The Analytical Profile Index (API) test system consists of
strips which contain miniaturised biochemical tests which assay for
enzymatic activity in bacterial species. These tests are routinely
used in the characterisation of novel strains. API 50 CHL testing
was carried out to examine carbohydrate metabolism in MRx0554. As
per manufacturer's instructions, bacteria were cultured in 10 mL
YCFA broth for 16-18 hours at 3.degree. C. in an anaerobic
workstation. This culture was diluted in 10 mL API CHL Medium so as
to achieve a density roughly equivalent to McFarland standard No.
2, and 110 .mu.l of this mixture was used to inoculate each cupule
on a set of API 50 CH test strips. Test strips were incubated in a
humidified incubation box at 37.degree. C. in an anaerobic
workstation for 48 hours, following which the colour of each cupule
was recorded and assigned a value of negative, intermediate
positive, positive or doubtful.
[0356] Using API 50 CHL analysis, MRx0554 tested positive for
fermentation of L-arabinose, D-ribose, D-xylose, D-galactose,
D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalin,
arbutin, esculin, salicin, D-cellobiose, D-maltose, D-saccharose
(sucrose), D-trehalose, gentiobiose, D-tagatose, and potassium
gluconate (FIG. 18). Intermediate reactions were observed for
D-mannitol, methyl .alpha.-D-glucopyranoside, D-lactose,
D-raffinose, amidon (starch), and D-turanose.
Example 11--TLR9 Activation
[0357] To further elucidate the mechanism by which MRx0518
stimulates the immune system, HEK-Blue.TM. human TLR9 reporter
cells (InvivoGen, San Diego, Calif., USA) were used to test the
effect of MRx0518 on TLR9 activation.
Maintenance of Cell Lines and Bacterial Strains
[0358] HEK-Blue.TM. human TLR9 reporter cells (InvivoGen, San
Diego, Calif., USA) were grown in DMEM supplemented with 10% (v/v)
foetal bovine serum (FBS), 4 mM L-glutamine, 4.5 mg/mL glucose, 100
U/mL penicillin, 100 .mu.g/mL streptomycin, 100 .mu.g/mL
Normocin.TM. (InvivoGen), 10 .mu.g/mL blastocidin (InvivoGen) and
100 .mu.g/mL zeocin (InvivoGen) to 90% density. Cells were
maintained at 37.degree. C. and 5% CO2. For assays, cells were
washed once with phosphate-buffered saline (PBS) (Sigma-Aldrich,
Gillingham, England, UK) and resuspended in antibiotic-free growth
media at a density of 450,000 cells/mL. All reagents were supplied
by Sigma Aldrich unless otherwise stated. E. gallinarum MRx0518 was
routinely cultured in Yeast extract, Casitone, Fatty Acid media
(YCFA, E&O Laboratories, Bonnybridge, Scotland, UK) at
37.degree. C. in an anaerobic cabinet (Don Whitley Scientific,
Shipley, England, UK).
TLR9 Reporter Assays
[0359] The following compositions were examined for their ability
to induce TLR9 activation: [0360] 1. Live fraction of MRx0518
(MRx0518LV)--Late log phase bacterial cultures were centrifuged at
5,000.times.g for 5 min at room temperature to generate bacterial
fractions. Pelleted bacteria were washed once in PBS and
re-suspended in antibiotic-free cell culture media to the
appropriate dilution.
[0361] 2. MRx0518 supernatant fraction (MRx05185N)--Culture
supernatants were harvested and filtered through a 0.22 .mu.m pore
size filter and diluted in water.
[0362] 3. Heat-killed fraction of MRx0518 (MRx0518HK)--Bacterial
cultures were heat-inactivated for 40 min at 80.degree. C. and
prepared as described above for the live fraction.
[0363] MRx0518LV and MRx0518HK were used at a multiplicity of
infection (MOI) of 100:1. A 100:1 MOI equivalent volume was used
for MRx0518SN. The synthetic CpG oligonucleotide ODN2006
(InvivoGen) was used as an assay positive control at a
concentration of 5 .mu.M. YCFA was used as a negative control.
Viable cells counts were determined by plating.
[0364] HEK-Blue.TM. human TLR9 reporter cells were incubated with
the above treatments for 22 hours at 37.degree. C. in a 5% CO2
atmosphere. Assays were developed using QUANTI-Blue.TM. (InvivoGen)
as per manufacturer's recommendations for 2 h. The results depicted
in FIG. 19 are an average from at least three independent
experiments. Statistical significance was determined using the
ordinary one-way ANOVA and Tukey's multiple comparisons tests.
[0365] The results demonstrate that the living and supernatant
fractions were able to activate TLR9.
Example 12--T Cell Differentiation
[0366] The ability of MRx0518 to induce T-cell differentiation was
explored in vitro on peripheral blood mononuclear cells (PBMCs,
Stemcell, Cat: 70025). Briefly, PBMCs were plated in 96-well plates
plated with anti-CD3 (Ebioscience, anti-CD3 monoclonal antibody
(OKT3 clone), functional grade, cat. No. 16-0037-81) at
400,000/well in 50 .mu.l cRPMI medium per well (cRPMI contains RPMI
1640 (+L-Glut, 21875-034) 2 mM final conc. Stock 200 mM; 10% HI FBS
(Gibco life technologies, 10082-147); 50.mu.m mercaptoethanol
(Gibco life technologies, 21985-023); and 1% pen/strep (P4333, 10
mg/mL). Heat-killed MRx0518 (prepared by incubation at 80.degree.
C. for 30 minutes, after which the cultures were washed with PBS
and resuspended in appropriate cell culture medium and viable
counts were confirmed by plating) was then added to each well,
4,000,000 in 100 .mu.l/well. Following 3 days in a 37.degree. C.
incubator, the cells were removed and re-suspended in a medium
containing PMA- (Sigma, Cat no. P8139), Ionomycin (Sigma, Cat no.
13909) and GolgiSTOP (BD, Cat no 554724) for 5 hours. PMA stock was
1 mg/mL in DMSO which was further diluted in 100 .mu.g/mL (each
sample required 50 ng/mL in cRPMI), Ionomycin stock was 1 mM in
DMSO (1 .mu.M in cRPMI was used) and GolgiStop concentration was
used at 4 .mu.l/6 mL. Supernatants were passed through a 0.22 .mu.m
filter and diluted appropriately in co-culture medium.
[0367] The cells were then subjected to a flow cytometry
staining:
[0368] After washing, the cells were incubated with viability dye
(Viobility 405/520 Fixable Dye from Miltenyi biotec 1
.mu.l/sample)+human Fc block, cat. 564219 (1 .mu.l/sample) in PBS
for 10 mins in the dark at room temperature. The surface antibodies
(2 .mu.l of each) were then added directly to the wells for 10 mins
in the dark at room temperature--CD3-APC-Vio 770 (Miltenyi, cat.
No. 130-113-136), CD4-VioBlue (Miltenyi, cat. No. 130-114-534) and
CD25-VioBright FITC (Miltenyi, cat. No. 130-113-283). The cells
were then washed twice in PBS and spun down at 300g/5min/RT.
[0369] The eBioscience FoxP3 transcription factor staining buffer
was then used to fix and permeabilise the cells (cat. No. 00-5523).
Following the eBioscience protocol, a perm/fix buffer was prepared
using 1 part of concentrate solution and 3 parts of diluent. The
cells were fixed for 1 h at RT and then washed 2.times. in
1.times.Perm wash and spun down at 300 g/5min/RT. The following
intracellular staining or transcription factor antibodies were
added to the samples in perm wash (1.times.) for 45 min/in the
dark/at room temperature or in the fridge overnight (up to 18h),
followed by washing the antibodies 2.times. using Perm wash (300
.mu.) and re-suspension in PBS (250 .mu.l) to acquire on the
cytometer:
TABLE-US-00014 Intracellular markers Transcription factors 2 ul
anti-IL10-PE 5.5 ul anti-FoxP3-PE- Cy7 2 ul anti-IFN.gamma.-PE 9 ul
anti-Tbet-APC Vio770 10 ul anti-IL17a-APC 9 ul anti-RoRyt-PE
[0370] Anti IFN.gamma.-PE Vio770 human antibodies (Miltenyi, cat.
No. 130-114-025) [0371] Anti IL10-PE human antibodies (Miltenyi,
cat. No. 130-112-728) [0372] Anti IL17a-APC human antibodies
(Miltenyi, cat. No. 130-099-202) [0373] Anti RORyt-PE human
antibodies (Miltenyi, cat. No. 130-103-837) [0374] Anti Tbet-APC
human antibodies (Miltenyi, cat. No. 130-098-655 [0375] Anti-Foxp3
monoclonal antibody (236A/E7), PE-Cy7 (ebioscience) cat. No.
25-4777-41
[0376] As can be seen in FIG. 20A-B, both supernatant of MRx0518
(SP 518) and heat-killed MRx0518 (HK 518) were able to induce
differentiation of T helper cells and cytotoxic T cells,
respectively, even in the absence of cytokines to induce
differentiation (no cyto)
Example 13--MRx0518 Induced Cytokine Signature
[0377] Splenocytes were isolated from C57BL/6 mice and plated in 96
well plates at a density of 900,000 cells/well in RPMI1640
supplemented with 10% FBS, 2 mM L-glutamine, 100 U/mL Pen/Strep
(Sigma-Aldrich) and 55 .mu.M .beta.-mercaptoethanol (Gibco). Cells
were treated with different concentrations of blank media
(YCFA.sup.+) or bacteria supernatant from stationary phase for 72
hrs. Cell free supernatants were collected after each time point
and stored at -80.degree. C. for cytokine analysis. Cytokines were
measured using multiplex procartaplex MO Th1/Th2/Th9/Th17/Th22/Treg
17plex kit (Invitrogen). Cell proliferation of untreated
splenocytes or splenocytes treated by 10% YCFA medium or 10%
MRx0518 bacteria supernatant was measured using MTT assay
(Millipore), as depicted in FIG. 21F.
[0378] Live, growing MRx0518 bacteria were incubated for up to 2h
with the human intestinal epithelial cell line
[0379] CaCo-2 and with the human monocyte/macrophage cell line
THP-1. The host response was analysed immediately (CaCo-2) or after
a further 24 h incubation (THP-1).
[0380] Frozen healthy human PBMCs were purchased from Stem Cells
Technologies (Cambridge UK). The cells were thawed and left to rest
overnight in full growth media (RPMI 1640 with 10% FBS, 55 .mu.M
13-mercaptoethanol, 2mM L. Glutamine and 100 U/mL penicillin, 100
.mu.g/mL streptomycin) in CO.sub.2 incubator at 37.degree. C. For
the experiment, cells were plated at a density of 750,000
cells/well in 48 well plates and treated in full growth media with
10% bacteria supernatants in the presence or absence of 1 ng/mL
LPS. Cell culture media was added to untreated wells. Cells were
left to rest for 72 h, thereafter cell free supernatants were
collected and spun down for 3 minutes at 10,000 g at 4.degree. C.
Samples were stored at -80.degree. C. for cytokine analysis.
Cytokine quantification was conducted using a ProcartaPlex
multiplex immunoassay following the manufacturers recommendations
(Thermo Fischer Scientific). Briefly, 50 .mu.l of cell-free
co-culture supernatants were used for cytokine quantification using
a MAGPIX.RTM. MILLIPLEX.RTM. system (Merck) with the xPONENT
software (Luminex, Austin, Tex., USA). Data was analysed using the
MILLIPLEX.RTM. analyst software (Merck) using a 5-parameter
logistic curve and background subtraction to convert mean
fluorescence intensity to pg/mL values.
[0381] Data are expressed in FIGS. 21A-D as an average of two
technical replicates of 10 biological replicates (PBMC) or three
biological replicates (splenocytes) and show production of
cytokines in (A) PBMCs; (B) Splenocytes; (C) THP-1 cells; and (D)
Caco-2 cells, following treatment with YCFA blank media ("Vehicle")
or MRx0518 bacteria/MRx0518 cell-free bacterial supernatant
("MRx0518"). FIG. 21E depicts additional data relating to cytokine
secretion from splenocytes (N=3), from cells that were either
untreated ("Untreated"), treated with YCFA blank media ("10% YCFA")
or treated with MRx0518 cell-free bacterial supernatant ("10%
MRx0518").
[0382] As can be seen in FIGS. 21A-D, treatment of different cells
with a supernatant of MRx0518 bacteria resulted in
immunostimulation as evident by an increase in cytokine
production.
Example 14--NF-.kappa.B Activation
[0383] The activation of the NF-KB promoter was tested in HEK293
cells co-expressing an NF-.kappa.B inducible secreted embryonic
alkaline phosphatase (SEAP) reporter gene with either the human
NOD2 gene, TLR4, TLR9 or TLR5 genes (HEK-Blue.TM.-hNOD2,
HEK-Blue.TM.-hTLR5, HEK-Blue.TM.-hTLR9 and HEK-Blue.TM.-hTLR4
cells, respectively, by InvivoGen, San Diego, Calif., USA).
[0384] Briefly, HEK-TLR4 cells were maintained in DMEM 4.5 g/L
D-glucose supplemented with 10% (v/v) heat-inactivated FBS, 4 mM
L-Glutamine, 100U/ml penicillin, 100 .mu.g/ml streptomycin, 100
.mu.g/ml normocin, 1.times.HEK-Blue selection media; for HEK-TLR5
and HEK-TLR9 same media was used with the exception of the use of 2
mM L-Glutamine. HEK-TLR5 and HEK-TLR9 were selected using 30
.mu.g/ml and 10 .mu.g/ml blasticidin respectively and 100 .mu.g/ml
zeocin media for both cell lines into the culture.
[0385] For the experiment, cells were washed with PBS, dissociated
in PBS and collected in growth media. Cells were plated in 96-well
plates at a density of 25,000 cells/well for HEK-TLR4 and HEK-TLR5,
80,000 cells/well for HEK-TLR9 and 50,000 cells/well for
HEK-NOD2.
[0386] To evaluate the responsiveness of the cells to their
ligands, the cells were treated with 1 ng/ml LPS (HEK-TLR4), 1
ng/.mu.l ultra-pure flagellin from Salmonella typhimurium
(HEK-TLR5), 1 .mu.M ODN2006 CPG (HEK-TLR9 positive control) or 1
.mu.M ODN2006 GPC (HEK-TLR9 negative control), 1 ng/ml of L18-MD
and incubated in a CO.sub.2 incubator at 37.degree. C. Treatments
proceeded for 22 h at 37.degree. C. and 5% CO2, after which the
detection of Secreted embryonic alkaline phosphatase (SEAP)
activity from cell culture supernatant was performed using
QUANTI-blue solution according to manufacturer's instructions.
Briefly, 20 .mu.l of cell culture media was collected and analysed
for the presence of SEAP by mixing with 200 .mu.l of QUANTI-Blue
detection media. After 2h (HEK-TLR4 and HEK-TLR5) or 4 h (HEK-TLR9
and HEK-NOD2) incubation at 37.degree. C., optical density was
measured at 655 nm on a microplate reader (iMark microplate,
Bio-Rad).
[0387] As can be seen in FIGS. 22A-D (showing results from averaged
technical replicates for three independent experiments),
NF-.kappa.B promoter activation was measured in cells which were
either untreated ("Untreated"), treated with YCFA+ medium ("YCFA")
or treated with MRx0518 ("MRx0518"). The following positive
controls (1 ng) were used--L18-MDP (for HEK-Blue.TM.-hNOD2 cells,
FIG. 22A), Lipopolysaccharide, LPS (for HEK-Blue.TM.-hTLR4, FIG.
22B), CPG or negC (for HEK-Blue.TM.-hTLR9, FIG. 22C) or recombinant
flagellin from S. typhimurium, FLA (for HEK-Blue.TM.-hTLR5, FIG.
22D). The cells were incubated with the various treatment at
37.degree. C. in a 5% CO2 atmosphere for 22 h. To measure
NF-.kappa.B promoter activation (N=3), QUANTI-Blue.TM. (InvivoGen)
was mixed with cell supernatants, the plates were incubated for 2 h
and optical density was measured at 655 nm.
Sequences
TABLE-US-00015 [0388] (Enterococcus gallinarum l6S rRNA
gene-AF039900) SEQ ID NO: 1 1 taatacatgc aagtcgaacg ctttttcttt
caccggagct tgctccaccg aaagaaaaag 61 agtggcgaac gggtgagtaa
cacgtgggta acctgcccat cagaagggga taacacttgg 121 aaacaggtgc
taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgctt 181
ttgcgtcact gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc
241 aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac
tgagacacgg 301 cccagactcc tacgggaggc agcagtaggg aatcttcggc
aatggacgaa agtctgaccg 361 agcaacgccg cgtgagtgaa gaaggttttc
ggatcgtaaa actctgttgt tagagaagaa 421 caaggatgag agtagaacgt
tcatcccttg acggtatcta accagaaagc cacggctaac 481 tacgtgccag
cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt 541
aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg
601 gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca
tgtgtagcgg 661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg
cggctctctg gtctgtaact 721 gacgctgagg ctcgaaagcg tggggagcga
acaggattag ataccctggt agtccacgcc 781 gtaaacgatg agtgctaagt
gttggagggt ttccgccctt cagtgctgca gcaaacgcat 841 taagcactcc
gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 901
ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc
961 ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa
gtgacaggtg 1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt
taagtcccgc aacgagcgca 1081 acccttattg ttagttgcca tcatttagtt
gggcactcta gcgagactgc cggtgacaaa 1141 ccggaggaag gtggggatga
cgtcaaatca tcatgcccct tatgacctgg gctacacacg 1201 tgctacaatg
ggaagtacaa cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc 1261
ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat
1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg
cccgtcacac 1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt
tttggagcca gccgcctaag 1441 gtgggataga tgattggggt gaagtcgtaa
caaggtagcc gtatcggaag gtgcggctgg 1501 at ca cc (consensus 16S rRNA
sequence for Enterococcus gallinarum strain MRx0518) SEQ ID NO:2
TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGTGGCGAACGGG-
TGAGTA
ACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAATACCGTATAACACTATTTTC-
CGCATG
GAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACG-
GCTCAC
CAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTAC-
GGGAGG
CAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAAGGTTTTCGGA-
TCGTAA
AACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCCTTGACGGTATCTAACCAGAAAGCCAC-
GGCTAA
CTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAG-
GCGGTT
TCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAG-
AGGAGA
GTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGGCTCTCTGGTC-
TGTAAC
TGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGT-
GCTAAG
TGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGG-
TTGAAA
CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTA-
CCAGGT
CTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTGCATGGTTGTC-
GTCAGC
TCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGG-
CACTCT
AGCGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCT-
ACACAC
GTGCTACAATGGGAAGTACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCG-
GATTGT
AGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCC-
GGGCCT
TGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCC-
GCCTAA GGTG (16S rRNA gene for Enterococcus gallinarum strain
MRx0554) SEQ ID NO:3 1 taatacatgc aagtcgaacg ctttttcttt caccggagct
tgctccaccg aaagaaaaag 61 agtggcgaac gggtgagtaa cacgtgggta
acctgcccat cagaagggga taacacttgg 121 aaacaggtgc taataccgta
taacactatt ttccgcatgg aagaaagttg aaaggcgctt 181 ttgcgtcact
gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc 241
aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac tgagacacgg
301 cccagactcc tacgggaggc agcagtaggg aatcttcggc aatggacgaa
agtctgaccg 361 agcaacgccg cgtgagtgaa gaaggttttc ggatcgtaaa
actctgttgt tagagaagaa 421 caaggatgag agtagaacgt tcatcccttg
acggtatcta accagaaagc cacggctaac 481 tacgtgccag cagccgcggt
aatacgtagg tggcaagcgt tgtccggatt tattgggcgt 541 aaagcgagcg
caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg 601
gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgtgtagcgg
661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg cggctctctg
gtctgtaact 721 gacgctgagg ctcgaaagcg tggggagcga acaggattag
ataccctggt agtccacgcc 781 gtaaacgatg agtgctaagt gttggagggt
ttccgccctt cagtgctgca gcaaacgcat 841 taagcactcc gcctggggag
tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 901 ccgcacaagc
ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 961
ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg
1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc
aacgagcgca 1081 acccttattg ttagttgcca tcatttagtt gggcactcta
gcgagactgc cggtgacaaa 1141 ccggaggaag gtggggatga cgtcaaatca
tcatgcccct tatgacctgg gctacacacg 1201 tgctacaatg ggaagtacaa
cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc 1261 ttctctcagt
tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat 1321
cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac
1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca
gccgcctaag 1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc
gtatcggaag gtgcggctgg 1501 atcacc
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Sequence CWU 1
1
411506DNAEnterococcus gallinarum 1taatacatgc aagtcgaacg ctttttcttt
caccggagct tgctccaccg aaagaaaaag 60agtggcgaac gggtgagtaa cacgtgggta
acctgcccat cagaagggga taacacttgg 120aaacaggtgc taataccgta
taacactatt ttccgcatgg aagaaagttg aaaggcgctt 180ttgcgtcact
gatggatgga cccgcggtgc attagctagt tggtgaggta acggctcacc
240aaggccacga tgcatagccg acctgagagg gtgatcggcc acactgggac
tgagacacgg 300cccagactcc tacgggaggc agcagtaggg aatcttcggc
aatggacgaa agtctgaccg 360agcaacgccg cgtgagtgaa gaaggttttc
ggatcgtaaa actctgttgt tagagaagaa 420caaggatgag agtagaacgt
tcatcccttg acggtatcta accagaaagc cacggctaac 480tacgtgccag
cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt
540aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca
accggggagg 600gtcattggaa actgggagac ttgagtgcag aagaggagag
tggaattcca tgtgtagcgg 660tgaaatgcgt agatatatgg aggaacacca
gtggcgaagg cggctctctg gtctgtaact 720gacgctgagg ctcgaaagcg
tggggagcga acaggattag ataccctggt agtccacgcc 780gtaaacgatg
agtgctaagt gttggagggt ttccgccctt cagtgctgca gcaaacgcat
840taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat
tgacgggggc 900ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac
gcgaagaacc ttaccaggtc 960ttgacatcct ttgaccactc tagagataga
gcttcccctt cgggggcaaa gtgacaggtg 1020gtgcatggtt gtcgtcagct
cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1080acccttattg
ttagttgcca tcatttagtt gggcactcta gcgagactgc cggtgacaaa
1140ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg
gctacacacg 1200tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg
ctaagctaat ctcttaaagc 1260ttctctcagt tcggattgta ggctgcaact
cgcctacatg aagccggaat cgctagtaat 1320cgcggatcag cacgccgcgg
tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380cacgagagtt
tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgcctaag
1440gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag
gtgcggctgg 1500atcacc 150621444DNAEnterococcus
gallinarumEnterococcus gallinarum strain MRx0518 2tgctatacat
gcagtcgaac gctttttctt tcaccggagc ttgctccacc gaaagaaaaa 60gagtggcgaa
cgggtgagta acacgtgggt aacctgccca tcagaagggg ataacacttg
120gaaacaggtg ctaataccgt ataacactat tttccgcatg gaagaaagtt
gaaaggcgct 180tttgcgtcac tgatggatgg acccgcggtg cattagctag
ttggtgaggt aacggctcac 240caaggccacg atgcatagcc gacctgagag
ggtgatcggc cacactggga ctgagacacg 300gcccagactc ctacgggagg
cagcagtagg gaatcttcgg caatggacga aagtctgacc 360gagcaacgcc
gcgtgagtga agaaggtttt cggatcgtaa aactctgttg ttagagaaga
420acaaggatga gagtagaacg ttcatccctt gacggtatct aaccagaaag
ccacggctaa 480ctacgtgcca gcagccgcgg taatacgtag gtggcaagcg
ttgtccggat ttattgggcg 540taaagcgagc gcaggcggtt tcttaagtct
gatgtgaaag cccccggctc aaccggggag 600ggtcattgga aactgggaga
cttgagtgca gaagaggaga gtggaattcc atgtgtagcg 660gtgaaatgcg
tagatatatg gaggaacacc agtggcgaag gcggctctct ggtctgtaac
720tgacgctgag gctcgaaagc gtggggagcg aacaggatta gataccctgg
tagtccacgc 780cgtaaacgat gagtgctaag tgttggaggg tttccgccct
tcagtgctgc agcaaacgca 840ttaagcactc cgcctgggga gtacgaccgc
aaggttgaaa ctcaaaggaa ttgacggggg 900cccgcacaag cggtggagca
tgtggtttaa ttcgaagcaa cgcgaagaac cttaccaggt 960cttgacatcc
tttgaccact ctagagatag agcttcccct tcgggggcaa agtgacaggt
1020ggtgcatggt tgtcgtcagc tcgtgtcgtg agatgttggg ttaagtcccg
caacgagcgc 1080aacccttatt gttagttgcc atcatttagt tgggcactct
agcgagactg ccggtgacaa 1140accggaggaa ggtggggatg acgtcaaatc
atcatgcccc ttatgacctg ggctacacac 1200gtgctacaat gggaagtaca
acgagttgcg aagtcgcgag gctaagctaa tctcttaaag 1260cttctctcag
ttcggattgt aggctgcaac tcgcctacat gaagccggaa tcgctagtaa
1320tcgcggatca gcacgccgcg gtgaatacgt tcccgggcct tgtacacacc
gcccgtcaca 1380ccacgagagt ttgtaacacc cgaagtcggt gaggtaacct
ttttggagcc agccgcctaa 1440ggtg 144431506DNAEnterococcus
gallinarumEnterococcus gallinarum strain MRx0554 3taatacatgc
aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag 60agtggcgaac
gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg
120aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg
aaaggcgctt 180ttgcgtcact gatggatgga cccgcggtgc attagctagt
tggtgaggta acggctcacc 240aaggccacga tgcatagccg acctgagagg
gtgatcggcc acactgggac tgagacacgg 300cccagactcc tacgggaggc
agcagtaggg aatcttcggc aatggacgaa agtctgaccg 360agcaacgccg
cgtgagtgaa gaaggttttc ggatcgtaaa actctgttgt tagagaagaa
420caaggatgag agtagaacgt tcatcccttg acggtatcta accagaaagc
cacggctaac 480tacgtgccag cagccgcggt aatacgtagg tggcaagcgt
tgtccggatt tattgggcgt 540aaagcgagcg caggcggttt cttaagtctg
atgtgaaagc ccccggctca accggggagg 600gtcattggaa actgggagac
ttgagtgcag aagaggagag tggaattcca tgtgtagcgg 660tgaaatgcgt
agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact
720gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt
agtccacgcc 780gtaaacgatg agtgctaagt gttggagggt ttccgccctt
cagtgctgca gcaaacgcat 840taagcactcc gcctggggag tacgaccgca
aggttgaaac tcaaaggaat tgacgggggc 900ccgcacaagc ggtggagcat
gtggtttaat tcgaagcaac gcgaagaacc ttaccaggtc 960ttgacatcct
ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg
1020gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc
aacgagcgca 1080acccttattg ttagttgcca tcatttagtt gggcactcta
gcgagactgc cggtgacaaa 1140ccggaggaag gtggggatga cgtcaaatca
tcatgcccct tatgacctgg gctacacacg 1200tgctacaatg ggaagtacaa
cgagttgcga agtcgcgagg ctaagctaat ctcttaaagc 1260ttctctcagt
tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat
1320cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg
cccgtcacac 1380cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt
tttggagcca gccgcctaag 1440gtgggataga tgattggggt gaagtcgtaa
caaggtagcc gtatcggaag gtgcggctgg 1500atcacc 1506415DNAArtificial
SequenceDescription of Artificial Sequence Synthetic biotype
identifying repetitive sequence 4gtggtggtgg tggtg 15
* * * * *