OA20782A - Compositions comprising bacterial strains. - Google Patents

Abstract

The invention provides compositions comprising bacterial strains for stimulating the immune system and treating and preventing diseases.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS TECHNICAL FIELD

This invention is in the field of compositions comprising bacterial strains isolated from the mammalian digestive tract and the use of such compositions in the treatment of disease, in particular in stimulating the immune System in the treatment of disease.

BACKGROUND TO THE INVENTION

The human intestine is thought to be stérile in utero, but it is exposed to a large variety of maternai and environmental microbes immediately after birth. Thereafter, a dynamic period of microbiai colonization and succession occurs, which is influenced by factors such as delivery mode, environment, diet and host génotype, ail of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently, the microbiota stabilizes and becomes adult-like [1], The human gut microbiota contains more than 500-1000 different phylotypes belonging essentîally to two major bacterial divisions, the Bacteroidetes and the Firmicutes [2], The successful symbiotic relationships arising from bacterial colonization of the human gut hâve yîelded a wide variety of metabolic, structural, protective and other bénéficiai fonctions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host. Similarly, the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animais which hâve an impaired immune System that is functionally reconstituted following the introduction of commensal bacteria [3-5].

Dramatic changes in microbiota composition hâve been documented in gastrointestinal disorders such as inflammatory bowel disease (IBD). For example, the levels of Clostridium cluster XIVa bacteria are reduced in IBD patients whilst numbers of E. coli are increased, suggesting a shift in the balance of symbionts and pathobionts wîthin the gut [6-9]. Interestingly, this microbiai dysbiosis is also associated with imbalances in T effector cell populations.

In récognition of the potential positive effect that certain bacterial strains may hâve on the animal gut, various strains hâve been proposed for use in the treatment of various diseases (see, for example, [10-13]). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, hâve been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [14] and [15] for reviews). Certain Streptococcus and Veillonella strains, and to a lesser extent, Enterococcus and Lactobaccillus strains hâve been suggested to hâve îmmunomodulatory effects, with varying effects on different cytokines in vitro, suggesting that data obtained in vitro with individual strains are unlikely to adéquately represent immune responses to mixtures of gut microbiota communities in vivo [88], However, the relationship between different diseases and different bacterial strains, and the précisé effects of particular bacterial strains on the gut and at a systemic level and on any parti cul ar types of diseases, are poorly characterised.

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 thérapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors hâve 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 hâve îdentified 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.

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.

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.

Preferably, the bacteria used in the invention is the strain deposited under accession number 42488 atNCIMB.

In further preferred embodiments, the bacteria used in the invention is the strain deposited under accession number 42761 at NC 1MB.

BRIEF DESCRIPTION OF DRAWINGS

Figure IA: 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.

Figure IB: 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).

Figure IC: Mouse model of breast cancer - infiltrating immune cells. Scatter plots represent cell counts of different immune markers from individual animais from each treatment group.

Figure 1D: Mouse model of breast cancer - Cytokine production in tumour lysâtes. 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.

Figure 1E: Mouse model of breast cancer - Cytokine production in blood plasma. Columns represent the mean pg/mL from each treatment group (+/- SEM).

Figure 1F: Représentative images of ileum cryosections from vehicle, MRx0518 and antiCTLA-4-treated mice immuno-labelled with antibodies against CD8a (lower panels) and counter-stained with DAPI (upper panels).

Figure IG: Plot quantifying animal study subsets with more than 3 CD8a+ cells per field taken from the ileum crypt région of mice treated with vehicle, MRx0518 or anti-CTLA-4,

Figure 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.

Figure 3A: Mouse model of lîver cancer - liver weight.

Figure 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.

Figure 4A: Cytokine levels (pg/mLmL) in immature dendritic cells (No bacteria).

Figure 4B: Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of LPS.

Figure 4C: Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of MRx0518MRx0518.

Figure 4D: Cytokine levels (pg/mLmL) in immature dendritic cells after the addition of MRx0518MRxO518 and LPS.

Figure 5A: Cytokine levels in THP-1 cells (No bacteria).

Figure SB: Cytokine levels in THP-l cells after addition of bacterial sédiment.

Figure 5C: Cytokine levels in THP-1 cells after the addition of MRx0518MRx0518 alone or in combination with LPS.

Figures 6 and 7: Immunostimulatory response - TNFa

Figure 8: Immunostimulatory response - IL-12p70

Figure 9: Immunomodulatory response - IL-10

Figure 10: Immunostimulatory response - IL-8

Figure 11 : Immunostimulatory response - IL-23

Figure 12: Immunostimulatory response - IL-1 β

Figure 13: Immunostimulatory response - IL-6

Figure 14: Mechanism of action - activation of NFkB

Figure 15: Mechanism of action-- activation of TLR5

Figure 16A: A schematîc représentation of the treatment schedule of the different groups used in Example 8 described hereîn below.

Figure 16B: Mean tumour volume in mîce bearing a tumour fonned by EMT-6 cells. The mice were either untreated or treated with a YCFA vehicle (Vehicle), MRxO518 bacteria in YCFA medium (MRxO518), 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.

Figure 17: Mouse model of breast cancer - tumour volume.

Figure 18: API 50 CHL profile of MRxO554.

Figure 19: Mechanism of action — TLR9 activation by MRxO518 (MRxO518lv), heat-killed MRx0518 (MRx05I8_Hk) and MRx05I8 culture supematant (MRx0518_Sn) in HEK-Blue™ hTLR9 reporter cell lines. ODN2006 was used as a positive control and YCFA medium was included as a négative control for MRxO518sn- 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 signifïcant différences with the relevant control are shown on the graphs as **** (p < 0.0001 ).

Figures 20A-B: Induction of T-cell différentiation in a population of (A) T-helper cells and (B) Cytotoxic T Lymphocytes (CTL), using heat-killed MRxO518 (HK 518), Supematant from MRx0518 culture or RPMI medium, wîthout addition of cytokines (no cyto). * = p< 0.05; **= p< 0.01; ***=p< 0.001; ****= p< 0.0001.

Figures 21A-D: ln-vitro cytokine production b y (A) PBMC cells; (B) Splénocytes; or (C) THP1 cells; which were treated with YCFA+ medium (“Vehicle”) or cell-free bacterial supematant of MRx0518 (“MRx0518”). Figure 21D shows fold change in cytokine expression following treatment of CaCo-2 cells with live bacteria (“MRx0518”) relative to untreated cells.

Figure 21E: In-vitro cytokine production by splénocytes (N=3), from cells that were either untreated (“Untreated”), treated with YCFA blank media (“10% YCFA”) or treated with MRx0518 cell-free bacterial supematant (“10% MRx0518”).

Figure 21F: Vîability of splénocytes 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 MRxO518 cell-free bacterial supematant (“10% MRxO518”).

Figures 22A-D: NF-κΒ promoter activation in (A) HEK-Blue™-hNOD2 cells; (B) HEKBlue™-hTLR4 cells; (C) HEK-Blue™-hTLR9 cells or (D) HEK-Blue™-hTLR5 cells. Cells were either untreated, treated with YCFA medium (“YCFA”), treated with MRx0518 (“MRx0518”) or treated with positive Controls.

Figure 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

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 dîsease.

Enterococcus gallinarum fonns 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 = NBRC 100675 =

NCIMB 702313 (formerly NCDO 2313) = NCTC 12359 [16]. The GenBank accession number for a 16S rRNA gene sequence of Enterococcus gallinarum is AF0399Û0 (disclosed herein as SEQ ID NO:1). An exemplary Enterococcus gallinarum strain is described in [16].

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 MRxO518 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, AB 21 9 Y A, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 16th November 2015 as “Enterococcus sp” and was assigned accession number NCIMB 42488.

The genome of strain MRxO518 comprises a chromosome and plasmid. A chromosome sequence for strain MRxO518 is provided in SEQ ID NO:3 OF WO2017/085520. 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.

The Enterococcus gallinarum bacterium deposited under accession number NCIMB 42761 was also tested in the Examples and is also referred to herein as strain MRxO554, References to MRxO554 and MRxO554 are used interchangeably. Strain MRx0554 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Aberdeen, AB21 9 Y A, Scotland) by 4D Pharma Research Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 22 May 2017 as “Enterococcus gallinarum MRxO554” and was assigned accession number NCIMB 42761. The genome sequence of this bacterium is disclosed herein as SEQ ID NO:2 OF WO2018/215782. 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 nucléotide. 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 présent in the assembly, rather than a consensus of the five 16S genes présent in MRx0554.

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 b y 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.

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 simîlar physiological and biochemical characteristics.

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 nucléotide 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 hâve at least 95%, 96%, 97%, 98%, 99%. 99.5% or 99.9% sequence îdentity 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 répétitive sequences such as BOX, ERIC, (GTG)s, or REP or [17]. Biotype strains may hâve sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42488. In some embodiments, a biotype strain has a sequence with ai least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRxO518 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.

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. 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 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:3 OF WO2017/085520. For example, the bacterial strain for use in the invention may hâve a chromosome with at least 90% sequence identity to SEQ ID NO:3 OF WO2017/085520 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 WO2017/085520 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 WO2017/085520 across 100% of SEQ ID NO:3 OF WO2017/085520.

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. ai 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 W02017/085520. For example, the bacterial strain for use in the invention may hâve 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 identîty to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520, or at least 95% sequence îdentity to SEQ 1D NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO2017/085520, or at least 95% sequence îdentity to SEQ ID NO:4 OF WO2017/085520 across 100% of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence îdentity to SEQ ID NO:4 OF WO2017/085520 across 70%of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence îdentity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence îdentity to SEQ ID NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence identîty to SEQ ID NO:4 OF WO2017/085520 across 100% of SEQ ID NO:4 OF WO2017/085520.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identîty to SEQ ID NO:3 OF WO2017/085520 and a plasmid with sequence identîty to SEQ ID NO:4 OF WO2017/085520.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identîty to SEQ ID NO:3 OF WO2017/085520, for example as described above, and a 16S rRNA sequence with sequence identîty 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 îdentity to SEQ ID NO:4 OF WO2017/085520, as described above.

In certain embodiments, the bacterial strain for use in invention has a chromosome with sequence identîty to SEQ ID NO:3 OF WO2017/085520, for example as described above, and optionally comprises a plasmid with sequence identîty to SEQ ID NO:4 OF WO2017/085520, as described above, and is effective for stimulating the immune System.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identîty to SEQ ID NO:3 OF WO2017/085520, for example as described above, and a 16S rRNA sequence with sequence îdentity to any of SEQ ID NOs: 1 or 2, for example as described above, and optionally comprises a plasmid with sequence identîty to SEQ ID NO:4 OF WO2017/085520, as described above, and is effective for stimulating the immune System.

In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA sequence that îs 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 WO2017/085520 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.

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 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.

In certain embodiments, the bacterial strain for use in the invention is a Enterococcus gallinantm 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.

Alternative! y, strains that are biotypes of the bacterium deposited under accession number NC 1MB 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 amplîfied fragment length polymorphism (FAFLP) and répétitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identify other Enterococcus gallinarum strains.

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 anaiysed by amplîfied ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[18]). Altematively, biotype strains are identified as strains that hâve the same carbohydrate fermentation patterns as the bacterium deposîted under accession number NCIMB 42488. In sonie embodiments, the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMérieux). In some embodiments, the bacterial strain used in the invention is:

(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 ail of): L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetyl glucosamine, amygdalin, arbutin, salicin, Dcellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and potassium gluconate; and/or (ii) intermediate for fermentation of at least one of (e.g. at least 2, 3, 4 or ail of): Dmannitol, Methyl-uD-glycopyranoside, D-lactose, starch, and L-fucose;

preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMérieux).

Other Enierococcus gallinarum strains that are use fui 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 perfonned in the examples. In particular, bacterial strains that hâve 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 hâve 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 admînistered in the method of the invention.

In some embodiments, the bacterial strain used in the invention is:

(i) Positive for at least one of (e.g. at least 2, 3, 4, 5, 6, 7 or ail of): mannose fermentation, glutamic acid decarboxylase, arginine arylamidase, phenylalanine arylamidase, pyro glutamic acid arylamidase, tyrosine arylamidase, histidine arylamidase and serine arylamidase; and/or (ii) Intermediate for at least one of (e.g. at ieast 2 or ail of): β-galaciosidase-ôphosphate, β-glucosidase and N-acetyl-β-glucosaminidase; and/or (iii) Négative for at least one of (e.g. at least 2, 3, 4, 5, 6 or ail of): Raffmose fermentation, 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 bioMérieux).

In some embodiments, the bacteriai strain used in the invention is:

(î) Négative for at least one of (e.g. at least 2, 3, or ail 4 of) glycine arylamidase, raffmose fermentation, proline arylamidase, and leucine arylamidase, for example, as determined by an assay of carbohydrate, amino acid and nitrate metabolism, preferably as detennined by Rapid ID 32A analysis (preferably using the Rapid ID 32A System from bioMérieux); and/or (ii) Intennediate positive for fermentation of L-fucose, preferably as determined by API 50 CHL analysis (preferably using the API 50 CFIL panel from bioMérieux).

In some embodiments, the bacteriai strain used in the invention is an extracellular ATP producer, for example one which produces 6-6.7 ng/μΐ (for example, 6.1-6.6 ng/μΐ or 6.2-6.5 ng/μΐ or 6.33 ± 0.10 ng/μΐ) of ATP as measured using the ATP Assay Kit (Sigma-Aldrich, MAKI 90). Bacteriai extracellular ATP can hâve pléïotropie effects including activation of T cell-recep tor mediated signalling (Schenk et al., 2011), promotion of intestinal Thl7 cell différentiation (Atarashi et al., 2008) and induction of sécrétion of the pro-inflammatory mediator IL-Ιβ by activating the NLRP3 inflammasome (Karmarkar et al., 2016). Accordingly, a bacteriai strain which is an extracellular ATP producer is useful for stimulating the immune System in the context of the method of the invention.

In some embodiments, the bacteriai 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 bacteriai 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 élément protein, Xylose ABC transporter, permease component, and F1G00632333: hypothetical protein.

A particularly preferred strain of the invention is the Enterococcus gallinarum strain deposited under accession number NCIMB 424S8. This is the exemplary MRxO518 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 4248S, or a derîvative thereof A dérivative of the strain deposited under accession number NCIMB 42488 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original.

A dérivative of a strain of the composition comprîsed in the invention may be modifted, for example ai the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeutically active. A derîvative strain will hâve comparable immune modulatory activity to the original NCIMB 42488 strain. In particular, a derîvative strain will elicit comparable effects on the cancer disease models when which may be identifïed by using the culturing and administration protocols described in the Examples. A derîvative of the NCIMB 42488 strain will generally be a biotype of the NCIMB 42488 strain.

References to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 encompass any cells that hâve the same safety and therapeutic eftîcacy characteristics as the strains deposited under accession number NCIMB 42488, and such cells are encompassed b y the the invention. Thus, in some embodiments, référencé 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, référencé to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42488 refers to cells that hâve 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.

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 sîmilar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited under accession number NCIMB 42761 and that are suitable for use in the invention may be identifïed by sequencing other nucléotide 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 hâve at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenee 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% sequenee identity across at least 98% of its genome or at least 99% sequenee identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or répétitive sequences such as BOX, ERIC, (GTG)s, or REP or [19]. Biotype strains may hâve sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenee identity to the corresponding sequenee of the bacterium deposited under accession number NCIMB 42761. In some embodiments, a biotype strain has a sequenee with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenee identity to the corresponding sequenee of strain MRx0554 deposited as NCIMB 42761. In some embodiments, a biotype strain has a sequenee with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenee identity to the corresponding sequenee of strain MRxO554 deposited as NCIMB 42761 and has a 16S rRNA gene sequenee that is at least 99% identieal (e.g. at least 99.5% or at least 99.9% identieal) to SEQ ID NO:3. In some embodiments, a biotype strain has a sequenee with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequenee identity to the corresponding sequenee of strain MRx0554 deposited as NCIMB 42761 and has the 16S rRNA gene sequenee of SEQ ID NO:3.

Alternative!y, 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 polymorphîsm (FAFLP) and répétitive DNA élément (rep)-PCR fingerprinting, or protein profiling, or partial 16S or 23s rDNA sequencing.

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,[20]). Altematively, biotype strains are identified as strains that hâve 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 (bioMérieux). In some embodiments, the bacterial strain used in the invention is:

(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 ail of): L-arabinose, D-ribose, D-xylose, D-galactose, D-glucose, D-fructose, D-mannose, N-acetylglucosamine, amygdalîn, arbutîn, salicin, Dcellobiose, D-maltose, sucrose, D-trehalose, gentiobiose, D-tagatose and potassium gluconate; and/or (îv) intermediate for fermentation of at least one of (e.g. at least 2, 3, 4 or ail of): Dmannitol, Methyl-aD-glycopyranoside, D-lactose, starch, and L-fucose;

preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMérieux).

In some embodiments, the bacterial strain used in the invention is:

(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 ail of): L-arabinose, D-ribose, D-xylose, D-galactose, Dglucose, D-fructose, D-mannose, N-acetylglucosamîne, amygdalin, arbutin, esculin, salicin, D-cellobiose, D-maltose, D-saccharose (sucrose), D-trehalose, gentiobiose, D-tagatose and potassium gluconate;

(îi) intermediate for fermentation of at least one of (e.g. at least 2, 3, 4, 5 or ail of): Dmannitol, Methyl-aD-giycopyranoside, D-lactose, D-raffinose, amidon (starch), and D-turanose; and/or (iii) négative for fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, II, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or ail of): glycerol, erythritol, Darabinose, L-xylose, D-adonitol, methyl-pD-xylopryranoside, L-sorbose, L-rhamnose, dulcitol, inositol, D-sorbitol, Methyl-aD-mannopyranoside, D-melibiose, inulin, Dmelezîtose, 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 bioMérieux, and preferably using the conditions described in Example 10).

Other Enterococcus galllnarum 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 approprîate method or strategy, including the assays described in the examples. For instance, strain for use in the invention may be identified by culturing in anaérobie YCFA and/or administering the bacteria to the type II collagen-induced arthritis mouse model and then assessing cytokine levels. In particular, bacterial strains that hâve similar growth patterns, metabolic type and/or surface antigens to the bacterium deposited under accession number NCIMB 42761 may be useful in the invention. A useful strain will hâve 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.

A dérivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeutically active. A dérivative strain will hâve comparable immune modulatory activity to the original NCIMB 42761 strain. In particular, a dérivative 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 dérivative of the NCIMB 42761 strain will generally be a biotype of the NCIMB 42761 strain.

Référencés to cells of the Enterococcus gallinarum strain deposited under accession number NCIMB 42761 encompass any cells that hâve 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 MRxO554 strain deposited under NCIMB 42761 and does not refer to a bacterial strain that was not deposited under NCIMB 42761.

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 îeast 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 WO2018/215782. For example, the bacterial strain for use in the invention may hâve a genome with at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 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 WO20I8/215782 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 WO20] 8/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 99.5% sequence identity to SEQ ID NO:2 OF 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 O F 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.

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 I6S 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.

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 stîmulating the immune System.

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 stîmulating the immune System.

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.

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% identîcal 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.

In preferred embodiments, the bacterial strains in the compositions of the invention are viable and capable of partially or totally colonising the intestine.

In alternative aspects of every embodiment of the invention, the bacterial strain in the composition of the invention is of the species Enterococcus casellijlavus. Enterococcus caselliflavus is highly similar to Enterococcus gallinarum and is also flagellated.

Therapeutic uses

Stimulating the immune svstem

The ex amples 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 hâve an immunostimuiatory effect, compositions of the invention may be usefui in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an mcreased 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.

Compositions of the invention may be usefui 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 usefui 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 usefui for treating or preventing diseases characterised by an increase in the percentage of CD4+CD25+CDI27- 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 sélective réduction 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.

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.

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⁺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 merease 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.

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.

Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of CD8⁺ aetivated 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⁺ aetivated 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 ’ aetivated cells in cell populations, wherein the increase in number or percentage of CD8⁺ aetivated 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⁺ aetivated cells.

The examples show that administration of the compositions of the invention can lead to an increase in expression of pro-inflammatory molécules, such as pro-inflammatory cytokines. Examples of pro-inflammatory molécules that showed an increase in expression levels upon administration of compositions of the invention include IL-8, IL-12p70, IL-23, TNF-a, IL-Ιβ, and IL-6. Since administration of the compositions of the invention were shown to increase the expression of pro-inflammatory molécules, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molécules, 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 molécules, 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-α, IL-1 β,- 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-a, IL-Ιβ, 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-a, IL-Ιβ, and/or IL6.

The examples also show that administration of the compositions of the invention can lead to an increase in expression of IL-Ιβ. IL-Ιβ is a pro-inflammatory cytokine [21]. The production and sécrétion of IL-1 β is régulât ed b y the inflammasome, a protein complex which is associated with activation of the inflammatory response [22]. Since administration of the compositions of the invention were shown to increase the expression of IL-Ιβ, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-Ιβ. In a particular embodiment, the compositions of the invention are for use in treatîng or preventing diseases characterised by a decrease in the expression and/or activity of IL-Ιβ. 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-Ιβ.

The ex amples 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 [23,24]. The proposed fonctions of IL-23 in the immune response include promoting the prolifération of CD4⁺ memory T cells and promoting the sécrétion of IFN-γ by dendritic cells (DCs) [25]. Since administration of the compositions of the invention were shown to increase the expression of IL-23, compositions of the invention may be usefol 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 treatîng 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.

The ex amples show that administration of the compositions of the invention can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-a). TNF-α is a pro-inflammatory cytokine which is known to be involved in varions signalling pathways to promote cell death. TNF-α initiâtes apoptosis by binding to its cognate receptor, TNFR-1, which leads to a cascade of cleavage events in the apoptotic pathway [26]. TNF-α can also trigger necrosis via a RIP kinase-dependent mechanism [27]. Since administration of the compositions of the invention show an increase in TNF-α expression, compositions of the invention may be usefol in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF-α. In one embodiment, the compositions of the invention are for use in treating diseases characterised by decreased TNF-α 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-α. In one embodiment, the compositions of the invention may be usefol for treating or preventing diseases by increasing the expression and/or activity of TNF-α. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of TNF-a.

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 différentiation of naïve CD4 T ceils and the différentiation of CD8⁺ T cells into cytotoxîc T cells [28]. Since administration of the compositions of the invention were shown to increase the expression of IL-6, compositions of the invention may be usefiil in the treatment of diseases characterised by a decrease in expression of IL-6. In a 5 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 10 activity of IL-6.

Bettelli et al.[29] 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 15 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.

In some embodiments, stimulating the immune System according to the présent invention comprises TLR5 activation or upregulation of TLR5 activation. In some embodiments, stimulating the immune System according to the présent invention comprises TLR9 activation or 20 upregulation of TLR9 activation. In some embodiments, stimulating the immune System according to the présent invention comprises activation of TLR5 and TLR9 or upregulation of TLR9 and TLR5 activation. In some embodiments, stimulating the immune System according to the présent invention comprises inducing and/or upregulating différentiation of T cells such as, but not limited to, T helper cells and T cytotoxic cells.

TLR signalling pathways culminate in the activation of the transcription factor nuclear factorkappaB (NF-κΒ). NF-kB Controls the expression of an array of inflammatory cytokine genes, including TNF-α. Immune stimulation causes, for example, the dimerization of TLR5, which subsequently recruits MyD88 and activâtes protein kinases, including IRAK1, IRAK2, IRAK4 and IRAK-M. The activation of these kinases leads to the nuclear localization of NF-κΒ, which 30 is a proinflammatory cytokine [30],

As demonstrated in the examples, compositions of the invention lead to an increase in expression of NF-κΒ. Since administration of the compositions of the invention increase the expression of the proin fl ammatory cytokine NF-κΒ, 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 5 invention are for use as an immune stimulant by increasing the level and/or actîvity of NF-κΒ. 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-κΒ. 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-kB.

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-κΒ. 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-κΒ

The activation of NF-κΒ is important for eliciting innate immune responses and the subséquent 15 development of adaptive immune responses. Thus, agonists of TLRs, 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 [30], 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, 20 allergies and/or tumours by increasing the level and/or activity of NF-kB.

The examples also demonstrate that the compositions of the invention promote the différentiation of T-helper cells and cytotoxic T lymphocytes. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the différentiation of T-helper cells and/or cytotoxic T lymphocytes.

In certain embodiments, the disease to be treated by the compositions of the invention is not cancer.

Use as a vaccine adjuvant

The ex amples show that administration of the compositions of the invention can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-ol). TNF-α is known to be 30 important for vaccine responses. For example, TNF-α has been shown to be required for an efficient vaccine response in a tlu vaccination of the elderly population [31]. Since administration of the compositions of the invention were shown to increase TNF-α 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 increasmg the level and/or activity of TNF-α. 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 infiuenza 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.

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 [32], Also, the data in the examples confirm that MRxO518 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 âge), who may hâve reduced immune System activity. TLR5 signal ling also plays a key rôle in age-associated innate immune responses [33]. 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 ail iront known pathogens and/or synthetic. In contrast, the compositions of the invention comprise commensal bacteria.

The examples also show that administration of the compositions of the invention can lead to an încrease in expression of IL-6. Increased IL-6 expression has been assocîated with vaccine responses for many diseases. For example, IL-6 was produced by CD14+CD16- inflammatory monocytes after adults were administered an infiuenza vaccine [34], and higher ievels of IL-6 were associated with achieving a vaccine response to an infiuenza vaccine [35], Furthermore, IL6 was produced after injection of the AS03 adjuvant System [36] and downregulation of IL-6 in mice was shown to reduce the helper T cell response after administration of a tuberculosis vaccine [37].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.

Furthermore, IL-6 and TNF-α expression hâve been shown to be correlated with the efficacy of a therapeutic HIV vaccine [Huang et al] a tuberculosis vaccine and a chlamydia vaccine [38]. Su et al. [39] showed that co-inoculation of IL-6 or TNF-α with the FMDV DNA vaccine resulted in increased IFN-γ expression by CD4^h and CD8 T cells, higher expression of IL-4 in CD4 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-α expression, compositions of the invention may be usefui as a vaccine adjuvant. In one embodiment, the compositions of the invention may be usefui as a vaccine adjuvant by încreasing the level and/or activity of TNF-a. In one embodiment, the compositions of the invention may be usefui as a vaccine adjuvant by încreasing the level and/or activity of IL-6. In a particular embodiment, the compositions of the invention may be usefui as a vaccine adjuvant by încreasing the level and/or activity of TNF-u and IL-6. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant în HIV therapy. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant in chlamydia therapy.

The examples also show that administration of the compositions of the invention can lead to an increase in expression of IL-Ιβ. Li et «/.[40] showed that the adjuvant aluminium hydroxide activated the sécrétion of IL-Ιβ, and suggested that IL-Ιβ itself can act as an adjuvant. Since administration of the compositions of the invention were shown to increase IL-Ιβ expression, compositions of the invention may be usefui as a vaccine adjuvant. The examples show that administration of the compositions of the invention can increase the ratio of CD8⁺ T cells to Tregs. Adjuvants hâve been shown to stimulate CD8^h T cells [41] and since administration of the compositions of the invention were shown to increase the ratio of CD8 T cells to Tregs, compositions of the invention may be usefui 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 încreasing the ratio of CD8⁺ T cells to Tregs.

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, aGalCer ail increase CXCL9 and 10 [42,43], which suggests the compositions of the invention will be effective as adjuvants. Also, CXCL9 and 10 are associated with IFNy/Thl responses and promote antîbody responses [44].In certain embodiments, the compositions of the invention are for use in promoting an antîbody response against an antigen, in particular a pathogenic or cancer antigen. Also, CXCL9 is a more sensitive measure than IFN γ of vaccine induced T-celi responses in volunteers receiving investigated malaria vaccines [45].In certain embodiments, the compositions of the invention are for use in promoting an Tcell response against an antigen, in partîcular 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.

The ex amples 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 [46], 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.

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.

The compositions of the invention may be used for enhancing an immune response to any useful antigen. Exemplary antigens for use with the invention inciude: 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, respîratory syncytia] virus, cytomégalovirus, 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 inciude glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wîlms 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, aïcohol or opiates.

Preferred antigens for use with the invention inciude pathogen antigens and tumour antigens. An antigen will elicit an immune response spécifie 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.

The invention also provides the use of: (i) an aqueous préparation of an antigen; and (ii) a composition comprising a bacteriai strain of the species Enterococcus gallinarum, in the manufacture of a médicament for raîsïng an immune response in a patient.

The immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response.

In some embodiments, a bacteriai strain of the species Enterococcus gallinarum is engineered to présent an antigen. Presenting an antigen on the bacteriai strain of the invention may maximise the immunostimulatory activities and further enhance the protective immune response générât ed against the antigen. In addition, manufacturing and delivering therapeutîcs 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 bacteriai strain are manufactured and administered separately. Therefore, in some embodiments, the invention provides a composition comprising a bacteriai strain of the species Enterococcus gallinarum that présents an antigen, for example on its cell surface. In some embodiments, the composition comprising the bacteriai strain that présents an antigen is for use as a vaccine antigen. In some embodiments, the antigen is derived from HIV, hookwonn, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomégalovirus, 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), Carcînoembryonic antigen (CEA), MUC-1, HER2, sîalyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-I25, 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.

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 bacteriai 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 gallmarum, wherein the cell expresses a heterologous antîgen. 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.

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 giycoprotein and lîpoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonîc 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.

The invention may also be useful for enhancing the response to vaccines against noncommunicable diseases such as elevated cholestérol (e.g. via the PCSK9 antigen).

The invention may also be useful for enhancing the response to vaccines against habit forming substances, for example nicotine, alcohol or opiates.

Cell thérapies

Chimeric Antigen Receptor T cell (CAR-T) therapy

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 sérum IL-6 was associated with CAR-T cell expansion, whereas inhibition of IL-6 was associated with inhibition of CAR-T cell prolifération [47]. 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.

Sélective déplétion of Tregs has been shown to enhance the efficacy of cytotoxic lymphocytes [48]. CAR-T cells are a subset of cytotoxic lymphocytes, and therefore it is thought that sélective déplétion 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 partîcular CAR-T cell therapy.

Therefore, the compositions of the invention may be useful in cell therapy, in partîcular in enhancing the response to a cell therapy.

Mesenchymalstem cell (MSC) therapy

Mesenchymal stem cell (MSC) therapy has been reported to hâve immunostimulatory properties. When MSCs are treated with LPS, they upregulate pro-inflammatory cytokines IL-6 and IL-8 which causes increased B cell prolifération [49], 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

It has been reported that, instead of using undifferentiated stem cells in stem cell transplantation therapy, it may be bénéficiai to differentiate stem cells to some extent prior to transplantation. For example, Heng et «/.[50] reported that cardiomyo génie différentiation of stem cells may be bénéficiai by having a higher engraftment efficiency, enhanced régénération of myocytes and increased reste ration of heart function. Since administration of the compositions of the invention initiated neuronal différentiation in undifferentiated neuroblastoma cells, compositions of the invention may be useful for stem cell différentiation in stem cell transplantation therapy.

Hematopoietic stem cell transplantation

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 casse liflavus) 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 [51].Therefore, the immunomodulatory effect shown in the examples may be useful in hematopoietic stem cell transplantation therapy. In certain embodîments, the compositions of the invention may be useful in improving survival after hematopoietic stem cell transplantation and in partîcular after allogenic hematopoietic stem cell transplantation.

The compositions of the invention may be usefiil in combination with allogenîc 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.

Imimmosenescence

Fulop et al. [52] identifïed that an increase in Treg cell number and a decrease in B cell number are associâted with aging in the adaptive immune System. Therefbre, 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 treatîng 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.

Furthermore, it has been proposed that vaccine adjuvants may overcome immunosenescence [53]. Sînce 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,

Diseases that are associated with immunosenescence include cardiovascular disease, cancer, diabètes mellitus type 2 [54] and autoimmune disorders [55].

Modes of administration

Preferably, the compositions of the invention are to be administered to the gastrointestinal tract în order to enable delîvery 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 (includîng sublingual), but they may be administered rectally or intranasally.

In certain embodiments, the compositions of the invention may be administered as a foam, as a spray or a gel.

In certain embodiments, the compositions of the invention may be administered as a suppository, such as a rectal suppository, for ex ample in the form of a theobroma oil (cocoa butter), synthetic hard fat (e.g. suppocire, witepsol), glycero-gelatin, polyethylene glycol, or soap glycerin composition.

In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a tube, such as a nasogastric tube, orogastric tube, gastric tube, jejunostomy tube (J tube), percutaneous endoscopie gastrostomy (PEG), or a port, such as a chest wall port that provides access to the stomach, jéjunum and other suitable access ports.

The compositions of the invention may be administered once, or they may be administered sequentially as part of a treatment regimen. In certain embodiments, the compositions of the invention are to be administered daily.

In certain embodiments of the invention, treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be repeated if delîvery 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 delîvery and / or partial or total colonisation is successfi.il and efficacy is observed.

In certain embodiments, the composition of the invention may be administered to a prégnant animal, for example a mammal such as a human in order to reduce the likelihood of disease developing în her chîld in utero and / or after it is born.

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.

The compositions of the invention may be administered to a patient that has been diagnosed with déficient immune activity, or that has been identified as being at risk of déficient immune activity. For example, the patient may hâve reduced or absent colonisation by Enterococcus, and in parti cul ar Enterococcus gallinarum.

The compositions of the invention may be administered as a food product, such as a nutrîtional supplément.

Generally, the compositions of the invention are for the treatment of humans, although they may be used to treat animais including monogastrîc mammals such as poultry, pigs, cats, dogs, horses or rabbits. The compositions of the invention may be useful for enhancing the growth and performance of animais. If administered to animais, oral gavage may be used.

Compositions

Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried fonn. 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.

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, référencés [56,58].

Alternative!y, the composition of the invention may comprise a live, active bacterial culture.

In preferred embodiments, the composition of the invention is encapsulated to enable delivery of the bacterial strain to the intestine. Encapsulation protects the composition from dégradation until delivery at the target location through, for example, rupturing with Chemical or physical stimuli such as pressure, enzymatic activity, or physical disîntegratîon, 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 solîd carrier surfaces, self-aggregation by flocculation or with cross-linking agents, and mechanical coniainment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the invention is available in, for example, references [59] and [60].

The composition may be administered orally and may be in the fonn of a tablet, capsule or powder. Encapsulâted products are preferred because Enterococcus are anaerobes. Other ingrédients (such as vitamin C, for example), may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo. Altematively, 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 phannaceutical product.

The composition may be formulated as a probiotic.

A composition of the invention includes a therapeutîcally effective amount of a bacterial strain of the invention. A therapeutîcally effective amount of a bacterial strain is sufficient to exert a bénéficiai effect upon a patient. A therapeutîcally effective amount of a bacterial strain may be sufficient to resuit in delivery to and / or partial or total colonisation of the patient’s intestine.

A suîtable daily dose of the bacteria, for example for an adult human, may be from about l x 10³ to about 1 x 10¹¹ colony forming units (CFU); for example, from about 1 x 10⁷ to about 1 x 10¹⁰ CFU; în another example from about 1 x 10⁶ to about 1 x 10¹⁰ CFU; in another example from about 1 x 10⁷ to about 1 x 10¹¹ CFU; in another example from about 1 x 10⁸ to about 1 x 10¹⁰ CFU; in another example from about 1 x 10$ to about 1 x 10¹¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10⁹ cells per day, such as at least 10^l°, at least 10¹¹, or at least I0¹² cells per day.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 10⁶ to about 1 x 10¹¹ CFU/g, respect to the weight of the composition; for example, from about 1 x 10⁸ to about 1 x 10¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

In certain embodiments, the invention provides the above phannaceutical composition, wherein the amount of the bacterial strain is from about 1 x 10³ to about I x 10^H colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the invention provides the above phannaceutical composition, wherein the composition is administered at a dose of between 500mg and lOOOmg, between 600mg and 900mg, between 700mg and 800mg, between 50Ûmg and 750mg or between 750mg and lOOOmg. 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 500mg and lOOOmg, between 600mg and 900mg, between 700mg and 800mg, between 500mg and 750mg or between 750mg and lOOOmg.

Typîcally, a probiotic, such as the composition of the invention, is optionally combined with at least one suitable prebiotîc compound. A prebiotîc 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-olîgosaccharides.

In certain embodiments, the probiotic composition of the présent invention includes a prebiotîc 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 FO S), short-chain fructo-oligosaccharides, inulin, isomalt-oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chitosan-oligosaccharides (or COS), beta-glucans, arable gum modified and résistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructooligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained b y the conversion of the beet sugar and includîng a saccharose molécule to which three glucose molécules are bonded.

The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the référencé [61]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [62]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol and the like. Examples of suitable diluents include éthanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, 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 stéarate, magnésium stéarate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided în the pharmaceuticai composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antîoxidants and suspendîng agents may be also used.

The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplément. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by beîng more similar to a common food item, rather than to a pharmaceuticai composition. In certain embodiments, the composition of the invention is formulated as a milk-based product. The term milk-based product means any liquîd 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 processîng, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

In certain embodiments, the compositions of the invention contain a single bacterial strain or species and do not contain any other bacterial strains or species. Such compositions may comprise only de minimis or biologically irrelevant amounts of other bacterial strains or species. Such compositions may be a culture that is substantially frec from other species of organism.

The compositions for use in accordance with the invention may or may not require marketing approval.

In some cases, the lyophilised bacterial strain is reconstituted prior to administration. In some cases, the reconstitution is by use of a diluent described herein.

The compositions of the invention can comprise pharmaceuticai 1 y acceptable excipients, diluents or carriers.

In certain embodiments, the invention provides a pharmaceuticai 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.

In certain embodiments, the invention provides pharmaceutical composition comprîsing: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount suffi ci ent to treat or prevent a disease or condition.

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 suffi ci ent to treat or prevent a disease or condition.

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.

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 β, TNF-α, ΜΙΡ-3α, 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-a.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 x 10³ to about 1 χ 10¹¹ colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered at a dose of 1 g, 3 g, 5 g or 10 g.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the composition is administered by a method selected from the group consisting of oral, rectal, subcutaneous, nasal, buccal, and sublingual.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a carrier selected from the group consisting of lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol and sorbitol.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a diluent selected from the group consisting of éthanol, glycerol and water.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising an excipient selected from the group consisting of starch, gelatin, glucose, anhydrous lactose, free-flow lactose, beta-Iactose, corn sweetener, acacia, tragacanth, sodium alginate, carboxymethyl cellulose, polyethylene glycol, sodium oleate, sodium stéarate, magnésium 5 stéarate, sodium benzoate, sodium acetate and sodium chloride.

In certain embodiments, the invention provides the above pharmaceutical composition, further comprising at least one of a preservative, an antioxidant and a stabilizer.

In certain embodiments, the invention provides the above pharmaceutical composition, comprising a preservative selected from the group consisting of sodium benzoate, sorbic acid 10 and esters of p-hydroxybenzoic acid.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein said bacterial strain is lyophilised.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein when the composition is stored in a sealed container at about 4°C or about 25 °C and the 15 container is placed in an atmosphère having 50% relative humîdity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

Culturing methods

The bacterial strains for use in the présent invention can be cultured using standard microbîology 20 techniques as detailed in, for example, référencés [63,,65].

The solid or liquid medium used for culture may be YCFA agar or YCFA medium. YCFA medium may include (per lOOmL, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09 g), MgSO₄ · 7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1 25 mg), biotîn (1 pg), cobalamin (1 pg), p-aminobenzoic acid (3 pg), folie acid (5 pg), and pyridoxamine (15 pg).

Bacterial strains for use in vaccine compositions

The inventors hâve identified that the bacterial strains of the invention are useful for treating or preventing diseases or conditions associated with reduce immune activity. This is likely to be a 30 resuit of the effect that the bacterial strains of the invention hâve on the host immune System.

Therefore, the compositions of the invention may also be useful for preventing diseases or conditions, when administered as vaccine compositions. In certain such embodiments, the bacterial strains of the invention may be killed, inactivated or attenuated. In certain such embodiments, the compositions may comprise a vaccine adjuvant. In certain embodiments, the compositions are for administration via injection, such as via subcutaneous injection.

General

The practice of the présent invention will employ, unless otherwise îndicated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, wîthin the skill of the art. Such techniques are explained fully in the literature. See, e.g., référencés [66] and [67,73], etc.

The term “comprising” encompasses “including” as well as “consistîng” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

The tenu “about” in relation to a numerical value x is optîonal and means, for example, a+10%.

The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the définition of the invention.

Référencés to a percentage sequence identity between two nucléotide sequences means that, when aligned, that percentage of nucléotides are the same in comparing the two sequences. This 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. [74], A preferred alignment is determined by the Smith-Waterman homology search algorîthm 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. [75].

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 intervenîng steps. Also, steps may be combined, omitted or performed in an alternative order, if appropriate.

Various embodiments of the invention are described herein. It will be appréciaied 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 rnodels of cancer

Summary

This study tested the efficacy of compositions comprising bacterial strains according to the invention in four tumor models.

Materials

Test substance - Bacterial strain #MRx0518.

Reference substance - Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: Syrian Hamster IgGl, Bioxcell).

Test and reference substances vehïcles - Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each day of injection to mi ce, antibody was diluted with PBS (ref: BE14-516F, Lonza, France),

Treatment doses - Bacteria: 2xl0⁸ in 200 pL. 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 pL of test substance will be administered) according to the most recent body weight of mice.

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).

Culture conditions of bacterial strain - The culture conditions for the bacterial strain were as follows:

• Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles) into Hungate tubes • Seal the tubes and flush with CO2 using a syringe input and exhaust System • Autoclave the Hungate tubes • When cooied, inoculate the Hungate tubes with 1 mL of the glycerol stocks • Place the tubes in a static 37°C incubator for about 16 hours.

• The following day, take 1 mL of this subculture and inoculate 10 mL of YCFA (prewarmed flushed Hungate tubes again, ail in duplicate) • Place them in a static 37°C incubator for 5 to 6h

Cancer cell line and culture conditions The cell lines that were used are detaîled in the table below:

Cell line	Type	Mouse strain	Origin
EMT-6	Breast carcinoma	BALB/c	ATCC
LL/2 (LLCI)	Lung carcinoma	C57BL/6	ATCC CRL1642
Hepa 1-6	Hepatocellular carcinoma	C57BL/6	IPSEN INNOVATION
RENCA	Rénal adenocarcinoma	BALB/c	ATCC

The EMT-6 cell line was established from a transplantable murine mammary carcinoma that 5 arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule [76].

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 [77].

The Hepa 1-6 cell line is a derîvative of the BW7756 mouse hepatoma that arose in a C57/L 10 mouse [78].

Cell culture conditions - Ail cell lines were grown as monolayer at 37°C in a humidified atmosphère (5% CO₂, 95% air). The culture medium and supplément are îndicated in the table below:

Cell line	Culture medium	Supplément
EMT6	RPMI 1640 containing 2mM L-glutamine (ref: BE12-702F, Lonza)	10% foetal bovine sérum (ref: #3302, Lonza)
LL/2 (LLCI)	RPMI 1640 containing 2mM L-glutamine (ref: BE12-702F, Lonza)	10% foetal bovine sérum (ref: #3302, Lonza)
Hepa 1-6	DMEM (ref: 11960-044, Gibco)	10% foetal bovine sérum (ref: #3302, Lonza) 2mM L-GIutamine penicillin-streptomycin (Sigma G-6784)

RENCA

DMEM

10% fêtai bovine sérum, 2mM Lglutamine, lug/mL puromycin

For experimental use, adhèrent tumour cells were detached from the culture flask by a 5 minute treatment with trypsîn-versene (ref: BEI7-161E, Lonza), in Hanks' medium without calcium or magnésium (ref: BE10-543F, Lonza) and neutralized by addition of complété culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.

Use of animais Healthy female BALB/C (BALB/cByJ) mi ce, of matching weight and âge, were obtained from CHARLES RIVER for the EMT6 and RENCA model experiments.

Healthy female C57BL/6 (C57BL16J) mîce, of matching weight and âge, were obtained from CHARLES RIVER (L’Arbresles) for the LL/2(LLC1) and the Hepal-6 model experiments.

Animais were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Régulations and NRC Guide for the Care and Use of Laboratory Animais were followed [79,80]. Animais were maintained in housing rooms under controlled environmentai conditions: Température: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod ( 12h light/12h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Animal enclosures were provided with stérile and adéquate space with bedding matériel, food and water, environmental and social enrichment (group housing) as described: 900 cm² cages (ref: green, Tecniplast) in ventilated racks, Epicéa bedding (SAFE),10 kGy Irradiated diet (A04-10, SAFE), Complété food for immuno-competent rodents - R/M-H Extrudate, water from water bottles.

Experimental design and treatments

Antitumor activity, EMT6 mode!

Treatment schedule - The start of fïrst dosing was considered as D0. On D0, non-engrafted mice were randomîzed according to their individual body weight into groups of 9/8 using Vivo manager® software (Biosystemes, Couternon, France). On D0, the mîce received vehicle (culture medium) or bacterial strain. On DI4, ail 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.

The treatment schedule is summarized in the table below:

Group	No. Animais	T reatment	Dose	Route	Treatment Schedule
1	8	Untreated	-	-	-
2	8	Vehicle (media)	-	PO	QlDx42
3	9	Bacteriai strain #1 (MRx0518)	2x108 bacteria	PO	QlDx42
4	8	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was perfonned as described below.

Induction of EMT6 tumours in animais - On D14, tumours were induced by subcutaneous injection of 1x10⁶ EMT-6 cells in 200 pL RPMI 1640 into the right flank of mice.

Euthanasia - Each mouse was euthanized when it reached a humane endpoînt as described below, or after a maximum of 6 weeks post start of dosing.

Antitumor activity, LL/2 (LLC1) model

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 10 manager® software (Biosystemes, Couternon, France). On DO, the mice received vehicle (culture medium) or bacteriai strain. On D14, ail 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.

The treatment schedule is summarized in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	8	Untreated	-	-	-
2	9	Vehicle (media)	-	PO	QlDx42
3	9	Bacteriai strain #1 (MRx0518)	2x108 bacteria	PO	QlDx42
4	8	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was perfonned as described below.

Induction of LL/2 (LLC1) tumors in animais - On DI4, tumors were induced by subcutaneous injection of IxlO⁶ LL/2 (LLC1) cells în 200 pL RPMI 1640 into the right flank of mice.

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

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 using Vivo manager® software (Biosystemes, Coutemon, France). On DO, the mice received vehicle 10 (culture medium) or bacterial strain. On D14, ail mice were engrafted with Hepa 1-6 tumor cells as described below. On DI6, mice from the positive control group received antî-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	9	Untreated	-	*	-
2	9	Vehicle (media)	-	PO	QlDx42
6	9	Bacterial strain #4 (MRx0518)	2xl08 bacteria	PO	QlDx42
7	9	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was perfonned as described below.

Orthotopic induction of Hepa 1-6 tumor cells in animais by intrasplenic injection - On D0, one million (Ix 10⁶) Hepa 1-6 tumor cells in 50 pL RTMI 1640 medium were transplanted via intrasplenic injection into mice. Briefly, a small left subcostal flank incision was made and the spleen was exteriorized. The spleen was exposed on a stérile gauze pad, and injected under Visual control with the cell suspension with a 27-gauge needle. After the cell inoculation, the spleen 20 was excised.

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.

Evaluation of tumour burden at euthanasia - At the time of termination, livers were collected and weighed.

Antitumor activity, RENCA model

Treatment schedule - The start of first dosîng was considered as DO. On DO, non-engrafted mice were randomized according to their indivîdual body weight into groups of 12 mice. On DO, the 5 mice received vehicle (culture medium) or bacterial straîn (2xl0⁸ in 100 pL, PO). On D14, ail 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-PDLl (clone 10F.9G2, 10 mg/kg, IP) was initiated from DI7.

The treatment schedule is summarized in the table below:

Group	No. Animais	T reatment	Dose	Route	Treatment Schedule
1	12	Untreated	•	-	-
2	12	Vehicle (media)	-	PO	QD
3	12	Bacterial strain (MRx0518)	2x108 bacteria	PO	QD
4	12	Paclitaxel	15 mg/kg	IP	Q4D (every four days)
5	12	Anti-CTLA4 + AntiPDLl	10 mg/kg + 10 mg/kg	IP	B1W (twice weekly) From day 3

The monitoring of animais was perfbrmed as described below.

On D14 (following 2 weeks of bacterial dosing/pre-treatment), 5x10⁵ viable cells in 100 pL of P BS 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 15 prior to cell implantation.

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.

Evaluation of tumour burden at euthanasia - At the time of termination, tumours were collected and their volume evaluated.

Animal monitoring

Clinical monitoring - The length and width of the tumour was measured 2-3 tîmes a week with callipers and the volume of the tumour was estimated by this formula [81]:

, width² x length umor volu me =-------------

Humane endpoints [82]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding 2000 mm\ Tumors interfering with ambulation or nutrition; Ulcerated tumour or tissue érosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, émaciation, cachexia, déhydration; Prolonged absence of voluntary responses to extemal stimuli; Rapid laboured breathing, anaemia, signifïcant bleeding; Neurologie signs: circling, convulsion, paralysie; Sustaîned decrease in body température; Abdominal distension.

Anaesthesia - Isoflurane gas anesthésia was used for ail procedures; surgery or tumour inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthésia was used for stereotaxia surgical procedure.

Analgesia - Carprofen or multimodal carprofen/buprenorphine analgesîa protocol were adapted to the severity of surgical procedure. Non-pharmacologîcal care was provîded for ail painful procedures. Additionally, pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinanan.

Euthanasia - Euthanasîa of animais was performed by gas anesthésia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

Results

Antitumor activity, EMT6 model

The results are shown in Figure IA. Treatment with the bacterial strain of the invention led to a clear réduction in tumour volume relative to both the négative Controls. The positive control also led to a réduction in tumour volume, as would be expected.

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 cote. To investigate whether MRx0518 treatment had influence on the degree of necrosis found within EMT6 tumeurs, paraffin sections from the mid-belly région of the tumours were stained with Haematoxylin and Eosin. MRxO518 treatment of a murine EMT6 breast carcinoma model showed a tendency towards increasing the cross-sectional area of necrosis within the tumour (Figure IB, upper panel). To investigate whether MRxO518 treatment had influence on dividing cells within the tumour, paraffin sections from the mid-belly région of the tumours were stained with the prolifération protein Ki67, along with DAPI counter stain, to estimate the percentage of cells dividing within the EMT6 tumour. MRxO518 treatment of a murine EMT6 breast carcinoma model significantly decreased the percentage of dividing cells seen within the tumour (Figure IB, lower panel, P=0.019).

Immune cell populations

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 eut into pièces. One piece was subjected to flow cytometry analysis. To assess the relative percentage of T lymphocytes, présent within the tumours, the foliowing markers were used; CD45, CD3, CD4, CD8, CD25 and FoxP3.

The preliminary flow cytometry data presented in Figure IC (and further supported by the below described data, presented in Figure 23) shows that the relative percentage of lymphocytes in tumours was slightly decreased in both the MRxO518 and anti-CTLA-4 treated groups, when compared respectively to vehicle or control animais. Likewise, the relative percentage of CD4^h cells appeared to be decreased in MRx0518 and anti-CTLA-4 treated animais, whilst the relative percentage of CD8⁺ cells followed an opposite trend in both groups, albeit with different magnitude. The relative percentage of CD4⁺FoxP3⁺ cells was lower in the anti-CTLA-4 treated group when compared to the slight decrease in MRxO518 treated animais; however, the réduction in the relative percentage of CD4'^kCD25⁺ 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 animais. These data presented here supports the hypothesis that antiCTLA-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.

Additional investigation of the tumour microenvironment was performed using NanoString analysis of the tumour tissues, to investigate whether the MRxO518 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 MRxO518-treated groups were collected. RNA was extracted from 5 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 1O 360 panel. Genes previously shown to be characteristic of varions cell populations were used to measure these populations' abundance:

Cell ty pe	Marker genes
NK CD56dim cells	I121r, Kir3dll, Kir3dl2
Exhausted CD8	Cd244, Eomes, Lag3, Ptger4
DC	Ccl2, Cd209e, Hsdllbl
Cytotoxic cells	Ctsw, Gzma, Gzmb, Klrb 1, Klrd 1, Klrk 1, Nkg7, Prfl
Macrophages	Cdl63, Cd68, Cd84, Ms4a4a
T-cells	Cd3d, Cd3e, Cd3g, Cd6, Sh2dla, Tratl
Mast cells	Cpa3, Hdc, Ms4a2
Neutrophils	Ceacam3, Csf3r, Fcgr4, Fprl
B-cells	Blk, Cdl9, Fcrlb, Ms4al, Pnoc, Spib, Tell, Tnfrsfl?
NK cells	Ncrl,XclI
CD45	Ptprc

Z-scores for each cell population were calculated using the linear cell type scores provided by the NanoString analysis (Figure 23, hcat map).

The NanoString data shows that the abundance of B cells, CD45, T cells, cytotoxic and NK cells were increased in the tumour tissue of MRxO518-treated group when compared to vehicletreated animais (Figure 23). The data presented here supports the hypothesis that MRxO518 has 15 an immunostimulatory effect by increasing leukocytes, in particular NK cells, T cells and cytotoxic cells in the tumour microenvironment.

Cytokine production

An additional tumour piece was used for total protein extraction and subséquent cytokine analysis, together with plasma samples. Protein levels of IL-10, CXCL1, CXCL2, CXCL10, ILIB, IL-17A, GM-CSF, TNF-α, IL-12p70 and IFN-γ in the tumour microenvironment were analysed by MagPix technology. While IL-17A and GM-CSF were below levels of détection, ail the other markers were expressed at reasonable levels (Figure ID). A signitîcance différence was observed between the vehicle and anti-CTLA-4 group for IFN-γ. The production of the IL-10 and IL-12p70 immune markers seemed reduced following MRx05I8 treatment compared to the control treatments.

Cytokine levels were also assessed in blood plasma of the same animais. Protein levels of IL-23, IL-6, IL-10, VEGF, CXCL1, CXCL2, CXCL10, IL-2, IL-Ιβ, IL-17A, GM-CSF, TNF-α, IL12p70 and IFN-γ were analysed by MagPix technology. Overall, little cytokine production was detected in the blood plasma of animais either before tumour induction or at the end of the study (Figure 1E). VEGF and CXCL10 were detected at substantial levels, while IL-23, IL-6, IL-10, CXCL1 and CXCL2 were detected ai low levels. 1L-2, IL-lb, 1L-17A, GM-CSF, TNF-α, IL12p70 and IFN-γ were not detected in the samples. MRxO518 significantly increased production of IL-6 at Day 0. MRxO518 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 différences in cytokine production in the tumour and plasma, between MRxO518 and ant-CTLA-4 suggests thaï each of them acts on a distinct and potentially complementary mechanism.

Localisation of CD8a Positive Cells in the Ileum pm cryo-sections of ileum were eut 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 ovemight with the primary antîbody (rat-anti-mouse-CD8a antîbody, Sigma-Aldrich, Millipore).

The next moming the slides were washed in PBS and stained with a secondary antîbody: goatantî-rat-antibody-Alexa488 (Molecular Probe, Invitrogen) for 1 hour at room température. 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 usîng a Zeiss Axioscope Microscope equîpped with a mercury vapour lamp, appropriate filters and a x20 apochromatic objective.

Examples of images obtained from slides from the vehicle, MRxO518, and anti-CTLA4 animais are shown (Figure 1F - upper panels: DAPÏ staining, lower panels: CD8a staining).

Fields of view were examined from 20 animais and imaged using inanual exposure time. The number of animais and fields analysed are shown in the following table:

Group	Number of fields analysed	Number of mice
Vehicle	53	5
MRxO518	70	7
Anti- CTLA4	71	8

The images were scored as foliow: fields with < 3 positive cells were scored as 0, whilst fields with more >3 cells were scored as 1. The results of this analysis are shown (Figure IG).

Ileum cryosections stained with anti-CD8α showed a higher number of CD8α positive cells localized in the crypt région tissues from animais treated with MRx0518 and anti-CTLA-4 compared to the vehicle group.

This observation is in line with CD8⁺ T cells being présent in the intestine in case of infection or inflammatory microenvironment, as part of the immune response.

Antitumor activîty, LL/2 (LLC1) model

The results are shown in Figure 2. Treatment with the bacterial strain of the invention led to a clear réduction in tumour volume relative to both the négative Controls.

Antitumor activîty, Hepal-6 model

The results are shown in Figure 3A. The untreated négative control does not appear as would be expected, because liver weight was lower in this group than the other groups. However, the vehicle négative control and the positive control groups both appear as would be expected, because mice treated with vehicle alone had larger lîvers than mice treated with anti-CTLA4 antibodies, reflecting a greater tumour burden in the vehicle négative control group. Treatment with the bacterial strain of the invention led to a clear réduction in liver weight (and therefore tumour burden) relative to the mice in the vehicle négative control group.

Antitumor activity, RENCA model

The results are shown in Figure 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) complété réduction in tumour sîze at DIS 5 and D22 compared to both the untreated and vehicle groups.

These data indicate that strain MRxO518 may be usefui for treating or preventing other diseases associated with reduced immune System activity.

Example 2 — PCR gene analysis

A pure culture of bacteria MRx0518 was studied in a PCR gene analysis. There were two anns 10 to the experiment: 1) MRx0518 was co-cultured with human colonie 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 scénarios were evaluated through gene expression analysis. The results are shown below:

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 Thl cell chemoattractant (inducible by IFN-g)
ILS	31.81	Cytokine, chemoattractant (especially neutrophils), many receptors including CXCR1 and CXCR2Z
CXCL1	16.48	CXCR2 ligand, stimulâtes cell prolifération as well as migration, overexpression is neuroprotective in EAE.
CD40	14.33	Co-stimulatory molécule, route of T cell dépendent DC activation.
TNF	13.50	Major proinflammatory cytokine
IL17C	12.18	Promotes antibacterial response from epthîelium, synergîstic with IL-22,
CXCL10	10.66	Close homology with CXCL9, think also CXCR3 ligand?
HSPA1B	10.19	Heat shock protein

NFKB1A	8.S7	NFkB signalling; PI3K
JUN	7.61	Antibacterial response; GPCR signalling.
TNFAIP3	6.63	TNF signalling
DUSP1	6.36	Anti-inflammatory phosphatase, inactivâtes MAPKs
JUNB	5.36	Transcription factor, JAK-STAT signalling
BIRC3	4.86	Adherens junctions, tight junctions
DUSP2	4.59	Anti-inflammatory, inactivâtes MAPK.
IL32	4.29	Proinflammatory cytokine, induced by IFN-g, IL-18
DUSP5	3.12	Anti-inflammatory, inactivâtes MAPK
FOS	3.03	Transcription factors, TLR signalling, forms part of AP-1
GADD45B	2.89	Cell growth and prolifération
CLDN4	2.61	Tight junctions
ADM	2.57	NFkB 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 prolifération. T helper cell différentiation
NOD2	-14.98	PRR, inflammasome activator, promûtes autophagy
INOS	-26.88	Proinflammatory, generator of nîtric oxide

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-y-type responses, also supported by IL-32, which is IFN-y-inducible. These data suggest that the compositions of the invention are useful for stimulating the immune System.

Example 3 - Stability testing

A composition described herein containing at least one bacterial strain described herein is stored in a sealed container at 25°C or 4°C and the container is placed in an aünosphere 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 MRxO518 compared to MRxO518 + LPS

Summary

This study tested the effect of the bacterial strain MRxO518 alone and in combination with lipopolysaccharide (LPS) on cytokine production in immature dendritic cells.

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 MRxO518 at a final concentration of 10⁷/mL, with the optional addition of LPS at a final concentration of lOOng/mL. The négative control involved incubating the cells with RPMI media alone and positive Controls incubated the cells with LPS at a final concentration of lOOng/mL. The cytokine content of the cells was then analysed.

Results

The results of these experiments can be seen in Figures 4a-d. The addition of MRx0518 alone leads to a substantial increase in the level of cytokines IL-6 and TNF-α compared to the négative control (Figure 4a and c). The addition of LPS (positive control) leads to an increase in the level of IL-6 and TNF-α compared to the négative control but not IL-Ιβ (Figure 4b). A combination of MRx0518 and LPS led to a synergistic increase in the level of IL-1 β produced (Figure 4d).

Conclusion

MRx0518 has the ability to înduce hîgher IL-6 and TNF-α cytokine production in immature dendritic cells. The combination LPS and MRx0518 can increase the levels of cytokines IL-1 β in immature dendritic cells. These data indicate that MRx0518 alone or in combination with LPS can increase inflammatory cytokines IL-1 β, IL-6 and TNF-α, which promûtes inflammation.

Example 5 — cytokine production in THP-1 cells induced by MRxO518 compared to MRxO518 + LPS

Summary

This study tested the effect of bacterial straîn MRx0518 alone and in combination with LPS on cytokine production in THP-1 cells, a model cell line for monocytes and macrophages.

THF-1 cells were differentiated into MO medium for 48h with 5ng/mL phorbol-12-myristate-13acetate (PMA). These cells were subsequently incubated with MRx0518 at a final concentration of 10⁸/mL, with or without the addition of LPS at a final concentration of lOOng/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 supematant was analysed for cytokine content.

Results

The results of these experiments can be seen in Figures 5a-c. The addition of MRxO518 without LPS leads to an increase in the cytokine levels of IL-1 β, IL-6 and TNF-α compared to the no bacterial and the bacterial sédiment Controls. The addition of LPS and MRx0518 leads to a synergistic increase in the production of cytokines.

Conclusion

MRxO518 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 β, IL-6 and TNF-οι, which promûtes inflammation.

Example 6 - Cytokine analysis

Introduction

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 MRxO518. 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.

Cytokines involved in the innate immune response (TNF-α, 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 β and IL-6).

Method

Bacterial s trains

MRx()518

LPS used as positive contre 1

Results

The results are shown in Figures 6-13. MRx05I8 induces a strong and characteristic immunostimulatory profde in THP-l-derived macrophages and DCs derived from monocytes. Cytokines involved in the innate immune response (TNF-α, 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. MRxO518 also induced IL-Ιβ.

Discussion

These data shows that MRx0518 has immunostimulatory properties, and may be an effective composition for immunostimulation.

Example 7 - ntechanism of action

Further experiments were performed to characterise the mechanism of action b y which MRx0518 stimulâtes the immune System. A TLR5 signalling reporter assay was selected and the data are presented in Figure 14 and 15. MRxO518 supematant was the most potent activator of TLR5 and NF-κΒ. Also, the supematant was treated with varions lytic enzymes and trypsin was found to abrogate the majority of activity.

Example 8 - adjuvant immunostimulatory activity of MRxO518 in a therapeutic combination with a CTLA-4 inhibitor

Summary

This study compared the anti-tumour activity of MRx0518, a CTLA-4 inhibitor and therapeutic combinations of MRx051S with the CTLA-4 inhibitor in mice bearing EMT-6 tumour cells.

Materials

Test and reference substances - Bacteriai strain #MRx0518; Anti-CTLA4 antibody (ref: BE0131, Bioxcell; clone: 9H10; reactivity: mouse; isotype: Hamster IgGl; storage conditions: +4°C).

Test and reference substances vehicies - The MRxO518 bacteria were grown in a bacteriai culture medium (Yeast ex tract, Casitone, Fatty Acid medium (YCFA)) and kept as a glycerol stock at -80°C. The animais were dosed with the bacteria according to the study protocol. The anti-CTLA-4 antibodies were diluted with PBS (ref: BEI4-516F, Lonza, France) on each day of injection to mice.

Treatment doses - Bacteria: 2x10⁸ in 200 pL. The anti CTLA4 antibodies were administered at 10 mg/kg body weight according to the most recent body weight ofrnice.

Routes of administration - The bacteriai composition was administered by oral gavage (per os, PO) via a gavage tube at a volume of 200 pL/inj. The anti CTLA-4 antibodies were injected into the peritoneal cavity of mice (Intraperitoneally, IP) at a volume of lOmL/kg adjusted to the most recent îndividual body weight of mice.

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.

Tumor cells were grown as monolayer at 37°C in a humidified atmosphère (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12- 702F, Lonza, Verviers, Belgium) supplemented with 10% fêtai bovine sérum (ref: 3302, Lonza). EMT-6 tumor cells are adhèrent 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 magnésium (ref: BE10-543F, Lonza) and neutralized by addition of complété culture medium. The cells were counted and their viability was assessed by 0.25% trypan blue exclusion assay.

Use of animais - Healthy female BALB/C (BALB/cByJ) mice, 5-7 weeks old, were obtained from CHARLES RIVER (L'Arbresles) and maintained in S PF health status according to the FELAS A guidelines. Animal housing and experimental procedures were realized according to the French and European Régulations and NRC Guide for the Care and Use of Laboratory Animais. Animais were maintained 3-4 per cage in housing rooms under controiled environmental conditions: Température: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod ( 12h light/12h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Animal enclosures were provided with stérile and adéquate space with bedding material, food and water, environmental and social enrichinent (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 (Ssnitï® Soest, Germany), Complété food for immunocompétent rodents R/M-H Extrudate, Stérile, filtrated at 0.2 pm water and Environmental enrichment (SIZZLE-dri kraft - D20004 SERLAB, France). Animais are individually identified with RFID transponder and each cage was ladled with a spécifie code. Treatment of the animais started after one week of acclimation for batches 2 and 3, or after three weeks of acclimation for batch 1.

Experimental design and treaiments

On day -14 (D-14), non-engrafted mice were randomized according to their individual body weight into 3 groups of 30 animais and 2 groups of 10 animais using Vivo Manager® software (Biosystemes, Couternon, France). The mice were separated into 3 batches of 10 animais per treatment group (batch 1:10 animais of groups 1, 2 and 3; batch 2: 10 animais of groups 1, 2 and 3 and batch 3: 10 animais of groups 1 to 5) with different termination points from the start of the study: D-14 or D0.

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 animais had 5 animais per treatment group (4 animais from cage one and one animal from cage 2). Based on the ethical criteria, if the tumor volume were higher than 1500mm³, the sélection of the animais to be sacrifice on D24 and D25 is based on tumor volume instead of the cage. The experimental design is depicted in Fig. I6A and summarized below:

) Batch 1 (groups 1, 2 and 3) started treatment on D0 and was culled at DI4 (10 animais form groups 1 to 3). These did not receive tumor cells and constituted the baseline group.

2) Batch 2 (group 1, 2 and 3) started treatment on D-14 and was culled at D7 (10 animais form groups I to 3).

3) Batch 3 (groups 1 to 5) started treatment on D-14 and was culled at D24/25 (10 animais form groups 1 to 5). The treatment of Anti CTLA-4 started on D10.

On day 0 (DO) ail 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 IxIO⁶ EMT-6 cells in 200 pL 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):

Group	No. Animais	T reatment	Dose	Route	Treatment Schedule
1	30 = 10 batch 1 10 batch 2 10 batch 3	Untreated (+ Tumour)	-	-	-
2	30 = 10 batch 1 10 batch 2 10 batch 3	Vehicle (YCFA)	-	PO	Daily -14 to D0 Daily -14 to D7 Daily -14 to D24/25
3	30 = 10 batch 1 10 batch 2 10 batch 3	MRx051S	2x108	PO	Daily-14 to D0 Daily -14 to D7 Daily -14 to D24/25
4	10 batch 3	Anti-CTLA-4 + YCFA	10 mg/kg	IP + PO	TWx2fromD10 YCFA Daily -14 to D24/25
5	10 batch 3	Anti- CTLA-4 + MRxO518	10 mg/kg + 2x108 bacteria	IP +PO	TWx2 fromDIO Bacteria Daily -14 I to D24/25

Animal monitoring

The viability and behaviour of the animais 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 ofthe tumour was estimated by the following formula:

Width² x Length.

Tumour volume = ------------The treatment efficacy was assessed in tenns of the effects of the test substance on the tumour volumes of treated animais relative to controi animais. The following évaluation criteria of antitumor efficacy were determined using Vivo Manager® software (Biosystemes, Coutemon, France).Mean tumour volumes of groups I to 5 are depicted in Fig. 16B. Throughout the course of the study, a progression in tumour growth was observed in ail groups, with the exception of the MRxO518 + Anti-CTLA-4-treated group where a régression of tumour growth occurred from Day 14 post tumour induction. MRxO518 + 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 MRxO518 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 efïect.

Example 9 - Efficacy of bacterial inocula in mouse models of cancer

Summary

This study tested the efficacy of compositions comprising bacterial strain according to the invention in a tumor mode!.

Materials

Test substance - Bacterial strain #MRxO554.

Reference substance - Anti-CTLA-4 antîbody (clone: 9H10, catalog: BE0131, isotype: Syrian Hamster IgGl, Bioxcell).

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).

Treatment doses - Bacteria: 2xlO^s in 200 pL 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 pL of test substance will be administered) according to the most recent body weight of mice.

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).

Culture conditions of bacterial strain - The culture conditions for the bacterial strain were as follows:

• Pipette 10 mL of YCFA (from the prepared 10 mL E&O lab bottles) into Hungate tubes • Seal the tubes and flush with CO2 using a syringe input and exhaust System • Autoclave the Hungate tubes • When cooled, inoculate the Hungate tubes with 1 mL of the glycerol stocks • Place the tubes in a static 37°C incubator for about 16 hours.

• The following day, take 1 mL of this subculture and inoculate 10 mL of YCFA (prewarmed flushed Hungate tubes again, ail in duplicate) • Place them in a static 37°C incubator for 5 to 6h

Cancer cell line and culture conditions The cell Unes that were used are detailed in the table below;

Ceil line	Type	Mouse strain	Origin
EMT-6	Breast carcinoma	BALB/c	ATCC

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 [83].

Cell culture conditions - Ail cell Unes were grown as monolayer at 37°C in a humidified atmosphère (5% CO2, 95% air). The culture medium and supplément are indicated in the table below:

Cell line	Culture medium	Supplément
EMT6	RPMI 1640 containing 2mM L-glutamine (ref: BE12-702F, Lonza)	10% fêtai bovine sérum (ref; #3302, Lonza)

For experimental use, adhèrent tumor cells were detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BEI 7-161E, Lonza), in Hanks' medium without calcium or magnésium (ref: BE10-543F, Lonza) and neutralized by addition of complété culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.

Use of animais Healthy female BALB/C (BALB/cByJ) mice, of matching weight and âge, were obtained from CHARLES RIVER (L'Arbresles) for the EMT6 model experiments.

Animais were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Régulations and NRC Guide for the Care and Use of Laboratory Animais were followed [84,85]. Animais were maintained in housing rooms under controlled environmental conditions: Température: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod (12h light/12h dark), HEPA filiered air, 15 air exchanges per hour with no recirculation. Animal enclosures were provided with stérile and adéquate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm² cages (ref: green, Tecniplast) in ventilated racks, Epicéa bedding (SAFE),10 kGy Irradiated diet (A04-10, SAFE), Complété food for immuno-competent rodents - R/M-H Extrudate, water from water bottles.

Experimental design and treatments

Antitumor activity, EMT6 model

Treatment schedule - The start of first dosîng was considered as D0. On D0, non-engrafted mice were randomîzed according to their individual body weight into groups of 8-9 using Vivo manager® software (Biosystemes, Coutemon, France). On D0, the mice received vehicle (culture medium) or bacterial strain. On D14, ail 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.

The treatment schedule is summarized in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	8	Untreated	-	-	-
2	8	Vehicle (media)	-	PO	38 days EMT6
3	8	MRxO554	2x10K bacteria	PO	38 days EMT6
4	8	Anti-CTLA4	lOmg/kg	IP	TWx2, D10, D13, D17 and D20 for EAAT6

The monitoring of animais was performed as described below.

Induction of EMT6 tumours in animais - On D14, tumors were induced by subcutaneous injection of 1x10⁶ EMT-6 cells in 200 pL RPMI 1640 into the right flank of mice.

Euthanasia - Each mouse was euthanîzed when it reached a humane endpoint as described 5 below, or after a maximum of 6 weeks post si art of dosing.

Animal monitoring

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 [86]:

, width² x length

Tumor volume =--2

Humane endpoints [87]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding 2000 mm³; Tumors interfering with ambulation or nutrition; Ulcerated tumor or tissue érosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, émaciation, cachexia, déhydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleedîng; Neurologie signs: circling, convulsion, paralysîs; Sustained decrease in body température; Abdominal distension.

Anaesthesia - Isoflurane gas anesthésia was used for ail procedures: surgery or tumor inoculation, i.v. injections, blood collection. Ketamine and Xylazine anesthésia was used for stereotaxia surgical procedure.

Anaigesia - Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severîty of surgical procedure. Non-pharmacological care was provided for ail paînful procedures. Additionally, phannacological care not interfering with studies (topic treatment) were provided at the recommendation of the attendîng veterinarian.

Euthanasia - Euthanasia of animais was performed by gas anesthésia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

Results

Antitumor activîty, EMT6 model

The results are shown in Figure 17. Treatment with the bacterial strain of the invention led to a clear réduction in tumour volume relative to both the négative Controls. The positive control also led to a réduction in tumour volume, as would be expected.

These data îndicate that strain MRx0554 may be useful for treating or preventing other diseases associated with reduced immune System activîty.

Example 10 -Analysis of carbohydrate metabolism - API 50 CHL analysis of MRxO554

The Analytical Profile Index (API) test System consists of strips which contain miniaturised biochemical tests which assay for enzymatic activîty in bacterial species. These tests are routinely used in the characterisation of novel strains. API 50 CHL testing was carried ont to examine carbohydrate metabolism in MRxO554. As per manufacturer’s instructions, bacteria were cultured in 10 mL YCFA broth for 16-18 hours at 37 °C in an anaérobie workstation. This culture was diluted in 10 mL API CHL Medium so as to achieve a density roughly équivalent to McFarland standard No. 2, and 110 pi 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 °C in an anaérobie workstation for 48 hours, following which the colour of each cupule was recorded and assigned a value of négative, intermediate positive, positive or doubtfal.

Using API 50 CHL analysis, MRxO554 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 (Figure 18). Intermediate reactions were observed for D-mannitol, methyl α-D-glucopyranoside, D-lactose, D-raffinose, amidon (starch), and D-turanose.

Example 11 -TLR9 activation

To further elucidate the mechanism by which MRx0518 stimulâtes the immune System, HEKBlue™ human TLR9 reporter cells (InvivoGen, San Diego, CA, USA) were used to test the effect of MRx0518 on TLR9 activation.

Maintenance of cell Unes and bacterial strains

HEK-Blue™ human TLR9 reporter cells (InvivoGen, San Diego, CA, USA) were grown in DMEM supplemented with 10 % (v/v) foetal bovine sérum (FBS), 4 mM L-glutamine, 4.5 mg/mL glucose, 100 U/mL penicillin, 100 pg/mL streptomycin, 100 pg/mL Normocin™ (InvivoGen), 10 pg/mL blastocidin (InvivoGen) and 100 pg/mL zeocin (InvivoGen) to 90 % density. Cells were maintained at 37 °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. Ail reagents were supplîed by Sigma Aldrich unless otherwise stated. E. gallînarum MRx0518 was routinely cultured in Yeast extract, Casitone, Fatty Acid media (YCFA, E&O Laboratories, Bonnybridge, Scotland, UK) at 37 °C in an anaérobie cabinet (Don Whitley Scientific, Shipley, England, UK).

TLR9 Reporter Assays

The foliowing compositions were examined for their ability to induce TLR9 activation:

Live fraction of MRx0518 (MR.xO518LV) - Late log phase bacterial cultures were centrîfuged at 5,000 x g for 5 min at room température to generate bacterial fractions. Pelleted bacteria were washed once in PBS and re-suspended in antibiotic-free cell culture media to the appropriate dilution.

MRx0518 supernatant fraction (MRx0518SN) - Culture supernatants were harvested and filtered through a 0.22 pm pore size filter and diluted in water.

Heat-killed fraction of MRx0518 (MRx0518HK) - Bacterial cultures were heat-inactivated for 40 min at 80 °C and prepared as described above for the live fraction.

MRx0518LV and MRxO518HK were used at a multiplicity of infection (MOI) of 100: LAI 00:1 MOI équivalent volume was used for MRxO518SN. The synthetic CpG oligonucleotide ODN2006 (InvivoGen) was used as an assay positive control at a concentration of 5 pM. YCFA was used as a négative control. Viable cells counts were determined by plating.

HEK-Blue™ human TLR9 reporter cells were incubated with the above treatments for 22 hours at 37 °C in a 5 % CO2 atmosphère. Assays were developed using QUANTI-Blue™ (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.

The results demonstrate that the living and supematant fractions were able to activate TLR9.

Example 12 — T cell différentiation

The ability of MRx0518 to induce T-cell différentiation 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), functîonal grade, cat. No. 16-0037-81) at 400,000/well in 50μ1 cRPMl medium per well (cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2mM final conc. Stock 200mM.; 10% HI FBS (Gibco life technologies, 10082-147); 50pm mercaptoethanol (Gibco life technologies, 21985023); and 1% pen/strep (P4333, lOmg/mL). Heat-killed MRxO518 (prepared by incubation at 80 °C for 30 minutes, after which the cultures were washed with PBS and resuspended in appropriai e cell culture medium and viable counts were confirmed b y plating) was then added to each well, 4,000,000 in 100 pl/well.

Following 3 days in a 37° C încubator, the cells were removed and re-suspended in a medium containing PMA- (Sigma, Cat no. P8139), lonomycin (Sigma, Cat no. 13909) and GolgiSTOP (BD, Cat no 554724) for 5 hours. PMA stock was Img/mL in DMSO which was further diluted in lOOug/mL (each sample required 50ng/mL in cRPMI), lonomycin stock was ImM in DMSO (1 μΜ in cRPMI was used) and GolgîStop concentration was used at 4pl/6mL. Supernatants were passed through a 0.22 pm fi lier and diluted appropriât ely in co-culture medium.

The cells were then subjected to a flow cytometry staining:

After washîng, the cells were incubated with viability dye (Viobility 405/520 Fixable Dye from Miltenyi biotec Ιμΐ/sample) + human Fc block, cat. 564219 (Ιμΐ/sample) in PBS for 10 mins in the dark at room température. The surface antibodies (2μ1 of each) were then added directly to the wells for 10 mins in the dark at room température - CD3-APC-Vio 770 (Miltenyi, cat. No. 130-1 13-136), 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.

The eBioscience FoxP3 transcription factor staining buffer was then used to fix and permeabitise 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 tîxed for Ih at RT and then washed 2x in Ix Perm wash and spun down at 30Üg/5min/RT. The following intracellular staining or transcription factor antibodies were added to the samples in perm wash (Ix) for 45min/in the dark/at room température or in the fridge overnight (up to 18h), followed by washing the antibodies 2x using Perm wash (300μ1) and re-suspension in PBS (250pl) to acquire on the cytometer:

Intracellular markers	Transcription factors
2ul anti-IL10-PE	5.5ul antî-FoxP3-PE- Cy7
2ul anti-IFNy-PE Vio770	9ul anti-Tbet-APC
lOul anti-IL17a-APC	9ul anti-RoRyt-PE

• Anti IFNy-ΡΕ Vio770 human antibodies (Miltenyi, cat. No. 130-114-025) ♦ Anti IL10-PE human antibodies (Miltenyi, cat. No. 130-112-728) • Anti IL17a-APC human antibodies (Miltenyi, cat. No. 130-099-202) • Anti RORyt-PE human antibodies (Miltenyi, cat. No. 130-103-837) • Anti Tbet-APC human antibodies (Miltenyi, cat. No. 130-098-655 • Anti-Foxp3 monoclonal antibody (236A/E7), PE-Cy7 (ebioscience) cat. No. 25-4777-41

As can be seen in Fig. 20A-B, both supematant of MRx0518 (SP 518) and heat-killed MRx0518 (HK 518) were able to induce différentiation of T helper cells and cytotoxîc T cells, respectively, even in the absence of cytokines to induce différentiation (no cyto)

Example 13 - MRxO518 induced cytokine signature

Splénocytes were isolated from C57BL/6 mice and plated in 96 well plates at a density of 900,000 cells/well in RPMI164Û supplemented with 10% FBS, 2mM L-glutamine, 100 U/mL

Pen/Strep (Sigma-Al drich) and 55 pM β-mercaptoethanol (Gibco). Cells were treated with different concentrations of blank media (YCFA⁺) or bacteria supematant from stationary phase for 72hrs. Cell free supematants were collected after each time point and stored at -80 DC for cytokine analysis. Cytokines were measured using multiplex procartaplex MO Thl/Th2/Th9/Thl7/Th22/Treg 17plex kit (Invitrogen). Cell prolifération of untreated splénocytes or splénocytes treated by 10% YCFA medium or 10% MRx0518 bacteria supematant was measured using MTT assay (Millipore), as depicted in Fig. 21F.

Live, growing MRxO518 bacteria were incubated for up to 2h with the human intestinal épithélial cell line CaCo-2 and with the human monocyte/macrophage cell line THP-1. The host response was analysed immediately (CaCo-2) or after a fiirther 24h incubation (THP-1).

Frozen healthy human PBMCs were purchased from Stem Cells Technologies (Cambridge UK). The cells were thawed and left to rest ovcmight in full growth media (RPMI 1640 with 10% FBS, 55 pM β-mercaptoethanol, 2mM L. Glutamine and 100 U/mL penicillin, lOOpg/mL streptomycin) in CO2 incubator at 37°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 supematants 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 supematants were collected and spun down for 3 minutes at 10,000g at 4°C. Samples were stored at -80°C for cytokine analysis. Cytokine quantification was conducted using a ProcartaPlex multiplex immunoassay following the manufacturers recommendations (Thermo Fischer Scientific). Briefly, 50 μΐ of cell-free co-culture supematants were used for cytokine quantification using a MA GP IX® MILLIPLEX® system (Merck) with the xPONENT software (Luminex, Austin, TX, USA). Data was analysed using the MILLIPLEX® analyst software (Merck) using a 5-parameter logistic curve and background sub traction to concert mean fluorescence intensif y to pg/mL values.

Data are expressed in Figs. 21A-D as an average of two technical replicates of 10 biological replicates (PBMC) or three biological replicates (splénocytes) and show production of cytokines in (A) PBMCs; (B) Splénocytes; (C) THP-1 cells; and (D) Caco-2 cells, following treatment with YCFA blank media (“Vehicle”) or MRx0518 bacteria/MRx0518 cell-free bacterial supematant (“MRx0518”). Fig. 21E depicts additional data relating to cytokine sécrétion from splénocytes (N=3), from cells that were either untreated (“Untreated”), treated with YCFA blank media (“10% YCFA”) or treated with MRxO518 cell-free bacterial supematant (“10% MRx0518”).

As can be seen in Figs. 21A-D, treatment of different cells with a supematant of MRx0518 bacteria resulted in immunostimulation as évident by an increase in cytokine production.

Example 14 - NF-κΒ activation

The activation of the NF-κΒ promoter was tested in HEK293 cells co-expressing an NF-kB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene with either the human NOD2 gene, TLR4, TLR9 or TLR5 genes (HEK-Blue™-hNOD2, HEK-Blue™-hTLR5, HEKBlue™-hTLR9 and HEK-Blue™-hTLR4 cells, respectively, by InvivoGen, San Diego, CA, USA).

Briefly, HEK-TLR4 cells were maintained in DMEM 4.5g/L D-glucose supplemented with 10% (v/v) heat-inactivated FBS, 4mM L-Glutamine, 1 OOU/ml penicillin, 100 pg/ml streptomycin, 100 pg/ml normocin, Ix HEK.-Blue sélection media; for HEK-TLR5 and HEK-TLR9 saine media was used with the exception of the use of 2mM L-Glutamine. HEK-TLR5 and HEK-TLR9 were selected using 30 pg/ml and 10 pg/ml blasticidin respectively and lOOpg/ml zeocin media for both cell fines into the culture.

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 densîty 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.

To évaluaie the responsiveness of the cells to their ligands, the cells were treated with l ng/ml LPS (HEK-TLR4), 1 ng/μΐ ultra-pure flagellin from Salmonella typhimurium (HEK-TLR5), 1 μΜ ODN2006 CPG (HEK-TLR9 positive control) or 1 μΜ ODN2006 GPC (HEK-TLR9 négative control), Ing/ml of L18-MDP and încubated in a CO₂ incubator at 37°C. Treatments proceeded for 22h at 37°C and 5% CO2, after which the détection of Secreted embryonic alkaline phosphatase (SEAP) activity from cell culture supematant was performed using QUANTI-blue solution according to manufacturer’s instructions. Briefly, 20 μΐ of cell culture media was collected and analysed for the presence of SEAP by mixing with 200 μΐ of QUANTI-Blue détection media. After 2h (HEK-TLR4 and HEK-TLR5) or 4h (HEK-TLR9 and HEK-NOD2) incubation at 37°C, optical densîty was measured at 655nm on a microplate reader (iMark microplate, Bio-Rad).

As can be seen in Figs. 22A-D (showing results from averaged technîcal replicates for three independent experiments), NF-κΒ 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 (Ing) were used - L18-MDP (for HEK-Blue™hNOD2 cells, Fig. 22A), Lipopolysaccharide, LPS (for HEK-Blue™-hTLR4, Fig. 22B), CPG or negC (for ElEK-Blue™-hTLR9, Fig. 22C) or recombinant flagellin from S. typhimurium, FLA (for HEK-Blue™-hTLR5, Fig. 22D). The cells were incubated with the various treatment at 37 °C in a 5% CO₂ atmosphère for 22 h. To measure NF-κΒ promoter activation (N=3), QUANTI-Blue™ (InvivoGen) was mîxed with cell supematants, the plates were incubated for 2h and optical density was measured at 655 nm.

Sequences

SEQ ID NO: 1 (Enterococcus gallinarum 16S rRNA gene - AF039900) taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag 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

SEQ ID NO:2 (consensus 16S rRNA sequence for Enterococcus gallinarum strain MRx0518)

TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAAAAAGAGT GGCGAACGGGTGAGTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGG TGCTAATACCGTATAACACTATTTTCCGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTG

ATGGATGGACCCGCGGTGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCCACGATGCAT AGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG CAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCAACGCCGCGTGAGTGAAGAA GGTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTTCATCCC TTGACGGTATCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGT GGCAAGCGTTGTCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTG AAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGAGACTTGAGTGCAGAAGAGGAGA GTGGAATTCCATGTGTAGCGGTGAAATGCGTAGATATATGGAGGAACACCAGTGGCGAAGGCGG CTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT GGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGCTGCAG CAAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGT CTTGACATCCTTTGACCACTCTAGAGATAGAGCTTCCCCTTCGGGGGCAAAGTGACAGGTGGTG CATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTA TTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTGACAAACCGGAGGAAGG TGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGAAG TACAACGAGTTGCGAAGTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGT AGGCTGCAACTCGCCTACATGAAGCCGGAATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCG GTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGTG

SEQ ID NO:3 (16S rRNA gene for Enterococcus gallinarum strain MRxO554) taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag 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

1Ü 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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Claims (13)

1. A composition comprising a bacterial strain of the species Enterococcus gallinarum, (a) for use in treating, preventing or delaying immunosenescence; or (b) for use as a vaccine adjuvant; or (c) for use in enhancing a cell therapy.

5

2. The composition for use according to claim 1, wherein the cell therapy is CAR-T.

3. The composition for use according to claim 1 or 2, for use in increasing the expression level and/or activity of IL-12p70, IL-8, IL-1 β, IL-6, IL-23 and/or TNF-ct.

4. The composition for use of any preceding claim, for use in increasing the level and/or activity of NF-kB

10

5. The composition for use of any preceding daim, wherein the bacterial strain 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:2 or wherein the bacterial strain has a 16s rRNA gene sequence represented by SEQ ID NO:2.

6. The composition for use of any preceding claim, wherein the bacterial strain has a 16s

15 rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:3 or wherein the bacterial strain has a 16s rRNA gene sequence represented by SEQ ID NO:3.

7. The composition for use of any preceding claim, wherein the bacterial strain is the strain deposited under accession number 42488 at NCIMB.

20

8. The composition for use of any preceding claim, wherein the bacterial strain is the strain deposited under accession number 42761 at NCIMB.

9. The composition for use of any preceding claim, wherein (a) the composition is for oral administration;

(b) the composition comprises one or more phamiaceutically acceptable excipients or

25 carriers; and/or (c) the bacterial strain is lyophilised.

10. A food product comprising the composition of any preceding claim, for the use of any preceding claim.

H. A composition comprising a cell of the bacterial strain defined in any of daims 1 to 10, 30 wherein the cell expresses one or more heterologous antigens.

12. The composition according to claim 11, wherein the cell présents the one or more heterologous antigens.

13. The composition according to claim 11 or daim 12, for use as a vaccine.

14. A cell of the bacterial strain defined in any of daims 1 to 10, wherein the cell expresses one or more heterologous antigens; optionally wherein the cell présents the one or more heterologous antigens and/or for use as a vaccine.