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 intest ine is thought to be stenle in ntcro but it is exposed to a large variety of maternal and environmental microbes immediately after birth Thereafter a dynamic period of microbial colonization and succession occurs, which is influenced by factors such as delivery mode, environment diet and host genotype all of which impact upon the composition of the gut microbiota, particularly during early life. Subsequently the microbiota stabilizes and becomes adult-like [ i ]. The human gut microbiota contains more than 500- 1000 different phylotypes belonging essentially to two major bacterial divisions the Bactcroidetes and the Einnicuies [2] The successful symbiotic relationships arising from bacterial colonization of the human gut have yielded a wide variety of metabolic, structural, protective and other beneficial functions. The enhanced metabolic activities of the colonized gut ensure that otherwise indigestible dietary components are degraded with release of by-products providing an important nutrient source for the host. Similarly the immunological importance of the gut microbiota is well-recognized and is exemplified in germfree animals which have an impaired immune system that is functionally reconstituted following the introduction of commensal bacteria [3-5]

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

in recognition of the potential positive effect that certain bacterial strains may hav e on the animal gut. c arious strains have been proposed for use in the treatment of c arious diseases (see. for example [ 10- 13] ). Also certain strains including mostly Lactobacillus and Bifidobacterium strains have been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines (see [ 14] and | 15] for rev iew s ) Certain Streptococcus and Veillonellu strains, and to a lesser extent. Ent rococcus and LactohucciHus strains have been suggested to have immunomodulatory effects with v arying effects on different cytokines in vitro , suggesting that data obtained in vitro with individual strains are unlikely to adequately represent immune responses to mixtures of gut microbiota communities in vivo [88] However the relationship between different diseases and different bacterial strains and the precise effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases, are poorly characterised. There is a requirement in the art for new methods of treating diseases. There is also a requirement for the potential e fleets of gut bacteria to be characterised so that new ihetapies using gut bacteria can be developed

SUMMARY OF THE INVENTION

The im entors have dev eloped new compositions comprising a bacterial strain of the species Enterococcus gall inarum that can be used in stimulating the immune system and treating and prev enting disease. The inventors have identified that strains of the species Enterococcus ga/limtnim can potently activ ate the immune system and can treat cancer which indicates that they may able to also treat other diseases where activ ation 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 prov ides a composition comprising a bacterial strain of the species Enterococcus uUinarnm. for use m treating preventing or delaying immunosenescenee.

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 inv ention is the strain deposited under accession number 42488 at NCIMB.

In further preferred embodiments, the bacteria used in the invention is the strain deposited undei accession number 42761 at NCIMB.

BRIEF DESC RIPTION OF DRAWINGS

Figure 1A: Mouse model of breast cancer - changes in tumour v olume post tumour induction and a table indicating the statistical significance between each two treatments at each time point.

Figure I B: Upper panel: Area of necrosis in EMT6 tumours (Untreated n=6. Vehicle n= 6. MRxOS I S 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, MRxO. S n=6. total number cells counted = 33539).

Figure 1C: Mouse model of breast cancer - infiltrating immune cells. Scatter plots represent cell counts of different immune markers from individual animals from each treatment group.

Figure ID: Mouse model of breast cancer - Cytokine production in tumour lysates. Columns represent the mean pg/mL of total protein from each treatment group. *p < 0.05 between groups using one-way ANOVA followed by Dimnett ^' s multiple comparisons test Figure I E: Mouse mode! of breast cancer - Cytokine production in blood plasma. Columns represent the mean pg/mL from each treatment group ( h - SEM).

Figure I F: Representative images of ileum cryosections from vehicle. MRx05 18 and anti-CTLA-4- treated mice immuno-!abelled with antibodies against CD8a (lower panels) and counter-stained with DAP1 (upper panels).

Figure 1G: Plot quantifying animal study subsets with more than 3 CD8a+ cells per field taken from the ileum crypt region 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 liver 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

MRxOS 18MRx0518.

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

MRx0518MRx0518 and LPS.

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

Figure 5B: Cytokine levels in THP- l cells alter addition of bacterial sediment.

Figure 5C: Cytokine levels in THP-l cells after the addition of MRxOS l8MRx0518 alone or in combination with LPS.

Figures 6 and 7: Imimmostimulatory response - TNFo

Figure 8: Immunostimulatory response - IL-12p7Q

Figure 9: Immunomodulatory response - 1L- 10 Figure 10: Immunostimulatory response - IL-8 Figure 1 1 : Immunostimulatory response - IL-23

Figure 12: Immunostimulatory response - IL-Ib Figure 13: Immunostimulatory _’ response - IL-6 Figure 14: Mechanism of action activation of NFtcB Figure 15: Mechanism of action - activation of TLR5

Figure 16A: A schematic representation of the treatment schedule of the different groups used in Example 8 described herein below

Figure 16B: Mean tumour volume in mice bearing a tumour formed by EMT-6 cells. The mice were either untreated or treated with a YCFA v ehicle (Vehicle). MRx05 18 bacteria in YCFA medium (MRx05 l8), 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.

Figu e 17: Mouse model of breast cancer - tumour volume.

Figure 18: API 50 CHL pro tile of Rx0 4.

Figure 19: Mechanism of action - TLR9 acti ation by MR\0518 (MRx05 ! Si _{\ )} . heat-killed MR\0518 ( M RX05 1 S _{] [K} ) and MRx05 I 8 culture supernatant (MRxO. SyO in HER- Blue™ hTLR9 reporter cell lines ODN2006 was used as a positive control and YCFA medium was included as a negative control for MRx05 1 S _s\ . The bar graph represents an average of at least three biological replicates Statistical analysis was performed using GraphPad Prism (ordinary one-way ANOVA analysis followed by Tukey’s Multiple comparison test). Statistically significant differences with the relevant control are shown on the graphs as **** (p < 0.0001).

Figures 20A-B: induction of T-cell differentiation in a population of (A ) T-helper cells and (B ) Cytotoxic T Lymphocytes (CTL ) using heat-killed MRx05 i 8 ( HK 18). Supernatant from MRx0 l 8 culture or RPM1 medium without addition of cytokines (no cyto). * - p< 0.05: **= p< 0.01 ; ***= p<

0.001 ; ****= p< 0.0001.

Figures 21A-D: In-vitro cytokine production by ( A ) PBMC cells: ( B) Splenocytes: or (C) THP- 1 cells: which were treated with YCFA+ medium (“Vehicle”) or cell-free bacterial supernatant of MRx051 8 (“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 splenocytes (N=3), from cells that were either untreated (“Untreated”), treated with YCFA blank media (“10% YCFA”) or treated with MRx0518 cell-free bacterial supernatant (“10% MRx05 l8”).

Figure 21 F: Viabi lity of splenocytes extracted from mice ( =4) as measure by an MTT assay. Cells were either untreated (“Untreated ^” ), treated with YCFA blank media (“10% YCFA ^” ) or treated with MRx0518 cell-free bacterial supernatant (“10% MRx0518”). Figures 22A-D: NF-kB promoter activ ation in (A) HEK-Biue™-hNOD2 cells; (B) H EK-Blue™- liTLR4 cells, (C) HEK-Blue™-hTLR9 cells or ( D) HEK-B!ue™-hTLR5 cells. Cells were either untreated treated with YCFA medium (“YCFA ^” ) treated with MRx0518 (“MRx05 18 ^" ) ortreated with positive controls.

Figure 23: Heat map representing NanoString analysis of EMT6 tumour microenvironment following treatment with Y CFA vehicle (“Vehicle”) or M R.\0518 (“M Rx05 I S”).

DISCLOSURE OF THE INVENTION

Bacterial strains

T1 l compositions of the inv ention comprise a bacterial strain of the species Enterococcus caIIϊί _ΐ apiiii. The examples demonstrate that bacteria of this genus are useful for stimulating the immune system and for treating disease.

Enterococcus gallinanim forms eoccoid cells mostly in pairs or short chains. It is motile and colonies on blood agar or nutrient agar are circular and smooth. Enterococcus guUinarntn reacts with Lancefieid group D antisera. The type strain of Enterococcus %alfinanan is FS7 276 = PB21 = ATCC 49573 = CCUG 18658 = CIP 103013 = JCM 8728 = LMG I 3 129 = NBRC 100675 = NCI MB 702313 {formerly CDO 2313 ) ^~ NCTC 12359 [ 16] The GenBank accession number for a 16S rRNA gene sequence of Enterococcus ^allinaru is AF039900 (disclosed herein as SEQ I D NO: l ) An exemplary Enterococcus al/inurum strain is described in [ 16]

The Enterococcus ^ullinarum bacterium deposited under accession number NCIMB 42488 was tested in the Examples and is also referred to herein as strain MRx05 I S. References to MRx05 l8 and MR.\05 1 8 are used interchangeably. A 16S rRNA sequence for the MRx05 18 strain that was tested is prov ided in SEQ I D NO:2. Strain MRx05 18 was deposited with the international depositary authority NCIMB Ltd. ( Ferguson Building Aberdeen AB2 I 9YA. Scotland ) by 4D Pharma Research Ltd. ( Life Sciences Innov ation Building. Aberdeen AB25 2ZS. Scotland) on 16th November 2015 as ' Enterococcus s/E and was assigned accession number NCI B 42488.

The genome of strain MR\0518 comprises a chromosome and plasmid. A chromosome sequence for strain MRx05 I S is provided in SEQ ID NO:3 OF WO2017/085520. A plasmid sequence for strain MRx05 ! S is pro ided in SEQ I D NO;4 OF WO201 7/085520. These sequences were generated using the PacBio RS 11 platform.

The Enterococcus xallinctntm bacterium deposited under accession number NCI MB 42761 was also tested in the Examples and is also referred to herein as strain MRx0554. References to MRx0554 and MRx0554 are used interchangeably Strain MRx0554 was deposited with the international depositary authority NCIMB. Ltd. (Ferguson Building. Aberdeen, AB21 9YA. Scotland) by 4D Pharma Research

Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) on 22 May 2017 as "Enterococcus galtinarum MRx0554" and was assigned accession number NCIMB 42761 The genome sequence of this baclei ium is disclosed herein as SEQ ID NO:2 OF WO2018/2 15782. The genome sequence was assembled from multiple conti s. Ns in the sequence represent gaps between the contigs ‘N ^" may represent an A G C or T nucleotide. A 16S rRNA gene sequence for the MRx0554 strain is provided in SEQ ID NO:3 SEQ ID NO:3 represents the full length sequence present in the assembly, rather than a consensus of the five 16S genes present in MRx0554.

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 I D NO: I or 2 Preferably the sequence identity is to SEQ I D NO:2. Preferably the bacterial strain for use in the invention has the 16s rRNA gene sequence represented by SEQ ID NO:2. In certain embodiments, the bacterial strain for use in the invention has a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:3.

Bacterial strains that are biotypes of the bacterium deposited under accession number 42488 are also expected to be effective for stimulating the immune system. A biotype is a closely related strain that has the same or very similar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 4248S. For example substantial ly the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95% 96%. 97% 98%. 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). For example, in some embodiments, a biotype strain has at least 98% sequence identity across at least 98% of its genome or at least 99% sequence identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitive sequences such as BOX, ERIC. (GTG)s, or REP or [ i 7] Biotype strains may have sequences with at least 95% 96%, 97%. 98%. 99%. 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42488. In some embodiments, a biotype strain has a sequence with at least 95%. 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRX0518 deposited as NCIMB 42488 and comprises a 16S rRNA gene sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ I D 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 MR\0518 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 inv ention 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 NOD 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 WO20 I 7 085520. For example the bacterial strain for use in the invention may har e a chromosome with at least 90% sequence identity to SEQ ID NOD 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 W02017/085520 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 W 02017 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 NOD OF WO2017/085520. or at least 95% sequence identity to SEQ ID NO:3 OF WO2017O85520 across 90% of SEQ ID NO:3 OF WO2017 O85520 or at least 95% sequence identity to SEQ ID NOD OF WO2017/085520 across 100% of SEQ ID NOD OF WO2017/085520. or at least 98% sequence identity to SEQ ID NOD OF WO2017/085520 across 70% of SEQ ID NOD OF WO2017/085520, or at least 98% sequence identity to SEQ ID NOD OF WO2017/085520 across 80% of SEQ ID NOD OF WO2017/085520, or at least 98% sequence identity to SEQ ID NOD OF W02017/085520 across 90% of SEQ ID NOD OF WO2017/085520, or at least 98% identity to SEQ ID NOD OF WO2017/085520 across 95% ofSEQ ID NOD OF WO2017/085520, or at least 98% sequence identity to SEQ ID NOD OF WO2017/085520 across 100% of SEQ ID NOD OF WO2017 085520. or at least 99.5% sequence identity to SEQ ID NOD OF WO2017Ό85520 across 90% of SEQ ID NOD OF W020 I 7 085520. or at least 99.5% identity to SEQ ID NOD OF WO2017/085520 across 95% of SEQ ID NOD OF WO2017/085520. or at least 99.5% identity to SEQ I D NO: 3 OF WO2017/085520 across 98% of SEQ ID NOD OF WO20 I 7/085520, or at least 99.5% sequence identity to SEQ ID NOD OF WO2017/085520 across 100% of SEQ ID NOD OF WO2017/085520.

In certain embodiments the bacterial strain for use in the invention has a plasmid with sequence identity to SEQ ID NOD OF WO20 I 7/085520 In preferred embodiments, the bacterial strain for use in the invention has a plasmid with at least 90% sequence identity (e.g. at least 92%, 94% 95% 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NOD 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 NOD OF WO2017/085520. For example, the bacterial strain for use in the invention may have a plasmid with at least 90% sequence identity to SEQ ID NOD OF WO2017/085520 across 70% of SEQ ID NOD OF WO2017/085520, or at least 90% sequence identity to SEQ ID NOD OF WO2017/085520 across 80% of SEQ ID NOD OF WO2017/085520. or at least 90% sequence identity to SEQ ID NOD OF WO2017/085520 across 90% of SEQ ID NOD OF WO2017/085520. or at least 90% sequence identity to SEQ ID NOD OF W020I 7/085520 across 100% of SEQ ID NOD OF WO2017/085520. or at least 95% sequence identity to SEQ ID NOD OF WO2017 085520 across 70% o SEQ ID NOD OF WO2017/085520, or at least 95% sequence identity to SEC) ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520. or al least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO201 7/085520, or at least 95% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 100% of SEQ ID NO 1 OF WO2017/085520, or at least

98% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 70% of SEQ ID NO:4 OF WQ2017/085520, or at least 98% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 80% of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence identity to SEQ ID NO:4 OF WO2017/085520 across 90% of SEQ ID NO:4 OF WO2017/085520, or at least 98% sequence identity 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 identity to SEQ ID NO:3 OF WO2017/085520 and a plasmid with sequence identity to SEQ ID NO:4 OF WO2017/085520.

In certain embodiments, the bacterial strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO2017/085520, for example as described above, and a 16S rRNA sequence with sequence identity to any of SEQ ID NO: I or 2, for example as described above, preferably with a 16s rRNA sequence that is at least 99% identical to SEQ ID NO: 2, more preferably which comprises the 16S rRNA sequence of SEQ ID NO:2, and optionally comprises a plasmid with sequence identity to SEQ I D NO:4 OF WO2017/085520, as described above.

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

In certain embodiments the bacteria! strain for use in the invention has a chromosome with sequence identity to SEQ ID NO:3 OF WO201 7/085520. for example as described above, and a I 6S rRNA sequence with sequence identity to any of SEQ ID NOs: 1 or 2. for example as described above and optionally comprises a plasmid ith sequence identity to SEQ ID NO:4 OF WO2017/085520, as described abo\ e. and is effective for stimulating the immune system.

In certain embodiments the bacterial strain tor use in the invention has a 16s rRNA sequence that is at least 99%. 99.5% or 99.9% identical to the 16s rRNA sequence represented by SEQ I D 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 5 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 WO201 7/085520 across at least 98% (e.g. across at least 99%. or at least 99.5%) of SEQ ID NO:3 OF WO201 7/085520. and optionally comprises a plasmid with sequence identity to SEQ ID O: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 gallinarum and has a 16s rRNA sequence that is at least 99%, 99 5% or 99.9% identical to the l6s 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.

Alternatively, strains that are biotypes of the bacterium deposited under accession number NCIMB 42488 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42488 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism ( FAFLP) and repetitiv e DMA element (rep )-PCR fingerprinting, or protein profiling or partial 1 &S or 23s rDN A sequencing. In preferred embodiments such techniques may be used to identify other Enterococcus gallhutni 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 inv ention are strains that prov ide the same pattern as the bacterium deposited under accession number NCIMB 424SS when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example,[18]). Alternatively, biotype strains are identified as strains that have the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42488. In some embodiments, the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMerieux). In some embodiments, the bacterial strain used in the invention is:

(i) positive for fermentation of at least one of (e g at least 2, 3, 4 5, 6, 7, S, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17 or all of): L-arabinose, D-ribose, D-xyiose, D-gaiactose, D-glucose, D- fructose, D-mannose, N-acetylglucosamine, ygdalin arbutin, salicin D-cellobiose, 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 all of): D-mannitol, 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 bioMerieux). Other Ente ococcus aίίίhappp strains that are useful in the compositions and methods of the inv ention, such as biotypes of the bacterium deposited under accession number NC !MB 42488. ma be identified using any appropriate method or strategy including the assays described in the examples. For instance strains for use in the invention may be identified by assessing their effects on cytokine levels as performed in the examples In particular, bacterial strains that have similar growth patterns metabolic type and/or surface antigens to the bacterium deposited under accession number NC1MB 42488 may be useful in the invention A useful strain will have comparable immune modulatory activity to the NC1MB 42488 strain. In particular, a biotype strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples, which may be identified by using the culturing and administration protocols described in the Examples. According to some embodiments, a biotype strain that may be used in the invention is a strain which is able to elicit comparable effects on the cancer disease models shown in the Examples when administered in the method of the invention.

In some embodiments, the bacterial strain used in the im emion is:

(i) Positive for at least one of (e.g. at least 2 3. 4. 5. 6, 7 or all of): mannose fermentation, glutamic aci decarboxylase arginine arylamidase. phenylalanine aryiamidase, pyroglutamic acid arylamidase tyrosine arylamidase, histidine arylamidase and serine arylamidase; and/or

(ii) Intermediate for at least one of (e.g. at least 2 or all of): p-galactosidase-6-phosphate, b-glucosidase and N-acetyl-p-glucosammidase; and/or

(iii) Negative for at least one of (e.g. at least 2, 3, 4, 5, 6 or all of): Raffinose 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 determine by Rapid ID 32A analysis (preferably using the Rapid ID 32A system from bioMerieux).

In some embodiments the bacterial str in used the invention is:

(i) Negative for at least one of (e.g. at least 2. 3, or all 4 of) glycine arylamidase. raffinose fermentation, proline aryiamidase, and leucine arylamidase for example as determined by an assay of carbohydrate amino acid and nitrate metabolism, preferably as determined by Rapid ID 32A analysis (preferably using the Rapid ID 32A system from bioMerieux); and/or

(ii) Intermediate positive for fermentation of L-fiicose, preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMerieux).

In some embodiments, the bacterial strain used in the invention is an extracellular ATP producer for example one which produces 6-6.7 ng/mΐ (for example, 6.1-6.6 ng/mΐ or 6.2-6.5 ng/mΐ or 6.33 ± 0.10 ng/mΐ) of ATP as measured using the ATP Assay Kit (Sigma-Aldrich, MAK190). Bacterial extracellular ATP can have pleiotropic effects including activation of T cell-receptor mediated signalling ( Schenk el aL 201 1 ) promotion of intestinal Til l 7 cell di fferentiation (Alarashi et al.. 2008) and induction of secretion of the pro-inflammatory mediator 1 L- 1 (1 by activating the NLRP3 inflammasome (Karmarkar et ah. 2016) Accordingly a bacterial strain which is an extracellular ATP producer is useful for stimulating the immune system in the context of the method of the invention.

In some embodiments, the bacterial strain for use in the invention comprises one or more of the following three genes: Mobile element protein; Xylose ABC transporter, permease component: and FIG00632333: hypothetical protein. For example, in certain embodiments the bacterial strain for use in the invention comprises genes encoding Mobile element protein and Xylose ABC transporter permease component; Mobile element protein and FIG00632333: hypothetical protei n; Xylose ABC transporter, permease component and FIG00632333: hypothetical protein; or Mobile element protein. Xylose ABC transporter permease component, and FIG00632333; hypothetical protein

A particularly preferred strain of the invention is the Enterococcus gallinamm strain deposited under accession number NCIMB 42488. This is the exemplary MRx0518 strain tested in the examples and shown to be effective for treating disease. The invention provides, according to some embodiments, a bacterial composition as part of the invention, comprising a cell of the Enterococcus gallinamm strain deposited under accession number NCIMB 42488, or a derivative thereof. A derivative of the strain deposited under accession number NCIMB 42488 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original.

A derivative of a strain of the composition comprised in the invention may be modified for example at the genetic level, without ablating the biological activity. In particular, a derivative strain of the invention is therapeutically active. A derivative strain will have comparable immune modulatory activity to the original NCIMB 42488 strain. In particular, a derivative strain will elicit comparable effects on the cancer disease models when which may be identified by using the culturing and administration protocols described in the Examples. A derivative of the NCIMB 42488 strain will generally be a biotype of the NCIMB 42488 strain.

References to cells of the Entcmcoca/s gallinamm strain deposited under accession number NCIMB 42488 encompass any cells that have the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42488. and such ceils are encompassed by the the invention. Thus in some embodiments, reference to cells of the Enterococcus gallinamm strain deposited under accession number NCIMB 42488 refers only to the MRx0518 strain deposited under NCIMB 42488 and does not refer to a bacterial strain that was not deposited under NCIMB 42488. In some embodiments, reference to cells of the Enterococcus gallinamm strain deposited under accession number NCIMB 42488 refers to cells that bas e the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCI B 42488. but which are not the strain deposited under NCI B 42488. Bacterial strains that are biotypes of the bacterium deposited under accession number 42761 are also expected to be effectiv e lot stimulating the immune system. A biotype is a closely related strain that has the same or v ery similar physiological and biochemical characteristics.

Strains that are biotypes of the bacterium deposited under accession number NC1MB 42761 and that are suitable for use in the invention may be identified by sequencing other nucleotide sequences for the bacterium deposited under accession number NCIMB 42761. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may have at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). For example in some embodiments, a biotype strain has at least 98% sequence identity across at least 9S% of its genome or at least 99° ₀ sequence identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or repetitiv e sequences such as BOX, ER IC. <GTG) _?, or REP or [19] Biotype strains tnay have sequences with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of the bacterium deposited under accession number NCIMB 42761. In some embodiments a biotype strain has a sequence with at least 95%. 96° o. 97%. 98%. 99%. 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0554 deposited as NCIMB 42761 . In some embodiments, a biotype strain has a sequence with at least 95%. 96%. 97%. 98% 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MR.\0554 deposited as NCIMB 42761 and has a 16S rRNA gene sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:3. In some embodiments, a biotype strain has a sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of strain MRx0554 deposited as NCIMB 42761 and has the 16S rRNA gene sequence of SEQ ID NO:3.

Alternativ ely, strains that are biotypes of the bacterium deposited under accession number NCIMB 42761 and that are suitable for use in the inv ention may be identified by using the accession number NCIMB 42761 deposit and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism ( FAFLP) and repetitive DNA element (rep)-PCR fingerprinting or protein profiling or partial 16S or 23s rDNA sequencing.

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]). Alternatively, biotype strains are identified as strains that hav e the same carbohydrate fermentation patterns as the bacterium deposited under accession number NCIMB 42761. In some embodiments, the carbohydrate fermentation pattern is determined using the API 50 CHL panel (bioMerieux). In some embodiments, the bacterial strain used in the in ention is: ( in) positive for fermentation of at least one of (e.g. at least 2. 3. 4. 5. 6 7. 8. 9. 10. I I . 12. 13 14. 15 16. 17 or all of): L-arabinose D-ribose. D-xylose. D-galaclose, D-ghicose, D- fructose D-mannose. N-acetyiglucosamine amygda!in arbutin. saliein D-eellobiose. D- maitose. sucrose. D-treha!ose. gentiobiose. D-tagatose and potassi um gluconate: and/or (iv) intermediate for fermentation of at least one of (e.g. at least 2. 3. 4 or all of): D-mannitol.

ethyl-aD-g!ycopyranoside. D-lactose. starch and L-fucose:

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

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

(i) posith e for fermentation of at least one of (e.g. at least 2 3 4. 5 6 7 8 9 10 1 1. 12 13.

14. 15. 16. I 7. 1 8 or all of): L-arabinose. D-ribose. D-xylose. D-galactose. D-glucose. D- fmctose, D-mannose N-acetylghicosamine, amygdalin. arbutin. escu!in. saliein. D- cellobiose. D-maltose. D-saccharose (sucrose), D-trehalose gentiobiose, D-tagatose and potassium gluconate;

(ii) intermediate for fermentation of at least one of (e.g. at least 2, 3, 4, 5 or all of): D-mannitol,

Methyl-aD-glycopyranoside, D-lactose, D-rafftnose, amidon (starch), and D-turanose; and/or

(iii) negative for fermentation of at least one of (e.g. at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,

14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or all of): glycerol erythritol, D-arabinose, L-xylose, D-adonitol, methyl-PD-xylopryranoside L-sorbose L-rhamnose, dulcitol. inositol, D- sorbitol, Methyl-aD-mamiopyranoside, D-melibiose, inu!in, D-melezitose, glycogen, xylitol, D-lyxose, D-fucose, L-fucose, D-arabitoI, L-arabitoI, potassium 2-ketogluconate and potassium 5-ketogluconate;

preferably as determined by API 50 CHL analysis (preferably using the API 50 CHL panel from bioMerieux, and preferably using the conditions described in Example 10).

Other Enterococcus gaUinunnn strains that are useful in the compositions and methods of the invention, such as biotypes of the bacterium deposited under accession number NCIMB 42761 , may be identified using any appropriate method or strategy including the assays described in the exam les. For instance, strain for use in the invention may be identified by culturing in anaerobic YC ^’ FA and or administering the bacteria to the type P collagen-induced arthritis mouse model and then assessing cytokine levels. In particular, bacterial strains that have similar growth patterns metabolic type and/or surface antigens to the bacterium deposited under accession number NCI B 42761 may be useful in the invention. A useful strain will have comparable immune modulatory activity to the NCIMB 42761 strain. In particular a biotype strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples which may be identified by using the culturing and administration protocols described in the Examples. A derivative of a strain of the invention may be modified for example at the genetic lex el without ablating the biological activity. In pai ticular. a derivative strain of the inv ention is ilieiapeutically active. A deriv ative strain wil l have comparable immune modulatory activity to the original NCiMB 42761 strain. In particular a deriv ative strain will elicit comparable effects on the cancer disease models to the effects shown in the Examples which may be identified by using the culturing and administration protocols described in the Examples. A derivative of the NCIMB 42761 strain will generally be a biotype of the NCIMB 42761 strain.

References to cells of the Eni rucoc s ga imirum strain deposited under accession number NCIMB 42761 encompass any cells that hav e 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 V.nlerococvus gul/iminim strain deposited under accession number NCIMB 42761 refers only to the MRx0554 strain deposited under NCIMB 42761 and does not refer to a bacterial strain that was not deposited under NCIMB 42761.

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 inv ention has a genome with at least 90% sequence identity (e.g. at least 92% 94% 95%, 96%, 97%. 98% 99% or 100% sequence identity) to SEQ ID NO:2 OF WO2018/215782 across at least 60% (e.g. across at least 65%, 70%, 75%. 80%, 85% 95%, 96%. 97%. 98%, 99% or 100%) of SEQ ID NO:2 OF WO2018/215782. For example the bacterial strain for use in the invention may have a genome with at least 90% sequence identity to SEQ I D NO:2 OF WQ2018/215782 across 70% of SEQ ID NO: 2 OF WO2018/215782, or at least 90% sequence identity to SEQ ID NO:20F WO2018/215782 across 80% of SEQ ID NQ:2 OF WO20I 8/215782, or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ ID NO:2 OF WO2018/215782, or at least 90% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 70% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 80% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ ID NO:2 OF WO2018/215782, or at least 95% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 98% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 70% of SEQ ID NO:2 OF WO2018/2 ! 5782, or at least 98% sequence identity to SEQ ID NO:2 OF WO2018/21 5782 across 80% of SEQ ID NO 2 OF WO20I 8/215782. or at least 98% sequence identity to SEQ ID NO:2 OF WO2018/215782 across 90% of SEQ ID NO: 2 OF WO20 I 8/215782. or at least 98% identity across 95% of SEQ ID NO:2 OF WO2018/2 I 5782, or at least 98% sequence identity to SEQ 1D NO:2 OF WO2018/215782 across 100% of SEQ ID NO:2 OF WO2018/215782, or at least 99.5% sequence identity to SEQ ID NO:2 OF 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% o† ^' SEQ ID NO:2 OF WO201 8/2 1 5782. or at least 99.5% sequence identity to SEQ ID NO:2 OF WO 201 8/2 1 5782 across 100% ot ^' SEQ ID NO 2 OF WO201 8/2 15782.

In certain embodiments, the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 OF WO20 I S 2 1 5782. for example as described above, and a 16S rRNA gene sequence with sequence identity to SEQ ID NO: l 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 stimulating the immune system.

In certain embodiments, the bacterial strain for use in the invention has a genome w ith sequence identity to SEQ ID NO:2 OF WO20 I 8/2 1 5782. for example as described above, and a 16S rRNA gene sequence with sequence identity to SEQ ID NO: 1 or 3. for example as described above and is effective for stimulating the immune system.

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 WO20I8/215782 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 ga!!inantm and has a 16S rRNA gene sequence that is at least 99%, 99.5% or 99.9% identical to the 1 6S 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 caselliflavus. Enterococcus caseUiflavus is highly similar to Enterococcus gallinanim and is also flagellated. Therapeutic uses

Stimulating the immune system

The examples show that administration of the compositions of the invention can lead to immune stimulation. Since administration of the compositions of the invention were shown to have an im unostimulatory effect, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an increased immune response. In certain embodiments the compositions of the invention are for use in stimulating the immune system. In certain embodiments the compositions of the invention are for use 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 inv ention may be useful in the treatment of diseases characterised by an increase in the percentage of Tregs in a cell population. In one embodiment the compositions of the inv ention may be useful for treating or prev enting diseases characterised by an increase in the percentage of Tregs in a cell population. In one embodiment the compositions of the invention may be useful for treating or preventing diseases characterised by an increase in the percentage of C D4+CD25+CD127- cells in a cell population. In one embodiment, the compositions of the i ention are for use in treating or preventing diseases by decreasing the percentage of Tregs in cell populations. In one embodiment, compositions of the invention are for use in reducing suppression of the immune response by Tregs. In one embodiment, compositions of the invention are for use in stimulating the immune response by the selective reduction of Tregs. In one embodiment, compositions of the invention are for use in immunostimulation, wherein the compositions of the invention reduce the number or percentage of T egs.

Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the ratio of CDB/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 CDB/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 CDB/Treg cells In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the ratio of CDB/Treg cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the ratio of activated CDB/Treg cells.

Compositions of the inv ention 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 inv ention 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 inv ention are for use in treating or preventing diseases by increasing the number or percentage of B cells in cell populations wherein the increase in number or percentage of B cells results in immune stimulation. In one embodiment compositions of the inv ention are lot 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 CDS T-eytotoxie 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 CDS 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 CDS 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 CDS 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 ⁺ activated cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of CDS ⁴ activated cells. In one embodiment, the compositions of the invention are for use in treating or pre enting diseases by increasing the number or percentage of CDS ⁴ activated cells in cell populations wherein the increase in number or percentage of CDS ⁴ activated cells results in immune stimulation In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of CDS ⁴ activated cells.

The examples show that administration of the compositions of the invention can lead to an increase in expression of pro- inflammatory molecules, such as pro- inflammatory cytokines. Examples of pro- inflammatory molecules that showed an increase in expression levels upon administration of compositions of the invention include IL-8, IL-12p70, IL-23, TNF-a, IL-l p, and IL-6. Since administration of the compositions of the invention were shown to increase the expression of pro- inflammatory molecules compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molecules such as pro-inflammatory cytokines. In one embodiment the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of pro-inflammatory molecules, in particular diseases characterised by a decrease in the expression and or activity of pro-inflammatory cytokines. In a particular embodiment, the compositions of the inv ention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-8, IL~l2p7G, IL- 23, TNF-a, IL-Ib,- 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-Ib, 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-12p7Q, IL-23, TNF-a, IL-Ib, and/or IL-6. The examples also show that administration of the compositions of the invention can lead to an increase in expression of iL- 1 b. IL- 1 b is a pro- inflammatory cytokine [21 ] The production and secretion of IL- ί b is regulated by the inflam asome, 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-Ib, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL- Ib. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL- 1 b . 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-lp.

The examples also show that administration of the compositions of the invention can lead to an increase in expression of 1L-23. 1L-23 has been linked to inflammation [23 24] The proposed functions of 1L-

23 in the immune response include promoting the proliferation of CD4 ⁺ memory T cells and promoting the secretion of IFN-g 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 useful in the treatment of diseases characterised by a decrease in expression of IL-23. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-23. In one embodiment the compositions of the invention are for use in treating or preventing diseases by increasing die 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 examples show that administration of the compositions of the invention can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-a). TNF-a is a pro-inflammatory cytokine which is known to be involved in various signalling pathways to promote cell death. TNF-a initiates apoptosis by binding to its cognate receptor. TNFR- I which leads to a cascade of cleavage ev ents in the apoptotic pathway [26]. T F-a can also trigger necrosis via a RIP kinase-dependent mechanism [27] Since administration of the compositions of the invention show an increase in TN F-u expression compositions of the inv ention may be useful in the treatment of diseases in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF-a. In one embodiment, the compositions of the inv ention are for use in treating diseases characterised by decreased TNF-a expression. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of TNF-a. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of TN F-a. In one embodiment compositions of the invention are for use in promoting the immune response by increasing the expression and· or activity of TNF-a.

The examples also show that administration of the compositions of the invention can lead to an increase in expression of IL-6. IL-d a pro-inflammatory cytokine that is produced during inflammation, and promotes the differentiation of naive CD4 T cel Is and tiie differentiation of CDS T cells into cytotoxic T cells [2 S] . Since administration of the compositions of the inv ention were shown to increase the expression of IL-6, compositions of the invention may he useful in the treatment of diseases characterised by a decrease in expression of IL-6. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-6. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-6. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-6.

Bettelli et if/. [29] reported that EL-6 inhibits the expansion of Tregs Since the examples show that compositions of the invention increase the expression of IL-6, compositions of the invention may selectively decrease the number or percentage of Tregs by increasing the expression of IL-6 In one embodiment, compositions of the invention are for use in immunostimulation by increasing the expression of IL-6. In another embodiment, compositions of the invention are for use in immunostimulation by decreasing the number or percentage of Tregs.

In some embodiments stimulating the immune system according to the present invention comprises TLR5 activation or upregulation of TLR5 activation. In some embodiments, stimulating the immune system according to the present inv ention comprises TLR9 activation or upregulation of TLR9 activation. In some embodiments, stimulating the immune system according to the present invention comprises activation of TLR5 and TLR9 or upregulation of TLR9 and TLR5 activation. In some embodiments, stimulating the immune system according to the present invention comprises inducing and/or upregulating differentiation of T cells such as, but not limited to, T helper cells and T cytotoxic cells.

TLR signalling pathways culminate in the activation of the transcription factor nuclear factor-kappaB (NF-kB). NF-KB controls the expression of an array of inflammatory cytokine genes, including TNF- a. Immune stimulation causes for example the dimerization of TLR5. w hich subsequently recruits MyDSS and activ ates protein kinases including I RAK I . IRAK2. IRAKT nd IRAK-V!. The activ ation of these kinases leads to the nuclear localization of NF-kB, which is a proinflammatory cytokine [30]

As demonstrated in the examples, compositions of the invention lead to an increase in expression of NF-kB. Since administration of the compositions of the invention increase the expression of the proinflammatory cytokine NF-kB, compositions of the invention may be useful in stimulating the immune response. In addition, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and/or diseases treatable by an increased immune response. In one embodiment, the compositions of the invention are for use as an immune stimulant by increasing the level and/or activity of NF-kB. 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 N F-kB. In one embodiment t e compositions of the int ention are for use in treating diseases treatable by an increased immune response by iucteasiug the lev el and or activity of N F-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-kB. 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-KB

The activation of NF-KB is important for eliciting innate immune responses and the subsequent development of adaptive immune responses. Thus agonists of TLRs such as compositions of the invention are likely to be useful as adjuv ants to treat infectious diseases allergies and tumours by promoting both innate and adaptiv e 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 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 differentiation of T-helper cells and cytotoxic T lymphocytes. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the differentiation of T-helper cells and/or cytotoxic T lymphocytes.

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

Use as a vaccine adjuvant

The examples show that administration of the compositions of the invention can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-a). TNF-a is known to be important for vaccine responses. For example, TNF-a has been shown to be required for an efficient vaccine response in a flu vaccination of the elderly population [31] Since administration of the compositions of the invention were shown to increase TNF-a expression, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the inv ention are for use as a vaccine adjuvant by increasing the level and/or activity of TNF-a. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant. In one embodiment the compositions of the invention are for use as a vaccine adjuvant in influenza therapy in certain embodiments, the compositions of the invention are for use in enhancing an immune response against an antigen. I 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.

Ent rococcus uUinunun and in particular strain VlRx05 l 8 is flagellated and fl age 11 ms 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 MRx05 ! 8 flage!liu is a TLR5 agonists Therefore, the compositions of the invention may be useful as vaccine adjuvants in particular for vaccine administered to elderly patients (e g. over 40. 50, 60. 70 or 80 years of age), who may have reduced immune system activity. TLR5 signalling also plays a key role in age- associated innate immune responses [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 all from 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 increase in expression of IL-6 Increased IL-6 expression has been associated with vaccine responses for many diseases. For example, I L-6 was produced by CD I 4H-CD 16- inflammatory monocytes after adults were administered an influenza vaccine [34], and higher lev els of IL-6 were associated with achieving a v accine response to an influenza vaccine [35] Furthermore, IL-6 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-a expression have been shown to be correlated with the efficacy of a therapeutic HIV vaccine [Huang et al] a tuberculosis vaccine and a chlamydia vaccine [38] Su ct al. [39] showed that co- inoculation of IL-6 or TMF-ec with the FMDV DNA vaccine resulted in increased lFN-g expression by CD4 ⁺ and CDS 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-a expression, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TNF-a. In one embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of IL-6. In a particular embodiment, the compositions of the invention may be useful as a vaccine adjuvant by increasing the level and/or activity of TN F-ct and IL-6 in one embodiment, the compositions of the invention are for use as a vaccine adju\ ant in 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-Ib. Li el til. [40] showed that the adjuvant aluminium hydroxide activated the secretion of IL- 1 b, and suggested that IL-I b itself can act as an adjuvant. Since administration of the compositions of the invention were shown to increase IL- 1 p expression, compositions of the invention may be useful as a vaccine adjuvant. The examples show that administration of the compositions of the inv ention can increase the tatio of CDS T cells to Treys Adjuvants have been shown to stimulate CD8 ⁺ 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 useful as a vaccine adjuvant.

In one embodiment, compositions of the invention are for use as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the ratio of CD8 ⁺ T cells to Tregs.

The examples also show that administration of the compositions of the invention can lead to an increase in expression or lev els of CXCR3 ligands CXCL9 and CXCL I 0. Known adjuv ants such as AS03. CpG. GLA-SE, c GalCer all increase CACL9 and 10 [42.43]. which suggests the compositions of the invention will be effective as adjuvants. Also. CXCL9 and 10 are associated with IFNyTh I responses and promote antibody responses [44] In certain embodiments the compositions of the inv ention are for use in promoting an antibody response against an antigen in particular a pathogenic or cancer antigen. Also, CXCL9 is a more sensitiv e measure than iFX-y of v accine induced T-cell responses in v olunteers receiv ing inv estigated malaria v accines [45] In certain embodiments the compositions of the invention are for use in promoting an T-cell response against an antigen in particular a pathogenic or cancer antigen. In one embodiment the compositions of the inv ention are for use as a vaccine adjuvant by increasing the level and Or activ ity of CXCL9 and CXCL 10 in certain embodiments the compositions are for rise in protecting against malaria .

The examples also show that administration of the compositions of the invention can lead to an increase in expression or levels of IL-l2p70. This effect has been associated with vaccine adjuvant efficiency and IL-I2 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 adjuv ant 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 include: viral antigens such as viral surface proteins; bacterial antigens, such as protein and/or saccharide antigens; fungal antigens; parasite antigens; and tumour antigens. The invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomegalovirus, Staphylococcus aureus, chlamydia, SARS coronavims, varicella zoster virus. Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, human papillomavirus, etc. Further antigens for use with the invention include glycoprotein and lipoglyean antigens archaea antigens melanoma antigen E (MAGE). Carciuoembi vonic antigen (CEA ). MUC- L HER2. sialy!-Tn ( STn ), luimau leionierase reverse transcriptase (liTERT). Wilms tumour gene ( WT1 ). CA- I 25 prostate-specific antigen ( PSA). Epstein- Barr virus antigens neoantigens oncoproteins amyloid-beta. Tau, PCSK9 and habit forming substances for example nicotine alcohol or opiates.

Preferred antigens for use with the invention include pathogen antigens and tumour antigens. An antigen will elicit an immune response specific for the antigen that will be effective for protecting against infection with the pathogen or attacking the tumour. Antigens may be, for example peptides or polysaccharides.

The invention also provides the use of: (i ) an aqueous preparation of an antigen: and (ii) a composition comprising a bacteria! strain of the species Enterococcus gallinarum, in the manufacture of a medicament for raising 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 bacterial strain of the species Enterococcus gallinarum is engineered to present an antigen. Presenting an antigen on the bacterial strain of the invention may maximise the immunostimulatory activities and further enhance the protectiv e immune response generated against the antigen. In addition manufacturing and deliv ering therapeutics comprising an antigen and a bacteria of the inv ention may be more efficient and effectiv e this w y than when each of the antigen and the composition comprising the bacterial strain are manufactured and administered separately. Therefore, in some embodiments the invention provides a composition comprising a bacterial strain of the species Enterococcus gallinarum that presents an antigen for example on its cell surface. In some embodiments, the composition comprising the bacterial strain that presents an antigen is for use as a v accine antigen in some embodiments the antigen is deriv ed from HIV. hookworm hepatitis B virus herpes simplex virus rabies, respiratory syncytial virus cytomegalovirus. Staphylococcus aureus chlamydia, SARS coronavirus. v aricella zoster virus. Streptococcus pneumoniae. Neisseria meningitidis , Mycobacterium tuberculosis , Bacillus authracis, Epstein Barr v irus or human papillomavirus. In some embodiments, the antigen is a glycoprotein antigen lipoglyean antigen, archaea antigen, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA- 125, prostate-specific antigen (PSA), Epstein-Barr vims antigens, neoamigens. 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 bacterial strain of the species Enterococcus gallinarum that expresses a heterologous antigen. The antigen may be part of a fusion polypeptide expressed with one or more polypeptides homologous to the bacteria. In some embodiments the bacteria express the antigen as a non-fusion polypeptide in some embodiments the invention prov ides a composition comprising a cell of a bacterial strain of the species Enterococcus gullinantm wherein the cell expresses a heterologous antigen. In some embodiments the composition is for use as a vaccine. In some embodiments the invention provides a cell of a bacterial strain of the species Enterococcus gall inarum , 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 bacteria! strain of the species Ent rococcus gallmar m include glycoprotein and lipoglycan antigens archaea antigens, melanoma antigen E (MAGE). Carcinoembryonic antigen (CEA ). MUC- 1. HER2. sia!yl-Tn ( STn), human telo erase rev erse transcriptase (hTERT). Wilms tumour gene ( WT! ) CA- 125, prostate-specific antigen ( PSA), Epstein-Barr v irus 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 non-communicable diseases such as elevated cholesterol (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 therapies

Chimeric Antigen Receptor T cell (CAR-T) therapy

The examples also s how that administration of the compositions of the invention can lead to an increase in expression of 1L-6. Increased 1L-6 expression has been correlated with response to CD19 CAR-T therapy of chronic lymphocyte leukaemia. An increase in serum IL-6 was associated with CAR-T cell expansion, whereas inhibition of IL-6 was associated with inhibition of CAR-T cell proliferation [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.

Selective depletion of Tregs has been shown to enhance the efficacy of cytotoxic lymphocytes | 48j. CAR-T cells are a subset of cytotoxic lymphocytes and therefore it is thought that selective depletion of Tregs is effective in CAR-T cell therapy. Since administration of the compositions of the invention were shown to deplete Tregs, compositions of the invention may be useful in cell therapy, in particular CAR-T cell therapy. Therefore the compositions of the invention may be useful in cell therapy in particular in enhancing the response to a cell therapy.

Mesenchymaktem cell (MSC) therapy

Mesenchymal stem cell ( MSC) therapy has been reported to have immunostimulatory properties. When MSCs are treated with IT’S, they upregulate pro- inflammatory cytokines IL-6 and IL-8 which causes increased B cell proliferation [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 beneficial to differentiate stem cells to some extent prior to transplantation. For example Menu et a/. [50] reported that cardiomyogenic differentiation of stem cells may be beneficial by having a higher engraftment efficiency enhanced regeneration of myocytes and increased restoration of heart function. Since administration of the compositions of the invention initiated neuronal di fferentiation m undifferentiated neuroblastoma cells compositions of the invention may be useful for stem cell differentiation 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 casseliflavus) prior to allogenic hematopoietic stem cell transplantation has been shown to lead to a significantly improved the 2 -year survival of patients after due to decreased nonrelapse mortality [51] Therefore, the immunomodulatory effect shown in the examples may be useful in hematopoietic stem cell transplantation therapy. In certain embodiments, the compositions of the invention may be useful in improving survival after hematopoietic stem cell transplantation and in particular after allogenic hematopoietic stem cell transplantation.

The compositions of the invention may be useful in combination with allogenic hematopoietic stem cell transplantation. The compositions of the invention may be effective in boosting successful patient response to allogenic hematopoietic stem cell transplantation in certain embodiments the compositions of the invention are administered prior to hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are for administration to a patient scheduled to receive hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are administered following hematopoietic stem cell transplantation. In certain embodiments, the compositions of the invention are for administration to a patient that has received hematopoietic stem cell transplantation. Immunosenescence

Fulop at t// [52] identified that an increase in Treg cell number and a decrease in B cell number are associated with aging in the adaptive immune system. Therefore compositions of the invention may be used to prevent or delay immunosenescence. In one embodiment compositions of the invention are for use in preventing immunosenescence. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by a decrease in B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number and a decrease in B cel! 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 inv ention are for use in delaying immunosenescence by decreasing Treg cell number and increasing B cell number. In one embodiment compositions of the invention are for use in treating diseases caused by immunosenescence. In one embodiment compositions of the invention are for use in treating aging- related diseases by delaying and/or preventing immunosenescence.

Furthermore it has been proposed that vaccine adjuvants may overcome immunosenescence [53] Since the compositions of the invention are suitable for use as a vaccine adjuvant compositions of the invention may be useful for preventing or delaying immunosenescence. In another embodiment compositions of the invention are for use in delaying and/or preventing immunosenescence as a vaccine adjuvant. In another embodiment, compositions of the invention are for use as a vaccine adjuvant wherein the compositions delay and/or prevent immunosenescence.

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

Modes of administration

Preferably, the compositions of the inv ention are to be administered to the gastrointestinal tract in order to enable delivery to and / or partial or total colonisation of the i testine with the bacterial strain of the invention. Generally, the compositions of the invention are administered orally ( including sublingual ) but they may be administered rectally or intranasally.

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

In certain embodiments the compositions of the invention may be administered as a suppository such as a rectal suppository for example in the form of a theobroraa oil (cocoa butter) synthetic hard fat (e.g. suppocire. vvitepsol ). glycero-geiatin. polyethylene glycol, or soap glycerin composition. In certain embodiments, the composition of the invention is administered to the gastrointestinal tract via a lube such as a nasogasli ic tube, orogasu ic tube, gaslrie tube, jejuuostomy tube (J lube) percutaneous endoscopic gastrostomy ( PEG) or a port such as a chest wall port that provides access to the stomach jejunum and other suitable access ports

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

In certain embodiments of the invention treatment according to the invention is accompanied by assessment of the patient’s gut microbiota. Treatment may be repeated if delivery of and or partial or total colonisation with the strain of the invention is not achieved such that efficacy is not observed or treatment may be ceased if delivery and / or partial or total colonisation is successful and efficacy is observed

In certain embodiments, the composition of the invention may be ad inistered to a pregnant animal for example a mammal such as a human in order to reduce the likelihood of disease dev eloping in her child in niero and / or after it is bom.

The compositions of die 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 i mune acti\ ity in a healthy patient.

The compositions of the inv ention may be administered to a patient that has been diagnosed with deficient immune activity, or that has been identified as being at risk of deficient immune activity For example the patient may have reduced or absent colonisation by Enterococcus. and in particular

Enterococcus gaUinarum

The compositions of the invention may be administered as a food product such as a nutritional supplement

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

Compositions

Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention the composition is formulated in freeze-dried form. For example the composition of the invention may comprise granules or gelatin capsules for example hard gelatin capsules, comprising a bacterial strain of the invention. Preferably the composition of the invention comprises lyophi!ised bacteria. Lyophil isation of bacteria is a well-established procedure and relevant guidance is available in for example references [56,58]

Alternatively, the composition of the invention may comprise a l ive 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 degradation until delivery at the target location through, for example rupturing with chemical or physical stimuli such as pressure enzymatic activity or physical disintegration, which may be triggered by changes in pH Any appropriate encapsulation method may be used. Exemplary encapsulation techniques include entrapment within a porous matrix, attachment or adsorption on solid carrier surfaces self-aggregation by flocculation or with cross-linking agents, and mechanical containment behind a microporous membrane or a microcapsule. Guidance on encapsulation that may be useful for preparing compositions of the i m ention is avai lable in. for example references [59] and [60]

The composition may be administered orally and may be in the form of a tablet, capsule or powder. Encapsulated products are preferred because Enterococcus are anaerobes. Other ingredients (such as vitamin C for example) may be included as oxygen scavengers and prebiotic substrates to improve the delivery and / or partial or total colonisation and survival in vivo. Alternatively, the probiotic composition of the invention may be administered orally as a food or nutritional product, such as milk or whey based fermented dairy product, or as a pharmaceutical product

The composition may be formulated as a probiotic.

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

A suitable daily dose of the bacteria, for example for an adult human, may be from about 1 x 10 ³ to about 1 x 10 ⁿ colony forming units (CPU); for example, from about l x 10 ⁷ to about 1 x 10 ¹⁰ CFU; in another example from about 1 x 10 ⁶ to about 1 x 10 ¹⁰ CFU; in another example from about 1 x 10 ⁷ to about 1 x 10 ¹¹ CFU; in another example from about 1 x 10 ^s to about 1 x 10 ¹⁰ CFU; in another example from about 1 x l0 ^s to about 1 x 10* ¹ CFU.

In certain embodiments, the dose of the bacteria is at least 10 ⁹ cells per day, such as at least 10 ⁵⁰ , at least Ί0 ¹¹ , or at least 10° cells per day.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1 x 10 ⁶ to about 1 x !0 ^U CFU/g, respect to the weight of the composition; for example, from about 1 x 10 ⁸ to about 1 x 1G ¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and lOg. hi certain embodiments the invention provides the above pharmaceutical composition w herein the amount of the bacterial strain is from about l * 10’ to about 1 ' 10" 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 between 500mg and lOOGmg, between bOOmg and 900mg, between 700mg and 800mg. between 500mg and 750mg or between 750mg and l OOGmg. In certain embodiments, the inv ention prov ides the above pharmaceutical composition, wherein the !yoplii lised bacteria in the pharmaceutical composition is administered at a dose of between 500mg and l OOOmg. between 6()0mg and 900mg. between 700mg and 800mg. between 500mg and 750mg or between 750mg and lOOOmg.

Typically, a probiotic, such as the composition of the invention is optionally combined with at least one suitable prebiotic compound. A prebiotic compound is usually a non-digestible carbohydrate such as an oligo- or polysaccharide, or a sugar alcohol, which is not degraded or absorbed in the upper digestive tract. Known prebiotics include commercial products such as inulin and transgalacto- oligosaecharides.

In certain embodiments, the probiotic composition of the present inv ention includes a prebiotic compound in an amount of from about 1 to about 30% by weight respect to the total weight composition (e.g. from 5 to 20% by weight). Carbohydrates may be selected from the group consisting of: fructo- oligosaccharides (or FOS), short-chain fructo-oligosaccharides, inulin, isomalt- oligosaccharides, pectins, xylo-oligosaccharides (or XOS), chi osan-oligosacchartdes (or COS), beta- glucans, arable gum modified and resistant starches, polydextrose, D-tagatose, acacia fibers, carob, oats, and citrus fibers. In one aspect, the prebiotics are the short-chain fructo-oligosaccharides (for simplicity shown herein below as FOSs-c.c); said FOSs-c.c. are not digestible carbohydrates, generally obtained by the conversion of the beet sugar and including a saccharose molecule to which three glucose molecules are bonded.

The compositions of the invention may comprise pharmaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [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 magnesium stearate, mannitol sorbitol and the like. Examples of suitable diluents include ethanol glycerol and water. The choice of pharmaceutical carrier excipient or di luent 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, com sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleale, sodium stearate, magnesium stearate sodium benzoate, sodium acetate sodium chloride and the like. Preservatives stabilizers dyes and even flavouring agents may be provided in the pharmaceutical composition. Examples of preservatives include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid. Antioxidants and suspending agents may be also used.

The compositions of the invention may be formulated as a food product. For example, a food product may provide nutritional benefit in addition to the therapeutic effect of the invention, such as in a nutritional supplement. Simi larly a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item rather than to a pharmaceutical composition In certain embodiments the composition of the invention is formulated as a milk-based product. The term "milk-based product" means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milk- based product can be, e.g. cow's milk, goafs milk sheep's milk skimmed milk whole milk, milk recombined from powdered milk and whey without any processing, or a processed product, such as yoghurt, curdled milk, curd, sour milk, sour whole milk, butter milk and other sour milk products. Another important group includes milk beverages, such as whey beverages, fermented milks, condensed milks, infant or baby milks; flavoured milks, ice cream; milk-containing food such as sweets.

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

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

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

The compositions of the invention can comprise pharmaceutically acceptable excipients, diluents or carriers.

In certain embodiments the inv ention prov ides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient carrier or di luent; 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 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 inv ention; and a pharmaceutically acceptable excipient, cai rier or diluent; w herein the bacteria! 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.

In certain embodiments the inv ention prov ides pharmaceutical composition comprising: a bacterial strain of the inv ention; and a pharmaceutically acceptable excipient carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prev ent a disease or condition mediated by pro- inflammatory cytokines such as IL- I p. TNF-u. MiP-3a, 1L-23 or iL-6. In a preferred embodiment, the inv ention 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 10 ³ to about 1 x 10" colony forming units per gram with respect to a weight of the composition.

In certain embodiments, the in ention provides the above pharmaceutical composition wherein the composition is administered at a dose of ! 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 abo e pharmaceutical composition comprising a carrier selected from the group consisting of lactose starch glucose methyl cellulose magnesium stearate mannitol and sorbitol.

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

In certain embodiments, the invention provides the abov e pharmaceutical composition comprising an excipient selected from the group consisting of starch gelatin glucose, anhydrous lactose free-flovv lactose beta-lactose corn sweetener acacia tragacanth sodium alginate carboxy methyl cellulose polyethylene glycol sodium oleate. sodium stearate magnesium stearate, 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 abo\ e pharmaceutical composition comprising a pieservulive selected fro the group consisting of sodium benzoate sorbic acid 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 container is placed in an atmosphere having 50% relative humidity, at least 80% of the bacterial strain as measured in colony forming units, remains after a period of at least about: 1 month, 3 months, 6 months. 1 year, 1.5 years, 2 years, 2.5 years or 3 years.

Culturing methods

The bacterial strains for use in the present invention can be cultured using standard microbiology techniques as detailed in. for example references [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), NaHC0 ₃ (0.4 g), cysteine (0.1 g), K ₂ HP0 ₄ (0.045 g), K¾P0 ₄ (0.045 g), NaCl (0.09 g), (NH^SCb (0.09 g), MgS0 ₄ - 7H ₂ 0 (0.009 g), CaCfe (0.009 g), resazurm (0.1 mg), hemin (1 mg), biotin (1 pg), cobalamin (1 pg), -aminobenzoic acid (3 pg), folic acid (5 pg), and pyridoxamine (15 pg).

Bacterial strains for use in vaccine compositions

The inventors have identified that the bacterial strains of the invention are useful for treating or preventing diseases or conditions associated with reduce immune activity. This is likely to be a result of the effect that the bacterial strains of the invention have on the host immune system. Therefore, the compositions of the invention may also be useful for preventing diseases or conditions, when administered as vaccine compositions. In certain such embodiments the bacterial strains of the invention may be killed inactivated or attenuated. In certain such embodiments the compositions may comprise a vaccine adjuvant. In certain embodiments the compositions are for administration via injection such as via subcutaneous injection.

(ienenil

The practice of the present invention wil l employ unless otherwise indicated, conventional methods of chemistry biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See c.g.. references [66] and [67 73] etc.

The term “comprising ^' encompasses ^“ including ^" as well as “consisting ^" c.g. a composition “comprising” X may consist exclusi ely of X or may include something additional e g. X - Y.

The term“about” in relation to a numerical value x is optional and means, for example, JC+10%. The word ^" substantially ^" does not exclude "completely ^" e.g. a composition which is "substantially free" fi otu Y may be completely free fiom Y. Where necessary the word“substantial ly ^" ’ may be omitted front the definition of the inv ention.

References to a percentage sequence identity between two nucleotide sequences means that, when aligned, that percentage of nucleotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [74]. A preferred alignment is determined by the Smith- Waterman homology search algorithm using an affine gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. The Smith- Waterman homology search algorithm is disclosed in ref [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 intervening 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 appreciated that the features specified in each embodiment may be combined with other specified features to provide further embodiments. In particular, embodiments highlighted herein as being suitable, typical or preferred may be combined with each other (except when they are mutually exclusive).

MODES FOR CARRYING OUT THE INVENTION

Example 1 Efficacy of bacterial inocula in mouse models of cancer

Summary

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

Materials

Test substance - Bacterial strain # Rx05 18.

Reference substance - Anti-CTLA-4 antibody (clone: 9H10, catalog: BE0131, isotype: Syrian

Hamster I G l , 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: BE 14- 5 16F Lonza. France).

Treatment doses - Bacteria: 2xl0 ⁸ 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 ga’t age (per o.s. PO) via a cannula. Cannulas were decontaminated eveiy day. Aiili-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 botles) into Hungate tubes

• Seal the tubes and flush with C(¾ 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 (pre-wamied flushed Hungate tubes again, all 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 detailed in the table below:

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

The LL/2 (LLC l ) 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 derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [78]

Cell culture conditions - Al l cell lines were grown as monolayer at 37 ^:' C in a humidified atmosphere (5% C0 ₂ , 95% air). The culture medium and supplement are indicated in the table below:

For experimental use, adherent tumour cells were detached from the culture flask by a 5 minute treatment with trypsin- versene (ref: BE 17- 16 IE, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.

Use of animals -

Healthy female BALB C ( BALB cByJ ) mice of matching weight and age, were obtained from CHARLES RIVER for the EV1T6 and RENCA model experiments.

Healthy female C57BL/6 (C57BL16J ) mice of matching weight and age, were obtained from CHARLES R IVF.R ( i .'Arhresles) for the I .L/2U .LC 1 ) and the Hepa l -6 model experiments

Animals were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals were followed [79.80] Animals were maintained in housing rooms under controlled environmental conditions: Temperature: 22 ± 2 ^C C, Humidity 55 ± 10%, Photoperiod ( ! 2h light/ ! 2h dark), HEPA filtered air, 1 5 air exchanges per hour with no recirculation. Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cnr cages (ref: green, Tecniplast) in ventilated racks, Epicea bedding (SAFE), 10 kGy Irradiated diet (A04-10, SAFE), Complete food for immunocompetent rodents - R/M-H Extrudate, water from water bottles. Experimental design and treatments

Antitumor activit , EMT6 model

Treatment schedule - The start of first dosing was considered as DO. On DO. noil-engrafted mice were randomized according to their individual body weight into groups of 9'8 using Vi\ o manager E software (Biosystemes. Couternon. France) On DO the mice reced ed vehicle (culture medium} or bacterial strain. On D 14. all mice were engrafted with EMT-6 tumor cel ls 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:

The monitoring of animals was performed as described below.

Induction of EMT6 tumours m animals - On D 14, tumours were induced by subcutaneous injection of 1\ 10" F.VIT-6 cells in 200 p i . RPM 1 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, LL/2 (LLC1) model

Treatment schedule - The start of first dosing was considered as DO. On DO, no -engrafted mice were randomized according to their individual body weight into 7 groups of 9/8 using Vivo managers software (Biosystemes, Coutemon, France). On DO, the mice received vehicle (culture medium) or bacterial strain. On D14, all mice were engrafted with LL 2 tumor cells as described below. On D27. mice from the positive control group received anti-CTLA-4 antibody treatments. The treatment schedule is summarized in the table below:

The monitoring of animals was performed as described below.

Induction of LL/2 (LLC1) tumors in animals - On D14, tumors were induced by subcutaneous injection of IxlO ⁶ LL/2 (LLC1) cells in 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, Hepa 1-6 model

Treatment schedule - The start of fust dosing was considered as DO. On DO. lion-engrafted mice were randomized according to their individual body weight into 7 groups of 9 using Vivo manager® software (Biosystemes. Conternon. France). On DO. the mice received vehicle (culture medium) or bacterial strain. On D M. all mice were engrafted with Hepa 1 -6 tumor cells as described below. On D I 6, mice from the positix e control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarize in the table below:

The monitoring of animals was performed as described below.

Orthotopic induction of Hepa 1-6 tumor cells in animals by intrasplenic injection - On DO, one million Hepa 1-6 tumor cells in 50 pL RPMI 1640 medium were transplanted via intra-splenic injection into mice. Briefly a small left subcostal flank incision was made and the spleen was exteriorized. The spleen was exposed on a stei ile gauze pad. and injected under \ isua! control with the cel l suspension with a 27-gauge needle. After the cell inoculation the spleen 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 dosing was considered as DO. On DO, n on-engrafted mice were randomized according to their individual body weight into groups of 12 mice. On DO, the mice received vehicle (culture medium ) or bacterial strain (2x 10 in 100 pL. PO). On D I 4, all mice were engrafted with RENCA tumour cells injected SC into the left hind flank as described below. Treatment with anti- C 1 LA-4 (clone 000 10 mg kg. IP ) and anti-PDL I (clone 10F. G2. 10 mg/kg, IP) was initiated from D17.

The treatment schedule is summarized in the table below:

The monitoring of animals was performed as described below.

On D 14 (following 2 weeks of bacterial dosing'pre-treatment), 5x 10' viable cells in 100 pL of PBS were injected subcutaneously into the left hind flank of each mouse (which was sterilised with surgical spirit). 1 syringe and needle per mouse. The implantation sites were shaved the day prior to cell implantation. 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 thei volume evaluated.

Animal monitoring

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

_ , width

Tumor 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 m : Tumors interfering with ambulation or nutrition: Ulcerated tumour or tissue erosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, emaciation, cachexia, dehydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleeding; Neurologic signs circling com uision, paralysis; Sustained decrease in body temperature Abdominal istension.

Anaesthesia - Tsoflurane gas anesthesia was used for all procedures: surgery or tumour inoculation, i.v. injections blood collection. Ketamine and Xylazine anesthesia was used for stereotaxis surgical procedure.

Analgesia - Carprofen or multimodal carprofen buprenorphine analgesia protocol were adapted to the severity of surgical procedure Non-pharmacological care was provided for all painful procedures. Additionally pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending veterinarian.

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

Results

Antitumor activity, EMT6 model

The results are shown in Figure 1 A. Treatment with the bacterial strain of the invention led to a clear reduction in tumour volume relative to both the negative controls. The positive control also led to a reduction in tumour volume, as would be expected.

To further elucidate the mechanisms through which MRx05 l8 conveys its therapeutic effects in syngeneic tumour models ex vivo analysis was performed on the syngeneic EMT6 tumour model studies. While tumour volume is the primary measurement in preclinical oncology studies, tumours often consist of actively dividing tumour cells along with a necrotic core. To investigate whether MR.\05 I S treatment had influence on the degree of necrosis found within EMT6 tumours, paraffin sections from the mid-belly region of the tumours were stained with Haematoxylin and Eosin. MRx0 1 S treatment of a murine EMT6 breast carcinoma model showed a tendency towards increasing the cross-sectional area of necrosis within the tumour (Figure 1 B. upper panel) To investigate whether MRx05 1 8 treatment had influence on dividing cells within the tumour, paraffin sections from the mid belly region of the tumours were stained with the proliferation protein Ki67, along with DAPI counter stain, to estimate the percentage of cells dividing within the EMT6 tumour. MRx05 18 treatment of a murine EMT6 breast carcinoma model significantly decreased the percentage of dividing cells seen within the tumour (Figure 1B, lower panel, P=0.019). immune ceil populations

Further investigation of the tumour microenvironment was performed through flow cytometry of the tumour, to investigate the hypothesis that the MRx05 i 8 bacteria! strain has the ability to regulate the immune system into inducing an anti-tumour effect. Tumours excised from the different treatment groups were cut into pieces. One piece was subjected to flow cytometry analysis. To assess the relative percentage of T lymphocytes, present within the tumours, the following markers were used: CD45, CD3, CD4, CDS, CD25 and FoxP3.

The preliminary flow cytometry data presented in Figure 1 C (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 MR\05 ! S and ami-OTL.A-4 treated groups when compared respectively to vehicle or control animals. Fikewise. the relative percentage of CD4 cells appeared to be decreased in MRx0518 and anti-CTLA-4 treated animals whilst the relativ e percentage of CDS 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 \1Rx05 I S treated animals; howev er the reduction in the relativ e percentage of CD4 CD25 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 M Rx0518 animals. These data presented here supports the hypothesis that anti-CTLA-4 antibody targets regulatory T cells (Tregs) by reducing their cell numbers or attenuating their suppressive activity in tumour tissue whilst suggesting a different mode of action for MRx05 18.

Additional investigation of the tumour microenvironment was performed using NanoString analysis of the tumour tissues, to investigate whether the MRx05 I 8 bacterial strain has the ability to regulate the immune system into inducing an anti-tumour effect in the FMT6 model. Tumours excised from vehicle and MRx0518-treated groups were collected. RNA was extracted from tumour tissue using TRIzol reagent (ThermoFisher) followed by a clean-up using the RNeasy Mini kit (Qiagen) including a DNase I treatment (Qiagen). RNA was then used for Nanostring analysis using the Pan Cancer Mouse 10 360 panel. Genes pre\ iously shown to be characteristic of various cel! populations were used to measure these populations' abundance:

Z-scores for each cell population were calculated using the linear cell type scores provided by the NanoString analysis ( Figure 23. heat 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 MRx05 18-treated group when compared to vehicle-treated animals (Figure 23). The data presented here supports the hypothesis that MRx0518 has an irmnunostimulatory 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 subsequent cytokine analysis together with plasma samples. Protein le els of !L- 10. CXCL I CXCL2, CXCL 10. IL- l B IL- I 7A. GV1-CSF. TNF-a, !L- 12p70 and IFN-y in the tumour microem ironment were analysed by MagPi technology. While I L- 17A and GM-CSF were below lex els of detection all the other markers were expressed at reasonable lev els ( Figure I D). A significance difference was observed between the v ehicle and anti-CTLA-4 group for IFN-y. The production of the IL-10 and IL-12p70 immune markers seemed reduced following MRx0518 treatment compared to the control treatments.

Cytokine levels w ere also assessed in blood plasma of the same animals Protein levels of 1L-23. iL-b. IL- 10. VEGF. CXCL I , CXCL2. CXCL ! 0, 1L-2. IL- I b. 1L- 1 7A, GM-CSF, TNF-a. IL- 1 2p70 and IFN- y were analysed by MagPix technology. Ov erall little cytokine production was detected in the blood plasma of animals either before tumour induction or at the end of the study (Figure I E ). VEGF and CXCL 10 were detected at substantia! levels whi le IL-23. IL-6 IL- 10 CXCL I and CXCL2 were detected at low lev els. IL-2. iL- l b. IL- I 7A. GM-CSF. TNF-a. IL- ! 2p70 and IFN-y were not detected in the samples. MRx05 1 S significantly increased production of 1 L-6 at Day 0. M Rx0518 also seemed to increase 1 L-23 production. VEGF and CXCL 10 were significantly down regulated in tlie anti-CLTA- 4 group at Day 22. Similarly to the results shown for the immune cell populations, the differences in cytokine production in the tumour and plasma, between MRx05 1 8 and ant-CTLA-4 suggests that each of them acts on a distinct and potentially complementary mechanism.

Localisation of CD8a Positive Cells in the Ileum

10 pm cryo-sections of ileum were cut in cryostat (CM 1950 Leica), picked up onto poly-L Lysine slides The sections were then air-dried for 1 hour, fixed for 10 minutes in ice-cold methanol, washed in PBS. blocked in 10% BSA in PBS pH 7.2 before being incubated overnight with the primary antibody (rat-anti-mouse-CDSa antibody, Sigma- Aldrich, Millipore).

The next morning the slides were washed in PBS and stained with a secondary antibody: goat-anti-rat- antibody-Aiexa488 (Molecular Probe lnvitrogen) for I hour at room temperature. After another washing step, the slides were counterstained with 4 ^’ .6-diamidino-2-phenyIindo!e dihydrochloride (DAP1) (Sigma- Aldrich. Mi llipore) and mounted in Vectashield (Vector Laboratories). The slides were viewed and imaged using a Zeiss Axioscope M icroscope equipped with a mercury vapour lamp, appropriate filters and a x20 apochromatic objective. Examples of images obtained from slides from the vehicle, MRx0518, and anti-CTLA4 animals are shown (Figure IF - upper panels: DAP! staining, lower panels: CD8« staining).

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

The images were scored as follow: 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 1G).

Ileum cryosections stained with anti-CD8a showed a higher number of CD8a positive cells localized in the crypt region tissues from animals treated with MRx0518 and anti-CTLA-4 compared to the vehicle group.

This observation is in line with CDS T cells being present in the intestine in case of infection or inflammatory microenvironment, as part of the immune response. Antitumor activity, LL/2 (LLC1 ) model

The results are shown in Figure 2. Treatment with the bacterial strain of the invention led to a clear reduction in tumour volume relative to both the negative controls.

Antitumor activity, Hepal-6 model

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

Antitumor activity, RENCA model

The results are shown in Figure 3B. Treatment with MRx05 1 8 monotherapy reduced tumour volume with Test/Control of 51 % (day 1 8) compared with the vehicle-treated groups. Paclitaxel and anti- CTLA-4 4 anti-PDL- 1 showed an (almost) complete reduction in tumour size at D I B and D22 compared to both the untreated and vehicle groups.

These data indicate that strain MRx05 l8 may be useful for treating or preventing other diseases associated with reduced immune system activity.

Example 2 - PCR gene analysis

A pure culture of bacteria MRx05l8 was studied in a PCR gene analysis. There were two arms to the experiment: 1) MRx05 l8 was co-cultured with human colonic cells (CaCo2) to investigate the effects of the bacteria on the host, and 2) MRx05 1 8 was co-cultured on CaCo2 cells that were stimulated with IL1 to mimic the effect of the bacteria in an inflammatory environment. The effects in both scenarios were evaluated through gene expression analysis. The results are shown below:

These data appear to show two gene expression signatures - CXCR 1 '2 ligands (CXCL3. CXCL2. CXCL I IL-8), which is associated with pro-inflammatory cell migration, and CXCR3 ligands (CXCL9, CXCL I O) which is more specifically indicative of IFN-y-type responses also supported by 1L-32, which is IFN-y-indueible. 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 atmosphere having 30%. 40%, 50% 60%, 70%. 75%. 80%. 90% or 95% relativ e humidity. After l month. 2 months. 3 months. 6 months, 1 year 1 .5 years 2 years. 2.5 years or 3 years, at least 50%. 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols

Example 4 cytokine production in immature dendritic cells induced by MRx0518 compared to

MRxOSIS + EPS

Summary

This study tested the effect of the bacterial strain MRx0518 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 MRx05 I S at a final concentration of 10 ⁷ /mL, with the optional addition of LPS at a final concentration of 1 OOng 'mL. The negative control involved incubating the cells with RPM1 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 M Rx0518 alone leads to a substantial increase in the level of cytokines IL-6 and TNF-a compared to the negative control (Figure 4a and c). The addition of LPS (positive control ! leads to an increase in the level of TL-6 and TNF-a compared to the negativ e control but not 1L- ! b ( Figure 4b). A combination of MRx051 S and LPS led to a synergistic increase in the lev el of l L- 1 b produced (Figure 4d).

Conclusion

MRx0 18 has the ability to induce higher 1L.-6 and TNF-a cytokine production in immature dendritic cells. The combination LPS and MRx05 18 can increase the lev els of cytokines IL- I in immature dendritic cells. These data indicate that MRx0518 alone or in combination with LPS can increase inflammatory cytokines IL-lp, IL-6 and TNF-a, which promotes inflammation.

Example 5 - cytokine production in THP-l cells induced byMMxdSlS compared to MRx0518 + LPS

Summary

This study tested the effect of bacterial strain MRx05 I 8 alone and in combination with LPS on cytokine production in THP-l cells, a model cell line for monocytes and macrophages.

THF- I cells were differentiated into 0 medium for 48h with 5ng/mL phorboi- 12-mynstate- 13- acetate ( PMA ) These cells were subsequently incubated with MRx0 18 at a final concentration of i O ml . with or without the addition of LPS at a final concentration of l OOng/mL. The bacteria were then washed off and the cells allowed to incubate under normal growing conditions for 24 h. The cells were then spun down and the resulting supernatant was analysed for cytokine content.

R sults

The results of these experiments can be seen in Figures 5a-c. The addition of MRx0518 without LPS leads to an increase in the cytokine lev els of IL- 1 [L IL-6 and TNF-a compared to the no bacterial and the bacterial sediment controls. The addition of LPS and MR.\05 ! 8 leads to a synergistic increase in the production of cytokines.

Conclusion

MRx05!8 has the ability to induce cytokine production in THP- l cells which can be synergistically increased with the addition of LPS. These data indicate that M x0 18 alone or in combination with LPS can increase inflammatory cytokines 1L- 1 b. IL-6 and TNF-a. which promotes inflammation. Example 6 Cytokine analysis

Introduction

The inventors sought to further analyse the immimostimulatory 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 MRx05 1 8 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 nate immune response ( TNF-CL IL- 12 and lL- 10) were analysed and also cytokines involved in the recruitment and activation of adaptive immune cells (IL-8, IL-23, IL- I b and

IL-6).

Method

Bacterial strains

MRx0518

LPS used as positive control

Results

The results are shown in Figures 6- 13. MRx05 I 8 induces a strong and characteristic immuno- stimulatory profile in THP- 1 -derived macrophages and DCs derived from monocytes. Cytokines involved in the innate immune response (TNF-a, IL- 12 and !L- 10) are significantly induced by Rx05 18 in both DCs and macrophages. MRx05 18 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. MRx05 18 also induced IL- I b.

Discussion

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

Example 7 mechanism of action

Further experiments were performed to characterise the mechanism of action by which M R\0 18 stimulates the immune system. A TLR5 signalling reporter assay was selected and the data are presented in Figure 14 and 15. MRx05 18 supernatant was the most potent activator of TLR5 and NF- KB Also the supernatant was treated with various lytic enzymes and trypsin was found to abrogate the majority of activity .

Summary

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

Materials

Test and reference substances - Bacterial strain 4 MRx05 1 S; Anti-CTLA4 antibody {ref: BEO 13 1 . Bioxcell; clone: 9H 10: reactivity: mouse; isotype: Hamster IgG I ; storage conditions: -4T).

Test and reference substances vehicles - The Rx0 I S bacteria were grown in a bacterial culture medium (Yeast extract, Casitone. Fatty Acid medium (YCFA )) and kept as a glycerol stock at -80°C. The animals were dosed with the bacteria according to the study protocol. The anti-CTLA-4 antibodies were diluted with PBS (ref: BE 14-516F. Lonza. France) on each day of injection to mice.

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

Routes of administration - The bacterial 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 (IntraperitoneaUy, IP) at a volume of lOmL/kg adjusted to the most recent individual 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 ^U C in a humidified atmosphere ( 5% C02. 95% air) The culture medium was RPM1 1640 containing 2 mM L-glutamine (ref: BE 12- 702F. Lonza, Verviers. Belgium) supplemented with 10% fetal bovine serum (ref 3302 Lonza). EMT-6 tumor cells are adherent to plastic flasks. For experimental use. tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE02- 007E. Lonza). in Hanks’ medium w ithout calcium or magnesium (ref BE I 0-543F, Lonza) and neutralized by addition of complete culture medium. The cel ls were counted and their viability was assessed by 0.25% trypan blue exclusion assay. Use of animals - Healthy female BALB/C (BALB cByj ) mice. 5-7 w eeks old were obtained from CHARLES RIVER ( L'Arbresles) and maintained in SPF health status according to the FELASA guidelines. Animal housing and experimental procedures were realized according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. Animals were maintained 3-4 per cage in housing rooms under controlled environmental conditions: Temperature: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod ( ! 2h light/ 12h dark ). HEPA filtered air 1 air exchanges per hour with no recirculation. Animal enclosures were provided with sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type 111 or IV cages. Corn cob bedding (ref: LAB COB 12.

SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunocompetent rodents - R/M-H Extrudate, Sterile, filtrated at 0.2 pm water and Environmental enrichment ( SIZZLE-dri kraft - D20004 SERLAB, France). Animals are individually identified with RFID transponder and each cage was ladled with a specific code. Treatment of the animals started after one week of acclimation for batches 2 and 3, or after three weeks of acclimation for batch 1.

Experimental design and treatments

On day -14 (D-14), non-engrafted mice were randomized according to their individual body weight into 3 groups of 30 animals and 2 groups of 10 animals using Vivo Manager® software

(Biosystemes, Coutemon, France). The mice were separated into 3 batches of 10 animals per treatment group (batch 1 : 10 animals of groups 1, 2 and 3; batch 2: 10 animals of groups 1, 2 and 3 and batch 3: 10 animals of groups 1 to 5) with different termination points from the start of the study: D-14 or DO.

At termination batch 3 was split into 2 cohorts due to termination and FACS analyses schedules; these were staggered over I day: D24'D25. Therefore, every cohort of animals had 5 animals per treatment group (4 animals from cage one and one animal from cage 2). Based on the ethical criteria, if the tumor volume were higher than 1500mm ³ , the selection of the animals to be sacrifice on D24 and D25 is based on tumor volume instead of the cage. The experimental design is depicted in Fig.

16A and summarized below:

1 ) Batch 1 (groups 1 , 2 and 3) started treatment on DO and was culled at D14 (10 animals form groups 1 to 3). These did not receive tumor cells and constituted the baseline group.

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

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

On day 0 (DO) all mice of batches 2 and 3 (termination at day 7 and 24/25, respectively) were engrafted with EMT-6 tumour cells by a subcutaneous injection of l l 0^ EMT-6 cells in 200 pL RPM1 1640 into the right flank (the 30 mice from batch I that were sacrificed on D 14 did not receive the tumour injection ) The mice were treated according to the foliowing treatment schedule groups (TWx2 = twice a week):

A nitnai moniiorimi

The viability and behaviour of the animals was recorded every day. Body weights were measured twice a week The length and width of the tumour was measured twice a week with callipers and the volume of the tumour was estimated by the following formula:

The treatment efficacy was assessed in terms of the effects of the test substance on the tumour volumes of treated animals relative to control animals. The following evaluation criteria of antitumor efficacy

d)

were determined using Vivo Manager software (Biosystemes, Coutemon, FranceJ.Mean tumour volumes of groups 1 to 5 are depicted in Fig. l bB. T hroughout the course of the study a progression in tumour growth was observed in all groups with the exception of the MRx05 1 8 + Anti-CTLA-4- treated group where a regression of tumour growth occurred from Day 14 post tumour induction MRx05 l 8 -+ Anti-CTLA-4 treatment significantly reduced tumour growth compared to the Vehicle- treated group on Day 21 and Day 24 post tumour induction. The combination treatment of MRx0518 with Anti-CTLA-4 was the most efficacious for reducing tumour growth in BALB/c mice bearing subcutaneously grafted EMT6 tumours. These data demonstrate that MRx0518 has an immunostimu latory effec t

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

Summary

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

Materials

Test substance - Bacterial strain ?MRx()5 4.

Reference substance - Anti-CTLA-4 antibody (clone: 9H 10 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- 5 I 6F Lonza, France)

Treatment doses - Bacteria: 2x10 ^s in 200 pL YCFA. The anti-CTL.4-4 was injected at 10 mg kg inj Anti-CTLA-4 w as 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 ora! gavage (per os. PO) via a cannula. Cannulas were decontaminated ev ery 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 CO; using a syringe input and exhaust system

• Autoclave the Hungate tubes

• When cooled, inoculate the FI ungate 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 I mL of this subculture and inoculate 10 mL of YCFA (pre-wamted flushed Hungate tubes again all 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 detailed in the table below:

The EMT-6 cell line was established from a transplantable murine mamma ry carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule [S3].

Cell culture conditions - All cell lines were grown as monolayer at 37°C in a humidified atmosphere (5% CO:. 95% air). The culture medium and supplement are indicated in the table below:

For experimental use. adherent tumor cells were detached from the culture flask by a 5 minute treatment w ith trypsin-versene (ref: BE I 7- 161 E, Lonza). in Hanks' medium without calcium or magnesium (ref: BE I 0-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hetnocy to meter and their viability will be assessed by 0.25% trypan blue exclusion assay.

Use of animals -

Healthy female BALB/C (BALB/cByJ ) mice of matching weight and age. w ere obtained from CF1ARLES RIVER (L'Arbresles) for the EMT6 model experiments.

Animals were maintained in SPF health status according to the FELASA guidelines, and animal housing and experimental procedures according to the French and European Regulations and N RC Guide for the Care and Use of Laboratory Animals were followed [84.85] Animals were maintained in housing rooms under controlled environmental conditions: Temperature: 22 i 2°C. Humidity 55 - 10%, Photoperiod ( I 2h light/ 12h dark). HEPA filtered air. 1 5 air exchanges per hour with no recirculation. Animal enclosures w ere provided with sterile and adequate space wdth bedding material, food and water, environmental and social enrichment (group housing) as described: 900 cm ² cages (ref: green Tecniplast) in ventilated racks, Epicea beddin (SAFE) 10 kGy irradiated diet (A04-10, SAFE). Complete food for i uno-competent rodents - R M-H Extrudate. water from water bottles.

Experimental design and treatments

Antitumor activity, EMT6 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 groups of 8-9 using Vivo manager® software ( Biosystemes Coutemon. France) On DO. the mice received vehicle (culture medium) or bacterial strain. On D 14. all mice wete 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:

1 he monitoring of animals was performed as described below.

induction of EMT6 tumours in animals - On D I4. tumors were induced by subcutaneous injection of 1 x 10 ^fr EV1T-6 cells in 200 pL RPYJ i 1640 into the right flank of mice.

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

Animal monitoring

Clinical monitoring - The length and width of the tumor was measured twice a week ith callipers and the volume of the tumor was estimated by this formula [86]:

_^ , width ² x length

Tumor volume = - ² —

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 m ; Tumors interfering with ambulation or nutrition; Ulcerated tumor or tissue erosion; 20% body weight loss remaining for 3 consecutive days; Poor body condition, emaciation, cachexia, dehydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia significant bleeding; Neurologic signs: circling, convulsion, paralysis; Sustained decrease in body temperature; Abdominal distension.

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

Analgesia - Carprofen or multimodal carprofen/buprenorphine analgesia protocol were adapted to the severity of surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally pharmacological care not interfering with studies (topic treatment) were provided at the recommendation of the attending v eterinarian.

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

Results

Antitumor activity, EMT6 model

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

These data indicate that strain Rx0554 may be useful for treating or preventing other diseases associated with reduced immune system activity.

Example 10 - Analysis of carbohydrate metabolism - API 50 CHE analysis of MRxO 554

The Analyt ical Profile Index (API ) test system consists of strips which contain miniaturised biochemical tests which assay for enzymatic activity in bacterial species. These tests are routinely used in the characterisation of novel strains. API 50 CH L testing was carried out to examine carbohydrate metabolism in MRx0554. As per manufacturer’s instructions, bacteria were cultured in 10 mL YCFA broth for 16-18 hours at 37 °C in an anaerobic workstation. This culture was diluted in 10 ml, API CHL Medium so as to achieve a density roughly equivalent to McFarland standard No. 2, and 1 10 til 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 anaerobic workstation for 48 hours following which tiie colour of each cupule was recorded and assigned a value of negative, intermediate positive, positive or doubtful.

Using API 50 CHL analysis, MRx0554 tested positive for fermentation of L-arabinose, D-ribose, D-xylose, D- alactose, D-glucose, D- fructose, D-mannose, N-acetylglucosamine, amygdalin, arbutin, esculin salicin. D-ce!lobiose, D-maltose, D-saccharose (sucrose), D-trehalose, gentiobiose D-tagatose, and potassium gluconate (Figure 18). Intermediate reactions were observed for D- mannitol, methyl a-D-glucopyranoside, D-lactose, D-raffinose, amidon (starch), and D-turanose. Example II - TLR9 activation

To further elucidate the mechanism by which MRx05l8 stimulates the immune system, HEK-Blue™ human TLR9 reporter cells (InvivoGen, San Diego, CA, USA) were used to test the effect of MRx0518 on TLR9 activation

Maintenance of cell lines and bacterial strains

HEK-RIue ^, human I LR reporter cells ( InvivoGen. San Diego CA. USA ) were grown in DM EM supplemented with 10 % (v/v) foetal bovine serum ( FBS). 4 mM L-glmamine. 4 5 mg/mL glucose. 100 U-mL penicillin 100 pg'rnL streptomycin 100 pg- rnL Normocin™ (InvivoGen). 10 pg inL b!astocidin (InvivoGen ) and 100 pg/mL zeocin ( InvivoGen) to 90 Co density. Cells were maintained at 37 ^~ C and 5 % C02. 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 inL. All reagents w ere supplied by Sigma Aldrich unless otherwise stated. /·.. gailiminim MRx05 18 was routinely cultured in Yeast extract. Casitone. Fatty Acid media (YCFA, E&O Laboratories. Bonnybrtdge. Scotland. UK) at 37 °C in an anaerobic cabinet (Don Whitley Scientific. Shipley, England, UK).

TL.R9 Reporter Assays

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

1. Live fraction of MRx0518 (MRx0518LV) - Late log phase bacterial cultures were centrifuged at 5,000 x g for 5 min at room temperature to generate bacterial fractions. Pelleted bacteria were washed once in PBS and re-suspended in antibiotic-free cell culture media to the appropriate dilution.

2. MRx05 I S supernatant fraction (MRx0 I SSN ) - Culture supernatants were harvested and filtered through a 0.22 pm pore size filter and diluted in water.

3. 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 MRx05 f 8HK were used at a multiplicity of infection (MOI) of 100: 1. A 100: 1 MOI equivalent volume was used for MRx0518SN. The synthetic CpG oligonucleotide ODN2006 (InvivoGen) was used as an assay positive control at a concentration of 5 mM. YCFA was used as a negative control. Viable cells counts were determined by plating. HEK-B!ue™ human TLR9 reporter cells were incubated w ith the above treatments for 22 hours at 37 ^' C in a 5 ° o C02 atmosphere Assays were dev eloped using QUANTI-Biue™ (4n\ iv oGen) 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 supernatant fractions were able to activate TLR9.

Example 12 - T cell differentiation

The ability of MRx05I8 to induce T-cell differentiation was explored in vitro on peripheral blood mononuclear cells (PBMCs, Stemcell, Cai:70025). Briefly, PBMC ^' s were plated in 96-well plates plated with anti~CD3 (Ebioscience, anti-CD3 monoclonal antibody (OKT3 clone) functional grade, cat. No. 16-0037-81 ) at 400,000/well in 50m1 cRPMl medium per well (cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2 M final cone. Stock 200tnM.; 10% H I FBS (Gihco life technologies, 10082-147); 50mih mercaptoethanol (Gibco life technologies, 21985-023); and 1 % pen/strep (P4333, lOmg/mL). Heat-killed MRx05 18 (prepared by incubation at 80 °C for 30 minutes after which the cultures were w ashed with PBS and resuspended in appropriate cell culture medium and viable counts were confirmed by plating) was then added to each well, 4,000,000 in 100 mΐ/well. Following 3 days in a 37° C incubator, the cells were remov ed and re-suspended in a medium containing PM A- (Sigma Cat no P8139). ionomycin (Sigma. Cat no 13909) and GolgiSTOP (BD. Cat no 554724) for 5 hours. PM A stock was I mg/mL in DMSO which was further diluted in l OOug/mL (each sample required 50ng/mL in cRPMl) Ionomycin stock was I mM in DMSO ( I mM in cRPM I was used) and GoIgiStop concentration was used at 4pl/6mL. Supernatants were passed through a 0.22 pm filter and diluted appropriately in co-culture medium.

The cells were then subjected to a flow cytometry staining:

After washing, the cells were incubated with viability dye (Viobility 405/520 Fixable Dye from Miltenyi biotec lpl/sample) + human Fc block, cat. 564219 (Imΐ/sample) in PBS for 10 mins in the dark at room temperature. The surface antibodies (2m1 of each) were then added directly to the wells for 10 mins in the dark at room temperature - D3-APC-Vio 770 (Miltenyi, cat. No. 130-113-136), CD4-VioBlue (Miltenyi, cat. No. 130- 1 14-534 ) and CD25-VioBright FITC {Miltenyi. cat No. 130- 1 1 -283) The ceils were then washed twice in PBS and spun down at 300g miivRT.

The eBioscience FoxP3 transcription factor staining buffer was then used to fix and permeabilise the cells (cat. No 00-5523 ) Follow ing the eBioscience protocol a perm/fix buffer was prepared using 1 part of concentrate solution and 3 parts of diluent The cells were fixed for l b at RT and then washed 2x in l x Perm wash and spun down at 300g '5min RT. The following intracellular staining or transcription factor antibodies w ere added to the samples in perm wash ( ! x) for 45min in the dark at room temperature or in the fridge overnight ( up to I 8h). followed by washing die antibodies 2x using Perm wash ( 300m1 ) and re-suspension in PBS (250mI ) to acquire on the cytometer:

• Anti ! FNY-PE Vio770 human antibodies ( Mi ltenvi, cat. No. 130- 1 14-025)

• Anti IL 10-PE human antibodies ( Miltenyi. cat. No. 130- i 12-728)

• Anti lL I 7a-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 supernatant of MRx05 I 8 (SP 518 ) and heat-killed MRx05 18 ( H K 5 18) were able to induce differentiation of T helper cells and cytotoxic T cells respectively even in the absence of cytokines to induce differentiation (no cyto)

Example 13 - MRxOSIS induced cytokine signature

Splenocytes were isolated from C57BL 6 mice and plated in 96 well plates at a density of 900.000 cells/well in RPMI 1640 supplemented with 10% FBS. 2mM L-glutamine. 100 U niL Pen Strep (Sigma- Aldrich) and 55 mM [3-mercaptoethanol (Gibcof Cells were treated with different concentrations of blank media ( YCFA ) or bacteria supernatant from stationary phase for 72hrs. Cell fee supernatants were collected after each time point and stored at -S0 'C for cytokine analysis. Cytokines were measured using multiplex procarta pi ex MO Th i 'Th2/Th9.Th 17/Th22/T reg I 7plex kit (Invitrogen). Cell proliferation of untreated splenocytes or splenocytes treated by 10% YCFA medium or 10% MRx.0518 bacteria supernatant was measured using MTT assay (Millipore). as depicted in Fig. 21F.

Lit e growing MRx051 S bacteria were incubated for up to 2h with the human intestinal epithel ial 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 further 24h incubation (THP- I ). Frozen healthy human PBMCs were purchased from Stem Cells Technologies (Cambridge UK). The cells were thawed and left lo l est overnight in full growth media ( RPM1 1640 wit!i 10% FBS. 55 mM b-mercaptoethanol. 2m M L. Glutamine and 100 ' ml penicillin l OOug/mL streptomycin) in CO: 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 supernatants in the presence or absence of I ng/mL LPS. Cell culture media was added to untreated wells. Cells were left to rest for 72 h, thereafter cell free supernatants were collected and spun down for 3 minutes at 10,000g 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 mΐ of cell-free co-culture supernatants were used for cytokine quantification using a MAGPIX® MILLIPLEX® system (Merck) with the xPOXENT software (Luminex. Austin. TX, USA). Data was analysed using the ILLI PLEX ® analyst software ( Merck ) using a 5-parameter logistic curve and background subtraction to convert mean fluorescence intensity to pg mL values.

Data are expressed in Figs. 2IA-D as an average of tw o technical replicates of 10 biological replicates ( PBMC) or three biological replicates (splenocytes) and show production of cytokines in ( A) PBMCs: ( B ) Splenocytes: (C) TH P- 1 cells; and ( D) Caco-2 cells fol lowing treatment with YCFA blank media { ^‘ Vehicle ^” ) or MRx05 ! 8 bacteria MRx05 I S cell-free bacterial supernatant (“MRx0 1 8"). Fig. 21 E depicts additional data relating to cytokine secretion from splenocytes ( N=3 ). from cells that were either untreated (“ Untreated") treated ith YC FA blank media (“10% YCFA ^” ) or treated with MRx0518 cell- free bacterial supernatant (“10% MRx0518”).

As can be seen in Figs. 21A-D, treatment of different cells with a supernatant of MRx0518 bacteria resulted in immunostimuiation as e\ ident by an increase in cytokine production.

Example 14— NF-kK activation

The activation of the NF- B promoter was tested in 11EK293 cells co-expressing an N F-kB inducible secreted embryonic alkaline phosphatase (SEAP) reporter gene with either the human NOD2 gene, TLR4, TLR9 or TLR5 genes ( HEK-Blue™-hNOD2, HEK-BIue™-hTLR5, HEK-Blue™-hTLR9 and HEK-Blue™-hTLR4 cells, respectively, by InvivoGen San Diego. CA, USA).

Briefly, HEK-TLR4 cells were maintained in DMEM 4.5 /L D-g!ucose supplemented with 10% (v/v) heat- inactivated FBS, 4mM L-Glutamine. l OOU/ml penicillin, 100 pg/ml streptomycin 100 pg/ml normocin, lx HEK-Blue selection media: for HEK-TLR5 and HEK-TLR9 same 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 100pg/ml zeocin media for both cell lines into the culture. For die experiment cells were waslied with PBS. dissociated in PBS and collected in growth media. Cells were plated in 96-wel l plates at a density of 25.000 cells· well for H EK-TLR4 and FI EK-TLR5. 80,000 cells/well for HEK-TLR9 and 50,000 cells/well for HEK-NOD2.

To evaluate the responsiveness of the cells to their ligands, the cells were treated with 1 ng/ml EPS (HEK-TLR4), 1 ng/mΐ ultra-pure flagellin from Salmonella typhimurium (HEK-TLR5), 1 mM

ODN2006 CPG (HEK-TLR9 positive control ) or I mM ODN2006 GPC ( HEK-TLR9 negative control ), 1 ng/ml of LI8-MDP and incubated in a CO incubator at 37 C. Treatments proceeded for 22h at 37°C and 5% COv after which the detection of Secreted embryonic alkaline phosphatase (SEAP) activity from cell culture supernatant was performed using QUANTI-b!ue solution according to manufacturer ^' s instructions. Briefly. 20 mΐ of cell culture media was collected and analysed for the presence of SEAP by mixing with 200 mI of QUANTl-Blue detection media. After 2h (F1 EK-TLR4 and HEK-TLR5) or 4h (HEK-TLR9 and HEK-NOD2) incubation at 37°C. optical density was measured at 655nm on a microplate reader (iMark microplate, Bio-Rad).

As can be seen in Figs. 22A-D (showing results from averaged technical replicates for three independent experiments). NF-kB promoter activation was measured in cells which were either untreated ( ^‘ Untreated ^” ) treated with YCFA+ medium (“YCFA ^” ) or treated with MRx051 S ( ^“ VI Rx0518 ^” ). The follow ing positive controls ( 1 ng) were used - L 18-M DP ( for H EK-B!ue™-hNOD2 cells. Fig 22A), Lipopolysaccharide, EPS (for HEK-Blue™-hTLR4, Fig. 22B), CPG or negC (for FlEK-Blue™-hTLR9. Fig. 22C) or recombinant flagellin from S. !yphiimtmmi . FLA ( for HEK-BIue™- hTLR5, Fig. 22D). The cells were incubated w ith the v arious treatment at 37 ^" C in a 5% CO _] atmosphere for 22 h To measure NF-kB promoter activ ation (N ^~ 3). QUANTI-Biue™ (Im iv oGen) was mixed with cell supernatants the plates were incubated for 2h and optical density was measured at 655 nm.

Sequences

SEQ ID NO: I (Enterococcus gallinarum 16S rRNA gene - AF039900)

1 taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag 61 agtggcgaac gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg

121 aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgc t

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 tcatcecttg acggtatcta accagaaagc cacggctaac

481 taegtgccag cagccgcggt aatacgtagg tggcaagcgt tgtccggatt tattgggcgt

541 aaagcgagcg caggcggttt cttaagtctg atgtgaaagc ccccggctca accggggagg

601 gtcattggaa actgggagac ttgagtgcag aagaggagag tggaattcca tgrgtagcgg 661 tgaaatgcgt agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact

721 gacgctgagg ctcgaaagcg tggggagcga acaggattag ataccctggt agtccacgcc

731 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 gtgaeaggag

1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca

1031 acccttattg ttagttgcca tcatttagtt gggcactcta gcgagactgc cggtgacaaa

1141 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgaectgg gctzacacacg

1201 tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg ctaagctaat ctctraaagc

1261 ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat

1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg CCCQ ^' CdCSC 1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccg craag

1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg

1501 atcacc

SEQ ID NO:2 (consensus ! 6S rRNA sequence for Enterococcus gallinarum strain MRx05 18)

TGCTATACATGCAGTCGAACGCTTTTTCTTTCACCGGAGCTTGCTCCACCGAAAGAA AAAGAGTSGCGAACGGGTGA

GTAACACGTGGGTAACCTGCCCATCAGAAGGGGATAACACTTGGAAACAGGTGCTAA TACCGTATAACACTATTTTC

CGCATGGAAGAAAGTTGAAAGGCGCTTTTGCGTCACTGATGGATGGACCCGCGGTGC ATTAGCTAGTTGGTGAGGTA

ACGGCTCACCAAGGCCACGATGCATAGCCGACCTGAGAGGGTGATCGGCCACACTGG GACTGAGACACGGCCCAGAC

TCCTACGGGAGGCAGCAGTAGGGAATCTTCGGCAATGGACGAAAGTCTGACCGAGCA ACGCCGCGTGAGTGAAGAAG

GTTTTCGGATCGTAAAACTCTGTTGTTAGAGAAGAACAAGGATGAGAGTAGAACGTT CATCCCTTGACGGTATCTAA

CCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGC GTTGTCGGGATTTATTGGGC

GTAAAGCGAGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGG GGAGGGTCATTGGAAACTGG

GAGACTTGAGTGCAGAAGAGGAGAGTGGAATTCCATGTGTAGCGGTGAAATGCGTAG ATATATGGAGGAACACCAGT

GGCGAAGGCGGCTCTCTGGTCTGTAACTGACGCTGAGGCTCGAAAGCGTGGGGAGCG AACAGGATTAGATACCCTGG

TAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTGGAGGGTTTCCGCCCTTCAGTGC TGCAGCAAACGCATTAAGCA

CTCCGCCTGGGGAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGACGGGGGCCCG CACAAGCGGTGGAGCATGTG

GTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTTTGACCACTC TAGAGATAGAGCTTCCCCTT

CGGGGGCAAAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGC

GCAACCCTTATTGTTAGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGT GACAAACCGGAGGAAGGTGG

GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGG GAAGTACAACGAGTTGCGAA

GTCGCGAGGCTAAGCTAATCTCTTAAAGCTTCTCTCAGTTCGGATTGTAGGCTGCAA CTCGCCTACATGAAGCCGGA

ATCGCTAGTAATCGCGGATCAGCACGCCGCGGTGAATACGTTCCCGGGCCTTGTACA CACCGCCCGTCACACCACGA

GAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTTGGAGCCAGCCGCCTAAGGT G

SEQ ID NO:3 (16S rRNA gene for Enterococcus gallinarum strain MRx0554)

1 taatacatgc aagtcgaacg ctttttcttt caccggagct tgctccaccg aaagaaaaag

61 agtggcgaac gggtgagtaa cacgtgggta acctgcccat cagaagggga taacacttgg

121 aaacaggtgc taataccgta taacactatt ttccgcatgg aagaaagttg aaaggcgctt 1S1 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 actctgttgn 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 tgcgtagcgg 661 tgaaargcgt agatatatgg aggaacacca gtggcgaagg cggctctctg gtctgtaact 121 gacgctgagg ctcgaaagcg tggggagcga acaggattag araccctggt agtccacgcc 381 gtaaacgatg agtgctaagt gttggagggt ttccgcactt cagtgctgca gcaaacgcat 841 taagcactcc gcctggggag tacgaccgca aggttgaaac tcaaaggaat tgacgggggc 901 ccgcacaagc ggtggagcat gtggtttaat tcgaagcaac gcgaagaacc ttaccaggrc 961 ttgacatcct ttgaccactc tagagataga gcttcccctt cgggggcaaa gtgacaggtg 1021 gtgcatggtt gtcgtcagct cgtgtcgtga gatgttgggt taagtcccgc aacgagcgca 1081 acccttattg ttagrtgcca tcanttagtt gggcactcta gcgagactgc cgglgacaaa 1141 ccggaggaag gtggggatga cgtcaaatca tcatgcccct tatgacctgg getacacacg 1201 tgctacaatg ggaagtacaa cgagttgcga agtcgcgagg ctaagetaat ctcttaaagc I 61 ttctctcagt tcggattgta ggctgcaact cgcctacatg aagccggaat cgctagtaat 1321 cgcggatcag cacgccgcgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1381 cacgagagtt tgtaacaccc gaagtcggtg aggtaacctt tttggagcca gccgcccaag 1441 gtgggataga tgattggggt gaagtcgtaa caaggtagcc gtatcggaag gtgcggctgg 1501 atcacc

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