OA20494A

OA20494A - Compositions comprising bacterial strains

Info

Publication number: OA20494A
Application number: OA1202100063
Authority: OA
Inventors: Emma RAFTIS; Imke Elisabeth MULDER; Amy Beth HOLT; Suaad AHMED; Anna ETTORRE; Emma Elizabeth Clare HENNESSY; Delphine Louise Claudette LAUTE-CALY; Philip COWIE; Marsilio ADRIANI; Maria CHRISTOFI
Original assignee: 4D Pharma Research Limited
Priority date: 2018-08-17
Filing date: 2019-08-19
Publication date: 2022-09-30

Abstract

The invention provides a composition comprising a bacterial strain for use in stimulating the immune system in a subject.

Description

COMPOSITIONS COMPRISING BACTERIAL STRAINS

TECHNICAL FIELD

This invention is in the field of compositions comprising bacterial strains isolated from the mammalîan digestive tract and the use of su ch compositions in the treatment of disease, in particular in stimulating the immune system in the treatment of disease.

BACKGROUND TO THE INVENTION

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

Dramatic changes in microbiota composition hâve been documented in gastrointestinal disorders such as inflammatory bowel disease (1BD). For example, the levels of Clostridium cluster XlVa bacteria are reduced in IBD patients whilst numbers of E. coli are increased, suggestîng a shift in the balance of symbionts and pathobionts within the gut [6-9]. Interestingly, this microbial dysbiosis is also associated with imbalances in T effector cell populations.

In récognition of the potential positive effect that certain bacterial strains may hâve on the animal gut, various strains hâve been proposed for use in the treatment of various diseases (see, for example, [10-13]). Also, certain strains, including mostly Lactobacillus and Bifidobacterium strains, hâve been proposed for use in treating various inflammatory and autoimmune diseases that are not directly linked to the intestines, for example through anti-inflammatory mechanisms (see [14] and [15] for reviews). Certain Streptococcus and Veillonella strains, and to a lesser extent, Enterococcus and Lactobaccillus strains hâve been suggested to hâve immunomodulatory effects, with varying effects on different cytokines in vitro. However, the relationship between different diseases and different bacterial strains, and tire précisé effects of particular bacterial strains on the gut and at a systemic level and on any particular types of diseases, are poorly characterised.

Recently, varions Bifidobacterium species hâve been investigated for their anti-inflammatory properties and several human trials hâve demonstrated that the therapeutic properties of bifidobacteria can be successfully translated from lab to clinic, in particular for inflammatory and autoimmune disorders. For instance, B. longum CECT 7347 and B. infantis NLS hâve both ehcited protective effects in patients with coeliac disease [16,17]). Studies hâve also demonstrated the ability of bifidobacteria to modulate diseases beyond the gut. A three-strain formulation composed ofÆ longum BB536, B. infantis M-63, and A. breve M-16V alleviated the symptoms associated with allergie rhinitis and mild intermittent asthma in children [18], Furthermore, administration of B. longum 35624 to patients with ulcerative colitis, psoriasis, and chronic fatigue syndrome resulted in decreased plasma levels of C-reactive protein in ail three disorders [19], suggesting that this straîn might be capable of modulating systemic immunity.

Organisms from the Bifidobacterium breve species hâve been proposed for use in preparing immunostimulatory suppléments by metabolising linoleic acid in, for example, [20] and [21]. However, there is nothing in those documents to suggest that such organisms, when administered to subjects, could elicit an immunostimulatory response.

Reference [22] suggests that nutrîtional compositions comprising beta-galacto-oligosaccharides A and B may elicit an immunostimulatory effect. It is stated that compositions comprising those sugars may additionally comprise varions other components, including Bifidobacterium breve, but there is no suggestion that Bifidobacterium breve organisms would contribute to or enhance any immunostimulatory properties of the beta-galacto-oligosaccharides.

The therapeutic use of organisms from the species Bifidobacterium breve is proposed in [23]. However, there is no suggestion that the bacterial strains disclosed therein had an immunostimulatory effect.

A study to investigate the potential effect of a strain of Bifidobacterium on immunogenicity of an oral choiera vaccine was reported in [24], However, the outcome of that study was inconclusive; it was reported in that paper that the tested strain while being well tolerated, did not exhibit an évident post-vaccinal immunostimulatory effect.

There is a requîrement in the art for new methods of treating diseases. There is also a requirement for the potential effects of gut bacteria to be characterised so that new thérapies using gut bacteria can be developed.

SUMMARY OF THE INVENTION

The inventors hâve developed new compositions comprising a bacterial strain of the species Bifidobacterium breve that can be used in stimulating the immune System and treating and preventing disease in a subject. The inventors hâve identified that strains of the species Bifidobacterium breve can potently activate the immune System.

The invention therefore provides a composition comprising a bacterial strain of the species Bifidobacterium breve, for use in stimulating the immune System in subject. Preferably, the invention provides a composition comprising the strain deposited under accession number 423 SO at NCIMB, or a dérivative or biotype thereof, for use in stimulating the immune System.

Additîonally, the invention provides a method of stimulating the immune System, comprising admînistering a composition comprising a bacterial strain of the species Bifidobacterium breve to the subject. Furthennore, the invention provides a use of a composition comprising a bacterial strain of the species Bifidobacterium breve for the manufacture of a médicament for stimulating the immune System in a subject.

In further aspects, the invention provides a composition comprising a bacterial strain of the species Bifidobacterium breve, for use as a vaccine adjuvant. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use as a vaccine adjuvant.

In further aspects, the invention provides a composition comprising a bacterial strain of the species Bifiidobacterium breve, for use in treating, preventing or delaying immunosenescence. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in treating, preventing or delaying immunosenescence.

In further aspects, the invention provides a composition comprising a bacterial strain of the species Bifidobacterium breve, for use in enhancing a cell therapy, such as CAR-T. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in enhancing a cell therapy, such as CAR-T.

The inventors hâve also characterised a strain of Bifidobacterium brève that is particularly potent at stimulating the immune System and hâve identified that its potency may be mediated by its exopolysaccharide (EPS). The invention therefore preferably uses a composition comprising a bacterial strain of the specîes Bifidobacterium brève that comprises a complété EPS locus and/or expresses EPS on its surface, for stimulating the immune System in subject.

Preferably, the bacteria used in the invention is the strain deposited under accession number 42380 at NCIMB.

In preferred embodiments, the invention provides a composition, for use in increasing the expression level and/or activity of IL-12p70, IL-12p70, IFNy, IL-4 and/or TNF-α in the treatment or prévention of disease, as demonstrated in the examples. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in increasing the expression level and/or activity of IL-12p70, IL-12p70, IFNy, IL-4 and/or TNF-α in the treatment or prévention of disease.

In preferred embodiments, the invention provides a composition, for use in increasing the expression level and/or activity ofTL-12p70, IL-12p70, IFNy, IL-4, TNF-α and/or IL-17a in the treatment or prévention of disease, as demonstrated in the examples. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in increasing the expression level and/or activity of IL-12p70, IL-12p70, IFNy, IL-4, TNF-α and/or IL-17a in the treatment or prévention of disease.

In preferred embodiments, the invention provides a composition, for use in stimulating TLR2. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in stimulating TLR2.

In preferred embodiments, the invention provides a composition, for use in stimulating NFkB. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for use in stimulating NFkB.

In preferred embodiments, the bacterial strain of the invention expresses pullulanase, which is shown in the examples to be hîghly expressed by potent B. breve straîns and may be involved in adhesion.

In further aspects, the invention provides a composition comprising a bacterial strain of the species Bifidobacterium breve for treating or preventing bacterial infections in a subject. The examples demonstrate that B. breve, and particularly the B. breve strains of the invention, hâve potent anti-microbial activity. Additionally, the invention provides a method of treating or preventing bacterial infections in a subject, comprising administering a composition a bacterial strain of the species Bifidobacterium breve. Furthermore, the invention provides a use of a composition comprising a bacterial strain of the species Bifidobacterium breve for the manufacture of a médicament for treating or preventing bacterial infections in a subject.

In preferred embodiments, the infection is a gastro-intestinal infection. In preferred embodiments, the infection is a Gram-negative bacterial infection. In preferred embodiments, the composition of the invention is for use in treating or preventing gastrointestinal E. coli infection. In preferred embodiments, the composition of the invention is for use in treating or preventing gastrointestinal 5. enterica infection. In some embodiments, the composition of the invention is for use in reducing the viability of a bacteria in the treatment of a bacterial infection. The bacteria of the invention may be used to restore the level of pathogenic bacteria to asymptomatîc levels or to eliminate the pathogenic bacteria entirely from a subject, thereby treating the bacterial infection, in addition to alleviating the symptoms associated with the elevated level of the bacteria.

Strains closely related to the B. breve strain tested in the examples are expected to be particularly effective at stimulating the immune system. In preferred embodiments, the invention provides a composition wherein the bacterial strain has a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or wherein the bacterial strain has a 16s rRNA gene sequence represented by SEQ ID NO:1.

In certain embodiments, the composition of the invention is for oral administration. Oral administration of the strains of the invention may be effective for stimulating the immune system. Also, oral administration is convenîent for patients and practitioners and allows delivery to and/or partial or total colonisation of the intestine.

In certain embodiments, the composition of the invention comprises one or more pharmaceutically acceptable excipients or carriers.

In certain embodiments, the composition of the invention comprises a bacterial strain that has been lyophilised. The composition of the invention can also comprise a lyophilised bacteria strain of the species Bifidobacterium breve. Lyophilisation is an effective and convenient technique for preparing stable compositions that allow delivery of bacteria.

In certain embodiments, the invention pro vides a food product comprising the composition as described above. The invention also provides a food product comprising a bacteria! strain of the species Bifidobacterium breve as described herein.

In certain embodiments, the invention provides a vaccine composition comprising a bacterial strain as described above. The invention also provides a vaccine composition comprising a composition according to the invention,

Additionally, the invention provides a method of treating or preventing a disease or condition associated with reduced immunostimulation, comprising administering a composition comprising a bacterial strain of the species Bifidobacterium breve to a patient in need thereof.

BRIEF DESCRIPTION OF DRAWINGS

Figure I: Mouse mode! ofbreast cancer —tumor volume.

Figure 2: Mouse model of lung cancer — tumour volume.

Figure 3: Mouse model of Itver cancer- liver weight.

Figure 4: Rapid ID 32 A profile of MRX004 alone (A) and in comparison with B. breve type strains (B). White — négative reaction (no colour change), Downwards cross-hatched = intermediate positive reaction (weak colour change) and Black = positive reaction (strong approprîate colour change).

Figure 5: API® 50 CH analysis of MRX004. Upward cross-hatched = négative reaction (no colour change), Downward cross-hatch = intermediate positive reaction (weak colour change), Black = positive reaction (strong approprîate colour change) and White = doubtful reaction (unexpected colour change).

Figure 6: Attachment of MRX004 and B. breve type strains to human cells.

Figure 7: Stimulation of NFkB and TLR2 by MRx0004 treatments. THP-1-NFkB (A) and HEK-TLR2 (B) reporter cell fines were treated with treatments of live MRx0004 (MRx0004_Lv), MRx0004 culture supernatant (MRx0004_SN) and heat-inactivated MRx0004 (MRx0004_Hk) at MOI 100:1 for 22 hours. Data are représentative of three biological replicates. Statistîcal analysis was carried out using ordinary one-way ANOVA and Tukey’s Multiple Comparisons Test.

Statistical ly significant différences are presented as * p < 0.05, *** p <0.001 and **** _p0.0001.

Figure 8: Transcriptional response of MRx0004 in vitro and in response to lECs. The expression of ten MRx0004 genes was analysed and compared between cultures in late log phase growth and after contact (3 hours) with lECs. Data are presented as the fold change (2 ^ΔΔΓ’) value calculated between named conditions and nonnalised to groEL, and are représentative of three independent biological replicates. Statistical analysis was carried ont using ordinary one-way ANOVA and Tukey’s Multiple Comparisons Test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** ^<0.0001.

Figure 9: Sequence diversity within EPS loci of MRx0004 and related strains

Figure 10: Phenotypic properties of an EPS-negative dérivative strain of MRx0004. Transmission électron microscopy (TEM) was carried out on MRx0004 (A) and its EPS-negative strain (EPS^neg) (B). (C) Bacterial adhesion to lECs was analysed after co-incubating HT29-MTX cells and bacterial strains at MOI 100:1 for 3 hours. Data presented are the average of three biological replicates of the percentage of adhèrent CFU from the initial inoculum. Statistical analysis was carried out using ordinary one-way ANOVA and Tukey’s Multiple Comparisons Test. *p < 0.05, **p< 0.01.

Figure 11: Impact of EPS déplétion on MRx0004 surface protein détection. Venn diagram showing the number of proteins identified in (A) MRx0004 shaved and shed proteins fractions, (B) EPS^neg shaved and shed proteins fractions and (C) MRx0004 and EPS^neg shaved protein fractions.

Figure 12. Elucidating the rôle of EPS in immunomodulation by MRx0004. Live treatments (lv) and culture supematants (sn) were added to HEK-TLR2 reporter cells (A) and ΤΗΡ-1-NFkB reporter cells (B) at an MOI of 100:1, and incubated for 22 hours. Data presented for MRx0004_Lv and MRx0004_Sn bave been previously presented in Figure 7. Data are représentative of three independent replicates. (C) HT29-MTX cells were co-incubated with live bacteria for 3 hours, following which TNFa was introduced at a concentration of 10 ng/ml for 24 hours. IL-8 levels were analysed in co-culture supematants using ELISA. Data are représentative of five biological replicates. Statistical analysis was carried out using ordinary one-way ANOVA and Tukey’s Multiple Comparisons Test. * p < 0.05, ** p < 0.01, *** p < 0.001, **** _p < 0.0001.

Figure 13: Identification of immune cell subsets by flow cytometry. PBMCs from six healthy donors were incubated for 72 hours with one of the following treatments; RPMI media alone, MRx0004_Hk, or EPS^neg _HK- Box and whisker plots are shown with their minimum and maximum represented b y vertical whiskers. Expression of the cellular activation marker CD25 is shown as a percentage of CD8⁺ and CD4^h cells (A-B). Tregs (CD25⁺ (high)/ CD 127' (low)) are shown as a percentage of CD4⁺ cells (C). Independent Treg/CD8 ratios were calculated from each donor (D) and B-cells (CD 19) as a percentage of CD3' cells (E). and statistical analysis was carried out using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01***p < 0.001 ****/?< 0.0001.

Figure 14: Cytokine profiles produced by peripheral blood mononuclear cells (PBMCs). Cells were incubated for 72 hours with one of the following treatments; RPMI media alone, MRx0004_Hk, or EPS^neÊHK· Box and whisker plots are shown with their minimum and maximum represented by vertical whiskers. PBMCs were obtained from 6 healthy donors and cytokine concentrations were determined using a multiplex assay (A-G). Independent IL-12p70/IL-4, IL10/ILlp70 and IL-10/IL12p7O ratios were calculated from each donor (H-J) and statistical analysis was carried out using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01 ***p < 0.001 ****p < 0.0001.

Figure 15: Venn diagram, generated with InteractiVenn, showing the number of proteins identifred in MRx0004 shaved and shed proteins fractions (listed in Table 2).

Figure 16: (A) Génération of an EPS négative strain of MRx0004 by insertional mutagenesis. (B) Autoaggregation of MRx0004 and its dérivative strains EPS^neg, EPS^vec and EPS^C0II,p.

Figure 17: Venn diagram comparîng the number of proteins identified in MRx0004SN and EPSnegSN.

Figure 18: Gating strategy used for seven colour panel flow cytometry experiment. An acquisition threshold was set using the live cell population at 1 x 10⁵ events. Shown are représentative pseudocolour plots of human peripheral blood mononuclear cells (PBMCs) from an untreated sample. Gates were set using isotype Controls and FMOs in FloJo. Forward and side scatter were used to identify lymphocytes before gating out the doublet population and utilîsing a viability dye to exclude dead cells. CD3 cells were sub-gated on with CD19 to identify B-cells, whilst the CD3⁺ cell population was used to further distinguish both the CD4⁺ and CD8⁺ cells. CD25 was then used to look at the percentage of activated cells and in the case of the CD4⁺population it was also used in conjunction with CD 127 to identify Tregs (CD25VCD127 ).

Figure 19: Identification of immune cell subsets by flow cytometry. PBMCs from six healthy donors were incubated for 72 hours with one of the following treatments; RPMI media alone, MRx0004_HK, or EPS^neg _HK. Box and whisker plots are shown with their minimum and maximum represented by vertical whiskers. CD8⁺ and CD4⁺ cells are shown as a percentage of CD3⁺ (AB) and activated B-cells as a percentage of CD19⁺ (C). Statistical analysis was carried out using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01***/? < 0.001****^ < 0.0001.

Figure 20: Identification of immune cell subsets by flow cytometry. PBMCs from six healthy donors were incubated for 72 hours with one of the following treatments; MRx0004_Hk, ΕΡ5^η%_Κ;EPS^veCHK or EPS^u™^1phk. Scatter plots are shown with their standard déviations represented by vertical bars. Expression of CD8⁺ and CD4 cells are shown as a percentage of CD3⁺ cells (A, C). Expression of the cellular activation marker CD25 is shown as a percentage of CD4 and CD8 cells (B, D). Tregs (CD25⁺ (high)/ CD127’(low)) are shown as a percentage of CD4⁺ cells (E) and B-cells (CD 19 ) as a percentage of CD3 cells (F) and activated B-cells as a percentage of CD19¹ (G). Independent Treg/CD8⁺ ratios were calculated from each donor (H) and statistical analysis was carried out using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01 ***p < 0.001 ****/? < 0.0001.

Figure 21: Cytokine profiles produced by peripheral blood mononuclear cells (PBMCs). Cells were incubated for 72 hours with one of the following treatments; MRx0004_HKh EPS^negHK, EPS^vetHK or EPS^comp _HK. Box and whisker plots are shown with their minimum and maximum represented by vertical whiskers. PBMCs were obtained from 6 healthy donors and cytokine concentrations were determined using a multiplex assay (A-G). Independent IL-12p70/IL-4, IL10/ILlp70 and IL-lp/IL12p70 ratios were calculated from each donor (H-J). Statistical analysis was carried ont using ordinary one-way ANOVA followed by Tukey’s multiple comparisons test. *p < 0.05, **p < 0.01 ***p < 0.001 ****p < 0.0001.

Figure 22: Example of a co-culture antimicrobial plate assay. Shown are the indicator strain, the test strains and the inhibition zone.

Figure 23: API 32A test results for the test and reference B. breve strains. White = négative reaction (no colour change), Star = întermediate positive reaction (weak colour change) and Black = positive reaction (strong appropriate colour change).

Figure 24: PFGE Spel Digest for the test and reference B. breve strains.

Figure 25; Spel digested 1% PFGE gel run in 0.5% TBE, 18 hrs, 6 V/cm, 1-15 sec ST. Black arrows indicate DNA size standards. Lanes 1 and 2 were grouped with lanes 3-7 from different parts of the same gel. Lane 1 = λ, Lane 2 = MRx0004, Lane 3 = B.breve REF 7, Lane 4 = B.breve REF6, Lane 5 = B.breve REF1, Lane 6 = B.breve REF2, Lane 7 = λ

Figure 26: UPGMA dendrogram based on the PFGE patterns of R breve included in this study (A). A similarity matrix generated from the PFGE banding pattern represented in panel B.

Figure 27: Induction of T-cell différentiation in a population of T-helper cells using heat-kîHed MRx0004 (HK 4), supematant from MRx0004 culture (SP 4) or RPMI medium, without addition of cytokines (no cyto). **= p< 0.01.

Figure 28: Induction of T-cell différentiation in a population of Cytotoxic T Lymphocytes (CTL) using heat-killed MRx0004 (HK 4), supematant from MRx0004 culture (SP 4) or RPMI medium, without addition of cytokines (no cyto). * = p< 0.05; ***= p< 0.001; ****= p< 0.0001.

Figure 29; Viability of splénocytes.

Figure 30: Cytokine profdes produced by splénocytes after treatment with MRx004.

Figure 31: Frequency of CD8+IFNy+ and CD4+IFNy+ cells and per cell IFNy production in spleen.

DISCLOSURE OF THE INVENTION

Bacterial strains

The compositions of the invention comprise a strain of the species Bifidobacterium breve. The ex amples demonstrate that such bacterial strains are useful for stimulatîng the immune System. The preferred bacterial strain of the invention is the bacterium deposited under accession number NCIMB 42380.

The Bifidobacterium breve bacterium deposited under accession number NCIMB 42380 was tested in the Examples and is also referred to herein as strain 751, MRX004 or MRx0004. A partial 16S rRNA sequence for the MRX004 strain that was tested is provided in SEQ ID NO:1. Bifidobacterium breve strain MRX004 was deposited with the international depositary authority NCIMB, Ltd. (Ferguson Building, Craibstone Estate, Bucksbum, Aberdeen, AB21 9YA, Scotland) by GT Biologics Ltd. (Life Sciences Innovation Building, Aberdeen, AB25 2ZS, Scotland) under identification reference 751 on 12th March 2015 and was assîgned accession number NCIMB 42380. GT Biologics Ltd. subsequently changed its naine to 4D Pharma Research Limited. These deposits were publîshed in WO2016/203223.

A genome sequence for strain NCIMB 42380 is provided in SEQ ID NO:2 of WO2016/203223.

Bacterial stiains closely related to the strain tested in the examples are also expected to be 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 95%, 96%, 97%, 98%, 99%, 99.5% _Or 99.9% identical to SEQ ID NO:1. Preferably, the bacterial strain has the 16s rRNA sequence represented by SEQ ID NO: 1. Most preferably, the bacterial strain is the Bifidobacterium brève strain deposited under accession number NCIMB 42380.

In certain embodiments, the bacterial strain for use in the invention has a genome with sequence identity to SEQ ID NO:2 of WO2016/203223. In preferred embodiments, the bacterial strain for use in the invention has a genome with at least 90% sequence identity (e.g. at least 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity) to SEQ ID NO:2 of WO2016/203223 across at least 60% (e.g. at least 65%, 70%, 75%, 80%, 85%, 95%, 96%, 97%, 98%, 99% or 100%) of SEQ ID NO: 2 of WO2016/203223. For example, the bacterial strain for use in the invention may hâve a genome with at least 90% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 70% of SEQ ID NO: 2 of WO2016/203223, or at least 90% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 80% of SEQ ID NO: 2 of WO2016/203223, or at least 90% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 90% of SEQ ID NO: 2 of WO2016/203223, or at least 90% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 100% of SEQ ID NO: 2 of WO2016/203223, or at least 95% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 70% of SEQ ID NO: 2 of WO2016/203223, or at least 95% sequence identity to SEQ ID NO: 2 ofWO2016/203223 across 80% of SEQ ID NO: 2 of WO2016/203223, or at least 95% sequence identity to SEQ ID NO: 2 ofWO2016/203223 across 90% of SEQ ID NO: 2 ofWO2016/203223, or at least 95% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 100% of SEQ ID NO: 2 of WO2016/203223, or at least 98% sequence identity to SEQ ID NO: 2 ofWO2016/203223 across 70% of SEQ ID NO: 2 ofWO2016/203223, or at least 98% sequence identity to SEQ ID NO: 2 ofWO2016/203223 across 80% of SEQ ID NO: 2 ofWO2016/203223, or at least 98% sequence identity to SEQ ID NO: 2 of WO2016/203223 across 90% of SEQ ID NO: 2 of WO2016/203223, or at least 98% sequence identity to SEQ ID NO: 2 ofWO2016/203223 across 100% of SEQ ID NO: 2 of WO2016/203223.

In prefened embodiments, the composition of the invention comprises live bacteria. In preferred embodiments, the composition of the invention comprises live bacteria in an active state, preferably lyophihsed. The examples demonstrate that administration of live bacteria is more effective than heat killed bacteria or supematants.

In preferred embodiments, the bacteria of the invention activate TLR2, for example in a HEKTLR2 reporter assay such as described in the examples. In further embodiments, the bacteria of the invention do not activate TLR4, TLR5 or TLR9. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes TLR2 and is for use as a vaccine adjuvant. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes TLR2 and is for use in enhancîng a cell therapy. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes TLR2 and is for use in treating, preventing or delaying immunosenescence.

In preferred embodiments, the bacteria of the invention activate NFkB, for example in a THP-1NFkB reporter assay such as described in the examples. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes NFkB and is for use as a vaccine adjuvant. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes NFkB and is for use in enhancîng a cell therapy. In a preferred embodiment, the composition of the invention comprises a bacteria that activâtes NFkB and is for use in treating, preventing or delaying immunosenescence.

In certain embodiments, the bacteria of the invention express one or more genes selected from the group consi sting of: oppA, pullulanase, serpin and tadA at a higher level in stationary phase compared to late log phase, for example, when grown in liquid culture, such as shown in the examples. The examples demonstrate that these genes may médiate useful therapeutic effects.

In certain embodiments, the bacteria of the invention express one or more genes selected from the group consisting of: eftU, enolase and pGTF at a higher level in late log phase compared to stationary phase, for example, when grown in liquid culture, such as shown in the examples. The examples demonstrate that these genes may médiate useful therapeutic effects.

In certain embodiments, the bacteria of the invention express one or more genes selected from the group consisting ot: enolase, pGTF, oppA, serpin and transaldolase at a higher level after contact with intestinal épithélial cells compared to late log phase, for example, when grown in liquid culture, such as shown in the examples. The examples demonstrate that these genes may médiate useful therapeutic effects.

In certain embodiments, the bacteria of the invention expresses and sécrétés into the culture supematant one or more of pullulanase, NlpC/P60 family proteins, FtsI, transaldolase, GAPDH, DnaK, GroEL and enolase. In certain embodiments, the bacteria of the invention expresses and sécrétés into the culture supematant one or more, such as 2, 3, 5, 10, 15, or ail, of the proteins in Table 2. The examples demonstrate that these proteins may médiate useful therapeutic effects.

In certain embodiments, the bacteria of the invention expresses on its cell surface one or more of pullulanase, a type I polyketide synthase, transaldolase, GAPDH, DnaK, GroEL, enolase and EfTu, In certain embodiments, the bacteria of the invention expresses and sécrétés into the culture supematant one or more, such as 2, 3, 5, 6, 8, or ail, of the proteins in Table 3. The examples demonstrate that these proteins may médiate useful therapeutic effects.

In certain embodiments, the bacteria of the invention express pullulanase. In a preferred embodiment, the composition of the invention comprises a bacteria that expresses pullulanase and is for use as a vaccine adjuvant. In a preferred embodiment, the composition of the invention comprises a bacteria that expresses pullulanase and is for use in enhancing a cell therapy. In a preferred embodiment, the composition of the invention comprises a bacteria that expresses pullulanase and is for use in treating, preventing or delaying immunosenescence.

In preferred embodiments, the bacteria of the invention comprises a complété EPS locus. The examples demonstrate that a complété EPS locus may contribute to increased therapeutic potency. In such embodiments, the EPS locus comprises a priming glycosyltransferase, one or more addîtîonal glycosyltransferases, a thiamine pyrophosphate binding protein, a membrane spanning protein, a flippase and a chain-length déterminant. In preferred embodiments, the EPS locus is over 30 Kb in sîze (including flanking hypothetical proteins). The examples demonstrate that such an EPS locus is adéquate for immunostimulatory function. In preferred embodiments, the EPS locus is 25-60 Kb, 30-50 Kb, 30-40 Kb, 30-35 Kb, or 30-32 Kb in size. In a preferred embodiment, the composition of the invention comprises a bacteria with a complété EPS locus and is for use as a vaccine adjuvant. In a preferred embodiment, the composition of the invention comprises a bacteria with a complété EPS locus and is for use in enhancing a cell therapy. In a preferred embodiment, the composition of the invention comprises a bacteria with a complété EPS locus and is for use in treating, preventing or delaying immunosenescence.

In preferred embodiments, the bacteria of the invention has a EPS locus with a high level of nucléotide identity to the EPS locus of strain MRX004, such as at least 90, 92, 94, 96, 98, 99 or 99.5 % nucléotide identity, for example as determined in the examples. The examples demonstrate that the EPS locus of strain MRX004 is genetically distinct from other B. breve strains and may contribute to potency and therapeutic utility.

In preferred embodiments, the bacteria of the invention carries EPS on its surface. The examples demonstrate that EPS modulâtes the exposure of proteins on the cell surface. In a preferred embodiment, the composition of the invention comprises a bacteria that cames EPS on its surface and is for use as a vaccine adjuvant. In a preferred embodiment, the composition of the invention comprises a bacteria that carries EPS on its surface and is for use in enhancing a cell therapy. In a preferred embodiment, the composition of the invention comprises a bacteria that carries EPS on its surface and is for use in treatîng, preventing or delaying immunosenescence,

Preferably, the bacteria used in the invention is able to ferment raffmose, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours. The examples suggest that the most effective B. breve strains are able to ferment raffmose, and it is involved in EPS production. In further preferred embodiments, the bacteria used in the invention is able to ferment one or more, such as 2, 3, 4, 5, 6 or ail 7 of: agalactosidase, β-galactosidase, ct-glucosidase and β-glucosidase, α-arabinose, mannose and raffinose, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours. In further preferred embodiments, the bacteria used in the invention is able to ferment one or more, such as 2, 3, 4, 5, 6 or ail 7 of: arginine, proline, phenylalanine, leucine, tyrosine, glycine and histidine. Any suîtable assay known in the art may be used to assess the ability of a bacterium to ferment a carbohydrate source or amino acid. Preferebly, the Rapid ID 32A analysis is used (preferably using the Rapid ID 32A System from bioMérieux).

Preferably, the bacteria used in the invention exhibit intermediate fermentation of β-glucosidase or intermediate fermentation of α-arabinose, or more preferably intermediate fermentation of βglucosidase and intermediate fermentation of α-arabinose, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that both B. breve strains MRX004 and Test 3 hâve useful activity and both exhibit intermediate fermentation of β-glucosidase and α-arabinose. In particularly preferred embodiments, the bacteria used in the invention exhibit intermediate fermentation of β-glucosidase, intermediate fermentation of aarabinose, and positive fermentation of rabinose.

Preferably, the bacteria used in the invention do not exhibit positive fermentation of N-acetyI-βglucosaminîdase, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The bacteria may exhibit only intermediate or no fermentation of N-acetyl-βglucosaminidase. The examples demonstrate that both B. breve strains MRX004 and Test 2 hâve useful activity and neither exhibits positive fermentation of N-acetyl-β-gIucosaminidasc. In particularly preferred embodiments, the bacteria used in the invention do not exhibit positive fermentation of N-acetyl-β-glucosaminidase and do exhibit positive fermentation of rabinose.

Preferably, the bacteria used in the invention exhibit intermediate fermentation of agalactosidase or intermediate fermentation of α-arabinose, or more preferably intermediate fermentation of α-galactosidase and intermediate fermentation of α-arabinose, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that B. breve strains MRX004 and Test S both hâve useful activity and both exhibit intermediate fermentation of α-galactosidase and α-arabinose. In particularly preferred embodiments, the bacteria used in the invention exhibit intermediate fermentation of α-galactosidase and intermediate fermentation of α-arabinose, and positive fermentation of rabinose.

In alternative embodiments, the bacteria used in the invention ferment serine arylamidase but not leucyl glycine arylamidase and not alanine arylamidase, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for ex ample when subjected to the Rapid ID 32 A analysis. The examples demonstrate that B. breve strains Test 11 and Test 12 both hâve useful activity and both ferment serine arylamidase but not leucyl glycine arylamidase and not alanine arylamidase. In particularly preferred embodiments, the bacteria used in the invention also ferment rabinose.

In alternative embodiments, the bacteria used in the invention exhibit intermediate fermentation of serine arylamidase, for example when cultured in an appropriate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that B. breve strains Test 3 and Test 7 both hâve potent anti-microbial activity and both exhibit intermediate fermentation of serine arylamidase. In particularly preferred embodiments, the bacteria used in the invention also ferment rabinose.

Any suitable assay known in the art may be used to assess the ability of a bacterium to ferment a carbohydrate source or amino acid. Preferebly, the Rapid ID 32A analysis is used (preferably using the Rapid ID 32A system from bioMérieux).

Preferably, the bacteria used in the invention produce the pattern shown in Figure 24 or Figure 25 for MRX004 when subjected to pulsed-field gel electrophorcsis using standard conditions, such as those used in Example 10.

In alternative preferred embodiments, the bacteria used in the invention is able to ferment one or more, such as 2, 3, 4, 5, 10, 15, 20, 25 or ail of: amidon (starch), amygdalin, arbutin, cellobiose, esculin, galactose, gentiobiose, glucose, glycogen, fructose, fucose, lactose, maltose, mannose, mannitol, melibiose, melezitose, methyl α-D-glucopyranoside, N-acetyl glucosamine, ribose, saccharose (sucrose), salicin, sorbitol, trehalose, turanose and xylitol. In such embodiments, any suitable assay known in the art may be used to assess the ability of a bacterium to ferment a carbohydrate source. Preferebly, the API 50 CH analysis is used from bioMérieux.

In certain embodiments, the compositions of the invention comprise a strain of Bifidobacterium breve that exhibits reduced attachment to human cells, in particular when tested in YCFA medium, in particular under the conditions of Example 5.

In certain embodiments, a composition of the invention comprises a biotype of the bacterium deposited under accession number NCIMB 42380. Bacterial strains that are biotypes of the bacterium deposited under accession number NCIMB 42380 are also expected to be effective for stimulating the immune system. A biotype will hâve comparable immune modulatory activity to the original NCIMB 42380 strain. A biotype is a closely related strain that has the same or very physiologîcal and biochemical characteristics.

A biotype will elicît comparable effects on the immune System to the effects shown in the examples, which may be identified by using the culturing and administration protocol s described in the examples. In particular, a biotype will elicit an effect on T cells and cytokines comparable to NCIMB 42380.

Strains that are biotypes of a bacterium deposited under accession number NCIMB 42380 and that are suitable for use in the invention may be identified by sequencing other nucléotide sequences for a bacterium deposited under accession number NCIMB 42380. For example, substantially the whole genome may be sequenced and a biotype strain for use in the invention may hâve at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity across at least 80% of its whole genome (e.g. across at least 85%, 90%, 95% or 99%, or across its whole genome). For example, in some embodiments, a biotype strain has at least 98% sequence identîty across at least 98% of its genome or at least 99% sequence identity across 99% of its genome. Other suitable sequences for use in identifying biotype strains may include hsp60 or répétitive sequences such as BOX, ERIC, (GTG)₅, or REP [25],

Biotype strains may hâve such sequences with at least 95%, 96%, 97%, 98%, 99%, 99,5% or 99.9% sequence identity to the corresponding sequence of a bacterium deposited under accession number NCIMB 42380. In some embodiments, a biotype strain may bave a I6S rRNA sequence with at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% sequence identity to the corresponding sequence of a bacterium deposited under accession number NCIMB 42380. In some embodiments, a biotype strain may comprises a 16S rRNA sequence that is at least 99% identical (e.g. at least 99.5% or at least 99.9% identical) to SEQ ID NO:1. In some embodiments, a biotype strain has the 16S rRNA sequence of SEQ ID NO:1.

Alternatively, strains that are biotypes of a bacterium deposited under accession number NCIMB 42380 and that are suitable for use in the invention may be identified by using the accession number NCIMB 42380 deposit, and restriction fragment analysis and/or PCR analysis, for example by using fluorescent amplified fragment length polymorphism (FAFLP) and répétitive DNA element (rep)-PCR fingerprinting, or protein profiling, or partial I6S or 23s rDNA sequencing. In preferred embodiments, such techniques may be used to identity other Bifidobacterium breve strains.

In certain embodiments, strains that are biotypes of a bacterium deposited under accession number NCIMB 42380 and that are suitable for use in the invention are strains that provide the same pattern as a bacterium deposited under accession number NCIMB 42380 when analysed by amplified ribosomal DNA restriction analysis (ARDRA), for example when using Sau3AI restriction enzyme (for exemplary methods and guidance see, for example [26]). Alternatively, biotype strains are identified as strains that hâve the same carbohydrate fermentation patterns as a bacterium deposited under accession number NCIMB 42380.

Other Bifidobacterium breve strains that are useful in the compositions and methods of the invention, such as biotypes of a bacterium deposited under accession number NCIMB 42380, may be identified using any appropriate method or strategy, including the assays described in the examples. In particular, bacterial strains that hâve similar growth patterns, metabolic type and/or surface antigens to a bacterium deposited under accession number NCIMB 42380 may be useful in the invention.

In certain embodiments, a composition of the invention comprises a dérivative of the bacterium deposited under accession number NCIMB 42380. A dérivative of the strain deposited under accession number NCIMB 42380 may be a daughter strain (progeny) or a strain cultured (subcloned) from the original. A dérivative of a strain of the invention may be modified, for example at the genetic level, without ablating the biological activity. In particular, a dérivative strain of the invention is therapeuticaiiy active. A dérivative strain will hâve comparable immune modulatory activity to the original NCIMB 42380 strain. A dérivative strain will hâve comparable microbiota modulatory activity to the original NCIMB 42380 strain. A dérivative strain will therefore be effective in stimulating the immune System.

A denvative strain will elicit comparable effects cancer models to the effects shown in the examples, which may be identified by using the culturing and administration protocols described in the examples. In particular, a dérivative strain will elicit an effect cytokines and gene expression comparable to those of a bacterium deposited under accession number NCIMB 42380. In particular, a dérivative strain will elicit an effect on immune stimulation comparable to that of a bacterium deposited under accession number NCIMB 42380. A derivatîve of the NCIMB 42380 strain will generally be a biotype of the NCIMB 42380 strain.

The bacterial strain may also be a strain that has the same safety and therapeutic efficacy characteristics as the strain deposited under accession number NCIMB 42380, and such cells are encompassed by the invention. The composition can therefore comprise a Bifidobacterium breve strain that is not the strain deposited under accession number NCIMB 42380 but has the same safety and therapeutic efficacy characteristics as the strains deposited under accession number NCIMB 42380. The safety characteristics of a strain can be established for example by testing the résistance of the strain to antibiotics, for example distinguishing between intrinsic and transmissible résistance to antibiotics. The safety characteristics of a strain can also be established by evaluating the pathogenic properties of a strain in vitro, for example the levels of toxin production. Other safety tests include testing the acute or chronic toxicity of the bacterial strain in rat and mice models. The therapeutic efficacy of a strain can be established by functional characterization of the bacterial strain in vitro and in vivo using a relevant model.

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

In a preferred embodiment, the bacterial strain for use in the invention both has low adhérence to human intestinal épithélial cells, in particular Caco-2 cells, in YCFA compared to one or more of the B. breve strains listed in Figure 9 (such as adhérence of iess than 1 % of total culture, such as preferably Iess than 0.5% or less than 0.3%), and produces more bound surface exopolysaccharides compared to one or more of the B. breve strains listed in Figure 9.

In certain preferred embodiments, the bacterial strain for use in the invention is able to ferment the polysaccharide raffinose, for ex ample when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours.

In certain embodiments, the bacterial strain for use in the invention has reduced abilîty to ferment α-glucosidase and/or β-glucosîdase compared to Bifidobacteria, in particular B. breve, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours.

In certain embodiments, the bacterial strain for use in the invention comprises one or more of the genes listed in Table 1 ofWO2016/203223, which is herein incorporated by reference, such as 5, 10, 20, 50 or ail of the genes in Table 1 of WO2016/203223. In certain embodiments, the bacterial strain for use in the invention comprises one or more of the genes listed in Table 1 of WO2016/203223 that are highlighted with single underlining, such as Transmembrane component BL0694 of energizing module of predicted ECF transporter and/or Duplicated ATPase component BL0693 of energizing module of predicted ECF transporter. In certain embodiments, the bacterial strain for use in the invention comprises one or more of the genes listed in Table 1 of WO2016/203223 that are highlighted with double underlining and in bold, such as 1, 2, 3, 4 or 5 genes selected from: maltodextrin glucosidase (EC 3.2.1.20), putative galactosidase, cellulose synthase (UDP-forming) (EC 2.4.1.12), chitinase (EC 3.2.1.14) and sensory box/GGDEF family protein. In certain embodiments, the bacterial strain for use in the invention comprises one or more of the genes listed in Table 1 of WO2016/203223 that are highlighted with italics, such as 1, 2, 3, 4, 5, 6, 7, 8 or 9 genes selected from: omega-3 polyunsaturated fatty acid synthase subunit PfaA, Type I polyketîde synthase, putative glycosyl hydrolase of unknown function (DUF1680), ATPase component BioM of energizing module of biotin ECF transporter, Cation-transporting ATPase E1-E2 family, Ribose ABC transport system permease protein RbsC (TC 3.A.1.2.1), Ribose ABC transport system ATP-binding protein RbsA (TC 3.A. 1.2.1), 3’-to-5’ oligoribonuclease (om), membrane protein related to Actinobacillus protein (1944168).

In preferred embodiments, the bacterial strain for use in the invention comprises one or more (such as 5, 10, 15, 20, 25, 30, 40, 45, 50 or ali) genes selected from: 2-succînyl-5-enolpyruvyi-6 hydroxy-3- cyclohexene-l-carboxylic-acid synthase (EC 2.2.1.9); 3'-to-5' oligoribonuclease (orn); Alpha-galactosidase (EC 3.2.1.22); ATPase comportent of general energizing module of ECF transporters; ATPase component STY3233 of energizing module of queuosine-regulated ECF transporter; ATP-dependent DNA helîcase recG (EC 3.6.1.-); Beta-glucosidase (EC 3.2.1.21); Cellulose synthase (UDP-formîng) (EC 2.4.1.12); Chitinase (EC 3.2.1.14); COG1309: Transcriptional regulator; D-alanyl-D-alanine carboxypeptidase (EC 3.4.16.4); Duplicated ATPase component BL0693 of energizing module of predicted ECF transporter; Fructokinase (EC 2.7.1.4); Glucose/mannose:H+ symporter GlcP; Glycosyltransferase (EC 2.4.1.-); GMP synthase [glutamine-hydrolyzing] (EC 6.3.5.2); Hypothetical sugar kinase in cluster with indigoidine synthase indA , PfkB family of kinases; inosine-uridine preferring nucleoside hydrolase (EC 3.2.2.1); LSU ribosomal protein L3lp @ LSU ribosomal proteîn L31p, zincîndependent; LSU ribosomal protein L33p @ LSU ribosomal protein L33p, zinc-independent; Maltodextrin glucosidase (EC 3.2.1.20); Membrane protein, related to Actinobacillus protein (1944168); Membrane-bound lytic murein transglycosylase D precursor (EC 3.2.L-); Methyltransferase (EC 2.1.1.-); NADH-dependent butanol dehydrogenase A (EC 1.1.1.-); Phosphoglycolate phosphatase (EC 3.1.3.18); Phosphoribosylanthranilate isomerase (EC 5.3.1.24); Putative glycosyl hydrolase of unknown function (DUF1680); Rhamnose-contaîning polysacharide translocation permease; Ribokinase (EC 2.7.1.15); Ribose ABC transport System, ATP-binding protein RbsA (TC 3.A.1.2.1); Ribose ABC transport System, ATP-bînding protein RbsA (TC 3.A.L2.1); Ribose ABC transport System, high affïnity permease RbsD (TC 3.A. 1.2.1); Ribose ABC transport System, periplasmic ribose-binding protein RbsB (TC 3.A. 1.2.1); Ribose ABC transport system, permease protein RbsC (TC 3.A. 1.2.1); Ribose ABC transport System, permease protein RbsC (TC 3.A. 1.2.1); Sorbitol dehydrogenase (EC 1.1.1.14); SSU ribosomal protein S14p (S 2 9e) @ S SU ribosomal protein S14p (S 29e), zinc-independent; Substrate-specîfic component STY3230 of queuosine-regulated ECF transporter; Sucrose-6phosphate hydrolase (EC 3.2.1.B3); Teichoic acid export ATP-binding protein TagH (EC 3.6.3.40); Transmembrane component BL0694 of energizing module of predicted ECF transporter; Transmembrane component STY3231 of energizing module of queuosine-regulated ECF transporter; Two-component response regulator colocalized with HrtAB transporter; Type I restriction-modification System, DNA-methyltransferase subunit M (EC 2.1.1.72); Type I restriction-modification System, restriction subunit R (EC 3.1.21.3); Type I restrictionmodification system, specificity subunit S (EC 3.1.21.3); Type I restriction-modification system, specificity subunit S (EC 3.1.21.3); Type I restriction-modification System, specificity subunit S (EC 3.1.21.3); Xylitol dehydrogenase (EC 1.1.1.9); and Xylose ABC transporter, periplasmic xylose-binding protein XylF. In preferred embodiments, the bacterial strain for use in the invention comprises one or more (such as 5, 10, 15, 20, 25, 30, 35 or ail) genes that are listed in thepreceding sentence and that are not highlighted in Table 1 of WO2016/203223.

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 hâve an immunostimulatory effect, compositions of the invention may be useful in the treatment of disease, in particular diseases characterised by reduced immune activation and diseases treatable by an increased immune response. In certain embodiments, the compositions of the invention are for use in stimulating the immune system. In certain embodiments, the compositions of the invention are for use in treating disease by stimulating the immune system. In certain embodiments, the compositions of the invention are for use in promoting an immune response. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

Immunodeficiency is a state in which a patient’s immune system is compromised or entirely absent. An immunodeficiency disease is an example of disease that is characterised by reduced immune activation and where it would be advantageous to stimulate the patient’s immune System in order to treat the disease. In some embodiments, the compositions of the invention are for use in treating or preventing an immunodeficiency disease.

There are two types of immunodeficiency diseases. Primary immunodeficiency diseases are inherited immune disorders resulting from genetic mutations that are usually present at birth and diagnosed in childhood. Secondary immunodeficiency diseases are acquired immunodeficîencies that are the resuit of a disease or an environment source, such as a toxic Chemical. In some embodiments, the compositions of the invention are for use in treating or preventing a primary immunodeficiency disease or a secondary immunodeficiency disease.

Examples of primary immunodeficiency disorders and examples include X-linked agammaglobulinemia (XLA), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID) and severe combined immunodeficiency (SCID), which is also known as alymphocytosis or “boy in a bubble” disease. Secondary immunodeficiency disorders can be caused by for example, severe bums, chemotherapy, radiation, diabètes, malnutrition. Examples of secondary immunodeficiency disorders include AIDS, cancers of the immune

System, such as leukaemia, immune-complex diseases, such as viral hepatitis multiple myeloma [2 7], Interferon-γ is an approved therapy for the immunodeficiency disease CGD [28], The examples demonstrate that the compositions of the invention can încrease the production of IFNγ, therefore the compositions of the invention may be particularly effective at treating immunodeficiency diseases, including primary and secondary immunodeficiency diseases.

In preferred embodiments, the composition of the invention is for use in stimulating the immune System through stimulating TLR2. In certain embodiments, the composition of the invention is for stimulating TLR2 in the treatment of disease. In certain embodiments, the composition of the invention is for use in treating a disease associated with decreased TLR2 activity, or is for use in treating a patient identified as having decreased TLR2 activity. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

In certain embodiments, treatment with compositions of the invention drives a Thl cell response. In certain embodiments, the compositions of the invention are for use in driving a Thl cell response in the treatment of disease. In certain embodiments, the composition of the invention is for use in treating a disease associated with decreased Thl cell activity, or is for use in treating a patient identified as having decreased Thl cell activity. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

In preferred embodiments, the composition of the invention is for use in stimulating the immune System through activatîng NFkB. In certain embodiments, the composition of the invention is for stimulating NFkB in the treatment of disease. In certain embodiments, the composition of the invention is for use in treating a disease associated with decreased NFkB activity, or is for use in treating a patient identified as having decreased NFkB activity. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

Compositions of the invention may be useful in the treatment of diseases characterised by decreased levels of activated CD8⁺ cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasîng the activity or levels of CD8⁺ cells. In one embodiment, composition of the invention are for use in treating disease by increasîng the activity or levels of CD8⁺ cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by activating CD8⁺ cells.

Compositions of the invention may be useful in the treatment of diseases characterised by decreased levels of B cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the activity or levels of B cells. In one embodiment, composition of the invention are for use in treating disease by increasing the activity or levels of B cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by activating B cells.

Compositions of the invention may be useful in the treatment of diseases characterised by decreased levels of activated CDS⁺CD25⁺ cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the activity or levels of CD8 CD25^h cells. In one embodiment, composition of the invention are for use in treating disease by increasing the activity or levels of CD8⁺CD25 cells. In one embodiment, compositions of the invention are for use in stimulating the immune response by activating CD8⁺CD25⁺ cells.

Compositions of the invention may be useful in the treatment of diseases characterised by a decrease in the number or percentage of B cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by decrease in the number or percentage of B cells. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the number or percentage of B cells in cell populations, wherein the increase in number or percentage of B cells results in immune stimulation. In one embodiment, compositions of the invention are for use in stimulating the immune response by increasing the number or percentage of B cells.

The examples show that administration of the compositions of the invention can lead to an increase in expression of pro-inflammatory molécules, such as pro-inflammatory cytokines. Examples of pro-inflammatory molécules that showed an increase in expression levels upon administration of compositions of the invention include IL-I2p70, TNF-α, IL-4, IFNy and IL17α. Since administration of the compositions of the invention were shown to increase the expression of pro-inflammatory molécules, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of pro-inflammatory molécules, such as pro-inflammatory cytokines. In one embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of pro-inflammatory molécules, in particular diseases characterised by a decrease in the expression and/or activity of pro-inflammatory cytokines. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-12p70, TNF-α, IL-4 and/or IFNy. 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-12p70, TNF-α, 1L-4 and/or IFNy. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-12p70, TNF-α, IL-4 and/or IFNy. 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-17α IL-12p70, TNF-α, IL-4, IFNy and/or IL-17a. 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-12p70, TNF-α, IL-4 IFNy and/or IL-17α. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of !L-12p70, TNF-α, IL-4 IFNy and/or IL-17α.

The examples also show that administration of the compositions of the invention can lead to an increase in expression of IL-Ιβ. IL-1 β is a pro-inflammatory cytokine [29]. The production and sécrétion of IL-1 β is regulated by the inflammasome, a protein complex which is associated with activation of the inflammatory response [30]. Since administration of the compositions of the invention were shown to increase the expression of IL-1 β, compositions of the invention may be useful in the treatment of diseases characterised by a decrease in expression of IL-Ιβ. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-Ιβ. In one embodiment, the compositions of the invention are for use in treating or preventing diseases by increasing the expression and/or activity of IL-1 β.

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-α is a pro-inflammatory cytokine which is known to be involved in varions signalling pathways to promote cell death. TNF-α initiâtes apoptosis by binding to its cognate receptor, TNFR-1, which leads to a cascade of cleavage events in the apoptotic pathway [31]. TNF-α can also trigger necrosis via a RIP kinase-dependent mechanism [32], Since administration of the compositions of the invention show an increase in TNF-α expression, compositions of the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by TNF-α. In one embodiment, the compositions of the invention are for use in treating diseases characterised b y decreased TNF-α expression. In a particular embodiment, the compositions of the invention are for use in treating or preventing diseases characterised by a decrease in the expression and/or activity of TNF-α. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of TNF-α. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of TNF-a.

Since administration of the compositions of the invention show an increase in IL-4 expression, compositions of the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by IL-4. In one embodiment, the compositions of the invention are for use in treating diseases characterised by decreased IL-4 expression. In a particular embodiment, the compositions of the invention are for use în treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-4. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of IL-4. In one embodiment, compositions of the invention are for use in promoting the immune response b y increasing the expression and/or activity of IL-4.

Since administration of the compositions of the invention show an increase în IL-17α expression, compositions of the invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by IL-17a. In one embodiment, the compositions of the invention are for use in treating diseases characterised by decreased IL-17a 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 IL-17α. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of IL-17α. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-17α.

Since administration of the compositions of the invention show an increase in IL-12p70 expression, compositions of ihe invention may be useful in the treatment of diseases, in particular for use in treating or preventing diseases characterised by a decrease in expression of by IL-12p70. In one embodiment, the compositions of the invention are for use in treating diseases characterised by decreased IL-12p70 expression. In a particular embodiment, the compositions of the invention are for use în treating or preventing diseases characterised by a decrease in the expression and/or activity of IL-12p70. In one embodiment, the compositions of the invention may be useful for treating or preventing diseases by increasing the expression and/or activity of IL-12p70. In one embodiment, compositions of the invention are for use in promoting the immune response by increasing the expression and/or activity of IL-12p70.

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

In certain embodiments, the disease to be treated by the composition of the invention is not mediated by IL-17 or the Th 17 pathway. In certain embodiments, the compositions of the invention increase expression or activity of IL-17 and/or the Th 17 pathway.

In preferred embodiments of the invention, the subject to whom the composition of the invention is administered is not taking lînoleic acid suppléments and / or does not hâve a dîet ri ch in linoleic acid. Additionally or altematîvely, the composition of the invention does not comprise lînoleic acid.

In embodiments, the composition of the invention does not comprise beta-galactooligosaccharides A and / or B.

Patients in need of immune stimulation may be at risk of bacterial infections. The examples demonstrate that the compositions of the invention hâve anti-mi crobi al activity. Therefore, in certain embodiments, the compositions of the invention are for use in stimulating the immune System and treating or preventing a bacterial infection. In certain embodiments, the compositions of the invention are for use in treating a bacterial infection by stimulating the immune System and înhibiting growth of the bacterial infection. In certain embodiments, the compositions of the invention are for use in promoting an immune response against a pathogénie bacteria and înhibiting growth of the bacteria. Preferably, the bacterial infection is of the gastrointestinal tract. Preferably the bacterial infection is of Gram-negative bacteria.

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

Use as a vaccine adjuvant

The examples show that administration of the compositions of the invention stimulate the immune system and can lead to an increase in expression of Tumour Necrosis Factor alpha (TNF-α) and activation of TLR2. TNF-α is known to be important for vaccine responses. For example, TNF-α has been shown to be requîred for an efficient vaccine response in a flu vaccination of the elderly population [33]. Similarly, TLR2 is an important target for vaccine adjuvants to improve responses [34], Since administration of the compositions of the invention were shown to increase TNF-α expression and TLR2 activity, compositions of the invention may be useful as a vaccine adjuvant. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of TNF-α. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of TLR2. 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 enhancîng an immune response against an antigen. In certain embodiments, the invention provides a composition to be administered in combination with an antigen. In certain embodiments, the compositions of the invention are for administration to a patient shortly prior to or after vaccination. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use as a vaccine adjuvant.

The examples demonstrate that the bacteria of the invention activate TLR2. TLR agonists are in development as vaccine adjuvants across a range of antigen types, particularly in the elderly population [35].Therefore, the compositions of the invention may be useful as vaccine adjuvants, in particular for vaccine administered to elderly patients (e.g. over 40, 50, 60, 70 or 80 years of âge), who may hâve reduced immune System activity. TLR2 signalling also plays a key rôle in age-associated innate immune responses [36]. In certain embodiments, the compositions are for use in enhancîng an innate immune response. Although TLR2 agonists are in development as vaccine adjuvants, these are ail 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-Ιβ. Li et al. [37] showed that the adjuvant aluminium hydroxîde activated the sécrétion of IL-1 β, and suggested that IL-β itself can act as an adjuvant. Since administration of the compositions of the invention were shown to increase IL-1 β expression, compositions of the invention may be useful as a vaccine adjuvant.

The examples demonstrate that the compositions of the invention can increase IFNy levels and promote a Thl cell response, both of which are associated with increase antibody responses against antigents [38].In certain embodiments, the compositions of the invention are for use in promoting an antibody response against an antigen, in particular a pathogenic or cancer antigen.

Also, is a IFN-γ measure of vaccine induced T-cell responses in volunteers receiving investigated malaria vaccines [39], In certain embodiments, the compositions of the invention are for use in promoting an T-cell response against an antigen, in particular a pathogenic or cancer antigen. In one embodiment, the compositions of the invention are for use as a vaccine 5 adjuvant by increasing the level and/or activity of IFN-γ. In certain embodiments, the compositions are for use 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-12p70. This effect has been associated with vaccine adjuvant efficiency and IL· 12 has been proposed as an adjuvant itself [40], which suggests the 10 compositions of the invention will be effective as adjuvants. In one embodiment, the compositions of the invention are for use as a vaccine adjuvant by increasing the level and/or activity of IL-12p70.

In some embodiments, when used as a vaccine adjuvant, the compositions of the invention will be administered on their own to provide an adjuvant effect for an antigen that has been 15 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 proteîn and/or saccharide antigens; fungal antigens; 20 parasite antigens; and tumor antigens. The invention is particularly useful for vaccines against influenza virus, HIV, hookworm, hepatitis B virus, herpes simplex virus, rabies, respiratory syncytial virus, cytomégalovirus, Staphylococcus aureus, chlamydia, SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus, human papillomavirus, etc. Further antigens 25 for use with the invention include glycoprotein and lipoglycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for example nicotine, alcohol or 30 opiates.

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

The invention also provides the use of: (i) an aqueous préparation of an antigen; and (ii) a composition comprising a bacterial strain of the species B, breve, in the manufacture of a médicament for raising an immune response in a patient. Preferably, the bacterial strain is the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof.

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

In some embodiments, a bacterial strain of the species Bifidobacterium brève is engineered to présent an antigen. Presenting an antigen on the bacterial strain of the invention may maximise the immunostimulatory activities and further enhance the protective immune response generated against the antigen. In addition, manufacturing and delivering therapeutics comprising an antigen and a bacteria of the invention may be more efficient and effective this way than when each of the antigen and the composition comprising the bacterial strain are manufactured and administered separately. Therefore, in some embodiments, the invention provides a composition comprising a bacterial strain of the species Bifidobacterium breve that présents an antigen, for example on its cell surface. In some embodiments, the composition comprising the bacterial strain that présents an antigen is for use as a vaccine antigen. In some embodiments, the antigen is derived from HIV, hookworm, hepatitis B virus, herpes simplex virus, rabîes, respiratory syncytial virus, cytomégalovirus, Staphylococcus aureus, chlamydia, SARS coronavirus, varicella zoster virus, Streptococcus pneumoniae, Neisseria meningitidis, Mycobacterium tuberculosis, Bacillus anthracis, Epstein Barr virus or human papillomavirus. In some embodiments, the antigen is a glycoprotein antigen, lipoglycan antigen, archaea antigen, melanoma antigen E (MAGE), 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-BaiT virus antigens, neoantigens, oncoproteîns, amyloid-beta, Tau, PCSK9 or a habit forming substance, such as, alcohol, opiates and the like.

In some embodiments, the bacteria of the invention expresses 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 Bifidobacterium breve 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 expresses the antîgen as a non-fusion polypeptide. In some embodiments, the invention provides a composition comprising a cell of a bacterial strain of the species Bifidobacterium breve, 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 Bifidobacterium breve, wherein the cell expresses a heterologous antigen. In some embodiments, the cell is for use as a vaccine.

Exempïary antigens for use with the invention include: viral antigens, such as viral surface proteins; bacterial antigens, such as proteïn and/or saccharide antigens; fungal antigens; parasite antigens; and tumor antigens. Further antigens for expressing in a bacterial strain of the species Bifidobacterium breve include glycoprotein and lipogiycan antigens, archaea antigens, melanoma antigen E (MAGE), Carcinoembryonic antigen (CEA), MUC-1, HER2, sialyl-Tn (STn), human telomerase reverse transcriptase (hTERT), Wilms tumour gene (WT1), CA-125, prostate-specific antigen (PSA), Epstein-Barr virus antigens, neoantigens, oncoproteins, amyloid-beta, Tau, PCSK9 and habit forming substances, for ex ample nicotine, alcohol, opiates, or the like.

The invention may also be useful for enhancing the response to vaccines agamst noncommunicable diseases such as Alzheimer’s Dîsease and other neurodegenerative disorders, in which case the antigen for use with the invention may be amyloid-beta or Tau. Other such antigens for non-communicable diseases include PCSK9 (for the treatment of elevated cholestérol).

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

The examples also demonstrate that the compositions of the invention hâve anti-microbial activity. Therefore, the compositions of the invention may be particularly effective for use in vaccines against bacterial infections, in particular Gram-negative bacterial infections. The compositions of the invention may exert an anti-microbial effect against the bacterial infection whilst also stimulating the immune system to tackle the infection. Therefore, in certain embodiments, the compositions of the invention are treating a bacterial infection and preventing future bacterial infections.

Cell thérapies

Chimeric Antigen Receptor T cell (CAR-T) therapy

The examples also show that administration of the compositions of the invention can lead to an increase in activation of TLR2. TLR2 stimulation potentiates the efficacy of CAR-T therapy [41], Therefore, compositions ofthe 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. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

The ex amples also show that administration of the compositions of the invention can lead to an increase in activation of NFkB. NFkB activation improves the potency of CAR-T therapy [42]. Therefore, compositions of the invention may be usefi.il in cell therapy, in particular CAR-T cell therapy.

In certain embodiments, the compositions of the invention are administered to a patient before T cell adoptive transfer during CAR-T therapy.

In certain embodiments, the compositions of the invention are administered to a patient after T cell adoptive transfer during CAR-T therapy.

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

Mesynchymal stem cell (MSC) therapy

Mesynchymal stem cell (MSC) therapy has been reported to hâve immunostimulatory properties. When MSCs are treated with LPS, they upregulate pro-inflammatory cytokines IL-8 which causes increased B cell prolifération [43]. Therefore, since compositions of the invention were shown to increase the expression of B cell prolifération, they may be useful in combination with MSC cell therapy.

Stem Cell Transplantation Therapy

It has been reported that, instead of using undifferentiated stem cells in stem cell transplantation therapy, it may be bénéficiai to differentiate stem cells to some extent prior to transplantation. For example, Heng et al. [44] reported that cardiomyogenic différentiation of stem cells may be bénéficiai by having a higher engraftment efficiency, enhanced régénération of myocytes and increased restoration of heart function. Also, studies hâve shown that GI colonisation with certain commensal strains of bacteria can improve survival following allogeneic haematopoietic cell transplant [45], Since administration of the compositions of the invention stimulated cells, compositions of the invention may be useful for stem cell différentiation in stem cell transplantation therapy.

Immunosenescence

Fulop et al. [46] identified that an increase in Treg cell number and a decrease in B cell number are associated with aging in the adaptive immune System. Therefore, compositions of the invention may be used to prevent or delay immunosenescence. In one embodiment, compositions of the invention are for use in preventing immunosenescence. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by a decrease in B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence characterised by an increase in Treg cell number and a decrease in B cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by decreasing Treg cell number. In one embodiment, compositions of the invention are for use in delaying immunosenescence by increasing B cell number. In another embodiment, compositions of the invention are for use in delaying immunosenescence b y 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. Preferably, the invention provides a composition comprising the strain deposited under accession number 42380 at NCIMB, or a dérivative or biotype thereof, for any such use.

Furthermore, it has been proposed that vaccine adjuvants may overcome immunosenescence [47], 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, neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, cancer, diabètes mellitus type 2 [48] and autoimmune disorders [49].

Subjects suffering from immunosenescence may be susceptible to bacterial infections. The examples show that the compositions of the invention hâve anti-microbial activity. Therefore, in certain embodiments, the compositions of the invention are for use in treating or preventing a bacterial infection in a patient exhibiting immunosenescence, such as an elderly patient, or a patient over 50, 55, 60, 65, 70 or 75 years of âge.

Treatins and preventing bacterial infections

The examples demonstrate that B. breve and particularly the B. breve strains of the invention hâve potent anti-microbial activity. Therefore, in certain embodiments, the compositions of the invention are for treating or preventing a bacterial infection.

In preferred embodiments, the infection is a gastro-intestinal infection. B. breve strains, and in particular B. breve strains of the invention hâve been shown to hâve potent effects when administered to the gastro-intestinal tract (see the examples and WO2016/2032). In preferred embodiments, the infection is a Gram-negative bacterial infection. In especially preferred embodiments, the infection is a Gram-negative gastro-intestinal infection, such as a Hélicobacter pylori, Salmonella enteritidis, Salmonella typhi or E. coli infection. Generally, the bacterial infection is a pathogenic bacterial infection. In preferred embodiments, the composition of the invention is for use in treating or preventing gastrointestinal E. coli infection. In further preferred embodiments, the composition of the invention is for use in treating or preventing gastrointestinal 5. enierica infection.

In some embodiments, the bacterial infection is of a genus selected from the list consisting of: Escherichia, Klebsiella, Salmonella, and Bacillus. In some embodiments, the bacterial infection for treatment or prévention is E. coli infection. In some embodiments, the bacterial infection for treatment or prévention is Klebsiella pneumoniae infection. In some embodiments, the bacterial infection for treatment or prévention is 5. Typhimurium infection. In some embodiments, the bacterial infection for treatment or prévention is B. subtilis infection. The compositions of the invention are shown to hâve potent anti-microbial activity against these bacteria.

In some embodiments, the bacterial infection for treatment or prévention is Pseudomonas aeruginosa infection. In some embodiments, the bacterial infection for treatment or prévention is Neisseria gonorrhoeae infection. In some embodiments, the bacterial infection for treatment or prévention is, Chlamydia trachomatis infection. In some embodiments, the bacterial infection for treatment or prévention is Yersinia pestis infection. In some embodiments, the bacterial infection for treatment or prévention is Neisseria meningitidis infection. In some embodiments, the bacterial infection for treatment or prévention is Moraxella catarrhalis infection. In some embodiments, the bacterial infection for treatment or prévention is Haemophilus influenzae infection. In some embodiments, the bacterial infection for treatment or prévention is Légionella pneumophila infection. In some embodiments, the bacterial infection for treatment or prévention 5 is Pseudomonas aeruginosa, infection. In some embodiments, the bacterial infection for treatment or prévention is Proteus mirabilis infection. In some embodiments, the bacterial infection for treatment or prévention is Enterobacter cloacae infection. In some embodiments, the bacterial infection for treatment or prévention is Serratia marcescens infection. In some embodiments, the bacterial infection for treatment or prévention is Hélicobacter pylori infection.

In some embodiments, the bacterial infection for treatment or prévention is Salmonella Enterîtidis infection. In some embodiments, the bacterial infection for treatment or prévention is Salmonella Typhî infection. In some embodiments, the bacterial infection for treatment or prévention is Salmonella Paratyphi infection. These bacteria are Gram-négative so may be susceptible to the compositions of the invention, as shown in the examples.

In some embodiments, the composition of the invention is for use in reducing the viability of a bacteria in the treatment of a bacterial infection. The bacteria of the invention may be used to restore the level of pathogénie bacteria to asymptomatîc ievels or to eliminate the pathogenic bacteria entirely from a subject, thereby treating the bacterial infection, in addition to alleviating the symptoms associated with the elevated level of the bacteria. In some embodiments, the 20 composition of the invention is for use in inhibiting the growth of a bacteria in the treatment of prévention of a bacterial infection. In other words, the composition may hâve cytostatic activity with respect to bacteria causing an infection.

In certain embodiments, the composition delays the onset of a récurrent infection or prevents a récurrent infection. In certain embodiments, the subject to be treated is at risk of developing a 25 bacterial infection, such as is an asymptomatîc carrier of the bacteria. In other embodiments, the compositions of the invention are for use in treating a patient exhîbiting symptoms of a bacterial infection.

Preferably, the bacteria used in the invention for treating or preventing a bacterial infection is able to ferment raffinose, for example when cultured in an appropriaie suspension medium (such 30 as API suspension medium) at 37°C for 4 hours.

Preferably, the bacteria used in the invention for treating or preventing a bacterial infection exhibit intermediate fermentation of β-glucosidase or întermediate fermentation of a-arabinose, or more preferably intermediate fermentation of β-glucosidase and intermediate fermentation of α-arabinose, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that both B. breve strains MRX004 and Test 3 hâve useful activity and both exhibit intermediate fermentation of β-glucosidase and a-arabinose.

Preferably, the bacteria used in the invention for treating or preventing a bacterial infection do not exhibit positive fermentation of N-acetyl-β-glucosaminidase, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The bacteria may exhibit only intermediate or no fermentation of N-acetyl-β-gIucosaminidase. The examples demonstrate that both B. breve strains MRX004 and Test 2 hâve useful activity and neither exhibits positive fermentation of N-acetyl-p-glucosaminidase.

Preferably, the bacteria used in the invention for treating or preventing a bacterial infection exhibit intermediate fermentation of α-galactosîdase or intermediate fermentation of a-arabinose, or more preferably intermediate fermentation of α-galactosîdase and intermediate fermentation of α-arabinose, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that B. breve strains MRX004 and Test 8 both hâve useful activity and both exhibit intermediate fermentation of α-galactosidase and a-arabinose.

In alternative embodiments, the bacteria used in the invention for treating or preventing a bacterial infection ferment serine arylamidase but not leucyl glycine arylamidase and not alanine arylamidase, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that B. breve strains Test II and Test 12 both hâve useful activity and both ferment serine arylamidase but not leucyl glycine arylamidase and not alanine arylamidase.

In alternative embodiments, the bacteria used in the invention for treating or preventing a bacterial infection exhibit intermediate fermentation of serine arylamidase, for example when cultured in an approprîate suspension medium (such as API suspension medium) at 37°C for 4 hours, and for example when subjected to the Rapid ID 32A analysis. The examples demonstrate that B. breve strains Test 3 and Test 7 both hâve potent anti-microbial activity and both exhibit intermediate fermentation of serine arylamidase.

Any suitable assay known in the art may be used to assess the ability of a bacterium to ferment a carbohydrate source or amino acid. Preferebly, the Rapid ID 32A analysis is used (preferably using the Rapid ID 32A System from bioMérîeux).

Modes of administration

Preferably, the compositions of the invention are formulated to be administered to the gastrointestinal tract in order to enable delivery to and/or partial or total colonisation of the intestine with the bacterial strain of the invention. In some embodiments, the term “total colonisation of the intestine’’ means that bacteria hâve colonised ail parts of the intestine (i.e. the small intestine, large intestine and rectum). In further embodiments of the invention, the term “total colonisation” or “partial colonisation” means that the bacteria are retained permanently or temporarily in the intestine, respectively. Generally, the compositions of the invention are administered orally, but they may be administered rectally, întranasally, or via buccal or sublingual routes.

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

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

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

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

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

In certain embodiments, the composition of the invention may be administered to a prégnant animal, for example a mammal such as a human in order to reduce the likelihood of disease developing in her child in utero and ! or aller it is bom.

The compositions of the invention may be administered to a patient that has been diagnosed with a disease or condition mediated reduced immune activity, or that has been identified as being at risk of a disease or condition mediated by reduced immune activity. The compositions may also be administered as a prophylactic measure to prevent the development of diseases or conditions mediated by reduced immune activity in a healthy patient.

The compositions of the invention may be administered to a patient that has been diagnosed with déficient immune activity, or that has been identified as being at risk of déficient immune activity. For example, the patient may hâve reduced or absent colonisation by B. breve,

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

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

Compositions

Generally, the composition of the invention comprises bacteria. In preferred embodiments of the invention, the composition is formulated in freeze-dried form. For example, the composition of the invention may comprise granules or gelatin capsules, for example hard gelatin capsules, comprising a bacterial strain of the invention.

Preferably, the composition of the invention comprises lyophilised bacteria. Lyophilisation of bacteria is a well-established procedure and relevant guidance is available in, for example, references [50,52]. The examples demonstrate that lyophilised compositions are partîcularly effective.

Altematively, the composition of the invention may comprise a live, active bacterial culture.

In preferred embodiments, the composition of the invention is encapsulated to enable delîvery of the bacterial strain to the intestine. Encapsulation protects the composition from dégradation untîl 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 useiul for preparing compositions of the invention is availabié in, for example, references [53] and [54],

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

In some embodiments, the composition does not comprise hydrolysed cow’s whey.

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

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

In certain embodiments, the dose of the bacteria is at least 10⁹ cells per day, such as at least 10¹⁰, at least 10¹¹, or at least 10¹² cells per day.

In certain embodiments, the composition contains the bacterial strain in an amount of from about 1x10 to about I x 10¹¹ CFU/g, respect to the weight of the composition; for example, from about 1 x 10⁸ to about 1 x 10¹⁰ CFU/g. The dose may be, for example, 1 g, 3g, 5g, and 10g.

A dose ofthe composition may comprise the bacterial strain from about 1 x 10⁶to about 1 x I0¹¹colony forming units (CFU) /g, respect to the weight of the composition. The dose may be suitable for an adult human. For example, the composition may comprise the bacterial strain from about 1 x 10³ to about 1 x 10¹¹ CFU/g; for example, from about 1 x 10⁷ to about 1 x 10ⁱ⁰

CFU/g; in another exampie from about 1 x 10⁶ to about 1 x 10¹⁰ CFU/g; in another example from about 1 x 10⁷ to about 1 x 10¹¹ CFU/g; in another example from about 1 x 10⁸ to about 1 x 10^lüCFU/g; in another example from about 1 x 10⁸ to about 1 x 10^H CFU/g, The dose may be, for example, 1 g, 3g, 5g, and 10g.

In certain embodiments, the invention provides the above pharmaceutical composition, wherein the amount of the bacterial strain is from about 1 x 10³ to about 1 x 10^lt colony forming unîts 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 lOOOmg, between 600mg and 900mg, between 700mg and 800mg, between 500mg and 750mg or between 750mg and lOOOmg. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the lyophilised bacteria in the pharmaceutical composition is administered at a dose of between 500mg and lOOOmg, between 600mg and 900mg, between 700mg and 800mg, between 500mg and 750mg or between 750mg and lOOOmg.

The composition may be formulated as a probiotic. A probiotic is defined by the FAO/WHO as a live microorganism that, when administered in adéquate amounts, confers a health benefit on the host.

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 transgai acto-oligosaccharides.

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

The compositions of the invention may comprise phannaceutically acceptable excipients or carriers. Examples of such suitable excipients may be found in the reference [55]. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art and are described, for example, in reference [56]. Examples of suitable carriers include lactose, starch, glucose, methyl cellulose, magnésium stéarate, mannitol, sorbitol and the like. Examples of suitable diluents include éthanol, glycerol and water. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as, or in addition to, the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s), solubilising agent(s). Examples of suitable binders include starch, gelatin, natural sugars such as glucose, anhydrous lactose, free-flow lactose, beta-lactose, com sweeteners, natural and synthetic gums, such as acacia, tragacanth or sodium alginate, carboxymethyl cellulose and polyethylene glycol. Examples of suitable lubricants include sodium oleate, sodium stéarate, magnésium stéarate, sodium benzoate, sodium acetate, sodium chloride and the like. Preservatives, stabilizers, dyes and even flavouring agents may be provided 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 supplément. Similarly, a food product may be formulated to enhance the taste of the composition of the invention or to make the composition more attractive to consume by being more similar to a common food item, rather than to a pharmaceutical composition. In certain embodiments, the composition of the invention is formulated as a milk-based product. The term milk-based product means any liquid or semi-solid milk- or whey- based product having a varying fat content. The milk-based product can be, e.g., cow's milk, goat's milk, sheep's milk, skîmmed 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. In certain embodiments, the compositions of the invention contain a single bacterial species and do not contain any other bacterial species. In certain embodiments, the compositions of the invention contain a single bacterial strain and do not contain any other bacterial strains. For example, the compositions of the invention may comprise bacteria only of the species Bifidobacierium breve. 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. In some embodiments, such compositions may be a lyophilisate that is substantially free from other species of organism.

In some embodiments, the compositions of the invention comprise more than one bacterial strain or species. For example, in some embodiments, the compositions of the invention comprise more than one strain from within the same species (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40 or 45 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise less than 50 strains from within the same species (e.g. less than 45, 40, 35, 30, 25, 20, 15, 12, 10, 9, 8, 7, 6, 5, 4 or 3 strains), and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise 1-40, 1-30, 1-20, 1-19, 1-18, 1-15, 1-10, 1-9, 1-8, 17, 1-6, 1-5, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 strains from within the same species and, optionally, do not contain bacteria from any other species. In some embodiments, the compositions of the invention comprise more than one species from within the same genus (e.g. more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, 23, 25, 30, 35 or 40 species), and, optionally, do not contain bacteria from any other genus. In some embodiments, the compositions of the invention comprise less than 50 species from within the same genus (e.g. less than 50, 45, 40, 35, 30, 25, 20, 15, 12, 10, 8, 7, 6, 5, 4 or 3 species), and, optionally, do not contain bacteria from any other genus. In some embodiments, the compositions of the invention comprise 1-50, 1-40, 1-30, 1-20, 1-15, 1-10, 1-9, 1-8, 1-7, 1-6, 15, 1-4, 1-3, 1-2, 2-50, 2-40, 2-30, 2-20, 2-15, 2-10, 2-5, 6-30, 6-15, 16-25, or 31-50 species from within the same genus and, optionally, do not contain bacteria from any other genus. The invention comprises any combination of the foregoing.

In some embodiments, the composition comprises a microbial consortium. For example, in some embodiments, the composition comprises the Bifidobacierium breve bacterial strain as part of a microbial consortium. For example, in some embodiments, the Bifidobacierium breve bacterial strain is présent in combination with one or more (e.g. at least 2, 3, 4, 5, 10, 15 or 20) other bacterial strains from the genus Blautia and/or other généra with which it can live symbiotîcally in vivo in the intestine. For example, in some embodiments, the composition comprises a bacterial strain of Bifidobacterium breve in combination with a bacterial strain from a different genus. In another example, the composition comprises a bacterial strain of Bifidobacterium breve in combination with a bacterial strain from the genus Bifidobacterium or the composition comprises a bacterial strain of Bifidobacterium breve in combination with a bacterial strain from the genus Bifidobacterium and a bacterial strain from a different genus. In some embodiments, the microbial consortium comprises two or more bacterial strains obtained from a faeces sampie of a single organism, e.g. a human. In some embodiments, the microbial consortium is not found together in nature. For example, in some embodiments, the microbial consortium comprises bacterial strains obtained from faeces samples of at least two different organîsms. In some embodiments, the two different organisms are from the same species, e.g. two different humans. In some embodiments, the two different organisms are an infant human and an adult human. In some embodiments, the two different organisms are a human and a nonhuman mammal.

In some embodiments, the composition of the invention additionally comprises a bacterial strain that has the same safety and therapeutic efficacy characteristics as the Bifidobacterium breve strain deposited under accession number NCIMB 42380, but which is not the Bifidobacterium breve strain deposited under accession number NCIMB 42380.

In some embodiments in which the composition of the invention comprises more than one bacterial strain, species or genus, the individual bacterial strains, species or généra may be for séparai e, simultanée us or sequential administration. For example, the composition may comprise ail of the more than one bacterial strain, species or généra, or the bacterial strains, species or généra may be stored separately and be administered separately, simultaneously or sequentially. In some embodiments, the more than one bacterial strains, species or généra are stored separately but are mixed together prior to use.

In some embodiments, the bacterial strain for use in the invention is obtained from human adult faeces. In some embodiments in which the composition of the invention comprises more than one bacterial strain, ail of the bacterial strains are obtained from human adult faeces or if other bacterial strains are présent, they are présent only in de minimis amounts. The bacteria may hâve been cultured subséquent to being obtained from the human adult faeces and being used in a composition of the invention.

In some embodiments, the one or more Bifidobacterium breve bacterial strains is/are the only therapeutically active agent(s) in a composition of the invention. In some embodiments, the bacterial strain(s) in the composition is/are the only therapeutically active agent(s) in a composition of the invention.

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

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 said bacterial strain is spray dried. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is live. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is capable of parti ally or totally colonising the intestine. In certain embodiments, the invention provides the above pharmaceutical composition, wherein the bacterial strain is lyophilised or spray dried and wherein it is viable and capable of partiaily or totally colonising the intestine.

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

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

In certain embodiments, the invention provides a pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficîent to treat a disorder when admmistered 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 sufficîent to treat or prevent a disease or condition.

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

In certain embodiments, the invention provides pharmaceutical composition comprising: a bacterial strain of the invention; and a pharmaceutically acceptable excipient, carrier or diluent; wherein the bacterial strain is in an amount sufficient to treat or prevent a disease or condition mediated a reduced immune response.

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

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

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

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

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

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

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

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

In certain embodiments, the invention provides the above pharmaceutical composition, wherein saîd bacteria] strain is lyophilised.

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

In some embodiments, the composition of the invention is provided in a sealed container comprising a composition as described herein. In some embodiments, the sealed container is a sachet or bottle. In some embodiments, the composition of the invention is provided in a syringe comprising a composition as described herein.

The composition of the présent invention may, in some embodiments, be provided as a pharmaceutical formulation. For example, the composition may be provided as a tablet or capsule. In some embodiments, the capsule is a gélatine capsule (“gel-cap”). The capsule can be a hard or a soft capsule. In some embodiments, the formulation is a soft capsule. Soft capsules are capsules which may, owing to additions of soft en ers, such as, for example, glycerol, sorbitol, maltitol and polyethylene glycols, présent in the capsule shell, hâve a certain elasticity and softness. Soft capsules can be produced, for example, on the basis of gélatine or starch. Gelatinebased soft capsules are commercially available from varions suppliers. Depending on the method of administration, such as, for example, orally or rectally, soft capsules can hâve various shapes, they can be, for example, round, oval, oblong or torpedo-shaped. Soft capsules can be produced by conventional pro cesses, such as, for example, b y the Scherer process, the Accogel process or the droplet or blowing process.

In some embodiments, the compositions of the invention are administered orally. Oral administration may involve swallowing, so that the compound enters the gastrointestinal tract.

Pharmaceutical formulations suitable for oral administration include solid plugs, solid microparticulates, semi-solid and liquid (including multiple phases or dispersed Systems) such as tablets; soft or hard capsules containing multi- or nano-particulates, liquîds (e.g. aqueous solutions), émulsions or powders; lozenges (including liquid-filled); chews; gels; fast dispersing dosage forms; films; ovules; sprays; and buccal/mucoadhesive patches.

In some embodiments the pharmaceutical formulation is an enteric formulation, i.e. a gastrorésistant formulation (for example, résistant to gastric pH) that is suitable for delîvery of the composition of the invention to the intestine by oral administration. Enteric formulations may be particularly useful when the bacteria or another comportent of the composition is acidsensitive, e.g. prone to dégradation under gastric conditions.

In some embodiments, the enteric formulation comprises an enteric coating. In some embodiments, the formulation is an enteric-coated dosage form. For example, the formulation may be an enteric-coated tablet or an enteric-coated capsule, or the like. The enteric coating may be a conventional enteric coating, for example, a conventional coating for a tablet, capsule, or the like for oral delivery. The formulation may comprise a film coating, for example, a thin film layer of an enteric polymer, e.g. an acid-insoluble polymer.

In some embodiments, the enteric formulation is intrinsically enteric, for example, gastrorésistant without the need for an enteric coating. Thus, in some embodiments, the formulation is an enteric formulation that does not comprise an enteric coating. In some embodiments, the formulation is a capsule made from a thermogelling material. In some embodiments, the thennogelling material is a cellulosîc material, such as methylcelhdose, hydroxymethylcellulose or hydroxypropylmethyl cellulose (HPMC). In some embodiments, the capsule comprises a shell that does not contain any film forming polymer. In some embodiments, the capsule comprises a shell and the shell comprises hydroxypropylmethylcellulose and does not comprise any film forming polymer (e.g. see [57]). In some embodiments, the formulation is an intrinsically enteric capsule (for example, Vcaps® from Capsugel).

Culturing methods

The bacterial strains for use in the présent invention can be cultured using standard mîcrobiology techniques as detailed in, for example, référencés [58-60],

The solid or liquid medium used for culture may be YCFA agar or YCFA medium. YCFA medium may include (per 100ml, approximate values): Casitone (1.0 g), yeast extract (0.25 g), NaHCO₃ (0.4 g), cysteine (0.1 g), K₂HPO₄ (0.045 g), KH₂PO₄ (0.045 g), NaCl (0.09 g), (NH₄)₂SO₄ (0.09 g), MgSO₄ 7H₂O (0.009 g), CaCl₂ (0.009 g), resazurin (0.1 mg), hemin (1 mg), biotîn (1 pg), cobalamin (1 pg), p-aminobenzoic acid (3 pg), folie acid (5 pg), and pyridoxamine (15 pg).

Bacterial strains for use in vaccine compositions

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

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

General

The practice of the présent invention will employ, unless otherwise indîcated, conventional methods of chemistry, biochemistry, molecular biology, immunology and pharmacology, within the skill of the art. Such techniques are explained fully in the literature. See, e.g., référencés [61] and [62,68], etc.

The terni “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include soinething additional e.g. X + Y.

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

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

Référencés to a percentage sequence identity between two nucléotide sequences means that, when aligned, that percentage of nucléotides are the same in comparing the two sequences. This alignment and the percent homology or sequence identity can be determined using software programs known in the art, for example those described in section 7.7.18 of ref. [69], 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. [70].

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.

Varîous 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).

AU patent and literature référencés cited in the présent spécification are hereby incorporated by reference in their entirety.

Any reference to a method for treatment comprising administering an agent to a patient, also covers that agent for use in said method for treatment, as well as the use of the agent in said method for treatment, and the use of the agent in the manufacture of a médicament.

The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the présent invention in any way.

MODES FOR CARRYING OUT THE INVENTION

Example 1

Summary

The objective of this study was to characterise the in vitro immunomodulatory properties of MRx0004. In addition, a combination of genomics, transcriptomics and proteomics was used to identify potential key effectors which could be responsable for mediating tire host response to MRx0004.

Materials and Methods

Bacterial strains, plasmids andprimers

Ail bacterial strains and plasmids and primers used to generate strains in this study are listed in Table î. B. breve strains were routinely cultured in yeast extract-casein hydrolysate-fatty acids (YCFA) broth (E&O Labs, Bonnybridge, UK) at 37 °C in an anaérobie workstation (Don Whitley Scientîfïc, Shipley, UK) unless otherwise stated. E. coli strains were routinely cultured in Luria Bertani (LB) broth [71] at 37 °C with agitation. Where approprîate, growth media was supplemented with tétracycline (10 pg/ml), chloramphenîcol (10 pg/ml for E. coli or 3 pg/ml for B. breve), erythromycin (100 pg/ml for E. coli or 1 pg/ml for B. breve), spectinomycin (100-300 pg/ml), or kanamycin (50 pg/ml) (ail antibiotics from Sigma-Aldrich, Gillingham, UK). Recombinant E. coli cells containing pORI19 or pWSK29 were selected on LB agar supplemented with 40 pg/ml X-gal (5-bromo-4-chIoro-3-indolyl-p-D-galactopyranoside) and 0.1 M IPTG (isopropyl-p-D-galactopyranoside) (both supplied by Sigma-Aldrich).

Routine culture of immortalised cells

HT29-MTX-E12 cells (Public Health England, Saiisbury, UK) were routinely cultured in Dulbecco’s Minimal Eagle’s Medium (DMEM) with high glucose modification, supplemented with 10 % (v/v) foetal bovine sérum (FBS), 4 mM L-glutamine, l X non-essentîal amino acid solution and 1 X antibiotic antîmycotic solution. Cells were seeded into assay vessels and cultured for nine days, following which they were washed twice with Hank’s Balanced Saline Solution and placed into co-culture medium (DMEM supplemented with 4 mM L-glutamine, 1 X non-essential amino acid solution, 5 pg/ml apo-transferrin and 200 ng/ml sodium selenite) prior to the beginning of treatments.

Préparation of bacterial treatments for co-culture assays

For co-culture experiments, bacteria were cultured until they reached log phase. Lîve bacterial cells and supematant were separated by centrifugation, following which live bacteria (designated lv) were washed once with PBS ( Sigma-Aldri ch) and resuspended in the appropriate cell culture medium for downstream use. Supernatants (designated _Sn) were passed through a 0.22 pm fïlter and diluted appropriately in co-culture medium. Heat-inactivated bacteria (designated _Hk) were prepared by incubation at 80 °C for 30 minutes, followed by washing with PBS and resuspension în appropriate cell culture medium. Viable counts were confirmed by plating.

Reporter assays

HEK-Blue™-hTLR2 and THPl-Blue™ NF-κΒ cells were grown to 90% density, washed once with PBS and resuspended in culture media without antibiotic at a density of 280,000 and 500,000 cells/ml, respectively. Bacterial treatments (lîve, heat-killed and supernatants) were added to cells at a multiplicity of infection (MOI) of 100:1. Positive assay Controls, PamCS3K4 (Invivogen) and heat-killed L. monocytogenes (HKLM) (Invivogen) were used at 10 ng/ml concentrations and MOI of 200:1, respectively. Négative Controls, media and vehîcles, were prepared to provide équivalents for each treatment. Cells were then incubated at 37°C and 5% CO₂ for 22 h. Medium from co-cultures was diluted ten-fold in QUANTI-Blue™ (Invivogen), and incubated for 1 hour (NFkB) or 2 hours (TLR2) and optical density at 655 nm was recorded.

Large scale co-cultures with HT29-MTX cells

HT29-MTX cells were cultured as described previously, in the upper chamber of 10 cm dîameter Transwells® (Thermo Fisher Scîentific, Waltham, MA, USA). Bacteria were cultured to late log phase, washed and resuspended as described previously. Bacteria were added to cells at an MOI of 100:1, and co-cultures were incubated for 3 hours in anaérobie conditions at 37 °C. Medium containing bacteria was collected from the upper chamber of the transwell, and centrifuged at 5000 x g for 5-10 minutes to collect bacterial cells for downstream applications.

Bacterial qPCR analysis

Bacteria were collected for RNA isolation from in vitro culture at late log and stationary growth phases, and post-large scale co-culture with HT29-MTX. Bacteria were collected and stored using RNAProtect Bacteria Reagent as per manufacturer’s instructions (QIAGEN, Hilden, Germany). Bacterial cells were lysed by incubation with lysozyme (15 mg/ml) (Sigma-Aldrich) and protéinase K (6 mAU) (QIAGEN) at 37 °C for 30 minutes, and subsequently homogenised using a FastPrep 24 instrument (2 x cycles of 6 m/s for 20 s), and Lysing Matrix B (both from MP Biomedicals, Santa Ana, CA, USA). Total RNA was isolated using an RNeasy Mini Kit (QIAGEN), and genomic DNA was removed using RNase-Free DNase in an on-column digest (QIAGEN), both as per manufacturer’s instructions. cDNA was synthesîsed using a Superscript IV kit (Thermo Fisher Scientific) as per manufacturer’s instructions. Primers were designed using Primer3Plus software [72], qPCR reactions were set up Power SYBR™ Green PCR Master Mix (Thermo Fisher Scientific) as per manufacturer’s recommendations, and assays were carried out on a 7500 Fast Real-time PCR System (Thermo Fisher Scientific), using the following cycle: 95 °C for 10 mins, followed by 40 cycles of 95 °C for 15 s, 60 °C for 1 min. Data was analysed was carried out using double delta Ct analysis and expression of test genes was nonnalised to the housekeepergraEL.

Bacterial cell shaving

Bacterial cells were harvested by centrifugation in late log growth phase or following contact with HT29-MTX (see co-culture section above for details), as appropriaie. Cells were then washed and resuspended in 50 mM TEAB buffet pH 8.5 (Sigma-Aldrich) at a 1/20 dilution. Shaved protein fractions were generated by incubating cells with sequencing grade modified trypsin (Promega, Madison, WI, USA) for 30 min at 37°C in 50 mM TEAB buffer supplemented with 1 mM DTT (Sigma-Aldrich). For each sample, a tube without trypsin was incubâted in parai lel, as a control for shed proteins (shed protein fraction). Shaved and shed protein fractions were harvested by centrifugation for 15 min at 4000 x g at 4°C and syringe- filtered through a Millex-GV 0.22 pm low protein binding membrane (Millipore). Total protein concentrations were measured using Pierce™ BCA Protein Assay Kit according to the manufacturer’s instructions (Thermo Fisher Scientific) and sample quality was assessed by SDS-PAGE (BioRad, Hercules, CA, USA). Viable cell counts were performed before and after the trypsin treatment by plating on YCFA agar. For each assay, samples from three biological replaçâtes were then analyzed by nano-LC-MS/MS.

Protêtn identification by LC-MS/MS

In brief, culture supematants were concentrated down to 0.5 ml and washed with ultrapure water, proteins were precipitated using a ReadyPrep 2-D Cleanup Kit (Bio-Rad) and resuspended in 100 μΐ 50 mM ammonium bicarbonate. Samples were then incubated with porcine trypsin (Promega) for 16 h ai 37°C and the resultmg supematants were dried by vacuum centrifugation and dissolved in 0.1% trifluoroacetic acid. Peptides were further desalted using p-C18 ZipTips (Merck, Keniloworth, NJ, USA) and eluted into a 96-weli microtiter plate, dried by vacuum centrifugation and dissolved in 10 μΐ LC-MS loading solvent (2% acetonitrile, 0.1% formic acid). Peptides were separated and identified by nanoLC-MS/MS (Q Exactive hybrid quadrupole-Orbitrap MS System) (Thermo Fisher Scientifïc) using a 15-cm PepMap column, 60minute LC-MS acquisition method and an injection volume of 5 μΐ. For the shaved and shed protein fractions, 70 μί 50 mM ammonium bicarbonate were directly added to 30 μΐ of sample. Samples were then incubated with porcine trypsin (Promega) ovemight at 37°C and the resulting supematants were frozen at -70°C, dried by vacuum centrifugation and dissolved in 20 pL of LC-MS loading solvent. Peptides were separated and identified by nanoLC-MS/MS (Q Exactive hybrid quadrupole-Orbitrap MS System, Thermo Scientifïc) using a 25-cm PepMap column, 60minute LC-MS acquisition method, and an injection volume of 2 pL. Data analysis was performed with Proteome Discoverer (Thermo Fisher Scientifïc). The Mascot Server was used as the search engine with the following parameters: enzyme = trypsin, maximum mixed cleavage sites = 2, precursor mass tolérance = 10 ppm, dynamic modifications = oxidation (M), statîc modifications = carbamidomethyl (C). Identified peptides were matched against a strain-specific protein sequence database, which was constmcted based on the sequenced genome of B. breve MRx0004 (2,047 sequences). A protein identification was considered valid when at least five peptides were identified in ail three biologîcal replicates.

Complémentation of B. breve MRxOOOFEPS^

A DNA fragment encompassing the primary glycosyl transferase encoding gene pGTF and its assumed promoter was generated by PCR amplification from B. breve MRx0004 chromosomal DNA using Q5 High-Fidel ity Polymerase (New Engl and BioLabs, Herefordshîre, United Kingdom) and primer pairs: pGTFcompF and pGTFcompR. The resulting fragment was digested with HinDIII and Xbal (both from New England Biolabs, Ipswich, MA, USA) and ligated to the similarly digested pBCI.2. The ligation mixture was introduced into E. coli EC101 by electrotransformation and transformants were then selected based on Cm résistance. The plasmid content of a number of Cm-resistant transformants was screened by restriction analysis. The integrity of the cloned insert in a number of the recombinant plasmids was confirmed by sequencîng prior to theîr introduction into E.coli EC101 pWSK29-MRX-M+S to facilitate méthylation. Methylated pBC1.2 or pBC1.2-pGTF was transformed into MRx0004-EPS^nce by electroporation with sélection on Reinforced Clostridial Agar (RCA; Thermo Fisher Scientific) supplemented with Tet and Cm. Transformants were checked for plasmid content using colony PCR, restriction analysis of plasmid DNA, and verified by sequencîng. The resulting strains were designated B. brève MRx0004-EPS'-pBC1.2 and MRx0004- EPS’-pBC1.2-pGTF.

Cytokine analysis from HT29-MTX cells

Live bacteria (prepared as described previously) were co-incubated with HT29-MTX cells in 24 well plates at an MOI of 100:1 for 3 hours at 37 °C and 5 % COa- Human Recombinant TNFa (PeproTech, Rocky Hill, NJ, USA) was then added to cells at 10 ng/ml, following which cocultures were incubâted for a further 24 hours, and subsequently supernatants were collected and centrîfuged at 12000 x g for 3 min at 4 °C to remove cell débris. IL-8 levels in supernatants were analysed using a Human IL-8 (CXCL8) Standard ABTS ELISA Development Kit (PeproTech) as per manufacturer’s recommendations.

Co-culture with PBMCs

Healthy frozen human peripheral blood mononuclear cells (PBMCs) were purchased from STEMCELL Technologies (Cambridge, UK). Cells were thawed and left to rest ovemight in full growth media; RPMI 1640 with 10% FBS, 2mM L. Glutamine and 100 U/ml penicillin, 100pg/ml streptomycin at 37°C and 5 % CO? (ail reagents from Sîgma-Aldrich). Bacterial treatments were prepared as previously described. For co-incubations, cells were plated at a density of 750,000 cell/wells in 48 well plates and co-incubated with heat-inactivated bacteria, and bacterial supernatants at an MOI of 10:1, with appropriate vehicles and 5ug/ml PHA (SîgmaAldrich) as a control. Co-cultures were încubated for 72 h at 37°C and 5 % CO2, following which cells were collected and centrîfuged at 10000 x g for 3 minutes at 4°C. Cell-free supernatants were collected and stored at -80 °C for cytokine analysis. Cell pellets were washed once and then resuspended in PBS on ice.

Flow cytometry

Treatment wells were pooled to give 1.5 x 10⁶ PBMCs per group, resuspended in 150ul PBS and transferred to a 96 V-bottom plate ready for staîning. Cells were first stained with the Viobility 405/520 Fixable Dye (Miltenyi Biotec Ltd. Bergisch Gladbach, Germany) to discriminate between live and dead cells for 10 min in the dark at room température. They were then stained with a cocktail of antibodies for CD3, CD4, CD8, CD25, CD127 and CD19 to détermine cell phenotype (Mîltenyi REA antibodies) and incubated for a further 10 min at room température. Cell s were then washed and resuspended in PB S and immediately analysed via flow cytométrie analysis. Isotypes were used for ail antibodies during the first experiment to help set gates and FMO Controls were included throughout ail the experiments. Ail experiments were performed using a BD FAC S Aria II with the stopping gâte for acquisition set on 100,000 ceiis in the “Live” gâte using FACSDiva software (BD Biosciences, Reading, UK). The analysis was conducted using Flowjo version 10.4.2 software (FlowJo LLC, Oregon, USA) and was based on live cells identified with the viability dye.

Cytokine analysis

Cytokine quantification in cell-free supematants from PBMC co-cultures was carried ont using custom ProcartaPlex multiplex immunoassays (Thermo Fisher Scientific) following the manufacturer’s recommendations. Briefly, 50 μΐ of supematants were processed using a MAGPIX® MILLIPLEX® system (Merck) with the xPONENT software (Luminex, Austin, TX, USA). Data was analysed using the MILLIPLEX® analyst software (Merck) using a 5-parameter logistic curve and background subtraction to couvert mean fluorescence intensity (MFI) to pg/ml values.

Data analysis

Statistical analyses were carried out using GraphPad Prism version 7.00 for Windows, GraphPad Software, La Jolla CA USA. Data were analysed using one-way ANOVA and Tukey’s Multiples Comparîsons Test. Venn diagrams were generated using Interactivenn [73],

Résulta

MRx0004 stimulâtes NFkB and TLR2 reporter cells

A THP-1-NFkB reporter cell line was employed to examine the impact of MRx0004 on activation of the pro-infiammatory transcription factor NFkB, due to its intégral rôle in the régulation of innate immunity. Heat-killed Listeria monocytogenes (HKLM) (InvivoGen) was used as a positive control for this assay. In order to identify the effective fraction(s) of this strain, THP-I-NFkB cells were co-incubated with treatments of live bacteria (MRx0004lv)> bacterial culture supematant (MRx0004sn) and heat-inactîvated bacteria (MRx0004_Hk)· AU three bacterial treatments significantly activated NFkB in comparison to négative Controls of untreated cells and bacterial growth medium (YCFA) (p < 0.0001 for ail comparîsons) (Figure 7A). MRx0004_Lv was the most effective treatment and was significantly more stimulatory than MRx0004_Sn and MRx0004_Hk (p < 0.0001 for both comparisons). MRx0004_Hk was in tum significantly more active than MRx0004_Sn (p = 0.006).

As MRx0004 was shown to activate NFkB, the ability of MRx0004 to stimulate the upstream receptors TLR2, TLR4, TLR5 and TLR9 was investigated. Preliminary data suggested that MRx0004 did not activate TLR4, TLR5 and TLR9 (data not shown). A HEK-TLR2 reporter assay was treated with positive control Pam3CSK4 and the same MRx0004 treatments and négative Controls as described above (Figure 7B). Ail MRx0004 treatments stîmulated TLR2 in comparison to négative Controls (p < 0.0001 for ail comparisons). MRx0004_LV was the most stimulatory treatment compared to MRx0004sn (p < Ü.0001) and MRx0004hk (p < 0.0001), the latter of which was the least effective treatment. Based on these data, MRx0004 is capable of potently stimulatîng the innate immune response in a TLR2-associated manner.

Transcriptional and proteomic profiling of MRx0004 reveals potential host response effectors

In order to identify potential effectors of the host response in MRx0004, three approaches were employed. A targeted transcriptional assay was employed to analyse ten MRx0004 genes with predicted identities as known effectors of bifidobacterial-host interaction (data not shown). These included genes codîng for predicted adhesins and moonlighting proteins (oppA, enolase, transaldolase, tadA, eftU, ptdlulanase (reviewed extensively in [74]), and genes with putative rôles in colonisation and immune modulation (the primary glycosyltransferase (pGTF) of the MRx00Û4 EPS locus [75], luxS [76] and serpin [77]) and putative therapeutic effects (pks [78]). Expression of these genes was analysed in RNA isolated from MRx0004 grown to late log phase and stationary phase in liquid culture, and after 3 h contact with HT29-MTX cells cultured in a large-scale transwell. qPCR analysis (Figure 8) demonstrated that eftU, enolase and pGTF were significantly upregulated in late log phase compared to stationary phase, whereas the expression of oppA, ptdlulanase, serpin and tadA was significantly higher in stationary phase than in late log phase. Six of the genes (eftU, enolase, pGTF, oppA, serpin and transaldolase) were significantly upregulated in response to intestinal épithélial cells (lECs) relative to in late log phase. It was évident from this analysis that gene expression of MRx0004 was altered by contact with lECs, inferring a potential rôle for the upregulated genes in MRx0004-host interactions. Most of the significantly upregulated genes in this qPCR analysis hâve predicted rôles in adhesion to lECs, which suggests that this might be an important functional property of MRx0004.

The expression of additional host-response effectors by MRx0004 was further characterised by identifying proteins présent in culture supematant and on the cell surface. Nano-LC-MS/MS analysis identified 64 proteins in MRx0004sn (Table 2). The proteins identified with the highest number of peptides matched (PSM) were pullulanase ((351.33 ± 33.62), two NlpC/P60 family proteins (82 ± 15.62 and 71.67 ± 13.61), a solute-binding protein of ABC transporter System (56 ± 7) and the cell division protein FtsI (56 ± 4.36). Several moonlighting proteins involved in host-interaction in B. breve and other bacterial species were identified, which included transaldolase (32.67 ± 3.79), GAPDH (30 ± 3.61), DnaK (17.67 ± 3.21), GroEL (12.67 ± 0.58) and enolase (5.67 ± 0.58) [74, 79-84].

Identification of proteins présent on the surface of MRx0004 cells was performed using an enzymatic cell-shaving approach. Bacterial cells were shaved using trypsîn and cleaved surfaceassociated proteins were identified using LC-MS/MS (shaved protein fraction). Proteins from no-trypsin Controls were also harvested and analysed by LC-MS/MS, allowing identification of proteins loosely bound to the surface (shed protein fraction). A total of 106 shaved proteins were identified, 44 of which were predîcted to be anchored in the cell wall (i.e. présent in the shaved protein fraction and absent from the shed protein fraction), (Table 3, Figure 15). As observed for MRxÛ004sn, the most abundant shaved protein identified was pullulanase (136.67 ± 17.47), which was also detected in the shed proteins fractions (79 ± 10.54). Interestingly, a type I polyketide synthase was also identified in both cell shaving fractions, although more matching peptides were identified in the shaved protein fraction than in the shed protein fraction (54.67 ± 10.69 and 11.67 ± 6.43 respectively). Ail moonlighting proteins présent in MRx0004_Sn and listed above were also detected in MRx0004 cell shaving fractions (Table 3). In addition, EfTu was identified in both the shaved and shed protein fractions (PSM = 32 ± 5 and 24.33 ± 8.39 respectively). Five of the genes analysed by qPCR (eftU, enolase, luxS, pullulanase and transaldolase) were also detected in one or both of the cell shaving and supematant datasets, thus confirming the translation of these genes. The data from transcriptional and proteomic datasets collectively ailowed us to identify potential novel effectors of interest in MRx0004.

Interestingly, the amylolytic enzyme pullulanase, the most abundant protein in the cell shaving dataset, is a moonlighting protein which has been reported to be involved in the adhesion of Streptococcus pyogenes to glycoproteins and host cells in vitro [85,86], In addition, DnaK and enolase from B. animalis subsp. lactis [82,83] and EfTu from B. longum [87] hâve been reported to adhéré to plasminogen in vitro. Furthermore, recombinant expression of the glycolytic enzyme transaldolase of B. bifidum A8 in L. lactis increased the adhérence of this strain to mucin [84].

The production of these moonlighting proteins by MRx0004 suggests that they may play a rôle in the adhesive capacity of MRx0004, and thus facilitate interaction with the host cell surface. The effects of bifidobacterial moonlighting proteins on spécifie host cell receptors and signallîng pathways hâve not yet been described, and these may represent novei regulatory pathways for MRx0004 through which MRx0004 interacts with the host.

Investigating the characteristics of modulation of the innate immune response by MRx()004

Further experiments were performed to characterise the host response towards MRx0004.

To assist this study, a strain (EPS^nes) was constructed whereby the pGTF gene of the EPS locus was inactivated through insertional mutagenesis (Figure 16A). This strain was constructed by utîlising the methodology described in [88], but rather than manipulating a Type II restrictionmodification (RM) System, the methylase and specificity subunits from the MRx0004 Type I RM system were expressed and used to methylate plasmid DNA prior to transformation. A complemented EPS^neg strain (EPS^roT’^ip) and an EPS^nee empty vector strain (EPS^vec) were also generated as Controls.

The impact of EPS on TLR2 and NFkB activation was assessed by co-incubating HEK-TLR2 and THP-1-NFkB reporter cells with live and supernatant treatments of MRx0004, EPS^nes, EPS^vec and EPS^corap. Ail live bacterial treatments activated TLR2 to a comparable extent (Figure 12A). In contrast, the activation of NFkB by MRx0Û04LV was significantly lower than that of EPS^negLV (p = 0.003) and EPS^vecLv (p = 0.009), but not EPS^ÛOmpLv (Figure 12B). There was no différence in TLR2 and NFkB activation between MRx0004_SN and EPS^corapSN) demonstrating that in this instance the wild type and complément displayed a similar phenotype. EPS^negSN and EPS^vec _SN were significantly more stimulatory towards TLR2 (p < 0.0001 for both) and NFkB (p = 0.002 and 0.0013 respectively) than MRx0004sn. These data suggest that TLR2 activation by MRx0004 is not medîated directly through its EPS. In contrast, the activation of NFkB is increased in response to the exposure of the MRx0004 cell surface in the absence of EPS. These data suggest that effective Îmmunomodulation by MRx0004 is best achieved using the whole, intact cell, and that the MRx0004 ligand for TLR2 is primarily cell surface-associated, but may also be shed or secreted.

The impact of MRx0004 and its dérivative strains on an in vitro model of IEC inflammation was also investigated. HT29-MTX cells were primed with MRx0004 and its dérivatives for 3 hours, foliowing whîch TNFa was added to wells as an înflammatory stimulant for a further 24 hours. Using this model, MRx0004 dîd not reduce TNFa-mediated IL-8 sécrétion in comparison with a no bacteria control (Figure 12C). However, 1L-8 sécrétion in non-TNFa-stimulated cells was decreased by MRx0004 treatment compared to a baseline control. EPS^neg treatment resulted in a significant réduction of TNFct-induced 1L-8 sécrétion compared to that of MRx0004 (p < 0.0001). IL-8 sécrétion in EPS^vec- and EPS^coinp-treated cells was also significantly lower than in response to MRx0004 (p < 0.0Ü01 for both comparisons). Interestingly, it appeared that the unshielding of surface-associated antigens had an anti-inflammatory effect on IEC s, in contrast to the effects demonstrated by EPS^neg in reporter assays.

Impact of MRx0004 on the adaptive immune response

To examine the effect of MRx0004 on the adaptive immune System, peripheral blood mononuclear cells (PBMCs) from healthy human donors were used to characterise cell populations and cytokine sécrétion profiles. PHA was used as a positive control in this assay (data not shown). PBMCs were co-incubated with heat-inactivated bacterial cells and cell-free culture supematants from MRx0004 and its dérivative strains for 72 hours. The expression of Tcell (CD3⁺CD4⁺ and CD3'CD8*), Treg (CD3⁺CD4⁺CD25⁺CD127) and B-cell (CD3‘CD19⁺) surface markers were analysed (along with activation marker CD25) by flow cytometry (refer to Figure 18 for gating strategy). Heat-inactivated rather than live bacteria were used as treatments in this model due to the probability that live bacteria would grow and outcompete human cells for nutrients during the 72 h incubation period. The expression of both the cell surface markers and cytokines in response to bacterial supematants from ail strains tested was not significantly different compared to that observed in response to the vehicle (YCFA) (data not shown). Data for EPS^vec and EPS^comp are illustrated in Figures 20 and 21.

MRx0004hk treatment resulted in a significant increase in activated CD8⁺CD25^h subsets compared to the untreated control (p = 0.0038, Figure 13A), whilst EPS^negHKdid not significantly increase the percentage of activated CD8^rCD25⁺ cells compared to Controls. Neither MRx0004hk nor EPS^negHK significantly încreased the percentage of CD8⁺, CD4⁺ or CD4⁺CD25 T-cell populations compared to Controls (Figure 19A and B, Figure 13B). The B cell population was increased by a similar, statistically significant extent by both MRx0004hk and EPS^negHK in comparison to untreated cells (p = 0.001 and 0.0013 respectively, Figure 13E). B-cell activation (CD19⁺CD25⁺) was not significantly affected by any of the applied treatments (Figure 19-C). Within the CD4⁺ cell population, the proportion of Treg cells was analysed using the CD25XD127* surface markers. An increase in the relative percentage of Tregs was observed in the EPS^negHK treated PBMCs but not the MRx0004hk PBMCs (p = 0.0014, Figure 13C) when compared to untreated cells. EPS^negHK increased Tregs relative to MRxOOO4hk. (p = 0.0196). A skew in the Treg/CD8 ratio towards a regulatory T cell response was observed in for MRx0004hk treatment in comparison to untreated cells (p = 0.0008, Figure 13D. Furthermore, the increased CD8⁺ positive skew in response to MRx0004_Hk was significantly higher than that towards EPS^negnK (p = 0.0272, Figure 13D). Taken together, these data confirm the îmmunostimulatory effect of MRx0004 and suggest that the loss of EPS may resuit in an increased stimulation of Tregs and the Treg/CD8⁺ ratio and a reduced îmmunostimulatory effect. Whilst EPS may play a rôle in the activation of CD8^T cells it does not seem to be involved in significantly modulating the B-cell population.

The secreted cytokine signature of PBMCs treated with MRx0004hk and EPS^negHK was also detennined, by quantifying cytokines mostly associated with Thl (IL-12p70, IFNy, TNFa), Th2 (IL-4), Th 17 (IL-I7a, IL-Ιβ), and Treg (IL-10) populations. TNFa, IL-12p70, IFNy, IL-4 and IL-17a were significantly increased by MRxÜ004hk treatment compared to untreated cells (p = 0.0038, 0.0025, 0.0036, 0.027 and 0.0316 respectively, Figure 14A-D). Treatment with EPS^negHK induced a significant response in TNFa, IFNy, IL-1 β, IL-10 and IL-17a (p = 0.0001, 0.0267, < 0.0001, 0.0004, and 0.0103 respectively, Figure 14A, C, E-G). In contrast to MRx0004hk, EPS^negHK did not significantly increase IL-12p70 or IL-4 sécrétion in comparison to untreated cells. MRx0004hk also significantly increased IL-12p70 in comparison to EPS^neg _HK (p = 0.0118, Figure 14B), whilst conversely EPS^negHK treatment was found to produce a higher concentration ofIL-Ιβ and IL-10 than MRx0004hk (p = 0.0008 and 0.014 respectively, Figure 14E-F).

Cytokine ratios were analysed in order to infer whether bacterial treatments skewed the T-helper cell response towards a particular subtype using cytokines produced by each individual T-helper cell subtype as indicators (Thl or Th2; IL-12p70/IL-4, Treg; IL-10/ILlp70, Thl7; ILlp/IL12p70, Figure 14H-J). MRx0004_Hk treatment appeared to significantly skew the immune response towards a Thl phenotype compared to untreated cells (p = 0.0172, Figure 14H), whilst EPS^negHK treatment appeared to induce a skew towards both a Treg and Thl7 response in comparison to untreated cells (p = 0.0312 and 0.0005, Figure I4I-J). The EPS^negHK Treg and Thl 7 skew was also significantly increased in comparison to MRx0004hk (p = 0.0423 and 0.0008, Figure 14J). A clear distinction was observed between MRx0004hk and EPS^neSHK treatments in their ability to drive different subsets of the T cell response.

Taken together, the observations in this study demonstrate that MRx0004 régulâtes the proinflammatory amis of the innate and adaptive immune response. Therefore, MRx0004 and other B. brève strains may be useful for stimulating the immune System and treating diseases associated with decreased immune activity.

Example 2 - characterising the effect of the MRx0004 eps locus on potency

Summary

The objective of this study was to characterise the rôle of MRx0004 exopolysaccharide (EPS) in the immunostimulatory and therapeutîc properties of MRx0004.

Materials and Methods

Expérimenta were performed as described in Example 1, with the additional procedures described below.

Routine culture of immortalised cells

HEK-Blue™-hTLR2 cells (InvivoGen, San Diego, CA, USA) were grown in DMEM supplemented with 10% (v/v) FBS, 4 mM L-glutamine, 4.5 mg/ml glucose, 100 U/ml penicillin, 100 pg/ml streptomycin, 100 pg/ml Normocin™ (InvivoGen), 30 gg/ml blastocydin and 100 pg/ml zeocin to 90% density. THPl-Blue™ NF-kB cells (InvivoGen) were grown in RPMI 1640 supplemented with 10 % (v/v) heat-inactivated FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 pg/ml streptomycin, 25 mM HEPES, 100 pg/ml Normocin™, 10 pg/ml blastocydin (cRPMI). Cell lines were cultured at 37 °C and 5 % CCh- Ail reagents were supplied by SigmaAldrich unless otherwise stated.

Comparative analysis of MRx0004 EPS locus and related strains ofB. breve

Genomes of Bifidobacterium strains available in the GenBank database used for the in silico analysis of EPS clusters and physical maps of the putative exopolysaccharide gene clusters from Bifidobacterium strains.

Differential expression analysis ofthe MRx0004 EPS locus

Total RNA was extracted late log phase cultures of strain MRx0004 with RNAprotect (Qiagen) and the RNeasy Mini kit (Qiagen), according to the manufacturer’s protocol with minor modifications. Mechanical cell lysis was performed using Lysing Matrix B and a MP Fast-Prep24 tissue and cell homogenizer (MP Biomedicals, Santa Ana, CA, USA) with oscillations set at 6 m/s. Cells were disrupted for two 20 s cycles with a 1 min rest on ice between cycles. RNA quality was checked on a Tapestation (Agitent Technologies, Santa Clara, CA, USA) with the Agîlent RNA Screentape (Agilent Technologies). The absence of RNA dégradation was checked, and ail samples had a minimum RNA Integrity Numbers > 9. MICROBExpress kit (Thermo Fisher Scientific) was used to deplete rRNA species. The absence of 16S and 23S rRNA species was assessed on were checked on an Agitent Bioanalyzer with the Agitent RNA RNA Screentape (Agilent Technologies). RNA samples depleted in rRNA were sent to GATC Biotech for strand-specific library préparation and sequenced on an Illumina sequencing to produce 150 bp singie-end reads. An average of 22,3878,08 (late log samples) and 18627178.6 (stationary phase samples) raw reads per RNA-Seq library were obtained totaling over 10.07 Gbp.and 8.38 Gbp respectîvely. Raw reads were trimmed using Trimmomatic (1) and quality filtered (98.36 % late log samples and 98.26 % (stationary phase samples) reads passed QC and were aligned 99.11% (LL) and 98.72% (SP) of clean reads mapped) to the MRx0004 genome using Bowtie (2). The expression levels of the replicate samples of each growth phase were calculated for each gene in the MRx0004 EPS locus using XX and DeSeq2 v X (Love et al, 2014) and subsequently visualized using Geneious RI 1 (Biomatters, Auckland, New Zealand). Differential expression between the two growth phases are represented. The base 2 logarithm of the ratio of the nomialized values between the two samples and when one sample has no or very low expression, the log2 ratio is capped at +/- 1,000,000.

Transmission électron microscopy (TEM)

Bacteria were diluted 1:5 in fixative solution (0.5 M sucrose in 0.1 M Na-phosphate buffer, 2% paraformaldéhyde and 0.16 % glutaraldehyde) and fixed for 2 hours at room température. Thereafter, Fomivar-carbon-coated copper grids were floated on 100 μΐ droplets of the B. breve suspensions for 1 hour, washed three times with 0.02 M glycine in PBS. The cells were negatively stained with 1.0 % ammonium molybdate. The grids were examined, and micrographs visualized, using a JEM-1400 transmission électron microscope (JEOL Ltd., Tokyo, Japan).

Bacterial adhesion assays

Live bacteria (prepared and resuspended in co-culture medium as described previously) were applied to HT29-MTX cells in 24 well plates at an MOI of 100:1, and co-incubated for 3 hours at 37 °C in anaérobie conditions. Cells were washed twice with PBS to remove unbound bacteria, and lysed with 0.1 % (v/v) Triton X-100 (Sigma-AIdrich). Lysate was plated, and the number of colony forming units (CFU) recovered was used to détermine the percentage of adhesion.

Results

The eps locus of MRx0004 is genetically distinct from other strains of B. breve and is highly expressed during late log growth.

Genome sequencing of strain MRx0004 indicated that it harboured a 2S Kb EPS locus which was found to encode 27 genes, representing the full complément of functions predicted to be requîred for EPS biosynthesis in B. breve. This région includes; a priming glycosyltransferase, four additional glycosyltransferases, a thiamine pyrophosphate binding protein which is encoded downstream of a membrane spannîng protein, a flippase and a ehain-length déterminant (Fig. 9). The majority of B. breve EPS loci, including that of strain MRx0004 are flanked by hypothetical proteins (extendîng the MRx0Û04 région to 31.5 Kb), which hâve been excluded from the représentation of the B. breve EPS loci represented in Figure 9. The majority (16/19) of strains illustrated in Figure 9 are infant isolâtes, the genomes of which are over-represented in public databases. B. breve EPS régions which exceed 50 Kb are thought to represent complété loci which encode ail the functions required to produce EPS-positive phenotypes [89]. In contrast, when these régions are < 30 Kb, they are thought to represent incomplète or remuant loci [89].

Comparative analyses identified the EPS locus as a major région of genetic divergence between the genome of strain MRx0004 (data not shown) and other strains of B. breve. The MRx0004 EPS locus was compared to those of the publicly available B. breve genomes. Strains of R. breve whose EPS loci displayed high ievels of sequence identity (ID) and gene synteny with that of strain MRx0004 are illustrated in Figure 9, and are ordered according to their similarity (mean % nt ID over the length of the operon) to strain MRx0004. B. breve NRBB51, an infant (breast fed) isolate shared the hîghest level of nucléotide identity (ID), 91.5 %, with strain MRx0004 over the complété length of the two loci. A central 1.3 Kb région, encoding predicted transposases in both strains, represents the primary région of diversity between strains MRx0004 and NRBB51. The genes encoded at the start and end of the MRx0004 locus displayed the highest level of sequence conservation with other B. breve isolâtes (Fig. 9). ThepGTF of MRx0004, which is essential for the primary step of EPS biosynthesis, shared 92.2 % pairwise ID (aa) with homologs in the comparator strains. A 4 Kb région encompassing a chain length regulator, and genes encoding a hypothetical and a membrane spanning protein share 98.3 % nt ID among the strains examined. Genes encoding hypothetical proteins and transposases accounted for the major régions of sequence diversity between strain MRx0004 and the ten most closely related EPS loci (Fig. 9). In contrast, the B. breve EPS loci that were found to be more dîssimilar to that of strain MRx0004 (the bottom ni ne strains in Figure 9) display variance in the number and order of genes that are of central importance to the production of EPS, including gtf, polymerase and acetyltransferase genes (Fig. 9).

In order to détermine whether the EPS locus of MRx0ü04 was more highly transcribed during late log or stationary phase growth, differential expression analysis was carried out on monocultures of strain MRx0004 grown in YCFA. (Fig. 9. B). The majority of genes which are predicted to be primarily responsable for MRx0004 EPS synthesis (described above), were upregulated during late log phase growth. The MRx0004 EPS locus encodes ten hypothetical proteins and five transposases, which represented the only categories of genes which were upregulated during stationary phase growth of MRx0004 (Fig. 9). The rôle of the hypothetical proteins in the synthesis of MRx0004 EPS is as yet unknown.

Génération ofan EPS négative strain of MRx0004

The rôle of MRx004 EPS in host-microbe interactions and immunomodulation was investigated and a strain (EPS^neg) was constructed, as discussed above, whereby the pGTF gene of the EPS locus was inactivated through insertionai mutagenesis (Figure 16A). This strain was constructed by utilising the methodology described in [88], but rather than manipulaiing a Type II restrictionmodification (RM) System, the methylase and specificîty subunits from the MRx0004 Type 1 RM System were expressed and used to methylate plasmîd DNA prior to transformation. A complemented EPS^neg strain (EPS^comp) and an EPS^neg empty vector strain (EPS^vec) were also generated as Controls. EPS^ne§ and EPS^vec displayed an increased autoagreggative phenotype compared with MRx0004 (Figure 16B). EPS^comp was less aggregative than EPS^neg and EPS^vec, but its autoagreggation appeared to be increased compared to MRx0Ü04, suggestîng that this strain may not be fully reverting to a wild type EPS phenotype.

The EPS phenotype of MRxQ004 and EPS^neg was investigated using transmission électron microscopy (TEM). The absence of EPS in the EPS^neê strain compared with MRx0004 is illustrated in Figure 10A and 10B. The adhesive capabilities of wild type MRx0004 and its dérivative strains were analysed using an in vitro IEC model. EPS^neg was over twice as adhèrent to lECs as MRx0004 (47.5 % adhérence vs 18.7 % respectively, p = 0.006) (Figure 10C), suggestîng that the absence of EPS increased the adhesive capacity of EPS^neg. The increased adhesion phenotype seen in EPS^neg was maintained with EPS^vec (40.1 % adhesion,/? = 0.03 vs MRx0004), but the adhesion of EPS^u'^mp (34.1 %) was not significantly different when compared to any other strain, further implying that the reversion of EPS^t0,np to a wild type phenotype was incomplète.

EPS déplétion in MRx0004 exposes surface proteins involved in host stimulation

The increased adhesion of EPS^neg to lECs suggested that the déplétion of EPS may hâve resulted in the increased exposure of surface-associated proteins, or “unshielding”. The composition of MRx0004 and EPS^lieg surface-associated proteins was analysed after contact with lECs. Foliowing 3 h contact with HT29-MTX cells, bacterial cells were shaved with trypsin, as described previously, and the resulting shaved and shed protein fractions were analysed b y LCMS/MS. MRx0004 cell shaving after contact with lECs yielded 55 shaved proteins (34 of which were predicted to be surface-anchored) and 24 shed proteins (Figure 1 IA). The shaving of EPS^neË cells after contact with lECs contained considerably more proteins than that of MRx0004, with 101 proteins in the shaved protein fraction and 45 in the shed protein fraction (Figure 1 IB). With the exception of three proteins identified in the MRx0004 shed protein fraction (enzymes involved in carbohydrates, lîpid and protein metabolîsm), ail proteins identified were présent in the shaved protein fraction for both strains.

Comparison of MRx0004 and EPS^neg shaved protein fractions identified 54 proteins that were présent in both samples and 47 proteins that were spécifie to the EPS^neë strain (Figure 1 IC ). The only protein that was uniquely identified in the MRx0004 shaved protein dataset was an NlpC/P60 family protein. The number of proteins harvested by cell shaving was higher for the EPS^neg strain than for MRx0004, inferring that EPS déplétion facilitated better access to surface proteins for trypsin cleavage. Addîtionally, proteins known to be involved in host-interaction in bifidobacteria and other généra were more abundant (as assessed by the number of peptides identified/gg total protein) in the EPS^neg shaved protein fraction than in that of MRx0004 (Table 4). These results add further credence to the hypothesis that EPS déplétion resulted in an unshielding effect, exposing surface proteins and potential MRx0004 immunogens which would be otherwise masked by MRx0004 EPS.

The protein content of EPS^neg culture supematant was also analysed by LC-MS/MS and confirmed that the lack of EPS resulted in an increase in the numbers of proteins potentially shed and secreted in the extracellular milieu by EPS^neg strain (Figure 17). A total of 146 proteins were identified in EPS^negsN in contrast to only 64 in MRx0004sn, of which 87 were detected only in EPS^neg$N and 59 in both samples. Moonlighting proteins identified in MRx0004sn and discussed above were ail detected in higher abundance in EPS^negsN, with the exception of GAPDH which was comparable between both samples (30 ± 3.61 and 28.33 ± 0.58 in MRx0004sn and EPS^negsN)· Interestingly, choloylglycine hydrolase (bile sait hydrolase) and EfTu, which hâve been shown to play a rôle in human plasminogen binding in B. lactis and B. longum, were detected exclusively in EPS^nLgsN [80,87], Bile sait hydrolase may also protect commensal species from environmental stresses in the gut [90]. The increased détection of proteins in EPS^neë _SNcompared to MRx0004_Sn suggests that unshieldîng in the absence of EPS may resuit in the increased shedding or sécrétion of MRx0004 surface-associated proteins.

Conclusions

Comparative genomic analyses demonstrated that the locus responsible for EPS synthesis in MRx0004 is genetically distinct from other B. breve strains. The genetic variance observed in this région may contribute to the enhanced potency and therapeutic utility of MRx0004 and related strains.

The potentîal importance of EPS as a mediator of MRx0004:host interactions and as an effector of immune responses was supported by the relative increase in expression of its pGTF upon contact with lECs. In addition to pGTF, most of the other significantly upregulated genes in qPCR analysis had predicted rôles in adhesion to lECs, implying that this might be an important functional property of MRx0004. The Type IV pii us-associated gene tadA of MRx0004 was expressed in in vitro culture, which was interesting due to the previous observation that the Tad pilus of B. breve UCC2003 was not produced under in vitro conditions [91], The expression of serpin was also significantly increased in response to lECs. A serpin from B. longum NCC2705 has recently been reported to reduce the infdtration of intra-epithelial lymphocytes in the small intestine of an in vivo coeliac disease model [92], thus indu ci ng an immunostimulatory and protective effect, which suggests that the serpin of B. breve, and MRx0004 in particular, might hâve similar immunomodulatory effects.

Proteomics analysis of supematants and cell shavings detected a large number of proteins with predicted rôles in carbohydrate metabolism. Interestingly, the amylolytîc enzyme pullulanase, the most abundant protein in the cell shaving dataset, is a moonlighting protein which has been reported to be involved in the adhesion of Streptococcus pyogenes to glycoproteins and host cells in vitro [85,86]. In addition, DnaK and enolase from B. animctlis subsp. lactis [82,83] and Effu from B. longum [87] hâve been reported to adhéré to plasminogen in vitro. Furthermore, recombinant expression of the glycolytic enzyme transaldolase of B. bifidum A8 in L. lactis increased the adhérence of this strain to mucin [84], The production of these moonlighting proteins by MRx0004 suggests that they may play a rôle in the adhesive capacity of MRx0004, and thus facilitate interaction with the host cell surface. These bifidobacterial moonlighting proteins may hâve particular effects on spécifie host cell receptors and signalling pathways that médiate the enhanced effects of MRx0004 on the immune System.

MRx0004 induced a significant increase in activated CD8⁴ subsets, which appeared to be partially associated with the presence of EPS.

EPS^neg treatment significantly increased Tregs in comparison to MRx0004. This suggests that the removal of EPS exposes another bacterial surface component capable of interacting with host cells and promoting a Treg response. Fluctuations were évident in both the CD8+ and Treg populations. EPS^neg induced a significant skew towards a Treg response as illustrated by the significantly increased Treg/CD8 ratio in comparison to baseline. This implies that unshieldîng of the cell surface in EPS^neg results in an anti-inflammatory effect, and the presence of EPS in MRx0004 supports its immunostimulatory potency.

The EPS of MRx0004 was found to be directly involved in sécrétion of IL-12p70. In addition, sécrétion of ail three of the Thl cytokines tested (IL-12p70, IFNy, TNFa) were significantly upregulated by MRx0004_Hk, suggesting that this strain induces a skew towards a Thl response which is partially mediated through its EPS. MRx0004hk also significantly induced the Th2 cytokine IL-4, and upregulated IL-10, IL-1 β and IL-17α, though not significantly, thus inferring that this strain may induce shifts in the T-helper cell microenvironment. The induction of a Thl may improve intestinal barrier stability in vivo and be bénéficiai for maintenance of immune homeostasis.

The EPS of MRx0004 may hâve spécifie immunoregulatory effects, namely, régulation of the CD8+, Treg and Thl responses, and these effects may provide enhanced potency and therapeutic effîcacy.

Example S - Effîcacy of bacterial inocula in mouse models of cancer

Summary

As set out in the preceding examples, the inventors hâve identified a new immunostimulatory effect of B. breve, and in particular strain MRX004. In light of the new data presented above, compositions comprising B. breve, and in particular strain MRX004, are expected to be effective for stimulating the immune system and treating diseases that are associated with decreased immune system aefivity or that benefit from increased immune System activity.

Cancer is a disease that may benefit from increased immune system activity attacking tumours. Consistent with the new data presented above and the new immunostimulatory effect of MRX004, MRX004 is shown in the study below to potently reduce tumour volume in mouse tumour models, which demonstrates that administration of MRX004 is effective to treat disease.

This study tested the efficacy of compositions comprising bacterial strains according to the invention in four tumor models, and compared the efficacy to an anti-CTLA antibody.

Materials

Test substance - Bacterial strain &MRX004, Bifidobacterium breve.

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

Test and reference substances vehicles - Bacterial culture medium (Yeast extract, Casitone, Fatty Acid medium (YCFA)). Each day of injection to mice, antibody was diluted with PBS (ref: BEI4-516F, Lonza, France).

Treatment doses - Bacteria: 2x10⁸ in 200 pL. The a-CTLA-4 was injected at 10 mg/kg/inj. Anti-CTLA-4 was administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse weighing 20 g, 200 pL of test substance will be administered) according to the most recent body weight of mice.

Routes of administration - Bacterial inoculum was administered by oral gavage (per os, PO) via a cannula. Cannulas were decontaminated every day. Anti-CTLA-4 was injected into the peritoneal cavity of mice (Intraperitoneally, IP).

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

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

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

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

Cell line	Type	Mouse strain	Origin
EMT-6	Breast carcinoma	BALB/c	ATCC
LL/2 (LLC1)	Lung carcinoma	C57BL/6	ATCC CRL1642
Hepa 1-6	Hepatocellular carcinoma	C57BL/6	IPSEN INNOVATION

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 [93],

The LL/2 (LLC1) cell line was established from the lung of a C57BL mouse bearing a tumor resulting from an implantation of primary Lewis lung carcinoma [94],

The Hepa 1-6 cell line is a dérivative of the BW7756 mouse hepatoma that arose in a C57/L mouse [95].

Cell culture conditions - Ail cell lines were grown as monolayer at 37°C in a huinidified 10 atmosphère (5% CO2, 95% air). The culture medium and supplément are indicated in the table below:

Cell line	Culture medium	Supplément
EMT6	RPMI 1640 containing 2mM L-glutamine (ref: BE12-702F, Lonza)	10% fêtai bovine sérum (ref: #3302, Lonza)
LL/2 (LLC1)	RPMI 1640 containing 2mM L-glutamine (ref: BE12-702F, Lonza)	10% fêtai bovine sérum (ref: #3302, Lonza)
Hepal6	DMEM (ref: 11960-044, Gibco)	10% fêtai bovine sérum (ref: #3302, Lonza) 2mM L-Glutamine penicillin-streptomycin (Sigma G6784)

For experimental use, adhèrent tumor ceils were detached from the culture flask by a 5 minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnésium (ref: BE10-543F, Lonza) and neutralized by addition of complété culture medium. The cells were counted in a hemocytometer and their viability will be assessed by 0.25% trypan blue exclusion assay.

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

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

Animais were maintained in S PF health status according to the FELAS A guidelines, and animal housing and experimental procedures according to the French and European Régulations and NRC Guide for the Care and Use of Laboratory Animais were followed [96,97], Animais were maintained in housing rooms under controlled environmental conditions: Température: 22 ± 2°C, Humidity 55 ± 10%, Photoperiod (12h light/12h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Animal enclosures were provided with stérile and adéquate space with bedding material, food and water, environmental and social enrichment (group housing) as A described: 900 cm cages (ref: green, Tecniplast) in ventilated racks, Epicéa bedding (SAFE),10 kGy Irradiatcd diet (A04-10, SAFE), Complété food for immuno-competent rodents - R/M-H Extrudate, water from water bottles.

Experimental design and treatments

Antitumor activity, EMT6 mode!

Treatment schedule - The start of first dosing was considered as D0. On D0, non-engrafted mice were randomized according to their îndividual body weight into groups of 9/8 using Vivo manager® software (Biosystemes, Coutemon, France). On D0, the mice received vehicle (culture medium) or bacterial strain. On D14, ail mice were engrafted with EMT-6 tumor cells as described below. On D24, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	8	Untreated	-	-	-
2	8	Vehicle (media)	-	PO	QlDx42
3	9	Bacterial strain #1 (MRX004)	2x108 bacteria	PO	QlDx42
4	8	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was perfonned as described below.

Induction of EMT6 tumors in animais - On DI4, tumors were induced by subcutaneous injection of IxlO⁶ EMT-6 cells in 200 pL RPMI 1640 into the right flank of mice.

Euthanasîa - Each mouse was euthanized when it reached a humane endpoint as described 5 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 consîdered as DO. On DO, non-engrafted mice were randomized according to their individual body weight into 7 groups of 9/8 using Vivo manager® software (Biosystemes, Couternon, France). On DO, the mice will received vehicle 10 (culture medium) or bacterial strain. On DI4, ail mice were engrafted with LL/2 tumor cells as described below. On D27, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarîzed in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	8	Untreated	-	-	-
2	9	Vehicle (media)	-	PO	QlDx42
3	9	Bacterial strain #1 (MRX004)	2xl0⁸ bacteria	PO	QlDx42
4	8	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was performed as described below.

Induction of LL/2 (LLC1) tumors in animais - On DI4, tumors were induced by subcutaneous injection of 1 xlO⁶ LL/2 (LLC1) cells in 200 pL RPMI 1640 into the right flank of mice.

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

Antitumor activity, Hepal-6 model

Treatment schedule - The start of first dosing was considered as DO. On DO, non-engrafted mice were randomized according to their individual body weight into 7 groups of 9 using Vivo manager® software (Biosystemes, Coutemon, France). On DO, the mice received vehicle (culture medium) or bacterial strain. On D14, ail mice were engrafted with Hepa 1-6 tumor cells as described below. On DI6, mice from the positive control group received anti-CTLA-4 antibody treatments.

The treatment schedule is summarized in the table below:

Group	No. Animais	Treatment	Dose	Route	Treatment Schedule
1	9	Untreated	-	-	-
2	9	Vehicle (media)	-	PO	QlDx42
4	9	Bacterial strain #2 (MRX004)	2x10⁸ bacteria	PO	QlDx42
7	9	Anti-CTLA4	10 mg/kg	IP	TWx2

The monitoring of animais was performed as described below.

Orthotopic induction of Hepa 1-6 tumor cells in animais by intrasplenic injection - On D14, one million (1x10⁶) Hepa 1-6 tumor cells in 50 pL RPMI 1640 medium were transplanted via intrasplenic injection into mice. Briefly, a small left subcostal flank incision was made and the spleen was exteriorized. The spleen was exposed on a stérile gauze pad, and injected under visual control with the cell suspension with a 27-gauge needle. After the cell inoculation, the spleen 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 tumor burden at euthanasia - At the time of termination, livers were collected and weighed.

Animal monitoring

Clinical monitoring - The length and width of the tumor was measured twice a week with callipers and the volume of the tumor was estimated by this formula [98]:

, width ² x length

Tumor volu me =----------⁵—2

Humane endpoints [99]: Signs of pain, suffering or distress: pain posture, pain face mask, behaviour; Tumor exceeding 10% of normal body weight, but non-exceeding 2000 mm³; Tumors interfering with ambulation or nutrition; Ulcerated tumor or tissue érosion; 20% body weight loss remaîning for 3 consecutive days; Poor body condition, émaciation, cachexia, déhydration; Prolonged absence of voluntary responses to external stimuli; Rapid laboured breathing, anaemia, significant bleeding; Neurologie signs: circling, convulsion, paralysis; Sustained decrease in body température; Abdominal distension.

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

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

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

Results

Antitumor activity, EMT6 model

The results are shown in Figure 1. Treatment with the bacterial strain of the invention led to a clear réduction in tumour volume relative to both the négative Controls. The positive control, which is known to activate the immune System, also led to a réduction in tumour volume, as would be expected.

Antitumor activity, LL/2 (LLC1) model

The results are shown in Figure 2. The négative and positive Controls do not appear as would be expected, because tumour volume was greater in the mi ce treat ed with the positive control than in the négative control groups. Nevertheless, tumour volume in the mice treated with the bacterial strain of the invention was comparable to the positive control group, which is consistent with a useful therapeutic and immunostimulatory effect.

Antitumor activity, Hepal-6 model

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

These data demonstrate that MRX004 is effective for treating cancer and, in light of the data in Ex amples 1 and 2, these data support that strain MRX004 may be useful for treating or preventing other diseases assocîated with reduced immune System activity.

Example 4 - Characterisation of enzymatic activity

The Analytîcal Profile Index (API®) test System consists of strips that contain miniaturised biochemical tests that assay for enzymatic activity in bacterial species. MRX004 (the bacterium deposited under accession number NCIMB 42380) was characterised usîng two API test Systems: Rapid ID 32A - This System is designed specifically for anaérobie species and encompasses tests for carbohydrate, amino acid and nitrate metabolism as well as alkaline phosphatase activity; and API® 50 CH - This System tests for the fermentation of 49 carbohydrate sources, and can be utilised in conjunction with API® CHL Medium for analysis of anaérobie species.

Rapid ID 32A testing was carried out on bacterial colonies as per manufacturer’s instructions. Briefly, bacteria were cultured on YCFA agar for 24 hours at 37 °C in an anaérobie workstation. Colonies were removed from plates usîng a stérile 5 μΐ inoculating loop and resuspended in a 2 ml ampoule of API® Suspension Medium until a density roughly équivalent to that of McFarland standard No. 4 was achieved. Fifty-five microlitres of bacterial suspension was added to each cupule on a Rapid ID 32A strip, and the urease test was overlayed with two drops of minerai oil. Strips were covered with a plastic lid and incubated aerobically at 37 °C for 4 hours, following which the bottom row of cupules were developed usîng the following reagents: NIT: 1 drop each of NIT1 and NIT2; IND: 1 drop of James reagent; ail remaining cupules: 1 drop of FastBlue reagent. Strips were incubated at room température for 5 minutes, following which the colour of each cupule was recorded and assigned a value of négative, intennediate positive or positive.

The results of the Rapid ID 32A analysis are shown in Figure 4. MRX004 tested positive for fermentation of several carbohydrate sources, namely α-galactosidase and β-galactosidase, aglucosidase and β-glucosidase, α-arabinose, mannose and raffinose, as well as the amino acids arginine, proline, phenylalanine, leucine, tyrosine, glycine and histidine.

Comparative Rapid ID 32A analysis was carried out between MRX004 and four B. breve type strains, which are annotated in Figure 4B as Bif Ref 1 (DSM 20091), Bif Ref 2 (DSM 20213), Bif Ref 6 (JCM 7017) and Bif Ref 7 (UCC2003). This analysis demonstrated that MRX004 was the only strain tested to ferment the polysaccharide raffinose, which may be significant, because raffinose is involved in the production of bacterial components such as exopolysaccharides, and raffinose fermentation can also reportedly confer effects on the host such as increased caecal butyrate, increased gastrointestinal prolifération and weîght loss.

API® 50 CH testing was carried out to further examine carbohydrate metabolism in MRX004. As per manufacturer’s instructions, bacteria were cultured in 10 ml YCFA broth for 16-18 hours at 37°C in an anaérobie workstation. This culture was dîîuted in 10 ml API® CHL Medium so as to achieve a densîty roughly équivalent to McFarland standard No. 2, and 110 pl of this mixture was used to inoculate each cupule on a set of API® 50 CH test strips. Test strips were incubated in a humidified incubation box at 37 °C in an anaérobie workstation for 48 hours, following which the colour of each cupule was recorded and assigned a value of négative, intennediate positive, positive or doubtfiil.

Using API® 50, MRX004 tested positive for utilisation of the following carbohydrate sources: amidon (starch), amygdalin, arbutin, cellobiose, esculin, galactose, gentiobiose, glucose, glycogen, fructose, fucose, lactose, maltose, mannose, mannitol, melibiose, melezitose, methyl α-D-glucopyranoside, N-acetylglucosamine, ribose, saccharose (sucrose), salicin, sorbitol, trehalose, turanose and xylitol (Figure 5). These results correlated with those obtained for Rapid ID 32A testing in that MRX004 demonstrated fermentation of galactose, glucose, mannose and raffinose in both test Systems.

Example 5 - Attachaient to human cells in YCFA medium

Summary

The level of binding of strain MRX004 and a number of other Bifidobacterium breve strains to human cells was detennined at 3 distinct time points in YCFA medium. The bacteria attached to the human cells were resuspended in medium and the optical density of the medium was then analysed - the higher the optical density, the higher the number of bacterial cells and thus, the higher the level of binding of the bacterial cells to human cells. The MRX004 strain was found to display reduced attachment to human cells compared to the Bifidobacterium breve reference strains.

Results and analysis

The results of the experiment are shown in Figure 6,

As shown in Figure 6, the reference Bifidobacterium breve strains show a high level of attachment to human cells at ail time points. On the other hand, the MRX004 strain has a drastically reduced level of attachment to human cells. Therefore, the low adhérence to human cells of strain MRX004 may increase the bénéficiai effect of the compositions of the invention on the immune system.

Exatnple 6 — 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 atmosphère having 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% relative humidity. After 1 month, 2 months, 3 months, 6 months, 1 year, 1.5 years, 2 years, 2.5 years or 3 years, at least 50%, 60%, 70%, 80% or 90% of the bacterial strain shall remain as measured in colony forming units determined by standard protocols.

Example 7 - antimicrobial activity

Introduction

The aim of this experiment was to test the antimicrobial activity potential of several B. breve strains derived from human infants against various indicator strains and to assess whether they produce bacteriocins in vitro.

Methods

A panel of strains were chosen as indicator strains (Table 5), which included closely-related Gram-positive, other Gram-positive and Gram-negative bacteria that hâve been previously shown to be inhibited by Bifidobacterium species [Error! Bookmark not defined.].

Co-culture assay

Straîns were grown for 16 h at 37°C (30°C for B. subtilis) under anaérobie conditions (aérobic conditions for E. coli, B. subtilis and S. aureus) from Research Cell Bank (for B. breve test and reference strains) or from bead stocks (for the indicator straîns). A lawn of indicator strain was made on a YCFA plate (E&O Labs, UK), left to dry and 10 pl of test strain culture were spotted on top of the lawn. Plates were incubated for 48 h at 37°C under anaérobie conditions (24 h at 37°C under anaérobie conditions followed by 24 h at 37°C under aérobic conditions for E. coli, B. subtilis and S. aureus). Each assay was performed in triplicate (except in duplicate for Bacillus subtilis NCIMB8045 with MRx0004, Test 1, Test 2, Test 3, Test 4, Test 5, Test 6, Test 7 and Test 8, and for Bifidobacterium breve DSM202I3, Lactobacillus plantarum NCIMB8826, Clostridium sporogenes ATCC3584, and Staphylococcus aureus NCIMB9518 with ail B. breve strains).

Antimicrobial activity was assessed by measuring the wîdth of the observed inhibition zone, a clear zone around the test strain spot (Figure 22). A score between 0 and 3 was given to each strain for each biological replicate.

Culture supematant

The agar diffusion method was used to test the antimicrobial potential of culture supernatants. In brief, 100 pl of filtered cell-free supematant were spotted on YCFA agar, pre-inoculated with a lawn of an indicator strain (as described above), into a well punched into the agar. The plate was left to stand for 1 h to allow diffusion and was then incubated for 48 h at 37°C under anaérobie conditions (aérobic conditions for E. coli, B. subtilis and S. aureus). Three biological replicates were performed.

Results

Co-culture

Mo st B. breve strains tested exhibited antagonism activity against E. coli, K. pneumoniae, S. Typhimurium and B. subtilis (Table 6). B. breve DSM 20091 was the only strain tested that inhibited the growth of B. breve DSM20213. MRx0004 and other test B. breve strains displayed antimicrobial activity against E. coli, K. pneumoniae, S. Typhimurium and B. subtilis (Table 6), with overall higher inhibition observed than with the B. breve reference stains. MRx0004, Test 1, Test 2, Test 3, Test 7, Test 8, Test 11 and Test 12 exhibited particularly potent antimicrobial activity. No antagonistic activity was detected against 5. aureus, C. sporogenes and L. plantarum in the conditions tested.

These data indicate that MRX0004 and the related test strains may be useful for treating bacterial infections.

Culture supernatant

The antimicrobial activity of cell-free supematants was tested against the same panel of indicator strains. No inhibition was observed for ail B. breve strains tested against any of the indicator strains (data not shown, n=3). This suggests that the inhibition observed in the co-culture assays was not due to the sécrétion of antimicrobial molécules.

Example 8 - Further characterisation of enzymatic activity

The API 32A test System described in Exampie 4 was used to characterise the B. breve strains tested în Example 7. Rapid ID 32A testing was carried out on bacterial colonies as per manufacturer’s instructions. Briefly, bacteria were cultured on YCFA agar for 24 hours at 37 °C in an anaérobie work station. Colonies were removed from plates using a stérile 5 μΐ inoculating loop and resuspended in a 2 ml ampoule of API® Suspension Medium until a density roughly équivalent to that of McFarland standard No. 4 was achieved. Fifty-five microlitres of bacterial suspension was added to each cupule on a Rapid ID 32A strip, and the urease test was overlayed with two drops of minerai oïl. Strips were covered with a plastic lid and incubâted aerobically at 37 °C for 4 hours, foliowing which the bottom row of cupules were developed using the following reagents: NIT: 1 drop each of NIT1 and NIT2; IND: 1 drop of James reagent; ail remaîning cupules: 1 drop of FastBlue reagent. Strips were incubated at room température for 5 minutes, following which the colour of each cupule was recorded and assigned a value of négative, intermediate positive or positive.

The results of the Rapid ID 32A analysis are shown in Figure 23. As found in Exampie 4,MRX004 tested positive for fermentation of several carbohydrate sources, namely agalactosidase and β-galactosidase, α-glucosidase and β-glucosidase, α-arabinose, mannose and raffinose, as well as the ami no acids arginine, proline, phenyl alanine, leucine, tyrosine, glycine and histidîne.

Rapid ID 32A analysis was also performed on the other test B. breve strains (Test 1 - Test 12) and the B. breve reference strains (DSM 20091, DSM 20213, JCM 7017, and NCIMB 8807/UCC2003) that were studied in Example 7. The test strains generally showed greater antimicrobial activity than the reference strains and showed metabolism patterns with similarity to MRX004.

Interestingly, MRX004 and the test strains ferment the polysaccharide raffmose, whilst the four reference strains do not. As noted above, raffmose is involved in the production of bacterial components such as exopolysaccharides.

Also, MRX004 and Test 3 both hâve potent anti-microbial activity and both exhibit intermediate fermentation of β-glucostdase and a-arabinose.

MRX004 and Test 2 both hâve potent anti-microbial activity and neither exhibits positive fermentation ofN-acetyl-p-glucosaminidase.

MRX004 and Test S both hâve potent antî-microbial activity and both exhibit intermediate fermentation of α-galactosidase and a-arabinose.

Test 11 and Test 12 both hâve potent anti-microbial activity and both ferment serine arylamidase but not leucyl glycine arylamidase and not alanine arylamidase.

Test 3 and Test 7 both hâve potent anti-microbial activity and both exhibit intermediate fermentation of serine arylamidase.

Example 9 - Pulsed-field Gel Electrophoresis

Pulsed-field Gel Electrophoresis (PFGE) was used to characterise the B. breve strains tested in Example 7. The results are shown in Figure 24. The test B. breve strains, which exhibited greater anti-microbial activity than the reference B. breve strains, were found to be grouped together with similar patterns and can be distinguished from the reference strains.

Example 10 - Pulsed-field Gel Electrophoresis

A further pulsed-field gel electrophoresis (PFGE) study was performed on MRx004 and the reference B.breve strains. PFGE is routinely applied as the “gold standard” for strain typing in clinical laboratories [100] and has been reported to be an effective method for discriminating human fecal Bifidobacterium isolâtes [101]. Indeed, many studies hâve explored the relationships of Bifidobacterium isolâtes using fingerprint analysis of PFGE-resolved fragments of genomic DNA, dîgested with either Xbal or Spel restriction enzymes [102,103]. Spel has proven particularly useful in plasmid profiling and intraspecific genotyping of Bifidobacterium breve [104,105].

PFGE plug préparations

Agarose gel plugs of high molecuiar weight DNA for PFGE were prepared according to a publîshed protocol [106].

Restriction ofPFGE plugs

Single slices (2 mm x 2 mm) were washed three times for 15 min in 1 ml 10 mM Tris.Cl, 0.1 mM EDTA (pH 8.0) at room température. Each slice was pre-incubated with 250 μΐ of restriction buffer recommended for the enzyme for 30 min at 4°C and then replaced with 250 μΐ of fresh buffer containing 20 units of restriction enzyme Smal. Restriction digests were carried out ovemight at 25oC, as recommended by the supplier (New England Biolabs (UK) Ltd).

PFGE

Treated (restriction enzyme) and untreated plugs of genomic DNA (gDNA) were examined under the followîng conditions. The λ ladder was heated at 45C prior to loading it into the gel. Running conditions were 6.0 V/cm at 14 °C for 20 h with puise times ramped from 1 to 20 s in 0.5 x TBE buffer. A lambda DNA ladder (Bio-Rad) was used as the size marker. The plugs were placed in wells of 1.0% agarose gels (Bio-Rad) made with 0.5xTBE (1 M Tris-borate, 0.5 M EDTA, pH 8.5), sealing with the same agarose. DNA fragments were resolved in 0.5* TBE running buffer maintained at 14°C using a CHEF-DR ΠΙ pulsed-field System (Bio-Rad Laboratories, Hercules, CA) at 6 V/cm for 18 h. Linear ramped puise times of were selected. A linear ramped puise times of ls-15s were employed for séparation of the fragments. Gels were stained in distilled water containing 0.5 pg/ml ethîdium bromîde for 120 min under light-limited conditions.

PFGE banding pattern analysis

Banding patterns were manually assessed using the guidelmes outlined by [107]. The PFGE image was processed in BioNumerics 7.6 (Applied Maths) to generate a band fingerprint for each strain. Cluster analysis on fmgerprints was carried out using the Jaccard similarity coefficient (recommended for fingerprint-type analysis) and Unweighted Pair Group Method with Arithmetic Mean (UPGMA).

Results

The DNA digestion and electrophoretic conditions employed in this study hâve previously been shown to be effective for subtyping species of B. breve [104] and were found to provide sufficient resolution (more than 10 observable bands) to distinguish strains of B. breve at a subspecies level. The restriction fragments of 4/5 strains were distinct and were well resolved (Figure 25). Cluster analysis (Figure 26) suggests that the génotype of strain MRx0004 is more closely related to that of B. breve REF7 than the other B. breve strains analysed in this study.

Example 11 - T cell différentiation

The ability of MRx0004 to induce T-cell différentiation was explored in vitro on perîpheral blood mononuclear cells (PBMCs, Stemcell, Cat:70025). Briefly, PBMCs were plated in 96-well plates plated with anti-CD3 (Ebioscience, CD3 monoclonal antibody (OKT3 clone), functional grade, cat. No. 16-0037-81) at 400,000/well in 50μ1 cRPMI medium per well (cRPMI contains RPMI 1640 (+L-Glut, 21875-034) 2mM final conc. Stock 200mM.; 10% HI FBS (Gibco life technologies, 10082-147); 50μιη mercaptoethanol (Gibco life technologies, 21985-023); and 1% pen/strep (P4333, lOmg/ml). Heat-killed MRx0004 (prepared by incubation at 80 °C for 30 minutes, after which the cultures were washed with PBS and resuspended in appropriate cell culture medium and viable counts were confîrmed by plating) was then added to each well, 4,000,000 in 100 μΙ/well. Following 3 days in a 37° C incubator, the cells were removed and resuspended in a medium containing PMA- (Sigma, Cat no. P8139), lonomycin (Sigma, Cat no. 13909) and GolgiSTOP (BD, Cat no 554724) for 5 hours. PMA stock was Img/ml in DMSO which was further diluted in lOOug/ml (each sample required 50ng/ml in cRPMI), lonomycin stock was ImM in DMSO (ΙμΜ in cRPMI was used) and GolgiStop concentration was used at 4μ!/6ιη1. Supematants were passed through a 0.22 μιη filter and diluted appropriately in coculture 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 bîotec, Ιμΐ/sample) + human Fc block, cat. 564219 (Ιμΐ/sample) in PBS for 10 mins in the dark at room température. The surface antibodies (2μ1 of each) were then added directly to the wells for 10 mins in the dark at room température - CD3-APC-Vio 770 (Miltenyi, cat. No. 130-113-136), CD4-VioBIue (Miltenyi, cat. No. 130-114-534) and CD25-VioBright FITC (Miltenyi, cat. No. 130-113-283). The cells were then washed twice in PBS and spun down at 300g/5min/RT.

The eBioscîence FoxP3 transcription factor staining buffer was then used to fix and permeabilise the cells (cat. No. 00-5523). Following the eBioscîence protocol, a perm/fix buffer was prepared using Ix concentrate and 3 diluent. The cells were fixed for lh at RT and then washed 2x in Ix Perm wash and spun down at 300g/5min/RT. The following intracellular staining or transcription factor antibodies were added to the samples in penn wash (Ix) for 45mis/dark/RT or in the fridge ovemight (up to 18h), followed by washing the antibodies 2x using Penn wash (300μ1) and re-suspension in PBS (250μ1) to acquire on the cytometer:

Intracellular

Transcription

markers	factors
2ul IL10-PE	5.5ul FoxP3-PE-Cy7
2ul IFNy-ΡΕ Vio770	9ul Tbet-APC
lOul IL17a-APC	9ul RoRyt-PE

• Anti IFNy-ΡΕ Vio770 human antibodies (Miltenyi, cat. No. 130-114-025) • Anti IL10-PE human antibodies (Miltenyi, cat. No. 130-112-728) • Anti IL17a-APC human antibodies (Miltenyi, cat. No. 130-099-202) · Anti RoRyt-PE human antibodies (Miltenyi, cat. No. 130-103-837) • Anti Tbet-APC human antibodies (Miltenyi, cat. No. 130-098-655 • Foxp3 monoclonal antibody (236A/E7), Pe cy7 (ebioscience) cat. No. 25-4777-41

As can be seen in Figures 27 and 28 both supematant of MRx0004 (SP 4) and heat-killed 10 MRx0004 (HK 4) were able to induce différentiation of T helper cells and cytotoxic T cells, respectively, even in the absence of cytokines to induce différentiation (no cyto).

Tables

Table 1. Strains, plasmids and primers used strain génération in this study

Strain

Description Reference

B, breve strains

MRx0004	B. breve human isolate This study
MRx0004 j9G7F::pORI19	MRx0004 pGTF insertion mutant This study
MRx0004 pG7F.-.pORI19 pBC1.2	MRx0004 pGTF insertion mutant harbouring the This study empty pBCl .2 vector
MRx0004 /7GTF;.-pORII9 pG7F-pBC1.2	MRx0004 pGTF insertion mutant complemented This study

E. coli strains

XI1 Blue

Clonîng host, tet^r

Stratgene, La

Jolla, CA, USA

ECI01 Cloning host, repA' km^r

EC101 pWSK29- E. coli strain for in vitro méthylation by MRx0004 This study

MRx-M+S Type I methylase

Plasmids

pORI19	Lactococcal ORr RepA' expression vector	[108]
pWSK29	Amp^r, low copy mumber E .coli cloning plasmid	[109]
pNZ8048	Cm^r, nisin inducible translational fusion factor	[HO]
pAM5	pBCl-pUC19-Tc^r	[Hl]
pPKCM	pblueCm harbouring rep pCIBA089	[112]
pSKEM	pblueEm harbouring rep pCIBA089	[112]
pNZEM	Em^r, nisin inducible	Margolles, unpublished
pDMl	pAM5 dérivative containing spectinomycîn résistance cassette	[H3]
pDM2	pDG7 dérivative containing spectinomycîn résistance cassette	[113]
Primers
Primer Name	Sequence (5’-3’)	Reference
PWSK_MRxM+ SFxbal	CGTCCGTCTAGAATAAGGAGGCACTCACCATG AATAAGCAGCAGCTTGC (SEQ ID NO:2)	This study
PWSK_MRxM+ SFxhol	GCTCTACTCGAGGCGATATGAGGCGAGCTTCA CG (SEQ ID NO:3)	This study
TetWconfirm F2	CAGGCATTGAAGGAATCG (SEQ ID NO:4)	This study
TetWconfïrm F2 +MRx-pGTFcomp	CTGGCTAAGCTTGTGCATGGGCTCAGTCCTTC (SEQ ID NO:5)	This study

Table 2. List of the 20 proteins with the highest peptide spectrum match (PSM) values identified in MRx0004 late log culture supernatants as identified by nanoLC-MS/MS,

This table lists ail proteins with a PSM value > 5 identified in three bîological replicates.

Annotation	Normalised PSM value ^a	Functiona 1 category b	Predicted localization (score)^c	MW (kDa) ^d	pl^d
Average	SD
Pullulanase	351.33	33.62	Carbohydr ates	E (9.98)	181.9	4.77
NlpC/P6Ü family protein	82.00	15.62	n.a.	E (9.72)	33.5	8.18
NlpC/P60 family protein	71.67	13.61	n.a.	n.d.(3.33)	24.9	6.18
Probable soluté binding protein of ABC transporter System	56.00	7.00	n.a.	n.d. (3.33)	32.8	4.69
Cell division protein FtsI	56.00	4.36	Cell Division and Cell Cycle	CM (9.68)	63.4	5.39
Multiple sugar ABC transporter, substrate-binding protein	54.67	4.16	Carbohydr ates	n.d.(3.33)	49.3	5.01
Xylulose-5-phosphate phosphoketolase	45.33	7.02	n.a.	n.d.(2.5)	92.3	5.26
Méthionine ABC transporter substrate-binding protein	38.67	2.08	Amino Acids and Dérivative s	CM (9.51)	35.2	5.17
Hypothetical protein	35.67	8.33	n.a.	n.d. (2.5)	34.6	7.47
Transaldolase	32.67	3.79	Carbohydr ates	C (7.5)	39.7	4.97
Maltose/maltodextrin ABC transporter	30.00	6.08	Carbohydr ates	n.d. (3.33)	43.9	4.48

N AD-dependent glyceraldehyde-3phosphate dehydrogenase	30.00	3.61	Carbohydr ates	C (9.97)	37.8	5.44
Hypothetical protein	29.67	4.93	n.a.	CW (9.21)	47.1	5.48
Soluté binding protein of ABC transporter system for peptides	26.67	1.15	n.a.	CW (9.2)	58.7	5.45
Dipeptide-binding ABC transporter, periplasmic substrate-binding component	24.00	4.58	Membrane Transport	n.d.(5.13)	59.1	5.31
NlpC/P60 family protein	21.00	1.00	n.a.	E (9.73)	24.5	6.30
FKBP-type peptidylprolyl cis-trans isomerase FkpA precursor	20.00	2.65	Potassium metabolis m	n.d. (5.15)	33.4	5.83
Glucose-6-phosphate isomerase	18.67	3.21	Carbohydr ates	C (9.97)	62.9	4.97
Multimodular transpeptidasetransglycosylase	18.00	2.65	Cell Wall and Capsule	CM (10)	81.9	5.72
Chaperone protein DnaK	17.67	3.21	Protein Metabolis m	C (9.97)	66.9	4.87

^a Average peptide spectrum match values from 3 biological replicates nonnalized to Ix 10^y cfu in and corresponding standard déviation values.

^b Subsystem category distribution as annotated by RAST [114, 115], n.a. not assigned.

^c Cellular localization as predicted using PSORTb v3.0 (Yu et al., 2010a). C: cytoplasmic, CM:

cytoplasmic membrane, CW: cell wall, E: extracellular, n.d.: not determined.

^d Parameters calculated by Proteome Dîscoverer (Thermo Scientific, Waltham, MA, USA). MW: molecular weight, pl: isoelectric point.

Table 3. List of the top 20 proteins with the highest peptide spectrum match (PSM) value identified in MRx0004 cell shavings as identified by nanoLC-MS/MS.

This table lists the 62 proteins identified in both MRx0004 shaved and shed protein fractions and the 44 proteins identified exclusively in MRx0004 shaved protein fraction. Proteins listed were 5 detected at PSM value > 5 and in three biological replicates.

Annotation	Shaved proteins	Shed proteins	Predicted localization (score) ^c
Average PSM^a	SD	Average PSM^a	SD	Functional category
Pullulanase	136.67	17.47	79.00	10.54	Carbohydrates	E (9.98)
Type I polyketide synthase	54.67	10.69	11.67	6.43	Unassigned	CM (9.78)
Xylulose-5phosphate phosphoketol ase	49.67	2.89	52.00	9.17	Unassigned	n.a. (2.5)
Cell division protein FtsI	34.33	9.24	33.00	10.00	Cell Division and Cell Cycle	CM (9.68)
Glucose-6-phosphate isomerase	32.33	2.31	30.33	5.86	Carbohydrates	C (9.97)
Translation élongation factor Tu	32.00	5.00	24.33	8.39	Protein Metabolism	C (9.97)
Maltose/maltodextrin ABC transporter, substrate binding periplasmic protein MalE	32.00	7.94	18.67	5.13	Carbohydrates	n.a. (3.33)
Glycogen phosphorylase	31.00	3.00	34.33	5.69	Carbohydrates	C (7.5)
Transaldolase	31.00	2.65	33.33	2.08	Carbohydrates	C (7.5)
Multiple sugar ABC transporter, substratebinding protein	30.00	6.93	36.00	11.79	Carbohydrates	n.a. (3.33)
Pyruvate formatelyase	26.67	8.33	21.67	7.77	Carbohydrates	C (9.97)
Soluté binding	26.00	0.00	12.67	2.89	Unassigned	CW (9.2)

protein of ABC transporter systemy for peptides

Transketolase	24.00	6.08	23.67	3.79	Carbohydrates	C (7.5)
Méthionine ABC transporter substratebinding protein	23.33	2.52	22.33	4.04	Amino Acids and Dérivatives	CM (9.51)
Translation élongation factor G	23.00	13.89	15.67	8.02	Protein Metabolism	C (9.97)
Polyribonucleotide nucleotidyltransferas e	22.67	5.03	12.33	5.69	Unassîgned	C (9.97)
PTS system, betagl ucoside-specî fi c IIB component	21.67	4.62	6.67	2.89	Unassigned	CM (10)
FKBP-type peptidylprolyl cis-trans isomerase FkpA precursor	21.33	5.86	7.67	3.06	Potassium metabolism	n.a. (5.15)
Chaperone protein DnaK	21.00	6.24	10.00	3.61	Protein Metabolism.	C (9.97)
Probable soluté	20.33	4.04	19.67	2.08	Unassigned	n.a. (3.33)

binding protein of

ABC transporter system

Average peptide spectrum match values from 3 biological replicates and corresponding standard déviation values, n.d.: not detected.

^b Subsystem category distribution as annotated by RAST [116,Error! Bookmark not defined.]. n.a. not assigned.

^c Cellular localization as predicted using PSORTb v3.0 [117], C: cytoplasmic, CM: cytoplasmic membrane, CW; cell wall, E: extraceliular, n.a.: not assigned.

Table 4. Comparison of host-interaction proteins identified in MRx0004 and EPS?^ strains shaved protein fractions.

This table lists selected proteins, identified by nanoLC-MS/MS in the shaved protein fractions, in three biological replicates and with a PSM value > 5.

Description

MRx0004^a

EPS^nega Functional

	Average normalise d PSM	SD	Average normalised PSM	SD	category ^b
Type I polyketide synthase	28.04	4.47	57.06	2.17	n.a.
Transaldolase	17.33	1.44	24.70	0.60	Carbohydrate s
Transketolase	14.18	2.50	25.40	0.60	Carbohydrate
Translation élongation factor Tu	13.23	1.64	26.10	4.55	Protein Metabolism
Chaperone protein DnaK	12.29	4.33	21.92	2.09	Protein Metabolism
NAD-dependent glyceraldehyde-3-	11.97	3.32	16.35	1.59	Carbohydrate
phosphate dehydrogenase					s
Heat shock protein 60 family chaperone GroEL	11.66	6.29	13.92	2.17	Protein Metabolism
Enolase	5.04	3.04	8.70	1.59	Carbohydrate s
Phosphoglycerate mutase	n.d.	n.d.	5.22	1.04	Carbohydrate s

Average peptide spectrum match values, normalized per pg of total protein, from 3 biological replicates and corresponding standard déviation values (SD), n.d: no peptides detected.

^b Subsystem category distribution as annotated by RAST [Error! Bookmark not defined.,Error! Bookmark not defined.], n.a, no subcategory assigned.

Table 5. List of indicator strains used in Example 7

Strain name	Gram s tain	Description
Escherichia coli ATCC 11775	Négative	Human gut commensal
Klebsiella pneumoniae NCIMB 10197	Négative	Human gut commensal, lung pathogen
Salmonella Typhimurium NCIMB 10248	Négative	Human gut commensal, opportunistic pathogen
Lactobacillus plantarum NCIMB 8826	Positive	Human gut commensal, closely-related bacteria
Clostridium sporogenes ATCC 3584	Positive	Ubîquitous human gut commensal
Bacillus subtilis NCIMB 8045	Positive	Human gut commensal

Staphylococcus aureus NCIMB 9518	Positive	Human commensal, opportunisme pathogen
Bifidobacterium breve DS M2 0213	Positive	B. breve type strain

Table 6. Antimicrobial activity of test and reference B. breve strains against a panel of indicator strains (n=3)

Strain	E. coli ATC C 11775	K. pneumoni ae NCIMB 10197	5. Typhimuriu m NCIMB 10248	B. subtili s NCIM B 8045	B. breve DSM 20213 »	5. aureu s NCIB M * 9518	C. sporogene s ATCC 3584*	L. plantarum NCIMB 8826*
MRxOOO 4	3	2	2	2*	0	0	0	0
Test 1	1	2	1	2’	0	0	0	0
Test 2	2	2	2	2’	0	0	0	0
Test 3	2	2	2	2’	0	0	0	0
Test 4	1	2	0	Γ	0	0	0	0
Test 5	1	1	1	Γ	0	0	0	0
Test 6	1	1	1	Γ	0	0	0	0
Test?	2	2	2	Γ	0	0	0	0
Test 8	2	1	1	2*	0	0	0	0
Test 9	1	I	1	1	0	0	0	0
Test 10	1	1	1	1	0	0	0	0
Test 11	2	2	2	2	0	0	0	0
Test 12	2	2	2	2	0	0	0	0
B. breve REF1 (DSM 20091)	I	I	1	I	1	0	0	0
B. breve REF2 (DSM 20213)	I	1	0	1	0	0	0	0

B. breve REF6 (JCM 7017)	1	1	1	2	0	0	0	0
B. breve REF7 (NCIMB 8807/ UCC200 3)	0	0	0	2	0	0	0	0

Example 12 - MRx004 has an immunostimulatory effect in the spleen.

Summary

The object of this study was to characterise the in vitro immunostimulatory properties of MRx0004 in the spleen.

Materials and methods

Treatments: Untreated, 10% YCFA and 10% Bifidobacterium breve strain MRx0004.

Préparation ofSplénocytes

Splénocytes were freshly prepared from spleen isolated from female C57BL/6 mice between 6 and 8 weeks old. Briefly, splénocytes wereplated at 900,000 cells/well in 96 well plates in RPMI 1640 with 10% FBS, 2mM L-GIutamine and 100 U/ml penicillin, lOOpghnl streptomycin, 55 μΜ of β-mercaptoethanol, resting or stimulâtes with 10% bacterial media YCFA+ (Blank media) or with 10% cell-free bacterial supematant from stationary MRx0518 culture and then incubated for 72h in a CO2 incubator at 37°C. Afterwards cell free supematants were collected, spun down for 5 minutes at 500g at 4°C. S amples were then collected and stored at -80°C for cytokine analysis.

MTT assav

The MTT assay kit was purchased from Merck Millipore (Cat n. CT01). After 72h incubation, ΙΟμΙ of MTT solution was added to each well, cells were incubated in CO2 incubator for 4h. Afterwards 100μ1 of isopropanol/0.04 M HCL solution was added to each well and the absorbance was measured at 560nm wavelength and a reference wavelength of 655 nm.

Cytokine analysis

Cytokine quantification was conducted using a 26-plex Mouse ProcartaPIex multiplex immunoassay following the manufacturer’s recommendations (Thermo Fischer Scientific). Briefly, 50 pl of cell-free co-culture supematants were used for cytokine quantification using a MAGPIX® MILLIPLEX® System (Merck) with the xPONENT software (Luminex, Austin, TX, USA). Data was analysed using the MILLIPLEX® analyst software (Merck) using a 5-parameter logistic curve and background subtraction to convert mean fluorescence intensity to pg/ml values.

Flow cytometry

Cells were first stained with the Viobîlîty 405/520 Fixable Dye (Miltenyi Biotec Ltd. Bergisch Gladbach, Germany) to discriminate between live and dead cells for 10 min in the dark at room température. They were then stained with a cocktail of antibodies for CD3, CD4, CD8 and IFN-γ to détermine cell phenotype (Miltenyi REA antibodies) and incubated for a further 10 min at room température. Cells were then washed and resuspended in PBS and immediately analysed via flow cytométrie analysis. Isotypes were used for ail antibodies during the first experiment to help set gates and FMO Controls were included throughout ali the experîments. Ail experiments were performed using a BD FACS Aria II with the stoppîng gâte for acquisition set on 100,000 cells in the “Live” gâte using FACSDiva software (BD Biosciences, Reading, UK). The analysis was conducted using Flowjo version 10.4.2 software (FlowJo LLC, Oregon, USA) and was based on live cells identified with the viability dye.

Results

The viability of the splénocytes after treatment was assessed using the MTT assay, which measured their metabolic activity. Figure 29 shows that splénocytes were viable after treatment with MRx0004.

Figure 30 shows that treatment with MRx0004 led to an increase in a variety of proinflammatory cytokines in the spleen, including IL-6, IL-17a IL-22, TNF-α, RANTES, IFN-γ, CCL3, CCL4 and CXCL2. These data indicate that live Bifidobacterium breve hâve a stimulatory effect on the immune System. The ability of MRx0004 to actîvate CD8+ and CD4+ T cells to produce IFNy is shown in Figure 31.

In combination with the PBMC data discussed in Examples 2 and 11, these data support the ability of bacterial strains from the species Bifidobacterium breve to stimulate the immune System, by activating T-cells and increasing the level of proinflammatory cytokines, in multiple tissues.

Séquences

SEQ ID NO:1 (consensus 16S rRNA sequence for Bifidobacterium breve strain deposited under accession number NCIMB 423 SO)

GGGACAGGCTCAGGATGAACGCCGGCGGCGTGCTTAACACATGCAAGTCGAACGGG ATCCATCGGGCTTTGCCTGGTGGTGAGAGTGGCGAACGGGTGAGTAATGCGTGACC GACCTGCCCCATGCACCGGAATAGCTCCTGGAAACGGGTGGTAATGCCGGATGCTC CATCACACCGCATGGTGTGTTGGGAAAGCCTTTGCGGCATGGGATGGGGTCGCGTCC TATCAGCTTGATGGCGGGGTAACGGCCCACCATGGCTTCGACGGGTAGCCGGCCTG AGAGGGCGACCGGCCACATTGGGACTGAGATACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCGACGCCGCGTGAGG GATGGAGGCCTTCGGGTTGTAAACCTCTTTTGTTAGGGAGCAAGGCACTTTGTGTTG AGTGTACCTTTCGAATAAGCACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGT AGGGTGCAAGCGTTATCCGGAATTATTGGGCGTAAAGGGCTCGTAGGCGGTTCGTC GCGTCCGGTGTGAAAGTCCATCGCTTAACGGTGGATCCGCGCCGGGTACGGGCGGG CTTGAGTGCGGTAGGGGAGACTGGAATTCCCGGTGTAACGGTGGAATGTGTAGATA TCGGGAAGAACACCAATGGCGAAGGCAGGTCTCTGGGCCGTTACTGACGCTGAGGA GCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACG GTGGATGCTGGATGTGGGGCCCGTTCCACGGGTTCCGTGTCGGAGCTAACGCGTTAA GCATCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGG CCCGCACAAGCGGCGGAGCATGCGGATTAATTCGATGCAACGCGAAGAACCTTACC TGGGCTTGACATGTTCCCGACGATCCCAGAGATGGGGTTTCCCTTCGGGGCGGGTTC ACAGGTGGTGCATGGTCGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTCGCCCCGTGTTGCCAGCGGATTGTGCCGGGAACTCACGGGG GACCGCCGGGGTTAACTCGGAGGAAGGTGGGGATGACGTCAGATCATCATGCCCCT TACGTCCAGGGCTTCACGCATGCTACAATGGCCGGTACAACGGGATGCGACAGCGC GAGCTGGAGCGGATCCCTGAAAACCGGTCTCAGTTCGGATCGCAGTCTGCAACTCG ACTGCGTGAAGGCGGAGTCGCTAGTAATCGCGAATCAGCAACGTCGCGGTGAATGC GTTCCCGGGCCTTGTACACACCGCCCGTCAAGTCATGAAAGTGGGCAGCACCCGAA GCCGGTGGCCTAACCCCTGCGGGAGGGAGCCKC

SEQ ID NO:2-5 - see Table 1

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Claims

1. A composition comprising a bacterial strain of the species Bifidobacterium breve, for use in stimulating the immune System in a subject.

2, The composition for use according to daim 1, wherein the composition is for use in treating an immunodeficiency disease in a subject, optionally the immunodeftciency disease is a primary immunodeficiency disease or a secondary immunodeficiency disease.

3. The composition for use according to claim 2, wherein:

(a) the primary immunodeficiency disease is selected from X-linked agammaglobulînemia (XLA), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID) and severe combined immunodeficiency (SCID), or (b) the secondary immunodeficiency disease is selected from AIDS, cancers of the immune System, such as leukemia, immune-compl ex dîseases, such as viral hepatitis multiple myeloma.

4. The composition for use according to claim 1, wherein the composition is for use:

(a) as a vaccine adjuvant, or (b) in treating, preventing or delaying immunosenescence, or (c) in enhancing a cell therapy, such as CAR-T.

5. The composition for use according to any preceding claim, wherein:

(a) the composition is for use in increasing the expression level and/or activity of IL-12p70, IL-12p70, IFNy, IL-4, TNF-α and/or IL-17α, and/or (b) the composition is for usein stimulating TLR2, and/or (c) the composition is for use in stimulating NFkB, and/or (d) the bacterial strain comprises a complété exopolysaccharide locus, and/or (e) the bacterial strain expresses pullulanase.

6. A composition comprising a bacterial strain of the species Bifidobacterium breve, for use in treating or preventing a bacterial infection.

7. The composition for use according to claim 6, wherein the composition is for use in treating or preventing a gastro-intestinal bacterial infection, optionally wherein the composition is for use in treating or preventing a Gram-negative bacterial infection.

8. The composition for use according to any preceding claim, wherein the bacterial strain:

(a) has a 16s rRNA gene sequence that is at least 95%, 96%, 97%, 98%, 99%, 99.5% or 99.9% identical to SEQ ID NO:1 or wherein the bacterial strain has a 16s rRNA gene sequence represented by SEQ ID NO:1, and/or (b) is able to ferment raffinose, and/or (c) is able to ferment one or more, such as 2, 3, 4, 5, 6 or ail 7 of: a-galactosidase, β-galactosidase, α-glucosîdase and β-glucosîdase, α-arabinose, mannose and raffinose, and/or (d) is the strain deposited under accession number 42380 at NCIMB.

9. The composition for use according to any preceding claim, wherein:

(a) the composition is for oral administration, and/or (b) the composition comprises one or more pharmaceutically acceptable excipients or carriers, and/or (c) the bacterial strain is lyophilised.

10. A composition comprising a cell of the bacterial strain defined in any of claims I to 9, wherein the cell expresses one or more heterologous antigens.

11. A cell of the bacterial strain defined in any of claims 1 to 9, wherein the cell expresses one or more heterologous antigens.

12. Tire composition according to claim 10 or the cell according to claim 11, wherein the cell présents the one or more heterologous antigens.

13. The composition according to claims 10 or claim 12 or the cell according to claim 11 or daim 12, for use as a vaccine.