HK1141554B - Probiotic bifidobacterium strains - Google Patents

Description

Probiotic bifidobacteria strains

Introduction to the design reside in

The present invention relates to bifidobacterium strains and their use as probiotics, in particular as immunomodulatory biotherapeutics.

The defense mechanisms that protect the human gastrointestinal tract from colonization by intestinal bacteria are quite complex and involve the fields of immunology and non-immunology (1). Innate defense mechanisms include low pH of the stomach, bile salts, peristalsis, mucin layers, and antimicrobial compounds such as lysozyme (2). Immunological mechanisms include idiopathic Peyer's patch, a basal M cell, also known as Peyer's patch, distributed throughout the small intestine and colon (3). Presentation of luminal antigens at these sites results in stimulation of appropriate T and B cell subtypes, thereby establishing a cytokine network and secreting antibodies into the gastrointestinal tract (4). Furthermore, antigens can be presented to both the epithelial lymphocytes and the basal lamina propria immune cells by epithelial cells (5). Thus, the host is considerably dependent on the immune defenses of the gastrointestinal tract. However, since the gastrointestinal mucosa is the largest surface of the host that interacts with the external environment, the specific control mechanisms that occur on it must be able to properly regulate the immune response to hundreds of tons of food handled by the gastrointestinal tract during the average lifetime. Moreover, the digestive tract is colonized with over 500 species of bacteria, the number in the colon being 10¹¹-10¹²(ii) in terms of/g. Thus, these control mechanisms must be able to distinguish non-pathogenic adherent bacteria from invasive pathogens that cause significant harm to the host. In fact, the intestinal flora is of great benefit to host defense by competing with newly ingested potentially pathogenic microorganisms.

Bacteria present in the human gastrointestinal tract can cause inflammation. Abnormal immune responses to the resident microbial flora may involve certain disease states, such as inflammatory bowel disease. Antigens associated with normal flora often lead to immune tolerance, and failure to achieve such tolerance is a major mechanism of mucosal inflammation (6). In patients with Inflammatory Bowel Disease (IBD), evidence of compromised tolerance includes elevated levels of antibodies to the gut flora.

The present invention relates to strains of bifidobacterium which may be shown to have immunomodulatory effects by modulating cytokine levels or by antagonizing and excreting pro-inflammatory microorganisms from the gastrointestinal tract.

Disclosure of Invention

The present invention provides bifidobacterium strain AH1205(NCIMB41387) or mutants or variants thereof.

The mutant is a genetically modified mutant. The variant is a naturally occurring variant of bifidobacterium.

The strain is a probiotic. It is in the form of a biologically pure culture.

The invention also provides an isolate of bifidobacterium NCIMB 41387.

In one embodiment of the invention, the bifidobacterium strain is in the form of viable cells. Or the Bifidobacterium strain is in the form of non-viable cells

In one embodiment of the invention, the bifidobacterium strain is isolated from infant faeces, which bifidobacterium strain is significantly immunomodulatory following oral consumption in humans.

The invention also provides a preparation containing the bifidobacterium strain.

In one embodiment of the invention, the formulation includes other prebiotic substances.

In one embodiment of the invention, the formulation includes a prebiotic substance.

Preferably, the formulation includes an ingestable carrier. The ingestable carrier may be a pharmaceutically acceptable carrier such as a capsule, tablet or powder. Preferably, the ingestible carrier may be a food product, such as acidified milk, yoghurt, frozen yoghurt, milk powder, milk concentrate, cheese spreads, dressings or beverages.

In one embodiment of the invention, the machine according to the invention also comprises proteins and/or peptides, in particular proteins and/or peptides rich in glutamine/glutamate, lipids, carbohydrates, vitamins, minerals and/or trace elements.

In one embodiment of the invention, the bifidobacterium strain is present at more than 10 per gram of delivery system⁶The amount of cfu is present in the formulation. Preferably, the formulation comprises one or more adjuvants, bacterial components, pharmaceutical entities or biological compounds.

In one embodiment of the invention, the formulation may be used in immunization and vaccination protocols.

The invention also provides a bifidobacterium strain or a formulation of the invention for use as a foodstuff, as a medicament for the prevention and/or treatment of undesirable inflammatory activity, for the prevention and/or treatment of undesirable respiratory inflammatory activity (e.g. asthma), for the prevention and/or treatment of undesirable gastrointestinal inflammatory activity (e.g. inflammatory bowel disease such as crohn's disease or ulcerative colitis, irritable bowel syndrome, chronic enteritis, or post-infectious colitis), for the prevention and/or treatment of gastrointestinal cancer, for the prevention and/or treatment of systemic diseases such as rheumatoid arthritis, for the prevention and/or treatment of autoimmune disorders caused by undesirable inflammatory activity, for the prevention and/or treatment of cancer caused by undesirable inflammatory activity, for the prevention and/or treatment of diarrhoeal disease caused by undesirable inflammatory activity (e.g. diarrhoea associated with Clostridium difficile (Clostridium difficile), a, Rotavirus associated diarrhea or post-infection diarrhea) for preventing and/or treating diarrhea diseases caused by infectious agents such as e.

The invention also provides a bifidobacterium strain or a formulation as described herein for use in the manufacture of an anti-inflammatory biotherapeutic agent for the prevention and/or treatment of undesirable inflammatory activity, or for use in the manufacture of a plurality of anti-inflammatory biotherapeutic agents for the prevention and/or treatment of undesirable inflammatory activity.

In one embodiment of the invention, the strains according to the invention may act by antagonizing and excluding proinflammatory microorganisms from the gastrointestinal tract.

The invention also provides a bifidobacterium strain or a formulation as described herein for use in the preparation of an anti-inflammatory biotherapeutic agent for reducing the level of pro-inflammatory cytokines.

The invention also provides a bifidobacterium strain as described herein for use in the preparation of an anti-inflammatory biotherapeutic agent for modulating IL-10 levels.

The invention also provides the use of a bifidobacterium strain as an anti-infective probiotic, due to its ability to antagonize the growth of pathogenic species.

The inventors have also found that specific bifidobacterium strains are capable of eliciting an immunomodulatory effect in vitro.

The invention therefore has great potential therapeutic value in the prevention or treatment of dysregulated immune responses, such as adverse inflammatory responses (e.g. asthma).

Bifidobacteria are symbiotic microorganisms. It is isolated from the microbial flora in the gastrointestinal tract of the human body. The immune system in the gastrointestinal tract does not respond significantly to members of this flora since the resulting inflammatory activity also destroys host cell and tissue function. Thus, there are mechanisms by which the immune system is able to recognize commensal nonpathogenic members of the gastrointestinal flora that are distinct from pathogenic organisms. This ensures that damage to host tissue is limited and still maintains a defensive barrier.

Bifidobacterium longum (longum) strain AH1205 was deposited at NCIMB on day 11 of 2006 under the accession number NCIMB 41387.

The bifidobacterium longum may be a genetically modified mutant strain or may be a naturally occurring variant strain.

Preferably, the bifidobacterium longum is in the form of viable cells.

Alternatively, the bifidobacterium longum may be in the form of non-viable cells.

It will be appreciated that a particular bifidobacterium strain of the invention may be administered to animals, including humans, in orally ingestible form in conventional formulations such as capsules, microcapsules, tablets, granules, powders, lozenges, pills, suppositories, suspensions and syrups. Suitable formulations may be prepared by conventional methods using conventional organic and inorganic additives. The amount of active ingredient in the pharmaceutical composition may be manipulated according to the desired therapeutic effect.

The formulation may also comprise a bacterial component, a pharmaceutical entity or a biological compound.

In addition, vaccines containing the strains of the invention may be prepared using any suitable known method and may include pharmaceutically acceptable carriers or adjuvants.

In the present specification, the terms mutant, variant and genetically modified mutant include strains of bifidobacterium which have altered genotypic and/or phenotypic properties compared to the parent strain. Naturally occurring variants of bifidobacterium longum include spontaneous alterations to the selected targeted properties isolated. Deliberate alterations to the properties of the parent strain are effected by conventional (in vitro) genetic manipulation techniques, e.g., gene disruption, conjugal transfer, etc. Genetic modification includes the introduction of exogenous and/or endogenous DNA sequences into the genome of a bifidobacterium strain, for example by insertion of a vector (including plasmid DNA or phage) into the genome of the bacterial strain.

Natural or induced mutations include changes of at least a single base, such as deletions, insertions, transformations, or other DNA modifications, which can result in changes in the amino acid sequence encoded by the DNA sequence.

The terms mutant, variant and genetically modified mutant also include strains of bifidobacterium which have undergone genetic alterations which accumulate in the genome at a rate consistent for all microorganisms in nature and/or which occur through spontaneous mutations and/or acquisition of and/or loss of genes, not through deliberate (in vitro) manipulation of the genome but through natural selection of the variant and/or mutant which can provide a selective advantage when exposed to environmental stresses such as antibiotics to support bacterial survival. Mutants can be generated by deliberate (in vitro) insertion of specific genes into the genome, which do not fundamentally alter the biochemical function of the organism in question, but whose products can be used for identifying or screening bacteria for, for example, antibiotic resistance.

It will be appreciated by those skilled in the art that mutants or variants of bifidobacteria may be identified by DNA sequence homology analysis with the parent strain. Strains of bifidobacterium having close sequence identity to the parent strain are considered to be mutants or variants. A strain of bifidobacterium having 96% or more sequence identity (homology) with a parent strain, for example 97% or more, or 98% or more, or 99% or more, is considered a mutant or variant. The homology of the sequences can be determined using the online homology algorithm "BLAST" program, disclosed in http:// www.ncbi.nlm.nih.gov/BLAST/.

Mutants of a parent strain also include those derived from a strain of bifidobacterium having at least 85% sequence homology, for example at least 90% sequence homology, or at least 95% sequence homology to the 16s-23s intergenic spacer polynucleotide sequence of the parent strain. These mutants also comprise DNA mutations of other DNA sequences in the bacterial genome.

Brief Description of Drawings

FIG. 1 is a BOX PCR (Bioanalyzer) bar code diagram of Bifidobacterium longum AH 1205. Base pair size was determined using Agilent2100 software.

Figure 2 illustrates fecal recovery of bifidobacterium longum AH1205 during the 8 day feeding period and demonstrates the ability of AH1205 to metastasize in the gastrointestinal tract of mice.

FIG. 3 is a bar graph showing the effect of Bifidobacterium longum AH1205 on IL-10 cytokine production by human PBMCs. Results are expressed as mean +/-SE (n ═ 6).

Figures 4A and B illustrate the effect of probiotic strain AH1205(a) and placebo (B) on total cell number in bronchoalveolar lavage fluid of sensitized animals following Ovalbumin (OVA) challenge (n 10/group versus OVA alone)^*＝p＜0.05)。

Figures 5A and B illustrate the effect of probiotic strain AH1205(a) and placebo (B) on methacholine airway reactivity, assessed by the change in increased quiescence (Penh) in Ovalbumin (OVA) -sensitized mice after 24 hours of intranasal challenge with OVA or saline. Each data point represents mean ± SEM (n-10/group, vs OVA alone^*＝p＜0.05)。

FIGS. 6A-B are graphical representations of IL-10(A), INF γ (B), TNF (C), IL-6(D) and CCL2(E) cytokine levels in bronchoalveolar lavage (BAL) from Ovalbumin (OVA) -sensitized mice. Each column represents mean ± SEM (n-10, relative to OVA challenged saline-out control)^*p＜0.05)。

FIG. 7 is a graph depicting CD4 from AH1205 fed mice⁺CD25⁺The cells significantly reduced proliferation of T cells in response to CD4 (n-7).

FIG. 8 shows a CD4⁺Group (also CD 25)⁺) The percentage of peyer's patches of cells assessed by flow cytometry.

FIGS. 9A and B show CD4/CD25 expressing the transcription factor Foxp3 in H1205-ingested sterile mice⁺The percentage of cells was significantly up-regulated. (A) Spleen cells, (B) ═ MLNC cells (n-4/group for spleen analysis, n-2/3 for MLNC analysis).

FIG. 10 is a graph showing the reduction in the levels of cytokines IL-6, MCP-1 and INF-gamma secreted in CD3/CD28 stimulated MLNC cultures after ingestion of Bifidobacterium longum AH1205 by sterile mice. Results are expressed as the mean +/-standard deviation of each group (n-4/group).

FIG. 11 is a graph showing the reduction in the levels of cytokines IL-6 and INF- α secreted in CD3/CD28 stimulated spleen cell cultures after ingestion of Bifidobacterium longum AH1205 by germ free mice. Results are expressed as mean/-standard deviation per group (n 4/group).

Figure 12 shows the stability of probiotic strain AH1205 after 3 months compared to Lactobacillus (Lactobacillus) GG.

Detailed Description

The inventors have found that bifidobacterium longum strain AH1205 is not only acid and bile tolerant and can be transported in the gastrointestinal tract (transit), but also surprisingly has an immunomodulatory effect by modulating cytokine levels or by antagonizing and excluding proinflammatory or immunomodulatory microorganisms from the gastrointestinal tract.

The probiotic bacteria are generally in the form of viable cells. However it may also extend to inactive cells, for example killed cultures or compositions containing beneficial factors expressed by probiotics. This may include heat killed microorganisms or microorganisms killed by exposure to altered pH or applied pressure. Considering that the preparation of non-viable cell products is simpler, such cells can be easily incorporated into pharmaceutical products and storage requirements are much more relaxed than for viable cells. Lactobacillus casei (Lactobacillus casei) YIT9018 provides an example of an effective use of heat killed cells as a means of treating and/or preventing tumor growth, see U.S. patent No.4,347,240.

It is not clear whether the intact bacteria are required for the implementation of the immunomodulating effect or whether the individual active ingredients of the invention can be used individually. Proinflammatory components have been identified for certain bacterial strains. The pro-inflammatory action of gram-negative bacteria is mediated by Lipopolysaccharide (LPS). LPS alone can induce pro-inflammatory networks, in part because LPS can bind to the CD14 receptor on monocytes. The components of the probiotic are generally considered to possess immunomodulatory activity due to the action of whole cells. Pharmaceutical grade manipulations cannot be expected until these components are separated.

IL-10 is produced by T cells, B cells, monocytes and macrophages. This cytokine can enhance proliferation and differentiation of B cells into antibody-secreting cells. IL-10 has the most prominent anti-inflammatory activity. It can up-regulate the expression of IL-1RA by monocytes and inhibit the inflammatory activity of most monocytes. IL-10 is able to inhibit the production of cytokines by monocytes through a feedback mechanism, with reactive oxygen and nitrogen intermediates, MHC class II expression, parasite killing, and IL-10 production (7). This cytokine has also been shown to interfere with PGE₂cAMP-dependent metabolic pathways to block the production of intestinal collagenase and collagenase type IV by monocytes and thus may serve as an important regulator of connective tissue destruction seen in chronic inflammatory diseases.

The host's response to infection can be described as innate and acquired cellular and humoral immune responses, aimed at limiting the spread of offensive organisms and reestablishing homeostasis of organs. However, to limit the aggressiveness of secondary injury to host tissues, a series of regulatory limitations may be activated. One such mechanism is achieved by regulatory T cells (Tregs). These cells may be from the thymus but may also be induced in peripheral organs, including in the mucosa of the digestive tract. Deliberate administration of Treg cells can suppress inflammatory diseases in a wide range of mouse models, including experimental autoimmune encephalomyelitis, inflammatory bowel disease, bacterially-induced colitis, collagen-induced arthritis, type I diabetes, airway eosinophilic inflammation, graft-host disease and organ transplantation. The forkhead transcription factor Foxp3 (forkhead box P3) is selectively expressed in Treg cells, is essential for Treg development and function, and is sufficient to induce Treg phenotype in conventional CD4 cells (19). Mutations in Foxp3 can cause severe multi-organ autoimmunity in humans and mice. The inventors describe strains of bifidobacterium capable of producing CD25 positive/Foxp 3 positive T regulatory cells in vivo.

The present invention can be more clearly understood from the following examples.

Example 1: bacteria isolated from infant feces were characterized. And (4) demonstration of probiotic characters.

Isolation of probiotics

Fresh faeces were obtained from breast-fed 3 day old male infants and serially diluted and inoculated in TPY (casein trypsin hydrolysate, peptone and yeast juice) and MRS (deMann, Rogosa and sharp) medium supplemented with 0.05% cysteine and mupirocin (mupirocin). Using CO₂Production kit (Anaerocult A, Merck) plates were incubated in anaerobic jar (BBL, Oxoid) at 37 ℃ for 2-5 days. Gram-positive, catalase-negative rod-shaped or bifurcated/polymorphic bacterial isolates were streaked on complex non-selective media (MRS and TPY). Unless otherwise stated, isolates were routinely cultured in MRS or TPY media under anaerobic conditions at 37 ℃. Putative bifidobacteria were stored in 40% glycerol and stored at-20 ℃ and-80 ℃.

After isolation of the pure bifidobacterium strain, designated as AH1205, its microbiological characteristics were evaluated and summarized in table 1 below. AH1205 was a gram-positive, catalase-negative polymorphic bacterium, which was fructose-6-phosphate phosphoketolase positive, confirming that it was a bifidobacterium. Using minimal medium with a single carbon source, AH1205 was able to grow on all carbon sources tested (glucose, lactose, ribose, arabinose, galactose, raffinose, fructose, malt extract, mannose, maltose, sucrose).

TABLE 1

^*Refers to the fructose-6-phosphate phosphoketolase analysis

Variety identification

Sequence determination of the 16s intergenic spacer (IGS) was performed to identify the isolated bifidobacteria. Briefly, DNA was extracted from AH1205 using 100. mu.l of the extract and 25. mu.l of tissue preparation (Sigma, XNAT2 kit). The samples were incubated at 95 ℃ for 5 minutes and then 100. mu.l of neutralising solution (XNAT2 kit) was added. Genomic DNA solutions were quantified using a Nanodrop spectrophotometer and stored at 4 ℃. Using IGS L based on SEQ ID NO. 1: 5'-GCTGGATCACCTCCTTTC-3' (SEQ ID NO.3), and IGS R based on SEQ ID NO. 2: 5'-CTGGTGCCAAGGCATCCA-3' (SEQ ID NO.4) was subjected to PCR. Cycling conditions were 94 ℃ for 3 minutes (1 cycle), 94 ℃ for 30 seconds, 53 ℃ for 30 seconds, 72 ℃ for 30 seconds (28 cycles). The PCR reaction contained 4. mu.l (50ng) of DNA, a PCR mix (XNAT2 kit), 0.4. mu.M IGS L and R primers (MWGBiotech, Germany). The PCR reaction was performed on an Eppendorf thermocycler. The PCR product (10. mu.l) was electrophoresed in 2% EtBr-stained agarose gel together with a molecular weight marker (100bp gradient marker, Roche) to determine the IGS profile. PCR products (single band) of bifidobacterium were purified using PromegaWizard PCR purification kit. The purified PCR products were sequenced using primer sequences for the intergenic spacer regions (as described above). The sequence data was then searched in the NCBI nucleotide database to determine the strain based on nucleotide homology. The resulting DNA sequence data was submitted to the NCBI Standard nucleotide-nucleotide homology BLAST search Engine (http:// www.ncbi.nlm.nih.gov/BLAST /). The closest match to the sequence was identified and the sequences were then compared and aligned using DNASTAR MegAlign software. The resulting sequences (SEQ ID NO.1[ IGS Forward sequence ] and SEQ ID NO.2[ IGS reverse sequence ]) are visible in the sequence Listing. Searching the NCIMB database revealed that AH1205 had a unique IGS (SEQ ID No.1[ forward sequence ] and SEQ ID No.2[ reverse sequence ]) sequence with the closest sequence homology to bifidobacterium longum.

To develop a barcode PCR map of AH1205, PCR was performed using BOX primers (8). Cycling conditions were 94 ℃ for 7 minutes (1 cycle), 94 ℃ for 1 minute, 65 ℃ for 8 minutes (30 cycles), and 65 ℃ for 16 minutes. The PCR reaction contained 50ng of DNA, a PCR mix (XNAT2 kit), 0.3. mu.M BOXA1R primer (5'-CTACGGCAAGGCGACGCTGACG-3') (SEQ ID NO.5) (MWG Biotech, Germany). The PCR reaction was performed on an Eppendorf thermocycler. Using DNA 7500The PCR product (1. mu.l) was manipulated with a molecular weight marker (7500bp gradient marker, Agilent, Germany) on an Agilent2100 bioanalyzer (Agilent, Germany). The barcode pattern (PCR product pattern,) was determined using Agilent bioanalyzer software, and the number of peaks (PCR products) and size were determined (fig. 1).

Antibiotic sensitivity profiles

The antibiotic sensitivity profile of the bifidobacterium longum was determined using a "paper sensitivity" assay. Cultures (100. mu.l) grown in the appropriate broth medium for 24-48 hours were poured onto agar medium and paper discs containing known concentrations of antibiotics were placed on the agar. The strains were tested for antibiotic susceptibility after incubation at 37 ℃ for 1-2 days under anaerobic conditions. The strain is considered to be sensitive if a zone of inhibition of 1mm or greater is visible. The Minimum Inhibitory Concentration (MIC) of each antibiotic was evaluated independently. The MICs for clindamycin, vancomycin and metronidazole were 0.032, 0.75 and > 256, respectively.

Intestinal metastasis

To determine whether bifidobacterium longum could survive under conditions corresponding to the low pH found in the stomach, bacterial cells were harvested from fresh overnight cultures, washed 2 times with phosphate buffer (pH6.5) and resuspended (using 1M HCl) in TPY broth adjusted to pH 2.5. Cells were incubated at 37 ℃ and survival was determined using plate counting at 5, 30, 60 and 120 minutes. AH1205 showed good viability within 5 minutes at pH2.5, but viable cells could not be recovered after 30 minutes.

After exiting the stomach, the putative probiotic will be exposed to bile salts in the small intestine. To determine the viability of bifidobacterium longum exposed to bile conditions, cultures were streaked onto TPY agar plates supplemented with 0.3% (w/v), 0.5%, 1%, 2%, 5%, 7.5% or 10% pig bile. Growth of bifidobacterium longum was observed on plates containing up to 0.5% bile.

In a murine model, the ability of bifidobacterium longum AH1205 to translocate in the gastrointestinal tract was evaluated. For daily intake of 1 × 10⁹AH1205 mice and fecal pellets were tested for the presence of feeding microorganisms. Detection of AH1205 was aided by isolation of spontaneously forming rifampicin resistant variants of the bifidobacteria-rifampicin was added to the TPY plates used to evaluate transfer, ensuring that only the fed rifampicin resistant bifidobacteria were cultured. Fecal samples were collected daily and confirmed the translocation of bifidobacterium longum through the gastrointestinal tract (fig. 2).

Antimicrobial activity

The indicative pathogenic microorganisms used in this study were amplified in the following medium under the following growth conditions: salmonella typhimurium (aerobic at 37 ℃) in tryptone Soy Broth/agar medium supplemented with 0.6% Yeast juice (TSAYE, Oxoid), Campylobacter jejuni (anaerobic at 37 ℃) and Escherichia coli O157: H7 (anaerobic at 37 ℃) in blood agar medium, Clostridium difficile (anaerobic at 37 ℃) in enhanced Clostridium medium (RCM, Oxiod). All strains were inoculated in fresh growth medium and grown overnight before the experiment.

Antimicrobial activity was measured using the delayed method (9). Briefly, Bifidobacterium longum AH1205 was incubated for 36-48 hours. The 10-fold serial dilutions were plated (100. mu.l) on TPY agar medium. After overnight incubation, indicator bacteria were overlaid on plates with completely separated colonies. The indicator lawn was prepared by pouring a thawed overlay inoculated with 2% (v/v) overnight indicator culture onto the surface of the inoculated TPY plate. The plates were again incubated overnight under conditions suitable for growth of indicator bacteria. An indicator culture with a zone radius of inhibition greater than 1mm may be considered sensitive to the detected bacteria. Bifidobacterium longum AH1205 inhibited growth of all pathogenic organisms tested, the hyaline circle determined was 8.67, > 80, 4.33 and 11.67mm for Salmonella typhimurium, Campylobacter jejuni, Escherichia coli O157: H7 and Clostridium difficile, respectively.

Example 2: production of cytokines by PBMC in response to Bifidobacterium longum

Peripheral Blood Mononuclear Cells (PBMCs) were isolated from healthy donors by density gradient centrifugation. PBMCs were stimulated with probiotic strains for 72 hours at 37 ℃. At this point, culture supernatants were collected, centrifuged, aliquoted and stored at-70 ℃ until the level of IL-10 was analyzed using a flow microbead array (BD BioSciences). AH1205 induced significant IL-10 secretion by human PBMC, suggesting that this probiotic strain was able to induce anti-inflammatory responses in vivo (fig. 3).

Example 3: bifidobacterium longum AH1205 in murine asthma model was able to alleviate respiratory tract disease

This study investigated whether the probiotic bifidobacterium longum AH1205 could inhibit the allergic reaction in Ovalbumin (OVA) sensitized mouse models of allergic airway inflammation. Briefly, adult male BALB/c mice were sensitized by intraperitoneal injection of OVA on day 0 and day 6. Intranasal challenge with OVA was performed on day 12 and 14. BAL procedure was performed after determination of airway reactivity of mice 24 hours after the last challenge (day 15). OVA/alum sensitized, saline challenged mice were used as controls. Animals received either probiotic or placebo throughout the duration of the experiment. Airway inflammation (cytokines and cell counts) was analyzed using inflammatory cell counts in bronchoalveolar lavage (BAL) fluid. Airway reactivity was also determined using a Buxco whole-body plethysmogram. Splenocytes were also isolated from OVA-sensitized mice and incubated in the presence of anti-CD 3 and anti-CD 28 antibodies, after which the cytokine levels in the supernatant were determined by flow cytometry.

Bifidobacterium longum AH1205 did not result in a significant reduction in the number of cells recovered from BAL fluid after OVA challenge compared to broth fed animals (figure 4). Airway reactivity was measured and challenge with OVA in sensitized mice increased AHR to methacholine when compared to saline-stimulated mice. AH1205 did not modulate this increased airway reactivity to methacholine as measured by changes in longer pauses (fig. 5).

BAL cytokine levels were determined by a bead array and demonstrated significantly reduced TNF- α levels in animals fed AH1205 compared to OVA controls (fig. 6C). There were no significant differences in IL-10, INF-gamma, IL-6 and CCL2 levels (FIG. 6).

Example 4: treg effector model

The present study investigated the effect of probiotic uptake on regulatory T cell number and activity in healthy mice. BALB/c mice (10/group) were fed bifidobacterium longum AH1205 or placebo for 3 weeks. Following probiotic/placebo intake, CD4+ CD25+ T regulatory cells were isolated and their inhibitory activity in vitro was determined by measuring proliferation of CFSE-labeled CD4+ responding T cells stimulated by anti-CD 3/CD28 using flow cytometry. CD4+ responsive T cells were co-incubated with CD4+ CD25+ T cells as controls. The percentage of CD4+ CD25+ cells (regulatory T cells) in mice splenocytes that were also FoxP3 positive was determined in spleens of probiotic or placebo-fed mice.

The percentage of CD4+ cells that proliferated upon co-incubation with CD4+ CD25+ cells from probiotic/placebo-fed mice was compared to the percentage of CD4+ cells that proliferated upon co-incubation with CD4+ CD 25-cells from identically treated mice. In each case, there was a decrease in T cell proliferation in cultures containing CD4+ CD25+ cells compared to cultures containing CD4 cells alone and depleted CD25+ cells (fig. 7).

The percentage of cells in the CD4+ population that were also CD25+ was determined (figure 8). Bifidobacterium longum AH1205 fed group had significantly more CD4+ T cells (i.e., T-regulatory cells) of CD25+ compared to their placebo-fed counterparts. This indicates a significant increase in the percentage of T-regulatory cells in the CD4+ population by feeding AH 1205.

The number of CD4+ CD25+ FoxP3+ cells in the whole splenocyte population of probiotic or placebo-fed mice was also determined. The number of CD4+ CD25+ T regulatory cells expressing FoxP3 in the spleen of probiotic-fed mice was unchanged compared to placebo-fed or non-fed mice.

Example 5: sterile model

Sterile mice at 6 weeks of age were purchased and maintained in sterile units at the UCC bio-service center. Animals consumed probiotic Bifidobacterium longum AH for 12059 days or maintained sterile status. Induction of T regulatory cells was analyzed by flow cytometry and cytokine levels were quantified by CBA.

Transfer of AH1205 was analyzed by determining the bifidobacterial count on selective agar during the study. Even when administered about 1X 10 per day⁹The organism, AH1205, was also not transferred in detectable amounts in the gut of germ-free mice. The results suggest that other microbial or host factors are required for bifidobacteria survival in the gut.

Although AH1205 did not migrate in detectable amounts in the gut, the bacteria did interact with the host's immune system. After 9 days of feeding, there was a significant increase in the number of CD4+ CD25+ FoxP3+ cells in both mesenteric lymph nodes and spleen of AH 1205-fed sterile animals (figure 9). The total CD3/CD4 or CD3/CD8 counts remained unchanged.

Isolated Mesenteric Lymph Node Cells (MLNC) and splenocytes were stimulated in vitro with anti-CD 3/CD28 antibody or LPS, or left unstimulated as negative controls. When mice were pre-fed AH1205, both IL-6 and INF- γ secretion from MLNC in culture supernatants were substantially (substantiaily) reduced, while MCP-1 levels were also significantly inhibited after CD3/CD28 stimulation (FIG. 10). IL-10 levels remained similar between groups. Both IL-6 and TNF- α splenocyte release were substantially but not significantly reduced when pre-fed AH1205 (FIG. 11). No significant differences were noted in unstimulated or LPS-stimulated cultures, but overall the inventors observed lower pro-inflammatory cytokine production in animals fed bifidobacterium AH 1205.

Example 6: stability results

The stability of the probiotic strain AH1205 was altered at 30 ℃ for 3 months (fig. 12).

Lactobacillus GG performed poorly in the assay phase with a two log unit reduction over a 3 month period, whereas strain AH1205 showed up to about a1 log unit reduction in viability in the same assay phase.

Immunomodulation

The human immune system plays an important role in the etiology and pathology of a variety of human diseases. High and low immune responses can lead to most disease states, or components thereof. A family of biological entities, called cytokines, is particularly important for the control of immune processes. Perturbation of these subtle cytokine networks is increasingly associated with a variety of diseases. These diseases include, but are not limited to, inflammatory diseases, immunodeficiency, inflammatory bowel disease, irritable bowel syndrome, cancer (particularly those of the gastrointestinal and immune systems), diarrheal diseases, antibiotic-associated diarrhea, pediatric diarrhea, appendicitis, autoimmune disorders, multiple sclerosis, Alzheimer's disease, rheumatoid arthritis, coeliac disease, diabetes, organ transplantation, bacterial infection, viral infection, fungal infection, periodontal disease, genitourinary disease, sexually transmitted diseases, HIV infection, HIV replication, HIV-associated diarrhea, surgery-associated trauma, surgery-induced metastatic disease, sepsis, weight loss, anorexia, fever control, cachexia (cachexia), wound healing, ulcers, gut barrier function, allergy, asthma, respiratory disease, circulatory disease, coronary heart disease, anemia, blood clotting system disease, digestive system disease, allergy, asthma, respiratory disease, and the like, Renal diseases, central nervous system diseases, liver diseases, ischemia, malnutrition, osteoporosis, endocrine disorders, epidermal diseases, psoriasis and acne vulgaris. The effect on cytokine production was specific for each probiotic strain tested. Thus, a particular probiotic strain may be selected for a specific cytokine dysregulation for a particular disease type. Customization of disease-specific treatment can be achieved using the AH1205 strain alone or mutants or variants thereof or selected ones of these strains.

Immunity education

The intestinal flora plays an important role in the development and proper function of the intestinal immune system. In the absence of gut flora, the gut immune system is underdeveloped, as demonstrated in sterile animal models, and certain functional parameters, such as macrophage phagocytosis and immunoglobulin production, are reduced (10). The importance of gut flora in stimulating non-nociceptive immune responses is also becoming more apparent. The increase in the incidence and severity of allergies in the western world has been associated with improvements in health care and public health and a reduction in the number and extent of infectious stimuli received by the host. This lack of immune stimulation may allow the host to react with non-pathogenic, but antigenic factors, resulting in allergy or autoimmunity. The deliberate uptake of a range of non-pathogenic immunomodulatory bacteria can provide the host with the necessary and appropriate educational stimuli to provide the appropriate development and control of immune function.

Inflammation(s)

Inflammation is a term used to describe the local accumulation of fluid, plasma proteins and leukocytes at the site of a physical insult, infection or ongoing immune response. Control of the inflammatory response can be performed in multiple levels (11). The controlling factors include cytokines, hormones (e.g., hydrocortisone), prostaglandins, reaction intermediates and leukotrienes. Cytokines are low molecular weight, biologically active proteins involved in immunology and the control and generation of inflammatory responses, while also regulating development, tissue repair and hematopoiesis. It provides a conduit for the communication of leukocytes both by themselves and with other cell types. Most cytokines are pleiotropic and can express multiple biologically overlapping activities. It is the cytokine cascade and network that controls the inflammatory response, rather than the effect of specific cytokines on specific cell types (12). The attenuation of the inflammatory response results in a lower concentration of the appropriate activation signal, while other inflammatory mediators result in the cessation of the inflammatory response. TNF α is a key proinflammatory cytokine, resulting in an inflammatory state as it initiates a range of cytokines and biological effects. Therefore, agents capable of inhibiting TNF α are currently used to treat inflammatory diseases, for example infliximab (infliximab).

Pro-inflammatory cytokines are thought to play an important role in a number of inflammatory diseases, including Inflammatory Bowel Disease (IBD). Current therapies directed at treating IBD are aimed at reducing the levels of these pro-inflammatory cytokines, including IL-8 and TNF α. Such therapies can also have important roles in the treatment of systemic inflammatory diseases such as rheumatoid arthritis.

The strains of the invention have potential use in the treatment of a range of inflammatory diseases, particularly in combination with other anti-inflammatory therapies such as non-steroidal anti-infective drugs (NSAIDs) or infliximab.

Cytokines and cancer

The production of multifunctional cytokines across a broad spectrum of tumor types suggests that a significant inflammatory response is ongoing in patients with cancer. It is not clear what protective effects of such responses would have an adverse effect on the growth and development of tumor cells in vivo. However, these inflammatory responses can adversely affect the tumor-bearing host. Complex cytokine interactions are involved in the regulation of cytokine production and cell proliferation in both tumor and normal tissues (13, 14). Weight loss (cachexia) has long been considered to be the single most common cause of death in cancer patients, and initial malnutrition also indicates a poor prognosis. For a tumor to grow and spread, it must induce the formation of new blood vessels and break down the extracellular matrix. The inflammatory response plays an important role in the implementation of the above mechanisms and thus has a role in the debilitation of the host and the development of tumors. Due to the anti-inflammatory properties of bifidobacterium longum infantis, these bacterial strains are able to reduce the rate of malignant cell transformation. Furthermore, enterobacteria are capable of producing substances from dietary compounds with genotoxic, carcinogenic and tumor promoting activities, while enterobacteria are capable of activating carcinogens to DNA reactive agents (15). Generally, species of bifidobacteria have lower heterotypic biomass-metabolizing enzymes than other species within the digestive tract, such as bacteroides, eubacteria and clostridia. Thus, an increase in the number of bacteria of the genus bifidobacterium in the gut could also beneficially modulate the levels of these enzymes.

Vaccine/drug delivery

Most pathogenic organisms enter through mucosal surfaces. These sites can be effectively inoculated with specific infectious agents to protect against infection. To date, oral vaccine strategies have focused on the use of attenuated or purified encapsulated antigens against live pathogenic organisms (16). Probiotics engineered to be able to produce antigens from infectious agents provide a promising alternative in vivo, as these bacteria are considered safe for human consumption (GRAS status).

Studies in mice have demonstrated that ingestion of probiotics expressing exogenous antigens can enhance the protective immune response. The gene encoding tetanus toxin fragment c (ttfc) can be expressed in lactobacilli and mice are immunized by the oral route. Such a system is capable of inducing antibody titers that are significantly high enough to protect mice from challenge with lethal toxins. In addition to antigen presentation, live bacterial vectors are also capable of producing biologically active compounds in vivo, such as cytokines with immunostimulatory effects. In intranasally immunized mice, lactobacilli secrete biologically active human IL-2 or IL-6 and TTFC capable of inducing serum IgG titers above 10-15 fold (17). However, with this particular bacterial strain, total IgA levels were not elevated by co-expression with these cytokines. Other bacterial strains, such as Streptococcus grignard (Streptococcus gordonii), have also been tested for their usefulness as mucosal vaccines. Recombinant S.grignard colonizes the murine oral cavity and the sheath cavity induces mucosal and systemic antibody responses to the antigens expressed by the bacteria (18). Thus, oral immunization using probiotics as carriers not only protects the host from infection, but also replaces immune stimulation normally caused by pathogens, thereby benefiting the host's immune education.

Prebiotics

The introduction of the probiotic organism is achieved by the ingestion of the micro-organism in a suitable carrier. The benefit is that it provides a medium that can promote the growth of these probiotic strains in the large intestine. The addition of one or more oligosaccharides, polysaccharides or other probiotics can promote the growth of lactic acid bacteria in the gastrointestinal tract. Prebiotics are any non-viable food ingredient that is specifically fermented in the colon by endogenous bacteria that are considered to be of positive value, such as bifidobacteria, lactobacilli. Types of prebiotics may include fructose, xylose, soy, galactose, glucose and mannose. The administration of a probiotic strain in combination with one or more prebiotic compounds enhances the growth of the administered probiotic in vivo, resulting in a more potent health benefit, and is known as a synergistic effect (synbiotic).

Other active ingredients

It will be appreciated that the probiotic strain may be administered prophylactically or as a method of treatment, either by itself or in combination with other probiotic and/or prebiotic substances as described above. Furthermore, the bacteria may be part of a prophylactic or therapeutic regimen using other active substances, such as those used to treat inflammation or other disorders, particularly immune-related disorders. Such combinations may be administered as a single formulation or as separate formulations at the same or different times and using the same or different routes of administration.

The invention is not limited to the embodiments described in the specification, which may vary in detail.

Reference to the literature

1.McCracken V.J.and Gaskins H.R.Probiotics and the immune system.In：Probiotics a critical review，Tannock，GW(ed)，Horizon Scientific Press，UK.1999，p.85-113.

2.Savage D.C.Interaction between the host and its microbes.In：MicrobialEcology of the Gut，Clark and Bauchop(eds)，Academic Press，London.1977，p.277-310.

3.Kagnoff M.F.Immunology of the intestinal tract.Gastroenterol.1993；105(5)：1275-80.

4.Lamm M.E.Interaction of antigens and antibodies at mucosal surfaces.Ann.Rev.Microbiol.1997；51：311-40.

5.Raychaudhuri S.，Rock KL.Fully mobilizing host defense：buildingbetter vaccines.Nat biotechnol，1998；16：1025-31.

6.Stallmach A.，Strober W，MacDonald TT，Lochs H，Zeitz M.Inductionand modulation of gastrointestinal inflammation.Immunol.Today，1998；19(10)：438-41.

7.de Waal Malefyt R，Haanen J，Spits H，Roncarolo MG，te Velde A，Figdor C，Johnson K，Kastelein R，Yssel H，de Vries JE.Interleukin 10(IL-10)and viral IL-10 strongly reduce antigen-specific human T cell proliferation bydiminishing the antigen-presenting capacity of monocytes via downregulation ofclass II major histocompatibility complex expression.J Exp Med 1991 Oct 1；174(4)：915-24.

8.Masco L，Huys G，Gevers D，Verbrugghen L，Swings J.Identification ofBifidobacterium species using rep-PCR fingerprinting.Syst Appl Microbiol.2003 Nov；26(4)：557-63.PMID：14666984.

9.Tagg，JR，Dajani，AS，Wannamaker，LW.Bacteriocins of Gram positivebacteria.Bacteriol Rev.1976；40：722-756.

10.Crabbe P.A.，H.Bazin，H.Eyssen，and J.F.Heremans.The normalmicrobial flora as a major stimulus for proliferation of plasma cells synthesizingIgA in the gut.The germ free intestinal tract.Into.Arch.Allergy Appl Immunol，1968；34：362-75.

11.Henderson B.，Poole，S and Wilson M.1998.In″Bacteria-Cytokineinteractions in health and disease.Portland Press，79-130.

12.Arai KI，Lee F，Miyajima A，Miyatake S，Arai N，Yokota T.Cytokines：coordinators of immune and inflammatory responses.Annu Rev Biochem1990；59：783-836.

13.McGee DW，Bamberg T，Vitkus SJ，McGhee JR.A synergisticrelationship between TNF-alpha，IL-1 beta，and TGF-beta 1 on IL-6 secretion bythe IEC-6 intestinal epithelial cell line.Immunology 1995Sep；86(1)：6-11.

14.Wu S，Meeker WA，Wiener JR，Berchuck A，Bast RC Jr，Boyer CM.Transfection of ovarian cancer cells with tumour necrosis factor alpha(TNF-alpha)antisense mRNA abolishes the proliferative response tointerleukin-1(IL-I)but not TNF-alpha.Gynecol Oncol 1994 Apr；53(1)：59-63.

15.Rowland LR.Toxicology of the colon：role of the intestinal microflora.In：Gibson G.R.(ed).Human colonic bacteria：role in nutrition，physiology andpathology，1995，pp 155-174.Boca Raton CRC Press.

16.Walker，R.I.New strategies for using mucosal vaccination to achievemore effective immunization.Vaccine，1994；12：387-400.

17.Steidler L.，K.Robinson，L.Chamberlain，K.M Scholfield，E.Remaut，R.W.F.Le Page and J.M.Wells.Mucosal delivery of murine interleukin-2(IL-2)and IL-6 by recombinant strains of Lactococcus lactis coexpressing antigen andcytokine.Infect.Immun.，1998；66：3183-9.

18.Medaglini D.，G.Pozzi，T.P.King and V.A.Fischetti.Mucosal andsystemic immune responses to a recombinant protein expressed on the surface ofthe oral commensal bacterium Streptococcus gordonii after oral colonization.Proc.Natl.Acad.Sci USA，1995；92：6868-72 McCracken VJ.and Gaskins H.R，′Probiotics a critical review′，Horizon Scientific Press，UK 1999，p.278.

19.Marson，A.，Kretschmer，K.，Frampton，G.M.，Jacobsen，E.S.，Polansky，J.K.，Maclsaac，K.D.，Levine，S.S.，Fraenkel，E.，von Boehmer，H and Young，R.A.Foxp3 occupancy and regulation of key target genes during T-cellstimulation.Letters to Nature，2007.

Claims (26)

1. A bifidobacterium isolate deposited under NCIMB accession number 41387.

2. A bifidobacterium strain as claimed in claim 1 in the form of viable cells.

3. A bifidobacterium strain as claimed in claim 1 in the form of non-viable cells.

4. A formulation comprising the bifidobacterium strain as claimed in claim 1.

5. The formulation of claim 4, further comprising a prebiotic.

6. A formulation according to any one of claims 4 or 5, which further comprises a prebiotic material.

7. The formulation of any one of claims 4 or 5, further comprising an ingestable carrier.

8. The formulation of claim 7, wherein the ingestable carrier is a pharmaceutically acceptable carrier.

9. The formulation of claim 8, wherein the ingestable carrier is a capsule, tablet or powder.

10. The formulation of claim 7, wherein the ingestable carrier is a food product.

11. The formulation of claim 10, wherein the ingestable carrier is selected from one or more of a milk powder, a sauce, or a beverage.

12. The formulation of claim 11, wherein the ingestable carrier is acidified milk.

13. The formulation of claim 11, wherein the ingestible carrier is concentrated milk.

14. The formulation of claim 11, wherein the ingestable carrier is a cheese spread.

15. The formulation of claim 12, wherein the acidified milk is yogurt.

16. The formulation of claim 12, wherein the acidified milk is frozen yogurt.

17. The formulation of claim 4, further comprising proteins and/or peptides, lipids, carbohydrates, vitamins, minerals and/or trace elements.

18. The formulation of claim 17, wherein the proteins and/or peptides comprise glutamine/glutamate rich proteins and/or peptides.

19. The formulation of claim 4, wherein the Bifidobacterium strain is present at greater than 10 per gram of the formulation⁶The amount of cfu is present.

20. The formulation of claim 4, further comprising an adjuvant.

21. The formulation of claim 4, further comprising a bacterial component for the treatment of inflammation.

22. The formulation of claim 4, further comprising a pharmaceutical entity for treating inflammation.

23. The formulation of claim 4, further comprising a biologically active compound for the treatment of inflammation.

24. The formulation of claim 4 for use in immunization and vaccination protocols.

25. A Bifidobacterium strain as claimed in claim 1 or a formulation as claimed in claim 4 for use in food.

26. Use of a bifidobacterium strain as claimed in any of claims 1 to 3 in the manufacture of a panel of biotherapeutic agents for modulating IL-10 levels.