WO2013190106A1

WO2013190106A1 - Use of a live bacterium for the treatment of intestinal inflammatory disorders

Info

Publication number: WO2013190106A1
Application number: PCT/EP2013/063028
Authority: WO
Inventors: Thien Nguyen; Christine Libon; Nathalie Castex-Rizzi
Original assignee: Pierre Fabre Medicament SA
Current assignee: Pierre Fabre Medicament SA
Priority date: 2012-06-21
Filing date: 2013-06-21
Publication date: 2013-12-27
Anticipated expiration: 2014-12-21

Description

USE OF A LIVE BACTERIUM FOR THE TREATMENT OF

INTESTINAL INFLAMMATORY DISORDERS

The present invention relates to the use of a bacterial strain isolated and characterized from groundwater, notably in the context of the treatment of inflammations.

More particularly, the present invention relates to novel compositions of interest in the treatment and the prevention of intestinal inflammatory disorders and to a method of treatment of intestinal inflammatory disorders.

Acute colitis, irritable bowel syndrome and Crohn's disease are increasing in developed countries and affect approximately 1.4 million Americans. (Arijs, I. et al., 2009. PLoS ONE.4:e7984, and Hill, D.A. and D. Artis. 2010. Annu. Rev. Immunol. 28:623-67 and Kaser, A. et al, 2010. Annu. Rev. Immunol. 28:573-621).

Acute colitis is defined as a sudden and self-limited inflammation of the colon, or the large intestine. Symptoms include abdominal pain, loss of appetite, fatigue, diarrhea, cramping, urgency and bloating. Various causes can be involved: infection, iatrogenic, idiopathic, autoimmune or vascular origins for example. Treatment depends on the identified etiology and may even include surgery in some severe cases.

Irritable bowel syndrome (IBS) is a chronic disorder of the bowel resulting in variable gastrointestinal discomfort, pain, diarrhea, spasms and sometimes limited intestinal inflammation. Even though less serious a condition than other diseases like inflammatory bowel disease, IBS often alters the patient's quality of life enough to require treatment. Current options usually target gastrointestinal motility and sensitivity, and sometimes psychological components of the disease.

Inflammatory bowel disease (IBD) is a collective term to define several chronic, and often severe, gastrointestinal inflammations, the two most important being ulcerative colitis and Crohn's disease. It is now generally acknowledged that the four major factors triggering IBD are genetic predisposition, environmental influence, immunological disturbances and unbalanced gastrointestinal microflora often causing intestinal wall barrier dysfunction. How these factors combine to result in more or less chronic inflammation is specific to each patient and disease etiology (Triantafillidis J. et al. 2011 Drug Design, Development and Therapy 5: 185-210). Crohn's disease, an inflammatory intestinal disease, affects segments of the digestive tract, but its preferential sites are the ileum (terminal portion of the small intestine) and the colon. The wall of the affected intestine is edematous. During its progression, this edema of the intestinal wall will cause a decrease in the diameter of the intestine. Evolution toward fibrosis, a source of stenosis (contraction), may also occur.

The disease is characterized by the presence of ulcerations, more or less wide and more or less deep, which pass through the wall (fissures) thus causing abscess and fistulas. This disease affects both sexes and appears in general between the ages of 20 and 40. In its typical form, it begins slowly and insidiously. Episodic diarrhea and indistinct abdominal pain summarize the symptomatology for months or years.

When the disease has firmly taken hold, diarrhea of moderate intensity, sometimes fatty and seldom bloody, is the principal symptom. Fixed and continuous pain in the right iliac fossa or paroxysmal or atypical pain is also associated with the disease. Weight loss and fever are other important symptoms. The signs vary according to the topography of the lesions.

The disease progresses by flare-ups that are variable in intensity and that often regress spontaneously.

Complications are, however, frequent and they may require multiple surgical procedures: intestinal obstruction, intestinal fistulas, intestinal perforations, fistulas (openings) in the skin or in intra-abdominal organs, anorectal complications (fissures, abscesses).

Aside from surgical procedures, treatments with monoclonal antibodies (anti- TNF, anti-IL-12/p40 or anti-IL-23/p40) exist to palliate Crohn's disease but they have the disadvantage of being very costly.

The invention of the present patent application, in this context, proposes a different, effective and much less costly approach for relieving patients suffering from this disease.

Crohn's disease is multifactorial and complex. One of the factors identified in this disease is immunological in nature. Recent publications have revealed that the immune system of the host is "disrupted": pro-inflammatory and inflammatory reactions have proven to be disproportionate and exacerbated. A deregulation of the immune system is suggested: Thl profile potentiated with production of IL-12, Thl7 profile potentiated with an increase in IL-23, disruption of the natural flora of the intestine and impaired tolerance, which leads to inappropriate local and systemic immune responses resulting in immune responses against the aberrant intestinal flora leading to pathogenesis (activation of T cells, inflammatory cytokines, antibodies against intestinal bacteria) (Abraham C. and Cho J.H., N Engl J Med 2009;361 :2066- 78).

Cenac et al., (Am J Pathol. 2002.161 : 1903-1915) discovered that the activation of proteinase-activated receptor-2 (PAR2) induced acute intestinal inflammation in animals. PAR2 is overexpressed in the gastrointestinal tract: endothelial cells, colonic myocytes, enterocytes, enteric neurons, immune cells, etc. Proteases (trypsin, tryptase) present in abundance in the gastrointestinal tract cleave the PAR2 at the N-terminal exposing a specific peptide which activates this same receptor (phenomenon of self- activation). Consequently, this activates the production of pro -inflammatory cytokines and triggers inflammation (Vergnolle, N. 2005. Gut. 54:867-874 and Vergnolle, N. 2009. Pharmacol. Ther. 123:292-309). This phenomenon is observed in the wild type mouse but does not appear in the KO mouse (PAR2 deficient). Treatment with an antiprotease and/or a PAR2 antagonist makes it possible to avoid this inflammation phenomenon.

Ulcerative colitis is the other major form of IBD. It is usually confined to the large intestine and more particularly characterized by ulcers or open sore. It is an intermittent disease with altering periods of exacerbated symptoms and other quite symptom-free. Current first-line treatments include anti-inflammatory drugs (mesalazine) and corticosteroids. For more severe and non-responding patients, immunosuppressants are used. It is thought that a dysregulation in bacterial growth and population profile might be involved in ulcerative colitis pathogenesis.

By contrast to Crohn's disease, ulcerative colitis seems to be characterized by a Th2 polarization, with particularly increased secretion of IL-5, and IL-13, along with a potentiation of Thl7 response (Triantafillidis J. et al. 2011 Drug Design, Development and Therapy 5: 185-210, Monteleone et al. 2011 Curr. Opin. Pharmacol.11 :640-645, Monteleone et al. 2011 BMC Medicine 9 : 122) .

In this context, the present invention provides a solution to the treatment of these inflammatory disorders by the isolation, the characterization of a novel bacterium named LMB64 or 1-4290 (according to the CNCM registration Number).

This bacterium LMB64, in addition to the fact of having been isolated, was characterized and defined as belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae, and probably of a novel genus not yet defined. Analysis of the gene sequence coding for 16S rRNA made it possible to place this bacterium close to the genera Chromobacterium, Paludimonas, Lutelia and Glubenkiana, with which it shares 95% sequence similarity.

This non-pathogenic bacterium is Gram-negative and will be described in greater detail in the examples. This bacterium also has the characteristic of being non- filamentous. Moreover, this bacterium has the advantage of being able to be cultured on a medium containing any type of water, and more particularly, ordinary water.

The gene coding for 16S rRNA has been almost completely sequenced (1487 bp). Bacterium LMB64 has a circular plasmid of 10948 bp. This plasmid was completely sequenced and the sequence is represented in sequence SEQ ID No. 2.

According to a first embodiment, the present invention relates to the use of a non-pathogenic Gram-negative live bacterium belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae, whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80%>, preferably 85%, 90 %>, 95% and 98%> identity with said sequence SEQ ID No. 1 for the treatment of intestinal inflammatory disorders.

The present invention thus relates to a non-pathogenic Gram-negative live bacterium belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae, whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80%>, preferably 85%, 90 %, 95%) and 98%> identity with said sequence SEQ ID No. 1, or to a composition comprising said Gram-negative live bacterium, for use in the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease.

In a preferred manner, the present invention relates to a non-pathogenic Gram- negative live bacterium belonging to the class of Betaproteobacteria, subfamily of

Neisseriaceae, characterized in that the nucleotide sequence of the 16S rRNA gene of said bacterium includes or comprises the sequence SEQ ID No. 1 for use in the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders- related disease.

The invention can also be described as a method for the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, wherein said method comprises a step of administration to a patient in need thereof of a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1 , or any nucleotide sequence with at least 80%, preferably 85%, 90 %, 95% and 98% identity with said sequence SEQ ID No. 1 , or a step of administration of a composition comprising said live bacterium.

The present invention is also directed to a method for the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, comprising the administration of a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1 , or any nucleotide sequence with at least 80%>, preferably 85%>, 90 %>, 95%> and 98% identity with said sequence SEQ ID No. 1 , or the administration of a composition comprising said live bacterium, in a therapeutically effective amount to an individual affected by an intestinal inflammatory disorders or intestinal inflammatory disorders- related disease, wherein said administration reduces inflammation or inflammatory effects in said individual, or induces anti-inflammatory effects.

The present invention is thus directed to a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1 , or any nucleotide sequence with at least 80%>, preferably 85%, 90 %, 95% and 98% identity with said sequence SEQ ID No. 1, or to a composition comprising said live bacterium, for use in the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, wherein said live bacterium or composition is administered to a patient in need thereof.

In a preferred embodiment, the administration of said live bacterium or composition comprising said live bacterium, is carried out in a therapeutically effective amount to an individual affected by an intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, and wherein said administration reduces inflammation or inflammatory effects in said individual, or induces anti-inflammatory effects. In the context of the present invention, "percentage identity" between two nucleic acid sequences refers to a percentage of identical nucleotides between the two sequences to be compared, obtained after the best alignment (optimal alignment), wherein this percentage is purely statistical and the differences between the two sequences are distributed randomly and over their entire length. Comparisons of sequences between two nucleic acid sequences are normally made by comparing these sequences after having aligned them in an optimal manner, wherein said comparison may be made per segment or per "comparison window." The optimal alignment of the sequences for the comparison can be carried out, in addition to manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2:482], by means of the local homology algorithm of Needleman and Wunsch (1970) [J. Mol. Biol. 48:443], by means of the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. The USA 85:2444] or by means of computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Group Computer, 575 Science Dr., Madison, WI, or the BLAST N or BLAST P comparison software).

The percentage identity between two nucleic acid sequences is determined by comparing these two aligned sequences in an optimal manner wherein the nucleic acid sequence to be compared may include additions or deletions in relation to the reference sequence for an optimal alignment between these two sequences. Percentage identity is calculated by determining the number of positions for which the nucleotide is identical between the two sequences, by dividing this number of identical positions by the total number of positions in the comparison window and by multiplying the result obtained by 100 to obtain the percentage identity between these two sequences.

For example, the "BLAST 2 sequences" program (Tatusova et ah, "Blast 2 sequences - a new tool for comparing protein and nucleotide sequences," FEMS

Microbiol Lett. 174:247-250), available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html, may be used with the default parameters (in particular for the parameters "open gap penalty": 5, and "extension gap penalty": 2; with the selected matrix being for example the "BLOSUM 62" matrix proposed by the program), with the percentage identity between the two sequences to be compared being calculated directly by the program. It is also possible to use other programs such as the "ALIGN" or "Megalign" software (DNASTAR).

According to another embodiment, the bacterium used according to the invention includes at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity with said sequence SEQ ID No. 2.

In a preferred manner, bacterium LMB64 includes at least one plasmid comprising sequence SEQ ID No. 2.

The invention can also be described as a method for the treatment of intestinal inflammatory disorders, wherein said method comprises a step of administration to a patient in need thereof of a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%>, preferably 85%, 90%, 95% and 98% identity with said sequence SEQ ID No. 2, or a step of administration of a composition comprising said live bacterium.

The present invention is also directed to a method for the treatment of intestinal inflammatory disorders, comprising the administration of a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%>, preferably 85%, 90%>, 95% and 98% identity with said sequence SEQ ID No. 2, or the administration of a composition comprising said live bacterium, in a therapeutically effective amount to an individual affected by an intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, wherein said administration reduces inflammation or inflammatory effects in said individual, or induces anti-inflammatory effects.

The present invention is thus directed to a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity with said sequence SEQ ID No. 2, or to a composition comprising said live bacterium, for use in the treatment of intestinal inflammatory disorders, wherein said live bacterium or composition comprising said live bacterium is administered to a patient in need thereof.

In a preferred embodiment, the administration of said live bacterium or composition comprising said live bacterium, is carried out in a therapeutically effective amount to an individual affected by an intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, and wherein said administration reduces inflammation or inflammatory effects in said individual, or induces anti-inflammatory effects.

According to a preferred embodiment of the invention, said live bacterium is the bacterium named LMB64.

According to a preferred embodiment of the invention, bacterium LMB64 is non- filamentous .

Other characteristics of said bacterium LMB64 will be detailed below in the examples.

Moreover, bacterium LMB64 of the present invention has been deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, Paris, on April 8, 2010, under the reference 1-4290. It must be understood that the invention also encompass the use of a homologue, a descendant or any other mutant of LMB64.

According to a preferred embodiment of the invention, said live bacterium is the bacterium named LMB64 deposited under the reference 1-4290.

The term "mutant" refers to any bacterium directly arising from strain 1-4290 and may comprise natural mutations or recombinations, such as, for example, any recombination related to cell proliferation, cell division (mutation due to errors occurring during bacterial division or DNA replication) or any other mechanism of natural selection or of selection in culture media, such as the selection of mutants that are resistant or that become resistant to a given compound. Included among these mutants are any bacteria arising from strain 1-4290 comprising one or more mutations in their genomic sequence (or that of their plasmid), in which the mutations were caused by radiation, by a virus, by transposons or by mutagenic chemicals.

According to a first embodiment of the invention, from a bacterial culture, the entire biomass may be isolated by various known methods such as, for example, by filtration, coagulation with an alcohol (ethanol, isopropanol, isobutanol), by drying on a cylinder with a scraped prelayer, etc., and then used in freeze-dried or heat-inactivated form.

A first advantageous aspect of the invention, related to immunomodulation, rests on the modulation property of pro -inflammatory cytokines. More particularly, the use of the bacterium according to the invention is able, in the case of a response strongly oriented toward a Thl or Thl7 profile as with Crohn's disease, to restore homeostasis.

Another advantage of the invention rests on the fact that, as will be apparent from the examples, the use of the bacterium according to the invention induces the production of antimicrobial peptides such as, for example but without being restrictive, peptides hBD-2, hBD-3, S1007A and LL-37. These peptides have an antimicrobial effect on pathogens that colonize the intestinal tract without affecting the normal growth of commensal microflora. As a result, their action restores normal microflora in the intestine.

The invention thus relates to the use of a live bacterium such as described above as an activator of TLR2, TLR4 and TLR5.

The invention relates to a method of activating TLR2 that comprises administering the live bacterium according to the invention.

The invention relates to a method of activating TLR4 that comprises administering the live bacterium according to the invention.

The invention relates to a method of activating TLR5 that comprises administering the live bacterium according to the invention.

The invention relates to a method of activating TLR2, TLR4 and TLR5 that comprises administering the live bacterium according to the invention.

The present invention thus relates to a live bacterium such as described above or a composition comprising said live bacterium for use as an activator of TLR2, TLR4 and/or TLR5, particularly when said live bacterium, or composition comprising the same, is administered to a patient in need thereof.

Said TLR5 activation activity is of significant interest in that TLR5 are known to induce certain antimicrobial peptides such as psoriasin (S100A7) and hBD-2 (Glaser et ah, Journal of Investigative Dermatology (2009) 129, 641-649). Moreover, TLR5 agonists act in synergy with those of TLR2 and TLR4, thus making it possible to potentiate the production of antimicrobial peptides. It has been shown that by blocking TLR5 with an antibody, the latter are produced little or not at all.

This aspect is thus particularly innovative in terms of immunomodulation applications for the bacterium according to the invention.

Surprisingly, it has been showed by the applicant that, for the use of the invention, the bacterium LMB64 must be a live bacterium.

In the present description, by the expression "live bacterium as described above" or "live bacterium as defined above" or "live bacterium according to the invention", it must be understood;

- a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80%, preferably 85%>, 90 %>, 95%> and 98%> identity with said sequence SEQ ID No. 1; or

- a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%>, preferably 85%>, 90%>, 95%> and 98%o identity with said sequence SEQ ID No. 2; or

- a live bacterium named LMB64; or

- a live non- filamentous bacterium LMB64; or

- a live bacterium LMB64 deposited with the CNCM, Institut Pasteur, Paris, on April 8, 2010, under the reference 1-4290, a homologue, a descendant or any other mutant of said deposited LMB64. By the expression "live bacterium" or "live bacteria", it must be understood a viable microorganism. Viability is defined as the microorganism can maintain itself or recover its potentialities. Usually, live bacterium is freshly harvest from culture medium at the end of fermentation. It can be stored at T: 4°C for few days but viability could drop dramatically after 4 days. For long term storage, freezing at -20°C to -80°C could be used but bacteria should be resuspended in medium containing for example Glycerol or DMSO as cryoconserving agent. Lyophilisation (Freeze-dry) is alternative method to preserve live bacteria at room temperature for long term storage. The bacteria should be resuspended in a lyoprotective media (for example sucrose) before freezing and drying.

Thus, the invention also has as an object a method for the treatment or prevention of pathology, in particular pathology related to an infection or to an immune response defect, wherein said pathology is associated with a defect in the activity of TLR2, TLR4 and TLR5, and wherein said treatment or prevention involves modulation of the activity, in particular an increase in the activity, of said TLR2, TLR4 and TLR5 by the administration of an activator of said receptors, wherein said method comprises the administration, to a patient who has or who is likely to have said pathology, of an effective quantity of a live bacterium according to the present invention.

In a preferred embodiment, the invention is directed to said live bacterium as described above, or composition comprising said live bacterium, for use in the treatment or prevention of pathology related to an infection or to an immune response defect, wherein said pathology is associated with a defect in the activity of TLR2, TLR4 and/or TLR5, and wherein said treatment or prevention involves modulation of the activity, in particular an increase in the activity, of said TLR2, TLR4 and/or TLR5 by the administration of an activator of said receptors, wherein said live bacterium or composition comprising said live bacterium is administered in an effective quantity to a patient who has or who is likely to have said pathology. Furthermore, in an unexpected manner, the Applicant has also demonstrated antagonistic activity toward PAR2. This activity is of significant interest in the context of anti-inflammatory treatments.

The invention thus relates, quite particularly, to the use of a live bacterium such as described above as a PAR2 activity antagonist.

The invention relates to a method of antagonizing PAR2 activity that comprises administering the live bacterium according to the invention.

The present invention thus relates to a live bacterium such as described above or a composition comprising said live bacterium for use as a PAR2 activity antagonist, particularly when said live bacterium, or composition comprising the same, is administered to a patient in need thereof.

The invention also has as an object a method for the treatment or prevention of pathology, in particular pathology related to inflammation, wherein said pathology is associated with a dysfunction of PAR2, and wherein said treatment or prevention involves modulation of the activity of said PAR2 particularly by the administration of an antagonist of said receptor, wherein said method comprises the administration, to a patient who has or who is likely to have said pathology, of an effective quantity of a live bacterium according to the present invention.

In a preferred embodiment, the invention is directed to said live bacterium as described above, or composition comprising said live bacterium, for use in the treatment or prevention of pathology related to inflammation, wherein said pathology is associated with a dysfunction of PAR2, and wherein said treatment or prevention involves modulation of the activity of said PAR2, wherein said live bacterium or composition comprising said live bacterium is administered in an effective quantity to a patient who has or who is likely to have said pathology.

The combination and the synergy of all these activities give this live bacterium LMB64 a high potential to treat inflammatory diseases and, quite particularly, inflammatory diseases in which PAR2 is involved and/or in which the immune system is weakened, disturbed or unbalanced.

The invention thus relates to the use of a live bacterium such as described above for the preparation of a composition intended for the treatment and/or the prevention of gastrointestinal inflammatory disorders.

The invention also relates to a live bacterium, or a composition comprising the same such as described above, or a composition comprising the same, for use in the treatment and/or the prevention of gastrointestinal inflammatory disorders.

In a preferred manner, said gastrointestinal inflammatory disorders consist of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or colitis.

According to another embodiment, the invention relates to a composition, preparation or formulation comprising, as an active ingredient, at least one live bacterium according to the invention.

The composition according to the invention relates to the treatment of gastrointestinal inflammatory disorders.

The invention thus relates to a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

In the present description, "pharmaceutically acceptable carrier" refers to a compound or a combination of compounds made part of a pharmaceutical composition that do not cause secondary reactions and that, for example, facilitate the administration of the active compounds, increase their lifespan and/or effectiveness in the body, increase their solubility in solution or improve their preservation. Said pharmaceutically acceptable carriers are well known and will be adapted by those persons skilled in the art according to the nature and the mode of administration of the active compounds selected.

Preferably, said compounds may be administered systemically by intramuscular, intradermal, intraperitoneal or subcutaneous route, or by oral route. The composition comprising the live bacterium according to the invention may be administered in several doses, spread out over time.

Their optimal modes of administration, dosing schedules and galenic forms may be determined according to criteria generally considered in the establishment of a treatment adapted to a patient such as, for example, the age or the weight of the patient, the seriousness of the patient's general health, tolerance to the treatment and side effects noted.

In another embodiment, the invention relates to a formulation for use as a probiotic.

Probiotics are generally defined as "living microorganism which, when administered in adequate amounts confer a health benefit on the host" (Joint FAO/WHO Working Group Report on Drafting Guidelines for the Evaluation of Probiotics in Food,

2002). They can exert their effects through various mechanisms, including: (i) restoration of the balance in gastrointestinal microflora, especially by preventing colonization by pathogens through simple competition or direct antagonism, and promoting beneficial species, (ii) improving the intestinal wall permeability properties and (iii) directly modifying the immune response of the host. (Rowland, I. et al. 2010

Gut Microbes 1 :436-439, Triantafillidis, J. et al. 2011 Drug Design, Development and Therapy 5 : 185-210). These properties have prompted a growing interest for the use of probiotics for the treatment and prevention of numerous diseases, among them inflammatory bowel diseases (IBD) including Crohn's disease and ulcerative colitis, and other gastrointestinal disorders.

Indeed, there is now a general consensus that gastrointestinal microflora alteration and disturbances in the innate and adaptative immune system are two of the major factors triggering gastrointestinal inflammation, and particularly Crohn's disease or colitis, along with environmental and genetic factors (Mack, D. R. 2011 Nutrients 3:245-264, Triantafillidis, J. et al. 2011 Drug Design, Development and Therapy 5 : 185- 210).

Accordingly, recent reviews show that the use of some selected probiotic strains might have beneficial effect as monotherapies or add-ons to usual treatments, on patients with ulcerative colitis, and to a lesser extent for patients with pouchitis, in particular to prevent new crises and to maintain remission (Mack, D. R. 2011 Nutrients 3:245-264, Sang L.-X. et al. 2011 World Journal of Gastroenterology 16: 1908-1915).

Some probiotic strains have also shown some efficacy in improving global discomfort in irritable bowel syndrome patients; particular relief was seen in abdominal pain and bloating (Rowland, I. et al. 2010 Gut Microbes 1 :436-439). The same review states the general consensus on the clinical benefit of certain probiotic strains in infectious colitis and diarrhea, particularly in children

As it is not possible to generalize the results found in the studies to all probiotics, there is always a particular interest in finding new strains with the right properties to develop new treatments.

The formulation according to the invention can include another probiotic material.

The formulation according to the invention can include a pharmaceutically acceptable carrier, preferably an ingestable carrier, said ingestable carrier being a pharmaceutically acceptable carrier such as a capsule, tablet or powder.

The formulation according to the invention can include an adjuvant.

The invention relates also to a foodstuff comprising a live bacterium according to the invention.

The invention also relates, in other words, to a non-pathogenic probiotic comprising the live bacterium of the invention. Particularly, the present invention is directed to a probiotic live bacterium, or a non-pathogenic probiotic composition comprising said live bacterium, wherein said live bacterium is selected from the group consisting of:

- a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80%, preferably 85%, 90 %, 95% and 98%> identity with said sequence SEQ ID No. 1; and

- a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%, preferably 85%, 90%, 95% and 98% identity with said sequence SEQ ID No. 2; and

- a live bacterium named LMB64; and

- a live non- filamentous bacterium LMB64; and

- a live bacterium LMB64 deposited with the CNCM, Institut Pasteur, Paris, on April 8, 2010, under the reference 1-4290, a homologue, a descendant or any other mutant of said deposited LMB64.

According to the method of the invention, the live bacterium can be frozen, lyophilized or conserved by any similar method known by the man skilled in the art. In a preferred embodiment, the bacterium is lyophilized.

According to the method of the invention, the freeze-drying process (also known as lyophilisation) can comprise the following steps.

centrifugation of the cultures bacteria and collection of the pellet resuspension in a lyoprotective medium, containing one or more lyoprotective agents

- freezing the resulting suspension

placing the frozen medium under vacuum

then primary drying i.e. increasing the temperature in order to sublimate the water contained in the samples.

secondary drying i.e. driving heat into the samples in order to remove the residual water

sealing the samples for conservation.

Lyophilization can be carried out with a manifold or a shelf lyophilizer. The lyoprotective agent can be chosen, without limitation, among skim milk, egg yolk, a solution of one or more sugars or disaccharides (such as sucrose, glucose, trehalose or lactose), sorbitol, or any other known substance by the person skilled in the art with the required properties.

The freezing step can be carried out manually of automatically, in a progressive manner of by flash- freezing the samples. The latter can be frozen at any temperature between -40 °C and -80°C.

Primary drying conditions can be optimized for various parameters such as, without limitation: temperature typically between 0°C and 20°C, heating time, vacuum pressure typically around 200 mTorr.

The liquid used to rehydrate the freeze dried bacteria can be chosen among a variability of mixtures, typically saline (0.9% NaCl), culture broth containing TSA (trypticase soy agar) or LB (Lysogeny broth) or another optimized mixture.

The invention will be better understood upon consideration of the examples below which illustrate the invention without limiting its scope.

Description of figures:

Figures 1A and IB present images of bacterium LMB64 under the transmission electron microscope (A) and the scanning electron microscope (B).

Figures 2A and 2B illustrate the effects of vehicle, Heat-killed LMB64 (HK-

LMB64), live LMB64 at doses of 10⁸ and 10⁹ bacteria/rat, Prednisolone and Remicade® on Macroscopic damage scores (A), and myeloperoxidase activity (B) in rats that received TNBS enema (30 mg/rat i.c). Results were expressed as means ± s.e.m. * p<0.05, ** p<0.01, *** p<0.001 (two-way ANOVA followed by Bonferroni post-test) all compared to TNBS/Vehicle-treated rats, and $$ p<0.01 for TNBS/Vehicle- treated rats compared to saline).

Figure 3 consists of a PCR amplification of the 16S rRNA-encoding gene Figure 4 illustrates the detection of LMB64 in rat faeces by amplification of the specific plasmid sequence naturally present in LMB64.

Figures 5A-5C show that LMB64 is detected only in the faeces of rats (R) receiving 10⁹ live LMB64 (A), not in the faeces of 10⁸ live LMB64- (B) nor in 10 Heat-Killed LMB64-treated rats (C). Example 1: Selection and characterization of bacterium LMB64

Bacterium LMB64 was isolated natural ecosystem.

More particularly, bacterium LMB64 is rod-shaped with a length of roughly 2.3 μιη (±0.3) and a width of roughly 1.0 μιη (±0.1). A distinctive characteristic of this bacterium is the presence of a polar flagellum (figures 1 A and IB). As can also be seen in these images, bacterium LMB64 is a nonfilamentous bacterium.

As mentioned above, bacterium LMB64 has a circular plasmid of roughly 11 kbp. This plasmid was completely sequenced (SEQ ID No. 2).

The gene coding for 16S rRNA was also sequenced (SEQ ID No. 1).

Example 2: Culture and preparation of live LMB64 sample:

In a 5 L volume shake flask, LMB64 bacteria cells were grown at T: 27°C, at 200 rpm, in 1 L medium composed of:

- 0.25 g/L NH₄C1,

- 0.4 g/L MgS0₄ 7H₂0,

- 2.5 g/L K₂HP0₄,

- 2 g/L Bacto Peptone,

- 3 g/L Yeast Extract,

- 6 g/L Glucose

The culture was stopped at OD₆oo of 2.5 and could be stored at 4°C up to 4 days. The culture medium was centrifuged at 3500 g's (15 mn, 4°C). Supernatant was discarded and bacteria were resuspended in saline buffer. The final concentration of 2.5.10¹⁰ live bacteria was adjusted in 10 mL of buffer.

Example 3: High Density Culture of live LMB64

The inoculum was prepared in 1L flask as described in the previous example. When the cell density reaches the desired criteria, the bacteria are inoculated in a 20L stirred fermentor. The basal culture medium used for the fed batch fermentation contained:

- 0.25 g/L NH₄C1, - 1.0 g/L MgSO₄ 7H₂0,

- 2.5 g/L K₂HP0₄,

- 2 g/L Bacto Peptone,

- 10 g/L Yeast Extract,

- 0.1 g/1 Antifoam

- 20 g/L Glucose

The temperature was 27°C during the grown phase. The pH was maintained at 7.0 by automatic pH controller with addition of amonia (NH₃). Disolved oxygen and pH were monitored by steam-sterilizable electrode. Aerobic conditions were maintained (p0₂ 40%) by aeration rate of 0.2 v.v.m and by agitation increase between 250 to 650 r.p.m.

Pressure was regulated at 0.2 bar to increase oxygen transfer and to prevent microbial contamination. The foam level was reduced by anti-foam addition.

Glucose was fed automatically when the initial glucose consumption was completed. The 50%> v/v glucose feeding is performed at constant flow rate. The culture growth was followed every hour by optical density measurement. All physico-chemical parameters were registered. After 25 h of cell growth, the culture was stopped by cooling (+4°C). The total final cell dry yield was determined by aliquot desiccation (100°C, 48h) at 25 g/1. The living cell density was estimated at 2 x 10¹⁰ UFC/ml. The cells harvesting was carried out with disc-stack centrifuge separator. Centrifuge speed rate was set at 1200 rpm with flow rate at 50 L/h. Automatic partial drains were performed to maintain supernatant below 15% of turbidity during the separation. After washing with phosphate buffer saline cells are removed from disc-stack and collected in plastic bag. 10%> v/v Glycerol was added to the biomass. The final viability was estimated before -20°C freezing.

Example 4: Evaluation of the potential therapeutic effects of the bacteria LMB64 in a model of inflammatory bowel diseases (IBP): the TNBS model

Six groups of 10 male rats were used for the colitis experiments and one extra group of 8 rats without colitis were used as control.

Colitis was induced by intraco Ionic instillation (through a catheter, 7 cm from the anus) of 2, 4, 6-trinitrobenzen-sulfonic acid (TNBS) (0.3 ml of a solution at 100 mg/ml of TNBS diluted in 50% ethanol) in 5 weeks old male Wistar rats.

The groups were divided as follows:

2 groups of 10 rats received treatments by oral gavage once daily (starting at 6 days before the TNBS intracolonic administration and stopping at day 0) with the LMB64 , at the doses of 10⁸ and 10⁹ respectively.

1 group of 10 rats received treatments by oral gavage once daily (starting at 6 days before the TNBS intracolonic administration and stopping at day 0) with the Heat-killed LMB64, at the doses of 10⁹.

1 group of 10 rats received treatments by oral gavage once daily (starting 6 days before the TNBS intracolonic administration and stopping day 0) with the vehicle of LMB64, namely saline.

1 group of 10 rats received treatments by oral gavage once daily (starting 1 hour before the TNBS intra colonic administration) with prednisolone at the dose of 3 mg/kg.

1 group of 10 rats received a single intraperitoneal injection of 5 mg/kg REMICADE® on DO (starting 1 hour before the TNBS intracolonic administration).

Control non-inflamed group

- 1 group of 8 rats received intra colonic administration of saline buffer and no TNBS intracolonic administration.

Colitis induction

Treatment with vehicule or LMB64 Treatment with prednisolone

Treatment with Rerricade

TIME-COURSE OF THE STUDY Compared to saline-treated rats, administration of TNBS increased significantly macroscopic damage scores (A), and myeloperoxidase (MPO) activity (B) (Figure 2, $$p<0.01 paired t test) measured 7 days post-TNBS administration.

Only Remicade® and live 10⁹ bacteria/rat LMB64 treatments were able to significantly reduce TNBS-induced macroscopic damage scores compared to vehicle- treated rats (Figure 2A), and LMB64 seemed to be more efficient to reduce this parameter than Remicade® (p<0.01 for LMB64, versus p<0.05 for Remicade®).

All the treatments were able to significantly reduce TNBS-induced increased MPO activity (Figure 2B), but 10⁹ live bacteria/rat LMB64 treatment seemed to be more efficient that any other treatment (***p<0.001 for 10⁹ bacteria/rat LMB64 treatment versus **p<0.01 for all the others, Figure 2B).

The present data demonstrated that heat-killed LMB64 have protective effects (reduced MPO activity), but most of the macroscopic inflammatory parameters are not modified by the killed bacteria, as opposed to strong anti-inflammatory effects for the live bacteria. Therefore, it can be concluded that the live bacteria hold better antiinflammatory properties than the killed bacteria.

The results from the present study showed that live LMB64 at the dose of 10⁹ bacteria/rat induced anti-inflammatory effects that are comparable to or better than Remicade® or Prednisolone for some parameters. Overall, and considering all the parameters of inflammation, LMB64 at the highest dose appeared as the most efficient treatment to reduce inflammation in the TNBS model of colitis, compared to drugs currently used in clinics.

Overall, the results generated in the present study confirm that:

- live LMB64 has anti-inflammatory properties

- the live bacteria is more potent that the heat-killed bacteria to induce antiinflammatory effects

- the anti-inflammatory effects of live LMB64 treatment in a rat model of colitis were comparable or better compared to the effects of drugs currently used in clinics. All together, these results show that live bacteria as well as heat-killed bacteria have an anti-inflammatory effect as determined by the drastic inhibition of MPO activity, the level of which is similar to that observed after Prednisolone or Remicade treatments. However, only live LMB64 bacteria are able to significantly reduce the macroscopic damage score, preferably at the dose of 10⁹/rat/day. It also shows that an anti-inflammatory effect is not the only mechanism leading to the prevention of TNBS- induced colon damages by LMB64.

Example 5: LMB64 is able to survive in the gastrointestinal tract of rats despite an optimal growth temperature of 27°C

Groups of ten WISTAR rats were orally treated by gavage with saline, 10⁸ live LMB64, 10⁹ LMB64 or 10⁹ Heat-killed LMB64 from DO to D7. On D8, faeces samples were collected and immediately frozen at -80°C until use. Since there is no selective growth media to identify LMB64, a molecular biology tool was used: the natural specific plasmid of LMB64 was amplified by PCR. DNA was first extracted from faeces samples (Kit Qiagen: DNA Stool and Dneasy Plant) and then quantified by spectrophotometry (Nanodrop). PCR amplification of the 16S rRNA-encoding gene was performed by using the universal primer pair UnivBactF (5' GAG TTT GAT YMT GGC TC 3', SEQ ID NO: 3) and UnivBActR (5' GYT ACC TTG TTA CGA CTT 3', SEQ ID NO: 4).

Primers used for PCR amplification of the LMB64 specific plasmid sequence Forward N°200 5' AACTGCATCTTTGACTGCCG 3' (SEQ ID NO: 5)

Reverse N°198 5' TGACCGTCAATACTTTGGGC 3' (SEQ ID NO: 6)

A single discrete PCR amplicon band of 1500 bp was observed when resolved on 2% agarose gel (Figure 3).

When LMB64 was added in known amounts to faeces from saline control rats, the strain was detected by amplification of the specific plasmid sequence naturally present in LMB64. As low as 10⁶ LMB64/g faeces could be detected (Figure 4).

LMB64 plasmid was then PCR amplified from the faeces of LMB64-treated rats. As shown in Figure 5A, LMB64 was effectively present in the faeces of all rats receiving 10⁹ live LMB64.

According to results presented in Figure 5 A , at least 10⁶ LMB64/g faeces could be detected in 10⁹ live LMB64-treated rats. In contrast, LMB64 could not be detected neither in the faeces of 10 live LMB64- (Figure 5C) nor in the faeces of 10⁹ Heat-Killed LMB64-treated rats (Figure 5B).

Thus, surprisingly, live LMB64 could survive in the gastrointestinal tract of rats, even though its optimal growth temperature is 27°C and optimal pH is 7.

Claims

1. A live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rR A comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80% identity with said sequence SEQ ID No. 1, or a composition comprising said live bacterium, for use in the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease.

2. A live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80% identity with said sequence SEQ ID No. 2, or a composition comprising said live bacterium, for use in the treatment of intestinal inflammatory disorders or intestinal inflammatory disorders-related disease.

3. A live bacterium, or a composition comprising said live bacterium, according to claim 1 or 2, wherein said live bacterium is selected from the group consisting of the bacterium LMB64 deposited with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, Paris, on April 8, 2010, under the reference 1-4290, and homologue, descendant or any other mutant of said bacterium LMB64.

4. A live bacterium, or a composition comprising said live bacterium, according to any of claims 1 to 3, wherein said live bacterium, or composition comprising said live bacterium, is administered to a patient in need thereof in a therapeutically effective amount to an individual affected by an intestinal inflammatory disorders or intestinal inflammatory disorders-related disease, and wherein said administration reduces inflammation or inflammatory effects in said individual, or induces anti-inflammatory effects.

5. A live bacterium, or a composition comprising said live bacterium, according to any of claims 1 to 4, wherein said intestinal inflammatory disorders is selected from the group consisting of Crohn's disease and ulcerative colitis.

6. A live bacterium as defined in one of claims 1 to 3, or composition comprising said live bacterium, for use in the treatment or prevention of pathology related to an infection or to an immune response defect, wherein said pathology is associated with a defect in the activity of TLR2, TLR4 and/or TLR5.

7. A live bacterium as defined in one of claims 1 to 3, or composition comprising said live bacterium, for use in the treatment or prevention of pathology related to inflammation, wherein said pathology is associated with a dysfunction of PAR2.

8. A live bacterium, or a composition comprising said live bacterium, according to any of claims 1 to 7, wherein said inflammatory disorder or inflammatory disorders-related disease is a gastrointestinal inflammatory disorders.

9. A live bacterium, or a composition comprising said live bacterium, according to claim 8, wherein said gastrointestinal inflammatory disorder is selected from the group consisting of inflammatory bowel disease (IBD), irritable bowel syndrome (IBS) or colitis.

10. A composition, preparation or formulation comprising, as an active ingredient, at least one live bacterium as defined in one of claims 1 to 3.

11. A composition, preparation or formulation for use as a probiotic comprising, as an active ingredient, at least one live bacterium as defining in one of claims 1 to 3, or a composition comprising said live bacterium as defining in one of claims 1 to 3.

12. The composition, preparation or formulation according to claim 10 or 11 which comprises another probiotic material.

13. The composition, preparation or formulation according to one of claims 10 to 12, which comprises a pharmaceutically acceptable carrier.

14. The composition, preparation or formulation according to claim 13, which comprises a pharmaceutically acceptable ingestable carrier.

15. A probiotic live bacterium, or a probiotic composition comprising said live bacterium, wherein said live bacterium is selected from the group consisting of:

- a live bacterium selected from bacterium whose nucleotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucleotide sequence with at least 80% identity with said sequence SEQ ID No. 1; and

- a live bacterium selected from bacteria comprising at least one plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80%> identity with said sequence

SEQ ID No. 2; and

- a live bacterium named LMB64; and

- a live non- filamentous bacterium LMB64; and