OA16463A

OA16463A - Novel bacterium and extracts of said bacterium and the use of same in dermatology.

Info

Publication number: OA16463A
Application number: OA1201300259
Authority: OA
Inventors: Philippe Lebaron; Muriel Bourrain; Nathalie Castex-Rizzi; Thien Nguyen
Original assignee: Pierre Fabre Dermo-Cosmetique; Universite Pierre Et Marie Curie (Paris 6); Centre National De La Recherche Scientifique (Cnrs)
Priority date: 2010-12-22
Filing date: 2011-12-22
Publication date: 2015-10-15

Abstract

The present invention relates to a novel bacterial strain isolated from groundwater. The invention also relates to bacterial extracts and to the use of same 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 inflammatory disorders, notably dermatological pathologies.

Description

The présent invention relates to a novel bacterial strain isolated from groundwatcr. The invention also relates to bacterial cxtracts and to the use of same in the contcxt of the treatment of inflammations,

More particularly, the présent invention relates to novel compositions of interest in the treatment and the prévention of inflammatory disorders, notably dermatological pathologies.

Dermatological diseases such as atopie dermatitis, pruritus, eczema and psoriasis arc increasingly frequent in young childrcn. The prevaience of atopie dermatitis has doubled or tripled in developed countries over lhe past 30 years: 15% to 30% of childrcn and 2% to 10% of adults are affected (Williams H. et al., JACI2006; 118:20913). Atopie dermatitis is the culaneous manifestation of atopy; it is a chronic inflammatory dermatosis or eczema, occurring due to a genetically determined set of circumstances. Il is now regarded as a major public health concem. Atopie dermatitis is often associated with other atopie disorders such as allergie rhinitis and asthma. This affection most often appears during early childhood and is characterized by repeated outbreaks over several years. It progresses with flarc-ups interrupted by spontaneous remissions.

The quality of life for patients suffering from atopie dermatitis is profoundly disturbed. Accepted treatments include topical corticosteroids and immunomodulators, systemic agents whose frequent side effects limit long-term use, and émollients. Current thérapies are reactive - treatment of outbreaks - but it is now believed that early intervention focused on the control of outbreaks and of cutaneous inflammation can be bénéficiai in terms of both control of the disease and the potential appearance of asthma and/or rhinitis (Bieber, T. 2008, Atopie dermatitis, The New England Journal of Mcdicine, vol. 358(14) 1483-1494), as atopie dermatitis is regarded as the initiai phase of atopie progression. In most cases, treatments include a local component in order to best provide relief to patients.

Standard treatments for atopie dermatitis notably use topical corticosteroids or &J immunosuppressants, although such treatments are not free of adverse effects in children in particular.

Atopie dermalitis is complex and multîfactorial. In the literature, some epidemïological studies hâve shown that the “hygiene factor in urban environments promote the discase like allergy and autoimmunily. On the other hand, in rural settings where man is in constant contact with microorganisms and/or allcrgcns, such cxposurc stimulâtes man’s défensive immune system from birth.

In atopie dermatitis, the borner fonction of the skin is weakencd and impaired, which promûtes the invasion and the colonization of pathogens (bacteria, viruses), in particular Staphylococcus aureus, which is known to predominate the commensal bacteria of the skin.

In terms of immunology, the issue is one of immune response imbalance. Atopy is often described as an allergie manifestation (IgE mediated, dominance of cytokines IL-4, IL-5, IL-13) orTh2 response. The lutter is ail the more accentuated in the presence of “antigenic stimuli” of Staphylococcus aureus. Immunomodulation consists in retuming immune homeostasis to a Thl/Th2 balance.

Innate immunity is the primary, rapid and nonspecifïc response of the immune response in mammals. The cell’s first barriers of defense are comprised of Toll-like receptors (TLRs). Each TLR specifically recognizes pathogen-associated molecular patterns (PAMPs) such as nucleic acids (TLR3), peptides, surface proteins, lipoteichoic acid (TLR2), flagella (TLR5) and lipopolysaccharides (TLR4) arising from foreign microorganisms. A spécifie interaction between a motif (agonist) and a TLR triggers a cascade of complex reactions resulting in the transcription of NFkB, followed by production of pro-inflammatory and anli-inflammatory cytokines and of chemokines (Kong et al., 2006). Other resulting pharmacological conséquences are the induction of antimicrobial peptides (AMPs), which hâve the ability to inhibit the growth of pathogens (bacteria, viruses, parasites) (Glaser, R, et al. 2005, Nat. Immunol. 6:57-64).

Atopie dermatitis is often accompanied by itching and pruritus, thus causing discomfort and annoyancc in daily life (scratching, slcep loss, etc.). One of the causes of this inflammatory pathology is duc to the activation of a G protcin-couplcd rcceptor called PAR2 (protease-activated receptor 2) (Steinhoff, M. et al. 2003 J Neurosci. 23:6176-6180). PAR2 is expressed on the surface of many cells, in particular &J i

kératinocytes, endothélial cells, colonie myocytes, enterocytcs, enteric neurons and immune cells. Proteases (trypsin, tryptase), présent in abundance in the epidermis, cleave the PAR2 at the N-terminal exposing a spécifie peptide which activâtes this same receptor (phenomenon of self-activation) (Vergnolle, N, 2009 Pharmacol. Ther.

123:292-309). This process involves activation of the NFkB gene, followed by the induction of pro-inflammatory cytokines, thus triggering inflammation. In this contcxt, the development of PAR2 antagonists and/or protease inhibitors has a high potcntial to treat the pathology of pruritus.

Psoriasis is also a cutaneous inflammatory disease with a chronic progression; it affects 2% of the population. Along with atopie dermatitis, psoriasis is one of the most common chronic cutaneous inflammatory diseases. It is characterized by abnormal growth of epidermal cells associated with an inflammatory reaction. The central mechanism of the inflammation phenomenon is related to the action of the immune system’s T cells, predominantly Thl cells (Wilsmann-Theis, D. et al., Eur J Dermatol., vol. 18(2) 172-180), which initiate and maintain the inflammatory process and stimulate the excessive prolifération of kératinocytes which then proceed through an accelerated and incomplète différentiation phase. Kératinocytes express receptors which make them sensitive to inflammatory signais and release pro-inflammatory mediators. Psoriatic inflammation is thus maintained by mutual stimulation of T cells and kératinocytes.

The disease must therefore be treated over the long term. There is thus a need and a high demand for therapeutic alternatives for thèse inflammatory dermatoses.

Mention may be made of patent document EP2018891 (Guéniche A., 2009) and the document by Guéniche A. et al., 2006 (European Journal of Dermatology, 16, 4, 380-384) which describe the use of a bacterial extract of Vitreoscilla filiformis (V.

5 filiformis) for the treatment of atopie dermatitis. Such an extract has the disadvantage of requiring the culture of said filamentous baclerium V. filiformis on a medium containing suifur-free minerai water.

In this context, the présent invention provides a solution to the treatment of these inflammatory disorders by the isolation, the characterization and the fractionation of a

0 novcl bactcrium never before described.

For the first time, and in a surprising manner, the Applicant succeeded in isolating a strain belonging to a novel bacterial species from groundwater, wherein said (jJ t

novel bacterial strain (or bacterium) is named LMB64.

This bacterium LMB64, in addition to the fact of having been isolated, was characterized and defined as bclonging to the class of Betaprotcobacteria, subfamily of Neisseriaceae, and probably of a novel genus not yet defined. Analysis of the gene sequence coding for I6S ribosomal RNA (rRNA) made it possible to place this bacterium close to the gcncra Chromobactcrium, Paludimonas, Lutclia and Glubcnkiana, with which it sharcs 95% sequence similarity.

This nonpathogenic bacterium is Gram-negative and will be described in greater detail in the cxamples. This bacterium also has the characteristic of being nonfilamentous. 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. As an cxample, in contrast to V. filiformis, the culture of bacterium LMB64 of the présent invention does not rcquire particular culture conditions and, more particularly, does not require a medium containing at least one sulfur-free type of minerai and/or thermal water. This represents a clear advantage in terms of both culture conditions and facilities and from an économie point of view.

The gene coding for 16S rRNA has been almost completely sequenced (1487 bp, corresponding to sequence SEQ ID No. 1). 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 présent invention relates to a nonpathogenic Gram-negative bacterium belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae, whose nucléotide sequence of the gene coding for 16S rRNA includes or comprises the sequence SEQ ID No. 1, or any nucléotide sequence with at least 80%, preferably 85%, 90 %, 95% and 98% identity with said sequence SEQ ID No. 1.

In a preferred manner, the présent invention relates to a nonpathogenic Gramnegative bacterium bclonging to the class of Betaproteobacteria, subfamily of Neisseriaceae, characterized in that the nucléotide sequence of the 16S rRNA gene of said bacterium includes or comprises the sequence SEQ ID No. 1.

In the contcxt of the présent invention, “percentage identity” between two nucleic acid sequences refers to a percentage of identical nucléotides between the two sequences to be compared, obtained after the best alignment (optimal alignment), wherein this percentage is purely statistical and the différences between (he 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 bc made per segment or per “comparison window.” The optimal alignment of the sequences for the comparison can bc 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 délétions 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 nucléotide 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 rcsult obtained by 100 to obtain the percentage identity between these two sequences.

For example, the “BLAST 2 sequences” program (Tatusova et al., “Blast 2 sequences - a new tool for comparing protein and nuclcotide 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 bc compared being calculated directly by the program. It is also possible to use other programs such as the “ALIGN” or “Megalîgn” software (DNASTAR).

According to another embodiment, the baclerium 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.

According to a preferred embodiment of the invention, bacterium LMB64 is characterized in that it is nonfilamentous.

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

Moreover, bacterium LMB64 of the présent invention has been deposited in accordance with the Budapest Treaty in the name of the Applicant with the Collection Nationale de Cultures de Microorganismes (CNCM), Institut Pasteur, Paris, on April 8, 2010, under the reference 1-4290.

Thus, onc object of the invention is the bacterium deposited with the CNCM on April 8, 2010, under the référencé 1-4290, or a homologue, a descendant or any other mutant.

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 prolifération, cell division (mutation due to errors occurring during bacterial division or DNA réplication) or any other mechanism of natural sélection, such as the sélection of mutants that arc résistant or thaï bccomc résistant to a given compound. Included among these mutants are any bacteria arising from strain ï-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 (éthanol, isopropanol, isobutanol), by drying on a cylindcr with a scrapcd prelayer, etc., and then used in frcczc-dricd or hcat-inactivatcd form.

According to another preferred embodiment, the invention relates in a general manner to a bacterial extract, also callcd a bacterial fraction, obtained from a suspension of bacteria as described above, namely bacterium LMB64.

The term “bacterial extract” refers to any extract or fraction of the bacterial biomass or any active fraction of said exlract. For example, such an extract may be obtained from a culture of bacterium LMB64 wherein the préparation method comprises at least one step of lysis of the bacteria and one step of séparation of the various fractions of which it is constitutcd by centrifugation or by filtration.

In a nonrcstrictive manner, the exlract according to the invention may consist of bacterial cells isolated from the culture medium which hâve been concentrated, for example by centrifugation; or concentrated bacterial cells which have undergone an operation in which the cell envclopc has been ruptured by any means known to those persons skilled in the art, such as by the action of ultrasound or autoclaving; or the supernatant obtained by filtration.

An important step of the extract préparation method according to the invention consists of the élimination of the various intracellular components such as, for example, nucleic acids (chromosomal DNA, extrachromosomal circular DNA, plasmids), ribosomes and intracellular stored substances such as glycogen, starch and poly-βhydroxybutyrate, etc.

In a preferred manner, the bacterial extract according to the invention is obtained after treatment of said bacterial suspension in such a way as to eliminate the intracellular components.

The rcsult is that the extract according to the invention primarily includes components arising from the membrane, from the periplasmic space and/or from the extracellular space.

More particularly, said intracellular components comprise at least the nucleic acids.

In addition to the élimination of intracellular compounds, and as a nonrcstrictive example, it is also easily possible for those persons skilled in the art to separate, after lysis of the bacteria and centrifugation, the components of the culture supernatant (hcrcaftcr fraction S0) and lhe components constituling the pcllct (hcrcaftcr E0). For cxamplc, it may bc suggested that the séparation thrcshold between the constitucnts of S0 and EO is around a molecular weight of 100 kDa, Consequently, the constitucnts of fraction S0 have, for the most pari, a molecular weight less than 100 kDa, whereas the components of fraction EO hâve, for the most part, a molecular weight greater than 100 kDa,

More particularly, it is thus possible by techniques known to those persons ski lied in the art to extract and separate the biomoleculcs found in the culture 5 supematant (SO) from those mainly comprised of surface proteins and proteins located in the pcriplasmic space of the bactcrium (EO).

According to one embodiment of the invention, the bacterial cxtract includes a fraction EO comprising at least membrane proteins, periplasmic proteins and proteins arising from the flagellum.

Periplasmic proteins include proteins lodgcd in the periplasmic space of Gramnegative bacteria which may be released by osmotic shock or by incubation in a medium containing a chaotropic agent or detergents (Molecular Cloning: A Laboratory Manual, 3¹ édition: Sambrook and Russell. CSHL Press).

Proteins arising from the flagellum include multimeric proteins of the flagellum or fragments of the flagellum, Methods for isolating and purifying whole bacterial flagella with detergents followed by ultracentrifugation séparations (in the presence of a CsCl gradient) are described in the literature. In the invention, the examples of extraction methods made it possible to recover flagella fragments.

Membrane proteins include proteins that are anchored in the membrane and of

0 which a part is exposed on the surface (outer membrane proteins, or Omp), proteins that arc adhcrcd to the surface of the membrane, lipoproteins and porins (Ward JB„ Microbial adhesion to surfaces, 1980).

In a preferred manner, said membrane proteins consist of porins, OmpA, lipopolysaccharides and/or lipoproteins.

According to another embodiment of the invention, it may be preferred to use fraction SO.

More particularly, the bacterial extract according to the invention includes a fraction SO comprising at least secreted peptides and proteins and secondary métabolites.

Sccrctcd peptides and proteins include peptides and proteins that arc naturally produced and secreted by bacterium LMB64 and which may be recovered by centrifugation or by filtration.^/

Secondary métabolites include the small molécules that bacterium LMB64 produces and sécrétés in the culture medium.

The presence of lipopolysaccharides within fraction SO should be mentioncd here. Indeed, lipopolysaccharides, although they are found primarily in fraction E0, are nevertheless also found in smaller quantities in fraction SO.

In an advantageous manner, fractions E0 and SO may be combined in such a way as to obtain a fraction ESO by lcaving, for exemple, the culture medium to incubatc and to react in basic medium (pH 9 to 11) for approximately 5 hours a température of 4 °C, by centrifuging and by filtcring at 0.2 pm in order to obtain a clear ESO solution.

Bacterial extract ESO is thus composed, among other things, of membrane proteins, lipopolysaccharides, periplasmic proteins, protein fragments of the flagellum and primary and secondary métabolites produced by the bacterium.

In a preferred way, extract ESO has a protein profile comprising at least, according to the SDS-PAGE technique, twelve bands including three principal bands corresponding, respectively, to molecular weights (approximate molecular weights given in relation to molecular standards, notably provided by Bio-Rad Laboratoires) rangîng between:

band 1: 30 kDa and 36 kDa, preferentially 34 kDa;

band 2: 41 kDa and 45 kDa, preferentially 43 kDa;

band 3:47 kDa and 51 kDa, preferentially 49 kDa.

According to another embodiment of the invention, the bacterial cxtract includes a fraction ESO comprising at least fraction E0 and fraction S0.

According to a preferred embodiment of the invention, the bacterial extract includes a fraction ESO with a protein profile, obtained by SDS-PAGE, which includes three principal bands corresponding to molecular weights ranging between 30 kDa and 36 kDa, 41 kDa and 45 kDa, and 47 kDa and 51 kDa, respectively.

According to a preferred embodiment of the invention, the bacterial extract includes a fraction ESO with a protein profile, obtained by SDS-PAGE, which includes three principal bands corresponding to molecular weights of 34 kDa, 43 kDa and 49 kDa, respectively.

According to another aspect, lhe invention describes a method for preparing a bacterial extract comprising the steps of: (/J

ΙΟ

a) culturing bacterium LMB64 in a suitable medium; and

b) eliminating the intracellular components.

According to another embodiment, the method according to the invention consists of a method for preparing a bacterial extract SO, wherein said method comprises the steps of:

a) culturing bacterium LMB64 in a suitable medium;

b) centrifuging said culture; and

c) recovering supematant SO.

According to another embodiment, the method according to the invention consists of a method for preparing a bacterial extract E0, wherein said method comprises the steps of:

a) culturing bacterium LMB64 in a suitable medium;

b) centrifuging said culture and eliminating the supematant;

c) treating the biomass resulting from step b) in such a way as to eliminate the intracellular components; and

d) recovering base E0.

In a preferred manner, step c) consists of ultrasonic treatment of the biomass resulting from step b) and then an initial centrifugation aimed at eliminating the pellet comprising said intracellular components and then a second centrifugation of the supematant.

a) culturing bacterium LMB64 in a suitable medium;

b) centrifuging said culture and eliminating the supematant;

c) treating with ultrasound the biomass resulting from step b);

d) centrifuging said biomass treated with ultrasound and eliminating the biomass obtained;

c) centrifuging the supematant resulting from step d); and

f) recovering the base E0.

ll should be noted that the various methods described above are provided for illustration only and that any methods known to those persons skilled in the art may be ίλ/ h

η used.

As will become apparent from the examples below, the Applicant has demonstrated, in addition to the activities expcctcd for this type of extract, several novel activities never before described.

A first advantageous aspect of lhe invention, related to immunomodulation, rests on the modulation property of pro-inflammatory cytokines. More particularly, the use of a bactcrium and/or an cxtract according to the invention significantly induccs cytokines IL-IO, IL-12 and TNF-α, which are preferentially involved in the Thl immune response, and significantly inhibits cytokines IL-4 and IL-6. The resuit is lhe activation of Langerhans cells and a retum to Thl/Th2 balance.

Furthermore, another observation demonstrated that the use of a bactcrium and/or an extract according to the invention makes it possible to greatly decrease the expression of IgE rcceptors, which is of interest in that IgE potentiates allergie phenomena.

Another advantage of the invention rests on the fact that, as will be apparent from the examples, the use of a bacterium and/or an extract according to the invention induces the production of antimicrobial peptides such as, for example, peptides hBD-2, hBD-3, S1007A andLL-31.

More particularly, as mentioned above, an extract of bacterium Vitreoscilla filiformis (Guéniche A. et al., Eur J Dermatol 2006; 16:380) has been known with activity on TLR2, duc to the présence of OmpA, and on TLR4, duc to the présence of lipopolysaccharides. Because of the absence of flagella in the V. filiformis bacterium, the extract obtained from V. filiformis has no TLR5 activity.

For the first time, the Applicant dcscribcs a bacterial exlract according to the invention which has, in addition to activity on TLR2 and TLR4, activity on TLR5.

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

In a preferred manner, said bacterial extract activator of TLR2, TLR4 and TLR5 consists of an cxtract comprising ail or part of the proteins arising from the flagcllum. In this case, as an example, said extract is preferentially cxtract E0 or cxtract ES0.

Said TLR5 activation activity is of signifîcant interest in that TLR5 are known to induce certain antimicrobial peptides such as psoriasin (S100A7) and hBD-2 (Glaser et £>/ ai., 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 potentiale the production of antimicrobial peptides. It has been shown that by blocking

TLR5 with an antibody, the latter arc produced little or not at ail.

This aspect is thus particularly innovativc in terms of immunomodulation applications for the bacterium and/or the cxtracts according to the invention.

Furthcrmorc, in an uncxpcctcd manner, the Applicant has also demonstrated, in contrasl to the bacterial extracts described to date, 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 bacterium and/or a bacterial extract such as described above as a PAR2 antagonist.

In a preferred manner, said PAR2 antagonist bacterial cxtract consists of extract S0 or ex tract ES0.

PAR2 is overexpressed in endothélial cells, colonie myocytes, enterocytes, enteric neurons, immune cells and kératinocytes. Proteases (trypsin, tryptase) présent in abundance in the environment cleave the PAR2 at the N-lerminal exposing a spécifie peptide which activâtes this same receptor (phenomenon of auto-activation). Consequently, this activâtes the production of pro-inflammatory cytokines and triggers inflammation (Vergnolle, N„ 2009 Pharmacol. Ther. 123:292-309). This phenomenon is observed in the wild mouse but does not appcar in the KO mouse (PAR2 déficient). Treatment with an antiprotcasc and/or a PAR2 antagonist makes it possible to avoid this inflammation phenomenon.

The combination and the synergy of ail these activities give this bacterium LMB64, or any extract arising from this same bacterium, 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 wcakened, disturbed or unbaianced.

The invention thus relates to the use of a bacterium such as described above and/or a bacterial extract arising from said bacterium for the préparation of a composition intended for the treatment and/or the prévention of dcrmatological inflammatory disorders.

In a preferred manner, said dermatological inflammatory disorders consist of atopie dermatitis, pruritus, eczema and psoriasis. cA

According to another embodiment, the invention of the présent patent application relates to a composition comprising, as an active ingrédient, at least one bacterium and/or one bacterial extract according to the invention.

The invention thus relates, in a preferred manner, to a cosmetic or dermatological composition.

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

In a preferred manner, said dermatological inflammatory disorders consist of atopie dermatitis, pruritus, eczema and psoriasis.

The composition according to the invention may in particular contain additives and formulation aids such as emulsifiers, thickeners, gelling agents, water binders, spreading agents, stabilizers, colorants, fragrances and preservatives.

The cosmetic or dermatological composition according to the invention further comprises one or more typical dermatologically-compatible excipients.

The composition according to the invention may be prepared in the form of a water-in-oil (W/O) or oil-in-water (O/W) émulsion, a multiple émulsion such as, for example, a water-in-oil-in-water (W/O/W) or oii-in-water-in-oil (O/W/O) émulsion, a microemulsion or in the form of a hydrodispersion or a lipodispersion, a gel or an aérosol.

The dermatologically or cosmetically compatible excipients may be any excipient among those known to those persons skilled in the art in order to obtain a composition for topical application in the form of a milk, a cream, a balm, an oil, a lotion, a gel, a foaming gel, a pomade, a spray, etc.

In addition to dermatological and cosmetic compositions, the invention also relates to pharmaceutical compositions for a use as drug.

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

In the présent description, “pharmaceutically acceptable carrier” refers to a compound or a combination of compounds made part of a pharmaceutical composition that do not cause sccondary réactions and that, for examplc, facilitatc the administration of the active compounds, increase their lifespan and/or effectiveness in the body, increasc their solubility in solution or improve their préservation. Said pharmaceutically (Xl * ' » 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 antibodies according to the invention may bc administered in several doses, spread out over time.

Their optimal modes of administration, dosing schedules and galcnic forms may be determined according to criteria generally considercd in the establishment of a treatment adapted to a patient such as, for example, the âge or the weight of the patient, the seriousness of the patient’s general health, tolérance to the treatment and side effects noted.

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

Description of figures:

Figure 1 illustrâtes the phylogenetic position of the sequence coding for the 16S rRNA of strain LMB64. The sequences appearing on this trec are sequences from the GenBank database closest to the sequence of LMB64.

Figures 2A and 2B présent images of bactcrium LMB64 under the transmission électron microscope (A) and the scanning électron microscope (B).

Figure 3 présents growth optima determined as a function of the température, pH and salinity of the R3 culture medium.

Figure 4 illustrâtes induction of cytokines IL-10 and IL-12 by cxlract E0 (dosedependent effect).

Figure 5 illustrâtes induction of surface molécules CD80, CD86, CD83 and CD54 by ex tract E0 (dosc-dependent effect).

Figure 6 illustrâtes inhibition of IgE reccptors by extract E0.

Figure 7 illustrâtes activation of TLR2 by cxtract ESO.

Figure 8 illustrâtes activation of TLR4 by cxtract ESO.

Figure 9 illustrâtes activation of TLR5 by extract ESO.

Figure 10 illustrâtes spécifie PAR2 antagonist activity by extract ESO. Cx/

Figure 11 illustrâtes induction of antimicrobial peptides and proteins by extract ESO.

Figure 12 consists of an SDS-PAGE gel of cxtract ESO.

Example 1; Sélection and characterization of bacterium LMB64

Bactcrium AV 13 was isolated from groundwater.

The taxonomie position of novel bactcrium LMB64 is proposcd in figure 1.

More partîcularly, bacterium LMB64 is rod-shaped with a length of roughly

2.3 pm (±0.3) and a width of roughly 1.0 pm (±0.1). A distinctive characteristic of this bacterium is the presence of a polar flagellum (figures 2A and 2B). As can also be seen in these images, bactcrium LMB64 is a nonfilamentous bacterium.

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

The genc coding for 16S rRNA was also sequenced (SEQ ID No. 1). The bacterium was cultured in a fermentor in a synthetic medium. The growth rate is higher when the medium has a low concentration of carbon substrates.

The culture media tested are R3, MS-glucose and LB media whose compositions are described below in tables la, lb and le, respectively.

20	COMPOSITION OF R3 MEDIUM
	Yeast cxtract	IgA
	Dïfco protcose peptone	IgA
	Casamino acids	IgA
25	Glucose	1 g/1
	Soluble starch	IgA
	Sodium pyruvate	0.5 g/1
	K2HPO4	0.6 g/1
	MgSO₄, 7H₂O	0.1 gA
30		Table la Ca/

* t

COMPOSITION OF MS-GLUCOSE MEDIUM
Glucose	6.0 g/1
Citric acid	0.84 g/1
MgSO₄, 7H₂O	0.25 g/1
NH₄C1	1.06 g/1
Anhydrous K2HPO4	8.75 g/1
Pyruvic acid sodium sali	0.5 g/1
Zinc sulfate, 7H2O	4 mg/1
Cobalt chloride, 6H₂O	3.5 mg/1
Sodium molybdate, 2H₂O	3.5 mg/1
Manganèse sulfate, 1H₂O	5 mg/1
Boric acid	2 mg/I
Concentrated hydrochloric acid	50 mg/1
Copper sulfate, 5H2O	4 mg/1
Iran chloride, 6H2O	27 mg/1

Table lb

COMPOSITION OF LB MEDIA
Tryptone	10 g/I
Yeast extract	5 g/1
NaCl	5 g/1

Table le (V

The growth rates of bacterium LMB64 as a function of culture medium are prcscntcd in table 2 below.

Growth rate (/h)

LB	0.25 (±0.05)
LB (½ dilution)	0.46 (±0.11)
LB (1/5 dilution)	0.60 (±0.14)
LB (1/10 dilution)	0.69 (±0.15)
MS-glucose	0.13 (±0.04)
R3	0.62 (±0.14)

Table 2

The growth optima were determined as a function of the température, pH and 15 salinity of the R3 culture medium (figure 3).

The sources of carbon assimilable by the bacterium were characterized using an API 50CH gallcry (incubation température: 25 °C). The results are summarized in table 3 below. CaI «

	Incubation time
4 days	5 days
1. Glycerol 2. Erythritol 3. D-arabinose 4. L-arabinose 5. D-ribose 6. D-xylose 7. L-xylose 8. D-adonitol 9. Methyl-p-D-xylopyranoside 10. D-galactose 11. D-glucose	+	+
12. D-fmctose	+	+
13. D-mannose 14. L-sorhose 15. L-rhumnose 16. Du Ici toi 17. Inositol	1	+
18. D-mannitol 19. D-sorbitol 20. Methyl-a-D-mannopyranoside 21. Methyl-a-D-ghtcopyranoside 22. N-acetyJglucosamine 23. Amygdaline 24. Arbutin 25. Esculin/iron citrate 26. Salicin 27. D-cellohiose 28. D-maltose	i	+
29. D-lactose (bovine origin) 30. D-melibiose 31. D-sucrose	+	+
32. D-trehalose	1	4-
33. Inulin 34. D-melezitose 35. D-raffinose 36. Starch 37. Glycogen 38. Xylitol 39. Geotiohiose 40. D-turanose	1	+
41. D-lyxose 42. D-tagatose 43. D-fucose 44. L-fucose 45. D-arabitol 46. L-arabitol 47. Potassium gluconate 48. Potassium 2-ketogluconate 49. Potassium 5-ketoglucanate +: usable substrate, 1: low use	Table 3 (W

*

The enzymatic aelivities demonstrated on the API ZYM gallery arc: alkaline phosphatase, esterase (C4), esterase/lipasc (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and a-glucosidase.

Bactcrium LMB64 is sensitive to ail the antibiotics tested as seen in table 4 5 below.

Antibiotics tested	Zone of inhibition diameter (mm)	Inhibitory activity
R3	LB 1/2	LB 1/5
Ampicillin (10 pg)	29	28	29	+
Chloramphenicol (30 pg)	29	26	24	+
Ciprofloxacin (5 pg)	38	34	34	+
Kanamycin (30 pg)	27	30	27	+
Penicillin (6 pg)	21	26	20	+
Polymyxin B (50 pg)	11	15	13	+
Rifampicin (30 pg)	20	19	15	+
Tétracycline (30 pg)	30	25	20	+
Streptomycin (10 pg)	25	25	24	+
Vancomycin (30 pg)	20	21	21	+

Table 4

Example 2: Method for extracting fractions EQ, SQ and ESO

Preculture: Strain AVI3 is inoculated in an Erlenmyer flask containing 250 ml of MS glucose pyruvate medium (see table 5 below), followed by incubation under stirring for roughly 40 hours at 30 °C (pH 7) and 200 rpm until an ODôœ-1.5 is obtained. Ca!

A

MS Glucose Pyruvate
Citric acid	0.84 g
MgSO₄.7H₂O	0.25 g
NH₄Cl	1.06 g
Anhydrous K2HPO4	8.75 g
Pyruvic acid sodium sait	0.5 g
Oligo mix	l ml
ddH₂O qsp	1000 ml
Verify pH	7
Autoclave	121°C 30 min
After autoclaving add: 20% glucose	30 ml

OLIGO MIX
Dissolve in 100 ml ofdistilled water:
Zinc sulfate, 7H₂O	4 g
Cobalt chloride, 6H₂O	3.5 g
Sodium molybdate, 2H₂O	3.5 g
Mangane.se sulfate, 1H₂O	5 g
Boric acid	2 g
Concentrated hydrochloric acid	50 g
Coppcr sulfate, 5H₂O	4 g
Dissolve in 50 ml of distilled water:
lron chloride, 6H₂O	27 g
ddH₂O qsp	1000 ml

Table 5 &J

Culture: The preculture is then inoculated in a fermentor (Applikon) containing 3.7 l of MS pyruvate medium + 114 ml of 20% glucose solution. A température sensor régulâtes the température preferably near 30 °C. An oxygen sensor (AppliSens) is used to maintain the concentration of dissolved oxygen in the medium at 18-25%. A pH sensor (AppliSens) is used to maintain the pH at 7 by the addition of 10% NH4OH via a fixed flow-ratc pump. A Wcdgcwood Analytical sensor is used to monitor changes in optical density in real time. The culture is programmed in fcd-batch mode; via a variable flow-rate pump the culture is supplied with 20% glucose solution. Fermentation is stopped when 00600==22-26, in general after roughly 30 hours.

Extraction SO: The supematant is separated from the biomass by centrifugation for 1 hour at 4 °C and 4000 g.

Extraction EO: The wet biomass is taken up in NaCI solution (IM). After centrifugation for 15 minutes at 4 °C and 9000 g, the supematant is discarded and the pellet is taken up in 1 M NaCI solution, The sample tube is then plunged into a cooled ultrasonic bath at a power setting of 50-60 W for several minutes. After centrifugation for 30 minutes at 4 °C and 6000 g, the pellet is discarded and the supematant is recovered, Two volumes of cold éthanol are added and the suspension is left overnight at 4 °C. After centrifugation for 30 minutes al 4 °C and 6000 g, the supematant is discarded and the pellet is taken up in 25 mM Tris buffer, pH 8,8.

Extraction ES0: The culture is brought to basic pH (pH 9-11) with a base buffer, The ncxl step is incubation under stirring for 5 hours at a température of 4 °C. After centrifugation, the supematant is prefiltcred to eliminate rcmaining biomass débris and then filtered on a 0,2 pm filter. A clear yellow solution is obtained (ES0).

Proteins are assayed according to the DC Protein Assay Kit Π (Bio-Rad) protocol, Sugars are assayed in glucose équivalent according to the phenol/sulfuric acid method (Dubois, M. et al„ 1956).

As an exemple, table 6 below présents certain spécifie charactcristics of extract ESO as obtained under the conditions described above.

i

	Test batch	Preclinical batch 1
Organoleptic characterlstics	Homogeneous and translucent yellow-orange liquid Dcnsîty near that of water
pH (in the presence of base buffer)	10.0	10.2
Dry residue (thermobalancc)	5.9%	5.1%
Protein profile (SDS-PAGE)	12 détectable bands (încluding 3 principal bands roughly 34 kDa, 43 kDa and 49 kDa in size, respectively)
Total protein assay (pBCA)	2.9 mg/ml	3.0 mg/ml

Table 6

It is clearly understood that the data above are presented here only for illustrative purposcs.

More precisely, the data relate to a protein profile obtained by SDS-PAGE exhibiting three principal bands.

SDS-PAGE protocol;

Extract ESO is taken up in buffer (20 mM Tris-HCl, pH 8.0; l mM EDTA; 2.5% SDS and 0.01% bromophenol blue) and 1 M DTT (1,4-dilhiothreitol). The sample and the mixture of molecular weight markers (WcstemC, Bio-Rad) were deposited respectively in wells of an 8-16% SDS-PAGE acrylamide gel (GeBaGel, Gene BioApplication). The migration buffer contains 2.5 mM Tris, 19.2 mM glycine and 0.01% SDS (w/v). Migration is allowed to procccd under a constant voltage of 160 V for approximately 1 hour (GeBaGel System). The protein bands were then stained with Coomassie Blue (Instant Blue, Expedeon). Sizes were calculated in relation to known standards (STD).

The gel obtained is presented in figure 12.

According to one embodiment of the invention, these three bands hâve molecular weights of approximately 34 kDa, 43 kDa and 49 kDa, respectively. &J * *

Example 3; Démonstration of the pharmacological activities of fractions EQ and ESO

Langerhans cells (LC) are generated in vitro from human monocytes isolated from Buffy-Coat pouchcs from the French National Blood Service (Etablissement Français du Sang (EFS) Pyrénées Méditerranée): isolation on a Ficoll gradient (Lymphocyte Séparation Medium, density 1.077 g/ml) and purification by magnctic immunoselection (Miltenyi Biotec); LC différentiation is carrîed out for 6 days in the presence of a cytokine cocktail (GM-CSF/lL-4fTGFP). LC distributed on 24-well plates 10 in RPMI-5% FCS culture medium are incubatcd for 24 hours with extract ESO.

Surface molécules are anaiyzed by flow cytometry (FACSCalibur, BD Biosciences) with triple or quadruple staining: CDla/CD54/CD80/CD83/CD86/FceRI; cytokines secreted in the culture supernatants are anaiyzed with the Cytometry Bead Array (cal. no. 550749, BD) in flow cytometry: IL-6, IL-8, TNF, IL-4, IL-10, IL-12.

3.1 Induction of key cytokines for Thl nolarization

Extract E0 induces according to a dosc-dependent effect the expression of cytokines IL-10 and IL-12 by Langerhans cells (figure 4). These cytokines promote the induction of TH1 polarity of naïve T lymphocytes.

3.2 Langerhans cell maturation and IgE receptor (FceRI) inhibition

Extract E0 induces the maturation of Langerhans cells observed by dosedependent induction of surface molécules CD80, CD86, CD83 and CD54 (figure 5). Similarly, extract E0 inhibits the expression of IgE receptors (FceRI) according to a dose-dependent effect (figure 6).

3.3 Activation of Toll-like receptors (TLRs)

The TLR activity of ESO was evaluated on TLR2, TLR4 and TLR5 using the model of HEK293 cells cotransfected by the gene for TLR2, TLR4 or TLRS and by the reporter gene NFkB-sAP (secreted alkaline phosphatase). The binding of a ligand to its TLR leads to the activation of the transcription factor NFkB; the sAP gene is placed under the control of a promoter that can bc induccd by NFkB. This reporter gcnc makes it possible to monitor cell signaling via TLRs: the release of sAP induccd by ESO and measured by colorimétrie assay makes it possible to détermine the activity of this active ingrédient as a TLR2, TLR4 or TLRS agonist. C-j

The study was carried out on the following human embryonic kidncy (HEK293) cell lines:

- HEK-Blue™-2 cells for TLR2,

- HEK-Blue™-4 cells for TLR4,

- HEK-Blue™-5 cells for TLR5,

These cell lines were maintained in HEK-Blue™ Sélection 10% FCS culture medium and then distributed in 96-wcll plates in HEK-Bluc™ Détection medium in the presence of ESO for 18 hours. The plates are rcad using calorimetry at 620 nm

3.3.1 Activation of TLR2

Extract ESO induces the activation of TLR2 according to a dosc-dependent effect with a maximum activity at 100 ng/ml (figure 7).

3.3.2 Activation of TLR4

Extract ESO induces lhe activation of TLR4 with a maximum activity at 10 ng/ml (figure 8).

3.3.3 Activation of TLR5

Extract ESO înduces the activation of TLR5 in a dose-dependent manner. This activity is inhibited in the presence of anti-TLR5 antibody, demonstraling the activation specificity of extract ESO on TLR5 (figure 9).

3.4 Inhibition of nrotease-activated receptor 2 (PAR2)

The inhibition of protease-activated receptors by extract ESO is evaluated on human kératinocytes from a cell line (HaCaT) by measuring the intraccllular calcium influx induccd after spécifie stimulation of PAR2 with stratum corncum tryptic enzyme (SCTE). The fluorescent probe Fluo-4/AM is used: its eslerifted form facilitâtes its pénétration by passive diffusion in the cell; only the deesterified form bound to calcium ions is excitable under 485 nm fluorescence and cmits at 535 nm.

The fluorescent probe is incorporated for 30 minutes in cells inoculated in 96well piales and then extract ESO is incubated for 30 minutes. Calcium flow is measured well by well in real time according to kinetics before and after injection of SCTE. The plates are read using a Mithras LB940™ reader (Berthold Technologies®).

Extract ESO inhibits in a dosc-dependent manner activation of PAR2 induced by human SCTE (figure 10). (λί

3.5 Modulation of targets of atopie dermatîtis on kératinocytes

The study was carried out on normal human epidermal kératinocytes (NHEK, KSFM culture medium) in the context of the induction of an atopie dermatîtis phenotype. The activity of ESO was studied on kératinocytes exhibiting an atopie dermatîtis 5 phenotype after stimulation for 24 hours with Poly I:C + IL-4 + IL-13 + TNF-α and analyzcd by PCR array on the expression of a panel of 32 selected gcncs.

On kératinocytes, cxlract ESO inhibited according to a dosc-dcpcndcnt cffcct 15 targets among the mediators involved in atopie dermatîtis pathology, as can be seen clearly in table 7 below (the results indicating for each target gene the percentage of 10 inhibition obtained).

	ESO	Dexaméthasone
10 pg/ml	30 pg/ml	60 pg/ml	2pM
Cytokines	TSLP	56%	75%	92%	91%
IL-la	35%	46%	59%	54%
IL-18	27%	44%	65%	44%
IFN-βΙ	66%	82%	90%	49%
Chemokines	IL-8	37%	55%	88%	75%
MlP-la	10%	43%	75%	76%
RANTES	15%	44%	65%	12%
MCP-3	43%	63%	88%	Pro 20%
TARC	58%	64%	39%	Pro 20%
MIP-3a	41%	61%	80%	40%
MDC	16%	44%	58%	45%
Skinkinc	28%	32%	39%	59%
Receptors	IL-4-R	30%	45%	69%	75%
RARRES3	30%	47%	63%	28%
TLR3	22%	50%	60%	pro 29%

Table 7

3.6 Induction of antimicrobial peptides

The activity of extract ESO on the expression of antimicrobial peptides and proteins is studied on the HaCaT kératinocyte cell line: after 3 hours of treatment in the presence of ESO, the cells arc recovered for an analysis of the expression of antimicrobial targets by quantitative RT-PCR; total RNA are extracted and assayed;£X/

after reverse transcription of mRNA into cDNA, the quantitative PCR amplification step is carried out in 96-wcll plates on an iCycler quantitative PCR systern (Bio-Rad). The results obtained are expressed as the relative quantity (RQ) of mRNA after treatment by ESO in relation to the control without lhe active ingrédient. IL-1β is used in parallel as a reference positive inducer of antimicrobial peptide expression. Expression of the gene of inlcrcst is considcrcd rcgulatcd when RQ>2 (induction) or RQ<0.5 (inhibition),

Extract ESO induces the expression of antimicrobial peptides and proteins hBD2, hBD3, S1007A, LL37, PI3, RNase 7 and NOD2 (figure 11).

Example 4: Formulation of a “body and face” cream comprising bacteria! extract ESO

Extract ESO:

0.1-5%

Evening primrose oil:	1-3%
Glycine:	0.1-0.4%
Ceramides:	0.1-0.3%
Humectants:	5-20%
Emulsifier:	2-7%
Capric/caprylic triglycérides:	1-10%
Preservatives

Water qsp 100%

Example 5: Formulation of a “body and face” cleansing gel comprising bacterial extract ESO

Extract ESO:

Evening primrosc oil: Glycine:

Ceramides:

Surfactants:

Humectants:

Preservatives

Water qsp 100%

0.1-5%

0.5-2%

0.1-0.4%

10-20% in active matter

5-15%

2 JUIN 2013

CABINET CAZENAVE ««ri ✓V Propriété Industrielle

B.P. 500 YAOUNDE , Cameroun

Tél. 22 21 32 89 - Fax: 22 20 64 14

E-mail: cabinetcazenave@iccnet.cm * *

Claims

1. Nonpathogenic Gram-negative bacterium belonging to the class of Betaproleobacteria, subfamily of Neisseriaceae, characterized in that the nucléotide sequence of the 16S rRNA gene of said bacterium includes sequence SEQ ID No, 1,

2. Bacterium according to claim 1, characterized in that it includes at least one

5 plasmid comprising sequence SEQ ID No. 2, or any sequence with at least 80% identity with said sequence SEQ ID No. 2.

3. Bacterium according to claim 1 or claim 2, characterized in that it is nonfilamentous.

4. Bacterium according to any one of claims 1 to 3, deposited with the CNCM 10 on April 8,2010, under the reference 1-4290, or any other mutant.

5. Bacterial exlract obtained from a suspension of bacteria according to any one of claims 1 to 4.

6. Bacterial exlract according (o claim 5, characterized in thaï il is obtained after treatment of said bacterial suspension in such a way as to eliminate the

15 intracellular components.

7. Bacterial extract according to claim 6, characterized in that said intracellular components include at least the nucleic acids.

8. Bacterial exlract according to claim 6 or claim 7, characterized in that it includes a fraction E0 comprising at least membrane proteins, periplasmic proteins and

2 0 proteins arising from the flagellum.

9. Bacterial cxtract according to claim 8, characterized in that said membrane proteins consist of porins, OmpA, lipopolysaccharides and/or lipoproteîns.

10. Bacterial extract according to claim 6 or claim 7, characterized in that it includes a fraction S0 comprising at least sccreted peptides and proteins and secondary

25 métabolites.

11. Bacterial cxtract according to claim 6 or claim 7, characterized in that it includes a fraction ESO comprising at least fraction E0 and fraction S0.

12. Bacterial extract according to claim 11, characterized in that it includes a fraction ESO with a protein profile, obtained by SDS-PAGE, which includes three principal bands corresponding to molecular weights ranging between 30 kDa and 36 kDa, 41 kDa and 45 kDa, and 47 kDa and 51 kDa, respectively.

13. Use of a bacterium according to any one of claims 1 to 4 and/or a bacterial extract according to any one of claims 5 to 9 and 12 for the préparation of a composition intended for the activation of TLR2, TLR4 and TLR5.

14. Use of a bacterium according to any onc of claims 1 to 4 and/or a bacterial cxtract according to any onc of claims 10 or 12 for the préparation of a PAR2 antagonist composition.

15. Use of a bacterium according to any one of claims 1 to 4 and/or a bacterial extract according to any one of claims 5 to 12 for the préparation of a composition intended for the treatment and/or the prévention of dermatological inflammatory disorders.

16. Use according to claim 13, characterized in that said dermatological inflammatory disorders consist of atopie dermatitis, pruritus, eczema and psoriasis.

17. Composition comprising, as an active ingrédient, at least one bacterium according to any one of claims 1 to 4 and/or one bacterial extract according to any one of claims 5 to 12.

18. Composition according to claim 15, for the treatment of dermatological inflammatory disorders.

19. Composition according to claim 18, characterized in that said dermatological inflammatory disorders consist of atopie dermatitis, pruritus, eczema and psoriasis.

20. Cosmetic or dermatological composition according to any one of claims 17 to 19, characterized in that it further includes one or more typical dermatologicallycompatible excipients.