# Patent

## Title
Bacterium and extracts of said bacterium and the use of same in dermatology

## 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.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a Continuation of co-pending application Ser. No. 13/997,029 filed on Jun. 21, 2013, which is a National Phase of PCT International Application No. PCT/EP2011/073747 filed on Dec. 22, 2011, which claims priority under 35 U.S.C. 119(a) to Patent Application No. 1061081 filed in France on Dec. 22, 2010. All of the above applications are hereby expressly incorporated by reference into the present application.


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.
Dermatological diseases such as atopic dermatitis, pruritus, eczema and psoriasis are increasingly frequent in young children. The prevalence of atopic dermatitis has doubled or tripled in developed countries over the past 30 years: 15% to 30% of children and 2% to 10% of adults are affected (Williams H. et al., JACI 2006; 118:209-13). Atopic dermatitis is the cutaneous manifestation of atopy; it is a chronic inflammatory dermatosis or eczema, occurring due to a genetically determined set of circumstances. It is now regarded as a major public health concern. Atopic dermatitis is often associated with other atopic disorders such as allergic rhinitis and asthma. This affection most often appears during early childhood and is characterized by repeated outbreaks over several years. It progresses with flare-ups interrupted by spontaneous remissions.
The quality of life for patients suffering from atopic dermatitis is profoundly disturbed. Accepted treatments include topical corticosteroids and immunomodulators, systemic agents whose frequent side effects limit long-term use, and emollients. Current therapies 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 beneficial in terms of both control of the disease and the potential appearance of asthma and/or rhinitis (Bieber, T. 2008, Atopic dermatitis, The New England Journal of Medicine, vol. 358(14) 1483-1494), as atopic dermatitis is regarded as the initial phase of atopic progression. In most cases, treatments include a local component in order to best provide relief to patients.
Standard treatments for atopic dermatitis notably use topical corticosteroids or immunosuppressants, although such treatments are not free of adverse effects in children in particular.
Atopic dermatitis is complex and multifactorial. In the literature, some epidemiological studies have shown that the “hygiene” factor in urban environments promote the disease like allergy and autoimmunity. On the other hand, in rural settings where man is in constant contact with microorganisms and/or allergens, such exposure stimulates man's defensive immune system from birth.
In atopic dermatitis, the barrier function of the skin is weakened and impaired, which promotes 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 allergic manifestation (IgE mediated, dominance of cytokines IL-4, IL-5, IL-13) or Th2 response. The latter is all the more accentuated in the presence of “antigenic stimuli” of Staphylococcus aureus. Immunomodulation consists in returning immune homeostasis to a Th1/Th2 balance.
Innate immunity is the primary, rapid and nonspecific 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 specific interaction between a motif (agonist) and a TLR triggers a cascade of complex reactions resulting in the transcription of NFκB, followed by production of pro-inflammatory and anti-inflammatory cytokines and of chemokines (Kang et al., 2006). Other resulting pharmacological consequences are the induction of antimicrobial peptides (AMPs), which have the ability to inhibit the growth of pathogens (bacteria, viruses, parasites) (Glaser, R. et al. 2005, Nat. Immunol. 6:57-64).
Atopic dermatitis is often accompanied by itching and pruritus, thus causing discomfort and annoyance in daily life (scratching, sleep loss, etc.). One of the causes of this inflammatory pathology is due to the activation of a G protein-coupled receptor 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 keratinocytes, endothelial cells, colonic myocytes, enterocytes, enteric neurons and immune cells. Proteases (trypsin, tryptase), present in abundance in the epidermis, cleave the PAR2 at the N-terminal exposing a specific peptide which activates this same receptor (phenomenon of self-activation) (Vergnolle, N. 2009 Pharmacol. Ther. 123:292-309). This process involves activation of the NFκB gene, followed by the induction of pro-inflammatory cytokines, thus triggering inflammation. In this context, the development of PAR2 antagonists and/or protease inhibitors has a high potential 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 atopic 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 Th1 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 proliferation of keratinocytes which then proceed through an accelerated and incomplete differentiation phase. Keratinocytes express receptors which make them sensitive to inflammatory signals and release pro-inflammatory mediators. Psoriatic inflammation is thus maintained by mutual stimulation of T cells and keratinocytes.
The disease must therefore be treated over the long term. There is thus a need and a high demand for therapeutic alternatives for these 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. filiformis) for the treatment of atopic dermatitis. Such an extract has the disadvantage of requiring the culture of said filamentous bacterium V. filiformis on a medium containing sulfur-free mineral water.
In this context, the present invention provides a solution to the treatment of these inflammatory disorders by the isolation, the characterization and the fractionation of a novel bacterium 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 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 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 ribosomal RNA (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 nonpathogenic bacterium is Gram-negative and will be described in greater detail in the examples. 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 example, in contrast to V. filiformis, the culture of bacterium LMB64 of the present invention does not require particular culture conditions and, more particularly, does not require a medium containing at least one sulfur-free type of mineral and/or thermal water. This represents a clear advantage in terms of both culture conditions and facilities and from an economic 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 present invention relates to a nonpathogenic Gram-negative 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.
In a preferred manner, the present invention relates to a nonpathogenic Gram-negative 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.
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, Wis., 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 aL, “Blast 2 sequences—a new tool for comparing protein and nucleotide sequences,” FEMS Microbiol Lett. 174:247-250), available at http://wwvv.ncbi.nlm.nih.gov/gorf/b12.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 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 present 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 Apr. 8, 2010, under the reference I-4290.
Thus, one object of the invention is the bacterium deposited with the CNCM on Apr. 8, 2010, under the reference I-4290, or a homologue, a descendant or any other mutant.
The term “mutant” refers to any bacterium directly arising from strain I-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, 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 I-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.
According to another preferred embodiment, the invention relates in a general manner to a bacterial extract, also called 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 extract. For example, such an extract may be obtained from a culture of bacterium LMB64 wherein the preparation method comprises at least one step of lysis of the bacteria and one step of separation of the various fractions of which it is constituted by centrifugation or by filtration.
In a nonrestrictive manner, the extract according to the invention may consist of bacterial cells isolated from the culture medium which have been concentrated, for example by centrifugation; or concentrated bacterial cells which have undergone an operation in which the cell envelope 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 preparation method according to the invention consists of the elimination 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-β-hydroxybutyrate, 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 result 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 elimination of intracellular compounds, and as a nonrestrictive 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 (hereafter fraction S0) and the components constituting the pellet (hereafter E0). For example, it may be suggested that the separation threshold between the constituents of S0 and E0 is around a molecular weight of 100 kDa. Consequently, the constituents of fraction S0 have, for the most part, a molecular weight less than 100 kDa, whereas the components of fraction E0 have, for the most part, a molecular weight greater than 100 kDa.
More particularly, it is thus possible by techniques known to those persons skilled in the art to extract and separate the biomolecules found in the culture supernatant (S0) from those mainly comprised of surface proteins and proteins located in the periplasmic space of the bacterium (E0).
According to one embodiment of the invention, the bacterial extract includes a fraction E0 comprising at least membrane proteins, periplasmic proteins and proteins arising from the flagellum.
Periplasmic proteins include proteins lodged in the periplasmic space of Gram-negative 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, 3rd edition: 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 separations (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 which a part is exposed on the surface (outer membrane proteins, or Omp), proteins that are adhered to the surface of the membrane, lipoproteins and porins (Ward J B., 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 S0.
More particularly, the bacterial extract according to the invention includes a fraction S0 comprising at least secreted peptides and proteins and secondary metabolites.
Secreted peptides and proteins include peptides and proteins that are naturally produced and secreted by bacterium LMB64 and which may be recovered by centrifugation or by filtration.
Secondary metabolites include the small molecules that bacterium LMB64 produces and secretes in the culture medium.
The presence of lipopolysaccharides within fraction S0 should be mentioned here. Indeed, lipopolysaccharides, although they are found primarily in fraction E0, are nevertheless also found in smaller quantities in fraction S0.
In an advantageous manner, fractions E0 and S0 may be combined in such a way as to obtain a fraction ES0 by leaving, for example, the culture medium to incubate and to react in basic medium (pH 9 to 11) for approximately 5 hours a temperature of 4° C., by centrifuging and by filtering at 0.2 μm in order to obtain a clear ES0 solution.
Bacterial extract ES0 is thus composed, among other things, of membrane proteins, lipopolysaccharides, periplasmic proteins, protein fragments of the flagellum and primary and secondary metabolites produced by the bacterium.
In a preferred way, extract ES0 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 Laboratories) ranging 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 extract includes a fraction ES0 comprising at least fraction E0 and fraction S0.
According to a preferred embodiment of the invention, the bacterial extract includes a fraction ES0 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 ES0 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, the invention describes a method for preparing a bacterial extract comprising the steps of:

    
    
        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 S0, wherein said method comprises the steps of:

    
    
        a) culturing bacterium LMB64 in a suitable medium;
        b) centrifuging said culture; and
        c) recovering supernatant S0.
    
    


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 supernatant;
        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 supernatant.
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 supernatant;
        c) treating with ultrasound the biomass resulting from step b);
        d) centrifuging said biomass treated with ultrasound and eliminating the biomass obtained;
        e) centrifuging the supernatant resulting from step d); and
        f) recovering the base E0.
    
    


It 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 used.
As will become apparent from the examples below, the Applicant has demonstrated, in addition to the activities expected for this type of extract, several novel activities never before described.
A first advantageous aspect of the invention, related to immunomodulation, rests on the modulation property of pro-inflammatory cytokines. More particularly, the use of a bacterium and/or an extract according to the invention significantly induces cytokines IL-10, IL-12 and TNF-α, which are preferentially involved in the Th1 immune response, and significantly inhibits cytokines IL-4 and IL-6. The result is the activation of Langerhans cells and a return to Th1/Th2 balance.
Furthermore, another observation demonstrated that the use of a bacterium and/or an extract according to the invention makes it possible to greatly decrease the expression of IgE receptors, which is of interest in that IgE potentiates allergic 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 and LL-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, due to the presence of OmpA, and on TLR4, due to the presence 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 describes a bacterial extract 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 extract comprising all or part of the proteins arising from the flagellum. In this case, as an example, said extract is preferentially extract E0 or extract ES0.
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 al., 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 and/or the extracts according to the invention.
Furthermore, in an unexpected manner, the Applicant has also demonstrated, in contrast 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 extract consists of extract S0 or extract ES0.
PAR2 is overexpressed in endothelial cells, colonic myocytes, enterocytes, enteric neurons, immune cells and keratinocytes. Proteases (trypsin, tryptase) present in abundance in the environment cleave the PAR2 at the N-terminal exposing a specific peptide which activates this same receptor (phenomenon of auto-activation). Consequently, this activates 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 appear in the KO mouse (PAR2 deficient). Treatment with an antiprotease and/or a PAR2 antagonist makes it possible to avoid this inflammation phenomenon.
The combination and the synergy of all 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 weakened, disturbed or unbalanced.
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 preparation of a composition intended for the treatment and/or the prevention of dermatological inflammatory disorders.
In a preferred manner, said dermatological inflammatory disorders consist of atopic dermatitis, pruritus, eczema and psoriasis.
According to another embodiment, the invention of the present patent application relates to a composition comprising, as an active ingredient, 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 atopic 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) emulsion, a multiple emulsion such as, for example, a water-in-oil-in-water (W/O/W) or oil-in-water-in-oil (O/W/O) emulsion, a microemulsion or in the form of a hydrodispersion or a lipodispersion, a gel or an aerosol.
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 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 antibodies 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.
The invention will be better understood upon consideration of the examples below which illustrate the invention without limiting its scope.



DESCRIPTION OF FIGURES
FIG. 1 illustrates the phylogenetic position of the sequence coding for the 16S rRNA of strain LMB64. The sequences appearing on this tree are sequences from the GenBank database closest to the sequence of LMB64.
FIGS. 2A and 2B present images of bacterium LMB64 under the transmission electron microscope (A) and the scanning electron microscope (B).
FIG. 3 presents growth optima determined as a function of the temperature, pH and salinity of the R3 culture medium.
FIG. 4 illustrates induction of cytokines IL-10 and IL-12 by extract E0 (dose-dependent effect).
FIG. 5 illustrates induction of surface molecules CD80, CD86, CD83 and CD54 by extract E0 (dose-dependent effect).
FIG. 6 illustrates inhibition of IgE receptors by extract E0.
FIG. 7 illustrates activation of TLR2 by extract ES0.
FIG. 8 illustrates activation of TLR4 by extract ES0.
FIG. 9 illustrates activation of TLR5 by extract ES0.
FIG. 10 illustrates specific PAR2 antagonist activity by extract ES0.
FIG. 11 illustrates induction of antimicrobial peptides and proteins by extract ES0.
FIG. 12 consists of an SDS-PAGE gel of extract ES0.



EXAMPLE 1
Selection and Characterization of Bacterium LMB64
Bacterium AV13 was isolated from groundwater.
The taxonomic position of novel bacterium LMB64 is proposed in FIG. 1.
More particularly, bacterium LMB64 is rod-shaped with a length of roughly 2.3 μm (±0.3) and a width of roughly 1.0 μm (±0.1). A distinctive characteristic of this bacterium is the presence of a polar flagellum (FIGS. 2A and 2B). 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 kpb. This plasmid was completely sequenced (SEQ ID No. 2).
The gene 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 1a, 1b and 1c, respectively.







TABLE 1a





COMPOSITION OF R3 MEDIUM


















Yeast extract
1 g/l



Difco proteose peptone
1 g/l



Casamino acids
1 g/l



Glucose
1 g/l



Soluble starch
1 g/l



Sodium pyruvate
0.5 g/l 



K2HPO4
0.6 g/l 



MgSO4, 7H2O
0.1 g/l 

















TABLE 1b





COMPOSITION OF MS-GLUCOSE MEDIUM



















Glucose
6.0
g/l



Citric acid
0.84
g/l



MgSO4, 7H2O
0.25
g/l



NH4Cl
1.06
g/l



Anhydrous K2HPO4
8.75
g/l



Pyruvic acid sodium salt
0.5
g/l



Zinc sulfate, 7H2O
4
mg/l



Cobalt chloride, 6H2O
3.5
mg/l



Sodium molybdate, 2H2O
3.5
mg/l



Manganese sulfate, 1H2O
5
mg/l



Boric acid
2
mg/l



Concentrated hydrochloric acid
50
mg/l



Copper sulfate, 5H2O
4
mg/l



Iron chloride, 6H2O
27
mg/l

















TABLE 1c





COMPOSITION OF LB MEDIA


















Tryptone
10 g/l 



Yeast extract
5 g/l



NaCl
5 g/l










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







TABLE 2





Growth rate (/h)


















LB
0.25 (±0.05)



LB (1⁄2 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)










The growth optima were determined as a function of the temperature, pH and salinity of the R3 culture medium (FIG. 3).
The sources of carbon assimilable by the bacterium were characterized using an API 50CH gallery (incubation temperature: 25° C.). The results are summarized in table 3 below.










TABLE 3









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-β-D-xylopyranoside



10. D-galactose



11. D-glucose
+
+



12. D-fructose
+
+



13. D-mannose



14. L-sorbose



15. L-rhamnose



16. Dulcitol



17. Inositol
l
+



18. D-mannitol



19. D-sorbitol



20. Methyl-α-D-mannopyranoside



21. Methyl-α-D-glucopyranoside



22. N-acetylglucosamine



23. Amygdaline



24. Arbutin



25. Esculin/iron citrate



26. Salicin



27. D-cellobiose



28. D-maltose
l
+



29. D-lactose (bovine origin)



30. D-melibiose



31. D-sucrose
+
+



32. D-trehalose
l
+



33. Inulin



34. D-melezitose



35. D-raffinose



36. Starch



37. Glycogen



38. Xylitol



39. Gentiobiose



40. D-turanose
l
+



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-ketogluconate







+: usable substrate,



l: low use






The enzymatic activities demonstrated on the API ZYM gallery are: alkaline phosphatase, esterase (C4), esterase/lipase (C8), leucine arylamidase, valine arylamidase, acid phosphatase, naphthol-AS-BI-phosphohydrolase, and α-glucosidase.
Bacterium LMB64 is sensitive to all the antibiotics tested as seen in table 4 below.










TABLE 4









Zone of inhibition diameter (mm)
Inhibitory











Antibiotics tested
R3
LB 1⁄2
LB 1⁄5
activity





Ampicillin (10 μg)
29
28
29
+


Chloramphenicol (30 μg)
29
26
24
+


Ciprofloxacin (5 μg)
38
34
34
+


Kanamycin (30 μg)
27
30
27
+


Penicillin (6 μg)
21
26
20
+


Polymyxin B (50 μg)
11
15
13
+


Rifampicin (30 μg)
20
19
15
+


Tetracycline (30 μg)
30
25
20
+


Streptomycin (10 μg)
25
25
24
+


Vancomycin (30 μg)
20
21
21
+









EXAMPLE 2
Method for Extracting Fractions E0, S0 and ES0
Preculture: Strain AV13 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 OD600≈1.5 is obtained.







TABLE 5







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 salt
0.5
g



Oligo mix
1
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 of distilled water:











Zinc sulfate, 7H2O
4
g



Cobalt chloride, 6H2O
3.5
g



Sodium molybdate, 2H2O
3.5
g



Manganese sulfate, 1H2O
5
g



Boric acid
2
g



Concentrated hydrochloric acid
50
g



Copper sulfate, 5H2O
4
g









Dissolve in 50 ml of distilled water:











Iron chloride, 6H2O
27
g



ddH2O qsp
1000
ml










Culture: The preculture is then inoculated in a fermentor (Applikon) containing 3.71 of MS pyruvate medium+114 ml of 20% glucose solution. A temperature sensor regulates the temperature 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-rate pump. A Wedgewood Analytical sensor is used to monitor changes in optical density in real time. The culture is programmed in fed-batch mode; via a variable flow-rate pump the culture is supplied with 20% glucose solution. Fermentation is stopped when OD600≈22-26, in general after roughly 30 hours.
Extraction S0: The supernatant is separated from the biomass by centrifugation for 1 hour at 4° C. and 4000 g.
Extraction E0: The wet biomass is taken up in NaCl solution (1 M). After centrifugation for 15 minutes at 4° C. and 9000 g, the supernatant is discarded and the pellet is taken up in 1 M NaCl 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 supernatant is recovered. Two volumes of cold ethanol are added and the suspension is left overnight at 4° C. After centrifugation for 30 minutes at 4° C. and 6000 g, the supernatant 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 next step is incubation under stirring for 5 hours at a temperature of 4° C. After centrifugation, the supernatant is prefiltered to eliminate remaining biomass debris and then filtered on a 0.2 μm filter. A clear yellow solution is obtained (ES0).
Proteins are assayed according to the DC Protein Assay Kit II (Bio-Rad) protocol. Sugars are assayed in glucose equivalent according to the phenol/sulfuric acid method (Dubois, M. et al., 1956).
As an example, table 6 below presents certain specific characteristics of extract ES0 as obtained under the conditions described above.










TABLE 6







Test batch
Preclinical batch 1

















Organoleptic
Homogeneous and translucent yellow-orange liquid


characteristics
Density near that of water









pH
10.0
10.2


(in the presence of


base buffer)


Dry residue
5.9%
5.1%


(thermobalance)








Protein profile
12 detectable bands (including 3 principal bands


(SDS-PAGE)
roughly 34 kDa, 43 kDa and 49 kDa in size,



respectively)









Total protein assay
2.9 mg/ml
3.0 mg/ml


(μBCA)









It is clearly understood that the data above are presented here only for illustrative purposes.
More precisely, the data relate to a protein profile obtained by SDS-PAGE exhibiting three principal bands.
SDS-PAGE Protocol:
Extract ES0 is taken up in buffer (20 mM Tris-HCl, pH 8.0; 1 mM EDTA; 2.5% SDS and 0.01% bromophenol blue) and 1 M DTT (1,4-dithiothreitol). The sample and the mixture of molecular weight markers (WesternC, Bio-Rad) were deposited respectively in wells of an 8-16% SDS-PAGE acrylamide gel (GeBaGel, Gene Bio-Application). The migration buffer contains 2.5 mM Tris, 19.2 mM glycine and 0.01% SDS (w/v). Migration is allowed to proceed 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 FIG. 12.
According to one embodiment of the invention, these three bands have molecular weights of approximately 34 kDa, 43 kDa and 49 kDa, respectively.
EXAMPLE 3
Demonstration of the Pharmacological Activities of Fractions E0 and ES0
Langerhans cells (LC) are generated in vitro from human monocytes isolated from Buffy-Coat pouches from the French National Blood Service (Etablissement Français du Sang (EFS) Pyrénées Méditerranée): isolation on a Ficoll gradient (Lymphocyte Separation Medium, density 1.077 g/ml) and purification by magnetic immunoselection (Miltenyi Biotec); LC differentiation is carried out for 6 days in the presence of a cytokine cocktail (GM-CSF/IL-4/TGFβ). LC distributed on 24-well plates in RPMI-5% FCS culture medium are incubated for 24 hours with extract ES0.
Surface molecules are analyzed by flow cytometry (FACSCalibur, BD Biosciences) with triple or quadruple staining: CD1a/CD54/CD80/CD83/CD86/FcεRI; cytokines secreted in the culture supernatants are analyzed with the Cytometry Bead Array (cat. no. 550749, BD) in flow cytometry: IL-6, IL-8, TNF, IL-4, IL-10, IL-12.
3.1 Induction of Key Cytokines for Th1 Polarization
Extract E0 induces according to a dose-dependent effect the expression of cytokines IL-10 and IL-12 by Langerhans cells (FIG. 4). These cytokines promote the induction of TH1 polarity of naive T lymphocytes.
3.2 Langerhans Cell Maturation and IgE Receptor (FcεRI) Inhibition
Extract E0 induces the maturation of Langerhans cells observed by dose-dependent induction of surface molecules CD80, CD86, CD83 and CD54 (FIG. 5). Similarly, extract E0 inhibits the expression of IgE receptors (FcεRI) according to a dose-dependent effect (FIG. 6).
3.3 Activation of Toll-Like Receptors (TLRs)
The TLR activity of ES0 was evaluated on TLR2, TLR4 and TLR5 using the model of HEK293 cells cotransfected by the gene for TLR2, TLR4 or TLR5 and by the reporter gene NFκB-sAP (secreted alkaline phosphatase). The binding of a ligand to its TLR leads to the activation of the transcription factor NFκB; the sAP gene is placed under the control of a promoter that can be induced by NFκB. This reporter gene makes it possible to monitor cell signaling via TLRs: the release of sAP induced by ES0 and measured by colorimetric assay makes it possible to determine the activity of this active ingredient as a TLR2, TLR4 or TLR5 agonist.
The study was carried out on the following human embryonic kidney (HEK293) cell lines:

    
    
        HEK-BlueTM-2 cells for TLR2,
        HEK-BlueTM-4 cells for TLR4,
        HEK-BlueTM-5 cells for TLR5,
    
    


These cell lines were maintained in HEK-BlueTM Selection 10% FCS culture medium and then distributed in 96-well plates in HEK-BlueTM Detection medium in the presence of ES0 for 18 hours. The plates are read using calorimetry at 620 nm
3.3.1 Activation of TLR2
Extract ES0 induces the activation of TLR2 according to a dose-dependent effect with a maximum activity at 100 ng/ml (FIG. 7).
3.3.2 Activation of TLR4
Extract ES0 induces the activation of TLR4 with a maximum activity at 10 ng/ml (FIG. 8).
3.3.3 Activation of TLR5
Extract ES0 induces the activation of TLR5 in a dose-dependent manner. This activity is inhibited in the presence of anti-TLR5 antibody, demonstrating the activation specificity of extract ES0 on TLR5 (FIG. 9).
3.4 Inhibition of Protease-activated Receptor 2 (PAR2)
The inhibition of protease-activated receptors by extract ES0 is evaluated on human keratinocytes from a cell line (HaCaT) by measuring the intracellular calcium influx induced after specific stimulation of PAR2 with stratum corneum tryptic enzyme (SCTE). The fluorescent probe Fluo-4/AM is used: its esterified form facilitates its penetration by passive diffusion in the cell; only the deesterified form bound to calcium ions is excitable under 485 nm fluorescence and emits at 535 nm.
The fluorescent probe is incorporated for 30 minutes in cells inoculated in 96-well plates and then extract ES0 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 LB940TM reader (Berthold Technologies®).
Extract ES0 inhibits in a dose-dependent manner activation of PAR2 induced by human SCTE (FIG. 10).
3.5 Modulation of Targets of Atopic Dermatitis on Keratinocytes
The study was carried out on normal human epidermal keratinocytes (NHEK, K-SFM culture medium) in the context of the induction of an atopic dermatitis phenotype. The activity of ES0 was studied on keratinocytes exhibiting an atopic dermatitis phenotype after stimulation for 24 hours with Poly I:C+IL-4+IL-13+TNF-α and analyzed by PCR array on the expression of a panel of 32 selected genes.
On keratinocytes, extract ES0 inhibited according to a dose-dependent effect 15 targets among the mediators involved in atopic dermatitis pathology, as can be seen clearly in table 7 below (the results indicating for each target gene the percentage of inhibition obtained).










TABLE 7









ES0
Dexamethasone












10 μg/ml
30 μg/ml
60 μg/ml
2 μM
















Cyto-
TSLP
56%
75%
92%
91%


kines
IL-1α
35%
46%
59%
54%



IL-18
27%
44%
65%
44%



IFN-β1
66%
82%
90%
49%


Chemo-
IL-8
37%
55%
88%
75%


kines
MlP-1α
10%
43%
75%
76%



RANTES
15%
44%
65%
12%



MCP-3
43%
63%
88%
Pro 20%



TARC
58%
64%
39%
Pro 20%



MIP-3α
41%
61%
80%
40%



MDC
16%
44%
58%
45%



Skinkine
28%
32%
39%
59%


Recep-
IL-4-R
30%
45%
69%
75%


tors
RARRES3
30%
47%
63%
28%



TLR3
22%
50%
60%
pro 29%









3.6 Induction of Antimicrobial Peptides
The activity of extract ES0 on the expression of antimicrobial peptides and proteins is studied on the HaCaT keratinocyte cell line: after 3 hours of treatment in the presence of ES0, the cells are recovered for an analysis of the expression of antimicrobial targets by quantitative RT-PCR; total RNA are extracted and assayed; after reverse transcription of mRNA into cDNA, the quantitative PCR amplification step is carried out in 96-well plates on an iCycler quantitative PCR system (Bio-Rad). The results obtained are expressed as the relative quantity (RQ) of mRNA after treatment by ES0 in relation to the control without the active ingredient. IL-1β is used in parallel as a reference positive inducer of antimicrobial peptide expression. Expression of the gene of interest is considered regulated when RQ≧2 (induction) or RQ≦0.5 (inhibition).
Extract ES0 induces the expression of antimicrobial peptides and proteins hBD2, hBD3, S1007A, LL37, P13, RNase 7 and NOD2 (FIG. 11).
EXAMPLE 4
Formulation of a “Body and Face” Cream Comprising Bacterial Extract ES0
Extract ES0: 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 triglycerides: 1-10%
Preservatives
Water qsp 100%
EXAMPLE 5
Formulation of a “Body and Face” Cleansing Gel Comprising Bacterial Extract ES0
Extract ES0: 0.1-5%
Evening primrose oil: 0.5-2%
Glycine: 0.1-0.4%
Ceramides: 0.1-0.4%
Surfactants: 10-20% in active matter
Humectants: 5-15%
Preservatives
Water qsp 100%

## Claims

1. A cosmetic or dermatological composition comprising effective amounts of a fractionated bacterial extract and a preservative,
wherein the fractionated bacterial extract is obtained froma Gram-negative non-filamentous bacterium belonging to the class of Betaproteobacteria, subfamily of Neisseriaceae which nucleotide sequence of the 16S rRNA gene comrprises SEQ ID NO: 1,
and wherein the fractionated bacterial extract is prepared by a method comprising incubating and reacting said bacterium in a basic medium of pH 9 to 11 for a suitable time, and removing intracellular components of the bacterium therefrom.

2. The composition according to claim 1, wherein said bacterium comprises at least one plasmid comprising SEQ ID NO: 2, or any sequence with at least 80% identity with said SEQ ID NO: 2.

3. The composition according to claim 1 wherein said bacterium has been deposited under deposit number 1-4290 in the Collection Nationale de Cultures de Microorganisms (CNCM).

4. The composition according to claim 1, wherein said intracellular components comprise nucleic acids.

5. The composition according to claim 1 wherein said fractionated extract comprises secreted peptides, secreted proteins and secondary metabolites.

6. The composition according to claim 1 wherein said fractionated extract comprises membrane proteins, periplasmic proteins, proteins arising from the flagellum, secreted peptides, secreted proteins and secondary metabolites.

7. The composition according to claim 1, wherein said fractionated extract comprises 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.

8. The composition according to claim 1 further comprising and one or more typical dermatologically-compatible excipients.

9. The composition according to claim 1 wherein said fractionated extract comprises membrane proteins, periplasmic proteins, and proteins arising from the flagellum.

10. The composition according to claim 9, wherein said membrane proteins are selected from porins, OmpA, lipopolysaccharides and lipoproteins.