WO2001087326A1

WO2001087326A1 - Use of an enterobacterium ompa protein as antimicrobial agent

Info

Publication number: WO2001087326A1
Application number: PCT/FR2001/001490
Authority: WO
Inventors: Pascale Jeannin; Yves Delneste; Thierry Baussant
Original assignee: Pierre Fabre Medicament
Priority date: 2000-05-16
Filing date: 2001-05-16
Publication date: 2001-11-22
Also published as: AU6242301A; FR2809014A1

Abstract

The invention concerns the use of an enterobacterium OmpA protein or one of its fragments, in particular of Klebsiella pneumoniae, as antimicrobial agent or for preparing an antimicrobial pharmaceutical composition for mucosal delivery. The invention further concerns said compositions, preferably antigen-free, and a device adapted for mucosal delivery characterised in that it contains the inventive composition.

Description

USE OF AN ENTEROMACTERY OmpA PROTEIN AS ANTIMICROBIAL AGENT

The invention relates to the use of an enterobacterium protein OmpA or one of its fragments, in particular of Klebsiella pneumoniae, as an antimicrobial agent or for the preparation of an antimicrobial pharmaceutical composition intended for administration. ) mucosally. The invention further relates to the compositions thus prepared, preferably free of antigen, as well as to a device suitable for administration by mucosal route, characterized in that it contains a composition according to the invention.

Many vaccine candidates composed of ribosomes or ribosomal RNA and proteoglycans of Gram-negative bacteria have already been described as an adjuvant of immunity or as an immunogenic agent for the prophylactic treatment of infection. Among these documents, we can cite European patent applications EP 0 238 407 and EP 0 035 429 as well as French patent applications FR 78/35649 and FR 73/46957 which mention that the strains from which the ribosomal fractions constituting these vaccines correspond to the strains responsible for the infection to be treated, these ribosomes or ribosomal RNA constituting the antigenic fraction of these vaccines.

Among these documents, there may be mentioned in particular French patent application FR 78/35649 which describes a vaccine composition for the prophylactic treatment of bronchial infections and of the ENT sphere comprising proteoglycans of Klebsiella pneumoniae and ribosomal RNAs of Klebsiella pneumoniae, Streptococcus pneumoniae, from Streptococcus pyogenes and aemophillus influenzae, such as the vaccine composition called D53. We can also cite the French patent application published under the number

FR 2 785 542 which describes in particular that the OmpA of K. pneumoniae binds to certain cells presenting antigens such as monocytes.

One can also cite the international patent application published under the number WO 96/14415 which relates to the use of the OmpA of K. pneumoniae called P40 at high concentrations as an adjuvant (cf. FIGS. 1A and IB of the document) . Finally, mention may be made of the international patent application published under the number WO 00/54790 describing, in particular in FIGS. 3 and 4 of this document, the production of TNF alpha and of IL-12 p70 by blood monocytes in the presence of l 'OmpA P40 from K. pneumoniae. This document indicates in particular (cf. FIG. 3) that at concentrations below 1.8 μg / ml of rP40 (recombinant P40), there is no production of TNF alpha.

The immune system and in particular the cells of innate immunity have adapted in order to recognize molecules expressed by groups of pathogens. These recognized molecules have among others the following characteristics: to be expressed in a significant way by a set of pathogens and not to be similar to components of the self. The binding of these molecules to cells of innate immunity generally results in the activation of these cells. When a microbial agent is introduced into the body, cells of innate immunity such as neutrophils, macrophages and NK lymphocytes (NK for "Natural Killer") are activated and produce proinflammatory mediators and chemokines which will promote the migration and activation of leukocytes at the site where the pathogen is located.

In addition, neutrophils and macrophages have phagocytic properties and will directly participate in the elimination of the pathogen by phagocytosis and destroy it. The phagocytic cells and in particular the macrophages present locally will therefore play an essential role in eliminating the pathogen and locally attracting other immune cells.

Besides an antipathogenic role by itself, the inflammatory response mediated by cells of innate immunity will contribute to activate the antigen presenting cells (CPAg) and by the same to induce the development of a specific response by lymphocytes. B and T.

Among the cytokines produced by macrophages which will participate in the development of a defense response of the organism against the pathogenic agent, mention may be made of proinflammatory mediators such as for example interleukin 1 (IL-1 ), the tumor necrosis factor TNFα (TNF for “Tumor Necrosis Factor”), nitric oxide (NO), 1TL-12 or chemokines. It has also been found that other cells present locally (at the level of the pathogen), for example epithelial cells (such as lung epithelium cells), or neutrophils also produce cytokines, such as those mentioned above and will thus also cause an antipathogenic effect.

LTL-1 and TNFα have a direct antipathogenic effect by activating neutrophils and macrophages and an indirect effect by promoting the expression by many cells of adhesion molecules which will promote the recruitment of leukocytes. In addition, these proinflammatory cytokines contribute to the activation of CPAg and to the development of the specific response which will follow the inflammatory response.

NO also modulates the migration of leukocytes as well as their activation, increases the phagocytic capacities of neutrophils and macrophages, and also has a powerful and direct antipathogenic role. IL-12 is produced mainly by CPAg, and in particular by macrophages. It induces the production of gamma interferon (IFNγ) and contributes to the activation of T lymphocytes and NK cells. In this way, IL-12 plays a crucial role in the development of a non-specific (dependent on NK cells) and specific (dependent on T lymphocytes) lymphocyte response. Thus, there is a need to have compounds which bind to the macrophages and induce their activation. Activated macrophages produce IL-1, TNFα, NO and IL-12. These compounds therefore activate cells of innate immunity and in particular macrophages and, through this, play an essential role in promoting and / or increasing the body's response against pathogens. Surprisingly, the inventors have demonstrated that a protein

Enterobacterium OmpA, in particular the protein OmpA of Klebsiella pneumoniae, is capable at low concentration of binding to macrophages and of inducing the secretion of IL-1, TNFα, NO and IL-12, and thus being used as an antimicrobial agent.

The present invention therefore relates to the use of an enterobacterium protein OmpA or a fragment thereof as an antimicrobial agent or for the preparation of an antimicrobial pharmaceutical composition intended to be administered by mucosal route. The main object of the present invention is thus the use of an enterobacterium protein OmpA, one of its fragments or one of its derived proteins, as an antimicrobial agent or for the preparation of an antimicrobial pharmaceutical composition. In a preferred embodiment, said enterobacterium OmpA is used at concentrations of between 0.08 μM and 1 μM (ie a concentration of 1 μM being approximately equivalent to 0.04 μg / μl for TOmpA P40 of Klebsiella pneumoniae) , preferably between 0.04 μM and 1 μM, and more preferably between 0.2 μM and 1 μM. The inventors have indeed shown in a completely surprising manner that an enterobacterium OmpA protein, in particular the OmpA protein of Klebsiella pneumoniae, exhibits antimicrobial activity even when the latter is used at such low concentrations, which has many advantages such as in particular that it can be easily used for treatment in humans, while making the cost of treatment affordable in view of the production costs of the recombinant proteins.

In the present invention, the term “protein” will also be understood to denote the peptides or polypeptides and the term “OmpA” (for “Outer Membrane Protein”), the proteins of the outer membrane of type A. By fragment of an OmpA protein is intended to denote in particular any fragment of amino acid sequence included in the amino acid sequence of the OmpA protein from which it is derived, and which is capable of generating or increasing an antimicrobial response, said fragment of the OmpA protein comprising at least 5 amino acids, preferably at least 10 amino acids or more preferably at least 15, 20, 25, 30, 40, 50, 75 and 100 consecutive amino acids of the sequence of said OmpA protein of which it is from.

The use as an antimicrobial agent or for the preparation of an antimicrobial pharmaceutical composition of proteins derived from said OmpA protein, the amino acid sequence of which has homology, as defined below, of at least 80%, preferably 85%, 90%, 95% and 99%, after optimal alignment with the sequence of the reference OmpA protein and which are capable of generating or enhancing an antimicrobial response is also included in the present invention.

By nucleic acid or amino acid sequence having a homology of at least 80% after optimal alignment with a determined nucleic acid or amino acid sequence, is meant a sequence which after optimal alignment with said determined sequence includes a percentage identity of at least 80% with said determined sequence.

By “percentage of identity” between two nucleic acid or amino acid sequences within the meaning of the present invention is meant a percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly and over their entire length. Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by "comparison window" to identify and compare the regions. sequence similarity locale. The optimal alignment of the sequences for comparison can be achieved, besides manually, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math. 2: 482], using the local homology algorithm of Neddleman and Wunsch (1970) [J. Mol. Biol. 48: 443], using the similarity search method of Pearson and Lipman (1988) [Proc. Natl. Acad. Sci. USA 85: 2444], using computer software using these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI, or by BLAST comparison software N or BLAST P).

The percentage of identity between two nucleic acid or amino acid sequences is determined by comparing these two optimally aligned sequences per comparison window in which the region of the nucleic acid or amino acid sequence to be compared. may include additions or deletions with respect to the reference sequence for optimal alignment between these two sequences. The percentage of identity is calculated by determining the number of identical positions for which the nucleotide or the amino acid residue 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 of identity between these two sequences.

For example, we could use the BLAST program, "BLAST 2 sequences", available on the site http://www.ncbi.nlm.nih.gov/gorf/bl2.html, the parameters used being those given by default (in particular for the parameters “open gap penaltie”: 5, and “extension gap penaltie”: 2; the chosen matrix being for example the “BLOSUM 62” matrix proposed by the program), the percentage of identity between the two sequences to be compared being calculated directly by the program. More preferably, by fragment or protein derived from OmpA protein capable of generating or increasing an antimicrobial response, is meant in particular a fragment or protein derived from OmpA protein capable of inducing or increasing the secretion of IL- 1, TNFα, NO and / or IL-12, preferably in an amount at least equal to 25%, more preferably at least equal to 50%, 75% and 90% of the amount obtained and such that measured in Examples 2 and 3 with the OmpA protein of sequence SEQ ID NO. 1, called P40, of Klebsiella pneumoniae.

The term “antimicrobial agent or antimicrobial pharmaceutical composition” is intended to denote an agent or a pharmaceutical composition which after administration, preferably in humans, is capable of generating or increasing an immune response making it possible to prevent or treat, partially or totally, an infection. by a bacterium, yeast, fungus, virus or parasite, preferably without the need for the addition of antigens from the infectious microorganism whose infection is sought to be prevented or treated.

Thus, by antimicrobial agent or antimicrobial pharmaceutical composition, preference is given to antimicrobial agents or antibacterial, anti-yeast, antifungal, antiviral or antiparasitic pharmaceutical compositions.

Another subject of the invention is the use of an enterobacterium protein OmpA, one of its fragments or one of its derived proteins, for, or for the preparation of a pharmaceutical composition intended to induce activation of macrophages, in particular inducing or increasing the secretion of IL-1, TNFα, NO and / or IL-12 by said macrophages. The invention also relates to the use of an enterobacterium protein OmpA, one of its fragments or one of its derived proteins, for, or for the preparation of a pharmaceutical composition intended to induce the activation of NK lymphocytes, epithelial cells and / or neutrophils. In a particular embodiment, the enterobacterium protein OmpA or one of its fragments, is obtained by an extraction process from a culture of said enterobacterium.

The methods for extracting proteins from bacterial membranes are known to those skilled in the art and will not be developed in the present description. Mention may be made, for example, but not limited to, of the extraction process described by Haeuw J.H. et al. (EUT. J. Biochem, 255, 446-454, 1998).

In another preferred embodiment, the invention also comprises the use of an enterobacterium OmpA protein, one of its fragments or one of its derived proteins according to the invention, characterized in that said protein Enterobacterium OmpA, said fragment or said derived protein, is obtained by recombinant means.

The methods for preparing recombinant proteins are today well known to those skilled in the art and will not be developed in the present description, however, reference may be made to the method described in the examples. Among the cells which can be used for the production of these recombinant proteins, mention must of course be made of bacterial cells (Olins PO and Lee SC, 1993, Recent advances in heterologous gene expression in E. coli. Curr. Op. Biotechnology 4: 520-525 ), but also yeast cells (Buckholz RG, 1993, Yeast Systems for the Expression of Heterologous Gene Products. Curr. Op. Biotechnology 4: 538-542), as well as animal cells, in particular cell cultures mammal (Edwards CP and Aruffo A., 1993, Current applications of COS cell based transient expression Systems. Curr. Op. Biotechnology 4: 558-563) but also insect cells in which methods can be used using example of baculo viruses (Luckow VA, 1993, Baculo virus Systems for the expression of human gene products. Curr. Op. Biotechnology 4: 564-572).

Most preferably, the use according to the invention is characterized in that said enterobacterium is Klebsiella pneumoniae. In particular, the invention relates to the use according to the invention, characterized in that the amino acid sequence of said OmpA protein of Klebsiella pneumoniae, one of its fragments or one of its derived proteins, comprises: a) the amino acid sequence of sequence SEQ ID NO. 1; b) the amino acid sequence of a sequence having a homology of at least 80%, preferably 85%, 90%, 95% and 99%, after optimal alignment with the sequence SEQ ID NO. 1; or c) the amino acid sequence of a fragment of at least 5 amino acids, preferably 10, 15, 20, 25, 30, 40, 50, 75 and 100 consecutive amino acids, of a sequence such as defined in a).

The invention also comprises the use of an enterobacterium protein OmpA, one of its fragments or one of the derived proteins as defined above, for the preparation of an antimicrobial pharmaceutical composition intended to be administered by the route mucosal, preferably by the oral, nasal, anal or vaginal route, more preferably by the oral or nasal route.

These compositions are especially intended for the prophylactic or therapeutic treatment, preferably prophylactic, of viral, bacterial, parasitic, fungal or yeast infections.

In a particularly preferred embodiment, the compositions according to the invention do not include antigens, in particular antigens originating from the infectious microorganism whose infection is sought to prevent or to be treated.

The present invention also relates to the use according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to ensure and or to improve its stability or its effectiveness.

The invention relates in particular to the use according to the invention characterized in that said pharmaceutical composition is conveyed in the form of a vector making it possible to ensure and or to improve the stability and / or the effectiveness of the protein OmpA, the one of its fragments or one of its derived proteins, in particular the vectors chosen from liposomes, virosomes, nanospheres, microspheres or microcapsules. The invention also relates to the use according to the invention, characterized in that said pharmaceutical composition contains a pharmaceutically acceptable medium, preferably chosen from pharmaceutically acceptable media for administration by mucosal route in humans, in particular consisting of, but without limitation, water, an aqueous saline solution or an aqueous solution based on dextrose and / or glycerol.

The concentrations of the enterobacterium protein OmpA, one of its fragments or one of its derived proteins used according to the invention are those which make it possible to obtain an antimicrobial effect. In a particular embodiment, the invention also relates to the use according to the invention, characterized in that said pharmaceutical composition also contains a detergent.

Among said detergents, any type of pharmaceutically acceptable surfactant is preferred, in particular anionic, cationic, nonionic or amphoteric surfactants. Preferably, Zwittergent 3-12 and Octylglucopyrannoside detergents are used, and even more preferably Zwittergent 3-14.

The invention further relates to the use according to the invention, characterized in that said pharmaceutical composition is in a form such as in the form of powder, suppository or ovum, or also in the form of an aerosol, in suspension , etc., preferably in a form suitable for administration by mucosal route, in particular by nasal route.

The invention also relates to a composition as defined above in the uses according to the invention, in particular the compositions not containing antigens, in particular resulting from the infectious micro-organism of which one seeks to prevent or to treat the infection.

In a last aspect, the subject of the invention is a device suitable for administration by mucosal route, characterized in that it contains a composition according to the invention as defined above.

Examples of devices within the meaning of the invention include an aerosol device including or not comprising a propellant.

The legends of the figures and examples which follow are intended to illustrate the invention without in any way limiting its scope. Legends of the figures:

Figures 1 A and IB: Attachment of the P40 protein to macrophages

Figure 1A: Human macrophages were incubated with 0.5 μM of protein P40 or glycophorin A labeled with Alexa and then were analyzed by FACS. The results are expressed in relative number of cells.

Figure IB: Human macrophages were incubated with different

488 concentrations of P40 or glycophorin A labeled with Alexa and then were analyzed by FACS. The results are expressed in mean fluorescence intensity (MFI). Figures 2A, 2B and 2C: Internalization of the P40 protein by macrophages Figures 2A and 2B: Human macrophages are incubated at 4 ° C with 0.5 μM of P40 marked with Alexa, washed then cytospine and observed with a confocal microscope before (Figure 2A) or after incubation at 37 ° C (Figures 2B).

Figure 2C: Human macrophages are incubated at 4 ° C with 0.5 μM of

488 P40 protein labeled with Alexa, washed and incubated at 37 ° C before being observed with a confocal microscope. Dimethyl amiloride (DMA) was added 10 minutes before adding P40 protein as well as in the FACS buffer used. Figure 3: Increased expression of mRNA coding for IL-1β, 1TL-8, 1TL-12 and TNFα by human macrophages in the presence of the P40 protein

Separation on agarose gel of the products obtained after RT-PCR for each of the target mRNAs transcribed in the presence of an RT-PCR validation control (GAPDH).

Line 0: unstimulated macrophages; LPS line: macrophages stimulated by lOng / ml LPS; Line P40: macrophages stimulated by 1 μM of protein P40. Figures 4A, 4B, 4C, 4D, 4E and 4F: Production of proinflammatory mediators and IL-8 by macrophages in the presence of the P40 protein by acting in synergy with LPS and IFNγ

Figures 4A, 4B, 4C, 4D and 4E: Production of TNFα (Figure 4A), IL-lβ (Figure 4B), IL-8 (Figure 4C), IL-12 (Figure 4D) and d 'IL-10 (Figure 4E) by human macrophages. The macrophages were stimulated by the protein P40 alone (JE) at different concentrations or: - either by P40 at different concentrations in the presence of 0.2 ng / ml of LPS (D);

- or by P40 at different concentrations, the macrophages having been previously stimulated by human IFNγ before being stimulated by P40 (X).

Cytokines were assayed in the supernatants by ELISA. The results obtained come from an experiment representative of several experiments carried out.

Figure 4F: Production of NO by murine cells RAW 264.7 The macrophages were stimulated by the protein P40 at different concentrations:

- either with the simultaneous addition of 0.2 ng / ml of LPS case with 0.2 ng / ml of LPS (B);

- either the RAW cells were primed (previously stimulated) for 6 hours with 5 ng / ml of murine IFNγ ().

Example 1: Fixation and internalization of P40 by macrophages Material and method Obtaining cells Human macrophages are differentiated from monocytes according to the method described below.

Mononuclear cells (CMN) from peripheral blood of healthy volunteers were purified on a Ficoll gradient. The monocytes are then purified from the CMN by positive selection with a magnetic cell separator (Milteny Biotex, Bergisch Gladbach, Germany). Monocytes are cultured for 5 to 7 days with 10 ng / ml of GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), (R & D Systems, Abingdon, UK) at 5 x 10 ⁶ cells / 5 ml / well of plate plate. 6-well culture in RPMI culture medium containing 10% fetal calf serum, 50 U / ml penicillin, 2 mM glutamine, 50 mg ml streptomycin, 10 mM HEPES buffer, and 0.1 mM amino acids non-essential (cRPMI). All the cell lines used come from ATCC.

Study of the binding of the protein P40 by the FACS method - On macrophages differentiated from monocytes The macrophages thus generated are incubated in FACS buffer (RPMI containing 0.1% bovine serum albumin) at 2 × 10 ⁵ cells per culture plate well 96 wells sharp bottom for 20 minutes in the presence of different

488 concentrations of protein P40 labeled with Alexa or with glycophorin 488

Has marked with Alexa. The P40 protein is the protein of sequence SEQ ID No. 1 obtained by the recombinant route, as described in the international application for

488 patent published on May 17, 1996 under number WO 96/14415. The Alexa marking is carried out as described in the technical data sheet for the product marketed by Molecular Probes. The cells are then washed in FACS buffer and analyzed using the FACSvantage cytofluorometer device.

- On different types of cells

Different cell lines as well as human monocytes and macrophages and mouse peritoneal macrophages were incubated with 0.5 μM of

488 P40 protein labeled with Alexa and then were analyzed by FACS. The results are expressed in relative index of mean fluorescence intensity (-, +, ++ and +++) corresponding to a binding intensity ranging from non-detectable to high (cf. Table 1 below).

Table 1: Study of the binding of the P40 protein by the FACS method on different types of cells.

Study of the internalization of the P40 protein by confocal microscopy Human macrophages are incubated in FACS buffer (RPMI containing

0.1% bovine serum albumin) at 2 x 10 ⁵ cells per well of 96-well culture plate sharp bottom for 20 minutes in the presence of 0.5 mM of protein P40 labeled with Alexa ⁴⁸⁸ , washed then incubated or not at 37 ° C. Macrophages are finally cytospine and observed with a confocal microscope using an inverted microscope LSM510 marketed by Zeiss provided with a 63 X apochromat plane objective. In certain experiments, 150 mM of dimethyl amiloride (DMA) were added 10 minutes before addition of the P40 protein as well as in the FACS buffer used. The

488 Alexa fluorescence was measured with a 530-30 nm filter after excitation with an argon ion laser at 488 nm. Results

The results obtained show that:

- After incubation at 4 ° C, human macrophages bind the P40 protein and do not bind the glycophorin A used as a control protein (see Figure 1 A); - human macrophages bind the P40 protein in a dose-dependent manner (cf.

Figure IB);

- certain myeloid lines and in particular the line of murine macrophages RAW 267.4 also bind the protein P40 (cf. Table 1);

- macrophages internalize the P40 protein at 37 ° C. After incubation at 4 ° C in the presence of azide, the P40 protein remains localized on the surface of the cell (see Figure

2A). Incubation at 37 ° C shows that the P40 protein is found inside the cell (cf. Figure 2B). DMA partially prevents this internalization, which suggests that the internalization of the P40 protein could be done by macropinocytosis (cf. Figure 2C).

EXAMPLE 2 Increase in the Production of Proinflammatory Mediators by Macrophages in the Presence of the P40 Protein Material and Method

The macrophages generated from peripheral blood monocytes as previously described are incubated at 5 × 10 ⁵ / ml in cRPMI in the presence of different concentrations of protein P40 or in the presence of lipopolysacharide (LPS), used as a positive control (cf. Table 2). Certain experiments were carried out in the presence of 10 ng / ml of polymixin B sulfate. The cytokines were assayed in the supernatant by the ELISA method. LTL-lβ, 1TL-8, and the biologically active IL-12 (IL-12 p40 / IL-12 p35) were measured using commercial kits (marketed by R & D Systems). IL-lβ and IL-8 were assayed at 24 hours. The IL-12 was dosed at 48 hours. TNFα was assayed using capture and detection antibodies marketed by R & D Systems.

Table 2: Production of proinflammatory mediators by macrophages in the presence of the P40 protein.

Stimulus Cytokines

(concentration) no P40 0.2 μM P40 lμM LPS (10 ng / ml)

TNFα (ng / ml) <0.3 ± 0.05 0.9 + 0.2 4 + 1

TNFα (ng / ml) * <0.3 ± 0.07 1.1 + 0.2 0.16 + 0.1

IL-lβ (ng / ml) <0.35 + 0.08 0.5 + 0.1 0.9 + 0.1

IL-8 (ng / ml) 10 ± 8 80 ± 11 95 + 15 n. d.

IL-12 (pg / ml)

1 ± 0.2 2 + 0.3 6 + 0.8

*: Experiment carried out in the presence of 10 ng / ml of polymixin B sulfate; <: not detectable; not. d. : not determined.

Transcription of mRNAs coding for TNFα, IL-lβ, 1TL-8, and 1TL-12 biologically active IL-12 p40 and IL-12 p35 (see Figure 3).

The expression of the mRNAs coding for the cytokines TNFα, IL-1β, IL-8, IL-12 p35 and IL-12 p40 was evaluated by RT-PCR. The sequences of the primers used have been reported previously in the literature (De Saint-Vis et al., J. Immunol. (1998) 160-166). Briefly, 8 hours after stimulation, the cell pellets are taken up in an extraction buffer containing guanidium isothiocyanate (Trizol®; Life technologies) at a rate of 1 ml for 10 × 10 cells. After two extractions with phenol / chloroform / isoamyl alcohol (25/24/1), the total RNAs are precipitated by adding an equal volume of isopropanol (18 hours at −20 ° C.) and then collected by centrifugation (13,000 rpm, 30 min., 4 ° C). The pellet is rinsed with 70% ethanol. Total RNA is resuspended in water containing an RNAse inhibitor (RNAsin®; Promega, Madison, WI), denatured by heating (5 min. At 65 ° C) then quantified by spectrophotometry (λ = 260 nm). The complementary DNAs (cDNAs) are synthesized using a commercial kit (First strand cDNA synthesis, Amersham Pharmacia Biotech, Uppsala, Sweden). Reverse transcription (2 mg total RNA) is carried out in 10 mM Tris-HCl buffer pH 8.3 containing 300 mg / ml of oligo-dT primer of sequence SEQ ID No. 2 (5'-AACTGGAAGAATTCGCGGCCGCAGGAA (T ) ι ₈ -3 ') ₅ 10 μM of dithiothreitol, 5 mM of the four nucleotides and 1 U / ml of reverse recombinant transcriptase. The reaction mixture is incubated for 1 hour at 37 ° C and the reaction is stopped by heating at 65 ° C for 5 minutes. The reaction mixture for RT-PCR is as follows: 1 μl of cDNA (corresponding to 25 ng of total RNA), 25 pmol of primer, dNTP 200 μM, 1.5 mM MgCl ₂ , 10 mM Tris-HCl pH 8.3, KC1 50 mM and 0.4 units of Taq polymerase (Perkin Elmer). The PCR conditions are as follows: 5 min. at 95 ° C, 30 cycles: 1 min. at 94 ° C, 1 min. at 55 ° C and 1 min. at 72 ° C, a final extension of 5 min. at 72 ° C. The samples are taken up in the sample buffer (0.25% bromophenol blue, 40% sucrose) and analyzed by electrophoresis in 1% agarose gel containing ethidium bromide. After migration, the samples are visualized by illumination of the gel at the UN. The integrity of the ARΝ is analyzed by evaluating the expression of the ADΝc encoding the actin β molecule using the following primers of sequence SEQ ID No. 3: 5'-TCCACCACCCTGTTGCTGTA-3 'and of sequence SEQ ID No. 4: 5'-ACCACAGTCCATGCCATCAC-3 '. Results

The results obtained (cf. Table 2 and Figure 3) which come from an experiment representative of 5 experiments carried out, show that:

- the P40 protein induces the production of IL-lβ, IL-8, IL-12 and TNFα by human macrophages (cf. Figure 3);

- the induction of TNFα induced by the P40 protein is not inhibited by polymixin B sulfate (see Table 1). This shows that the effect of the P40 protein is not mediated by contaminating endotoxins. Confirming these results, we also observed that the production of murine TNFα induced by the P40 protein is similar in normal / mutated mice at the receptor toll level 4 (C3H / HeN mice versus C3H / HeJ mice) (data not shown); - the protein P40 acts at the level of transcription and increases the expression of r mRNA coding for IL-1β, IL-8, IL-12 and TNFα (cf. FIG. 3).

EXAMPLE 3 The P40 protein increases the production of proinflammatory mediators and of IL-8 by macrophages by acting in synergy with LPS and IFN. Material and method

Macrophages were obtained and stimulated as described above with the following modifications to study the synergy with LPS and IFNy:

- either by adding 0.2 ng / ml of LPS at the time of stimulation with the P40 protein;

- Either the macrophages used were previously stimulated (awarded) 6 hours with 5 ng / ml of human IFNγ before stimulation with the P40 protein (cf. Figures 4A, 4B, 4C, 4D and 4E).

RAW 264.7 cells (murine macrophages) from ATCC were stimulated by the protein P40 and after 24 hours the nitrites were determined in the supernatants by the Griess method using NaNO ₂ as standard. In some cases the RAW cells were primed for 6 hours with 5 ng / ml of urine IFNγ, and in other cases with 0.2 ng / ml of LPS added at the same time as the P40 protein (cf. FIG. 4F). Results

The results show that:

B protein P40 acts in synergy with LPS and IFNγ to induce the production of proinflammatory mediators, of 1TL-8 by macrophages

(cf. Figures 4A, 4B, 4C, 4D and 4E); • the P40 protein induces the production of NO by murine macrophages and acts in synergy with IFNγ (see Figure 4F).

These results show in particular that the enterobacterium protein OmpA, in particular the Klebsiella pneumoniae OmpA, induces the production of cytokines by macrophages and is capable of acting in synergy with LPS and IFNy. LTFNγ is produced by T lymphocytes and NK cells. Thus, the effect of OmpA will most likely be amplified during an attack by gram-negative bacteria with LPS.

In the presence of OmpA, small amounts of bacteria are then sufficient to stimulate the cells of the year. In addition, locally present lymphocytes can also be activated by cytokines produced by macrophages stimulated by OmpA and amplify its effect by positive feedback, producing IFNγ. Thus, OmpA behaves as a stress signal which will act in synergy with other endogenous or exogenous signals to increase the defense responses of the immune system.

Claims

1. Use of an enterobacterium OmpA protein, one of its fragments or one of its derived proteins, for the preparation of an antimicrobial pharmaceutical composition in which said OmpA protein is at a concentration of between 0.08 μM and 1 μM.

2. Use according to claim 1, characterized in that the antimicrobial pharmaceutical composition is an antibacterial, anti-yeast, antifungal, antiviral or antiparasitic pharmaceutical composition.

3. Use of an enterobacterium protein OmpA according to claim 1 or 2, for the preparation of a pharmaceutical composition intended to induce the activation of macrophages.

4. Use of an enterobacterium protein OmpA according to one of claims 1 to 3, for the preparation of a pharmaceutical composition intended to induce the activation of NK lymphocytes, epithelial cells and / or neutrophils.

5. Use according to one of claims 1 to 4, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained by an extraction process from a culture of said enterobacterium.

6. Use according to one of claims 1 to 4, characterized in that said protein OmpA enterobacterium, one of its fragments or one of its proteins derived, is obtained by recombinant route.

7 Use according to one of claims 1 to 6, characterized in that said enterobacterium is Klebsiella pneumoniae.

8. Use according to claim 7, characterized in that the amino acid sequence of said OmpA protein, or one of its fragments, comprises: a) the amino acid sequence of sequence SEQ ID No. 1; b) the amino acid sequence of a sequence having at least 80% homology with the sequence SEQ ID No. 1; or c) the amino acid sequence of a fragment of at least 5 amino acids of a sequence as defined in a).

9. Use of an OmpA protein, one of its fragments or one of its derived proteins as defined in any one of claims 5 to 8, for the preparation of an antimicrobial pharmaceutical composition intended for administration mucosally.

10. Use according to claim 9, characterized in that the pharmaceutical composition is intended to be administered by oral, nasal, anal or vaginal route.

11. Use according to one of claims 1 to 10, intended for the prophylactic or therapeutic treatment of viral, bacterial, parasitic, fungal or yeast infections.

12. Use according to one of claims 1 to 11, characterized in that said composition does not contain an antigen, preferably an antigen from the infectious microorganism.

13. Use according to one of claims 1 to 12, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its effectiveness.

14. Use according to one of claims 1 to 13, characterized in that said pharmaceutical composition contains a pharmaceutically acceptable medium.

15. Use according to one of claims 1 to 14, characterized in that said composition also contains a detergent.

16. Use according to one of claims 1 to 15, characterized in that the composition is in the form of powder, suppository or ovum.

17. Composition as defined in any one of claims 1 to 16.

18. Device suitable for administration by mucosal route, characterized in that it contains a composition as defined in any one of claims 1 to 16.

19. Device according to claim 18, characterized in that it is chosen from an aerosol device comprising or not a propellant.