WO2004007540A1 - Klebsiella membrane extracts exhibiting advantageous properties, and method for producing said extracts - Google Patents

Abstract

The invention concerns bacterial extracts, in particular Klebsiella extracts, more particularly of Klebsiella pneumoniae, exhibiting advantageous properties, in particular for several applications, an increased activity as compared to known Klebsiella extracts. The invention also concerns the method for producing said extracts.

Description

 KLEBSIELLA MEMBRANE EXTRACTS HAVING ADVANTAGEOUS PROPERTIES, AND PROCESS FOR PRODUCING

THESE EXCERPTS

The invention relates to bacterial extracts, in particular extracts of Klebsiella, more particularly of Klebsiella pneumoniae, having advantageous properties, in particular, in several applications, increased activity compared to extracts of Klebsiella already known. The process for producing these extracts is also part of the present invention.

A drug consisting of extracts of Klebsiella pneumoniae, RU 41740, is marketed under the brand Biostim by Laboratoires Cassenne (France). This medication is composed of glycoprotein extracts obtained from a strain of Klebsiella p neumoniae (strain 01 K2 NCTC 5055). It is obtained after lysis of the bacterial walls, organic extraction, centrifugation and ultrafiltration.

It includes: - glyco-conjugates, in an amount greater than 60%;

- amino acids; and

- lipids.

The glyco-conjugate part is divided into 3 fractions: P1, F1, F2. P1, of capsular origin, represents approximately 50% of RU 41740, and has an average molecular weight of 95 kD,

F1 is of membrane origin and represents approximately 20% of RU 41740, and has an average molecular weight of 350 kD

F2 appears to be a part of P1.

RU 41740 does not cause a pyrogenic effect, as evidenced by the negativity of the Limulus test carried out with this compound. RU 41740 has immunomodulatory activity. Its activity in humans has been demonstrated, in particular to stimulate the host's defenses in the case of chronic bronchitis (Fietta, Bersani et al. 1988), or to avoid delayed hypersensitivity reactions in affected patients. lymphomas (Lang, Gastaut et al. 1986). More recently, trials in mice have shown that parenteral administration of RU 41740 is effective in preventing the formation of intra-abdominal abscesses following infection (Finlay-Jones, Boyden et al. 1993), and that oral administration of RU 41740 protected mice from experimental infections, mainly against Gram-negative bacteria (Nimier, Wolff et al. 1999). At the cellular level, Garin et al have shown the effect of RU 41740 on the modulation of molecules involved in the presentation of the antigen (HLA-DR and H LA-DQ), I has capture of endotoxin (CD 14) and l activation (CD23) on the surface of monocytes (Garin, Bernaud et al. 1996). Furthermore, Estaquier et al. have shown that RU 41740 prolongs the survival of monocytes in culture, by limiting their apoptosis (Estaquier, Bloy et al. 1998)

Biostim is currently mainly prescribed to stimulate the immune system in children with respiratory infection.

In addition, the Applicant has recently demonstrated two other advantageous properties of RU 41740. The addition of RU 41740 to the culture medium of immature dendritic cells obtained in vitro from monocytes allows their maturation. This property of RU 41740, described in patent application No. FR 0100275, is of great interest in the field of cell therapy.

Substantial modifications made to the production process of RU 41740, in particular with the aim of improving the sanitary quality of the product, led the applicant to a new product, designated under the name "LCOS 1013". Unexpectedly, the plaintiff put in evidence that this new analogous extract of RU 41740, compared to the latter, in addition to greater health safety, increased effectiveness in several of the indications mentioned above. LCOS 1013 in particular has an activity twice that of RU 41740 in the application mentioned above, namely the maturation of dendritic cells. These results show that new bacterial extracts, in particular of Klebsiella, can be advantageously used in several new applications. These bacterial extracts, as well as their production process, are the subject of the present invention.

In particular, LCOS 1013 is composed of glycoprotein extracts obtained from the strain O1 K2 NCTC 5055 from Klebsiella pneumoniae. It is obtained by a process comprising stages of lysis of the bacterial walls, organic extraction, centrifugation and ultrafiltration, as described in Example 1. With respect to the process for preparing RU 41740, the LCOS 1013 preparation protocol presents the advantage of not using mercurothiolate. This compound has, in addition to its lytic agent properties, antiseptic properties. Its elimination from the LCOS 1013 preparation protocol therefore leads to the need to carry out production under strict aseptic conditions, in order to protect the product from any bacterial contamination. Furthermore, the LCOS 1013 preparation protocol presents, with respect to the process for preparing RU 41740, an additional step of molecular sieving with concentration (step 6 of Example 1), between the lysis step and the lyophilization steps and extractions. This step is carried out for example by tangential filtration, with a cutoff threshold of 40 to 60 kDa.

The modifications in the preparation process of LCOS 1013 compared to that of RU 41740 lead to differences in composition between these two extracts. Indeed, the comparative study between RU 41740 and the new extract of Klebsiella pneumoniae LCOS 1013 shows differences at the structural level and at the activity level.

The SDS-PAGE electrophoretic analysis of these two active ingredients reveals a difference in composition due to the number of bands detected after staining with Coomassie blue and the relative intensity of each of the bands present in RU 41740 and LCOS. 1013 (example 2).

The immunological activity of these two active ingredients is also different. The immunonephelometry test shows that LCOS 1013 has a different reactivity from RU 417401 when these products are reacted against an anti-RU 41740 immune serum, which means that the antigenic composition of these two products are different, at equivalent concentration (example 3).

The above analyzes therefore demonstrate that LCOS 1013 is a different product from RU 41740.

The inventors then compared the activity of these two products as agents for maturing dendritic cells (Example 8).

The Applicant has also compared the activity of LCOS 1013 and RU 41740 in the maturation of dendritic cells obtained from monocytes (MODC) in vitro. Again, the LCOS 1013 proved to be significantly more efficient than the RU 41740, since (example 8).

All these results, presented in more detail in the examples below, clearly show that the activity of LCOS 1013 is greater than that of RU 41740 in several possible applications of bacterial extracts, in particular obtained from Klebsiella pneumoniae. The known biocompatibility and good tolerance of RU 41740 makes it possible to envisage an advantageous use of these compounds for the manufacture of medicaments intended to treat various pathologies, all the more so since some of the differences between the production protocols of LCOS 1013 compared to RU 41740 make the new compound a priori more efficient on the initial phase, since the manufacturing procedure does not require the use of antiseptic at during lysis.

The invention therefore relates firstly to a process for the preparation of bacterial extracts from encapsulated germs, comprising the following steps: a) culture of the bacteria, b) lysis of the bacteria, c) first molecular sieving, with concentration, d ) lyophilization of the concentrate e) extractions with organic solvents, f) re-solution, g) centrifugation, h) second molecular sieving, i) filtration j) lyophilization

In a preferred embodiment of the above method, the bacteria are Klebsiels, preferably Klebsiella pneumoniae.

In the methods of the invention, the culture of the bacteria is preferably carried out at a pH below 6.7, for example at pH 6.5.

In a preferred embodiment of the above methods, at least 90% of the bacteria have settled after step b). In addition, in this new process, the stage of lysis of bacteria preferably lasts between 4 and 8 days, for example 6 days. More preferably, the lysis of the bacteria is carried out without adding a lysing agent comprising an antiseptic component. This allows on the one hand, a less polluting production for the environment and, on the other hand, to ensure greater health safety of the product, since it does not risk keeping traces of an antiseptic derivative.

In a particular embodiment of the methods of the invention, the pH of the culture resulting from step a) is reduced to a value less than 6, for example at pH 5.8 ± 0.1 before the addition of the lysing agents .

The first molecular sieving step, with concentration (step c) is an important step in the process of the invention. Preferably, this step leads to a concentration of a factor of between 1.5 and 3 of the lysed bacterial suspension resulting from step b), for example to a concentration of a factor 2.

This step c) leads to the elimination of the small unbound molecules, with a cutoff threshold preferably situated at 20 kDa, 70 kDa, or 100 kDa and, more preferably, at 50 kDa or around this value. Examples of cartridges which can be used for this are CARBOSEP type cartridges with a cut-off threshold of approximately 40 to 60 kDa.

In a particular embodiment of the process of the invention, step e) comprises an extraction with a ketone and an alcoholic extraction. For example, step e) comprises an extraction with acetone and an extraction with methanol, the volume used for each of these solvents being proportional to the mass of lyophilisate obtained from step d), so that

10 x mass of lyophilisate (in kg) <volume of solvent (in I) <20 x mass of lyophilisate (in kg)

In a preferred embodiment of the methods of the invention, and always with the concern of good sanitary quality of the products obtained, step f) of re-solution of the product from step e) is carried out without adding a substance with an antiseptic component. The invention also relates to any product capable of being obtained by a process as described above.

A pharmaceutical composition comprising a product as described in the preceding paragraph, and intended to promote the maturation of dendritic cells (in vitro or in vivo), also forms part of the present invention.

The present invention also relates to a product capable of being obtained at the end of step c) of a process such as those described above. Such a product will certainly have a lower purity than LCOS 1013, but it can be advantageously used for example in the manufacture of a cosmetic composition or of a composition intended for veterinary use. Such pharmaceutical or veterinary compositions are also an integral part of the present invention.

The examples and figures presented below illustrate some of the differences between RU 41740 and LCOS 1013, in particular from the point of view of their composition and their immunological properties. These examples and figures are presented by way of non-limiting illustration, and will make it possible to highlight certain advantages and characteristics of the present invention.

Legend of figures

Figure 1: Summary diagram of the manufacturing steps of LCOS 1013. Figure 2: Electrophoretic profiles of the various products mentioned in Example 2. The electrophoresis is carried out in SDS-PAGE, on polyacrylamid gel 8-16% Tris-Glycine, 1.5mm, 15 wells. Wells 1, 6, 11: Markers (200 kDa to 6 kDa) Wells 2 and 3: RU 41740 PA lot N ° 457 Wells 4 and 5: LCOS 1014 (Atomized stage) Wells 7 and 8: LCOS 1012 (Stage 11 of LCOS 1013) Wells 9 and 10: LCOS 1013 (Freeze-dried stage) Example 1: Manufacturing process for LCOS 1013

A. GENERAL PRESENTATION OF LCOS 1013

Like RU 41740, LCOS 1013 consists of a set of substances extracted from a culture lysate of Klebsiella pneumoniae. Its manufacturing method is based on the succession of stages or stages, d have the general schema and is presented below. Media that can be used for cultivation, as well as an indicative presentation of the progress of each stage, are described in more detail after this diagram. The manufacturing technique described below, purely by way of illustration, corresponds to an operating unit based on a volume of bacterial culture obtained in a fermenter of 600 liters.

B. DESCRIPTION OF DIFFERENT MEDIA USED FOR THE CULTURE OF BACTERIA

The culture of the bacteria, with a view to producing LCOS 1013 according to the succession of steps specified above, can be carried out using the culture media containing the following components:

Nutritious Broth

- Soy Papain Peptone 4 +/- 2 g / l - Yeast autolysate 4 +/- 2 g / l

- Sodium chloride 5 +/- 2 g / l

- Sodium hydroxide qs for pH 7.4 ± 0.2

Red Box

- Sodium chloride 5 +/- 2 g / l - Anhydrous glucose 5 +/- 2 g / l

- Yeast autolysate 12 +/- 3 g / l - Soy Papain Peptone 5 +/- 2 g / l

- Agar 30 +/- 4 g / l

- Sodium hydroxide qs for pH 7.5 ± 0.1

- Dipotassium phosphate 4 +/- 2 g / l - Monopotassium phosphate 0.5 to 0.8 g / l

Preculture medium

- Soy Papain Peptone 20 +/- 2 g / l

- Yeast autolysate, 10 +/- 2 g / l

- Sodium chloride 5 +/- 2 g / l - Dipotassium phosphate 3.5 +/- 1 g / l

- Monopotassium phosphate 1 to 2 g / l

Culture medium for fermenter

- Bakery yeast autolysate 10 +/- 2 g / l

- Sodium Chloride 5 +/- 2 g / l - Papain Peptone of Soy 20 +/- 2 g / l

- Dipotassium phosphate 3.5 +/- 1 g / l

- Monopotassium phosphate 1 to 2 g / l

C. DETAILED DESCRIPTION OF THE DIFFERENT STAGES OF PRODUCTION OF LCOS 1013

STAGE 1: Preparation of the inoculum

The purpose of this stage is to carry out, on an agar medium in a Roux dish, the culture of Klebsiella pneumoniae necessary for the inoculation of a preculture in liquid medium, from a cryotube or a lyophilisate from the bank of job. The work bank is made for example from a strain of Klebsiella pneumoniae from the Institut Pasteur, reference CIP 52.145.

To do this, the strain is revitalized by carrying out a bacterial suspension in the nutritive broth, from which boxes of Roux are inoculated, which are then incubated at 37 ° C + 0.5 for 20 to 24 hours.

STAGE 2: Precultures

From the inoculum prepared in stage 1, a preculture is carried out in a 3-liter bottle, intended to inoculate a 35 I fermenter, which in turn is intended to inoculate a 600 I fermenter.

The first preculture is carried out in the preculture medium described above, to which a sterile glucose solution is added, at a rate of 30 ± 10 g of glucose per 3 liters. The second preculture, of 35 liters, is carried out in the culture medium for fermenter described above, to which 400 ± 100 g of glucose are added.

Satellite vials containing respectively a sterile 10 N sodium hydroxide solution, a sterile solution of orthophosphoric acid diluted with Y∑, and a sterile anti-foam solution, can be used for the second preculture, in a fermenter, in order to in particular to maintain the pH around 6.5, throughout the duration of the preculture, that is to say approximately 5 hours at approximately 37 ° C., with stirring.

STAGE 3: Culture in a fermenter

The object of this stage is to carry out in a fermenter, under defined conditions, a bacterial culture of content greater than or equal to 10 ⁹ germs / ml, to allow subsequent extraction of a satisfactory quantity of product.

For this, the suspension of germs contained in the 35 I fermenter is transferred to a 600 liter fermenter containing the fermenter culture medium described above, to which 5 kg of glucose have been added.

As for the 35 liter preculture, satellite tanks containing respectively a sterile 10 N sodium hydroxide solution and a sterile solution of orthophosphoric acid diluted to ^Λ A are used for the regulation of the pH around 6.5, thus than a satellite tank containing a sterile anti-foam solution. The culture is carried out for approximately 7 hours, at 37 ° C ± 1 ° C, with stirring.

STAGE 4: End of culture and addition of lysing agents

The purpose of this stage is to inactivate the culture by the action of lysing agents and heating from 55 to 75 ° C. for a period greater than or equal to 40 minutes. For this, the lysing agents used are the following:

- sterile polysorbate 80 solution,

- sterile EDTA solution,

- sterile lysozyme hydrochloride solution.

At the end of the culture, the pH is brought to 5.8 ± 0.3 using a sterile solution of orthophosphoric acid, then the above lysing agents are transferred to the culture.

The content of the fermenter is followed at a temperature of 65 ° C ± 10 ° C and maintained for at least 40 minutes at this temperature with stirring. The b iolysate o btenu e was then transferred to an industrial lysis tank (preheated to 65 ° C ± 10 ° C), and maintained at this temperature for at least 20 minutes, before being brought back to 37 ° C ± 2 ° C.

STAGE 5: Lysis The lysis step consists in breaking the wall of bacteria by enzymatic way, which involves the release of the cytoplasmic constituents of the microbial cells. It is carried out by maintaining the pre-lyosed suspension of the previous stage, at 37 ° C ± 2 ° C, in the lysis tank for at least 6 days

STAGE 6: Molecular sieving and concentration

In this stage, the volume of raw lysate is reduced by half to reduce the quantity to be treated in the following stages. The concentration is carried out for example on an ultrafilter equipped with a CARBOSEP type filter cartridge with a cutoff threshold of 20 to 100 kDa, thus eliminating small unbound molecules.

STAGE 7: Lyophilization of the lysate

In order to obtain the crude lysate from stage 6 in solid form allowing subsequent extraction with solvents, a lyophilization operation is carried out immediately after the concentration stage. This gives a brown powder, sticky to the touch.

STAGE 8: Extraction with acetone:

The lyophilized LCOS 1013 lysate from stage 7 contains lipids which are partially removed by solid-liquid extraction, using acetone at room temperature. The product is recovered by centrifugation, then dried. STAGE 9: Extraction with methanol:

After extraction with acetone, the product obtained in stage 8 still contains residual lipids and pigments which are eliminated by solid-liquid extraction, using methanol at room temperature.

The product is recovered by centrifugation, then dried.

STAGE 10: Re-solution

"The ethanol extract" from stage 9 is put back into aqueous solution, to allow the subsequent isolation of the product by centrifugation and ultrafiltration.

STAGE 11: Centrifugation

The crude suspension extract from stage 10 contains denatured proteins and water-insoluble matter which is removed by centrifugation.

The centrifuged product has a slightly colloidal appearance and is beige to brown in color.

STAGE 12: Ultrafiltration

This stage represents the essential stage of product isolation.

The product from stage 11 contains substances of different molecular sizes: mineral salts, proteins, glycoproteins, etc.

The macromolecules of PM> 300,000 constituting the product are isolated by ultrafiltration on a membrane with a cutoff threshold of 300 KD. The product, once introduced into the tank of the device, is continuously transported by means of a pump, in a closed circuit comprising an ultrafilter. The medium is kept homogeneous by means of stirring. The pressure created by the pump on the filter membrane forces part of the solute to pass through this membrane (permeate).

The retained part (retentate) remains in circulation. Knowing that we operate at constant volume, the loss of volume is constantly compensated for by the same volume of water.

At the end of the operation, the ultrafiltered solution.

The ultrafiltrate obtained is a slightly yellow translucent liquid solution.

STAGE 13: Filtration

The clarification of the centrifugation supernatant obtained in stage 11 is refined by filtration through a nominal retention membrane of 1.2 μm.

An opalescent "solution of glycoproteins", light beige to cream in color, is thus obtained.

STAGE 14: Lyophilization

In order to allow good preservation of the product, the solution from stage 13 is then lyophilized, by a process comprising a freezing step, then the lyophilization proper.

The lyophilized glycoproteins have the appearance of a fluffy powder of white to cream color, hygroscopic. STAGE 15: Mixing

The aim of this last step is to homogenize the lyophilized glycoproteins from stage 14.

Atomization can advantageously replace lyophilization and mixing (LCOS 1014).

Example 2: analysis of the electrophoretic profile of the new extract of Klebsiella pneumoniae LCOS 1013.

The objective of these experiments is to compare the electrophoretic profile of the lyophilized stage of the new extract of Klebsiella pneumoniae (LCOS 1013) and of RU 41740, the electrophoresis being carried out on SDS-PAGE gel.

1) Materials and reagents

- Polyacrylamide gel 8 - 16% Tris-Glycine, 1.5 mm, 15 wells (NOVEX, ref .: EC60485);

- Standard markers (NOVEX, ref .: LC5677); - Tris-Glycine SDS sample buffer (2x) (NOVEX, ref .: LC2676);

- Tris-Glycine SDS running buffer (1 Ox) (NOVEX, ref .: LC2675);

- Drying solution (NOVEX, ref .: LC4025);

- Generator LKB 2303 Multidrive XL (2WL57).

2) Products tested

The products tested are RU 41740 (wells 2 and 3), LCOS 1013 (wells 9 and 10), as well as intermediate products for the manufacture of LCOS 1013, namely LCOS1012 (corresponding to the product obtained after stage 11 of the process described in example 1) (wells 7 and 8) and LCOS1014 (atomized stage) (wells 4 and 5). 3) Results

The compared electrophoretic profile of the different products is shown in Figure 2.

A comparison of the well corresponding to RU 41740 (wells 2 and 3) and of the wells corresponding to LCOS 1013 (wells 9 and 10) reveals the following differences between the two products:

- RU 41740 has a triplet of bands between 97.4 and 66.3 Kd, a triplet which is reduced to a doublet for LCOS 1013;

- RU 41740 has a second triplet of bands between 55.4 and 36.5 Kd, a triplet which is also present in LCOS 1013 but whose first two bands have a lower intensity in LCOS 1013 compared to bands of the same triplet in RU 41740.

In order to refine and quantify this analysis, all of the gel tracks were scanned and then analyzed by ONE-SCAN software.

The following parameters are calculated for each of the bands of each track:

-% of RF;

- amplitude; - intensity (Int OD);

-% of relative intensity (% Int OD).

Analysis of the data concerning tracks 3 (RU 41740 lot no. 457) and 10 (LCOS 1013) is shown in tables 1 and 2 below. Table 1: Densitometric analysis of the bands of molecular weight 97.4 Kd / 66.3 Kd and 55.4 Kd / 36.5 Kd appearing on the gel of FIG. 2.

r

i-

-I g. α:

r

CM

H 111

_l α.

Tableau 2 : Analyse densitométrique des bandes de poids moléculaire inférieur à 36,5 Kd apparaissant sur le gel de la figure 2.

Table 2: Densitometric analysis of the bands of molecular weight less than 36.5 Kd appearing on the gel of FIG. 2.

An asymmetry in the migration of the gel means that the Rf percentages of each of the markers on track 6 are greater than the Rf percentages of each of the markers deposited on track 11 by approximately 3%. This asymmetry was taken into account to establish a correspondence between the bands of the tracks considered, that is to say track 3 (RU 41740) and track 10 (LCOS 1 013). So, for example, I ab and n ° 1 3 of track 3 (percentage Rf = 52.8) and the band n ° 15 of track 10 (percentage Rf = 49.8) both correspond to the first strip of the first triplet. Table 1 summarizes the results obtained for the bands of molecular weight between 97.4 KDa and 55.4 KDa:

a) TRIPLET 1:

Analysis of the relative intensity of each of the bands making up this triplet shows that:

- the first band of this triplet present in RU 41740 is almost non-existent in LCOS 1013;

- the second band of this triplet is present in RU 41740 with an intensity 2 times less compared to that present in LCOS 1013;

- the third band of this triplet is present in RU 41740 with an intensity 2 times higher than that present in LCOS 1013.

b) TRIPLET 2:

Analysis of the relative intensity of each of the bands making up this triplet shows that the first two bands making up this triplet are practically nonexistent in LCOS 1013 in comparison with the first two bands of triplet 2 from RU 41740.

Table 2, which relates to the bands with a molecular weight less than 36.5 KDa, reveals that no significant difference appears between the bands corresponding to LCOS 1013 and to RU 41740.

The electrophoretic profiles of RU 41740 and LCOS 1013 therefore have several differences, which indicates that the composition of these two products is different. Example 3: Comparison of the immunological activity of LCOS 1013 and RU 41740.

The objective of these experiments is to measure the antigenic similarity between the two products.

For this, the reactivity of antibodies produced against RU 41740 was measured comparatively with respect to RU 41740 and LCOS 1013.

This reactivate was measured by measuring the light scattered by a precipitate formed during the antigen-antibody reaction in a liquid medium, according to the technique.

According to this technique, the reactivity observed with respect to a product B identical to a product A against which the antibodies have been obtained must be greater than or equal to 75% of the reactivity observed with respect to product A; otherwise, this reflects a different chemical and / or structural composition between the two products.

Materials and reagents

- ICS nephelometer referenced 2 WF 19;

- M33 card;

- 6% anti-SB4 serum;

- Reference SB5, slopes = 0.995 RS 02 dosages = 0.956 RS 02

Results

The results obtained at equal concentration and on dry product (water content of 4.0%) show that the reactivity of anti-RU 41740 antibodies, vis-à-vis LCOS 1013, is only 33% compared to their reactivity vis-à-vis RU 41740. This reflects a significant difference in antigenic structure between the two products. BIBLIOGRAPHY

Estaquier, J., C. Bloy, et al. (1998). "The immunomodulating glycoprotein extract from Klebsiella pneumoniae RU 41740 exerts a suppressive effect on human monocyte death by apoptosis." Immunopharmacoloqy 39 (2): 157-64.

Fietta, A., C. Bersani, et al. (1988). "Double-blind trial RU 41740 vs. placebo: immunological and clinical effects in a group of patients with chronic bronchitis." Respiration 54 (3): 145-52.

Finlay-Jones, J. J., A. N. Boyden, et al. (1993). "The effects of RU 41.740, a glycoprotein immunomodulating agent derived from Klebsiella pneumoniae, on intra-abdominal abscess formation in mice." J Med Microbiol 38 (6): 454-8.

Garin, L, J. Bernaud, et al. (1996). "RU 41 740 (Biostim) and IL-4, or IL-13, have opposite effects on CD14, CD23, HLA-DR and HLA-DQ on monocytes." Int J Immunopharmacol 18 (1): 69-74.

Lang, J. M., J. A. Gastaut, et al. (1986). "Enhancement of delayed cutaneous hypersensitivity by oral administration of RU 41740 (Biostim) in lymphoma patients-a randomized double blind multicentric trial." Int J Immunopharmacol 8 (7): 687-90.

Nimier, K., F. Wolff, et al. (1999). "Protective effects of RU 41740, a bacterial immunomodulator, against experimental infections: induction of cytokine and immunoglobulin release in mice after oral administration." Int J Immunopharmacol 21 (9): 561-74.

Claims

1. Process for the preparation of bacterial extracts from encapsulated germs, comprising the following steps: a) culture of the bacteria, b) lysis of the bacteria, c) first molecular sieving, with concentration, d) lyophilization of the concentrate e) extractions by organic solvents, f) re-solution, g) centrifugation, h) second molecular sieving, i) filtration, j) lyophilization 2. Method according to claim 1, characterized in that the bacteria are Klebsiels, preferably Klebsiella pneumoniae. 3. Method according to claim 1 or 2, characterized in that the culture of the bacteria is carried out at pH less than 6.7. 4. Method according to claims 1 to 3, characterized in that at least 90% of the bacteria are lysed at the end of step b). 5. Method according to claims 1 to 4, characterized in that the step of lysis of bacteria has a duration of between 4 and 8 days. 6. Method according to claims 1 to 5, characterized in that the lysis of bacteria is carried out without adding a lysing agent comprising an antiseptic component. 7. Method according to claims 1 to 6, characterized in that the pH of the culture resulting from step a) is reduced to a value less than 6 before the addition of the lysing agents. 8. Method according to claims 1 to 7, characterized in that step c) leads to a concentration of a factor between 1, 5 and 3 of the lysed bacterial suspension resulting from step b), and preferably by a factor of 2. 9. Method according to claims 1 to 8, characterized in that step c) leads to the elimination of small unbound molecules, with a cutoff threshold of 20 kDa, 50 kDa, 70 kDa, or 100 kDa. 10. Method according to claims 1 to 9, characterized in that step e) comprises an extraction with a ketone and an alcoholic extraction. 11. Method according to claim 10, characterized in that step e) comprises an extraction with acetone and an extraction with methanol, the volume used for each of these solvents being proportional to the mass of lyophilisate obtained from the step d). 12. Product capable of being obtained by a process according to any one of claims 1 to 11. 13. Pharmaceutical composition comprising a product according to claim 12, and intended to promote the maturation of dendritic cells. 14. Cosmetic composition comprising a product capable of being obtained at the end of step c) of a process according to any one of claims 1 to 11. 15. Composition intended for veterinary use, characterized in that it comprises a product capable of being obtained at the end of step c) of a process according to any one of claims 1 to 11.