FR2789902A1 - Use of enterobacterial outer membrane protein as immunogenic carrier, particularly for contraceptive and anti-cancer vaccines, provides strong humoral response - Google Patents

Abstract

Use of an enterobacterial outer membrane protein A (OmpA), or its fragments, associated with an immunogen (I), to prepare a pharmaceutical composition for improving the immunological response to (I). (I) is at least one of cytokine, growth factor or hormone (or their receptors) and/or tumor-specific markers, or their fragments or analogs. Independent claims are also included for the following: (a) pharmaceutical composition containing OmpA, or its fragments, particularly from Klebsiella pneumoniae, associated with, as immunogen, a cytokine, growth factor and/or hormone, or their fragments; and (b) pharmaceutical composition containing a nucleic acid construct encoding OmpA, as in (a), associated with a nucleic acid encoding an immunogenic peptide as in (a).

Description

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 The invention relates to the use of a mixture or complex comprising an enterobacterium OmpA membrane protein, in particular Klebsiella pneumoniae, associated with an immunogen selected from phCG, a compound involved in the proliferation of tumor cells or in fertility. or a fragment thereof for the preparation of a pharmaceutical composition for increasing the immune response against said immunogen. The invention further comprises a pharmaceutical composition comprising said mixture or complex, intended in particular for the prevention and treatment of tumors, or the treatment of fertility.

 Vaccination is an effective way to prevent or reduce viral or bacterial infections. The success of vaccination campaigns in this area has helped to expand the concept of vaccine in the field of autoimmune diseases and cancer.

 Chorionic hormone human gonadotropin (hCG) is a dimeric hormone that belongs to the super family of cysteine heart growth factors (TGFp, NGF, PDGFp) and the gonadotropin family. This family includes on the one hand pituitary hormones LH (luteinizing hormone or interstitial cell stimulating hormone) and FSH (follicle stimulating hormone or follitropin) responsible for stimulating testicular and ovarian functions. On the other hand, the hormones of placental origin, among which hCG synthesized during pregnancy and whose physiological role is to maintain the growth of corpus luteus functional.

 All these hormones, despite the diversity of their physiological functions, are structurally close and consist of two α or β glycosylated chains or subunits at specific sites and linked by disulfide bridges. Within a given species, the sequence of the α-subunit is basically identical for all of these hormones. The hormonal activity of these ap hybrids is dictated by the particularity of the P subunit present (see Figure 1).

 In addition to its placental origin, a pituitary localization has been shown for hCG or hCG-like molecules. This pituitary source (Hoermann et al., J. Endocrinal Metab 1990, could be the cause of the very weak

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 concentrations of "hCG like" material present in the serum of healthy individuals of both sexes (Odell et al., N Engl J. Med 1987; 317: 1688-1691; Odcll et al.

Clin Endocrinol. metab. 1990 ; 71: 1318-1321).

 Moreover, hCG and its fragments appear to be products of malignant transformation. Indeed, while benign neoplasms do not express or produce this kind of material, several authors have shown that malignant tumors, but also embryonic and fetal cells, express hCG in vivo and in vitro. It has been observed that hCG immunoreactivity is present in 10 to 50% of patients with different non-trophoblastic tumors including endometrial tumors, ovarian, pancreas, stomach, colon, the liver, lung, breast, kidneys, bladder and some lymphomas (Braunstein et al Ann Intern Med 1973, Kuida et al Arch Pathol Lab Med 1988, 112: 282-285).

 In tumor cells, hCG, its subunits or fragments may be associated with the membrane, thereby constituting an insoluble pool of non-extractable sialoglycoproteins without destruction of the membrane. They can also constitute a hydrophilic soluble pool secreted by the cells and present in the blood and urine.

It should be noted that for certain tumor types such as lung or bladder, the existence of phCG activity is correlated with the stage of tumor progression (Yoshimura et al., Cancer 1994, Norman et al.
Endocrinol. 1985; 23: 25-34; Olivier et al. Molecular Biotherapy 1988; All of these data indicate that immunological targeting of sshCG or its fragments may be of particular interest in the treatment of certain types of cancer. Preclinical studies in mice (Acevedo et al.

Cancer Detection and Prevention 1987; Supp. 1: or in the rat (Kellen et al., Cancer 1982, respectively showed the efficacy of DT-CTP37 or TT-phCG conjugates on the growth of a lung tumor (Lewis LL / 2 carcinoma) or on the appearance of breast tumor metastases (R3230).

 The use of a carrier protein named P40, derived from the outer membrane type A (or OmpA for Outer Membrane protein type A Klebsiella

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 pneumoniae has been described in the patent applications WO 95 27787 and WO 96 14415 as making it possible to increase the immune response, in particular of the humoral type, when this one was associated with an immunogen derived from a viral protein of the respiratory syncytial virus ( RSV).

 Surprisingly, the authors of the present invention have demonstrated that the association between an enterobacteria OmpA protein and a compound involved in tumor cell proliferation or in fertility could not only induce a specific antibody response of the compound and high in the second injection with low concentrations of associated products injected, but moreover this association resulted in a significant decrease in the tumor mass in treated animals, greater than the decrease obtained with a carrier compound such as diphtheria toxin.

 Thus, the present invention relates to the use of an enterobacteria OmpA protein or a fragment thereof, associated with an immunogen selected from cytokines or their receptor, growth factors or their receptor, hormones or their receptor and / or tumor-specific markers, a fragment thereof or an analogue thereof, for the preparation of a pharmaceutical composition for improving the immunological response against said immunogen, especially in the absence of adjuvant of the immunity.

 Among the cytokines, growth factors and / or hormones, mention may be made in particular of, but not limited to: cytokines and / or growth factors and their receptor involved in tumor cell proliferation and / or specific markers tumors such as for example: G-CSF, IL-6 receptor, IGF-1, IGF-2, EGF, FGF, VEGF, CEA, PSA; - Growth hormones or hormones involved in fertility, such as La - hCG; as well as any peptide whose amino acid sequence has at least 80% homology with the sequences of said cytokines, growth factors and / or hormones according to the invention.

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 In the present invention, the term "protein" will also be understood to mean peptides or polypeptides and the term OmpA (for Outer Protein Membrane), the proteins of the outer membrane of type A.

 By fragment of an OmpA protein, is meant in particular any amino acid sequence fragment included in the amino acid sequence of the OmpA protein which, when associated with an immunogen, is capable of generating or increasing an immune response, especially humoral, directed against said immunogen and comprising at least 5 amino acids, preferably at least 10 amino acids or more preferably at least 15 amino acids.

 By fragment of a cytokine, of a growth factor or of a hormone, is meant in particular any amino acid sequence fragment included in the amino acid sequence of the cytokine, the growth factor or the the hormone which, alone or in combination with an adjuvant of the immunity, is capable of inducing an immune response, in particular a humoral response, directed against said fragment, comprising at least 5 amino acids, preferably at least 10 amino acids, or more preferably at least 15 amino acids of the amino acid sequence of said cytokine, said growth factor or said hormone.

 By analog of a cytokine type immunogen, growth factor or hormone, is meant in particular in the present description a compound having a structural analogy with said immunogen, capable of inducing an immunological response directed against said immunogen in a previously organism immunized with said analog.

 In a particular embodiment, the invention comprises the use of an enterobacterium OmpA protein or a fragment thereof according to the invention, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained by a process of extraction from a culture of said enterobacterium.

 The methods for extracting bacterial membrane proteins are known to those skilled in the art and will not be developed in the present description. For example, but not limited to the extraction method described by Haeuw J.F. et al. (Eur J. Biochem, 255, 466-454, 1998).

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 In another preferred embodiment, the invention also comprises the use of an enterobacterium OmpA protein or a fragment thereof according to the invention, characterized in that said enterobacterium OmpA protein, or the one of its fragments is obtained recombinantly.

 The methods of preparation of recombinant proteins are now well known to those skilled in the art and will not be developed in the present description, it will nevertheless be possible to refer to the method described in the examples. Among the cells that can be used for the production of these recombinant proteins, it is of course necessary to mention 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 Op Biotechnology 4: 538-542), as well as animal cells, in particular cell cultures. mammals (Edwards CP and Aruffo A., 1993, Current applications of COS cell based transient expression systems, Curr Op Op Biotechnology 4: 558-563) but also insect cells in which one can use methods using Baculoviruses, for example (Luckow VA, 1993, Baculovirus systems for the expression of human gene products, Curr Op 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, or one of its fragments, comprises: a) the sequence of amino acids of sequence SEQ ID N 2; b) the amino acid sequence of a sequence having a homology of at least
80, preferably 90%, with the sequence SEQ ID N 2; or c) the amino acid sequence of a fragment of at least 5 amino acids, preferably at least 10 amino acids, or more preferably at least 10 amino acids,
15 amino acids, of a sequence as defined in a) or b).

 By amino acid sequence of a sequence having a homology of at least 80% with a defined amino acid sequence, it is meant to designate a

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 sequence which after alignment with said determined sequence comprises at least 80% of amino acids in common with said determined sequence.

 The present invention also relates to the use according to the invention, characterized in that said immunogen is coupled by covalent bond, in particular by chemical coupling, with said OmpA protein or one of its fragments.

 The invention further comprises the use according to the invention, characterized in that said immunogen is selected from peptides.

 In a particular embodiment, the use according to the invention is characterized in that one or more binding elements are introduced into said OmpA protein, or one of its fragments, and / or in said immunogen to facilitate the chemical coupling, preferably, said introduced connecting element is an amino acid.

 According to the invention, it is possible to introduce one or more linking elements, in particular amino acids, to facilitate the coupling reactions between the OmpA protein, or one of its fragments, and the said immunogen. The covalent coupling between the OmpA protein, or one of its fragments, and said immunogen according to the invention can be carried out at the N- or C-terminal end of the OmpA protein, or one of its fragments. The bifunctional reagents allowing this coupling will be determined according to the end of the OmpA protein, or one of its fragments, chosen to perform the coupling and the nature of said immunogen to be coupled.

 In another particular embodiment, the use according to the invention is characterized in that said hybrid protein obtained after coupling between said immunogen and said OmpA protein, or one of its fragments, is prepared by genetic recombination.

 The conjugates derived from a coupling to said OmpA protein, or one of its fragments, may be prepared by genetic recombination. The chimeric or hybrid protein (conjugate) may be produced by recombinant DNA techniques by insertion or addition to the DNA sequence coding for said OmpA protein, or a fragment thereof, of a sequence coding for said immunogen of a protein nature.

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 Methods for synthesizing hybrid molecules include methods used in genetic engineering to construct hybrid polynucleotides encoding the desired polypeptide sequences. For example, the technique for obtaining genes coding for fusion proteins described by D.V. Goeddel (Gene expression technology, Methods in Enzymology, vol 185, 3-187, 1990) can be advantageously referred to.

 In another aspect, the invention relates to the use according to the invention, characterized in that the pharmaceutical composition contains a vector comprising a nucleic construct coding for said hybrid protein or a host cell transformed by said vector capable of expressing said protein hybrid.

 In a particular embodiment of the invention, said nucleic construct contained in said vector comprises a nucleic sequence chosen from the sequence SEQ ID No. 1, one of its fragments having at least 15 consecutive nucleotides of the sequence SEQ ID No. 1 , or a sequence having a homology of at least 80% with one of said sequences.

 By nucleic sequence of a sequence having a homology of at least 80% with a determined nucleic sequence, is meant a sequence which after alignment with said determined sequence comprises at least 80% nucleotides in common.

 The present invention particularly relates to the use according to the invention, characterized in that the immunogen is the hormone sshCG, or a fragment thereof, in particular the amino acid sequence peptide TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

 The subject of the invention is also the use according to the invention, characterized in that the immunogen is a cytokine or a growth factor involved in the proliferation of tumor cells.

 The invention further relates to the use according to the invention, for the preparation of a vaccine-type pharmaceutical composition intended to prevent or treat cancers such as pancreatic cancer, colon cancer, prostate cancer, bladder, lung or any other tumor.

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 In another aspect, the invention relates to the use according to the invention, characterized in that the immunogen is a hormone, or one of its fragments, involved in fertility.

 Thus, the present invention includes the use according to the invention for the preparation of a vaccine-type pharmaceutical composition for contraception.

 The invention also relates to a pharmaceutical composition characterized in that it comprises an enterobacterium OmpA protein or one of its fragments, in particular Klebsiella pneumoniae, combined with an immunogen selected from cytokines, growth factors and / or hormones, or one of their fragments.

 The invention furthermore relates to a pharmaceutical composition characterized in that it comprises a nucleic construct coding for an enterobacteria OmpA protein, or a fragment thereof, in particular of Klebsiella pneumoniae, associated with a nucleic construct coding for an immunogen peptide selected from cytokines, growth factors and / or hormones, or a fragment thereof.

 In a preferred embodiment of the invention, the immunogen is the hormone (3hCG, one of its fragments or analogous fragments, especially the peptide of amino acid sequence TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ.

 Of course, and in the same manner as described above for the uses according to the invention, said pharmaceutical composition may comprise said OmpA protein, or one of its fragments, and said associated immunogen in identical forms such as mixed, coupled, in the form of recombinant hybrid proteins or of a host cell capable of expressing said hybrid protein.

 The invention also comprises the use according to the invention, or pharmaceutical composition according to the invention, characterized in that said pharmaceutical composition is conveyed in a form making it possible to improve its stability and / or its immunogenicity, preferably in vivo.

 Preferably, said vehicle is a liposome, a viral vector containing a nucleic construct coding for said OmpA protein, or one of its fragments, said immunogen, or for said hybrid protein, or a transformed host cell.

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 capable of expressing said OmpA protein, or a fragment thereof, said immunogen, or said hybrid protein.

 Finally, the invention comprises the use or pharmaceutical composition according to the invention, characterized in that said pharmaceutical composition comprises an adjuvant of the immunity, mixed with or coupled to said OmpA protein, immunogenic audit, or one of their fragments, or to said hybrid protein.

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

Figure legends: Figure 1: Schematic representation of the heterodimeric structure of hCG.

Figure 2: Mass spectrum (ES-MS) of the purified peptide [ss-hCG (109-145)].

 FIGS. 3A and 3B: by SDS-PAGE electrophoresis (10% polyacrylamide gel) of the rP40-CTP37 (FIG. 3A) and DT-CTP37 (FIG. 3B) conjugates.

3A: - lane 1: standard molecular weights, - lane 2: rP40, - lane 3: rP40 activated with MCS, - lane 4: conjugate rP40-CTP37 before dialysis, - lane 5: conjugate rP40-CTP37 after dialysis, lane 6: conjugate rP40-CTP37 after purification and concentration.

3B: - lane 1: standard molecular weights, - lane 2: DT, - lane 3: dialysed DT, - lane 4: DT activated with MCS, - lane 5: DT-CTP37 conjugate before dialysis, - lane 6: DT-CTP37 conjugate after dialysis, lane 7: DT-CTP37 conjugate after purification and concentration.

 Figure 4: Humoral response anti-CTP37 induced after injection of variable doses of rP40-CTP37 in the C57 / B16 mouse.

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 FIG. 5: Anti-CTP37 humoral response induced after injection of 200 g of rP40-CTP37 conjugate in the absence or in the presence of adjuvant in the C57 / B16 mouse. Figure 6: Comparison of the anti-CTP37 IgG response in presensitized or non-presensitized mice with Klebsiella pneumoniae bacteria.

 FIGS. 7A and 7B: Evaluation of the Antitumor Activity of the rP40-CTP37 Conjugate in Non Presensitized C57 / B16 Mouse (FIG. 7A) or Presensitized Mouse (FIG. 7B) with Klebsiella pneunoniae before Treatment and Tumor Transplantation.

Example 1 Cloning of the rP40 Gene
The gene coding for the Klebsiella pneumoniae P40 protein was obtained by PCR amplification from genomic DNA of Klebsiella pneumoniae IP 1145 (Nguyen et al., Gene, 1998). The gene fragment encoding ptotein P40 is inserted into various expression vectors, in particular a vector under the control of the Trp operon promoter. The nucleotide sequence and the peptide sequence of rP40 are respectively represented by the sequence SEQ ID No. 1 and SEQ ID No. 2 of the list of sequences below. An E. coli K12 producing strain was transformed with a pvaLP40 expression vector. The recombinant P40 protein, referred to herein as rP40, is produced as an inclusion body with a high yield (> 10%, g protein / g dry biomass). This example is only an illustration of the expression of rP40, but it can be extended to other bacterial strains as well as other expression vectors.

Example 2: Process for Fermentation of Fusion Proteins rP40
In an Erlenmeyer flask containing 250 ml of TSB medium (Tryptic Soy Broth, Difco) containing ampicillin (100 g / ml, Sigma) and Tetracycline (8 g / ml, Sigma) was inoculated with the E. coli strain. recombinant described above.

Incubate overnight at 37 C and 200 ml of this culture are used to seed
2 liters of culture medium in a fermenter (Biolafitte, France). In a fairly conventional manner, the culture medium may be composed of chemical agents, supplemented with vitamins, yeast extracts known to have a high density growth of bacterial cells.

 Controlled parameters during fermentation are: pH, agitation, temperature, oxygenation rate, combined feed Glycerol or

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 Glucose. In general, the pH is regulated at 7.0, the temperature is fixed at 37 C. Growth is controlled by feeding glycerol (87%) at a constant rate (12 ml / h) to maintain the voltage signal of dissolved oxygen at 30%. When the turbidity of the culture (measured at 580 nm) reaches the value of 80 (after about 24 hours of culture), the production of the proteins is triggered by addition of indole acrylic acid (IAA) to the final concentration of 25 mg / 1. About four hours after induction, the cells are harvested by centrifugation. The amount of wet biomass obtained is about 200 gr.

Example 3: Process for Extraction and Purification of the rP40 Protein Extraction of the rP40
After centrifugation of the culture broth (4000 rpm, 10 min, 4 C), the cells are resuspended in 25 mM Tris-HCl buffer pH 8.5. Insolubles or inclusion bodies are obtained after treatment with lysozyme (0.5 g / liter, 1 hour room temperature / gentle agitation). The inclusion body pellet obtained by centrifugation (15 min at 10,000 g at 4 ° C.) is taken up in 25 mM Tris-HCl buffer at pH 8.5 and 5 mM MgCl 2 and then centrifuged (15 min at 10,000 g). ).

 The inclusion bodies are solubilized at 37 ° C. for 2 hours in a 25 mM Tris-HCl pH 8.5 buffer containing 7 M urea (denaturing agent) and 10 mM Dithiothreitol (reduction of disulfide bridges). Centrifugation (15 min at 10,000 g) removed insoluble particles.

 It is then resuspended in 13 X volumes of 25 mM Tris-HCl pH 8.5 buffer containing NaCl (8.76 g / l) and Zwittergent 3-14 (0.1%, w / v). The solution is left overnight at room temperature with gentle stirring in contact with the air (promotes the renaturation of the protein by dilution and reoxidation of the disulfide bridges).

 Purification of the rP40 protein a) Anion exchange chromatography step.

After a new centrifugation, the solution is dialyzed against a buffer
25 mM Tris-HCl pH 8.5 containing 0.1% Zwittergent 3-14 (100 X buffer volumes) overnight at 4 C.

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 The dialysate is deposited on a column containing a strong anion exchanger medium (Biorad Macro Prep High Q gel) equilibrated in the buffer described above at a linear flow rate of 15 cm / h. The proteins are detected at 280 nm. The rP40 protein is eluted, with a linear flow rate of 60 cm / h, at a concentration of 0.2 M NaCl in 25 mM Tris-HCl buffer, pH 8.5; 0.1% Zwittcrgent 3-14. b) cation exchange chromatography step.

 The fractions containing the rP40 protein are pooled and concentrated by ultrafiltration using an Amicon stirred cell system used with a YM10 type Diaflo membrane (cutoff threshold 10 kDa) for volumes of the order of 100 ml. , or using a Minitan Millipore tangential flow filtration system used with membrane plates with a 10 kDa cutoff for higher volumes. The fraction thus concentrated is dialyzed overnight at 4 ° C. against a 20 mM citrate buffer pH 3.0, 0.1% Zwittergent 3-14.

The dialysate is deposited on a column containing a strong cation-exchange-type support (Biorad Macro Prep High S gel) equilibrated in 20 mM citrate buffer pH 3.0, 0.1% Zwittergent 3-14. The rP40 protein is eluted (61 cm / h speed) at a concentration of 0.7 M NaCl. The electrophoretic profiles show a degree of purity of the order of 95%. The state of the protein is followed by SDS-PAGE. According to its denatured or native form, the P40 protein extracted from the membrane of Klebsiella pneumoniae has a characteristic electrophoretic behavior (migration). The native form (ss-sheet structure) has in fact a lower molecular weight than the denatured form (structure in helices a) under the action of a denaturing agent, such as urea or guanidine hydrochloride, or by heating at 100 C in the presence of SDS). The rP40 protein is not correctly renatured at the end of renaturation, whether this is carried out in the absence or in the presence of 0.1%; (p / v) Zwittergent 3-14. On the other hand, a complete renaturation is obtained after dialysis against a buffer 25 mM Tris / HCl pH 8.5 containing 0.1% (w / v)
Zwittergent 3-14. However, it should be noted that this renaturation is obtained only when the dilution step and the treatment at room temperature are themselves carried out in the presence of Zwittergent 3-14 (negative results in the absence of detergent).

 Example 4 Preparation of the fragment CTP37 derived from the sshCG

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T109CD D PRFQ DSSSSKAPPPSLPSPSRLPG PS DTPI LPQ 145
Il est obtenu par synthèse chimique sur phase solide et il est destiné à une conjugaison chimique à l'aide d'un réctif de couplage hétérobifonctionnel sur différentes protéines porteuses d'antigènes (protéine P40 et toxoïde diphtérique (DT)). Contrairement à l'usage, ce peptide ne nécessite pas l'addition d'une cystéine supplémentaire pour le couplage univoque dans la mesure ou la Cys110 présente du côté N-terminal peut être utilisée à cet effet. The peptide CTP37 is a peptide of 37 amino acids which corresponds to the C-terminal fragment 109-145 of the phCG (ss human chorionic gonadotropin) of sequence:

T109CD D PRFQ DSSSSKAPPPSLPSPSRLPG PS DTPI LPQ 145
It is obtained by solid-phase chemical synthesis and is intended for chemical conjugation using a heterobifunctional coupling reagent on different antigen-bearing proteins (P40 protein and diphtheria toxoid (DT)). In contrast to use, this peptide does not require the addition of additional cysteine for unambiguous coupling as the Cys110 present on the N-terminal side can be used for this purpose.

 The peptide was synthesized using a C-side solid-side peptide synthesizer to the N-terminal side (FMOC chemistry at 0.1, 0.25 or 1.0 mmol). The CTP37 peptide is synthesized from a glutamine preloaded on a HMP type resin, which after cleavage makes it possible to obtain a free acid function on the C-terminal side or a Rink amide MHBA type resin which, after cleavage, is used. to obtain an amide function on the C-terminal side. The reactive functions of the side chains of the amino acids used are protected by groups compatible with FMOC chemistry [Cys (Trt); Arg (Pmc); Gln (Trt); Lilies (Boc); Ser (tBu); Thr (tBu)]. A coupling cycle is carried out as follows: deprotection of the N-terminal amine function of the first amino acid with piperidine, activation of the acid function of the second amino acid to be coupled using HBTU / NMP and coupling.

 The CTP37 sequence has the particularity of containing 10 prolines on 37 amino acids. Prolines are known to form elbows in polypeptide sequences and thus strongly influence the secondary structure of these sequences (E.P. Rock et al., Am J Reprod Imm, 1996, 35, 156-162). From the synthesis point of view, this implies particular difficulties. To overcome this difficulty, particularly at the level of the 3 successive Pro124,125,126, we have introduced three double coupling steps for Pro124, Ala123 and Lys122 in order to optimize the quality of the crude peptide. In addition, all coupling cycles are followed by a

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 capping step using acetic anhydride to also improve the quality of the crude peptide and thus facilitate purification.

 At the end of the synthesis, the peptide is cleaved from the resin and the side chains are deprotected by reaction with a water / TFA mixture.

 The crude peptide is purified by semi-preparative reverse phase HPLC.

 The homogeneity of the purified peptide is verified by reverse phase IIPLC and capillary electrophoresis (FZCE). The theoretical structure is confirmed by comparing the mass compatibility measured by ES-MS type mass spectrometry with the mass calculated from the theoretical amino acid sequence to the nearest dalton (FIG. 2).

Characteristics of the CTP37 Peptide - Synthesis: Theoretical peptide-resin weight at the end of synthesis: 650 mg Weight measured after drying with nitrogen: 642 mg Mass of crude peptide cleaved after freeze-drying: 352 mg (75% of net peptide quantity, 77% % yield).

 Purification: Mass of purified peptide after lyophilization: 269 mg (75% net quantity, ie 58% yield).

Characterization: Homogeneity of the purified peptide:> 99% (RP-HPLC, UV 210 nm)
91% (PZCE / Microcoat ™, UV 200 nm).

 - Mass spectrometry (ES-MS): Calculated mass (C167H264N46O58S): 3876.28 Da Measured mass: 3876.00 Da 0.06 - Abbreviations: DT: Diphtheria toxoid; ES-MS: Electrospray - Mass spectrometry; FMOC: fluorenylmethoxycarbonyl; FZCE: Free Zone capillary electrophoresis (Free Zone Capillary Electrophoresis); HBTU: 2- (1H-Benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate; HF: hydrofluoric acid; HMPA: 4-hydroxymethylphenoxyacetic acid (resin); NMP: N-methyl-2-pyrrolidone;

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 MBHA: p-methylbenzydrylamine (resin); Pmc: 2,2,5,7,8-Pentamethylchroman-6sulfonyl; RP-HPLC: Reverse Phase High Performance Liquid Chromatography; tBOC: tbutyloxycarbonyl; tBu: tertiary butyl; TFA: tritluoroacetic acid; Trt: trityl; UV: ultraviolet.

Example 5 Synthesis, Purification and Characterization of Conjugates rP40-CTP37 and DT-CTP37 Synthesis and Purification of Conjugate rP40-CTP37
Coupling of the peptide is performed using the MCS reagent, or 6maleimidocaproic acyl N-hydroxysuccinimide ester (Lee et al., 1980, Mol Immunol 17, 779-756). This heterobifunctional reagent allows an activation of the Lysine residues of the protein, then a coupling of the peptide by the free thiol group of the Cysteine residue carried by the peptide CTP37 (Cys in position 2).

 Firstly, the rP40 protein is dialyzed against a 0.1M sodium phosphate buffer pH 6.6 containing 0.1% Zwittergent 3-14 overnight at +4 C. The dialyzed protein is then activated using the MCS reagent added at a rate of 340 g per mg of rP40, then the whole is stirred for 2 hours at room temperature and in the dark.

 The activated rP40 is desalted by molecular sieving chromatography on Pharmacia TMSephadex G75 support. The elution of the protein is carried out using a 0.1 M sodium phosphate buffer pH 6.6 containing 10 mM EDTA and 0.1% Zwittergent 3-14. The fractions containing the activated rP40 are pooled.

 For coupling, the activated protein solution is added to the lyophilized peptide at 1 mg peptide / mg rP40. After saturation under a stream of nitrogen, the mixture is stirred overnight at room temperature and in the dark.

After dialysis against a 0.1M sodium phosphate buffer pH 6.6 containing 0.1% Zwittergent 3-14 for 24 hours at +4 ° C., the unbound CTP37 peptide is removed by molecular sieving chromatography on a support column.
Pharmacia TMSephadex G50 eluted by the aforementioned buffer. The fractions containing the rP40-CTP37 conjugate are pooled, and the solution is concentrated by

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 ultrafiltration using an Amicon concentration cell on a YM10 filter (10 kDa cut-off).

Synthesis and purification of DT-CTP37 conjugate
After dialysis against an O, 1M pH 6,6 sodium phosphate buffer overnight at +4 ° C., the DT protein (SBL Vaccine AB, batch n U235) is activated using the MCS reagent added at a rate of 416 g per ml. mg of DT. The whole is then stirred for 1 hour at room temperature and in the dark.

 The activated protein is desalted by molecular sieving chromatography on Pharmacia TMSephadex G75 support. The elution is carried out using a 0.1M sodium phosphate buffer pH 6.6 containing 10 mM EDTA. Fractions containing activated DT are pooled.

 For coupling, the activated protein in solution is added to the lyophilized peptide at 1.25 mg peptide / mg DT. After saturation under a stream of nitrogen, the mixture is stirred overnight at room temperature and in the dark.

 After dialysis against a 0.1 M sodium phosphate buffer pH 6.6 for 24 hours at +4 ° C., the unbound CTP37 peptide is removed by molecular sieving chromatography on a Pharmacia TMSephadex G50 support column eluted with the same buffer. The fractions containing DT-CTP37 conjugate are pooled, and the solution is concentrated by ultrafiltration using an Amicon concentration cell on a YM10 filter (10 kDa cutoff).

Characterization of Conjugates, rP40-CTP37 and DT-CTP37
The rP40-CTP37 and DT-CTP37 conjugates are analyzed by SDS-PAGE electrophoresis (FIGS. 3A and 3B). An assay of the amino acids liberated after hydrolysis (6N HCl) is carried out by HPLC (Waters Pico Tag method) after derivatization using the PITC reagent. This assay makes it possible to determine the protein concentration of the conjugate solutions, and to precisely define the number of CTP37 peptides fixed per mole of rP40 or DT protein as well as the fixed peptide concentration.

 All the results obtained are collated in Table 1 below:

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Table 1: Characterization of the conjugates rP40-CTP37 and DT-CTP37

<Tb>
<tb> Conjugate <SEP> [protein] <SEP> mol <SEP> CTP-37 <SEP> / <SEP> [CTP37 <SEP> fixed] <SEP> CTP37 / portcur <SEP>% <SEP> CTP37
<tb> mg / ml <SEP> mol <SEP> carrier <SEP> mg / ml <SEP> (p: <SEP> p) <SEP> fixed
<tb> rP40-CTP37 <SEP> 5.4 <SEP> 12 <SEP> 3 <SEP> 1.25 <SEP> 55.6
<tb> DT-CTP37 <SEP> 8.4 <SEP> 10 <SEP> 3.1 <SEP> 0.59 <SEP> 37.3
<Tb>
Example 6 Immunogenicity of Adjuvated rP40-CTP37 Conjugates
Increasing concentrations of rP40-CTP37 conjugate are administered subcutaneously at the base of the neck to batches of six six week old C57B1 / 6 mice in the presence of an adjuvant mixture of N-acetylmuramyl-L-alanyl-Disoglutamine and squalene mannide monooleate. The mice receive four immunizations, respectively at OJ, J7, J14, and J22. The humoral response developed by these animals against the CTP37 peptide coupled to the rP40 carrier protein is evaluated, 7, 14, 22 and 35 days after the first immunization. The animals are also punctured at 0 to verify their seronegativity towards the peptide CTP37 and one when injected with a mixture of PBS (phosphate buffered saline) and adjuvant is added as a negative control.

 The results presented in Figure 4 show that rP40 behaves as a CTP37-bearing protein against which it induces a specific and high antibody response at the lowest concentrations of injected conjugate.

The response seems to be almost maximal from the second injection, whatever the dose. The level of antibodies reaches its maximum for a concentration of 100 g of conjugate.

Example 7 Immunogenicity of Non-Adjuvated rP40-CTP37 Conjugates
The rP40-CTP37 conjugate was also tested at the highest dose in the absence of adjuvant (Figure 5).

 The data presented in FIG. 5 show that, in the absence of adjuvant, the humoral response reached, after 4 immunizations, values of the same order as those observed in the presence of adjuvant. These results highlight the effectiveness of the

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 rP40 carrier to induce a humoral response against an antigen of protein nature in the absence of adjuvant.

Example 8 Immunogenicity of Conjugates After Sensitization with Klebsiella pneumoniae
Since the rP40 protein is isolated from the bacterium Klebsiella pneumoniae which is a pathogen of the human respiratory tract, the entire population has antibody titers against rP40. In order to mimic the human situation, C57B1 / 6 mice are presensitized twice 7 days apart with Kp intranasally before being immunized subcutaneously with a dose of 200 g of rP40- conjugate. CTP37.

 The results presented in FIG. 6 show that a presensitization of the mice with Klebsiella pneumoniae, which results in the existence in these animals of an anti-carrier titre of the order of 3 loglo, has no influence on the intensity of the response directed against CTP37. These results suggest a lack of epitopic suppression when rP40 is used as a carrier unlike data published with other carriers such as TT (Kaliyaperumal A and Eur.J.

Immunol. 1995; 25: 3375-3380).

Example 9: Tumor regression induced by rP40-phCG conjugates
In order to evaluate the efficacy of a rP40-CTP37 conjugate vaccination on the evolution of tumor growth in vivo, a positive line for sshCG, the Lewis carcinoma model, in the C57BI / 6 mouse , was selected after verification, by immunolabeling techniques, of the positivity of the cells injected into the animal for the phCG.

 For this experiment, the mice are immunized on day 0, day 7, day 14 and day 21 with 200 g of rP40-CTP37 conjugate. After verification of the anti-CTP37 antibody titer on D35, the mice receive, on D39, 106 LL / 2 cells by subcutaneous injection. 17 days after implantation of the cells, the mice are sacrificed and the tumors weighed individually. DT-CTP37 conjugate is used as reference control.

 The two experiments presented in FIGS. 7A and 7B show that the induction of an immune response against CTP37 by injection of rP40-CTP37 results in a significant decrease in the tumor mass in the treated animals.

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This response is specifically related to the targeting of a phCG activity of the tumor cell since the mice immunized with P40 alone in the presence of the adjuvant mixture do not show a significant reduction in the tumor mass at the end of the experiment. Presensitization of mice by two intranasal administrations of Klebsiella pneunoniae has no influence on the impact of rP40CTP37 treatment. These results are in agreement with the observations made above (FIG. 6) concerning the similarity of the anti-CTP37 IgG titers in the mice presensitized or not by Klebsiella pneunoniae.

 On the other hand, compared to the PBS + adjuvant control, the rP40-CTP37 conjugate is more effective than the DT-CTP37 conjugate.

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 SEQUENCE LIST (1) GENERAL INFORMATION: (i) DEPOSITOR: (A) NAME: FABRE MEDICAMENT (B) STREET: 45 PLACE ABEL GANCE (C) CITY: (E) COUNTRY: (F) POSTAL CODE: CEDEX (ii) TITLE OF THE INVENTION: Klebsiella pneumoniae OmpA protein associated with the hCG hormone P chain or a compound involved in tumor cell proliferation or fertility, useful in a pharmaceutical composition.

 (iii) NUMBER OF SEQUENCES: (iv) COMPUTER-DEPENDABLE FORM: (A) SUPPORT TYPE: Floppy disk (B) COMPUTER: Compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE : PatentIn Release fil.0, Version # 1.30 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: base pairs (B) TYPE: (C) NUMBER DE BRINS: simple (D) CONFIGURATION: linear (ii) TYPE OF MOLECULE: cDNA (vi) ORIGIN: (A) ORGANISM: Klebsiella pneumoniae (ix) CHARACTERISTIC: (A) NAME / KEY: exon (B) LOCATION: 1. .1032 (ix) CHARACTERISTIC: (A) NAME / KEY: intron (B) LOCATION: 1033..1035

<Desc / Clms Page number 21>

(ix) CHARACTERISTIC: (A) NAME / KEY: CDS (B) LOCATION: 1..1032 (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1: ATG AAA GCA ATT TTC GTA CTG AAT GCG GCT CCG AAA GAT AAC ACC TGG 48 Met Lys Ala Ile Phe Val Leu Asn Ala Ala Pro Lys Asp Asn Thr Trp 1 5 10 15 TAT GCA GGT GGT AAA CTG GGT TGG TCC CAG TAT CAC GAC ACC GGT TTC 96 Tyr Ala Gly Gly Lys Leu Gly Trp Ser Gln Tyr His Asp Thr Gly Phe
20 25 30 TAC GGT AAC GGT TTC CAG AAC AAC AAC GGT CCG ACC CGT AAC GAT CAG 144 Tyr Gly Asn Gly Phe Gln Asn Asn Asn Gly Pro Thr Arg Asn Asp Gln
35 40 45 CTT GGT GCT GGT GCG TTC GGT GGT TAC CAG GTT AAC CCG TAC CTC GGT 192 Leu Gly Ala Gly Ala Phe Gly Gly Tyr Gln Val Asn Pro Tyr Leu Gly
50 55 60 TTC GAA ATG GGT TAT GAC TGG CTG GGC CGT ATG GCA TAT AAA GGC AGC 240 Phe Glu Met Gly Tyr Asp Trp Leu Gly Arg Met Ala Tyr Lys Gly Ser
65 70 75 80 GTT GAC AAC GGT GCT TTC AAA GCT CAG GGC GTT CAG CTG ACC GCT AAA 288 Val Asp Asn Gly Ala Phe Lys Ala Gln Gly Val Gln Leu Thr Ala Lys
85 90 95 CTG GGT TAC CCG ATC ACT GAC GAT CTG GAC ATC TAC ACC CGT CTG GGC 336 Leu Gly Tyr Pro Asp Asp Asp Asp Asp Asp Asp Tyr Tyr Arg Arg Gly
100 105 110 GGC ATG GTT TGG CGC GCT GAC TCC AAA GGC AAC TAC GCT TCT ACC GGC 384 Gly Met Val Trp Arg Ala Asp Ser Lys Gly Asn Tyr Ala Ser Thr Gly
115 120 125 GTT TCC CGT AGC GAA CAC GAC ACT GGC GTT TCC CCA GTA TT GCT GGC 432 Val Ser Arg Ser Glu His Asp Thr Gly Val Ser Pro Val Phe Ala Gly
130 135 140
GGC GTA GAG TGG ACT GCT ACT CGT GAC CTA GCT ACC CGT CTG GAA TAC 480
Gly Val Glu Trp Ala Thr Val Arg Asp Asp Ala Thr Island Arg Leu Glu Tyr
145 150 155 160
CAG TGG GTT AAC AAC ATC GGC GAC GCG GGC ACT GTG GGT ACC CGT CCT 528
Gln Trp Val Asn Asn Gly Asp Ala Gly Thr Val Gly Thr Arg Pro
165 170 175
GAT AAC GGC ATG CTG AGC CTG GGC GTT TCC TAC CGC TTC GGT CAG GAA 576
Asp Asn Gly Met Leu Ser Leu Gly Val Ser Tyr Arg Phe Gly Gin Glu
180 185 190

<Desc / Clms Page number 22>

GAT GCT GCA CCG GTT GTT GCT CCG GCT CCG GCT CCG GCT CCG GAA GTG 624 Asp Ala Pro Ala Pro Val Pro Ala Pro Ala Pro Pro Ala Glu Val
195 200 205 GCT ACC AAG CAC TTC ACC CTG AAG TCT GAC GTT CTG TTC AAC TTC AAC 672 Ala Thr Lys His Phe Thr Leu Lys Asp Asp Val Leu Phe Asn Phe Asn
210 215 220 AAA GCT ACC CTG AAA CCG GAA GGT CAG AGC GCT CTG GAT CAG CTG TAC 720 Lys Ala Thr Leu Lys Pro Glu Gly Gln Gln Ala Leu Asp Gln Leu Tyr 225 230 235 240 ACT CAG CTG AGC AAC ATG GAT CCG AAA GAC GGT TCC GCT GTT CTG CTG 768 Thr Gln Ser Ser Asn Met Asp Pro Asp Lys Gly Ser Ala Val Val Leu
245 250 255 GGC TAC ACC GAC CGC ATC GGT TCC GAA GCT TAC AAC CAG CAG CTG TCT 816 Gly Tyr Thr Asp Arg Ile Gly Ser Glu Ala Tyr Asn Gln Gln Leu Ser
260 265 270 GAG AAA CGT GCT CAG TCC GTT GTT GAC TAC CTG GTT GCT AAA GGC ATC 864 Glu Lys Arg Ala Gln Ser Val Val Asp Tyr Leu Val Ala Lys Gly Ile
275 280 285 CCG GCT GGC AAA ATC TCC GCT CGC GGC ATG GGT GAA TCC AAC CGC GTT 912 Pro Ala Gly Lys Ser Island Arg Ala Arg Gly Glu Ser Asn Pro Val
290 295 300 ACT GGC AAC ACC TGT GAC AAC GTG AAA GCT CGC GCT GCC CTG ATC GAT 960 Thr Gly Asn Thr Cys Asp Asn Val Lys Ala Arg Ala Ala Leu Ile Asp 305 310 315 320 TGC CTG GCT CCG GAT CGT CGT GTA GAG ATC GAA GTT AAA GGC TAC AAA 1008 Cys Leu Ala Pro Asp Arg Arg Val Glu Island Glu Val Lys Gly Tyr Lys
325 330 335 GAA GTT GTA ACT CAG CCG GCG GGT TAA 1035 Glu Val Val Gln Gln Pro Ala Gly *
340 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 344 amino acids (B) TYPE: amino acid (D) CONFIGURATION: (ii) MOLECULE TYPE: (xi ) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2:

<Desc / Clms Page number 23>

Met Lys Ala Ile Phe Val Leu Asn Ala Ala Pro Lys Asp Asn Thr Trp 1 5 10 15 Tyr Ala Gly Gly Lys Leu Gly Trp Ser Gln Tyr His Asp Thr Gly Phe
20 25 30 Tyr Gly Asn Gly Phe Gln Asn Asn Asn Gly Pro Thr Arg Asn Asp Gln
35 40 45 Leu Gly Ala Gly Ala Phe Gly Gly Tyr Gln Val Asn Pro Tyr Leu Gly
50 55 60 Phe Glu Met Gly Tyr Asp Trp Leu Gly Arg Met Ala Tyr Lys Gly Ser
65 70 75 80 Val Asp Asn Gly Ala Phe Lilies Ala Gln Gly Gln Val Gln Leu Thr Ala Lys
85 90 95 Leu Gly Tyr Pro Asp Thr Asp Asp Asp Asp Asp Tyr Tyr Arg Arg Gly
100 105 110 Gly Met Val Trp Arg Ala Ser Ser Lys Gly Asn Tyr Ser Ala Ser Gly
115 120 125 Val Ser Arg Ser Asp Glu His Gly Val Ser Pro Val Phe Ala Gly
130 135 140 Gly Val Glu Trp Ala Val Thr Arg Asp Ala Thr Island Arg Leu Glu Tire 145 150 155 160 Gln Trp Val Asn Asn Gly Asp Island Ala Gly Thr Val Gly Thr Arg Pro
165 170 175 Asp Asn Gly Met Leu Ser Leu Gly Val Ser Tyr Arg Phe Gly Gln Glu
180 185 190 Asp Ala Ala Pro Ala Val Pro Ala Pro Pro Ala Pro Ala Pro Glu Val
195 200 205 Ala Thr Lys His Phe Thr Leu Lys Asp Asp Val Leu Phe Asn Phe Asn
210 215 220 Lys Ala Thr Leu Lys Pro Glu Gly Gln Gln Ala Leu Asp Gln Leu Tyr 225 230 235 240 Thr Gln Leu Ser Asn Met Asp Pro Lys Asp Gly Ser Ala Val Val Leu
245 250 255 Gly Tyr Thr Asp Arg Gly Ser Glu Ala Tyr Asn Gln Gln Leu Ser
260 265 270 Glu Lys Arg Ala Gln Ser Val Val Asp Tyr Leu Val Ala Lys Gly Ile
275 280 285
Pro Ala Gly Lys Ser Ser Ala Arg Gly Met Ser Gly Glu Asn Pro Val
290,295,300

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Thr Gly Asn Thr Cys Asp Asn Val Lys Ala Arg Ala Ala Leu Asp 305 310 315 320 Cys Leu Ala Pro Asp Arg Arg Val Glu Glu Val Lys Gly Tyr Lys
325 330 335 Glu Val Val Thr Gin Pro Ala Gly
340

Claims (27)

1. Use of an enterobacterium OmpA protein or fragment thereof, associated with an immunogen selected from cytokines or their receptor, growth factors or their receptor, hormones or their receptor and / or specific markers tumor, a fragment thereof, or an analogue thereof for the preparation of a pharmaceutical composition for improving the immunological response against said immunogen.  2. Use according to claim 1, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained by a method of extraction from a culture of said enterobacterium.  3. Use according to claim 1, characterized in that said enterobacterium OmpA protein, or one of its fragments, is obtained recombinantly.  4. Use according to one of claims 1 to 3, characterized in that said enterobacterium is Klebsiella pneumoniae.  5. Use according to claim 4, 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 N 2; b) the amino acid sequence of a sequence having a homology of at least 80% with the sequence SEQ ID N 2; or c) the amino acid sequence of a fragment of at least 5 consecutive amino acids of a sequence as defined in a) or b).  6. Use according to one of claims 1 to 5, characterized in that said immunogen is coupled or mixed with said OmpA protein or a fragment thereof.  7. Use according to claim 6, characterized in that said immunogen is coupled by covalent bonding with said OmpA protein or one of its fragments.  8. Use according to claim 7, characterized in that the coupling by covalent bond is a coupling produced by chemical synthesis.  9. Use according to one of claims 1 to 7, characterized in that said immunogen is selected from peptides. <Desc / Clms Page number 26>  10. Use according to claims 8 and 9, characterized in that there is introduced one or more binding elements in said OmpA protein, or one of its fragments, and / or in said immunogen to facilitate the chemical coupling.  11. Use according to claim 10, characterized in that said connecting element introduced is an amino acid.  12. Use according to claim 9, characterized in that the hybrid protein obtained after coupling between said peptide immunogen and said OmpA protein, or one of its fragments, is prepared by genetic recombination.  13. Use according to claim 12, characterized in that the pharmaceutical composition contains a vector comprising a nucleic construct coding for said hybrid protein or a host cell transformed by said vector capable of expressing said hybrid protein.  14. Use according to claim 13, characterized in that said nucleic construct comprises a nucleic sequence chosen from the sequence SEQ ID No. 1, one of its fragments having at least 15 consecutive nucleotides of the sequence SEQ ID No. 1, or a sequence having a homology of at least 80% with one of said sequences.  15. Use according to one of claims 1 to 14, characterized in that the immunogen is the hormone sshCG, or a fragment thereof.  16. Use according to claim 15, characterized in that the immunogen is the peptide of amino acid sequence TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ.  17. Use according to one of claims 1 to 14, characterized in that the immunogen is a cytokine or a growth factor involved in the proliferation of tumor cells.  18. Use according to one of claims 15 to 17 for the preparation of a vaccine-type pharmaceutical composition for preventing or treating cancers.  19. Use according to one of claims 1 to 16, characterized in that the immunogen is a hormone, or one of its fragments, involved in fertility. <Desc / Clms Page number 27>  20. Use according to claim 19, for the preparation of a pharmaceutical composition of vaccina type (intended for contraception.  21. A pharmaceutical composition characterized in that it comprises an enterobacteric OmpA protein or one of its fragments, in particular Klebsiella pneumoniae, combined with an immunogen selected from cytokines, growth factors and / or hormones, or one of their fragments.  22. Pharmaceutical composition characterized in that it comprises a nucleic construct coding for an enterobacteria OmpA protein, or one of its fragments, in particular Klebsiella pneumoniae, associated with a nucleic construct coding for a peptide immunogen chosen from cytokines. , growth factors and / or hormones, or a fragment thereof.  23. Pharmaceutical composition according to claim 21 or 22, characterized in that the immunogen is phCG hormone, or a fragment thereof.  24. Pharmaceutical composition according to claim 23, characterized in that the immunogen is the peptide of amino acid sequence TCDDPRFQDSSSSKAPPPSLPSPSRLPGPSDTPILPQ.  25. Use according to one of claims 1 to 21, or pharmaceutical composition according to one of claims 21 to 24, characterized in that said pharmaceutical composition is conveyed in a form to improve its stability and / or its immunogenicity.  26. Use or pharmaceutical composition according to claim 24, characterized in that said vehicle is a liposome, a viral vector containing a nucleic construct coding for said OmpA protein, or one of its fragments, said immunogen, or for said hybrid protein. , or a transformed host cell capable of expressing said OmpA protein, or a fragment thereof, said immunogen, or said hybrid protein.  27. Use or pharmaceutical composition according to claim 26, characterized in that said pharmaceutical composition comprises an adjuvant of the immunity, mixed with or coupled to said OmpA protein, said immunogen, or one of their fragments, or to said protein hybrid.