MXPA97007638A - Chlamy vaccine - Google Patents

Abstract

The present invention relates to a novel vaccine presented for the prevention of Chlamydia infections using the outer membrane protein and two immunostimulants, 3D-MPL and QS

Description

CHLAMYDIA VACCINES The present invention relates to a vaccine formulation for the prevention of Chlamydia infections. In particular, to a formulation containing recombinant PMEP auxiliaries with QS21 and 3D-MPL. The obligate intracellular bacterium Chlamydia trachomatis infects epithelial cells of the mucosa of the conjunctiva and the urogenital tract, causing a broad spectrum of human diseases such as trachoma and genital infections which can result in long-term sequelae. Trachoma, which is endemic in several developing countries, is the leading cause in the world of preventable blindness, genital infections, which represents around 3 million cases per year in the United States, making annually infertile to 200,000 women following the salpingits of Chlamydia (1). Therefore, this pathogen is a significant public health problem and efforts have been made to establish a vaccine against Chlamydia infections in humans. Tests on vaccines performed on humans and non-human primates using the whole organism as an immunogen gave serovar-specific protection, but some of those that were vaccinated showed that the pathology associated with Chlamydia infection is immunologically mediated (3); In addition, it was shown that 57 kDa (Hsp60) of purified Chlamydia produces a pathology similar to reinfection in previously infected animals (4, 5).

This observation leads to the conclusion that protection against Chlamydia trachomatis could only be achieved by using a subunit vaccine. The species of Chlamydia trachomatis is stereotyped in 15 serovars that are placed in 2 serogroups: the B complex (serovars B, Ba, D, E, L1 and L2), the intermediate complex (serovars F, G, K, L3) and the complex C (serovars A, C, H, I and J) (6). Sexually transmitted diseases (STDs) are caused by serovars D to K that cover all 3 serogroups. Therefore, a subunit vaccine against Chlamydia STD should protect against multiple serovars that are more or less antigenically related. For the design of a subunit vaccine, much attention has been focused on the serotyping antigen which consists of the 40 kDa main outer membrane protein (PMEP). This protein, which showed that it functions in vitro as a porin (7), is present throughout the life cycle of the bacterium (8), this main surface protein is highly immunogenic in humans and animals. PMEP exhibits 4 variable domains (DV) surrounded by five constant regions surrounded by five constant regions that are highly conserved among serovars (9, 10). B-cell epitopes of neutralization in vitro and in vivo have been downloaded in DVs (11, 12, 13, 14, 15) while T-cell epitopes have been identified in both variable and constant domains (16, 17). Recombinant PMEPs have been expressed in E. coli by different authors (18 19, 20); however, Manning et al. show that their recombinant protein does not react with a monoclonal antibody that recognizes a conformational PMEP epitope (18). Immunizations with recombinant or purified PMEP followed by homotypic or heterotypic Chlamydia, the comparison has been made in different animal models with variable effects on the parameters of the infection (21, 22, 23). An elegant experimental model of salpingitis has been developed in mice in which intrauterine inoculation of a human strain of Chlamydia trachomatis leads to long-term infertility (24, 25). In a heterotypic confrontation experiment, Tuffrey and others have shown that parenteral and mucosal immunization with PEMPr absorbed on the hydrogel reduced the severity of salpingitis and the duration of colonization of the lower genital tract, respectively. However, the preparation did not confer protection against the infertility that results from the infection (23). Both cell-mediated and humoral immunity seem to play a protective role in the genital diseases caused by Chlamydia trachomatis. However, Rank's group suggests that in mice, T-cell-mediated immunity is the main mechanism for controlling chlamydia genital disease (26, 27, 28) and CD4 and CD8 positive T cells have shown to contribute to anti-viral immunity in vivo (29, 30).

De-O-acylated monophosphoryl lipid A of GB2 220 211 (Ribi) is known. Chemically it is a mixture of lipid A of deacylated monophosphoryl 3 with 4, 5 or 6 acylated chains and is manufactured by Ribi Immunochem Montana. QS21 is a purified non-toxic fraction of Hplc from a saponin from the bark of the South American tree Quillaja saponaria molina and its method of production is described (as QA21) in U.S. Patent No. 5,057,540. Vaccines comprising both QS21 and 3D-MPL are described in International Patent Application No. WO094 / 00153. The present invention provides the first time a vaccine composition that is effective in conferring protection against infertility resulting from Chlamydia infections. Accordingly, the present invention provides a cradle formulation comprising 3D-MPL, QS21 and Chlamydia PMEP. In particular, the vaccine contains PMEP of the B complex serogroup. Preferably, the vaccine contains at least one PMEP of serovar L2, but may additionally contain antigens of other serovars, such as D and E. In a preferred embodiment, QS21 is present with a sterol since said compositions show decreased reactogenicity and improves the stability of QS21 for base-mediated hydrolysis. In preferred compositions of the invention, QS21 is associated with cholesterol-containing liposome structure (hereinafter referred to as DQ). Said auxiliary compositions are described in copending U.S. Patent Applications 9508326.7 and 9513107.4, the disclosure of which is incorporated herein by reference. The antigen and the MPL are, in this preferred embodiment, outside the structure of the liposome. The preparation of the vaccine is usually described in New Trends and Developments in Vaccines, edited by Voller et al., University Park Press, Baltimore, Maryland, U.S.A. 1978. Encapsulation within liposomes is described, for example, by Fullerton, Patent of E. U .A. 4, 235,877. The conjugation of proteins to macromolecules is described, for example, by Likhite, Patent of E. U.A. 4, 372, 985 and by Armor et al., Patent of E. U.A. 4,474,757. The amount of protein in each vaccine dose is selected as an amount that induces an immunoprotective response without significant adverse side effects in those who are normally vaccinated. Said amount will vary depending on which specific immunogen is used and how it is presented. Generally, it is expected that each dose will comprise from 1 to 1000 μg, more preferably from 4 to 40 μg. An optimal amount for a particular vaccine can be assessed by normal studies involving the observation of appropriate immune responses in subjects. Following an initial vaccination, subjects may receive one or several separate reinforcement immunizations appropriately.

The formulations of the present invention can be used for both prophylactic and therapeutic purposes.

Accordingly, in one aspect, the invention provides a method of treatment comprising administering an effective amount of a vaccine of the present invention to a patient. In one embodiment of the invention, the PMEP antigen is from Serovart 2 and is produced in E. coli by recombinant DNA techniques. In these circumstances. The protein is produced without its signal sequence. In the present invention, the help of the antigen with or without MPL + QS21 strongly influenced the ratio of IgG 1: IgG2a in immunized groups, immunization with MPL + QS21 was associated with low IgG 1: IgG2a ratios and partial protection while immunization without MPL + QS21 leads to a higher IgG 1: IgG2a ratio and gave no protection. Interestingly, the change to IgG2a antibody production by the B cell is mediated by interferon gamma that is produced by the Th 1 subgroup of the T helper cells while the production of lgG1 is mediated by interleukin-4 secreted by Th2 cells (40) Since the production of IgG2a is controlled by the Th 1 cell products, it might seem likely that the protected groups showed an activation of Th 1 cells driven by MPL + QS21 In human models and in mice, the Th 1, IL-2 and IFN-gamma cytokines are generally associated with resistance to infection with pathogens intracellular while the Th2, I L-4 and IL-10 cytokines are associated with progressive disease. This can be illustrated by the resistance or susceptibility of decreasing strains of mice to greater Leishmania that correlates with the induction of specific Th 1 or Th 2 (41) response. In addition, the inerferon gamma has anticlimaid activity in vitro and is involved in the resolution of infection in vivo (42, 43). Therefore, the inmnoestimulates that drive the Th 1 cytokines may be responsible for the protection observed in this vaccine experiment. The other two observations support the hypothesis of the installation of a protective cell-mediated immunity in the vaccinated groups of PEMPr + MPL + QS21, first, the absence of specific secretory IgA in vaginal secretions and the non-neutralizing nature of the response of strong serum antibodies exclude the possibility of having a humoral protection; Secondly, the heterotypic nature of the protection obtained using two different serovars that differ considerably at the level of the PMEP sequence DVs suggests that the T cell epitopes of conserved processed PMEP domains may be the agent protecting against infertility. The following illustrates the invention. 1 . MATERIAL AND METHODS 1 .1 Chlamydia strains and mammals Strain N 11 F of Chlamydia trachomatis serovar isolated by Tuffrey et al. And kindly provided by Dr. J. Orfila and strain 434 serovar L2 of Chlamydia trachomatis (ATCC, Rockville, Md) were used in this study. Chlamydia was inoculated into Me Coy cells (ATCC, Rockville, Md) and an inclusion concentration of approximately 106 units / ml (IFU / ml) in MEM supplemented with 10% fetal calf serum (SBF) (Gigbco BRL) . After 1 h of centrifugation (1500 g) and 2 h of incubation at 37 ° C (5% CO2), the inoculum was removed and the cells were refed with fresh medium supplemented with 0.5 μg / ml cycloheximide (Sigma). After incubation at 37 ° C for 48 h, the cells were disrupted with glass beads, cultured in 250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid, pH 7.2 (SPG) to a approximate concentration of 107 to 108 IFU / ml and stored at -70 ° C. Female C3H / HeOuJ (H-2k) 6-8 week old mice of Iffa Credo (France) were obtained. Males 8 to 19 weeks of age of the same strain (B &K, U.K.) were used for progenitors. 1.2 Amplification of PCR and constructions of plasmids Amplification. L2 DNA was obtained from serovar PMEP by lysis of 10 μl of the chlamydial inoculum in 240 μl of lysis buffer and PCR amplification as previously described by Denamur et al. (33). The synthetic oligonucleotides d'GAGACTCCCATGGATCCACTGCCTGTGGGGAATCCTGC-3 'and 5'-TTAGAAGCGGAATTGTGCATTTAC-3' (SB Biologicals, Belgium) were chosen from the published sequence (34). The 5 'oligonucleotide contained the nucleotide sequence encoding the amino terminus of the mature PMEP (underlined) preceded by a restriction endonuclease site of BamHl (bold). The amplification was carried out using Pfu DNA polymerase (Stratagene) and a Koch Light NBS Thermal Cycler (New Brunswick). A properly sized PCR product was purified from a 1% agarose gel using a Geneclean II kit (Bio 101). 1.3 Cloning The amplified ADM PMEP L2 serovar was blunted at one end with the Klenow fragment of DNA I polymerase, ligated into pGEM4Z (Promega) previously digested with Smal and transformed into E. coli JM109 using the normal CaCL2 protocol. Restriction analysis was carried out on resulting clones and a straight DNA construct was amplified and purified using a PC-100 nucleounion kit (Machery-Nagel). The PMEP DNA was then excised from pGEM4Z-PMEP by digestion with BamHI and in place of pET15 digested with BamHI (Novagen) downstream of the lilac promoter and the His.Tag sequence. Straight clones were selected by restriction analysis after transformation in the E. coli strain DH10B (stratagem); the complete nucleotide sequence of the cloned DNA was verified by the dideoxy chain termination method (35). A plasmid preparation of pET15-PMEP was finally used to transform strain BL21 (DE3) (Novagen) which is capable of promoting expression of the recombinant product since it has an IPTG-inducible T7 polymerase. 1.4 Production of immunogens, characterization, purification and formulation 1.4.1 Production and characterization. BL21 (DE3) bacteria transformed from pET15-PMEP were cultured in LB medium (Gibco BRL) supplemented with 200 μg / ml ampicillin (Sigma). Expression was induced by adding 1 mM IPTG when the optical density of the culture measured at 600 nm reached from 0.6 to 0.8. Cells were cultured 3H after induction, washed 3 times with PBS and used in pH buffer in the sample containing 2% SDS and 5% mercaptoethanol. The samples were heated for 3 minutes at 95 ° C and the total proteins were separated by 12% SDS-PAGE using separate molecular weight markers on the same gel (Gibco BRL). For immunostaining, proteins separated from 12% SD-PAGE were transferred into nitrocellulose and detected using mAbs L2 1-45 and L2 1-10 kindly provided by Dr. H. Caldwell and a goat anti-PMEP antiserum (Chemicon). The cellular location of the PEMPr was determined by the fractionation of cells as described in Maniatis et al. (36); the pellets and supernatants were resuspended or adjusted in sample pH buffer and analyzed as the total cell lysate. 1.4.2. Purification. A volume of 100 ml of culture induced by IPTG for 3 hrs. Was lysozyme and deoxycholate as previously described by Marston et al. (37). The cell lysate was centrifuged (12,000 g for 15 min) the pellet was resuspended in 2 ml of sample buffer from SDS PAGE containing 2% SDS but not mercaptoethanol and boiled for 3 min. The lysate was centrifuged (12,000 g for 15 min), the pellet was discarded and the supernatant was adjusted to 20 nM Tris-HCl pH 7.9, 0.5% SDS, 500 mM NaCl, 5 mM imidazole in final concentration. The sample was then loaded onto a chromatography column containing 2 ml of His resin. Bind (Novagen); metal ion affinity chromatography was then achieved according to the manufacturer's procedure. The identity and purity of the eluted product was estimated by SDS-PAGE under reducing conditions followed by staining of Coomassie blue or injected (see above). The concentration of the protein was determined by the Lowry analysis, PEMPr containing fractions were mixed and dialyzed overnight against PBS using the Cassette Cassettes from Slide A (Pierce). Formulation of the antigen. Three formulations were tested: 1) PMEP + QS21 3DMPL 2) Oil in water emulsion of PMEP + SB62 3) PMEP, SB62, QS21, 3DMPL This was carried out according to the procedure described in WO 95/17210 and / or WO94 / 01 153. PEMPr was diluted briefly purified and dialysed in PBS at 25 μg / ml (200 μl) for injection with 5 μg MPL + 10 μg QS21 or at 50 μg / ml (100 μl) to be mixed with 100 μl of oil emulsion in water named as S B62 with or if n 5 μg MPL / 10 μg QS21. For each of the formulations, the vaccines were prepared as follows: 1) MPL / QS21 formulated by adding MPL (as 100 nm particles) to PMEP antigen, followed by pH buffer and then QS21. 2) MPL / QS21 / SB62 formulated by adding antigen to H regulatory solution followed by SB62 followed by MPL as a 100 nm particle followed by QS21. In this formulation, it is thought that the antigen is outside (ie, inside the emulsion drop), the MPL is outside and most of the QS21 is outside. 3) SB62 formulated by adding SB62 to antigen in pH buffer. The antigen is outside. 1.5 Vaccine in mice model of salpingitis, fertility or serological follow-up. The groups of ten C3H / HeOuJ were immunized subcutaneously at the base of the tail with 2 x 5 μg M PL and 10 μg QS21. The inoculation was carried out in week 6 following the protocol previously described by Tuffrey and others (24). Briefly, the mice were given 2.5 mg of progesterone subcutaneously (Depo-Provera, Upjohn) 7 days before the challenge which was carried out by bilateral intrauterine inoculation with 5,105 IFU Chlamydia trachomatis N11 in 100 μl SPG or 100 μl of a Me Coy cell extract. In week 10, the treated mice were caged with 3-month-old males for fertility evaluation (1 male for 2 females, weekly male confrontation within each group). The parameters that were calculated during the reproductive period were the average number of newborn mice per group (M) and the average litter size (N). Blood was taken at 6 weeks and the serum was analyzed for antibodies specific for PEMPr, recognition and neutralization of chlamydial NI1 inclusions of the serovar F strain of in vitro heterologous infection (NI1). The vaginal washes were collected in week 6 by pipetting 50 μl of PBS in and out of the vagina several times and it was analyzed for secretory IgA antibodies specific for PEMPr. 1.6 Serological analyzes 1.6.1 Determination of anti-PEMPr antibodies. The titers of IgG or IgA specific for PEMP-r were determined using the PEMPr as antigen in an enzyme-linked immunosorbent assay (ELISA). Plates (Masisorp, Nunc) where it was coated overnight at 4 ° C with a solution of 5 μg / ml in carbonate / bicarbonate buffer of 10 mM, pH 9.6 buffer, washed with 0.1% PBS of Tween 20 (washing pH buffer) and blocked for 1 h at 37 ° C with 3% BSA PBS (Sigma). The test sera were serially diluted in the washing pH buffer solution containing 0.5% BSA (buffer solution for incubation pH) for 1 h at 37 ° C with a goat anti-goat IgG or IgA conjugated with horseradish peroxidase (Sigma ). After washing, the substrate orthophenylenediamine (Sigma) was added at room temperature for 20 minutes; the reaction was stopped by the addition of 2M H2SO4 and the absorbance at 492 nm was measured in a Multiskan Labsystems. The titration of IgG or IgA anti-PEMPr was expressed as the reciprocal of the dilution of the serum sample giving a mid-point absorbance value. For each serum sample, the specific IgG response to PEMP-r was excised at ratios of IgG2a, IgG2b and IgG1 specific for PMEMP in a direct ELISA as described above with some modifications. The test sera were incubated in triplicate. After 1 h at 37 ° C the plates were washed and incubated for 1 h with a complex of streptavidin radish peroxidase Amersham). The disclosure and its determination of titling were carried out as described above. The prevalence of each of the 3 subtypes of I gG expressed in percentage as the relationship between this titration of IgG subtype and the total of the titrations determined for the 3 subtypes. 1 .6.2. Heterotypic detection of chlamydial inclusions.

The Mac Coy cells were cultured in sterile flat-bottomed 96-well microplates (N unc) and the confluent monolayers were infected with approximately 5 IFU at 104 of the strain NI 1 serovar F of Chlamydia trachomatis, 24 hours after infection. they washed the cells with PBS and fixed 10 minutes with methanol. Washing was repeated and 100 μl of the serum samples diluted 1/100 with PBS were incubated for 1 h at 37 ° C. The plates were washed and treated with goat anti-mouse IgG conjugated with horseradish peroxidase (Sigma) for 1 h at 37 ° C. After washing with PBS. antibody binding was visualized by the addition of diaminobenzidine tetrahydrochloride (DAB), Sigma). The presence of revealed N 11 inclusions of anti-PEMPr IgG was evaluated using an inverted optical microscope. 1 .6.3 Heterotypic in vitro neutralizing activity. Complementary independent in vitro neutralization analysis was performed as described by Su et al. (38) with some modifications. Briefly, 50 μl of double dilution of SPG from sera of individual non-supplemented mice was adhered to I FU 105 of strain N I 1 serovar F diluted in 50 μl SPG. The mixture was incubated for 20 min at 37 ° C (5% CO2) and then 100 μl was inoculated into Syrian Hamster R iin cells (HaK, ATCC, Rockville, Md) washed with H BSS (Gibco BRL) and incubated for 2 h at 37 ° C, cells were fixed and the inclusions were immunochemically detected as described above using a commercial goat anti-PMEP antiserum (Chemicon) and an alkaline phosphatase conjugated rabbit anti-tear (Sigma). 5-Bromo-4-chloro-3-indolyl / nitro blue tetrazolyl phosphate (BCIP / NBT, Sigma) was used as the substrate for the enzymatic reaction. IFU were quantified by counting 5 fields at an increase of 200 x using an inverted microscope. The mean number of I FU per change obtained with the sample serum was expressed as a percentage reduction in the number of I F U med obtained with a negative control mixture that contained serum from native mice. The neutralization titers (NT 50) were calculated as the reciprocal of the dilution of the serum sample providing 50% reduction of the ineffectiveness. 1.7 Results 1.7.1 Expression and characterization of recombinant antigen A PCR amplified DNA fragment containing the nucleotide sequence for serovar L2 mature PMEP was inserted into the straight reading frame and the orientation in the expression vector of pET15; the nucleotide sequence of the chlamydia protein and the fusion junction with the polynucleotide extension encoding the 5'-terminal His-Tag peptide were as predicted by the design of the cloning strategy. After cell fractionation, the expression product was located in the insoluble fraction of E. coli which suggests that it was expressed as insoluble inclusion bodies. The pellet containing recombinant PMEP was solubilized in pH buffer solution of 2% SDS and run on a metal ion affinity chromatography column in which the immobilized nickel ions were used to chelate the histidine residues carried by the His peptide, tag fused with the recombinant PMEP. The purified protein that has been washed and eluted in pH-free buffer solutions of SDS exhibited the predicted molecular weight and was immunoreactive with monoclonal anti-PMEP and polyclonal antibodies as shown by SDS-PAGE and Western blot analysis. Respectively. After dialysis, the PEMPr concentration was between 500 μg / ml and 1 mg / ml and the purity of the recombinant product was estimated at 90%. 1. 7.2. Effect of immunization with PEMPr with ancillary serological response of mice and fertility after heterotypic confrontation. The results of the experiment designed to evaluate the prophylactic potential of recombinant PMEP differentially with helper was presented as described in Table 1. Groups 4 and 5 were immunized subcutaneously with PEMPr aided with 5 μg MPL and 10 μg of QS21; in group 4, the recombinant protein with helper was prepared in the emulsion of SB62 containing squalene and alpha tocopheron as the oil phase and Tween 80 as the surfactant. Group 6 was immunized subcutaneously with PEMPr combined with the same emulsion of SB62 as group 4 but without immunostimulants. Three control groups were also designed. A group of untreated animals (group 1) a group of mice with simulated immunization using both immunostimulant combined with emulsion of SB62 (group 2) and group 3 which was immunized as group 4 but was dedicated to imitation infection. Groups 2, 4, 5 and 6 were compared to the NI1 strain of heterotypic Chlamydia trachomatis. 1.7.3. Effect of immunization on the serological response.

In order to evaluate the immunogenicity of the different preparation, the IgG titrations were measured by serum ELISA extracted 4 weeks after the second dose of vaccine (day of confrontation) and arithmetic mean titrations (TMA) were calculated for each group. Immunization with two injections of 5 μg of PEMPr led to the appearance of high level anti-PEMPr IgG. As shown in table 1, AMT were virtually the same for groups 4 and 6 but the combination of emulsion and immunostimulants resulted in a double increase in the average IgG-specific titre for PEMPr. The animals with false immunization only with auxiliaries (group 2) did not have significant antibody titers against the recombinant chlamydia antigen. In any group, specific PEMPr secretory IgAs were detected in the vaginal washings collected just before the challenge. As shown in Table 1, a significant difference in the profile of IgG subclasses was observed between the groups containing immunostimulants (4 and 5) and group 6 that was immunized with emulsified PEMPr of SB62. It was shown that the use of MPL + QS21 significantly increases the relative level of IgG2a and decreased the relative level of IgG 1; the maximum effect of this phenomenon was achieved with the non-emulsified preparation. To evaluate whether the specific IgG for PEMPr were able to cross-react with Chlamydia of the heterotypic infectious strain, the sera were diluted to 1: 100 and tested individually in an enzyme-linked immunosorbent assay for their ability to recognize the inclusion of chlamydia in cells infected cells fixed with methanol. It was shown that all sera of mice from each immunized group contain IgG that reacts with the N 11 strain of Chlamydia trachomatis serovar F used for the challenge. Therefore, all were tested for neutralization activity independent of in vitro complement against this strain using sera from mice with false immunization as negative controls. The results were not consistent in comparison with those obtained by ELISA and immunoenzymatic analysis since none of the immune sera was able to significantly reduce the ineffectiveness of chlamydia. 1.7.4. Heterotypic immunity effect of fertility after confrontation. Eight times after the last immunization (or false immunization) with PEMPr and 4 weeks after intrauterine infection (or sham infection), the mice were mated with 2-month-old males. The result of the comparison with heterologous strain NI1 in fertility of C3H / HeOuJ mice was measured by the following parameters: the average number of newborns per group (N) and the average litter size (M). Compared with untreated mice, the fertility of mice inoculated with Chlamydia in group 2 (false immunization) were significantly altered; this result confirmed the validity of the animal model in this particular case. The animals immunized and with simulated infection (group 3) had reduced fertility parameters compared with the untreated ones; This group designed to take into consideration the non-pathological alteration of the animal's fertility as a control was used to evaluate the prophylactic potential of the PEMPr formulations. As shown in Table 1, significant differences were observed between the covaccinated groups with immunostimulants (groups 4 and 5) and group 6 that was immunized with PEMPr emulsified with SB62. Group 2 exhibited the best results with 7 of 10 mice giving birth to layers and parameters of maximum fertility; the value of N reached 50% of the value assigned to control group 3 and the value of M was comparable with that calculated for the same control group. On the contrary, the fertility parameters exhibited by group 6 lacking immunostimulants is comparable with that obtained in group 2 of infection control. Immunization of PEMPr by combining immunostimulants and the SB62 emulsion also resulted in protection against infertility but the use of the SB62 emulsion appeared to partially decrease the protection regimens. Therefore, the use of MPL plus QS was shown to offer partial protection against chlamydial infection of the upper genital tract and the maximum effect of this phenomenon was achieved with the non-emulsified preparation. 1.8 Conclusion Our results show that parenteral immunization with PEMPr aided with MPL + QS21 prevents infertility caused by a heterologous chlamydial infection of the mouse genital tract; On the contrary, the injection of PEMPr emulsified with SB62 without both immunostimulants does not induce any protection. On the other hand, all the preparations produced a specific homogeneous PMEP-specific total IgG response in the serum of immunized animals, those antibodies were able to cross-react with methanol-bound chlamydial inclusions of the heterotypic infection strain but were not able to reduce the ineffectiveness of chlamydia in vitro. Just before the confrontation, the specific IgA for PEMPr were not detectable in vaginal secretion that is consistent with the method of parenteral antigen administration. Therefore, a comparison of the total specific IgG titers or neutralization titers with the pathology result for all immunized groups did not reveal any significant correlation of any of this comparison. The results of the present investigation demonstrate that a PMEP combine with the immunostimulants of MPL + QSA21 is able to produce immune protection against infertility caused by Chlamydia trachomatis. 2. SECOND SERIES OF VARIOUS EXPERIMENTS AUXILIARY FORMULATIONS OF PMEP 2.1 Materials and methods were tested Chlamydia and strains of mice were identical to those used in the experiment described in example 1. 2.2 Production and formulation of PMEP The production and use of DNA construction pET15-PMEP for production of antigen were described in Example 1. The antigen purification protocol was modified in order to produce large amounts of endotoxin-free antigen. Briefly, 10 ml of his (Novagen) binding resin was washed with 25 volumes of 2% SDS, 6M urea in water before carrying out the purification step according to the manufacturer while 250 ml of induced ready formed in pellets by centrifugation was washed with 4M urea, 2M NaCl followed by 2% Zwitergen (Calbiochem) before solubilization in Tris-HCl with pH 7.9, 0.5% SDS, 500 mM NaCl, 5 mM imidazole. The antigen was eluted with 100 nM Imidazole Tris-HCl pH 7.9, extensively dialyzed against 5 mM Tris-HCl pH 7.4 and filtered on a 0.22 μm sterilization filter (Millipore). A test of amebocium lysate and limulus (Coatest, Chromogenix) was then used to evaluate the content of LPS. 2.3 Formulation The antigen was formulated as described in Example 1 except that the amount of MPL per dose was adjusted to 10 μg; a group using modified QS21 as described in co-pending UK applications 9508326.7, 9512107.4 (referred to as DQ) were also tested and added to the vaccine at the 10 μg per dose regimen. In greater detail, the vaccine was formulated by adding antigen to the buffer solution. MPL was then added as 100 nm particles. In a separate tube, QS21 was mixed with small unilamellar liposomes composed of dioleoylphosphatidylcholine (DOPC) and cholesterol (DOPC: cholesterol = 4: 1 w / w) so that the ratio of QS21 to cholesterol is 1: 5. (Under these conditions, QS21 was incorporated into the liposomal membrane). The mixture of QS21 / SUV (called DQ) was then added to the mixture of antigens / MPL. In this formulation, the antigen is outside, the MPL is outside, the QS21 is in liposomes. 2.4 Vaccination in the mouse model of salpingitis, fertility, immunological and histological follow-up. Immunization, experimental infection and sampling were programmed as described above except that the negative control group was falsely immunized with the emulsion containing 10 μg MPL and 10 μg of QS21 was added and that an extra group immunized with the antigen combined with MPL + DQ. The groups were composed of 15 mice; 0 of them mated during a period of 8 weeks while 5 were sacrificed 2 weeks after the confrontation for histopathological and immunocytochemical analysis. The parameters used to calculate the fertility of the group are: F (number of mice that gave one or more litters divided by the total number of mice), M (number of newborn mice (dead or alive) divided by the number of litters ) and N number of newborn mice (dead or alive) divided by the total number of mice). Serums and vaginal washes were analyzed for specific antibodies to PEMPr by ELISA, sera were also examined for the recognition of chlamydia inclusions of the NI1 strain of serovar F and neutralization of heterologous in vitro infection (NI1). All the techniques were described before. The upper middle genital tract (ovary, oviduct and upper part of the uterine tube) were embedded in the OCT compound (Tissue-TEK, Miles), the sections of instantaneous freezing and freezing (10 μm) were mounted on glass slides (Superforst, Menzal-glazer). The sections were air-dried, fixed in actone for 5 minutes and then stored at -70 ° C. For the histopathological analysis, rehydrated sections were stained with water with hematoxylin (H) and eosin (E). For immunocytochemical staining, sections were rehydrated in PBS, incubated for 60 minutes with 2 μg biotinylated anti-marathon CD4 or CD8 mAb (Serotec) in 100 μl PBS, washed 2 times with PBS and re-incubated 30 minutes with a 1/2000 dilution of HRP-estrepatavidin (Zymed). After washing, color was developed with a liquid CAB kit (Zymed), counter-assigned with hematoxylin and permanently mounted on acritol (Surgipath). 2.5 Range analysis of interferons Mice were injected subcutaneously at the base of the tail with 200 μl of formulation at weeks 0 and 2 the control group was falsely immunized with 10 μg MPL and 10 μg of QS21 combined with the emulsion. The animals were bled for serological analysis and then sacrificed in week 4, the spleens were removed aseptically, mixed and a single cell suspension was prepared for stimulation with 1 μg / ml PEMPr or with 4 / ml With Á ( Boerhinger Mannheim) as a control. Therefore, the cultures were plated in 24 well flat bottom culture plates using 106 responder cells per ml of RPMI 1640 with 10% fetal calf serum (Gibco-BRL). The supernatants cultured 96 hours after restimulation were analyzed for I FN-gamma using a commercial ELISA kit (Cytoscreen, Biosource). 2.6 Results 2.6.1. Antigen After dialysis, the concentration of PEMPr was calculated around mg / ml and endotoxins were below 0.05 EU / μg PEM Pr / 2.6.2. Effect of immunization with PEMPr aided in the humoral immune response of mice, in their fertility after heterotypic confrontation and in the inflammatory infiltrate resulting from the infection. The results of the experiment designed to evaluate the prophylactic potential of differentially assisted PEMPr are presented as described in Table 2. 2.6.3. Effect of immunization on the humoral response. The humoral response after vaccination was evaluated in serum and vaginal washings collected on the day of the confrontation. All formulations of the antigen gave similar geometric mean titers (TMG) of similar anti-PEMPr IgG of about 30,000. The animals falsely immunized with auxiliaries alone did not have significant antibody against the recombinant antigen of chlamydia. In any groups, no secretory IgA specific for PEMPr was detected in the vaginal washings collected just before the challenge. The biotyping of the specific IgG response to PEMPr revealed that the presence of MPL and QS21 or DQ increased the relative level of IgG2a when compared to the response evoked by emulsion PMEP alone. It was shown that all immune sera contain IgG that react with fixed inclusions with methanol of trachomatis strain FC serovar NI1 not used for the comparison. 2.6.4 Effect of heterotypic immunization on fertility after confrontation. To overcome the confrontation with heterologous strain NI1 in mice fertility was measured through the parameters F, N and M defined in the experimental procedures. For each group, the values of those parameters calculated on the duration of the mating period are presented in Table 1. In opposition to the group falsely infected, the infection of the immunized group falsely led to almost complete infertility, indicating that the observed infertility is induced by C. Trachomatis and not by the manipulation of the animals. Among the groups immunized with PEMPr adjuvant with both immunostimulants, the MPL + DQ formulation of the antigen led to partial protection in this group, the values of the parameters of F and N reach about 80% of those registered in the group falsely infected (positive control). On the contrary, immunization before the confrontation with PEMPr formulated in the emulsion led to very low values of the fertility parameters of F and N. 2. 6.5. Histopathological changes after the confrontation.

In the histopathological counterpart of the fertility experiment, the classical H E stain performed on tissue sections revealed no noticeable difference in ovarian oviduct inflammation classifications between falsely immunized and vaccinated groups. However, when observed at the frequency of subsets of T cells by immunocytochemical ancestry, CD4 positive T cells were only detected in the vaccinated group of MPL + DQ (4 out of 4 mice) while CD8 positive T cells were detected. they were detected in immunized groups as well as falsely immunized (table 3). Therefore, in this experiment, the positive infiltration T cells of C D4 were only found in the vaccinated group of MPL + DQ which was the only group that would be protected in the fertility counterpart of the experiment. 2.6.6 Effect of the formulation on gamma secretion of IFN on re-stimulation in vitro. Cell activation r induced by the antigens formulations (table 4) were analyzed in a separate experiment. On the one hand, the spleen cells were isolated from animals vaccinated with PEMPr combined with MPL + QS21 or MPL + DQ and in vitro were restimulated with the antigen exhibited gamma concentrations of I FN in their culture supernatants that are comparable to those stimulated during the same period with 4 μg / ml concanavalin A. On the other hand, cells isolated from animals vaccinated falsely or vaccinated with PEMPr devoid of immunostimulants did not produce detectable levels of IFN range while their counterparts co-cultured with ConA were all positive for that cytokine. The serological analysis performed in serum mixtures of each group revealed that gamma-IFN secretion was associated with an increase in the ratio of IgG2A specific for antigen. CONCLUSION Gathered, the data from the comparison assay and the IFN range screening analysis suggest that, in the mouse, the combination of the two MPL and QS21 or DQ helper with a recombinant PMEP induces a Th1-like immune response specific for a given antigen by the secretion of IFN range and raised the IgG2a ratios, which can result in protection against infertility resulting from Chlamydia infection. REFERENCES 1. Washington AE, Johnson RE and Sanders LL. Chlamydia trachomatis infections in the United States: what are they costing us. JAMA 1987, 257, 2070-2072. 2. Grayston JT and Wang SP. New knowledge of Chlamydiae and the diseases they cause. The Journal of Infectious Diseases 1975, 132: 87-105. 3. Grayston JT, Wang SP, Yeh LJ, and Kuo CC. Importance of reinfection in the pathogenesis of trachoma. Reviews of Infectious Diseases 1985, 7, 717-725 4. Morrison RP, RJ Belland, Lyng K and HD Caldwell. Chlamydial disease pathogenesis. 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Diversity of C? Lamydia trachomatis Major Guter Membrane Protein genes. Bacteriol. 1987, 169, 3879-3885. 10. Yuan Y, Zhang YX, Watkins NG, and Caldwell HD. Nucleotide and deduced amino acid sequences for the four variable domains of the major outer membrane proteins of the 15 Chlamydia trachomatis serovars. Infected Immun. 1989, 57, 1040-1049. 11. Baehr W, Zhang YX, Joseph 1, Su H, Nano FE, Evereít EK and Calwell HD. Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes. Proc. Nati Acad. Sci. USA 1988, 85, 4000-4004 12. Lucero ME and Kuo CC. Neutralization of Chlamydia trachomatis cell culture infection by serovar-specific monoclonal antibodies. Infect. Immun. 1985, 50, 595-597 13. Zhang YX, Síewari 5, Joseph T, Taylor HR and HD Caldwell. Protective monoclonal antibodies recognize epitopes localized on the major oufer membrane prolein of Chlamydia trachomatis. 3. Immunol. 1987, 138, 575-581. 14. 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Table 1: Serological and fertility analysis in immunized and monitored control mice during a period of 12 weeks.

Table 2: w Q? se: subcutaneous route TMG: geometric mean titration NR: not performed Table 3: 3 se: subcutaneous route NR: not performed The classifications of ftes. T cells positive for CD4 or CD8 graduated from cells without (-) to maximum infiltration (+++) of the cell type considered Table 4: se: subcutaneous route TMG: geometric mean titration í GD NR: not done

Claims (6)

1. .
2. REVIVAL DICTION IS 1. A vaccine composition comprising a PM EP protein from Chylamydia together with QS21 and 3DMPL. 2. A vaccine according to claim 1, wherein the protein is derived from a Serovar L2.
3. 3. A vaccine according to claim 1 or 2, further comprising a prolein of a different serovar.
4. 4. A vaccine according to claim 1, which additionally comprises sterol.
5. 5. A vaccine according to claim 4, wherein the sterol is choleslerol.
6. 6. A vaccine according to claim 5, wherein QS21 is associated with a choleslerol that contains a liposome.