VACCINE COMPOSITION AGAINST CHLAMYDIACEAE INFECTIONS

Abstract

The invention relates to vaccine compositions for treating and/or preventing infections by a bacterium of the Chlamydiaceae family, said compositions comprising bacteria of the Chlamydiaceae family, which have been previously treated by at least one peptidoglycan inhibitor, or extracts of said treated bacteria.

Claims

1. A vaccine composition comprising bacteria of the family Chlamydiaceae first treated with at least one peptidoglycan inhibitor, or extracts of said treated bacteria, for use in the treatment and/or prevention of infections with a bacterium of the family Chlamydiaceae.

2. A vaccine composition for treating and/or preventing the pathologies caused by an infection with a bacterium of the family Chlamydiaceae, said composition comprising bacteria of the family Chlamydiaceae first treated with at least one peptidoglycan inhibitor or extracts of said treated bacteria.

3. The vaccine composition of any one of claims 1 or 2, characterized in that said extracts of bacteria of the family Chlamydiaceae first treated with at least one peptidoglycan inhibitor are membrane extracts or cytoplasmic extracts.

4. The vaccine composition of any one of the preceding claims, characterized in that said petidoglycan inhibitor is a -lactam or a D-cycloserine.

5. The vaccine composition of any one of the preceding claims, characterized in that said -lactam is selected from the group consisting of amoxicillin, benzylpenicillin (penicillin G), phenoxymethylpenicillin (penicillin V), cloxacillin, cefadroxil, cefixime, imipenem, cefixime in combination with imipenem, mecillinam, clavulanic acid, tazobactam and sulbactam.

6. The vaccine composition of claim 5, characterized in that said -lactam is penicillin G.

7. The vaccine composition of any one of the preceding claims, characterized in that said bacterium of the family Chlamydiaceae is Chlamydia trachomatis or Chlamydia muridarum.

8. The vaccine composition of any one of claims 2 to 7, characterized in that the pathology is an ocular pathology, a genital pathology, a respiratory pathology, a cardiovascular dysfunction, a circulatory dysfunction, a joint disease or an inflammatory disease.

9. The vaccine composition of claim 8 characterized in that the genital pathology is selected from the group consisting of venereal lymphogranuloma, urethritis, orchiepididymitis, cervicovaginitis, cervicitis, endocervicitis, endometritis, perihepatitis and salpingitis.

10. A vaccine composition comprising bacteria of the family Chlamydiaceae first treated with at least one peptidoglycan inhibitor or extracts of said bacteria, and an adjuvant.

11. A process for preparing the vaccine composition of claim 10, said process comprising a step of treating said bacteria of the family Chlamydiaceae with a peptidoglycan inhibitor.

12. The process of claim 11, comprising a preliminary step of infecting a host cell with said bacteria of the family Chlamydiaceae prior to the treatment step with a peptidoglycan inhibitor.

13. A method for detecting a bacterium of the family Chlamydiaceae in a sample from a subject believed to be infected with said bacterium, comprising the steps consisting of: a) contacting said sample with antibodies specific for said bacterium generated against said bacterium first treated with a peptidoglycan inhibitor, said antibodies comprising a label producing a detectable signal, or being fixed to a reagent labeled in a detectable manner; b) allowing the bacterium present in said sample to bind thereto so as to form antigen/antibody complexes; and c) determining the presence of said bacterium in said sample by means of said detectable label.

14. A diagnostic kit for detecting infection with a bacterium of the family Chlamydiaceae, comprising said bacterium first treated with a peptidoglycan inhibitor together with substances for performing an analysis to determine humoral immunity against said bacterium, in a unit packaging container.

15. Use of a bacterium of the family Chlamydiaceae first treated with a peptidoglycan inhibitor as in vitro diagnostic reagent in a binding assay, optionally in a diagnostic kit, for detecting neutralizing antibody conferring immunity against said bacterium.

16. Use of antibodies specific for a bacterium of the family Chlamydiaceae generated against said bacterium first treated with a -lactam as in vitro diagnostic reagent in a binding assay, optionally in a diagnostic kit, for detecting said bacterium.

Description

FIGURE CAPTIONS

[0097] FIG. 1: Vaccine efficacy of the BL forms. The BL forms lead to a significant decrease in the number of infected mice.

[0098] FIG. 2: Proportion of animals from the various immunization groups (Mefs, b-Lac, b-Lac/3, Dc) with a vaginal loss of Chlamydia muridarum at various times post-infection. : p<0.08; * p<0.05; ** p<0.01; *** p<0.005 relative to the Mefs group (Mann-Whitney test).

[0099] FIG. 3: A-Characterization of cell loss in the lamina propria of the uterine horns of mice, normal uterine horn grade 0, (A), grade 1 (B), grade 2 (C), grade 3 (D), photographs taken at 10 magnification. BEvaluation of the severity of the microscopic damage of the uterine horns of animals from the various groups. Normal uterine horn (grade 0, A), grade 1 (B), grade 2 (C), grade 3 (D). *p=0.054 (Mann-Whitney test).

[0100] FIG. 4: Total number of splenic lymphocytes of mice after complete immunization with the vaccine or control forms. Isolated cells were counted in a Malassez cell and their number was calculated in relation to the number of viable cells analyzed by flow cytometry using a viability label (Fixable Viability Dye, BD Biosciences). Graph representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0101] FIG. 5: Total number of CD4.sup.+ T lymphocytes and number activated after complete immunization with the vaccine forms. The total number of CD4.sup.+ T lymphocytes (A) and the number of CD4+T lymphocytes having activation markers are represented with a focus on membrane expression of CD25 (B), CD44.sup.hi CD62L.sup.low (C), ICOS.sup.+ (D) and on intracellular expression of FoxP3 in order to show the population of regulatory T lymphocytes (E). Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols *, **, represent measurements of p-values derived from tests carried out with respect to the control MEF (* p0.05, ** p0.01,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0102] FIG. 6: Total number of CD8.sup.+ T lymphocytes and number activated after complete immunization with the vaccine forms. The total number of CD8.sup.+ T lymphocytes (A) and the number of CD8+T lymphocytes having activation markers are represented with a focus on membrane expression of ICOS (B) and of CD44.sup.hi CD62L.sup.low (C). Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols *, represent measurements of p-values derived from tests carried out with respect to the control MEF (* p0.05,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0103] FIG. 7: Total number of cells in the lymph nodes and the spleen of mice after complete immunization with the vaccine forms, and one-week post-intravaginal infection with Chlamydia muridarum. Isolated cells were counted in a Malassez cell and their number was calculated in relation to the number of viable cells analyzed by flow cytometry using a viability label (Fixable Viability Dye, BD Biosciences). Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols *, **, represent measurements of p-values derived from tests carried out with respect to the control MEF (* p0.05, ** p0.01,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0104] FIG. 8: Total number of splenic CD4.sup.+ T and CD8.sup.+ T lymphocytes and number activated after complete immunization with the vaccine forms and one-week post-intravaginal infection with Chlamydia muridarum. The total number of CD4.sup.+ (A) and CD8.sup.+ (D) T lymphocytes and the activation profile thereof are represented here with a focus on the number of CD25.sup.+ T lymphocytes among the CD4.sup.+ T (B) and CD8.sup.+ T (E), and on the number of CD44.sup.hi CD62L.sup.low CD4.sup.+ T and CD8.sup.+ T lymphocytes (C and F, respectively). Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols *, **, represent measurements of p-values derived from tests carried out with respect to the control MEF (* p0.05, ** p0.01,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0105] FIG. 9: Total number of lymph node CD4.sup.+ T and CD8.sup.+ T lymphocytes and number activated after complete immunization with the vaccine forms and one-week post-intravaginal infection with Chlamydia muridarum. The total number of CD4.sup.+ (A) and CD8.sup.+ (E) T lymphocytes and the activation profile thereof are represented here with a focus on the CD4+CD25.sup.+ (C) and ICOS.sup.+ (D) lymphocyte population, and on the number of CD44.sup.hi CD62L.sup.low CD4.sup.+ T and CD8.sup.+ T lymphocytes (B and F respectively). Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols *, **, represent measurements of p-values derived from tests carried out with respect to the control MEF (*p0.05, **p0.01,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0106] FIG. 10: The number of CD4.sup.+ T and CD8.sup.+ T lymphocytes proliferating in the lymph nodes draining the genital tract and the spleen after complete immunization with the vaccine forms and one-week post-intravaginal infection with Chlamydia muridarum. The total number of CD4.sup.+ (A) and CD8.sup.+ (B) T lymphocytes proliferating in the lymph nodes draining the genital tract (I) and the spleen (II) is presented here. Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

[0107] FIG. 11: Total number of CD4.sup.+ regulatory T lymphocytes and number proliferating in the lymph nodes draining the genital tract and the spleen after complete immunization with the vaccine forms and one-week post-infection. The total number of CD4.sup.+ regulatory T lymphocytes (Treg) (A) and the number proliferating (B) in the lymph nodes draining the genital tract (I) and the spleen (II) are presented here. Graphs representative of an experiment on groups of animals tested individually (PBS, n=3; MEF, n=4; b-Lac, n=5; Dc, n=5). MeansSEM. Unpaired t-test: the symbols represent measurements of p-values derived from tests carried out with respect to the control MEF (* p0.05,). The symbol ns or the absence of stars indicates the absence of statistically significant differences between the group of control animals immunized with sonicate of uninfected MEF cells and the group of control animals injected with PBS alone, or between MEF and b-Lac, or MEF and Dc groups.

EXAMPLES

Example 1: Immunization of C57BL/6J Mice

[0108] C57BL/6J mice were vaccinated with different compositions, then secondarily infected with bacteria of the family Chlamydiaceae.

[0109] In order to avoid a host immune reaction related to the recognition of alloantigens carried by eukaryotic cells, the various bacterial forms (infectious, BL) were produced in embryonic fibroblasts derived from C57BL/6J mice.

[0110] To that end, embryonic fibroblasts (MEF) derived from C57Bl/6 mice with Chlamydia muridarum (Cm) were infected in vitro. This strain is genetically very similar to Chlamydia trachomatis. It infects mice and guinea pigs but not humans, and causes the same physiopathological after-effects in the genital tract as in humans.

[0111] Several compositions were prepared [0112] C1composition comprising an infectious form of Cm, also called virulent form (72-hour in vitro cell infection and sampling of the cell lysate) [0113] C2composition comprising an infectious form inactivated 30 min at 56 C., also called heated form (idem 1 followed by a heat-inactivation step) [0114] C3composition comprising an antibiotic-inactivated form: (3-hour in vitro cell infection, then continuous incubation of the infected cells with penicillin G (100 IU/mL), sampling of the cell lysate after 90 hours). This is a BL form and, in particular, a b-Lac form.

[0115] It was confirmed by titration that form 1 was infectious, and that forms 2 and 3 were not.

[0116] 4 groups of 8-week-old female C57BL/6J mice were used.

[0117] Three groups were injected via the intraperitoneal (IP) route one or more times with one of the compositions, then the 4 groups were infected via the vaginal route with the infectious form of Cm.

[0118] The amount of composition used for each injection corresponded to about 10 cm.sup.2 of cells (MEF) cultivated in vitro, 100% infected, in a 150 L volume by IP injection. [0119] group 1 (4 animals): infection with Cm (1 million IFU). [0120] group 2 (3 mice): one IP injection (at time to) of form 1 of the bacterium. At t0+39d, vaginal infection with 1 million IFU of Cm. [0121] group 3 (3 mice): three IP injections (to, to+10d, to+21d) of form 2 of the bacterium. At t0+39d, vaginal infection with 1 million IFU of Cm. [0122] group 4 (4 mice): three IP injections (to, to+10d, to+21d) of form 3 of the bacterium. At t0+39d, vaginal infection with 1 million IFU of Cm.

[0123] After infection, the mice were kept isolated for 82 days, with no antibiotic treatment, in order to allow the physiopathological after-effects to develop. The mice were then sacrificed and examined: macroscopic appearance of the genital tract, investigation of tissue lesions (hydrosalpinx, deformations, fluid cysts,), measurement of the weight of the spleen, sampling of various organs (including the para-aortic lymph nodes draining the uterus) for subsequent analyses.

[0124] Results obtained: [0125] Group 1: 3/4 of the mice had tissue lesions of the upper genital tract on at least one of the two uterine horns (spleen 1063 g, normal para-aortic lymph nodes) [0126] Group 2: 1/3 of the mice had tissue lesions of the upper genital tract on at least one of the two uterine horns (spleen 1149 g, normal para-aortic lymph nodes) [0127] Group 3: 2/3 of the mice had tissue lesions of the upper genital tract on at least one of the uterine horns (spleen 28081 g, normal para-aortic lymph nodes). One mouse was sacrificed 60d post-infection because it exhibited skin wounds and substantial weight loss, potentially unrelated to the experiment. [0128] Group 4: no mouse had tissue lesions of the upper genital tract (spleen 11117 g, normal para-aortic lymph nodes)

[0129] This preliminary study shows that only the group of mice preinjected with the BL form of Cm was protected from developing tissue lesions of the upper genital tract, following infection with Cm.

Example 2: Vaccine Efficacy of the BL Forms

[0130] In order to confirm the vaccine efficacy of the BL forms, a new experiment was carried out under the same conditions as in example 1. [0131] Mice 1-10: immunization with MEFs at D0, D10 and D21 [0132] Mice 1-5: infection with 2.10.sup.6 IFU at D39 [0133] 3/5 mice (60%) exhibit after-effects (tissue lesions of the upper genital tract) [0134] Mice 6-10: infection with 2.10.sup.5 IFU at D39 [0135] 3/5 mice (60%) exhibit after-effects (tissue lesions of the upper genital tract) [0136] Mice 11-20: immunization with the infectious form first inactivated 10 min at 90 C. at D0, D10 and D21 [0137] Mice 11-15: infection with 2.10.sup.6 IFU at D39 [0138] 3/5 mice (60%) exhibit after-effects (tissue lesions of the upper genital tract) [0139] Mice 16-20: infection with 2.10.sup.5 IFU at D39 [0140] 3/5 mice (60%) exhibit after-effects (tissue lesions of the upper genital tract) [0141] Mice 21-30: immunization with the BL form at D0, D10 and D21 [0142] Mice 21-25: infection with 2.10.sup.6 IFU at D39 [0143] 0/5 mice (0%) presents after-effects (tissue lesions of the upper genital tract) [0144] Mice 26-30: infection with 2.10.sup.5 IFU at D39 [0145] 0/5 mice (0%) presents after-effects (tissue lesions of the upper genital tract) [0146] Mice 31-40: immunization with the infectious form Cm at D0, D10 and D21 [0147] Mice 31-35: infection with 2.10.sup.6 IFU at D39 [0148] 2/5 mice died one-week post-infection [0149] 2/3 surviving mice exhibit after-effects (tissue lesions of the upper genital tract [0150] Mice 36-40: infection with 2.10.sup.5 IFU at D39 [0151] 2/5 mice died one-week post-infection [0152] 2/3 surviving mice exhibit after-effects (tissue lesions of the upper genital tract)

[0153] The results show a significant decrease in the number of infected mice in the group first immunized with the BL form (FIG. 1). These results confirm that the administration of the BL form protects the mice from infection with C. muridarum.

Example 3: Effect of Intraperitoneal Injection of Various Immunizing Doses of b-Lac Forms (BL Forms, Treated with a -Lactam) or Dc Forms (BL Forms Treated with D-Cycloserine) of Chlamydia muridarum on Protection Against Vaginal Infection with C. muridarum and the Development of Tubal Pathology

[0154] 4 groups of 10 female mice were immunized via intraperitoneal injection at D0, D0+10, D0+21 either with an extract of embryonic fibroblasts of C57/BL6 mice (Mefs), or with the b-Lac form (b-Lac), or with the Lac form diluted to one-third of the usual dose (b-Lac/3), or with the Dc form (Dc). These vaccine forms were produced in Mefs.

[0155] At D32, the animals were cycled by subcutaneous injection of 50 L of Deprovera (50 mg/mL, Pfizer).

[0156] At D39 the animals were infected via the vaginal route with 10.sup.7 IFU of Chlamydia muridarum.

[0157] At D3, D6, D8, D10 and D13 post-infection, vaginal smears were carried out and the presence of Chlamydia muridarum was evaluated by titration of the infectious capacity of the Chlamydiae extracted from these smears (FIG. 2).

[0158] At 8-weeks post-infection the remaining animals (n=6) were sacrificed, the uterine horns dissected and macroscopic lesions (fluid cysts and translucent zones on the uterine horns or the oviduct) were investigated with the binocular magnifier. Only the mice of the b-Lac group have no macroscopic lesions. The uterine horns were fixed with paraformaldehyde, dehydrated and imbedded in paraffin. Longitudinal sections were stained with HPS (hematoxylin, phloxine, saffron) to show the various structures of the uterine horn (myometrium and endometrium comprising the epithelium and the lamina propria). We evaluated the severity of microscopic damage by the cell loss observed in the uterine lamina propria (FIG. 3).

[0159] A larger number of animals with minor tubal lesions (grade 1) or no lesions (grade 0) is observed in the b-Lac and Dc groups, compared with the Mefs and b-Lac/3 groups. The most severe damage visible in the Mefs group is absent in the b-Lac, b-Lac/3 and Dc groups.

[0160] These results thus confirm the protective effect of 3 intraperitoneal injections of b-Lac forms (with a confirmed dose-effect) and of Dc forms, with respect to vaginal infection with a virulent form of Chlamydia muridarum. This protection is expressed as a reduction in bacterial load from the beginning of the local infection, a specific antibody response which is detectable in the spleen of infected immunized mice, and a protection against inflammatory genital lesions post-infection in these same mice.

Example 4: Study of Lymphocyte Activation after Complete Immunization Followed or not Followed by Infection

[0161] 1. T Lymphocyte Activation Profile after Complete Immunization with the b-Lac and Dc Vaccine Forms In Vivo

[0162] One week after complete immunization (three immunizations one week apart), the splenocytes of individual mice were harvested as previously described and the CD4.sup.+ and CD8.sup.+ T lymphocyte activation profile was studied by flow cytometry with a focus on expression of the classical activation markers CD25, CD62L, CD44 and ICOS. The CD4.sup.+ T or CD8.sup.+ T lymphocyte population selection (gating) strategy employed was as follows: FSC/SSC (scattering of light emitted at small angles and at 90), Singlets (elimination of cell doublets), CD19 negative (exclusion of B lymphocytes), CD3.sup.+ (selection of T lymphocytes bearing a TCR) then CD4.sup.+ (CD4.sup.+ T lymphocytes) or CD8.sup.+ (CD8.sup.+ T lymphocytes). The study of the number of CD4.sup.+ regulatory T lymphocytes (Treg) was carried out after permeabilization of the splenocytes and intracellular staining of the FoxP3 molecule. The CD4.sup.+ regulatory T lymphocyte (Treg) gating strategy employed was as follows: FSC/SSC, Singlets, CD19 negative (exclusion of B lymphocytes), Fixable Viability Dye (selection of viable cells), CD3.sup.+ (T lymphocytes), CD4.sup.+ (CD4.sup.+ T lymphocytes) then FoxP3.sup.+ (selection of Tregs, a high expression of FoxP3 being a specific marker of Tregs). Statistical analyses were carried out using the GraphPad PRISM software.

[0163] In the spleen, the numbers of total lymphocytes or of CD4+ or CD8+T lymphocytes are not significantly different between the various groups (FIGS. 4, 5A, 6A). However, the number of CD44.sup.hi CD62L.sup.low CD4.sup.+ activated T lymphocytes is significantly increased following immunization with the b-Lac vaccine form alone (FIG. 35). The expression profile of ICOS on the CD4.sup.+ T lymphocytes also shows a very significant increase in the number of ICOS.sup.+ CD4.sup.+ T lymphocytes (ICOS being a marker reflecting the inflammatory potential of CD4.sup.+ T lymphocytes) (FIG. 5). In parallel, FoxP3 intracellular staining shows a decrease in the CD4.sup.+ regulatory lymphocyte population, significant in terms of number following immunization with the Dc vaccine form. The same trend is observed in the b-Lac group (FIG. 5E). The vaccine forms do not induce significant differences in terms of ICOS.sup.+ CD8.sup.+ T populations (FIG. 6B). A significant increase in the number of CD44.sup.hi CD62L.sup.low CD8.sup.+ activated T lymphocytes is noted after immunization with the b-Lac form (FIG. 6C).

[0164] 2. T Lymphocyte Activation Profile after Complete Immunization with the b-Lac and Dc Vaccine Forms Followed by Intravaginal Infection with 10.sup.7 IFU of Chlamydia muridarum.

[0165] After complete immunization (three immunizations one week apart), the mice were cycled by injection of progesterone (Deprovera, 50 L per mouse) and infected with 10.sup.7 IFU of Chlamydia muridarum via the intravaginal route. After one-week post-infection, the mice were sacrificed and tested individually. The splenocytes and lymph nodes draining the genital tract (inguinal/para-aortic/mesenteric) were harvested and isolated. The activation profile and the number of CD4.sup.+ and CD8.sup.+ T lymphocytes were studied by flow cytometry as indicated in the preceding paragraph (part 1) with a focus on expression of CD25, CD62L, CD44 and ICOS. The study of T lymphocyte proliferation and number of Tregs is carried out after permeabilization and intracellular staining of the molecule Ki67 (proliferation) and FoxP3 (Treg). Statistical analyses were carried out using the GraphPad PRISM software.

[0166] FIG. 7 presents the total number of lymphoid cells of the lymph nodes draining the genital tract and the spleen of the mice of the 4 groups PBS, MEF, b-Lac or Dc. It is quite clear that immunization with the b-Lac form followed by intravaginal infection of the mice leads to a clear increase in the number of lymphocytes of the draining lymph nodes (p0.05) and also a quite marked decrease in the spleen (p0.01).

[0167] In the spleen of these mice, one also observes, for the b-Lac group, a significant decrease in CD4+CD25+T lymphocytes (FIG. 8B) and a comparable trend for the CD44.sup.hi CD62L.sup.low CD4+ and CD4+T lymphocytes (FIGS. 8A and 8C, respectively) With regard to the CD8+T lymphocytes, in the b-Lac group, they are significantly decreased (p0.05, FIG. 8D), as well as the CD8+CD25+T (p0.01, FIG. 8E).

[0168] A mirror image is observed in the draining lymph nodes, with a significant increase in the same CD4+ and CD4+CD25+T populations (FIGS. 9A and C) in the b-Lac group, and a comparable trend for the CD4+ and CD44.sup.hi CD62L.sup.low CD4+ and CD4+ ICOS.sup.hi T lymphocytes (FIGS. 9 B and D). The same observation applies to the CD8+ and CD44.sup.hi CD62L.sup.low CD8+T lymphocytes, which are also significantly increased only in the b-Lac group (FIGS. 9 E and F).

[0169] Consistent with the results above, if the numbers of proliferating (carrying the nuclear marker Ki67) CD4+ and CD8+T lymphocytes are examined, a clear trend toward a decrease in the spleen and an increase in the lymph nodes is observed, again only in the b-Lac group (FIG. 10).

[0170] Analyses of the number of regulatory T lymphocytes (carrying markers CD4 and FoxP3) arrive at the same conclusions, again in the b-Lac group: trend toward a decrease in CD4+Ki67+ Treg in the spleen, and significant increases (p0.05) of CD4+ and CD4+Ki67+T reg in the draining lymph nodes (FIG. 11).

[0171] All of these results suggest that the immunizing injections of b-Lac forms induce an activation and a T immune response specific for Chlamydia muridarum in the spleen, with production of specific antibodies. In response to intravaginal infection, the activated T lymphocytes leave the spleen for the lymph nodes draining the genital tract. Thus, an immune basis for the drop in local bacterial load (effector lymphocytes) and for the reduction in the local inflammatory reaction generating tissue lesions (regulatory T-lymphocytes) is provided.