Vaccination with the recombinant major outer membrane protein elicits long-term protection in mice against vaginal shedding and infertility following a Chlamydia muridarum genital challenge

Abstract

Implementation of a vaccine is likely the best approach to curtail Chlamydia trachomatis infections. The aim of this study was to determine the ability of a vaccine formulated with the recombinant major outer membrane protein (MOMP) and Th1 and Th2 adjuvants, delivered by combinations of systemic and mucosal routes, to elicit long-term protection in mice against a genital challenge with Chlamydia muridarum. As a negative control, mice were vaccinated with the recombinant Neisseria gonorrhoeae porinB, and the positive control group was immunized with C. muridarum live elementary bodies (EB). The four vaccines formulated with MOMP, as determined by the titers of IgG and neutralizing antibodies in serum, proliferative responses of T-cells stimulated with EB and levels of IFN-γ in the supernatants, elicited robust humoral and cellular immune responses over a 6-month period. Groups of mice were challenged genitally at 60, 120, or 180 days postimmunization. Based on the number of mice with positive vaginal cultures, number of positive cultures, length of time of shedding, and number of inclusion forming units recovered, MOMP vaccinated groups were significantly protected. To assess fertility, when the vaginal cultures became negative, female mice were caged with male mice and the outcome of the pregnancy evaluated. As determined by the number of pregnant mice and the number of embryos, two of the vaccine formulations protected mice up to 180 days postimmunization. To our knowledge this is the first subunit of Chlamydia vaccine that has elicited in mice significant long-term protection against a genital challenge.

Keywords: Diseases; Immunology; Microbiology.

Fig. 1. Serum and vaginal C. muridarum-specific antibody titers the day prior to the genital challenge.

Mice were vaccinated with C. muridarum MOMP using different combinations of mucosal and systemic routes. CpG-1826 and Montanide ISA 720 (only systemically) were used as adjuvants. The day before the genital challenges at 60, 120 and 180 days postimmunization blood and one pool of vaginal washes were tested from each group and antibody titers determined using C. muridarum EB as antigen. IgG2a and IgG1 titers were determined in serum (a) and IgG and IgA titers in vaginal washes (b). Error bars represent geometric mean with 95% confidence intervals. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous.

Fig. 2. Binding of serum antibodies to synthetic C. muridarum MOMP peptides.

Serum samples from immunized mice were collected the day before each of the genital challenges and their reactivity to 25-mer peptides corresponding to the C. muridarum mature MOMP were analyzed by ELISA. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous.

Fig. 3. Serum C. muridarum-specific neutralization titers the day prior to the genital challenges.

Mice were vaccinated with C. muridarum MOMP, using different combinations of mucosal and systemic routes. CpG-1826 and Montanide ISA 720 (only systemically) were used as adjuvants. The day before the genital challenges at 60, 120, and 180 days postimmunization blood was collected and neutralizing antibody titers determined using C. muridarum EB as antigen. Error bars represent geometric mean titers with 95% confidence intervals. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous.

Fig. 4. Cell-mediated immune responses of vaccinated mice the day before the genital challenges at 60, 120 and 180 days following immunization.

Spleen T-cells, collected the day before the genital challenges, were stimulated with C. muridarum EB and the proliferative responses (a) and levels of IFN-γ (b) secretion were determined. As a negative background control, T-cells were stimulated with minimal essential medium. Error bars represent mean with one standard error. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous, SI stimulation index.

Fig. 5. Vaginal cultures of mice following intrabursal challenges with C. muridarum at 60, 120, and 180 days postimmunization.

Mice were immunized with recombinant C. muridarum MOMP, or with N. gonorrhoeae porB as a negative control antigen, using different combinations of mucosal and systemic routes. CpG-1826 and Montanide ISA 720 (only systemically) were used as adjuvants. At 60 (a), 120 (b) and 180 (c) days postimmunization, mice were challenged in the left ovarian bursa with C. muridarum and vaginal cultures were collected for a period of 6 weeks. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous. Each dot represents a mouse. Horizontal bars represent median IFU numbers. Numbers in brackets correspond to percentage of mice with positive vaginal cultures. Limit of detection (<2 IFU/culture).

Fig. 6. Cumulative number of C. muridarum IFU collected per mouse from vaginal cultures at 60, 120, and 180 days postimmunization.

The number of C. muridarum IFU collected from vaginal cultures at 60, 120, and 180 days postchallenge were totaled. Bars represent the median number of C. muridarum IFU/mouse and error bars represent range. Symbol * indicates P value is less than 0.05 by the Mann–Whitney U-test. EB elementary bodies, col colonic, i.n. intranasal, i.m. intramuscular, s.c. subcutaneous.