CTH1 Anti-Chlamydial Vaccine Figure 2. Vaccine-induced immune responses and protection against C. trachomatis serovar D. (A) Antigen specific responses by PBMC 5 weeks after the last immunization with CTH1 or the single components CT521 and CT443. C3H/HeN mice were immunized three times at 2 weeks intervals with either CT521 or CT443 or CTH1 emulsified in CAF01. Cell cultures pooled from 13 animals in each group were re-stimulated with 5 mg/ ml of CT521, CT443, CTH1and a negative control well (C) without antigen and the IFN-c responses were measured after 72 h of stimulation. Each point represents the mean of triplicate values6standard deviations. The experiment was performed twice with similar results. (B) Vaccine-induced protection. Six weeks after the final vaccination mice were infected i.vag. with 16107 IFU of C. trachomatis Serovar D/mouse. Median vaginal bacterial loads6interquartil ranges are compared for mice receiving CTH1 (n = 8), CT443 (n = 8), CT521 (n = 8), and controls consisting of na¨ıve and CAF01 immunized mice (n = 16) at day 3 post infection. Culture-negative mice were assigned the lower cut-off of the shedding assay (10 IFU/mouse) **, P,0.01; ***, P,0.001 compared to the control group (Kruskal-Wallis-test, Dunn’s posttest). doi:10.1371/journal.pone.0010768.g002 difference between the single components and CTH1 at day 3 post infection. CT521/CAF01 did not significantly reduce the vaginal bacterial shedding (P.0.05) whereas mice immunized with CT443/CAF01 had significantly reduced bacterial shedding with a median reduction of 0.9 log10 IFU/mouse (P,0.01). In comparison mice vaccinated with CTH1/CAF01 reduced the bacterial shedding with 1.46 log10 IFU/mouse (P,0.001) compared to the control group (from 2.46 log10 to 1.0 log10). At day 7 post infection there was no significant difference between vaccinated and non-vaccinated mice (results not shown). CTH1/CAF01 and MoPn infection induced a different clearance kinetic A Ct Serovar D infection is cleared fast in the mouse model resulting in a narrow window for analysing the protective efficacy of a vaccine. Since CT521 is 99% and CT443 97% homologous between Ct serovar D and MoPn, we continued by investigating the effect of this vaccine construct in the well established MoPn mouse challenge model. Compared with the brief course of infection with Ct serovar D, MoPn is significantly more virulent than Ct serovar D and gives rise to a fully developed infection that is cleared between 4–5 weeks post challenge and is associated with disease related pathology [31,32]. This model therefore allows more detailed studies of the dynamics and mechanisms involved in the vaccine promoted protection and importantly allows a comparison with a well established positive control (prior i.n. infection with MoPn), that provides high levels of protection against a genital challenge [33]. Two different mice strains representing different haplotypes, C3H/HeN (H-2k) and CB6F1 (H-2b and H-2d) were immunized 3 times with 5 mg of CTH1/ CAF01 or rendered immune by prior i.n. infection. Six and 8 weeks post vaccination and i.n. infection, respectively, the mice were challenged i.vag. with 16105 MoPn and vaginal swabs were taken at different time points from day 3 to 35 after challenge infection. Figure 3A and B shows the number of isolated bacteria in the vaginal swabs expressed as median log10 IFU/mice in C3H/ HeN and CB6F1 mice, respectively. Compared to the control group the CTH1/CAF01 immunized C3H/HeN mice displayed significantly reduced vaginal bacterial shedding at day 7, 14 and 21 post infection (P,0.01) with the most efficient protection expressed at day 14–21 (median IFU/mouse reduced by 1.1–1.9 log10) (Fig. 3A). The CTH1 based vaccine also significantly protected CB6F1 mice ranging from 1.5–2.4 log10 reduction in IFU’s (Fig. 3B). Mice immunized by prior i.n. infection with MoPn cleared the challenge infection in a highly accelerated manner. Whereas CTH1 vaccinated mice had a delay of one week before the first manifestation of protection the i.n. infected group induced a high level of protection immediately after challenge and already at day 7–14 post infection these mice had resolved the infection. CTH1/CAF01 subunit vaccination and MoPn infection promotes a different distribution of cytokine producing CD4+ T cell subsets Prophylactic vaccination with CTH1/CAF01 reduced the number of bacteria significantly compared to the control group, but with a lower level of protection and a delayed kinetic compared to a prior i.n. MoPn infection. We therefore characterized and compared the T cell and antibody responses generated by the CTH1/CAF01 vaccine and the i.n. MoPn infection. C3H/HeN mice were vaccinated three times with CTH1/CAF01 or infected i.n. with 5000 IFU of MoPn. As control we included a group of non-vaccinated mice. Five weeks after the last vaccination or 7 weeks after the i.n. MoPn infection, the spleens were isolated from 4 individual mice and splenocytes were stimulated in vitro with the homologous antigen preparations, CTH1 and MoPn EBs, respectively. Antigen-specific CD4+ and PLoS ONE | www.plosone.org 3 May 2010 | Volume 5 | Issue 5 | e10768