CTH1 Anti-Chlamydial Vaccine Figure 4. IcFACS analysis of cytokine expression in CD4+ and CD8+ T cells after CTH1/CAF01 immunization and i.n. MoPn infection. (A) Gating tree for phenotypic and functional characterization of CD4+ T cells in CTH1/CAF01 immunised mice by multiparameter flow cytometry. Splenic cells were analysed at 5 weeks after last vaccination or 7 weeks post i.n. MoPn infection by icFACS based on individual spleen cells (4 mice/ group) stimulated with either CTH1 or MoPn-EBs. Lymphocytes were gated based on their FSC vs. SSC profile, and the CD4+CD44high T cell population was further subdivided into various cytokine-producing subsets. (B) Cytokine-producing cells (IFN-c, TNF-a, IL-2) within the CD4+CD44high population were divided into seven distinct subpopulations based on their production of these cytokines in any combination. (C) Bar chart shows the frequency of each subset within the CD44high CD4+ and CD8+ T cell populations. The experiment was repeated twice with similar results. doi:10.1371/journal.pone.0010768.g004 of antigens recognized by CD4+ T cells [37–40] and delivery of these antigens to induce a strong CMI response is therefore a central goal when developing novel vaccines against Ct. In the present study we have genetically engineered a fusion molecule encoding two chlamydial proteins; CT521 that resides in the cytosol of reticulate bodies during the replicating cycle (data not shown) and CT443 (omcB) located in the outer cell wall [41,42]. Both proteins are frequently recognized by T cells from patients with a confirmed genital chlamydial infection [26,27] and CT443 is, in addition, a well known antibody target [26,43,44]. The CTH1 antigen was delivered in CAF01, an adjuvant system, known to induce both strong CMI and antibody response [28–30]. Our study demonstrates that this vaccine promotes significant protection against genital infection in mice infected with either Ct serovar D or MoPn. Ct serovar D has relatively low virulence in the mouse and gives rise to a transient infection. The protection promoted by the vaccine was therefore evident at day 3 post infection, but by day 7 the window between vaccinated and control animals became too narrow to detect any significant difference. Since the Ct Serovar D sequence of the CTH1 protein is 97% homologous to the MoPn sequence of CTH1 we decided to investigate the protective capacity of CTH1 using MoPn as challenge strain. Compared to the Ct serovar D challenge model we found the kinetic of bacterial growth and elimination to be different. There was no initial reduction in IFU in vaccinated animals, but from day 14–21 we could detect up to a 2 fold reduction in IFUs compared to a control group. The level of protection and the delayed clearance was not due to a lack of strain specific immunity as later experiments have demonstrated similar infection dynamics after vaccination with a MoPn version of CTH1 (results not shown). Importantly, the CTH1 mediated reduction of IFU was found to be mediated exclusively by CD4+ T cells. CMI-induced protection has been observed in other recent studies promoted by subunit vaccines based on CPAF or NrdB in PLoS ONE | www.plosone.org 5 May 2010 | Volume 5 | Issue 5 | e10768