CTH1 Anti-Chlamydial Vaccine Figure 5. Immune responses of CD4-depleted and non-depleted mice. (A) The density plot represents CD4+ and CD8+ T cells in PBMCs 2 days post challenge in non-depleted (top panel) and CD4-depleted mice (lower panel). (B) CTH1-specific and MoPn-specific IFN-c responses in depleted and non-depleted CTH1 vaccinated and i.n. MoPn infected C3H/HeN mice. The IFN-c responses were measured in triplicate cell cultures pooled from 3 animals/group 2 days post challenge. Each group was re-stimulated with 5 mg/ml of CTH1 and HIA-MoPn and IFN-c responses were measured after 72 h of stimulation. Each bar represents the mean of triplicate values6standard deviations, (C) CTH1-specific and MoPn-specific antibodies generated after CTH1 vaccination or i.n. MoPn infection in CD4 depleted and non-depleted mice Antibodies (IgG1, IgG2a and IgA) were measured in serum (diluted 5 fold from a 1:20 dilution) obtained 2 days after challenge infection, by ELISA. Reciprocal serum dilutions corresponding to 50% maximal binding (EC50) were computed using the Prism 4 software. Each bar represents the mean EC506SEM of 3 individual mice. The experiment was repeated twice with similar results. doi:10.1371/journal.pone.0010768.g005 which IL-12 or CpG and cholera toxin plus CpG were used as adjuvants, respectively. For both of these vaccines, protection was found to be dependent upon CD4+ T cells producing IFN-c [38,45,46]. IFN-c has often been used as a single readout for Th1 responses, but recent studies of other infectious diseases have emphasized the importance of polyfunctional T cells co-producing IL-2, IFN-c and TNF-a [14,47]. Here we demonstrated that CTH1/CAF01 immunized animals had a high percentage of antigen specific CD4+ T cells co-expressing IFN-c/TNF-a/IL-2 or TNF-a/IL-2. IL-2 co-expression has been demonstrated to play an important role in the long-term survival of primed CD4+ T cells in vivo [34,35]. Furthermore these polyfunctional cells have been demonstrated to correlate with efficient protection against other intracellular pathogens such as Leishmania major and Mycobacterium tuberculosis [14,48]. In the present study, the proportion of polyfunctional T cells are much higher in the CTH1/CAF01 immunized group compared to the i.n. MoPn infected group. This suggests that the CTH1 vaccine promotes a fully-adequate, high quality T cell response, as do other vaccines employing the liposome based adjuvant CAF01 [48]. It is therefore likely that the explanation for the lower levels of protection promoted by the subunit vaccine should be sought elsewhere than in the quality of the CMI response induced. In agreement with this, our data clearly demonstrate that whereas the protection in the subunit PLoS ONE | www.plosone.org 6 May 2010 | Volume 5 | Issue 5 | e10768