CTH1 Anti-Chlamydial Vaccine Figure 3. Vaccine-induced protection against MoPn. Mice were either vaccinated 3 times at 2-week intervals with CTH1/CAF01 or i.n infected with 56103–16104 MoPn. Six and 8 weeks post last vaccination or infection, respectively mice were i.vag. infected with 16105 IFU of MoPn. Median vaginal bacterial loads6interquartil ranges at different time points post infection are compared in C3H/HeN mice (A) or CB6F1 mice (B). Culture- negative mice were assigned the lower cut-off of the shedding assay (10 IFU/mouse). * P,0.05, ** P,0.01; *** P,0.001 compared to the control group (Kruskal-Wallis-test, Dunn’s posttest). doi:10.1371/journal.pone.0010768.g003 CD8+ T cells were characterized based on their ability to secrete the cytokines IFN-c, TNF-a and IL-2 at the single cell level by intracellular FACS staining. The CD4+ and CD8+ T cells were analyzed in terms of their CD44 expression and CD44high T cells were assessed for their cytokine-production to establish the proportion of CD4+ and CD8+ cells positive for IFN-c, TNF-a and IL-2 (Fig. 4A). The CD4+CD44high and the CD8+CD44high T cell populations was separated into seven distinct sub-populations based on their production of IFN-c, IL-2 or TNF-a in any combination and the representation of each of these sub- populations within the pool of T cells established (Fig. 4B). In the CTH1 vaccinated group, multifunctional CTH1-specific CD4+ T cells expressing TNF-a/IL-2 (double positive) and IFN- c/TNF-a/IL-2 (triple positive) CD4+ T cells, which has been associated with memory [34,35], constituted around 1/3 of the total CTH1 specific CD4+ T cell population. In comparison the i.n. MoPn infection only induced a very low level of these CD4+ T cell subsets and was dominated by CD4+ T cells producing IFN-c/ TNF-a (double positive) (Fig. 4C). Neither the vaccine nor the i.n. infection induced detectable numbers of CTH1 and MoPn EB specific CD8+ T cells (Fig. 4C) and non-vaccinated mice did not recognize the vaccine components (results not shown). CTH1/CAF01 mediated protection is CD4+ T cell dependent in the MoPn model We continued by investigating the contribution of the CD4+ T cells in the protective immune response promoted by CTH1/ CAF01 and i.n. MoPn infection. C3H/HeN mice were vaccinated 3 times with CTH1/CAF01 or infected once i.n. with 5000 IFU of MoPn. A non-vaccinated group was included as control. At three time points before challenge (Day -7, -5 and -3), 8 mice from each group were depleted of CD4+ T cells through injection of monoclonal anti-mouse CD4 antibodies (GK 1.5). Injection of a relevant rat IgG2b (isotype control) in a separate group had no impact on the number of CD4+ T cells (data not shown). Six weeks after the last immunization with CTH1/CAF01 or 8 weeks after the primary i.n. MoPn infection the mice were challenged i.vag. with 105 MoPn and vaginal swabs were taken at day 3, 7, 14, and 21 post infection. The depletion of CD4+ T cells was continued by injections of GK.1.5 every 3–4 days (Day +1, +4, +8, +11, +15, +18 and +22). FACS analysis on the blood 2 days after challenge showed that the CD3+/CD4+ cell population of the total CD3 population was between 0–0.23% in mice receiving GK1.5 compared to approximately 60% in non-depleted mice, indicating a very efficient depletion of the CD4+ T cell subset in treated animals (Fig. 5A) which was maintained throughout the infection period. The absence of CD4+ T cells was also seen in collagenase treated genital tract tissue of depleted mice (Figure S1). In agreement with this, CD4-depleted CTH1/CAF01 immunized and i.n. MoPn infected mice only induced marginal IFN-c responses to CTH1 or MoPn EBs, respectively compared to the non-depleted mice (Fig. 5B) The CD4-depletion had no effect on the level of specific antibody to neither CTH1 nor MoPn EB (Fig. 5C). CTH1 immunization induced high levels of IgG1 and IgG2a but no IgA, whereas the i.n MoPn infected mice induced high levels of all three types. Analysis of the vaginal bacterial shedding showed that the protection promoted by CTH1/CAF01 vaccination (Fig. 6A, top panel) was completely eliminated after CD4+ T cell depletion (Fig. 6B, top panel), suggesting that the protection from CTH1/CAF01 vaccination is solely CD4+ T cell dependent. In agreement with earlier observations (Fig. 3A) mice i.n. infected with MoPn reduced the level of bacterial shedding with 4 log10 as early as day 3 post challenge infection (Fig. 6A, lower panel). Depleting CD4+ T cells in this group resulted in impaired resistance and 1.5–3 log more bacteria throughout infection demonstrating a clear involvement of CD4+ T cells also in the protection promoted by prior i.n. infection with MoPn (Fig. 6B, lower panel). It was on the other hand clear that even in the absence of CD4+ T cells, bacterial shedding was reduced with around 3 log10, compared to the non-immunized group which suggests an effector mechanism independent of CD4+ T cells in the i.n. immunized group. CTH1/CAF01 induced high levels of specific antibodies but with limited binding potential for the chlamydial EBs We finally compared the capacity of antibodies generated after CTH1/CAF01 vaccination and i.n. MoPn infection to bind to the MoPn EBs. Vaginal secretions from CTH1/CAF01 immunized, i.n. MoPn infected, and control animals were tested by ELISA coated with CTH1 and chlamydial EBs. We found that CTH1/ CAF01 immunization induced very high levels of CTH1-specific IgG1 and IgG2a in the vaginal secretions (Fig. 7A, black bars). However, these CTH1/CAF01 specific antibodies only had minimal ability to bind MoPn EBs (Fig. 7B, black bars) or Ct serovar D (results not shown). No CTH1 or MoPn EB specific IgA could be detected after CTH1/CAF01 immunization. In contrast i.n MoPn infection was associated with high levels of IgA in the vaginal secretions directed to both CTH1 and MoPn EB (Fig. 7A and 7B (gray bars)). Discussion T helper-type 1 (Th1)-type CMI responses play a predominant role in anti-chlamydial protective immunity [12,13,36]. Identification PLoS ONE | www.plosone.org 4 May 2010 | Volume 5 | Issue 5 | e10768