CTH1 Anti-Chlamydial Vaccine Figure 6. Importance of CD4+ T cell immunity in vaccine-mediated protection. C3H/HeN mice depleted of CD4+ T cells received i.p. injections of 250 mg of anti–mouse CD4 monoclonal antibody (clone GK1.5) on days -7, -4, -3 and of 400 mg purified antibody at day +1, +4, +8, +11, +15,+18,+22 relative to the day of infection. Mice were vaccinated and challenged as described for Fig. 2. Swab samples were obtained on days 3, 7, 14, 21 and 35 post infection. Median vaginal MoPn loads6interquartile ranges were compared for (A), top panel: control mice (n = 20) vs. CTH1/ CAF01 vaccinated mice (n = 16). (A), lower panel: control mice (n = 20) vs. i.n. MoPn infected mice (n = 8) (B), top panel CD4-depleted control mice (n = 8) vs. CD4-depletion CTH1/CAF01 immunized mice (n = 8). (B), lower panel: CD4-depleted control mice (n = 8) vs. CD4-depleted i.n. MoPn infected mice (n = 8). 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 post test). doi:10.1371/journal.pone.0010768.g006 vaccinated mice was solely dependent on CD4+ T cells, the i.n. MoPn infected mice displayed a very substantial protective immune response even in the absence of CD4+ T cells. The CD4+ T cell independent effect is most likely not mediated by CD8+ T cells as CD8-depletion of i.n. infected mice had minimal influence on the level of protection (authors’ unpublished observations). This is in agreement with other observations from mouse models of genital tract infection where immunity is neither diminished in the absence of CD8+ T cells nor is any significant protection conferred by CD8+ T cells [12,49,50]. Another T cell subset that has been demonstrated to play a potential role in the immune response to intracellular pathogens is cd T cells. Similarly to CD4+ and CD8+ T cells, cd T cells secrete IFN-c, can lyse infected macrophages and can help contain bacterial growth (reviewed in [51]). Their role in Chlamydia infection, although still not completely resolved, seems to be very modest [13,52] and is therefore most likely not responsible for the CD4 independent protection we observe in the present study. However, a number of important studies from Morrison and colleagues have indicated a previously unrecognized protective role for antibodies and B cells against secondary genital chlamydial infection in mice [20,21,50,53]. The mechanisms of action for antibodies in immunity to Chlamydia remain unresolved and data from different laboratories collectively suggest a more complex role than just direct neutralisation of the invading pathogen. Mice deficient in activating Fc receptors (FcR2/2 mice) had impaired resistance to secondary chlamydial challenge [24,54]. This has been interpreted to suggest that the effects of antibody may occur via Fc receptor-dependent mechanisms that could accelerate the subsequent CMI response. That CMI and antibodies interact in protection against Chlamydia was also emphasized by the finding that antibody-mediated protection is highly dependent on CD4+ T cell-mediated adaptive changes that occur in the local genital tract tissues during primary infection [53]. Our study demonstrates that CTH1/CAF01 vaccination induced high levels of CTH1 specific IgG1 and IgG2a both in blood and vaginal secretions. However, the antibodies were only weakly reactive to chlamydial EBs of either MoPn (Fig. 7) or Ct serovar D. In contrast, mice i.n infected with MoPn induced high levels of IgA directed to EBs in both serum and vaginal wash. There are at least two explanations for this difference. It may relate to the recognition of multiple target antigens on the surface of EB in the i.n. vaccinated group as demonstrated elsewhere [55], compared to the focused response in CTH1 vaccinated animals directed to CT443 and CT521. The weak binding of CTH1-specific antibodies to the whole EB preparation could be explained by a relatively low concentration of CT443 and CT521 in the ELISA wells of EB coated plates compared to CTH1 coated plates. Alternatively, the weak recognition of the EB preparation in the CTH1 vaccinated mice may relate to differences in the conformation of the two vaccine antigens in their natural localization in EB’s compared to the recombinant vaccine construct. CT443 (OmcB) was originally identified as part of the outer membrane complex [41], and recent data have demonstrated that this molecule functions as a chlamydial adhesin [42], emphasizing its surface localization. CT443 may however suffer from the same problem as MOMP, where a number of recent studies have demonstrated that the optimal antibody response and protective efficacy depends on its native conformation-something that has so far not been possible to achieve with recombinantly expressed MOMP [23,56]. PLoS ONE | www.plosone.org 7 May 2010 | Volume 5 | Issue 5 | e10768