Antibodies have also attracted renewed interest and seem to play an important role during a secondary encounter with Ct [19–21]. Their role and the exact mechanism of action are however still not clear. In some vaccine studies, antibodies were found largely dispensable [22], whereas studies of vaccines based on the major outer membrane protein (MOMP) found the fine specificity of the humoral response to be critically important [23]. Others have suggested that serum antibodies do not operate through neutralization and that protection against Ct requires CD4+ T cell subsets in collaboration with antibodies [21,24]. It has become clear that an effective subunit vaccine against Ct must contain multiple epitopes to ensure broad coverage of a genetically heterogeneous population infected with multiple serovars of Ct (D to L). The strategy in our laboratory is therefore focused on the molecular engineering of recombinant fusion proteins containing several selected chlamydial vaccine antigens. We have previously demonstrated that fusion proteins can induce amplified responses to molecules with low inherent immunoge- nicity, leading to significantly higher protection compared to single components or mixtures [25]. The fusion protein approach offers the advantage of a more defined product, reducing the number of recombinant expression and purification steps required. This reduces in turn, the cost of production compared to production and mixing of multiple individual antigenic components. The purpose of the current study was to evaluate the potential of a subunit vaccine based on the fusion protein CTH1 consisting of the two immunodominant antigens CT443 (omcB) and CT521 (rl16). These antigens were chosen because they represent targets for both arms of the immune system. CT443 is a target for both strong humoral and CMI responses [26] and CT521 was recently discovered by our group to be a strong and frequent target for T cells during natural Ct infection in humans [27]. Both of these antigens are highly conserved (.97% homology) across the different serotypes and can be expected to provide substantial levels of cross protection. We examined the immunogenicity and protective efficacy of this fusion protein in comparison to an intranasal (i.n.) Chlamydia muridarum (MoPn) infection known to provide a strong protective immune response in the mouse model. CTH1 was combined with the liposomal adjuvant CAF01, known to promote the induction of both CMI and humoral immune CTH1 Anti-Chlamydial Vaccine responses [28,29]. Our data demonstrated that although this subunit vaccine induced a high quality protective CD4+ T cell response and high levels of CTH1-specific antibodies in both serum and the genital tract, it fails to provide complete protection. Results Experimental vaccine based on a recombinant fusion protein between CT521 and CT443 A fusion protein of CT521 and CT443 (CTH1) was recombinantly expressed in E. coli, affinity purified, subjected to ion exchange chromatography, and analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) together with the individual proteins CT521 and CT443 (Fig. 1A). Western blotting demonstrated that CTH1 retained the ability to bind antibodies against both components (Fig. 1B). The initial immunological investigations were done to compare the immu- nogenicity of the fusion protein with that of the single components and to clarify whether both components of the fusion proteins were recognized by the immune system after immunization. Groups of C3H/HeN mice were immunized subcutaneously three times at two weeks intervals with 5 mg of either the fusion protein or the single components combined with CAF01, an adjuvant known to promote the induction of a highly efficient Th1 and antibody responses [28–30]. The immune response to the fusion protein and the single components were investigated five weeks after the last immunization. Immunization with 5 mg of the fusion protein induced a strong IFN-c release in response to re- stimulation with either the fusion protein or the homologous proteins. Moreover the level of IFN-c release in response to both CT443 and CT521 restimulation was enhanced in the CTH1 immunized group compared to immunization with the single components (Fig. 2A). Neither mice receiving the adjuvant combination alone (Fig. 2A) nor non-vaccinated naive mice (results not shown) responded to restimulation with CTH1, CT443 and CT521. Six weeks after the last vaccination the mice were challenged intravaginally (i.vag.) with 16107 inclusion forming units (IFU) of Ct Serovar D. Vaginal swab samples were obtained at day 3 and 7 and the protective effects of CTH1/CAF01, expressed as median log10 IFU/mouse. Figure 2B shows the Figure 1. SDS-PAGE and Immunoblotting. A. SDS-PAGE analysis of purified recombinant Ct serovar D antigens. 1–1.5 mg of protein was loaded in each lane. Lane 1, CT521; Lane 2, CT443; Lane 3, CTH1; Lane 4, molecular weight standard. Protein bands were visualized by coomassie blue staining. B. Western blot of CTH1 was performed with 1 mg of protein pr. lane. The nitrocellulose membrane was reacted with specific anti-CT521 and anti-CT443 rabbit serum and anti-penta-His (Qiagen, Ballerup, Denmark) (Lane 2, 3 and 4 respectively). doi:10.1371/journal.pone.0010768.g001 PLoS ONE | www.plosone.org 2 May 2010 | Volume 5 | Issue 5 | e10768