Figure 7. CTH1-specific (A) and MoPn-specific (B) antibodies generated after vaccination or i.n MoPn infection. Mice were either immunized with CTH1/CAF01 or infected i.n. with MoPn. Non- immunized control mice were included as controls. Antibodies (IgG1, IgG2a and IgA) were measured in vaginal secretions (dilution shown 1:10) obtained 8 days after challenge infection, by ELISA. Each symbol and bar represents the mean Abs 4506SD of 4 individual mice. doi:10.1371/journal.pone.0010768.g007 In conclusion, our data suggest that the efficient protection promoted by i.n. infection is mediated partly by a CD4 independent mechanism. Our CTH1 construct efficiently induces both CD4 T cell and a surface targeted antibody response. While the CTH1 vaccine obviously needs further improvement this will not necessarily be achieved by the incorporation of additional antigens. CTH1 may face the same hurdles as often encountered in attempts to recombinantly produce biologically active proteins, where refolding into bioactive forms is cumbersome, results in poor recovery and accounts for the major cost and complexity. For a fusion molecule like CTH1 which incorporates the cysteine rich CT443, restoration of the native-like secondary structure or at least exposure of important linear B cell epitopes will obviously be very difficult and demand carefully targeted mutagenesis of selected cysteine residues. Work towards this goal is currently ongoing in our laboratory. Materials and Methods Ethics Statement The handling of mice was conducted in accordance with the regulations set forward by the Danish Ministry of Justice and animal protection committees by Danish Animal Experiments Inspectorate, and in compliance with European Community Directive 86/609 and the U.S. Association for Laboratory Animal CTH1 Anti-Chlamydial Vaccine Care recommendations for the care and use of laboratory animals. All the techniques/procedures have been refined to provide for maximum comfort/minimal stress to the animals. Organisms The C. muridarum strain MoPn/NiggII and C. trachomatis serovar D was purchased from the ATCC and propagated in HeLa-229 cells as described previously [27]. Chlamydial EBs were harvested, purified and quantified as described previously [27,30] and stored at 280uC in a 0.2 M sucrose, 20 mM sodium phosphate and 5 mM glutamic acid buffer (SPG). Cloning, gene expression and protein purification of recombinant proteins for vaccination Cloning of vaccine hybrid construct CTH1. DNA frag- ments containing the genes of ct521 and ct433 were amplified from Ct serovar D genomic DNA by overlap extension PCR [57]. Amplifications were carried out for 25 cycles each with denaturation at 94uC for 30 sec, annealing at 55uC for 30 sec, and extension at 72uC for 2 min, using Phusion polymerase (Finnzymes, Espoo, Finland). Nucleotide sequencing was performed directly on the PCR products by MWG-Biotech AG (Germany) using specific sequencing primers. The ct521-ct433 gene fusion was created using the specific primer Ct521_fw_1 (59- CAC CGG ATC CAT GTT AAT GCC TAA ACG AAC AAA ATT TC and Ct521_rev_1 (59- CAC CCC GCT AGC AAA TAA ACT TAC CCT TTC CAC ACG CTT AAC AAA) [ct521], Ct443_fw_1 (59-TTT GTT AAG CGT GTG GAA AGG GTA AGT TTA TTT GCT AGC GGG GTG) and Ct443_rev_1 59- GGA TCC CTA ATA GAT GTG TGT ATT CTC TGT ATC AGA AAC TG [ct433] in a first round PCR using chlamydial DNA extracted as described in [58] as the template. The respective products were used as templates in second round PCR using the primers Ct521_fw_1 and Ct443_rev_1. The resulting DNA fragment was cloned into pENTR/D-TOPO and subsequently into pDEST17 expression vector (Invitrogen, Copenhagen, Denmark) thereby creating an in frame fusion with 6*His tag. Recombinant gene expression and protein purifi- cation. Escheria coli BL-21 AI cells transformed with plasmid pDEST17 (Invitrogen, Copenhagen, Denmark) encoding CTH1 were grown at 37uC to reach the logarithmic phase OD600 ,0.5 and protein expression was induced by adding arabinose to total concentration of 0.2%. The protein expression was induced for 4 hours and cells were harvested by centrifugation (6,000 g for 15 min.). E. coli were lysed using Bugbuster (Novagen, Darmstadt, Germany) containing Benzonase, rLysozyme and Protease inhibitor Cocktail I (Calbiochem, San Diego, CA) to avoid unwanted degradation. Lysis was performed at room temperature for 30 min. during gentle agitation. Inclusion bodies were isolated by centrifugation (10,000 g for 10 min.) The pellet was washed once with 1:5 diluted Bugbuster solution in 3 M urea and then dissolved in 50 mM NaH2PO4, 0.4 M NaCl, 8 M Urea, 10% glycerol, 10 mM Imidazole pH 7.5. This solution was loaded onto a 5 ml HisTrap HP (Amersham Biosciences, Buckinghamshire, United Kingdom) and the bound proteins were eluted by applying a gradient of 50 to 500 mM imidazole. Fractions containing the desired recombinant protein were pooled, dialyzed against 20 mM ethanolamine, pH 9, 8 M urea and applied to a 5 ml HiTrap Q Sepharose HP (Amersham Biosciences, Buckinghamshire, United Kingdom). The recombinant protein was eluted by applying a gradient of 0 to 1 M NaCl over 10 column volumes. Analysis of all fractions was performed by SDS-PAGE. Protein concentrations were measured by the BCA protein assay (Pierce, Rockford, PLoS ONE | www.plosone.org 8 May 2010 | Volume 5 | Issue 5 | e10768