Illinois, USA). The purity was assessed by SDS-PAGE followed by coomassie staining and western blot with anti-penta-His (Qiagen, Ballerup, Denmark) and anti-E. coli antibodies to detect contaminants (DAKO, Glostrup, Denmark). CTH1 was refolded by a stepwise removal of buffer containing urea ending up in 20 mM Citrate-phosphate buffer pH 4, 10% glycerol, 1 mM cysteine which yielded soluble protein. Recombinant gene expression and protein purification of CT443 and CT521 was done as described in [26]. The purified recombinant proteins were stored at 220uC until use. SDS-page and Immunoblotting SDS-PAGE and western blot of CT521, CT443 and CTH1 was performed with 1 mg of protein pr. lane (Figure 1 A and B). The nitrocellulose membrane was reacted with specific anti-CT521, anti-CT443 rabbit serum and anti-penta-His (Qiagen, Ballerup, Denmark) (Lane 2, 3 and 4 respectively). Immunizations Female 6–8 weeks old C3H/HeN and CB6F1 mice were purchased from Harlan Scandinavia (Denmark). Animals were immunized subcutaneously with 1–5 mg/dose of CT521, CT443 or CTH1 in 100 ml sterile Tris-buffer (pH 7.4) mixed by vortexing with 100 ml CAF01 adjuvant (SSI) consisting of 50 mg/dose of the glycolipid trehalose 6,69-dibehenate (TDB) incorporated into 250 mg/dose of cationic liposomes composed of dimethyldiocta- decylammonium (DDA) bromide [28]. Mice received a total of three immunizations at two week intervals. Ten and 3 days before MoPn or Ct serovar D challenge, the oestrus cycle was synchronized by injection of 2.5 mg Medroxyprogesteronacetat (Depo-Provera; Pfizer). Six weeks after the final vaccination the mice were challenged i.vag. with 16105 IFU of MoPn or 16107 IFU of Ct serovar D in 10 ml SPG buffer. For i.n. infection with MoPn, mice received from 56103 to 16104 bacteria 8 weeks prior to i.vag. challenge. Specific antisera against CT443 and CT521 were generated by injecting recombinant proteins 50 mg into rabbits in combination with Montanide ISA720. Two booster doses were administered at 2-week intervals. Depletion of CD4+ T-cells Monoclonal anti-mouse CD4 IgG2b (clone GK1.5) was purified from hybridoma supernatants made in our lab, using HiTrap protein G HP columns (Amersham Biosciences). The purified IgG was dialyzed against PBS, sterile filtered and the protein concentration was determined. Mice were depleted of CD4+ T- cells by the i.p route with 3 injections of 250 mg purified antibody each at day -7, -5, -3 followed by 400 mg purified antibody at day +1, +4, +8,+11,+15,+18,+22 relative to the day of infection. The depletion of CD4+ T-cells was verified by FACS analysis on PBMCs at day, +2, +7, and day +16 relative to the day of infection using a FITC conjugated anti-CD4 antibody (clone RM4-4), a PE conjugated anti-CD8 antibody (clone 53-6.7) and a APC conjugated anti-CD3 (clone 145-2C11)(BD Biosciences). Likewise, genital tract tissue was removed collagenase (0.7 mg/ml)/DNAse (40 mg/ml) treated for 1 h and surface stained for FACS analysis. Injection of isotype control antibody was found to have no impact on the size of the CD4+ T-cell population. Measurement of antibody levels in serum and vaginal secretions Blood and vaginal secretion fluid were collected for quantifica- tion of vaccine-specific antibodies by ELISA. Blood were collected from the periorbital vein plexus and centrifuged 2500 c.p.m. to CTH1 Anti-Chlamydial Vaccine separate serum. Vaginal secretion samples were collected by flushing the vagina with 100 ml of sterile PBS. Maxisorb Plates (Nunc, Roskilde, Denmark) were coated with either CTH1 (1 mg/ ml) or heat inactivated (HIA) MoPn EBs (10 mg/ml) and the samples were serially diluted before added to ELISA-plates. Serum samples and vaginal secretion fluid were 5 fold diluted 8 times from a 1:20 and 1:10 dilution, respectively. Antigen-specific IgG1, IgG2a, and IgA were detected with isotype-specific HRP- conjugated rabbit anti-mouse IgG (Zymed) diluted 1:5000. Substrate was TMB-PLUS (Kem-En-TEC, Taastrup, Denmark). Reciprocal serum dilutions corresponding to 50% maximal binding (EC50) were computed using the GraphPad Prism 4. Chlamydia-specific cellular responses Blood lymphocytes were purified on a density gradient. Cells were pooled from 3–13 mice in each group. Single-cell suspensions were prepared from individual spleens (4 mice/group) and obtained by homogenisation through a metal mesh and washed twice in RPMI-1640 (Gibco Invitrogen, Taastrup, Denmark). All cell cultures were grown in Nucleon microtiter plates (Nunc, Roskilde, Denmark) containing 26105 cells/well in 200 ml RPMI- 1640 supplemented with 561025 M 2-mercaptoethanol, 1 mM glutamine, 1% pyruvate, 1% penicillin-streptomycin, 1% HEPES and 10% fetal calf serum (FCS) (Invitrogen, Taastrup, Denmark). The cells were restimulated with either HIA-MoPn (5 mg/ml), CTH1 (5 mg/ml), CT443 (5 mg/ml) or CT521 (5 mg/ml). Stimulation with Concavalin A (5 mg/ml) (results not shown) or media (C) were done as positive control for cell viability and negative control, respectively. After 72 h of incubation at 37uC/ 5% CO2, supernatants were harvested and stored at 220uC before use. The amounts of secreted IFN-c were determined by enzyme-linked immunosorbant assay (ELISA) as described elsewhere [59]. Intracellular cytokine staining procedure Splenocytes were stimulated over night with 5 mg/ml of CTH1 or HIA-MoPn at 37uC/5% CO2 and subsequently incubated for 5 h at 37uC with 10 mg/ml brefeldin A (Sigma-Aldrich, USA) and 0.7 ml/ml monensin/GolgiStop (BD Pharmingen). The cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before surface staining with rat anti-mouse antibodies. The intracellular cytokine staining procedure was done essentially as described in [48]. Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/CytopermTM (BD, San Diego, CA, USA). The following antibodies were used for surface staining: PerCP-Cy.5.5-anti-CD8a (53-6.7), APC-Cy7- anti-CD4 (GK1.5), and FITC-anti-CD44. The following antibod- ies were used for intracellular staining: PE-anti-TNF-a, APC-anti- IL-2, PE-Cy7-anti-IFN-c. All antibodies were purchased from BD Pharmingen (San Diego, USA) or eBiosciences (San Diego, USA). Vaginal chlamydial load Vaginal swabs were obtained at 3, 7, 14, 21, 28 and 35 days after infection. Swabs were vortexed with glass-beads in 1 ml SPG buffer and stored at 280uC until analysis. Infectious load was determined as described in [30]. Briefly, McCoy cell monolayers were infected with a titrated volume of the swab suspension. The plates were centrifuged at 7506g for 1 h at RT followed by incubation at 35uC for 2 h. Infection-media was then replaced with fresh media and the cells incubated at 37uC for 30 h. Inclusions were visualised by staining with polyclonal rabbit anti- MOMP serum made in our laboratory, followed by a FITC conjugated swine anti-rabbit Ig (DAKO, Glostrup, Denmark). Background staining was done with propidium iodide (Invitrogen, PLoS ONE | www.plosone.org 9 May 2010 | Volume 5 | Issue 5 | e10768