280uC in growth medium at ,56108 CFU/ml. Bacteria were thawed, sonicated, washed, and diluted in phosphate-buffered saline (PBS) for immunizations and infections. All bacterial work was performed at the Statens Serum Institut by authorized personnel. Construction of recombinant replication-deficient adenoviral 5 based vaccine Ad-H4 The M.tb fusion protein H4 consisting of the two antigens Ag85B and TB10.4 was amplified by overlapping PCR from plasmid template and cloned into a pacCMV-based shuttle vector. From the shuttle plasmid, human type 5 recombinant non-replicating adenovirus vectors were produced from homol- ogous recombination by standard methods [42]. After purifica- tion, adenoviral stocks were immediately aliquoted and frozen at 280uC in 10% glycerol, and the infectivity of the adenoviral stocks was determined with Adeno-X Rapid Titer kit (BD Clontech). Immunization Mice immunized with Ad-H4 were anesthetized and injected once with 26107 infectious units (IFU) s.c. in the right hind footpad. Mice immunized with the Ag85B-TB10.4 fusion protein (rH4) were immunized twice at two-week intervals s.c. at the base of the tail with a total volume of 200 ml containing 5 mg rH4 formulated in cationic liposomes (CAF01). For boosting studies, mice received a single dose of rH4 injected s.c. at the base of the tail and two weeks later the mice were given one booster vaccination of Ad-H4 in the right footpad. Non-vaccinated control mice received NaCl s.c. at the base of the tail, and the BCG control mice received a single dose of BCG Danish 1331 (2.56105 CFU) injected s.c. at the base of the tail. Experimental infections When challenged by the aerosol route, the animals were infected with ,50 CFU of M.tb Erdman/mouse with an inhalation exposure system (Glas-Col, Indiana, USA). Mice were challenged eight weeks after the first vaccination and killed either two weeks post-challenge for immune surveillance or six weeks post-challenge for bacterial counts. Numbers of bacteria in the lung were determined by serial three-fold dilutions of individual whole-organ homogenates in duplicate on 7H11 medium. Colonies were counted after two-three weeks of incubation at 37uC. Protective efficacies were expressed as log10 bacterial CFU. Lymphocyte preparation Splenocytes and lung lymphocytes were obtained by passage of the organs through a 100 mm nylon cell strainer (BD Pharmingen, USA) followed by two washing procedures using RPMI 1640. Cells in each experiment were cultured in sterile microtiter wells (96-well plates; Nunc, Denmark) containing ,26106 cells in 200 ml of RPMI 1640 supplemented with 1% (v/v) premixed penicillin-streptomycin solution (Invitrogen Life Technologies), 1 mM glutamine, and 5% (v/v) fetal calve serum (FCS) at 37uC/ 5% CO2. The lymphocyte cultures were further used for ELISA and FACS analysis. Antigens for in vitro stimulation Synthetic overlapping peptides (9- and 18-mers) covering the complete primary structure of Ag85B, TB10.4, and ESAT-6 were synthesized by standard solid-phase methods on a SyRo peptide synthesizer (MultiSynTech, New England) at the JPT Peptide Technologies (Berlin, Germany), or at Schafer-N (Copenhagen, A New TB Vaccine Strategy Denmark). Peptides were lyophilized and stored dry at 220uC until reconstitution in PBS. IFN-c enzyme-linked immunosorbent assay (ELISA) Microtiter plates (96 well; Maxisorb; Nunc, Denmark) were coated with 1 mg/ml monoclonal rat anti-murine IFN-c (clone R4-6A2; BD Pharmingen). Free binding sites were blocked with 2% (w/v) milk powder in PBS. Culture supernatants were harvested from lymphocyte cultures after 72 hours of in vitro antigen stimulation and tested in triplicates. IFN-c was detected with a 0.1 mg/ml biotin-labeled rat anti-murine antibody (mAb; clone XMG1.2; BD Pharmingen, USA) and 0.35 mg/ml horse- radish peroxidase-conjugated streptavidin (Zymed, Invitrogen, USA). The enzyme reaction was developed with 3.39, 5.59- tetramethylbenzidine, hydrogen peroxide (TMB plus; Kementec, Denmark) and stopped with 0.2 M H2SO4. rIFN-c (BD Pharmingen, USA) was used as a standard. Plates were read at 450 nm with an ELISA-reader and analyzed with KC4 3.03 Rev 4 software. Flow cytometric analysis Intracellular cytokine staining procedure: Splenocytes or lung lymphocytes were stimulated for 1 h with 2 mg/ml antigen at 37uC/5% CO2 and subsequently incubated for 5 h at 37uC with 10 mg/ml brefeldin A (Sigma-Aldrich, USA) at 37uC. The cells were washed in FACS buffer (PBS containing 0.1% sodium azide and 1% FCS) before surface staining with rat anti-mouse antibodies. Cells were washed with FACS buffer before fixation and permeabilization using the BD Cytofix/CytopermTM (BD, San Diego, CA, USA) according to the manufacturer’s protocol followed by intracellular staining. When staining with FITC-anti- BrdU an extra fixation and permabilisation step, as well as DNAse treatment, was introduced before the intracellular staining. Cells were washed and resuspended in formaldehyde solution 4% (w/v) pH 7.0 (Bie & Berntsen, Denmark) and samples were analyzed on a six-color BD FACSCanto flow cytometer (BD Biosciences, USA). Data analysis was done on FlowJo Software ( Tree Star, Asland, OR, 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. For intracellular staining the following antibodies were used: PE-anti-TNF-a, APC-anti-IL-2, PE-Cy7-anti-IFN-c, and FITC-anti-BrdU. All antibodies were purchased from BD Pharmingen (San Diego, USA) or eBios- ciences (San Diego, USA). In vivo cytotoxicity assessed by adoptive transfer of CFSE-labeled target cells Single splenocyte suspensions of CB6F1 mice were obtained by passage through a fine 100 mm nylon cell strainer (BD Pharmin- gen, USA). Erythrocytes were depleted by lysis in ammonium chloride solution, washed in PBS before resuspension in incomplete RPMI and stained with 5(6)-Carboxyfluorescein diacetate N-succinimidyl ester (CFSE) (Sigma-Aldrich, San Louis, USA) at CFSEhigh (40 mM) or CFSElow (4 mM) concentration for 10 min at 37uC. Excess CFSE was quenched with RPMI containing 10% FCS and subsequently washed in medium without FCS. Next, CFSEhigh labeled cells were pulsed with TB10.43-11, TB10.420-28, or TB10.474-88 peptides at a concentra- tion of 10 mg/ml for 1.5 hr at 37uC. After being washed and resuspended in PBS the CFSEhigh and CFSElow suspensions for each peptide was mixed at equal volumes. Finally, 106106 cells per target peptide per mouse were given intravenously into na¨ıve mice and mice vaccinated one week before. Four hours later, PLoS ONE | www.plosone.org 11 April 2009 | Volume 4 | Issue 4 | e5139