mycin B (BD Biosciences). Avian influenza H5N1 strain A/ Hanoi/30408/2005 (H5N1) was generously provided by Q. Mai Le and T. Hien Nguyen, National Institute of Hygiene and Epidemiology, Hanoi, Vietnam. Virus was propagated on MDCK cells cultured with virus diluent (MEM, 0.3% bovine serum albumin, and antibiotics) containing 3.0 mg/ml TPCK-treated trypsin (TPCK-trypsin) and titered by plaque assay. All infectious recombinant adenovirus constructs were propagated on HEK 293 or VR1BL E1 cells and purified by a cesium chloride density gradient. Vectors and construction of transfer plasmids The full complementary DNA (cDNA) sequence from the A/ Hanoi/30408/2005 H5N1 hemagglutinin (H05-HA) gene was obtained, codon optimized, and synthesized from overlapping oligonucleotide primers, as previously described [36]. The H05- HA gene was first cloned into the pCAGa plasmid, containing a chicken-b-actin (CAG) promoter, to generate the pCAGa-HA construct. The pCAGa-HA expression cassette was then excised and inserted into two shuttle vector systems: pShuttle2 (Clontech) and pPAV227 (VIDO, University of Saskatchewan). Insertion of the expression cassette replaced the existing pShuttle2 cytomeg- alovirus (CMV) promoter with the CAG promoter, resulting in the pShuttle2-HA construct. Transfer plasmid pPAV227 was also modified to include the SV40 polyadenylation signal from pShuttle2, generating pPAV227- HA-PolyA. The transgene cassettes from pShuttle2-HA and pPAV227-HA- PolyA were cloned into replication-deficient DE1DE3 adenoviral vectors: pAdenoX (AdHu5, Clontech) or pFPAV227 (PAV3, VIDO, University of Saskatchewan). pAdenoX-HA was generated through digestion of both pShuttle2-HA and pAdenoX by homing endonucleases I-CeuI/PI-SceI and ligation of cohesive ends using T4 DNA ligase (Invitrogen). pFPAV227-HA was generated through homologous recombination in Escherichia coli strain BJ5183 (recBC, sbcBC) [37] of linearized pPAV227-HA-PolyA (Eco47III/TthIII1) and pFPAV227 (PacI). Production of PAV3-HA and AdHu5-HA vaccine vectors To obtain AdHu5-HA vaccine, HEK 293 cells were transfected with 10 mg of linearized pAdenoX-HA DNA in calcium phosphate (BD Biosciences) solution and cells were cultured until the appearance of cytopathic effects (CPE). Similarly, VR1BL E1 cells were transfected with 10 ug of linearized pFPAV227-HA DNA combined with Lipofectin (Invitrogen) and cells were cultured until CPE were apparent. Amplified adenoviruses containing cell lysates were harvested, freeze-thawed three times, and purified by CsCl gradients. The integrity of the H05-HA transgene cassette was confirmed through EcoRI restriction digests and by sequencing (DNA Core, National Microbiology Labora- tory) with multiple primer sets. Total virus particles (vp) was determined by OD260 and total infectious particles was determined using anti-hexon antibodies against AdHu5 (AdenoX Rapid Titer kit, Clontech) or against PAV3 (VIDO, University of Saskatchewan). Four independent vector preparations were used for each vaccine. Total infectious particles and total viral particles were determined for both adenovirus vaccines, with ratios of 1:285, 1:333, 1:250, and 1:300 for PAV3-HA and 1:150, 1:250, 1:220, and 1:250 for AdHu5-HA. Expression of PAV3-HA and AdHu5-HA vaccines Protein expression of H05-HA by both vectors was confirmed in HEK 293, VRIBL E1, and AB12 cells using standard Western blotting techniques. To evaluate in vivo expression, groups of 6 BALB/c mice were vaccinated with 1010 vp of PAV3-HA or A Porcine Adenovirus Vaccine Vector against H5N1 AdHu5-HA and muscle tissue was harvested 4 days following immunization. Muscle tissues were homogenized and normalized per gram of muscle tissue in radioimmunoprecipitation (RIPA) buffer. Expression of each vaccine was detected from 75 mg of loaded muscle tissue. Immunization and viral challenge Groups of 10 BALB/c (Charles River Canada) were vaccinated with 108, 109, or 1010 vp of recombinant adenovirus PAV3-HA or AdHu5-HA by intramuscular (I.M.) administration. Each vaccine was diluted in 100 ml and 50 ml was administered in each of the right and left hind limbs. All mice were anesthetized and challenged after 28 days through intranasal inoculation with 100 times the dose of A/Hanoi/30408/2005 virus required to obtain 50% survival (100 Lethal Dose 50 or 100LD50) in 50 ml virus diluent. The LD50 for the H5N1-H05 virus was 1.05 plaque forming units (pfu), therefore 100LD50 was 105 pfu. Mice were monitored for 15 to 20 days following challenge and signs of disease including weight loss, labored breathing, ruffled fur, and death were observed according to an approved scoring chart. All animal procedures were approved by the Institutional Animal Care Committee at the National Microbiology Laboratory (NML) at the Public Health Agency of Canada (PHAC), according to the guidelines of the Canadian Council on Animal Care. All infectious work was performed in the high biocontainment laboratory at NML/PHAC. Enzyme-linked immunosorbent spot assay Groups of 4 BALB/c mice were vaccinated with 1010 vp of PAV3-HA or AdHu5-HA vaccines. The day before each experiment, ELISPOT-IFNc (BD Biosciences) plates were coated with purified mouse IFNc and incubated at 4uC, overnight. As well, overlapping 15mer peptides (Mimitopes, Australia) spanning the entire H05-HA protein were resuspended overnight in dimethyl sulfoxide (DMSO) and allocated in pools by matrix format. Spleens were harvested on days 8, 10, 14, and 21 post- immunization and splenocytes were plated at 56105 cells/well in RPMI 1640 (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 561023 M 2-b-mercaptoeth- anol, and antibiotics) and restimulated with each of the peptide pools (2.5 mg/ml per well). An individual 9mer peptide represent- ing the immunodominant H5N1 H05-HA (IYSTVASSL, con- served influenza A viruses) epitope was also evaluated, along with negative controls PR8-NP (TYQRTRALV, A/PuertoRico/8/34 (H1N1) nucleoprotein) and ZGP (TELRTFSI, Zaire Ebolavirus glycoprotein). ELISPOT-IFNc plates were incubated at 37uC overnight (18–20 hours) and washed the following day according to the manufacturer’s instructions. AEC Substrate Set (BD Biosciences) was used to develop spots formed by interferon gamma secreting cells. Spots were visualized and counted using an ELISPOT plate reader (AID ELISPOT reader, Cell Technology, Colombia, Maryland). Flow cytometric analysis Splenocytes obtained on day 10 post-vaccination, plated at 26106 cells/well, were restimulated with 9mer H05-HA, PR8-NP, or 8mer ZGP individual peptides in DMEM (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 561023 M b-mercaptoethanol, and antibiotics), IL2, and GolgiStop (Brefeldin A, BD Biosciences). Cells were stimulated for 5 hours and stained with anti-mouse CD8-FITC (fluorescein isothiocyanate) at 4uC for 30 minutes. BD Cytofix and permwash protocol was used to fix and permeabilized cells according to the manufacturer instructions. The following day, cells were stained PLoS ONE | www.plosone.org 9 December 2010 | Volume 5 | Issue 12 | e15301