for interferon gamma using anti-mouse IFNc-PE (phycoerythrin). All samples were read on the LSRII Flow cytometer (BD Biosciences). Data was analyzed using BD FACSDiva 6.0.1 software (BD Biosciences). Antibody Detection Serum for the detection of HA specific antibodies by neutralization or hemagglutination inhibition assays was collected from each mouse following immunization, as previously described [16,38]. Briefly, all samples were treated with receptor-destroying enzyme (RDE, 1:3 ratio, Accurate Chemical) overnight at 37uC and inactivated the following day for 45 minutes at 56uC. Starting with a 1:10 dilution, two-fold serial dilutions of serum were done in a 96-well round- bottom microtiter plate in virus diluent (MEM, 0.3% BSA, and antibiotics), mixed with 100 pfu/well of homologous A/Hanoi/ 30408/2005 virus and incubated at 37uC for 60 minutes. For hemagglutination inhibition (HI) assays, two-fold serial dilutions of each sample were performed in phosphate buffered saline (PBS) and 50 ml/well was added to a 96-well V-bottom microtiter plate. Four hemagglutinating doses of homologous A/Hanoi/30408/2005 were added to each well and the plates were incubated at room temperature for one hour. Horse red blood cells (0.5% erythrocytes in 0.85% saline) were added to each well and incubated for 60 minutes. Hemagglutination titer was determined as the reciprocal of the highest dilution where red blood cells did not agglutinate. For neutralization assays, the serum/virus mixture was added onto subconfluent MDCK cells in a 96-well flat-bottom microtiter plate and incubated for 5–10 minutes at room temperature. Virus diluent supplemented with 3.0 mg/ml TPCK-trypsin was added into each well (100 ml/well) and incubated at 37uC, 5% CO2, for 48 hours. Cells were monitored for the presence or absence of CPE using a light microscope. The highest serum dilution which did not exhibit CPE was scored as positive for neutralizing antibody and titers were reported as the reciprocal of the dilution. All infectious in vitro work was performed in the high containment laboratory of NML/PHAC. An enzyme-linked immunosorbant assay (ELISA) was per- formed to evaluate total IgG antibodies present in serum at 25 days and 1 year following vaccination. 96-well plates (NUNC) were coated with 1 ug/ml H5 HA antigen, overnight at 4uC. The following day, serial tenfold dilutions of serum were added to the References 1. Gao W, Soloff AC, Lu X, Montecalvo A, Nguyen DC, et al. (2006) Protection of mice and poultry from lethal H5N1 avian influenza virus through adenovirus- based immunization. J Virol 80: 1959–1964. 2. Fattori E, Zampaglione I, Arcuri M, Meola A, Ercole BB, et al. 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The absorbance was detected at 450 nm (OD450). The endpoint cut-off (A450 = 0.100) was determined as the last serum dilution which was greater than two times the optical density of the negative control. Endpoint titres were reported as the reciprocal of the first serum dilution that was higher than the cut-off. Rapid vaccination regimen and detection of viral load Groups of 15 BALB/c mice were vaccinated with a 1010 vp/ mouse dose of PAV3-HA or AdHu5-HA 5, 8, or 10 days prior to challenge with 100LD50 of H5N1-H05 virus. Nine animals were monitored for survival and lung tissue and blood were harvested from the remaining 6 mice three days post-infection. Virus load was determined by the tissue culture infectious dose which will result in cytopathic effects (CPE) of fifty percent of cell culture (TCID50). TCID50 was performed by added serial dilutions of homogenized lung tissue onto MDCK cells and monitoring the presence of CPE after 48 hours. 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