Groups of 4 BALB/c mice were vaccinated with 10 10 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 postimmunization and splenocytes were plated at 5610 5 cells/well in RPMI 1640 (supplemented with 10% FBS, L-glutamine, NaPyr, HEPES, non-essential amino acids, 5610 23 M 2-b-mercaptoethanol, and antibiotics) and restimulated with each of the peptide pools (2.5 mg/ml per well). An individual 9mer peptide representing the immunodominant H5N1 H05-HA (IYSTVASSL, conserved 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).