The cytotoxicity assay was performed using a Delfia EuTDA cytotoxicity detection kit (Perkin-Elmer, Waltham, MA, USA). Mice were sacrificed 1 week after the last vaccination for splenocyte extraction. Splenocytes were cultured in an IMDM medium (Invitrogen) supplemented with 5% FBS (Invitrogen), the corresponding target peptides (10 lg/mL), and IL-2 (10 U/mL, eBioscience) in 12-well plates (BD Bioscience) at 37°C for 5 days. They were seeded with the TDA-labeled, peptide-loaded T2 cells HLA-A2 T-cell epitopes in H5N1 nucleoprotein Vet. Res. (2010) 41:24 in ratios of 50:1, 10:1, and 5:1, respectively. The mice immunized with N220HHDpBudCE4.1 were used as an experiment control. The splenocytes seeded with N220-loaded T2 cells were used as a positive control while those seeded with N160-loaded T2 cells served as a negative control [2] . After incubating at 37°C for 1 h, the culture medium was collected and the fluorescent signals emitted by the dead peptide-loaded T2 cells were detected with the cytotoxicity reagents according to the procedures stated by the manufacturer. The cytotoxicity was determined by the following equation: