||The influenza A virus nucleoprotein (NP) is an attractive target for avian flu vaccine development because of its high conversancy in the evolutionary chain of the virus. Here I have identified two novel HLA-A*0201-restricted H5N1 NP epitopes, named NP373-381 AMDSNTLEL (NP373) and NP458-466 FQGRGVFEL (NP458), using computational bioinformatic analysis. These NP peptides showed a high binding affinity to HLA-A*0201 on T2 cells, and were able to induce activation of cytotoxic T lymphocytes (CTLs) in the human peripheral blood mononuclear cells. Therefore, the potential of using NP373 and NP458 peptide sequences supplemented with single-chain trimer (SCT) as potential DNA vaccine candidates was examined. After administration of the candidate constructs into the HHD transgenic mouse model the using gene gun delivery system, the amount of secreted interferon-gamma and number of cell deaths were measured by enzyme-linked immunosorbent spot assays and cytotoxicity assays, respectively. Results indicated that a significant amount of interferon-gamma was secreted by the T lymphocytes of the vaccinated mice, and the T lymphocytes were able to eliminate the corresponding peptide-loaded T2 cells. To improve this system, co-stimulatory molecules, CD80/CD86 and CD137L, were included in the SCT-based DNA vaccine construct. Animals immunized with DNA constructs carrying SCT plus CD80/CD86 alone, or both CD80/CD86 and CD137L provided additive NP373-specific CTL responses in the HHD transgenic mice. The discovery of these novel immunogenic NP peptides and improvement in the DNA construct design provide valuable information for avian flu vaccine development.