We have previously linked the sorting signals of the lysosome-associated membrane protein-1 (LAMP-1) to HPV-16 E7 antigen, creating a chimera, Sig/E7/LAMP-1. We found that both Sig/E7/LAMP-1-containing recombinant vaccinia virus (Vac-Sig/E7/LAMP-1) and Sig/E7/LAMP-1 DNA can generate strong antitumor immunity. To determine whether combination of Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 can further enhance immune responses, sequential vaccination with Sig/E7/LAMP-1 DNA and Vac-Sig/E7/LAMP-1 was given. We found that priming with Sig/E7/LAMP-1 DNA and boosting with Vac-Sig/E7/LAMP-1 generated the strongest E7-specific CD8 + T cell responses. Our results encourage the use of the DNA prime/vaccinia booster regimen in future clinical trials.