Abstract While both pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine and TA-HPV recombinant vaccinia viral vector-based vaccines have elicited HPV-specific CD8+ T cell responses in HPV16/E7-expressing tumor models, and been used as prime-boost regimen to enhance HPV-specific immune responses in humans (NCT00788164), the optimal route of administration for TA-HPV remains unclear. In a preclinical model, we examined the immunogenicity of priming with intramuscular pNGVL4a-Sig/E7(detox)/HSP70 followed by TA-HPV boost through different administration routes. We observed that priming twice with a pNGVL4a-Sig/E7(detox)/HSP70 followed by a single TA-HPV immunization boost through skin scarification generated the strongest antigen-specific CD8+ T cell response in C57BL/6 mice. These data translate to tumor control and prolonged survival of treated mice. Our results provide rationale for future clinical testing of intramuscular pNGVL4a-Sig/E7(detox)/HSP70 DNA vaccine prime, TA-HPV vaccine skin scarification boost immunization regimen for the control of HPV-associated diseases. Highlights • DNA prime/TA-HPV SS boost generates strong E6/E7-specific CD8+ T cell responses. • DNA prime/TA-HPV SS boost generates strong E6/E7-specific CD8+ antitumor responses. • Antitumor responses in TC-1-bearing mice is possibly vaccination site-specific. • DNA prime/TA-HPV SS boost enhances immune response against multiple antigens. • TA-HPV SS produces stronger E7-specific CD8+ T cell responses than IM injection. [ABSTRACT FROM AUTHOR]