Background: Despite the development of highly effective prophylactic vaccines against human papillomavirus (HPV) types 16 and 18, prevention of cervical dysplasia and cancer in women infected with high-risk HPV types remains an unmet medical need. We have previously reported data from a phase 1 dose escalation study for a therapeutic HPV16/18 vaccine, VGX-3100, delivered by in vivo electroporation (EP). Immunization in that study drove seroconversion as gauged by ELISA to at least one vaccine antigen in 100% of patients while 78% mounted an Interferon Gamma (IFNg)ELISpot response to the vaccine antigens.. Methods: Of the 18 patients enrolled in that trial, 13 were rolled into a second phase 1 immunogenicity trial entailing a single 6.0mg boost of VGX-3100 administered using EP. Results: Increases in immune reactivity following the boost were noted, as eleven patients showed boost-driven increases in ELISA titers and the mean ELISpot magnitude increased by at least 2-fold in seven patients irrespective of the dose of VGX-3100 they received in the first clinical trial. Additional Flow Cytometric analyses of cellular responses revealed increased HPV specific IFNg and TNFa production as compared to prior to the boost (0.34% to 0.57% and 0.27% to 0.60%, respectively) with contribution from both the CD4+ and CD8+ T cell compartments. Additionally, HPV specific CD8+ T cells expressing Fas Ligand concomitantly with either cytokine or the degranulation marker CD107a increased from 0.19% to 0.28% following the boost. Employment of staining for the activation marker CD137 (41BB) showed that patient CD8+ T cells continued to activate efficiently in response to HPV antigens following the boost, including a statistically significant increase in co-expression of Granzyme B and Perforin within the activated T cell subset (p=0.026). Conclusions: Taken together these data suggest that immunization with VGX-3100 drives immune responses that have the ability to be further boosted by additional administrations. Current studies are underway in an untreated HPV 16/18 associated CIN2/3 setting to assess whether these significant immune responses can translate into clinically effective treatment options to surgery. Clinical trial information: NCT01188850.