Abstract
Persistent infection with high-risk human papillomavirus, particularly HPV16, is a major cause of cervical cancer and orogenital malignancies. Therapeutic DNA vaccines targeting viral oncoproteins represent a promising strategy to induce antitumor immune responses. However, the impact of antigen combinations in bicistronic vectors and the immunization sequence on therapeutic efficacy remains poorly understood. Thus, this study aimed to characterize in silico previously described E5CP and E7CP vaccines, develop a new bicistronic construct, and evaluate its antitumor efficacy under different immunization regimens in vivo. The constructs were initially characterized regarding predicted physicochemical, immunological, and structural properties, followed by molecular confirmation of plasmid construction and qualitative assessment of transcript detection after transient transfection. In vivo, C57BL/6 mice bearing C3 tumors were immunized with five different regimens combining the monocistronic or bicistronic constructs. At the endpoint, tumors and spleens were collected for histopathological and immunological analyses. The results showed that regimens using the bicistronic construct or including E5 in the first dose were associated with lower mean tumor volumes or delayed tumor growth compared to the control groups. Histopathological and immunophenotypic changes were variable among regimens, and an exploratory analysis suggested an association between lower nitric oxide levels and smaller tumor volumes. Therefore, the order of antigen administration and the use of bicistronic constructs warrant further investigation as strategies that could be optimized to improve the therapeutic efficacy of HPV DNA vaccines.