Oncolytic virus-mediated remodeling of tumor microenvironment enhances efficacy of an HPV16 E6/E7 mRNA vaccine in an HPV-positive tumor.

Therapeutic vaccination targeting the viral oncoproteins E6 and E7 in HPV-positive tumors remains an attractive strategy; however, current HPV E6/E7 vaccines produced only limited clinical benefit. We developed mRNA-ubiquitin (UB)-E6/E7, an mRNA-based HPV16 E6/E7 construct incorporating an N-terminal UB tag to enhance antigen processing. UB tagging in the mRNA format significantly enhanced antigen-specific immune responses, leading to increased cytotoxic T lymphocyte (CTL) activity and higher frequencies of E7-specific CD8+ T cells. Consequently, mRNA-UB-E6/E7 exhibited enhanced antitumor activity compared with the control when administered as a monotherapy. However, tumor control remained incomplete. To improve therapeutic efficacy, we evaluated several combination strategies, including oncolytic virotherapy, immune modulator-encoding mRNAs, PD-1 checkpoint blockade, and a liver X receptor (LXR) antagonist. Among these approaches, only the combination with the oncolytic herpes simplex virus (FusOn-H2) showed improved therapeutic efficacy. This improvement was associated with increased intratumoral CD8+ T cell infiltration and enhanced CTL activity and IFN-γ production. These findings suggest that effective tumor vaccination requires not only robust systemic T cell responses but also efficient trafficking and function of effector T cells within tumor microenvironment. Collectively, our results support the combination of mRNA-UB-E6/E7 vaccination with FusOn-H2 as a promising strategy for enhancing therapeutic efficacy against HPV-associated malignancies.