Human papillomavirus (HPV)-associated malignancies remain a significant global health burden, particularly among individuals with established infection or limited access to prophylactic vaccination. Although the E6 and E7 oncogenes of high-risk HPV types represent attractive therapeutic targets, current vaccine approaches have shown limited clinical efficacy. In this study, we investigated multiple strategies to enhance the therapeutic activity of an HPV16 E6/E7 mRNA vaccine. To leverage pre-existing antiviral immune memory, we engineered an immunodominant CD4+ T-cell epitope derived from herpes simplex virus type 1 (HSV-1) glycoprotein D (gD) into a ubiquitin-tagged mRNA-UB-E6/E7 construct, generating an epitope-enhanced HPV16 therapeutic vaccine. The ubiquitin moiety was incorporated as an additional potentiation strategy to enhance antigen processing and presentation. Antitumor efficacy, antigen-specific T-cell responses, and tumor immune infiltration were assessed following vaccination alone or in combination with the histone deacetylase (HDAC) inhibitor entinostat or the STING agonist ADU-S100, two immunomodulatory agents known to remodel the tumor microenvironment. Incorporation of ubiquitin and HSV-1 gD CD4+ T-cell epitope significantly enhanced HPV-specific CD8+ T-cell responses and improved antitumor efficacy. Furthermore, combination therapy with either entinostat or ADU-S100 provided additional therapeutic benefit. These findings demonstrate that incorporation of a heterologous HSV-1 gD helper epitope can augment HPV E6/E7-targeted mRNA vaccination by harnessing pre-existing HSV-1-specific immune memory and enhancing CD4+ T-cell help. Together with ubiquitin-mediated enhancement of antigen presentation and modulation of the tumor microenvironment through HDAC inhibition or STING activation, these complementary potentiation strategies may further improve therapeutic outcomes and support the development of combinatorial immunotherapeutic approaches for HPV-associated cancers.
