Project Summary/Abstract: FDA-approved prophylactic human papillomavirus (HPV) vaccines are unable to clear pre-existing HPV infections, the primary etiologic factor for subsets of anogenital and oropharyngeal cancers, including essentially all cervical cancers. Therefore, therapeutic HPV vaccines are being developed and evaluated for their ability to generate HPV-specific CD8+ T cell responses to clear HPV antigen-expressing infected or malignant cells. For example, TA-CIN is a recombinant HPV16 E6, E7, and L2 fusion protein vaccine that can generate both HPV16 E6/E7-specific CD8+ T cell responses as well as L2-specific antibodies. Unfortunately, due to its low intrinsic immunogenicity, TA-CIN has lacked efficacy to date in clinical settings against HPV lesions, despite being well tolerated by vaccinated patients. FMS-like tyrosine kinase 3 ligand (Flt3L) is a cytokine that has the potential to enhance the immunogenicity of TA-CIN vaccination due to its central role in stimulating the expansion and differentiation of CD8+ and CD103+ dendritic cells (DCs) that play a crucial role in antigen cross-presentation for the activation of CD8+ T cell responses, including following vaccination. However, the efficacy of Flt3L treatment is limited by its lack of targeting and short half-life in vivo. We have overcome these disadvantages of Flt3L by fusing it to albumin (Alb), resulting in the development of a novel fusion protein adjuvant, Alb-Flt3L. Utilizing the Fc receptor (FcRn)-mediated in vivo half-life prolonging and the lymph node targeting effects of the Alb fusion, we have demonstrated in our preliminary studies that the Alb- Flt3L fusion protein maintains its bioactivity in activating DCs in vitro, and accumulates in the draining lymph node to a far greater extent than Flt3L following administration in vivo. The Alb-Flt3L fusion safely produces more effective DC expansion and differentiation, and subsequently, upon co-administration of a protein antigen, stronger CD8+ and CD4+ T cells and IgG2a antibody responses. We reasoned that due to its ability to enhance both T cell- and B cell-mediated immune responses, Alb-Flt3L is a promising adjuvant to enhance both the therapeutic and prophylactic immunogenicity of TA-CIN HPV protein vaccine. In this application, we will evaluate the ability of Alb-Flt3L co-administration with TA-CIN to enhance HPV antigen-specific CD8+ T cell, CD4+ T cell, and antibody responses elicited by vaccination, as compared to Flt3L co-administration. We will further assess how the Alb-Flt3L co-administration with TA-CIN vaccination impacts therapeutic antitumor efficacy against a preclinical HPV16+ cervical cancer model, as well as protection against intravaginal HPV1...