Background <p>Human papillomavirus (HPV) 16 infection is associated with several human malignancies. Developing therapeutic vaccines holds great potential for patients who do not benefit from standard care. Circular RNA (circRNA) is an emerging next-generation platform for cancer vaccine development owing to its superior stability and convenient manufacturing process. Herein, we report development of a synthetic circRNA encoding fused HPV16 E7/E6 antigens encapsulated with lipid nanoparticles (LNP) to treat HPV16-related solid tumors.</p> Methods <p>The immunogenicity and anti-tumor immune response of the LNP-circRNA vaccine was determined in naïve C57BL/6 mice and TC-1 tumor-bearing mice, respectively. Changes in immune cells were examined using flow cytometry and immunofluorescence assay. RNA sequencing was used to identify differentially expressed genes and changes in the tumor microenvironment (TME) of tumors treated with LNP-circRNA^E7E6 and empty LNP. Anti-tumor efficacy was further evaluated in LNP-circRNA^E7E6 vaccine combined with anti-PD-L1 antibody treatment.</p> Results <p>Prime-boost vaccination with LNP-circRNA^E7E6 induced a large pool of functional antigen-specific cytotoxic T cells in both the peripheral blood and spleen. This immunization led to profound changes in the TME, characterized by the upregulation of immune activation genes, heavy infiltration of immune cells, and polarization toward a proinflammatory state. Consequently, circRNA^E7E6 immunization could mediate complete tumor regression and prevent tumor growth. Moreover, vaccination sensitized non-inflamed tumors to immune checkpoint blockade therapy.</p> Conclusions <p>The present study results demonstrate that LNP-circRNA^E7E6 vaccine is capable of eliciting robust anti-tumor immunity in the periphery and TME, highlighting the potential for treating HPV16-related cancers and preventing tumor recurrence.</p>