Abstract
BACKGROUND: With limited therapeutic options for advanced stages, hepatocellular carcinoma (HCC) continues to be the primary cause of cancer-related deaths globally. Although still less than ideal in HCC, immunotherapy-especially immune checkpoint drugs targeting the PD-1/PD-L1 axis-shows promise. Combining direct tumor lysis with immune modulation provides a new strategy in oncolytic virotherapy using vaccinia virus. Designed to boost anti-tumor immunity through dual checkpoint inhibition and oncolysis, this study assessed the efficacy of the FilC/PD-1 recombinant vaccinia virus. MATERIALS AND METHODS: Homologous recombination was used to develop a recombinant vaccinia virus expressing FilC and PD-1 inhibitors. We evaluated viral infectivity and replication in HCC cell lines (HepG2, Huh7, Hepa1-6, PLC/PRF/5) and in VERO cells-a non-hepatic kidney epithelial cell line from Chlorocebus sabaeus (African green monkey)-commonly used in virology-to assess baseline viral tropism outside the liver. Using BALB/c nude mice (xenograft) and C57BL/6 mice (syngeneic model), in vivo efficacy was assessed in HCC murine models, evaluating tumor volume reduction, immune cell infiltration, survival rates, and systemic toxicity. FINDINGS: The FilC/PD-1 recombinant virus displayed high infection efficiency (88.4% in HepG2), robust viral replication, and substantial oncolytic activity in HCC cells. Compared to the PD-1 inhibitor virus alone, the virus greatly lowered tumor volume (84%) and raised CD8(+) T cell infiltration (42.8%), thereby prolonging survival (68 days). Histopathological study verified low toxicity in the main organs. CONCLUSION: Combining synergistic immune checkpoint inhibition with oncolytic virotherapy, the FilC/PD-1 recombinant vaccinia virus significantly increases anti-tumor immunity and slows the growth of HCC.