To investigate the synergistic antitumor effect and immunological mechanisms of a recombinant oncolytic influenza virus carrying programmed cell death 1 (PD-1) antibody (OVFlu-αhPD1) combined with radiotherapy (RT) in hepatocellular carcinoma (HCC).

A recombinant oncolytic virus was successfully rescued in the early stage using reverse genetics (RG) technology, and designated OVFlu-αhPD1, which was comprehensively characterized via chicken red blood cell hemagglutination assay and detection of replication kinetics in human hepatocellular carcinoma cells Huh7 and murine hepatocellular carcinoma cells Hepa1-6. In vitro, CCK-8 assays were used to assess the effects of OVFlu-αhPD1 at different multiplicities of infection (MOI=0.1, 1, and 3), alone or combined with radiotherapy (RT; 8 Gy), on the viability of hepatoma cells (Huh7 and Hepa1-6) and normal hepatocytes (LX2 and AML12), and a colony formation assay was performed to evaluate the inhibitory effect of OVFlu-αhPD1 plus RT on Huh7 cell proliferation. For in vivo experiments, female C57BL/6 mice (6 to 8 weeks old, weighing 17 to19 g; n=45) were subjected to establish a subcutaneous tumor-bearing model of Hepa1-6 cells, and then the model mice were randomized into 5 groups (n=9): PBS, wild-type PR8, OVFlu-αhPD1, RT (24 Gy in 3 fractions), and OVFlu-αhPD1+RT to evaluate the antitumor efficacy of the combination therapy. Tumor volumes were measured every 2 days during treatment. After the final treatment, the spleens were havested for CD4⁺CD69⁺ and CD8⁺CD69⁺ T-cell frequencies by flow cytometry. Major organs (heart, liver, lung, kidneys, and brain) were harvested for HE staining to assess histopathological changes, and the activities of serum AST and ALT were measured to evaluate the safety of oncolytic virus therapy.

The recombinant oncolytic influenza virus OVFlu-αhPD1 showed a hemagglutination titer of 2⁷ to 2⁸, and replicated efficiently in Huh7 and Hepa1-6 cells, with viral titers reaching 7 to 8 lgTCID₅₀/mL at 72 h post-infection. CCK-8 assay demonstrated that OVFlu-αhPD1 inhibited HCC cell viability in a dose-dependent manner (MOI=3 significantly decreased viability in Huh7 and Hepa1-6 cells, P<0.01), and the inhibitory effect was further enhanced after combination with RT (P<0.01). Whereas no significant toxicity to normal hepatocytes was observed. Colony formation assay indicated that combined treatment significantly reduced colony numbers of Huh7 cells (P<0.01). In the mouse tumor-bearing model, the combination group exhibited significantly smaller tumor volumes than any monotherapy group (P<0.01), accompanied by significantly increased proportions of splenic CD4⁺CD69⁺ and CD8⁺CD69⁺ T cells in spleen (P<0.01). HE staining revealed no evident pathological changes in major organs, and serum ALT and AST levels did not differ significantly among groups (P>0.05).

The combination of oncolytic influenza virus and radiotherapy significantly enhances the killing effect against HCC cells in vitro and markedly improves tumor suppression in HCC-bearing mice by activating antitumor immune responses, demonstrating a favorable synergistic antitumor effect and providing a theoretical basis for the clinical application of oncolytic virus-based combination therapy.