Over the past decade, oncolytic viruses (OVs) have been developed as a promising treatment alone or in combination in immuno-oncology but have faced challenges in late-stage clinical trials. Our retrospective reanalysis of vaccinia oncolytic virus (VOV) clinical trials indicates that lower doses—rather than the maximum tolerated dose (MTD)—are associated with better tumor response rates. Patients who responded well to lower doses generally had prolonged survival rates in the early phase clinical trial. The association between poor outcomes and an increase in OV-induced neutrophils (OV-N) but not baseline neutrophil counts suggests the need for a comprehensive characterization of OV-N. Although this reanalysis is limited by patient heterogeneity—including differences in cancer type and stage, treatment schedules, and administration routes—it remains informative given the complexities of translational studies in the tumor-bearing mouse models of vaccinia oncolytic viruses. Notably, while OV-N increases with higher viral doses, the immune state shaped by tumor progression likely amplifies this tendency. These findings highlight the importance of OV-N immune modulation as well as dose optimization for the successful clinical development of VOV.

Viral replication of thymidine kinase deleted (tk⁻) vaccinia virus (VV) is attenuated in resting normal cells, enabling cancer selectivity, however, replication potency of VV-tk⁻ appears to be diminished in cancer cells. Previously, we found that wild-type herpes simplex virus (HSV)-tk (HSV-tk) disappeared in most of the recombinant VV after multiple screenings, and only a few recombinant VV containing naturally mutated HSV-tk remained stable. In this study, VV-tk of western reserve (WR) VV was replaced by A167Y mutated HSV-tk (HSV-tk_418m), to alter nucleoside selectivity from broad spectrum to purine exclusive selectivity. WOTS-418 remained stable after numerous passages. WOTS-418 replication was significantly attenuated in normal cells, but cytotoxicity was almost similar to that of wild type WR VV in cancer cells. WOTS-418 showed no lethality following a 5 × 10⁸ PFU intranasal injection, contrasting WR VV, which showed 100% lethality at 1 × 10⁵ PFU. Additionally, ganciclovir (GCV) but not BvdU inhibited WOTS-418 replication, confirming specificity to purine nucleoside analogs. The potency of WOTS-418 replication inhibition by GCV was > 10-fold higher than that of our previous truncated HSV-tk recombinant OTS-412. Overall, WOTS-418 demonstrated robust oncolytic efficacy and pharmacological safety which may delegate it as a candidate for future clinical use in OV therapy.