Characterization of a Novel Oncolytic Virus for Cancer Therapy
Authors: Ravichandran Abirami , Nissly Ruth H. , Chothe Shubhada , Jakka Padmaja , Kuchipudi Suresh V. 
Published: May 2022

BackgroundOncolytic viruses (OVs) that selectively replicate (oncoselectivity) and lyse (oncotoxicity) cancer cells are emerging as promising cancer therapeutics. A range of DNA and RNA OVs are in different stages of human clinical trials. The presence of preexisting immunity against OVs and their potential pathogenic effects are significant limitations. Using animal viruses that are non‐pathogenic to humans is considered an attractive strategy. We identified a naturally occurring animal virus with potent oncolytic ability, hereafter referred to as oncolytic virus 1 (OV1). We tested the oncoselectivity and oncotoxicity of OV1 using a range of human cancer cells, immortalized cells, and normal non‐cancerous cells. We further evaluated the oncolytic properties of OV1 in vivo using the murine triple‐negative breast cancer (TNBC) model, 4T1.2.MethodsHuman renal (769P), mammary (HCC 1500), colorectal (HRT‐18G), lung (A549), and pancreatic (BxPC‐3) cancer cells, and leukemia (THP‐1) cells, human immortalized embryonic kidney cells (293T), and human primary bronchial epithelial cells (PBEC) were infected with OV1 at a range of multiplicity of infection (MOI) from 0.01 to 5. After 72h of infection, MTS assay to determine cell viability and Caspase‐Glo 3/7 assay to quantify apoptosis were performed. OV1 replication in cultured 4T1.2 cells was assessed by infecting the cells at an MOI of 0.1 and quantifying viral nucleic acid in the supernatants at 72h. Female BALB/c mice were injected with 4T1.2 cells into the fourth mammary gland on the left side. At 19 days post‐tumor implantation, 105TCID50 units of OV1 in 50µl was injected half intratumorally and half intraperitoneally. Mice in the mock treatment group received 50µl of vehicle control. Tumor sizes were measured regularly and at day 35, mice were sacrificed and the number of live cells in the tumor were quantified by flow cytometry.ResultsThere was a significant decrease in the viability of OV1 infected 769P cells (MOI ≥ 0.1), A549 (MOI ≥ 1), THP‐1 (MOI ≥ 2.5), and HCC 1500 (MOI ≥ 2.5) at 72h compared with controls. Notably, there was no significant change in the viability of primary cells (PBEC) and immortalized 293T infected with OV1 at MOI of 0.1 to 2.5 compared to controls. There was a significant increase in caspase activity in 769P cells (at MOI ≥ 0.1), A549 (at MOI ≥ 1), THP‐1 (at MOI ≥ 1), BxPC3 (at MOI ≥ 2.5), HRT‐18G (at MOI ≥ 2.5) and HCC 1500 (at MOI ≥ 0.1). There was no increase in the caspase activity of PBEC at all the tested MOI. OV1 viral replication was observed in 4T1.2 cells in vitro, as evidenced by increasing viral nucleic acid in culture supernatants. BALB/c mice injected with OV1 showed a significant decrease in tumor size and a decrease in the live cell numbers in the tumor compared with mock treated mice.Conclusions and future directionsThe results established the oncoselectivity and oncotoxicity of OV1 and its oncolytic ability in vivo. Further studies are underway to investigate OV1 replication in cells, immune stimulatory properties of OV1 in vivo, and the generation of a recombinant OV1 with increased oncolytic ability.