Oncolytic virotherapy with tumor selective viruses offers a promising treatment modality for cancer. In human medicine, vaccinia virus (VV) has shown encouraging results on tumor explants. This biotechnology is underused in veterinary oncology.

First objective of this study was to investigate the capacity of various species cell lines to support infection by a replication-selective oncolytic VV (VVTG17990). Our second objective was to assess replication potency of VVTG17990 on those cell lines.

A thymidine kinase and ribonucleotide reductase genes-deleted VV expressing green fluorescent protein (GFP) was designed (VVTG17990).

Non-tumoral canine (DKE1), feline (CrFK), porcine (PK-15) and human (293) cell lines were used as well as tumoral canine (A72) and human (HeLa) cell lines.

Susceptibility was evaluated 16 hours after infection with VVTG17990 using fluorescence microscopy and flow cytometry. Multiplicity of infection (MOI) of 0.1, 0.01 and 0.001 were tested.

Virus titers were evaluated 4 days after infection at a MOI of 0.0001 and 0.00001, by standard plaque forming assay on culture medium and cell lysate.

All experiments were performed in triplicate.

GFP expression was detected by fluorescence microscopy 16 hours after infection for all cell lines even with low infective doses. Flow cytometry allowed an assessment of a dose-dependent infection of cells. For all cell lines, more than 88% of cells were infected at a MOI of 0.1. Equivalent percentage of infection was noticed for HeLa, 293 and PK-15 at a MOI of 0.01. On the other hand, lower infection was assessed for DKE1 (55%), A72 (27%) and CrFK (21%). At a MOI of 0.001, higher percentage of infection was observed for 293 (32%) and PK-15 (25%). For the others, less than 13% of cells were infected. Tumoral status of cell lines didn't seem to influence susceptibility to VVTG17990.

Interestingly, 16 hours after infection VVTG17990 proved effective lytic potency on cell lines. Lytic potency was more important with higher viral doses.

A replication factor of 10⁶ to 10⁷ was determined 4 days after infection for all cell lines, except for feline cell lines (about 10³). Tumoral status of cell lines didn't seem to influence the replication factor.

This study proves that canine, porcine and human cell lines support infection by VVTG17990 with a high replication factor. In comparison, susceptibility and replication potency were lower for feline cell lines. An in vitro lytic potency was also noticed. These promising results support the use of replication-selective oncolytic VV on canine tumors.

Disclosures

Disclosures to report
This study was supported by Transgene.