Cytotoxicity of rVV towards mature DCs. (A) DCs were recultured at 1 × 105/200 μL of 5% human serum in 1 well of a 96-well flat-bottom well plate, and cell viability was tested with trypan blue staining for 5 days. Live and PLWUV-inactivated, rVV-infected, and uninfected DCs were compared. Cell viability of rVV-infected mature DCs decreased significantly by day 3 compared with PLWUV rVV-infected and uninfected DCs. (B) Viability of uninfected and infected mature DCs was monitored with Annexin V and PI staining (PI stain not shown). The VV1-6B6 antibody indicated the rate of infection. Mature DCs were infected with live and PLWUV-inactivated rVV-LMP-1 at an MOI of 2:1. Twenty-four to 30 hours later, Annexin V FITC staining was performed and immediately analyzed by FACScan. DCs infected with live virus showed greater than 30% Annexin V+ cells, whereas uninfected and PLWUV rVV-infected DCs showed less than 10% positive cells.
