2.4. Cell‐free HSV‐1 virus

Cell‐free HSV‐1 virus was made by plating 10 million Vero cells per T‐75 flask on day 0. On day 1 medium was removed, and about 10 million virus was added in 2 mL of medium per flask for a multiplicity of infection of about 1. This was incubated with rocking every 2 min for 1–2 h to allow viral adsorption to the cells. On day 4, the flasks were shaken to detach cells, the contents were then withdrawn and centrifuged at 1400g for 10 min to pellet cells and cell debris. The supernatant was collected and centrifuged at 23 000g for 2 h to pellet virus. The pellet was resuspended in 1 mL of Vero medium for each T‐75 harvested, and then frozen at −70°C. Virus prepared this way was found to have about 2.4 × 10⁹ pfu/mL.

Both the HSV‐1‐infected cell extracts and the cell‐free HSV‐1 virus were heated at 70°C for 30 min before adding them to the PBMC plates to heat inactivate virus to prevent it from killing the PBMCs in the plates.