THE PRIMARY PRODUCTION OF AN INFECTIOUS RECOMBINANT HERPES-SIMPLEX VIRUS-VACCINE

The production and extracellular release of a recombinant Herpes Simpl ex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are ident ified that provide adequate virus titers with cell seeding densities a nd viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/ ml) but leads to process stream contamination by cellular proteins thr ough the rupturing of cells (to 28 pg protein/pfu). By comparison, fre eze-thaw cycles and osmotic rupture by hypotonic saline or glycerol sh ock procedures yield only low virus recovery (typically <10% of that b y sonication), and are accompanied by yet higher levels of protein con tamination (up to 30-fold higher pg protein/pfu). Addition of the poly anionic polymers, heparin or dextran sulphate to a harvest using eithe r hypotonic saline, glycerol shock or isotonic phosphate buffered sali ne increased the yield of infectious virus in the supernatant. By cont rast, addition of polycationic poly-L-lysine resulted in negligible in crease in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/ml) were achieved following treatment of roller bottle cult ured cells displaying a high cytopathic effect with heparin at 50 mu g /mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold low er yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological stat e at harvest. (C) 1998 John Wiley & Sons, Inc.