Feasibility of infectious prion digestion using mild conditions and commercial subtilisin

Two serine protease enzymes, subtilisin 309 and subtilisin 309-v, were used to digest brain homogenates containing high levels of prion infectivity using mildly alkaline conditions to investigate prion decontamination methods. To establish that PrPres infectivity was eliminated, we utilized the Rocky Mountain Laboratory (RML) mouse-adapted scrapie model system for bioassay. Only one digestion condition (subtilisin 309 at 138 mAU/ml, 55 °C and 14 h digestion time pH 7.9) was considered to be highly relevant statistically (P < 0.001) compared to control, with 52% of challenged mice surviving until the end of the study period. In contrast, treatment of PrPres by autoclaving at 134 °C or treatment with hypochlorite at a concentration of 20,000 ppm completely protected mice from prionosis. Further, in vitro assays suggest that potential proteolytic based PrPres decontamination methods must use high enzyme concentration, pH values >9.0, and elevated temperatures to be a safely efficacious, thereby limiting applicability on delicate surgical instruments and use in the environment.