1. Proteolytic Inactivation of prions; a biological solution to TSE decontamination. Dickinson J*, Murdoch H*, Sutton JM*, Crabb, W. D.#, Bott, R#, Penet, C.#, and Raven N.D.H* *- Health Protection Agency, UK # -Genencor International

2. Decontamination of TSE agents; practical issues Are any of the available methods effective or applicable in the real world ? Issues of practicality Procedures time consuming Use of large amounts of caustic and hazardous chemicals Damaging to sensitive surgical instruments Operator safety Environmental considerations Not compatible with existing practice Balance of cost vs risk Genuine need for cheap, clean, “user and environment friendly” alternative.

3. Digestion of BSE by thermostable proteases Conditions, protease and standard of assessment critical: Proteases: Properase (~3-log reduction infectivity) and MC3 (>7-log reduction infectivity) Model: mouse-passaged BSE strain 301-V (infectious mouse brain homogenate (iMBH) >10 9 iu / mg) Assessment: Western blot (6H4 and PAb2) and bioassayed in VM mice Process: 10% iMBH digested at pH12, 60 0 C for 30 minutes

4. High molecular weight PrP isoforms are digested by MC3 but not Properase. MC3 PAb2 Properase PAb2 MC3 6H4 Properase 6H4 33kDa 19kDa 52kDa pH12 PrP pH12 Prt PrP 33kDa 19kDa 52kDa pH12 Prt PrP

5. MC3 digestion at 60 0 C pH 12 reduces infectivity by more than 7-logs MC3: 66% survival 500 days 0 10 20 30 40 50 60 70 80 90 100 150200250300350400450 Incubation time (days post inoculation) Percentage survival 1.0E-011.0E-03 1.0E-051.0E-06 1.0E-071.0E-08 Properase MC3 Infectivity

6. Validation of inactivation methods; raising the standard In vitro assessment of PrP Sc degradation not enough to validate performance Both Properase and MC3 completely remove all 6H4 immunoreactive material BUT significant difference between levels of observed infectivity Choice of model for bioassay important relevant combination of agent and animal model significant reduction in infectious dose

7. Decontamination of surgical instruments; summary Good progress made towards a practical solution to decontamination of instruments >7-log reduction in infectious dose, in vivo, with BSE-301V Further studies underway Compatible with current disinfection practice without major investment Safe, environmentally friendly process Demonstrates capabilities of protein engineering to address issue Enzyme (MC3) under development/scale up Work ongoing to evaluate MC3 for rendering/bone meal applic.

8. Acknowledgements: Neil Raven Joanne Dickinson Anne McLeod Heather Murdoch Dawn Taylor Clive Buswell Jean Carr Graham Hall Mike Dennis Biological Investigations Group HPA-Porton Down Mark Sutton Funding: EC/DEFRA Genencor International