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Proteolytic Inactivation of prions; a biological solution to TSE decontamination.

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. Decontamination of TSE agents; practical issues.

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Proteolytic Inactivation of prions; a biological solution to TSE decontamination.

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  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) >109 iu / mg) • Assessment: Western blot (6H4 and PAb2) and bioassayed in VM mice • Process: 10% iMBH digested at pH12, 600C for 30 minutes

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

  5. MC3 digestion at 600C pH 12 reduces infectivity by more than 7-logs 1.0E-01 1.0E-03 100 1.0E-05 1.0E-06 1.0E-07 1.0E-08 90 Properase 80 MC3 Infectivity 70 MC3: 66% survival 500 days 60 Percentage survival 50 40 30 20 10 0 100 150 200 250 300 350 400 450 Incubation time (days post inoculation)

  6. Validation of inactivation methods;raising the standard • In vitro assessment of PrPSc 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. 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 Acknowledgements:

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