Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant

• DOI: 10.1016/j.vaccine.2014.06.032
• Publication Date: 2014-06-18T13:46:46Z
• Authors: Bei Wang; Su Wang; Richard R. Rustandi; Feng Wang; Christopher D. Me...; Laura Hong; Adam Kristopeit; Susan Secore; Geethanjali Dorna...; Anthony Kanavage; Jon H. Heinrichs; Henryk Mach; Jeffrey T. Blue; David S. Thiriot
• Keywords: Reversion

The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders magnitude a combination targeted point mutations. However, TcdB mutant with five mutations (referred to herein as mTcdB) still has residual that detected in cell-based assays and in-vivo mouse assays. This effectively removed treatment formaldehyde solution. Storage the formaldehyde-treated mTcdB liquid result reversion over time back level toxicity, rate dependent on storage temperature. We found for both "forward" detoxification reaction formaldehyde, "reverse" reaction, correlated several biochemical including anion exchange chromatography retention appearance SDS-PAGE. Maintenance low concentration prevents formulations. when samples 0.016% (v/v) were lyophilized stored at 37 °C, continued react modify state. Lyophilization alone prevented formaldehyde-treated, formaldehyde-removed °C 6 months. Formaldehyde-treated, showed no evidence appeared stable assays, was immunogenic mice, even after months °C.