title: Detecting and preventing reversion to toxicity for a formaldehyde-treated C. difficile toxin B mutant.
abstract: The toxicity of Clostridium difficile large clostridial toxin B (TcdB) can be reduced by many orders of magnitude by a combination of targeted point mutations. However, a TcdB mutant with five point mutations (referred to herein as mTcdB) still has residual toxicity that can be detected in cell-based assays and in-vivo mouse toxicity assays. This residual toxicity can be effectively removed by treatment with formaldehyde in solution. Storage of the formaldehyde-treated mTcdB as a liquid can result in reversion over time back to the mTcdB level of toxicity, with the rate of reversion dependent on the storage temperature. We found that for both the "forward" mTcdB detoxification reaction with formaldehyde, and the "reverse" reversion to toxicity reaction, mouse toxicity correlated with several biochemical assays including anion exchange chromatography retention time and appearance on SDS-PAGE. Maintenance of a low concentration of formaldehyde prevents reversion to toxicity in liquid formulations. However, when samples with 0.016% (v/v) formaldehyde were lyophilized and stored at 37 °C, formaldehyde continued to react with and modify the mTcdB in the lyophilized state. Lyophilization alone effectively prevented reversion to toxicity for formaldehyde-treated, formaldehyde-removed mTcdB samples stored at 37 °C for 6 months. Formaldehyde-treated, formaldehyde-removed lyophilized mTcdB showed no evidence of reversion to toxicity, appeared stable by several assays, and was immunogenic in mice, even after storage for 6 months at 37 °C.
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Bei
authors.initials: B
authors.lastname: Wang
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Su
authors.initials: S
authors.lastname: Wang
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Richard R
authors.initials: RR
authors.lastname: Rustandi
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Wang
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Mensch
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Hong
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Adam
authors.initials: A
authors.lastname: Kristopeit
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Susan
authors.initials: S
authors.lastname: Secore
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Geethanjali
authors.initials: G
authors.lastname: Dornadula
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Anthony
authors.initials: A
authors.lastname: Kanavage
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Jon H
authors.initials: JH
authors.lastname: Heinrichs
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Henryk
authors.initials: H
authors.lastname: Mach
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA.
authors.forename: Jeffrey T
authors.initials: JT
authors.lastname: Blue
authors.affiliation: Merck Research Laboratories, 770 Sumneytown Pike, West Point, PA 19486, USA. Electronic address: david_thiriot@merck.com.
authors.forename: David S
authors.initials: DS
authors.lastname: Thiriot
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D001427:Bacterial Toxins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*; D001428:Bacterial Vaccines / Q000737:chemistry / Q000276:immunology / Q000528:radiation effects / Q000633:toxicity*; D002852:Chromatography, Ion Exchange; D004356:Drug Storage; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005557:Formaldehyde / Q000378:metabolism*; D005612:Freeze Drying; D008810:Mice, Inbred C57BL; D050505:Mutant Proteins / Q000737:chemistry / Q000276:immunology / Q000633:toxicity; D013696:Temperature; D013997:Time Factors; D014121:Toxoids / Q000737:chemistry / Q000276:immunology / Q000633:toxicity*
chemical_list: D001426:Bacterial Proteins; D001427:Bacterial Toxins; D001428:Bacterial Vaccines; D050505:Mutant Proteins; D014121:Toxoids; C031815:toxB protein, Clostridium difficile; D005557:Formaldehyde
keywords: Clostridium difficile toxin B; Toxoid vaccines