Extraction and inhibition of enzymatic activity of botulinum neurotoxins /B1, /B2, /B3, /B4, and /B5 by a panel of monoclonal anti-BoNT/B antibodies

Abstract

BackgroundBotulism is caused by botulinum neurotoxins (BoNTs), extremely toxic proteins which can induce respiratory failure leading to long-term intensive care or death. Treatment for botulism includes administration of antitoxins, which must be administered early in the course of the intoxication; therefore, rapid determination of human exposure to BoNT is an important public health goal. In previous work, our laboratory reported on Endopep-MS, a mass spectrometry-based activity method for detecting and differentiating BoNT/A, /B, /E, and /F in clinical samples. We also demonstrated that antibody-capture is effective for purification and concentration of BoNTs from complex matrices such as clinical samples. However, some antibodies inhibit or neutralize the enzymatic activity of BoNT, so the choice of antibody for toxin extraction is critical.ResultsIn this work, we evaluated 24 anti-BoNT/B monoclonal antibodies (mAbs) for their ability to inhibit the in vitro activity of BoNT/B1, /B2, /B3, /B4, and /B5 and to extract those toxins. Among the mAbs, there were significant differences in ability to extract BoNT/B subtypes and inhibitory effect on BoNT catalytic activity. Some of the mAbs tested enhanced the in vitro light chain activity of BoNT/B, suggesting that BoNT/B may undergo conformational change upon binding some mAbs.ConclusionsIn addition to determining in vitro inhibition abilities of a panel of mAbs against BoNT/B1-/B5, this work has determined B12.2 and 2B18.2 to be the best mAbs for sample preparation before Endopep-MS. These mAb characterizations also have the potential to assist with mechanistic studies of BoNT/B protection and treatment, which is important for studying alternative therapeutics for botulism.

The thioredoxin reductase‐thioredoxin system is involved in the entry of tetanus and botulinum neurotoxins in the cytosol of nerve terminals

“…This situation calls for the development of novel drugs that can counteract TeNT and BoNT action by affecting any of the four steps of the mechanism of intoxication: (a) binding; (b) endocytosis; (c) membrane translocation and (d) metalloprotease cleavage of SNAREs. Before their binding to the presynaptic membrane of neurons, the toxins are best neutralized by specific anti-toxin antibodies [20,21] which prevent the first step of the entry of TeNT and BoNT into their target neurons. Currently, several groups are attempting to block any of the three subsequent steps [22][23][24][25][26][27][28][29][30].…”

Implementing the Bruker MALDI Biotyper in the Public Health Laboratory for C. botulinum Neurotoxin Detection

“…As toxin concentrations were above the exempt maximum concentration of 0.5 mg, a Tier 1 Select Agent laboratory was necessary to perform all experiments. Previously described antibodies were provided by James D. Marks, MD, Ph.D. (University of California, San Francisco, CA, USA) and Suzanne Kalb, PhD (CDC, Atlanta, GA, USA) [19,20,21]. MS peptide calibrant was obtained from Bruker Daltonics (Billerica, MA, USA).…”

Botulinum Toxins, Diversity, Mode of Action, Epidemiology of Botulism in France

“…In contrast to the BoNT/F subtypes which cleave VAMP1 and VAMP2 at QK site, BoNT/F5 uses a distinct cleavage site (LE) [86] ( Table 1). Monoclonal antibodies against BoNT/B differently recognize the subtypes BoNT/B1 to BoNT/B5 [87]. Similarly, monoclonal antibodies against BoNT/A recognize and/or neutralize the distinct BoNT/A subtypes with variable efficiently [88,89].…”