Upon exposure to Botulinum neurotoxin (BoNT), the toxin enters the body and migrates to motor neurons, internalizes and delivers a protease which inactivates nerve function leading to flaccid paralysis and, in serious cases, death. Once the toxin is inside neurons, antitoxins become ineffective and no therapy exists beyond palliative care. The protease can persist within neurons for many months resulting in long-term paralysis. While this is a desirable trait for therapeutic applications, BoNT persistence is problematic when the exposure is accidental or malicious. The Department of Infectious Disease and Global Health is collaborating with both the Ichtchenko and Dong labs in the development of biomolecular antidotes for botulism that enter motor neurons and promote the rapid destruction of the intoxicating protease. These projects recently culminated in two cover story publications in Science Translational Medicine (see Miyashita et al and McNutt et al, below).
Our strategy for botulism antidote development employs camelid antitoxin heavy-chain-only Ab VH (VHH) binding domains that specifically recognize and neutralize intoxicating Botulinum neurotoxin (BoNT) proteases. After testing a variety of potential vehicles to specifically deliver BoNT protease neutralizing VHHs to the cytosol of intoxicated neurons, we have now focused on the use of atoxic BoNT biomolecules as the most effective. These atoxic BoNT delivery vehicles possess mutations that eliminate their toxicity but do not compromise their ability to deliver cargo to neurons. These VHH-based biomolecular antidotes reverse symptoms of botulism in three different animal models including primates (see McNutt et al, below). The Dong lab employs a non-toxic BoNT/X VHH delivery vehicle and these agents were shown to reverse symptoms of botulism from both major BoNT serotypes, A and B, in mouse models (see Miyashita et al, below).
We have also been exploring strategies to enhance the efficacy of biomolecular botulism antidotes. In one approach, VHHs would be genetically fused to an F-box domain to create a ‘targeted F-box’ or TFB which would promote the rapid degradation of the VHH-bound BoNT protease using the cell’s own natural protein turnover system (see Kuo et al, below).