Infectious Disease: Botulism antidotes

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 exposures are accidental or malicious. The Department of Infectious Disease and Global Health is developing biomolecular antidotes for botulism that enter motor neurons and neutralize the toxin protease. Toxin neutralization is effected by VHH-base agents which neutralize the BoNT protease within neurons. Ideally, our goal is to combine toxin neutralization with accelerated destruction of the protease. If successful, similar treatments may become possible for diseases caused by some other intracellular pathogens.

The largest technological challenge to a VHH-based botulism antidote is delivery of the agent into intoxicated neurons. Currently we are focused on a very promising delivery strategy that employs atoxic mutants of BoNT to serve as a ‘Trojan horse’ to specifically deliver the protease-neutralizing VHH agent to intoxicated neurons within botulism patients. This strategy is being developed through a collaboration with the Ichtchenko lab at NYU where the BoNT vectors are being developed. The VHH-based antidote agents created to date have been shown to be effective in mouse models of BoNT intoxication (publications pending).

We believe that an ideal botulism antidote will also target the pathogenic protease for accelerated destruction as a botulism cure. Towards this goal, in collaboration with Synaptic Research LLC, we are seeking to identify agents or motifs that can be genetically fused to the toxin-neutralizing VHH and promote the intraneuronal degradation of the protease itself. One early success employed an F-box motif to create a ‘targeted F-box’ or TFB which promotes the rapid degradation of the VHH-bound BoNT protease using the cell’s own natural protein turnover system (see Kuo et al). Due to difficulties in large scale expression of TFBs, alternative strategies to promote intraneuronal BoNT protease degradation are being developed for testing with funding from NIH NIAID.

Publications

  1. Shoemaker CB, Oyler GA. 2013. Persistence of botulinum neurotoxin inactivation of nerve function. Curr Top Microbiol Immunol.364:179-96. PMCID: PMC3528263.
  2. Krautz-Peterson, Oyler GA, Feng H, Shoemaker CB. 2012. Retargeting Clostridium difficile toxin B to neuronal cells as a potential vehicle for cytosolic delivery of therapeutic biomolecules to treat botulism. J. Toxicol. 2012:760142.
  3. Kuo CL, Oyler GA, Shoemaker CB. 2011. Accelerated neuronal cell recovery from botulinum neurotoxin intoxication by targeted ubiquitination. PLoS ONE. 6(5):e20352.
  4. Tsai YC, Maditz R, Kuo CL, Fishman PS, Shoemaker CB, Oyler GA, Weissman AM. 2010. Targeting botulinum neurotoxin persistence by the ubiquitin-proteasome system. Proc. Natl. Acad. Sci. USA. 107:16554-9. PMCID: PMC2944746.
  5. Tremblay JM, Kuo CL, Abeijon C, Sepulveda J, Oyler G, Hu X, Jin MM, Shoemaker CB. 2010. Camelid single domain antibodies (VHHs) as neuronal cell intrabody binding agents and inhibitors of Clostridium botulinum neurotoxin (BoNT) proteases. Toxicon. 56:990-8. PMCID: PMC2946066.