Next-generation protein neurotherapeutics for botulism poisoning

About This Grant

PROJECT SUMMARY/ABSTRACT Botulinum neurotoxins (BoNTs) are highly potent biological neurotoxins produced by Clostridium botulinum, causing botulism, a disease characterized by flaccid paralysis. Structurally, matured BoNTs comprise a ~ 100 kDa heavy chain (HC) and a ~50 kDa light chain (LC) linked by a disulfide bond. The HC consists of a receptor- binding domain (HCC) and a translocation domain (HCN), which mediate neuronal targeting and endosomal escape. At the same time, the LC is a zinc-dependent endopeptidase that disrupts synaptic transmission by cleaving SNARE proteins required for the synaptic fusion event. Despite the extreme toxicity of BoNT, no pharmacological antidote exists. The current treatment is post-exposure prophylaxis with equine-derived Heptavalent Botulinum AntiToxin (HBAT) combined with mechanical ventilation and supportive care. HBAT neutralizes toxins in the circulation but is ineffective once the toxin has been internalized into neurons, an event that marks the onset of clinical symptoms. The combination of its potency and lack of an antidote to reverse its effects after the onset of the symptoms has led to its classification as a Select Agent by the U.S. Centers for Disease Control and Prevention (CDC) due to its potential for bioterrorism. These factors underscore the urgent need to develop an effective antidote to mitigate the threat posed by BoNTs. One major challenge in developing effective antidotes is the existence of many BoNT isoforms, grouped in at least seven different serotypes (indicated by letters A, B, C, D, E, F, and G), each comprising multiple subtypes with varying degrees of amino acid sequence differences. The heterogeneity of the toxin’s molecular target, which influences both the degree of neurotoxicity and susceptibility to neutralization, together with the intracellular localization of the toxic LC metalloprotease, further complicates the design of broadly effective post-symptomatic anti-botulinum therapeutics.To overcome the barriers to developing BoNT antidotes, we propose to create Post-Symptomatic Anti-Botulinum Therapeutics (PSABT), which combine broad-spectrum nanobodies that potently inhibit the catalytic activity of BoNT LC for all subtypes of serotypes A and B, fused to a non-toxic toxin-based neuronal delivery vehicle that mimics the same trafficking pathway as the native toxin, and enables the intracellular delivery of immunotherapies for the treatment of BoNT intoxication. Given that BoNT/A1 and BoNT/B1 are the most long-lasting and prevalent subtypes in botulism cases, which are also present in commercially available products, we propose developing antitoxins targeting all eight subtypes of serotype A and all eight subtypes of serotype B.