Bivalent Recombinant Vaccine For Botulinum Neurotoxin Types A And B Based On A Polypeptide Comprising Their Effector And Translocation Domains That Is Protective Against The Predominant A And B Subtypes

The botulinum neurotoxins (BoNTs) are a large family of extremely potent, neuroparalytic, dichain proteins which act at the peripheral nervous system. The wide genetic diversity observed with this neurotoxin family poses a significant challenge for the development of an effective botulinum vaccine. The present study describes a vaccine development platform based on protein fragments representing the N-terminal two-thirds of each toxin molecule. These fragments, designated LHN, comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LHN fragments derived from BoNT serotypes A and B were expressed in Escherichia coli in high yield with >1 g of purified, soluble fragment recoverable from 4.5 liter-scale fermentations. The protective efficacy of LHN/A was significantly enhanced by treatment with formaldehyde, which induced intramolecular cross-linking but virtually no aggregation of the fragment. A single immunization of the modified fragment protected mice from challenge with a 10(3) 50% lethal dose (LD50) of BoNT/A(1) with an 50% effective dose (ED50) of 50 ng of the vaccine. In similar experiments, the LHN/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A(1), A(2), and A(3), which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LHN/B vaccine was also highly efficacious, giving an ED50 of similar to 140 ng to a challenge of 10(3) LD50 of BoNT/B-1. In addition, LHN/B provided single-dose protection in mice against BoNT/B-4 (non-proteolytic toxin subtype).