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 LH_N, comprise the light chain and translocation domains of each neurotoxin and are devoid of any neuron-binding activity. Using codon-optimized genes, LH_N 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 LH_N/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 10³ 50% lethal dose (LD ₅₀) of BoNT/A₁ with an 50% effective dose (ED ₅₀) of 50 ng of the vaccine. In similar experiments, the LH _N/A vaccine was shown to protect mice against challenge with BoNT/A subtypes A₁, A₂, and A₃, which is the first demonstration of single-dose protection by a vaccine against the principal toxin subtypes of BoNT/A. The LH_N/B vaccine was also highly efficacious, giving an ED₅₀ of ∼140 ng to a challenge of 10³ LD₅₀ of BoNT/B₁. In addition, LH_N/B provided single-dose protection in mice against BoNT/B₄ (non-proteolytic toxin subtype).