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

Abstract

ABSTRACT 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 10 3 50% lethal dose (LD 50 ) of BoNT/A 1 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 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 LH N /B vaccine was also highly efficacious, giving an ED 50 of ∼140 ng to a challenge of 10 3 LD 50 of BoNT/B 1 . In addition, LH N /B provided single-dose protection in mice against BoNT/B 4 (nonproteolytic toxin subtype).