Identifying the principal protective antigenic determinants of type A botulinum neurotoxin

The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction. As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (H-C; residues 861-1296), protects mice against lethal challenges with the intact toxin. To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-H-C were isolated and cloned. Specific binding of the mAbs to BoNt/A-H-C was demonstrated by surface plasmon resonance, with K(d)s in the range of 10(-10) to 10(-11) M. These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity. Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin H-C fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops. Based on these predictions, two 25-mer H-C-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs. Mice immunized with the H-C-peptides had high levels of antibodies that recognized BoNT/A-H-C. However; immunizations with only one of the H-C peptides protected when mice were challenged with BoNT/A. On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins. Published by Elsevier Science Ltd. All rights reserved.