The development of recombinant vaccines against clostridial neurotoxins was pioneered by Helting and Nau52 and Fairweather and colleagues53 using tetanus toxin (TeNT) as the model. They were the first to show that a C-terminal fragment of the TeNT heavy chain, referred to as the fragment C region, was able to protect mice from active challenge with tetanus toxin.
The high sequence and structural homology between TeNT and BoNT led scientists at USAMRIID to take a similar approach for BoNT recombinant vaccines. Several overlapping domains of the DNA sequence encoding the heavy chain of BoNT/A were isolated by PCR from the C. botulinum NCTC2916 strain. Expression and purification from Escherichia coli indicated that the most promising vaccine candidate encompassed the approximately 50-kDa domain from the C-terminus of the heavy chain (Hc), crude extracts of which were able to protect mice against BoNT challenge.53 54 This suggested that the domain chosen was folding correctly, thus allowing the correct formation of protective epitopes.
Published crystal structures of intact neurotoxins and the recombinant Hc domains have indicated that in both, the Hc domain comprises an N-terminal jelly roll motif and a C-terminal P-trefoil domain (see Chapter 2). This latter domain, comprising 150 amino acid residues, shows very little sequence homology among the different serotypes, suggesting that this domain may be principally responsible for determining serotype specificity. There is some evidence, however, that both halves of the Hc domain are required to elicit neutralizing antibodies and that accurate folding of the entire domain is essential. Vaccinating mice with only the N-terminal half, of the Hc domain, only the C-terminal half, or a mixture of the two failed to produce a notable protective immune response.55
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