The receptor binding domain of botulinum neurotoxin serotype A (BoNT/A) inhibits BoNT/A and BoNT/E intoxications in vivo

Abstract

The receptor binding domain of botulinum neurotoxin (BoNT), also designated the C terminus of the heavy chain (H(C)), is a promising vaccine candidate against botulism. In this study, a highly efficient expression system for the protein was developed in Escherichia coli, which provided yields that were 1 order of magnitude higher than those reported to date (350 mg H(C) per liter). The product was highly immunogenic, protecting mice from a challenge with 10(5) 50% lethal dose (LD(50)) after a single vaccination and generating a neutralizing titer of 49.98 IU/ml after three immunizations. In addition, a single boost with HC increased neutralizing titers by up to 1 order of magnitude in rabbits hyperimmunized against toxoid. Moreover, we demonstrate here for the first time in vivo inhibition of BoNT/A intoxication by H(C)/A, presumably due to a blockade of the neurotoxin protein receptor SV2. Administration of HC/A delayed the time to death from 10.4 to 27.3 h in mice exposed to a lethal dose of BoNT/A (P = 0.0005). Since BoNT/A and BoNT/E partially share SV2 isoforms as their protein receptors, the ability of H(C)/A to cross-inhibit BoNT/E intoxication was evaluated. The administration of H(C)/A together with BoNT/E led to 50% survival and significantly delayed the time to death for the nonsurviving mice (P = 0.003). Furthermore, a combination of H(C)/A and a subprotective dose of antitoxin E fully protected mice against 850 mouse LD(50) of BoNT/E, suggesting complementary mechanisms of protection consisting of toxin neutralization by antibodies and receptor blocking by H(C)/A.

Fig 1

DNA constructs for recombinant H_C fragment expression. The constructs were designed to obtain coexpression of TrxA and H_C. Two gene configurations were tested: trxA-H_C, in which the H_C fragment gene is located immediately downstream of trxA (A), and trxA-rbs-H_C, in which an additional rbs sequence precedes the H_C fragment gene (B). Both gene configurations were cloned into the expression vectors pET-9a (T7 promoter) and pET-22b(+) (T7lac promoter).

Fig 2

Purification of the receptor binding domain from E. coli BL21(DE3) carrying the plasmid pET-9a-trxA-rbs-H_C. SDS-PAGE (A) and Western blot (B) analyses were performed with samples withdrawn during the purification process. Lane 1, molecular mass markers (in kDa); lane 2, soluble cell fraction; lane 3, insoluble cell fraction; lane 4, flowthrough from loading of the soluble cell fraction on the IMAC column (unbound proteins); lane 5, eluted protein. The H_C fragment was detected using rabbit antibodies directed against amino acids 1279 to 1295 of BoNT/A. (C) The purity of the final product was analyzed using a UPLC system equipped with a C₁₈ column. The protein was found to be >99% pure. AU, absorbance units.

Fig 3

H_C/A delays the time to death for mice exposed to BoNT/A and BoNT/E but not BoNT/B. Mice (n = 6) were exposed to 5 MsLD₅₀ of BoNT/A (A), BoNT/E (B), or BoNT/B (C) in the presence (dashed line) or absence (solid line) of H_C/A, and survival was monitored. Time to death was significantly delayed as a result of coadministration of H_C/A for neurotoxins A and E (P = 0.0005 and P = 0.003, respectively, by the log-rank test) but not for neurotoxin B (P = 0.87).