Purification, potency, and efficacy of the botulinum neurotoxin type A binding domain from Pichia pastoris as a recombinant vaccine candidate

Abstract

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(Hc)], expressed in Pichia pastoris, was developed as a vaccine candidate for preventing botulinum neurotoxin type A (BoNT/A) intoxication. After fermentation and cell disruption, BoNT/A(Hc) was purified by using a three-step chromatographic process consisting of expanded-bed chromatography, Mono S cation-exchange chromatography, and hydrophobic interaction chromatography. Two pools of immunogenic product were separated on the Mono S column and processed individually. Both products were more than 95% pure and indistinguishable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blot analysis, and enzyme-linked immunosorbent assay (ELISA). Each protein was assayed for potency in mice at immunogen doses ranging from 2.4 ng to 10 microg, followed by challenge with 1,000 mouse intraperitoneal 50% lethal doses (i.p. LD50) of BoNT/A. The calculated 50% effective dose for both peaks was approximately 0.1 microg/mouse. Peak 1 was evaluated further in a mouse efficacy assay. Mice were injected either once, twice, or three times at five different doses and subsequently challenged with 100,000 mouse i.p. LD50 of BoNT/A. In general, multiple injections protected better than one, with complete or nearly complete protection realized at doses of >/=0.5 microg/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of >/=1, 600, as measured by ELISA, survived, but only 6 of 42 mice with titers of </=100 survived. This work shows that the purified BoNT/A(Hc) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.

FIG. 1

Modification of the synthetic gene encoding BoNT/A(H_c). (A) Gene construct as described by Clayton et al. (6). (B) Modified gene construct for the work presented in this report. In both sequences, only the regions modified are shown. The cloning restriction sites are shown above the sequence. The initiation and termination codons are shown in bold, and the one-letter amino acid codes are depicted below the gene sequences. The initiation codon represents amino acid 861 of the full-length BoNT/A sequence.

FIG. 2

Purification of BoNT/A(H_c) by sequential chromatography. (A) Mono S cation-exchange chromatography of the Streamline pool. Proteins were eluted with an increasing NaCl gradient, yielding two immunoreactive peaks. Peak 1 (B) and peak 2 (C) were pooled individually and subjected to HIC, and proteins were eluted with a decreasing ammonium sulfate gradient. Protein production was monitored by A₂₈₀ (left axis); elution conditions are recorded on the right axis, with a gradient trace laid over the chromatogram.

FIG. 3

(A) Reducing SDS-PAGE of FPLC-purified BoNT/A(H_c) separated on a 10% tricine gel and visualized by Coomassie blue. The gel shows Novex wide-range molecular weight markers (15 μl; lane 1), peak 1 (2 μg; lane 2), and peak 2 (2 μg; lane 3). (B) Western blot analysis of FPLC-purified BoNT/A(H_c) detected with MAb 6E9-11. The blot shows Novex prestained molecular weight markers (15 μl; lane 1), peak 1 (2 μg; lane 2), and peak 2 (2 μg; lane 3).

FIG. 4

Dependence of mouse survival on dose of purified BoNT/A(H_c) administered. Ten mice per group were vaccinated once with peak 1 and peak 2 at doses ranging from 2.4 ng to 10 μg per mouse. Mice were challenged with 1,000 i.p. LD₅₀ of BoNT/A toxin 21 days after vaccination.