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

Recombinant botulinum neurotoxin serotype A binding domain [BoNT/A(H-c)], 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(H-c) 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 mu g, 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 mu g/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 greater than or equal to 0.5 mu g/mouse. Serum neutralization and ELISA titers were also determined. Tellingly, 82 of 83 mice with antibody titers of greater than or equal to 1,600, as measured by ELISA, survived, but only 6 of 42 mice with titers of less than or equal to 100 survived. This work shows that the purified BoNT/A(H-c) produced was a highly effective immunogen, able to protect against a high challenge dose of neurotoxin.