title: 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.
authors.affiliation: Division of Toxinology, United States Army Research Institute for Infectious Diseases, Frederick, Maryland 21702-5011, USA.
authors.forename: M P
authors.initials: MP
authors.lastname: Byrne
authors.forename: T J
authors.initials: TJ
authors.lastname: Smith
authors.forename: V A
authors.initials: VA
authors.lastname: Montgomery
authors.forename: L A
authors.initials: LA
authors.lastname: Smith
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001428:Bacterial Vaccines / Q000191:economics / Q000627:therapeutic use*; D001665:Binding Sites; D019274:Botulinum Toxins, Type A / Q000096:biosynthesis / Q000235:genetics / Q000627:therapeutic use* / Q000633:toxicity; D001906:Botulism / Q000517:prevention & control*; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D015870:Gene Expression; D005813:Genes, Synthetic; D007115:Immunization Schedule; D007928:Lethal Dose 50; D051379:Mice; D009500:Neutralization Tests; D010843:Pichia / Q000235:genetics; D014614:Vaccines, Synthetic / Q000191:economics / Q000627:therapeutic use*
chemical_list: D000907:Antibodies, Bacterial; D001428:Bacterial Vaccines; D014614:Vaccines, Synthetic; D019274:Botulinum Toxins, Type A