Development of recombinant vaccines for botulinum neurotoxin

Synthetic genes encoding non-toxic, carboxyl-terminal regions (approximately 50 kDa) of botulinum neurotoxin (BoNT) serotypes A and B (referred to as fragment C or HC) were constructed and cloned into the methylotropic yeast, Pichia pastoris. Genes specifying BoNTA(HC) and BoNTB(HC) were expressed as both intracellular and secreted products. Recombinants, expressed intracellularly, yielded products with the expected molecular weight as judged by SDS PAGE and Western blot (immunoblot) analysis, while secreted products were larger due to glycosylation. Gene products were used to vaccinate mice and evaluated for their ability to elicit protective antibody titers in vivo. Mice given three intramuscular vaccinations with yeast supernatant containing glycosylated BoNTA(HC) were protected against an intraperitoneal challenge of 10(6) 50% mouse lethal doses (MLD50) of serotype A neurotoxin, a result not duplicated by its BoNTB(HC) counterpart. Vaccinating mice with cytoplasmically produced BoNTA(HC) and BoNTB(HC) protected animals from a challenge of 10(6) MLD50 of serotype A and B toxins, respectively. Because of the glycosylation encountered with secreted BoNT(HC), our efforts focused on the production and purification of products from intracellular expression.