Clostridium botulinum NEUROTOXIN TYPE E BINDING DOMAIN FROM Pichia pastoris AS A RECOMBINANT VACCINE CANDIDATE

Abstract

Botulinum toxin, one of the deadliest substances known to exist, is a complex protein produced by the bacterium Clostridium botulinum. The toxin prevents the release of acetylcholine from nerve terminals resulting in a severe and potentially fatal muscular paralysis. This occurs as the result of a series of molecular events orchestrated individually by each of the three toxin subunits: binding to the nerve cell, internalization into the cytoplasm, and catalysis of one of the key components of the exocytic fusion complex. Prevention of any step in the process of intoxication could prevent the onset of the disease. A pentavalent vaccine with Investigational New Drug (IND) status is currently available to protect individuals considered to be "at-risk" for toxin exposure. This group includes laboratory workers who handle the bacteria and military personnel on the battlefield who may face a threat with the toxin as a bio-weapon. The vaccine is a formalin-fixed toxoid produced from crude culture supernatants of five of the seven known serotypes of C. botulinum. Shortcomings associated with this vaccine include a limited supply, expensive cost of production, and undesirable side effects upon administration. The Department of Immunology and Molecular Biology, Division of Toxinology at the United States Army Medical Research Institute of Infectious Diseases (USAIVIRIED) has focused on a toxin subunit in an effort to produce a new-generation vaccine. Synthetic genes encoding non-toxic, carboxy—terminal fragments of the C botulinum neurotoxins (rBoNT/(Hc) A, B, C₁, and F have been expressed in Pichia pastoris and administration of these fragments have protected mice from challenge of the respective serotype. A synthetic gene encoding the fragment C of C. botulinum serotype E was transformed into P. pastoris. The expressed rBoNT/E(Hc) was purified and used to immunize three groups of mice once, twice, or three times respectively. Each vaccination group was subsequently subjected to 1000, 10,000, or 100,000 times the 50% mouse lethal dose (MLD₅₀) of C. botulinum type E toxin. Mice that were vaccinated three times with 1μg of rBoNT/E(Hc) survived the highest level of toxin challenge of 100,000 times the MLD₅₀. Pooled sera from the same vaccination group produced neutralizing antibodies of 48.2 IU/mL in mice. In a separate potency assay to determine the ability to elicit protective immunity, mice challenged with 1000 MLD₅₀ of BoNT/E toxin had an ED₅₀ of 193 ng with rBoNT/E(Hc) while 1.61 μg of the pentavalent toxoid vaccine was required for the same level of protection.