EFFICACY OF A DNA VACCINE FOR BOTULINUM NEUROTOXIN

Abstract

Clostridium botulinum is an anaerobic, spore forming bacterium producing a neurotoxin that is one of the most poisonous substances known. The toxin is synthesized as a single polypeptide chain of approximately 150 kilodaltons (kDa). Upon release from the bacterium, the polypeptide is processed by endogenous host proteases to generate two disulfide-linked fragments. The heavy chain (100kDa) is involved in cell binding and penetration, while the light chain (50kDa) is responsible for intracellular activity. The toxin exerts its effect at the neuromuscular junction by blocking the release of acetylcholine via a zinc-dependent protease activity, resulting in flaccid paralysis. There are seven different serotypes of botulinum neurotoxin, A-G. An Investigative New Drug (IND) vaccine is currently available to protect laboratory personnel and others at high risk from botulinum toxin poisoning, but a Food and Drug Administration (FDA) approved vaccine does not exist. The vaccine is a formaldehyde-inactivated culture supernatant from C. botulinum grown in fermentors. The IND vaccine is costly to produce and poses a risk to scientists producing the vaccine. In addition, only five of the seven serotypes are represented in the vaccine. Lastly, some of the serotypes are derived from strains that do not produce high levels of toxin in culture, making purification and toxoiding laborious. Therefore, the development of a new generation of botulinum vaccine is imperative. The botulinum toxins are the most potent natural toxins known (Mebs, D. and F. Hucho 1990). Because of its toxicity, botulinum neurotoxin research has focused on the development of a nontoxic fragment-based vaccine. A synthetic gene encoding the 50kDa carboxyl terminal fragment of botulinum type A (bntAC-1) has been synthesized and the protein has been expressed (Tic). Preliminary immunological studies have demonstrated that this protein confers protection to higher levels than those achieved with the currently available vaccine (Clayton, et al. 1995). Advantages of using the synthetic gene include ease of production, efficacy, and potency. However, the use of proteins requires purification. Recent research has demonstrated the possibility that DNA in a vector can serve as a vaccine candidate. Studies with influenza genes have shown that intramuscular injection of this "naked DNA" can be taken up by muscle cells of mice. The encoded protein is then expressed and antibodies to the viral protein are made by the animal. Animals subsequently challenged with a lethal dose of influenza A had a 90% survival rate (Ulmer, et al. 1993). Following the example of intramuscular injection of influenza genes, this research used the synthetic gene for the Fic fragment of botulinum type A, in an analogous expression system to determine the efficacy of this novel vaccine technology. The possibility that intramuscular injection of DNA encoding the Hc fragment of botulinum toxin would confer immunity to mice was explored. The bntAC-1 gene, after modification, was cloned into one of two separate regions of the pCMVint-BL vector, each of which was optimized for either intracellular or extracellular secretion. Mice were immunized with either construct, or with vector DNA only, and then challenged with botulinum toxin. As much as 82% survival was observed in mice immunized with the construct optimized for extracellular secretion. Results of these immunizations were compared to conventional immunizations with the Hc fragment obtained from heterologous expression systems. Protection levels following a challenge with botulinum toxin were not as high in mice immunized with DNA in comparison with mice immunized with the conventional fic fragment. In addition, antibody levels of immunized animals were measured against whole botulinum toxin and against purified Hc using enzyme-linked immunosorbent assay (ELISA). Previously, Hc had never been utilized in ELISA. Overall, titers were higher in the animals who survived a challenge with botulinum toxin.