374. Development of a Genetic Vaccine Conferring Protective Immunity Against α-Cobratoxin Following a Single Administration of an Adenovirus Vector Encoding a Modified, Non-Toxic Cobratoxin Variant

Abstract

Approximately 300,000 venomous snakebites occur throughout the world each year, almost 40,000 of which result in death. Little has changed over the past century regarding the medical approach to the risk of snakebite, and there are no vaccines available to protect against the common poisonous snakes. The present study presents a new paradigm for the development of vaccines to protect against snakebite, using knowledge of the structure and action of specific toxins combined with a gene-based strategy to deliver a toxin gene modified to render it non-toxic while maintaining its 3-dimensional structure and hence its ability to function as an immunogen. As a model for this approach, we developed a genetic vaccine to protect against α-cobratoxin (CTX), a potent, post-synaptic neurotoxin that is the major toxic component of the venom of Naja naja kathouia, the spectacled cobra. To develop the vaccine, substitutions in the CTX cDNA were introduced at 2 residues critical for binding to the nicotinic acetylcholine receptor (Asp27 to Arg, Arg33 to Gly) and a third substitution (Val37) which was silent at the amino acid level. The mutated CTX (mCTX) retained the overall antigenic structure of native CTX, while reducing receptor binding 10,000-fold (Fruchart-Gaillard et al. Proc Natl Acad Sci USA 2002; 99: 3216–3221). The mutated CTX expression cassette was delivered either as a plasmid (pmCTX) or in the context of a replication deficient adenovirus vector (AdmCTX). To assess whether expression of the mutated CTX in vivo leads to the development of protective immunity, BALB/c mice were challenged by IV administration of 2 μg of α-cobratoxin protein 21 days after IV injection of the mCTX plasmid vaccine. For animals receiving either no treatment (naive) or IV injection of Null plasmid, 80% died from the α-cobratoxin challenge. However, 100% of animals receiving a single IV dose of the mCTX plasmid vaccine survived, i.e., not only was it safe to express the mutated cobratoxin, but it evoked protective immunity. Similarly, BALB/c mice were challenged by IV administration of 2 μg of α-cobratoxin protein 21 days after IV injection of 109 particle units of AdmCTX (the vaccine in the context of an Ad vector) or AdNull (as a control). Animals receiving AdmCTX or AdNull and no challenge suffered no ill effects, but ≥80% of naive animals or those receiving AdNull died within10 min from the CTX protein challenge. In contrast, 100% of animals receiving a single IV dose of AdmCTX 21 days prior to challenge survived. The data demonstrates that both plasmid and adenovirus-based vaccines can be developed to protect against lethal challenge with α-cobratoxin. The effectiveness of this approach might serve as a basis to consider the development of a global public health program to protect the lives of those at risk for death by snakebite.