Vaccination of Rabbits with an Alkylated Toxoid Rapidly Elicits Potent Neutralizing Antibodies against Botulinum Neurotoxin Serotype B

New Zealand White (NZW) rabbits were immunized with several different nontoxic botulinum neurotoxin serotype B (BoNT/B) preparations in an effort to optimize the production of a rapid and highly potent, effective neutralizing antibody response. The immunogens included a recombinant heavy chain (rHc) protein produced in Escherichia coli, a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin. All three immunogens elicited an antibody response to BoNT/B, detected by enzyme-linked immunosorbent assay (ELISA) and by toxin neutralization assay, by the use of two distinct mouse toxin challenge models. The induction period and the ultimate potency of the observed immune response varied for each immunogen, and the ELISA titer was not reliably predictive of the potency of toxin neutralization. The kinetics of the BoNT/B-specific binding immune response were nearly identical for the formaldehyde toxoid and alkylated toxoid immunogens, but immunization with the alkylated toxoid generated an approximately 10-fold higher neutralization potency that endured throughout the study, and after just 49 days, each milliliter of serum was capable of neutralizing 10⁷ 50% lethal doses of the toxin. Overall, the immunization of rabbits with alkylated BoNT/B toxoid appears to have induced a neutralizing immune response more rapid and more potent than the responses generated by vaccination with formaldehyde toxoid or rHc preparations.Botulinum neurotoxin (BoNT), the causative agent of botulism, is the most potent of all the known toxins (7). BoNT is a secreted protein produced by the anaerobic soil organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum in seven distinct serotypes (serotypes A to G) (9, 23, 28). The BoNT serotypes are all synthesized as single-chain polypeptides with molecular masses of approximately 150 kDa. Posttranslational cleavage of the original polypeptide monomer results in the formation of a disulfide-linked dichain product composed of light chain (LC) and heavy chain (HC) domains. The HC is divided into two distinct functional domains; the first mediates toxin binding and uptake by peripheral neuronal cells, and the second mediates translocation of the LC subunit into the target cell cytosol. Once it is in the cytosol, the zinc metalloprotease of the LC specifically cleaves the soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptors (SNAREs) responsible for synaptic vesicle docking and neurotransmitter release at the neuromuscular synapse.Human botulism typically results from the ingestion of contaminated foods (often improperly prepared canned goods), although BoNT intoxication can also result from wound colonization by one or more species of Clostridium. Similarly, infant botulism results from exposure to actively secreted toxin following the germination of ingested Clostridium spores, which proliferate in the immature gastrointestinal tract. Regardless of the route of exposure, BoNT intoxication occurs by the same mechanism, once the toxin enters the circulation. Although there is no cure for botulism after the onset of symptoms, an effective circulating antibody response can completely neutralize an otherwise intoxicating dose of BoNT. Widespread immunization against the toxin is precluded by the growing number of clinical applications of BoNT for the treatment of various neuromuscular spasticity disorders, yet BoNT vaccine development continues for the purposes of immunizing at-risk populations, such as laboratory workers, first responders, and military personnel (26).A number of BoNT immunogens and a variety of vaccination strategies have successfully been used to elicit neutralizing antibody responses against individual BoNT serotypes (3, 19, 20, 29, 32). The immune responses to BoNT vary according to the animal species, the toxin serotype, and the antigen preparation. Additionally, the development of a potent neutralizing antibody response to BoNT serotype B (BoNT/B) has proven problematic, prompting a demand for alternative toxin-derived immunogens (25, 27).In the present study, we tested three BoNT/B immunogens in New Zealand White (NZW) rabbits using a rapid vaccination scheme to develop a potent toxin-neutralizing immune response in a short time period (12). Rabbits were immunized with BoNT/B recombinant heavy chain (rHc) or toxoid preparations derived from formaldehyde inactivation or urea- iodoacetamide alkylation of active toxin (15). All three immunogens elicited toxin-neutralizing antibody responses by the end of the study; however, vaccination with the alkylated toxoid preparation induced a more rapid and more potent BoNT/B-neutralizing response than the other immunogens.