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

Abstract

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 10 7 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 ...

New equine antitoxins to botulinum neurotoxins serotypes A and B

“…It is established in the botulinum toxin field that ELISA titers have poor correlation with protective titers when serum samples have been collected at a single time point from a diverse group of immunized individuals or when serum samples are screened for ELISA binding capacity to antigen or native toxin [28, 9]. In some cases, ELISA titers provide entirely misleading data regarding the magnitude of protective titers [36]. Our data do not dispute these previous observations.…”

DNA electroporation in rabbits as a method for generation of high-titer neutralizing antisera

“…For comparison, a titer as high as 220 IU/ml has been reported against toxin B in the rabbit after alkylated toxoid immunization. 13 Although it is known that it is difficult to induce a strong immune response against botulinum toxin B, this does not explain the difference we observed between the recombinant FcBoNT/B fragment and the DNA immunization. The reason why the botulinum toxin B Fc fragment has a different behavior than the two other fragments of toxins A and E after genetic immunization remains unclear and needs further investigation.…”

“…These conditions were then used for all rabbit immunizations. More preliminary experiments on different generally raised after animal immunization with formalin-detoxified toxins or more recently recombinant or chemically-altered derivatives of the toxins, [10][11][12][13] which requires recombinant protein purification or handling of the full toxins. Genetic immunization is an effective alternative to recombinant proteins immunization to raise an efficient immune response against an antigen.…”

Expression, Purification and Development of Neutralizing Antibodies from Synthetic BoNT/B LC and Its Application in Detection of Botulinum Toxin Serotype B

“…Held et al [39] compared three immunogens, a recombinant heavy chain (rHc) protein produced in E. coli, using a commercially available formaldehyde-inactivated toxoid, and an alkylated toxoid produced by urea-iodoacetamide inactivation of the purified active toxin to elicit the antibody response to BoNT/B. Detected by enzyme-linked immunosorbent assay (ELISA) and toxin neutralization assay.…”