Botulinum toxin is an extraordinarily potent molecule that has an unusually long duration of action. Despite this, there is little information available on natural mechanisms for metabolism or elimination and virtually no information on pharmacologically induced mechanisms for metabolism and elimination. Therefore, a number of experiments were performed on laboratory animals that addressed two major issues: 1) the effect of blood on the structure, function, and biologic half-life of the toxin, and 2) the effect of neutralizing antibodies on half-life and elimination of circulating toxin. In the first series of studies, the metabolic transformation of toxin was assessed by incubating it in blood for varying lengths of time. At each time point, aliquots were examined to determine the amount of toxin, the structure of toxin, the catalytic activity of toxin, and the neuromuscular blocking activity of toxin. This work demonstrated that blood did not alter any characteristic of the toxin molecule. Experiments were also done in which toxin was administered to mice and rats at doses that produced clinical poisoning. The results demonstrated that the elimination half-life for native (nonmetabolized) toxin in blood and serum was 230 to 260 min. During the second series of studies, the rate of elimination of circulating toxin was studied in the presence of antibodies directed against the carboxyl-terminal half of the toxin molecule. This work demonstrated that neutralizing antibodies 1) enhanced clearance of toxin from the circulation and 2) enhanced tissue accumulation of toxin, particularly in liver and spleen.

BackgroundBotulinum neurotoxins (BoNT) are a family of category A select bioterror agents and the most potent biological toxins known. Cloned antibody therapeutics hold considerable promise as BoNT therapeutics, but the therapeutic utility of antibodies that bind the BoNT light chain domain (LC), a metalloprotease that functions in the cytosol of cholinergic neurons, has not been thoroughly explored.Methods and FindingsWe used an optimized hybridoma method to clone a fully human antibody specific for the LC of serotype A BoNT (BoNT/A). The 4LCA antibody demonstrated potent in vivo neutralization when administered alone and collaborated with an antibody specific for the HC. In Neuro-2a neuroblastoma cells, the 4LCA antibody prevented the cleavage of the BoNT/A proteolytic target, SNAP-25. Unlike an antibody specific for the HC, the 4LCA antibody did not block entry of BoNT/A into cultured cells. Instead, it was taken up into synaptic vesicles along with BoNT/A. The 4LCA antibody also directly inhibited BoNT/A catalytic activity in vitro.ConclusionsAn antibody specific for the BoNT/A LC can potently inhibit BoNT/A in vivo and in vitro, using mechanisms not previously associated with BoNT-neutralizing antibodies. Antibodies specific for BoNT LC may be valuable components of an antibody antidote for BoNT exposure.

Most reports dealing with vaccines against botulinum toxin have focused on the injection route of administration. This is unfortunate, because a mucosal vaccine is likely to be more efficacious for patients and pose fewer risks to health care workers and to the environment. Therefore, efforts were made to generate a mucosal vaccine that provides protection against the botulinum serotypes that typically cause human illness (serotypes A, B, and E). This work demonstrated that carboxy-terminal peptides derived from each of the three serotypes were able to bind to and penetrate human epithelial barriers in vitro, and there was no cross inhibition of membrane binding and transcytosis. The three polypeptides were then tested in vivo as a trivalent vaccine that could be administered to mice by the intranasal route. The results indicated that the mucosal vaccine evoked high secretory titers of immunoglobulin A (IgA), as well as high circulating titers of IgG and IgA, and it also evoked a high level of resistance to challenge with toxin. The immunoglobulin responses and the levels of resistance to challenge were increased by coadministration of adjuvants, such as chitosan and vitamin E. At least three mechanisms were identified to account for the antibody-induced resistance: (i) blockade of toxin absorption across epithelial cells, (ii) enhanced clearance of toxin from the circulation, and (iii) blockade of toxin action at the neuromuscular junction. These results are a compelling demonstration that a mucosal vaccine against multiple serotypes of botulinum toxin has been identified.

The ability of botulinum toxin to poison cholinergic nerve transmission is a dynamic phenomenon that involves not only the actions of the toxin on the body but also the actions of the body on the toxin. The former has been the subject of intense research, whereas the latter has received almost no attention.

Botulinum toxin typically interacts with two types of cells to cause the disease botulism. The toxin initially interacts with epithelial cells in the gut or airway to undergo binding, transcytosis, and delivery to the general circulation. The toxin then interacts with peripheral cholinergic nerve endings to undergo binding, endocytosis, and delivery to the cytosol. The receptors for botulinum toxin on nerve cells have been identified, but receptors on epithelial cells remain unknown. The initial toxin binding site on nerve cells is a polysialoganglioside, so experiments were performed to determine whether polysialogangliosides are also receptors on epithelial cells. A series of single mutant and dimutant forms of the botulinum toxin type A binding domain (HC 50 ) were cloned and expressed. One of these (dimutant HC 50 A W1266L,Y1267S ) was shown to have lost its ability to bind nerve cells (phrenic nerve-hemidiaphragm preparation), yet it retained its ability to bind and cross human epithelial monolayers (T-84 cells). In addition, the wild-type HC 50 and the dimutant HC 50 displayed the same ability to undergo binding and transcytosis (absorption) in a mouse model. The fact that the dimutant retained the ability to cross epithelial barriers but did not possess the ability to bind to nerve cells was exploited to create a mucosal vaccine that was non-neurotropic. The wild-type HC 50 and non-neurotropic HC 50 proved to be comparable in their abilities to: 1) evoke a circulating IgA and IgG response and 2) evoke protection against a substantial challenge dose of botulinum toxin.