BoNT is the most toxic substance known to man. The only accepted detection method for BoNTs is mouse bioassay,which need to take four days to complete, and cannot meet clinical diagnostics need. We are working on development of biosensors to detect BoNT with the sensitivity similar or better than mouse bioassay, and a detection time of less than one hour.
Clostridium botulinum has many strains producing seven serotypes of BoNT and over two dozen subtypes. Even if the same serotype and subtype of BoNT is obtained its bacterial source may differ. The Center in collaboration with CDC has started a groundbreaking work in genomics for identifying different isolates of the same strain of C. botulinum, thus allowing forensic analysis of the pathogenic agent.
The long-lasting endopeptidase activity of BoNT is a critical biological activity inside the nerve cell, as it prompts proteolysis of the SNARE proteins involved in the exocytosis of the neurotransmitter acetylcholine. Thus, the BoNT endopeptidase activity is an appropriate clinical target for designing new small molecule antidotes against BoNT, with the potential to reverse the paralysis syndrome of botulism.
We have screened a library of natural compounds and their analogues, against the Zn2+- endopeptidase domain of BoNT/A. Using a progression of biochemical and cellular assays of increasing complexity, we have identified a nitrophenyl psoralen derivative as a selective and potent inhibitor of BoNT/A from the natural product library.
Botulinum neurotoxins (BoNTs) are produced by Clostridium botulinum pose a significant threat to the public as potential bioterrorist weapons because of their extreme potency, relative ease of production and transport, and the burden that affected individuals would place on the public health care system. Estimates suggest that as little as 1 to 3 ng/kg of neurotoxin type A is enough to kill 50 percent of a human population exposed by oral or inhalation routes, respectively. Thus, it is essential to develop an effective vaccine therapy for the prevention of these deadly diseases.
We have developed a double-mutant E224A/E262A full-length botulinum neurotoxin (BoNT) Type A with structural similarity to native BoNT/A but lacking the endopeptidase activity provides an ideal surrogate for testing pharmacokinetics and immunochemical characteristics of BoNT. drBoNT/A was found to be highly immunogenic when tested under various in vivo conditions in Balb/C mice model. For the first time we have demonstrated that a full length 150 kDa drBoNT/A, by administering via inhalation route in mice model, has evoked both circulating immunoglobulin levels of IgG and secretory IgA at the mucosal surface. The immunoglobulin levels were sufficient enough to protect against the challenge dose of native BoNT toxin in mice model. Tissue distribution of drBoNT/A seems to be similar to that of native toxin.