Development of a Highly Sensitive Cell-Based Assay for Botulinum Neurotoxin

Botulinum neurotoxin (BoNT), the most potent naturally occurring toxin, is a potential food contaminant but also an invaluable medical drug. Despite their effective use of BoNT/A and BoNT/B in clinical applications, the major adverse effect has been the formation of antibodies, which make patients refractory to treatment. Currently, patients are not routinely monitored for antibody formation during their treatment regime since a sensitive assay that measures antibodies in human sera (and in other animal species such as equine) is not commercially available. <P> The mouse bioassay is relatively insensitive and has well-known drawbacks including the need for animals and associated required facilities, the requirement for 2-4 days for results, nonspecific deaths, and other drawbacks as described in more detail below. Other detection platforms including ELISA and determination of catalytic activity in vivo have the drawback of high background and importantly that they measure only one biological property of BoNT activity. <P> The overall hypothesis of this research project is that a cell-based assay can be developed for sensitive and quantitative detection of BoNTs and serum antibodies or inhibitors to BoNTs by the specific evaluation of cell lines, and by over-expression of cell factors important for BoNT toxicity in the cell line. <P> The goal of this proposal is to design a continuous cell line that is highly sensitive to BoNT/A and which would measure all aspects of neuronal cell intoxication by BoNT. This cells line would provide a specific, sensitive, quantitative, relatively fast and low-cost alternative to the mouse bioassay. In addition to its use in screening of human serum for the presence of antibodies to BoNT/A, such a sensitive cell-based assay for BoNT will have many other potential applications, including large-scale screenings for new BoNT inhibitors, evaluating BoNT/A toxin potency, as well as serving as a research tool. Currently available antitoxins against BoNTs are scarce and have a high incidence of serious side effects. Therefore, there is a great need for the identification and production of new and safer BoNT antitoxins and inhibitors. In order to construct such a cell line, the effect of over-expression of the BoNT/A cell surface receptor (synaptic vesicle protein 2) in cell lines such as neuro-2a cells and PC12 cells will be examined. <P> One or two promising cell lines will be evaluated for the ability to enhance sensitivity by transient intracellular expression of several factors implicated in BoNT sensitivity, including SV2c, gD3-synthase (which induces formation of gangliosides), and synaptotagmin I/II. Assay conditions will be optimized to increase sensitivity. Optimization will include testing the sensitivity of fluorescent BoNT sensors inside the cells and possibly enhancing the cell line by stably expressing these sensors. Finally, the cell assay will be compared to the BoNT mouse assay. <P> PUBLIC HEALTH RELEVANCE: The goal of this proposal is to construct a cell line that is highly sensitive to biologically active BoNT/A. This cell line could then be used in large-scale screening projects designed to discover BoNT inhibitors, as well as numerous other potential applications in BoNT testing. While this assay will not entirely replace the in vivo mouse bioassay, it would be very valuable as a first step in screening projects and other research involving BoNT/A, and therefore would significantly reduce the number of animals used in BoNT assays. This cell line might also prove extremely useful in the detection of neutralizing antibodies in BoNT-treated patients, which is an ongoing problem with the medical use of BoNTs.