Development of a tetanus-sensitive cell line for assaying tetanus toxin activity

Tetanus and botulinum vaccines are used in both humans and livestock. During vaccine manufacture the product must be tested at several stages for unwanted residual neurotoxicity. Currently there are no simple sensitive quantitative assays to measure neurotoxin activity forcing manufacturers and regulatory agencies to use laboratory rodents for biological testing often with a lethal endpoint and unacceptable levels of animal suffering. An ideal animal replacement would be a cell line that could faithfully represent all the biological steps of in vivo tetanus and botulinum toxin action, including receptor binding, internalisation and cleavage of the final target of tetanus toxicity VAMP2, thereby blocking neurotransmission. We have recently demonstrated that unlike other widely used neuronal cell lines, differentiated SiMa cells are capable of binding the tetanus toxin receptor binding domain. However, immunoblotting experiments suggest that SiMa cells do not express the final target of tetanus and botulinum type D toxicity, VAMP2.

We have now generated a new stable SiMa cell-line carrying a VAMP2-Green Fluorescent Protein (VAMP2-GFP) fusion protein. Using lipofection, fluorescence microscopy and western immunoblotting, we have demonstrated that, analogous to the action of tetanus toxin in vivo, the tetanus endopeptidase domain can cleave VAMP2-GFP protein inside our novel cell line, thereby completing the missing step needed for intoxication. We propose to develop a sensitive assay for detection of tetanus activity. We plan to achieve this by first deriving fully clonal VAMP2-GFP SiMa cell lines providing a stable and genetically homogeneous cell model. Using antibodies against GFP and VAMP2, we will then develop a user friendly and robust sandwich ELISA detection assay for the detection of tetanus neurotoxin, a major threat to human health. Ultimately, this cell-based assay has great potential to stop animal suffering and reduce and replace animal assays globally.

• News: New cell line developed for testing botulinum toxin activity