Implementation of a Triple-Function-Analysis Method for In vitro Detection of Fully Active Tetanus Toxin to Replace In vivo Animal Models

Performance Period: 10/1/2020 to 4/30/2022

There is a continued international industry focus on replacing historical in vivo models with in vitro testing. However, animal models remain the regulatory “gold standard.” Attempts to replace these models with alternate testing methods have demonstrated poor correlation to the historical animal models due to the complex mechanisms of actions of bacterial toxins. In the case of tetanus toxin, there are three mechanisms that must all work to initiate disease: binding of toxin to peripheral neurons, transport into the cell cytoplasm, and finally destruction of synapotobrevin, a protein that controls muscle activity. Loss of any one function inactivates the toxin. Thus, measuring activity of one tetanus function through an in vitro test may not correlate to the animal model, which by default, measures the activity of all three functions. As a result, in vitro assays have not been widely implemented in place of in vivo tests.  

This project will work to change that paradigm. Recently, a binding and cleavage assay (BINACLE, 2013) advanced the development of an in vitro assay to replace the historical animal model for tetanus toxicity. The assay was designed to monitor two of the three functions of tetanus toxin: binding to the peripheral neurons and destruction (cleaving) of synapotobrevin. We propose to enhance the published method and improve correlation to the historical animal model by adding an additional step to measure transport into the cell cytoplasm. In this project, we will develop an antibody or VHH specific to the translocation domain of tetanus toxin produced using a MassBiologics licensed process (TDVAX, MassBiologics FDA License #1779) for the new assay step. In addition, we will provide a peptide substrate conjugated with a fluorescent probe and its quencher to improve quantification of the synaptobrevin-cleaving activity.