Engineered cell-based assays for detection of SNAP25-cleaving botulinum neurotoxins

This project proposes to establish quantitative animal-free assays for detection of Botulinum Neurotoxins (BoNTs) type A and C. Botulism is a severe paralytic disease affecting both humans and animals. The paralytic botulinum neurotoxins types A-G are produced by anaerobic bacteria which are ever-present in the soil, and also in decaying matter and poorly prepared food. Unlike other botulinum serotypes, type A and C BoNTs target specifically intraneuronal protein SNAP25 and cause the longest muscle paralysis lasting up to 6 months. Botulism type A was originally identified in humans but also afflicts animals, whereas type C botulism is an animal disease. There is a long-standing need for sensitive detection of toxic material in afflicted animals and environmental samples. To prevent botulism outbreaks managed animals receive botulism vaccines that incorporate inactivated BoNTs. These are being tested for the lack of active toxic material in live mouse toxicity assays. In addition, anti-toxins used in human and veterinary medicine are also tested for their potency in live mouse toxicity assays. Finally, due to the longest muscle paralysis type A BoNT is widely used in medicine for local muscle treatments and as an aesthetic, while BoNT/C is being investigated as an additional medical option in the cases where repeated BoNT/A use leads to immunological resistance. The manufacturing of pharmaceutical BoNT preparations unfortunately also relies on live mouse toxicity assays. The currently used live mouse toxicity assays are unethical due to the slow mode of animal death by asphyxiation.
There is a long-standing need, and recently governmental pressure, to replace live mouse toxicity assays with animal-free detection of botulinum activities. This project aims to develop cell-based assays which would accurately measure the activities of BoNT type A and C. This opportunity arose from our recently acquired detailed knowledge of molecular mechanisms of BoNT actions and our identification of a new continuous cell line that exhibits a robust binding of BoNTs. Using synthetic biology approaches we will engineer this cell line to report the activity of BoNT type A and C in a simple multiwell light-emitting assay. Successful implementation of this project will contribute to better animal health through easier production of veterinary and human medicines, will save hundreds of thousands of mice from unethical testing, and also provide a long-sought-after test for BoNT presence during botulism outbreaks both in farmed animals and in wild environments.

This project will utilise our knowledge of molecular mechanisms of Botulinum toxins (BoNTs) action and proven synthetic biology approaches to engineer sensitive cell-based assays to replace animal use in the field of botulinum medicine. Following ingestion, BoNTs enter the blood circulatory system and bind to presynaptic motor nerve terminals. BoNTs target specific receptors present on the neuronal surfaces - gangliosides and additionally a vesicular protein receptor for efficient internalization. Upon endocytosis, the acidic intravesicular pH triggers a conformational change in the toxin structure which causes vesicular membrane penetration and the release of the botulinum enzyme into the presynaptic cytosol. In the case of BoNT type A and type C, their released enzymes cleave intraneuronal protein called SNAP25 thereby causing months-long synaptic blockade, leading to death if untreated.
We will engineer a reporter SNAP25 molecule which - when cleaved by type A and C enzymes - could be captured on microplates using custom-made anti-cleaved SNAP25 antibodies. Since the engineered SNAP25 reporter carries a Nanoluc luciferase, the botulinum activity can then be revealed via a luminescent reaction with high sensitivity. To implement a biologically relevant readout of the botulinum activities we will transduce the Nanoluc-SNAP25 reporter into a newly identified botulinum-sensitive human neuroblastoma cell line. Our preliminary results demonstrated the feasibility of such an approach for the detection of SNAP25-cleaving BoNTs which extends our previous success with engineering of human neuroblastoma cells for detection of BoNT/B activity (1,2).
1. Rust A, Doran C, Hart R, Binz T, Stickings P, Sesardic D, Peden AA, Davletov B. A Cell Line for Detection of Botulinum Neurotoxin Type B. Front Pharmacol (2017) 8:796. This paper led to several industrial collaborations.
2. Patent 'Stable VAMP reporter assay' WO2018150177, Davletov B, Peden A, Rust A, Doran C.