A highly sensitive replacement assay for botulinum neurotoxin type B

Lead Research Organisation: University of Sheffield
Department Name: Biomedical Science

Abstract

Botulinum neurotoxins (BoNTs) are the most potent toxins known, with serotypes A, B, E causing human botulism and B, C, D causing animal botulism. Produced by Clostridium bacteria, BoNTs are highly lethal. However, in controlled conditions, some BoNTs variations (serotypes) can be used in a number of medical applications, such as neuromuscular spasm treatments. The main aim of this project is to establish a world-leading, quantitative in-vitro assay for detection of BoNT type B activity. This will allow replacement of the mouse lethality test currently used in production of pharmaceutical products related to BoNT type B. With previous support from the NC3Rs, we genetically engineered the first cell line that is sensitive to BoNT/B. We also incorporated a luminescent reporter system allowing convenient detection of BoNT/B activity using a user friendly microplate assay. Now, using our cell line and in partnership with the National Institute of Biological Standards and Control (NIBSC), we propose to both increase the sensitivity of the cell line and to develop a monoclonal antibody-based BoNT/B activity assay that outperforms the mouse bioassay on both specificity and sensitivity. Validation of our assay will be performed using pharmaceutical BoNT/B preparations and BoNT/B antitoxins. The successful implementation of this project will provide a firm incentive for BoNT/B manufacturers to replace the mouse LD50 testing with a faster, cheaper and more specific cell-based approach. We will also apply our technology to engineer an assay for detection of BoNT type D which is used for production of toxoids for veterinary care. We estimate that our assay can replace the use of thousands of mice in unethical testing during development and validation of pharmaceutical products. The availability of a BoNT/B-sensitive cell line and the quantitative assay for BoNT/B activity will aid development of new improved therapeutics for treatment of common neurological disorders.

Technical Summary

The proposed in-vitro assay aims to replace use of animals in the manufacture of pharmaceutical BoNT/B products. BoNT/B product testing involves the most severe levels of animal suffering due to death by slow asphyxiation being the assay endpoint. The LD50 assay also lacks specificity to distinguish between the different BoNTs serotypes, which all cause similar muscular paralysis.
With previous NC3Rs support, we engineered a human neuroblastoma SiMa cell line to carry a synthetic VAMP reporter molecule, the target of BoNT/B. We also developed a polyclonal antibody which recognises the BoNT/B-cleaved end of the VAMP reporter molecule. By selecting right capture plates we were able to design a refined ELISA capture assay which can detect the BoNT/B-cleaved VAMP via highly sensitive luminescent reaction. This advance was published in Frontiers in Pharmacology in 2017. To build a robust 3Rs legacy we must now create an assay for easy assimilation into a GMP-compliant environment, allowing widespread adoption by BoNT/B product manufacturers and regulators. In order to achieve this, we must produce monoclonal antibodies that can specifically detect VAMP2 cleaved by BoNT/B and devise a microplate-based assay for reproducible sensitive detection of BoNT/B activity. To make our ELISA assay most compelling, we will improve the sensitivity of our cell lines to further outperform the mouse bioassay by introducing the receptor with the highest known affinity for BoNT/B, mouse synaptotagmin 2 into our engineered cell lines. We will then screen for the most sensitive clones using industrial and in-house pharmaceutical BoNT/B and related BoNT/D. Finally, in partnership with our collaborators we will validate our highly specific and reproducible assay for suitability to test botulinum antitoxins and other pharmaceutical products.

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