A highly sensitive replacement assay for botulinum neurotoxin type B
Lead Research Organisation:
University of Sheffield
Department Name: School of Biosciences
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.
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.
Planned Impact
This project aims to directly replace the use of animals in Botulinum Neurotoxin type B (BoNT/B) product testing on an international scale. Currently, two BoNT types are available as commercial products, BoNT/A and BoNT/B. These are utilised both in the cosmetic industry and therapeutically, with BoNT/B currently being used primarily to treat cervical dystonia but also excessive sweating and salivation. Substantial investment is under way to expand their uses with a forecast annual growth rate of 8%. As a "biologic medicine", each time a toxin is produced, it is treated as a new drug. Therefore, in accordance with WHO guidelines, it must undergo rigorous potency, quality and safety testing. The gold standard assay is the LD50 animal lethality assay. This involves the most severe animal suffering with a lethal endpoint of slow asphyxiation of tested mice. An alternative replacement is urgently needed.
With previous NC3Rs support, we have engineered a cell line that, for the first time, is clearly sensitive to BoNT/B. We now propose to create a finalised, user friendly replacement assay that can outperform the animal LD50 assay both in sensitivity and specificity. Our project partners are the National Institute of Biological Standards and Control (NIBSC), who advise the WHO on botulinum testing guidelines for industry. NIBSC will validate our replacement assay in-house and provide a globally respected, international platform to launch our assay to industry. The principles of our assay also can be adopted to replace equivalent LD50 testing of other botulinum products and anti-tetanus vaccines. Given the extreme severity of the LD50 assay where animals die due to gradual asphyxation, manufacturers are not inclined to release the data on animal usage. However, recent estimates suggest that in the UK, around 70,000 mice are used each year for botulinum toxin testing; globally the figure is estimated to be more than 600,000 mice, with BoNT/B accounting for at least 10% market share, which is projected to grow further (Refs 2,3).
Since BoNT/B is a natural hazard and a potential threat as a bioweapon, all Western countries stockpile anti-BoNT/B antibodies known as antitoxins. Quality control of this antitoxins is done not only at the point of manufacture but also regularly to confirm their lasting potency. Unfortunately the same mouse LD50 assay is used by manufacturers and the regulatory bodies to determine the BoNT/B neutralisation potency by antitoxins. We will collaborate with NIBSC to confirm that our newly engineered cells combined with the convenient ELISA assay can provide adequate replacement for unethical mouse LD50 testing. The cell line and assay developed here will provide the basis for a non-animal botulinum toxicity gold-standard bioassay to reduce and replace the existing WHO/industrial standards. In addition to botulinum type B testing, the proposed cell line could be of value for manufacture of botulinum neurotoxin type D vaccine used for protecting livestock. This is due to both neurotoxins recognising the same VAMP2 molecule to elicit muscle paralysis.
Our clonal cells can be utilised also in academic research aimed at development of botulinum-based therapeutics or studying mechanisms of BoNT action, including our own laboratory and we already routinely use engineered SiMa cells replacing approximately 100 animals a year. We estimate from 2017 international meetings on botulinum neurotoxins and also from recently published literature that there are around 20 active research laboratories currently working on VAMP-cleaving botulinum neurotoxins. We anticipate that introduction of the ultimately sensitive and user friendly cell-based assay will help these research laboratories to avoid inhumane killing of several thousands of mice.
With previous NC3Rs support, we have engineered a cell line that, for the first time, is clearly sensitive to BoNT/B. We now propose to create a finalised, user friendly replacement assay that can outperform the animal LD50 assay both in sensitivity and specificity. Our project partners are the National Institute of Biological Standards and Control (NIBSC), who advise the WHO on botulinum testing guidelines for industry. NIBSC will validate our replacement assay in-house and provide a globally respected, international platform to launch our assay to industry. The principles of our assay also can be adopted to replace equivalent LD50 testing of other botulinum products and anti-tetanus vaccines. Given the extreme severity of the LD50 assay where animals die due to gradual asphyxation, manufacturers are not inclined to release the data on animal usage. However, recent estimates suggest that in the UK, around 70,000 mice are used each year for botulinum toxin testing; globally the figure is estimated to be more than 600,000 mice, with BoNT/B accounting for at least 10% market share, which is projected to grow further (Refs 2,3).
Since BoNT/B is a natural hazard and a potential threat as a bioweapon, all Western countries stockpile anti-BoNT/B antibodies known as antitoxins. Quality control of this antitoxins is done not only at the point of manufacture but also regularly to confirm their lasting potency. Unfortunately the same mouse LD50 assay is used by manufacturers and the regulatory bodies to determine the BoNT/B neutralisation potency by antitoxins. We will collaborate with NIBSC to confirm that our newly engineered cells combined with the convenient ELISA assay can provide adequate replacement for unethical mouse LD50 testing. The cell line and assay developed here will provide the basis for a non-animal botulinum toxicity gold-standard bioassay to reduce and replace the existing WHO/industrial standards. In addition to botulinum type B testing, the proposed cell line could be of value for manufacture of botulinum neurotoxin type D vaccine used for protecting livestock. This is due to both neurotoxins recognising the same VAMP2 molecule to elicit muscle paralysis.
Our clonal cells can be utilised also in academic research aimed at development of botulinum-based therapeutics or studying mechanisms of BoNT action, including our own laboratory and we already routinely use engineered SiMa cells replacing approximately 100 animals a year. We estimate from 2017 international meetings on botulinum neurotoxins and also from recently published literature that there are around 20 active research laboratories currently working on VAMP-cleaving botulinum neurotoxins. We anticipate that introduction of the ultimately sensitive and user friendly cell-based assay will help these research laboratories to avoid inhumane killing of several thousands of mice.
