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

Tetanus and botulinum vaccines are used in humans and livestock. Vaccines contain tetanus or botulinum toxoids, a detoxified form of each neurotoxin capable of illiciting an immunogenic response in the body. During vaccine production the product must be tested at several stages for unwanted residual neurotoxicity. Currently there are no appropriate non-animal assays to measure neurotoxin activity forcing manufacturers, regulatory agencies and academic researchers to use laboratory rodents for biological testing often with lethal endpoint and unacceptable levels of animal suffering. An ideal animal replacement would be a continuous cell line that could faithfully represent all the biological steps of tetanus and botulinum toxin action that occurs in humans and animals. This comprises sequentially (1) attachment of the toxin to the outer surface of cells, (2) uptake of the toxin into cells and (3) destruction of the final target of tetanus toxicity, the protein called VAMP2, ultimately causing muscle paralysis. We have recently demonstrated that a unique neuroblastoma cell line can recognise and internalise tetanus toxin (cell attachment and uptake). However, these cells do not carry the final target of tetanus toxicity, VAMP2. We have now engineered TetCell neuroblastoma line by introducing VAMP2 protein. We have demonstrated that, analogous to the action of tetanus toxin in humans and animals, tetanus can destroy the VAMP2 protein inside our novel cell line, thereby completing the final missing step mimicking the natural mechanism of intoxication. We now propose to develop a highly sensitive assay for detection of tetanus activity in our cell line. We plan to achieve this by using our VAMP2-cells to derive a clonal cell line that is genetically identical, which is critical for assay reproducibility. We will then develop a user friendly and robust detection assay that can detect tetanus neurotoxin activity. Ultimately, this cell-based assay has great potential to reduce and replace tens of thousands animal assays in tetanus vaccine testing.

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.

We envisage three impact streams following the successful completion of this project, which include 3Rs, scientific and economic benefits that have the potential to save thousands of animals and ultimately many human lives in the longer term.
The 3Rs impact will be huge as our cells can replace all animal lethality tests in tetanus vaccine manufacture. The traditional tools used to assess tetanus vaccine product safety (i.e. animal toxicology) have changed little over many decades and have largely not benefited from recent gains in scientific knowledge. Animal methods of vaccine potency testing lead to substantial animal suffering and have low reproducibility with ill-defined relevance to humans. The cell line and assays developed here will provide the basis for a non-animal tetanus toxicity gold-standard bioassay to reduce and replace the existing WHO/industrial standards. In addition to tetanus vaccine 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.
Following the completion of this work, new neural cell lines with useful characteristics will be available to the Scientific Community.To date, none of the widely used neuronal cell lines have been shown to be sensitive to tetanus toxin. Mouse embryonic stem cells could potentially be used as an in vitro model for testing toxoids of the wider family of botulinum neurotoxins but reproducible and homogeneous differentiation of embryonic stem cells into neurons is time consuming, laborious, expensive and inefficient compared to a neuronal cell line. Ultimately our engineered and characterised cells have the potential to provide an inexpensive, user-friendly tool for basic academic and pharmaceutical research into clostridial neurotoxins.
This project has the potential to have a major Economic and Social Impact, through the provision of cheaper and faster tetanus vaccine production. Tetanus vaccine is an essential global medicine and there will be an ongoing requirement for humans and livestock to be immunised against this disease. Currently, only about 70 % of humans are fully immunised against Tetanus, however the WHO aims to achieve full coverage in 10 years. The number of animals used in vaccine production is therefore set to rise as the global population increases (approaching 10 billion by 2050). Animal testing has to be replaced and therefore a robust, cheap and user-friendly cell-based assay would contribute substantially to allow immunisation coverage against Tetanus on a global scale, ultimately saving many human lives.