Cell Based Assay for Tetanus Vaccine and Antitoxin Production

Tetanus toxin is a member of the Clostridial Toxin family which also includes Botulinum Toxins. This family comprises some of the most potent toxins in existence. They also are used in a wide number of medical applications. Tetanus toxin is chemically treated to make Tetanus Toxoid, which is a non-harmful derivative of Tetanus that is used in Tetanus Vaccine. This vaccine is a WHO Essential Global Medicine. Indeed, it forms the backbone of immunisation schedules across the globe with 83% of the global population currently vaccinated to date. As the bacteria that causes tetanus is present in the soil, it can never be eradicated and therefore the requirement for vaccine will be continuous and expanding along with the global human and livestock populations. As part of the quality control process, tetanus-based products must be tested in animals for potency and, in the case of toxoid, for traces of remaining toxicity. Animal testing is associated with considerable suffering to large numbers of animals and there is an urgent and long recognised need to replace these tests.

Previously, we have genetically engineered a cell-line to be highly sensitive to the closely related Botulinum/B. We then developed a robust antibody-based assay which incorporates a luminescent reporter readout for ease of detection of Botulinum/B activity. The assay can detect the very tiny amounts of toxin necessary for potency testing/quality control purposes. Now, we have adapted our technology to be acutely sensitive to Tetanus Toxin and generated state of the art recombinant monoclonal antibodies that can be readily scaled up for global adoption of our technology. In collaboration with the MHRA, we will fully validate our assay for Tetanus Toxin, Antitoxin and importantly for quality control of commercial Toxoids from Vaccine Producers. This project will lay a solid foundation to then immediately translate the technology to end-users for in-house evaluation and ultimately adoption. We will also optimise and "test to destruction" the tolerance of our assay.

Many elements of the mechanism of action of tetanus toxin remain to be elucidated owing to the difficulty in studying the specific populations of cells that are sensitive to tetanus in vivo. Our unique portfolio of tools together with recent advances in RNA-sequencing technologies places us in an excellent position to answer these longstanding questions. We will use our engineered cell lines that can be rendered sensitive to tetanus through chemical differentiation, to probe the molecular substrates underlying tetanus sensitivity.

Tetanus and Botulinum/B are Clostridial toxins that exert their toxicity by cleaving a critical component of the exocytosis machinery, VAMP, at the same cleavage site. However, their method of intoxication is different and not fully elucidated, particularly in the case of tetanus. With previous NC3Rs funding, we have genetically engineered a cell-line to be highly sensitive to Botulinum/B. Importantly it recapitulates all the steps of in-vivo intoxication, namely cell surface binding, internalisation, endosomal escape and cleavage of VAMP. Following cleavage, VAMP is rapidly degraded in the cell and evades detection using immunochemical based methods. We overcame this historical hurdle by stabilising toxin-cleaved VAMP by fusing it to a luminescence reporter molecule. This, together with a polyclonal antibody specific for Tetanus and Botulinum/B cleaved VAMP, formed the basis of our robust and user friendly One Step ELISA Assay. We have now improved the sensitivity of our cells to Tetanus toxin making it possible to detect physiologically meaningful concentrations of the toxin. We also have developed a panel of novel recombinant capture antibodies specific for toxin-cleaved VAMP which can be used in the One Step ELISA assay. In collaboration with the MHRA, we propose to undertake a 2-phase study to translate our technology into a next generation cell-based assay for measuring residual Tetanus toxin activity. In Phase I, we will fully optimise and validate our assay using Tetanus Toxin, Antitoxin and commercial Toxoids supplied from vaccine manufacturers. In Phase 2, we will transfer our technology to commercial end-users and have them validate the technology in-house as a replacement for the animal bioassay. In parallel to these studies, we will also use a proteogenomic approach in combination with our unique Tetanus toxin sensitive models to identify the elusive molecular substrates that confer Tetanus sensitivity to specific populations of neurons.