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

破伤风和肉毒杆菌疫苗用于人类和牲畜。疫苗含有破伤风或肉毒杆菌类毒素，这是每种神经毒素的一种解毒形式，能够在体内引发免疫原性反应。在疫苗生产期间，产品必须在几个阶段进行测试，以确定是否存在不必要的残留神经毒性。目前还没有合适的非动物方法来测量神经毒素的活性，迫使制造商、监管机构和学术研究人员使用实验室啮齿动物进行生物测试，通常具有致命的终点和不可接受的动物痛苦程度。理想的动物替代品应该是一个连续的细胞系，它可以忠实地代表破伤风和肉毒杆菌毒素作用在人类和动物身上发生的所有生物学步骤。这包括依次(1)将毒素附着在细胞外表面，(2)将毒素吸收到细胞内，以及(3)破坏破伤风毒性的最终目标，称为VAMP2的蛋白质，最终导致肌肉瘫痪。我们最近证明了一种独特的神经母细胞瘤细胞系可以识别和内化破伤风毒素(细胞附着和摄取)。然而，这些细胞并不携带破伤风毒性的最终靶点VAMP2。我们现在已经通过导入VAMP2蛋白获得了Tetcell神经母细胞瘤细胞系。我们已经证明，类似于破伤风毒素对人和动物的作用，破伤风可以破坏我们新细胞系内的VAMP2蛋白，从而完成模仿自然中毒机制的最后缺失的步骤。我们现在建议开发一种高灵敏度的检测我们细胞系中破伤风活性的方法。我们计划通过使用我们的VAMP2-细胞来获得一个基因相同的克隆细胞系来实现这一点，这对检测的重复性至关重要。然后，我们将开发一种用户友好和强大的检测方法，可以检测破伤风神经毒素的活性。最终，这种基于细胞的检测方法在破伤风疫苗检测中有巨大的潜力，可以减少和取代数以万计的动物检测。

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.