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

开发用于测定破伤风毒素活性的破伤风敏感细胞系

• 批准号: NC/N001516/1
• 负责人: Bazbek Davletov
• 金额: $ 18.8万
• 依托单位: University of Sheffield
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=NC%2FN001516%2F1
• 关键词: Development; tetanus; sensitive; cell; line

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.

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解毒形式，能够在体内引起免疫原性反应。在疫苗生产过程中，必须在几个阶段测试产品是否存在不必要的残留神经毒性。目前，没有合适的非动物测定来测量神经毒素活性，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水平。理想的动物替代品是一个连续的细胞系，它可以忠实地代表人类和动物中发生的破伤风和肉毒杆菌毒素作用的所有生物步骤。这依次包括（1）毒素附着于细胞的外表面，（2）毒素被摄取到细胞中，和（3）破坏破伤风毒性的最终靶标，称为VAMP2的蛋白质，最终导致肌肉麻痹。我们最近已经证明，一个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，这些细胞不携带破伤风毒性的最终靶标VAMP2。我们现在已经通过引入VAMP2蛋白来工程化TetCell神经母细胞瘤系。我们已经证明，类似于破伤风毒素在人类和动物中的作用，破伤风可以破坏我们的新细胞系内的VAMP2蛋白，从而完成模拟自然中毒机制的最后一步。我们现在建议开发一种高灵敏度的检测破伤风活性在我们的细胞系。我们计划通过使用我们的VAMP2细胞来获得遗传上相同的克隆细胞系来实现这一目标，这对测定重现性至关重要。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活性。最终，这种基于细胞的检测方法具有很大的潜力，可以减少和取代破伤风疫苗检测中的数万种动物检测方法。