Development of a Highly Sensitive Cell-Based Assay for Botulinum Neurotoxin

说明(申请人提供)：肉毒杆菌神经毒素(BoNT)是最有效的天然毒素，是一种潜在的食品污染物，也是一种无价的医学药物。尽管BONT/A和BONT/B在临床上得到了有效的应用，但主要的不良反应是形成抗体，使患者对治疗难以治疗。目前，患者在治疗期间没有常规监测抗体形成，因为没有商业上可以获得的测量人类血清(以及其他动物物种，如马)中抗体的灵敏检测方法。小鼠生物测定相对不灵敏，具有众所周知的缺点，包括需要动物和相关的所需设施，结果需要2-4天，非特异性死亡，以及下面更详细描述的其他缺点。其他检测平台，包括ELISA和体内催化活性的测定，都存在本底高的缺点，更重要的是，它们只测量BONT活性的一个生物学特性。本研究项目的总体假设是，通过对细胞系的特异性评估，以及通过在细胞系中过度表达对BONT毒性至关重要的细胞因子，可以建立一种基于细胞的检测方法，用于灵敏和定量地检测BoNTs和针对BoNTs的血清抗体或抑制剂。这项建议的目标是设计一种对BONT/A高度敏感的连续细胞系，该细胞系将测量BONT对神经细胞中毒的各个方面。该细胞系将为小鼠生物检测提供一种特异、灵敏、定量、相对快速和低成本的替代方法。除了用于筛选人血清中是否存在抗BONT/A的抗体外，这种敏感的基于细胞的分析方法还将有许多其他潜在的应用，包括大规模筛选新的BONT抑制剂，评估BONT/A毒素的效力，以及作为研究工具。目前针对BoNTs的抗毒素药物很少，且严重副作用发生率高。因此，迫切需要鉴定和生产新的、更安全的BONT抗毒素和抑制剂。为了构建这样的细胞系，我们将检测BoNT/A细胞表面受体(突触囊泡蛋白2)在神经2a细胞和PC12细胞等细胞系中过表达的作用。一个或两个有希望的细胞系将通过在细胞内瞬时表达与BONT敏感性有关的几个因子来评估提高敏感性的能力，这些因素包括SV2c、GD3-合成酶(诱导神经节苷脂的形成)和突触素I/II。将优化检测条件以提高敏感性。优化将包括测试细胞内荧光BONT传感器的灵敏度，并可能通过稳定表达这些传感器来增强细胞系。最后，将细胞试验与BONT小鼠试验进行比较。公共卫生相关性：这项提议的目标是构建一种对生物活性的BONT/A高度敏感的细胞系，该细胞系随后可以用于旨在发现BONT抑制剂的大规模筛选项目，以及在BONT测试中的许多其他潜在应用。虽然这项检测不会完全取代体内的小鼠生物检测，但作为涉及BONT/A的筛选项目和其他研究的第一步，它将非常有价值，因此将显著减少BONT检测中使用的动物数量。这种细胞系在检测接受BONT治疗的患者中的中和抗体方面也可能被证明是非常有用的，这是BoNTs在医学上应用的一个持续问题。

DESCRIPTION (provided by applicant): Botulinum neurotoxin (BoNT), the most potent naturally occurring toxin, is a potential food contaminant but also an invaluable medical drug. Despite their effective use of BoNT/A and BoNT/B in clinical applications, the major adverse effect has been the formation of antibodies, which make patients refractory to treatment. Currently, patients are not routinely monitored for antibody formation during their treatment regime since a sensitive assay that measures antibodies in human sera (and in other animal species such as equine) is not commercially available. The mouse bioassay is relatively insensitive and has well-known drawbacks including the need for animals and associated required facilities, the requirement for 2-4 days for results, nonspecific deaths, and other drawbacks as described in more detail below. Other detection platforms including ELISA and determination of catalytic activity in vivo have the drawback of high background and importantly that they measure only one biological property of BoNT activity. The overall hypothesis of this research project is that a cell-based assay can be developed for sensitive and quantitative detection of BoNTs and serum antibodies or inhibitors to BoNTs by the specific evaluation of cell lines, and by over-expression of cell factors important for BoNT toxicity in the cell line. The goal of this proposal is to design a continuous cell line that is highly sensitive to BoNT/A and which would measure all aspects of neuronal cell intoxication by BoNT. This cells line would provide a specific, sensitive, quantitative, relatively fast and low-cost alternative to the mouse bioassay. In addition to its use in screening of human serum for the presence of antibodies to BoNT/A, such a sensitive cell-based assay for BoNT will have many other potential applications, including large-scale screenings for new BoNT inhibitors, evaluating BoNT/A toxin potency, as well as serving as a research tool. Currently available antitoxins against BoNTs are scarce and have a high incidence of serious side effects. Therefore, there is a great need for the identification and production of new and safer BoNT antitoxins and inhibitors. In order to construct such a cell line, the effect of over-expression of the BoNT/A cell surface receptor (synaptic vesicle protein 2) in cell lines such as neuro-2a cells and PC12 cells will be examined. One or two promising cell lines will be evaluated for the ability to enhance sensitivity by transient intracellular expression of several factors implicated in BoNT sensitivity, including SV2c, gD3-synthase (which induces formation of gangliosides), and synaptotagmin I/II. Assay conditions will be optimized to increase sensitivity. Optimization will include testing the sensitivity of fluorescent BoNT sensors inside the cells and possibly enhancing the cell line by stably expressing these sensors. Finally, the cell assay will be compared to the BoNT mouse assay. PUBLIC HEALTH RELEVANCE: The goal of this proposal is to construct a cell line that is highly sensitive to biologically active BoNT/A. This cell line could then be used in large-scale screening projects designed to discover BoNT inhibitors, as well as numerous other potential applications in BoNT testing. While this assay will not entirely replace the in vivo mouse bioassay, it would be very valuable as a first step in screening projects and other research involving BoNT/A, and therefore would significantly reduce the number of animals used in BoNT assays. This cell line might also prove extremely useful in the detection of neutralizing antibodies in BoNT-treated patients, which is an ongoing problem with the medical use of BoNTs.