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

描述（由申请人提供）：肉毒神经毒素（BoNT）是最有效的天然毒素，是一种潜在的食品污染物，但也是一种宝贵的医疗药物。尽管BoNT/A和BoNT/B在临床应用中有效使用，但主要的副作用是抗体的形成，这使得患者难以治疗。目前，在患者的治疗方案期间，不对患者的抗体形成进行常规监测，因为测量人血清（和其他动物物种如马）中抗体的灵敏测定法尚未商购获得。小鼠生物测定相对不敏感，并且具有众所周知的缺点，包括需要动物和相关的所需设施，需要2-4天的结果，非特异性死亡，以及下面更详细描述的其他缺点。包括ELISA和体内催化活性测定的其他检测平台具有高背景的缺点，并且重要的是它们仅测量BoNT活性的一种生物学性质。该研究项目的总体假设是，可以开发基于细胞的测定，通过对细胞系的特异性评价，以及通过对细胞系中BoNT毒性重要的细胞因子的过表达，来灵敏和定量检测BoNT和血清抗体或BoNT抑制剂。该提案的目标是设计一种连续的细胞系，其对BoNT/A高度敏感，并且将测量BoNT引起的神经元细胞中毒的所有方面。该细胞系将为小鼠生物测定法提供一种特异、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BoNT/A抗体的存在外，这种灵敏的基于细胞的BoNT测定将具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毒素效力，以及作为研究工具。目前可用的抗BoNT的抗毒素是稀缺的，并具有严重副作用的高发生率。因此，非常需要鉴定和生产新的和更安全的BoNT抗毒素和抑制剂。为了构建这样的细胞系，将检查BoNT/A细胞表面受体（突触囊泡蛋白2）在诸如neuro-2a细胞和PC 12细胞的细胞系中过表达的效果。将评价一种或两种有前途的细胞系通过瞬时细胞内表达与BoNT敏感性有关的几种因子（包括SV 2c、gD 3合酶（诱导神经节苷脂形成）和突触结合蛋白I/II）来增强敏感性的能力。将优化试验条件以提高灵敏度。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，将细胞测定与BoNT小鼠测定进行比较。公共卫生相关性：本提案的目标是构建对生物活性BoNT/A高度敏感的细胞系。然后，该细胞系可用于大规模筛选项目，旨在发现BoNT抑制剂，以及BoNT测试中的许多其他潜在应用。虽然这种测定法不会完全取代体内小鼠生物测定法，但作为筛选项目和涉及BoNT/A的其他研究的第一步，它将非常有价值，因此将显著减少BoNT测定法中使用的动物数量。这种细胞系也可能被证明在检测BoNT治疗患者的中和抗体方面非常有用，这是BoNT医疗用途的一个持续问题。

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.