Critical Analysis of Neuronal Cell and the Mouse Bioassay for Detection of Botulinum Neurotoxins

Abstract

Botulinum Neurotoxins (BoNTs) are a large protein family that includes the most potent neurotoxins known to humankind. BoNTs delivered locally in humans at low doses are widely used pharmaceuticals. Reliable and quantitative detection of BoNTs is of paramount importance for the clinical diagnosis of botulism, basic research, drug development, potency determination, and detection in clinical, environmental, and food samples. Ideally, a definitive assay for BoNT should reflect the activity of each of the four steps in nerve intoxication. The in vivo mouse bioassay (MBA) is the 'gold standard' for the detection of BoNTs. The MBA is sensitive, robust, semi-quantitative, and reliable within its sensitivity limits. Potential drawbacks with the MBA include assay-to-assay potency variations, especially between laboratories, and false positives or negatives. These limitations can be largely avoided by careful planning and performance. Another detection method that has gained importance in recent years for research and potency determination of pharmaceutical BoNTs is cell-based assays, as these assays can be highly sensitive, quantitative, human-specific, and detect fully functional holotoxins at physiologically relevant concentrations. A myriad of other in vitro BoNT detection methods exist. This review focuses on critical factors and assay limitations of the mouse bioassay and cell-based assays for BoNT detection. Key Contribution:This manuscript reviews two widely used botulinum neurotoxin detection assays, the mouse bioassay and cell-based assays, and the critical factors involved in their methodology as well as interpretation of results. Despite our ability to now reduce animal use and replace the mouse bioassay for certain aspects of botulinum neurotoxin detection, the mouse bioassay is sensitive and is the only in vivo assay considering all aspects of botulinum neurotoxin intoxication on a physiological level as well as pharmacokinetic aspects of the toxins, and thus remains important assay for botulinum neurotoxin detection.Toxins 2019, 11, 713 2 of 23 cells, with preferential entry into motor-neurons. Inside a neuron, the toxins block neurotransmitter release, leading to the long-lasting descending bilateral paralysis characteristic of botulism [6]. Even if applied intramuscularly for therapeutic purposes, a portion of the injected BoNTs, in particular at high doses, can diffuse away from the local injection site leading to distal neuronal effects and at very high doses systemic distribution [7][8][9][10]. BoNTs can also enter human or vertebrate circulation and cause botulism by different routes, including ingestion of contaminated foods, by wound infection with neurotoxigenic clostridia, and by the colonization of the intestinal tract by neurotoxigenic clostridia, causing infant botulism or adult intestinal botulism [11,12]. The latter is rare, as C. botulinum usually does not colonize a healthy intestine with a mature microbiota. The severe symptoms of botulism last from several days to up to a year, depending ...

Analysis of Motor Neurons Differentiated from Human Induced Pluripotent Stem Cells for the Use in Cell-Based Botulinum Neurotoxin Activity Assays

“…The gold standard for potency estimation is the mouse lethality assay in which mice are injected with multiple dilutions of BoNTs and the LD 50 is determined [15]. The mouse lethality assay has been criticized for being time-consuming, expensive, not always representative for humans, and having an intra-laboratory error of up to 20% and inter-laboratory error of more than 50% [16,17]. Injection with BoNTs causes severe distress in the test animals, which should be reduced according to the 3Rs principle (Reduction, Refinement, Replacement) described by Russel and Burch [18,19].…”

“…About 400,000 mice are still used in Europe annually [20]. The prerequisite for an in vitro assay that can reduce or replace the use of the mouse lethality assay is the ability to detect functionally active toxin and to consider the processes of toxin binding, internalization, release from neuronal vesicles, and target cleavage [4,17,21]. Cell-based assays utilizing neuronal cell lines, primary cultured neurons, or stem cell-derived neurons are capable of recapitulating several if not all of these aspects [22,23].…”

“…All BoNT serotypes have the same very well-defined mode of action, which results in the cleavage of a SNARE protein at a cleavage site characteristic for each serotype [7]. The quantification of a specific cleaved target is incorporated in many in vitro assays, which limits their applicability to single BoNT serotypes only [17]. Cell-based in vitro assays have been validated by BoNT producing companies for the analysis of BoNT/A1 and /B1 [20].…”

“…To detect all serotypes in one assay, the quantification of the neurotransmitter release inhibition, which is the toxicological endpoint at the cellular level for all BoNT serotypes, has been proposed in several neuronal cell line-based assays [26][27][28]. However, for basic research, mechanistic studies, and the development of BoNT inhibitors and antibodies, human MNs would be the most physiologically relevant cell type instead of cell lines and can recapitulate every step of BoNT intoxication including neurotransmitter release inhibition [17,23,[29][30][31]. Another factor to consider in terms of sensitivity is the interspecies variation.…”

Human-Relevant Sensitivity of iPSC-Derived Human Motor Neurons to BoNT/A1 and B1

“…In many in vitro assays, the toxin or the cleaved substrate is detected with analytical or immunological methods, which omits one of the steps that dictates the sensitivity towards the toxin, which is the binding to a cell [ 22 , 23 ]. Cell-based assays, however, utilize different types of (neuroblastoma) cell lines, primary cells or neurons differentiated from stem cells, which can recapitulate all steps of toxin entry and activity [ 24 , 25 ]. The most physiologically relevant cell-based assay would be based on human MNs, as these can reflect all steps of toxicity relevant for BoNT pharmaceuticals and additionally should represent the sensitivity of humans.…”