SiMa Cells for a Serotype Specific and Sensitive Cell-Based Neutralization Test for Botulinum Toxin A and E

Abstract

Botulinum toxins (BoNTs), of which there are seven serotypes, are among the most potent neurotoxins, with serotypes A, B and E causing human botulism. Antitoxins form the first line of treatment for botulism, and functional, highly sensitive in vitro methods for toxin neutralization are needed to replace the current in vivo methods used for determination of antitoxin potency. In this preliminary proof of concept study, we report the development of a neutralization test using the neuroblastoma SiMa cell line. The assay is serotype specific for either BoNT/A or BoNT/E, which both cleave unique sequences on SNAP-25 within SiMa cells. The end point is simple immunodetection of cleaved SNAP-25 from cell lysates with antibodies detecting only the newly exposed sequence on SNAP-25. Neutralizing antibodies prevent the toxin-induced cleavage of SNAP-25. The toxin neutralization assay, with an EC50 of ~2 mIU/mL determined with a standardized reference antiserum, is more sensitive than the mouse bioassays. Relevance was demonstrated with commercial and experimental antitoxins targeting different functional domains, and of known in vivo neutralizing activities. This is the first report describing a simple, specific, in vitro cell-based assay for the detection of neutralizing antibodies against BoNT/A and BoNT/E with a sensitivity exceeding that of the mouse bioassay.

A cell-based alternative to the mouse potency assay for pharmaceutical type E botulinum antitoxins

“…Several previous studies have used cell-based assays for in vitro quantification of neutralizing antibodies (Bak et al, 2017;Hall et al, 2004;Nuss et al, 2010;Pellett et al, 2007;Yadirgi et al, 2017). However, the vast majority of the in vitro values were not compared to respective PMNA data.…”

“…However, the vast majority of the in vitro values were not compared to respective PMNA data. To date, only one cell-based study has attempted to assess the in vitro neutralizing activity of type E botulinum antitoxins with respect to the PMNA, and its evaluation was limited since the in vivo potency was not precisely defined but was based on an assumed broad range (>50 IU/mL, stated by the antitoxin manufacturer) (Bak et al, 2017).…”

“…Alternatively, cell-based assays that include all of the intoxication steps represent the entire intoxication process, resembling the in vivo process, and have the potential to enable an unbiased measurement of toxin potency and antitoxin neutralizing activity. Indeed, several cell-based assays have been developed to measure BoNT activity for pharmaceutical and clinical purposes (Bak et al, 2017;Fernandez-Salas et al, 2012;Hall et al, 2004;Kiris et al, 2014;McNutt et al, 2011;Nuss et al, 2010;Pellett et al, 2011;Pellett et al, 2007;Pellett et al, 2017;Pellett et al, 2010;Rust et al, 2017;Yadirgi et al, 2017;Pathe-Neuschäfer-Rube et al, 2015;Pathe-Neuschäfer-Rube et al, 2018;Torgeman et al, 2017a). However, no systematic and direct quantitative comparison to PMNA values of cell-based assays developed to measure the potency of anti-BoNT/E preparations have been described.…”

A Cell Line for Detection of Botulinum Neurotoxin Type B

“…Our NanoLuc-VAMP2 SiMa neuroblastoma assay far exceeds the rat spinal cord cell sensitivity using a very simple 3-day differentiation protocol. It appears that the differentiated SiMa neuroblastoma cells are sensitive to BoNTs ( Fernández-Salas et al, 2012 ; Rust et al, 2016 ; Bak et al, 2017 ) due to the high level of expression of neuron-specific GT1b ganglioside ( Figure 1D ) which is essential for the initial binding of the toxins ( Montecucco and Schiavo, 1994 ; Binz and Rummel, 2009 ). If needed, sensitivity could be improved further by stably transfecting into the NanoLuc-VAMP2 SiMa neuroblastoma cells the mouse synaptotagmin 2 which has 10 times higher affinity to BoNT/B compared to human synaptotagmin ( Strotmeier et al, 2012 ).…”

Botulinum Neurotoxins (BoNTs)—Antibody and Vaccine

“…Fetweh H. Al-Saleem, Rashmi Sharma, Scott K. Dessain, and their co-workers from the United States of America, presented the development of a fusion protein for red blood cell adherence of immune complexes containing BoNT, to improve neutralization and macrophage uptake [13]. Nicola Bak, Dorothea Sesardic, and their team from the United Kingdom, reported on the application of SiMa cells for cell-based neutralization test for BoNT/A and BoNT/E [14]. On the vaccine development side, Robert P. Webb, Theresa J. Smith, and their team from USAMRIID, reported on the production of non-toxic holoproteins and recombinant BoNT toxin domain subunits as vaccine candidates against multiple serotypes [15], Denis Y. Otaka, Felipe M. Salvarani, and their team from Brazil, reported on the humoral response of buffalos to a recombinant vaccine against BoNT/C and D [16].…”