Development of a highly sensitive in vitro endothelial cell toxicity assay for evaluating ricin toxin A chain-based vaccines or therapeutics

• Published in: Toxicon (Oxford) Vol. 167; pp. 152 - 161
• Main Authors: Machesky, Nicholas J., Rusnak, Janice M., Moore, Evan H., Dorsey, Christopher B., Ward, Lucy A.
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01-09-2019
• Subjects: Drug Evaluation, Preclinical; Electric Impedance; Endothelial cell toxicity assay; Endothelial Cells - drug effects; Human Umbilical Vein Endothelial Cells; Humans; Ricin; Ricin - toxicity; Ricin toxin A chain; Toxicity Tests - methods; Transendothelial electrical resistance; Vaccine; Vascular leak; Ricin; Vaccine; Transendothelial electrical resistance; Endothelial cell toxicity assay; dgRTA; BSA; Vh; Vascular leak; ECT; rRTA; TEER; IT-RTA; VLS; Ricin toxin A chain; EGM-2; RTA; HUVECs
• DOI: 10.1016/j.toxicon.2019.06.015
• EISSN: 1879-3150
• ISSN: 0041-0101

The ricin toxin A chain (RTA) is responsible for ricin intoxication due to inhibition of protein synthesis. RTA is also known to cause endothelial toxicity [via a 3 amino acid sequence (x)D(y) motif that acts as a natural disintegrin] resulting in vascular leak syndrome (VLS) in humans. An in vitro endothelial cell toxicity (ECT) assay was developed to evaluate if the ricin vaccine candidate (RVEc) exhibited endothelial toxicity, determined by altered transendothelial electrical resistance (TEER) across human umbilical vein endothelial cell (HUVEC) monolayers. Timepoints at 2 and 4 h were included to evaluate HUVEC monolayers before the effects of RTA ribotoxic activity are observed. Both the 3 μM and 6 μM RTA positive controls consistently demonstrated significantly reduced TEER values, compared to their corresponding vehicle control, in a time- and concentration-dependent manner at 2, 4, and 24 h. Fluorescent imaging of HUVECs exposed to 3 μM RTA showed cell rounding at 2 and 4 h and gap formation at 24 h. No changes in TEER or fluorescent imaging were observed after exposure to endothelial cell growth medium-2 (EGM-2) exchange (mock control). The negative controls, which included 2 mutant RTA vaccine derivatives [RVEc with an (x)D(y) VLS sequence modification to V76M or D75N] and bovine serum albumin (BSA), demonstrated no evidence of HUVEC toxicity at 3 μM and 6  μM concentrations. Overall, the performance of the ECT assay was consistent, allowing for the development of acceptance criteria that were related to time- and concentration-dependent decreases in TEER between 2 and 24 h.
•RTA-containing immunotoxins are associated with vascular leak syndrome in humans.•RTA may cause endothelial toxicity due to a (x)D(y) motif acting as a disintegrin.•Assessment of RTA-based vaccines for human use has required demonstration of the absence of vascular leak activity.•A new in vitro assay detects RTA-induced endothelial toxicity at 2, 4 and 24 h.•RTA effects on vascular leak were consistent, and time and concentration dependent.