RTA-specific monoclonal antibodies neutralize ricin toxin intracellularly

Antibodies are extraordinary in their capacity to neutralize even the most potent toxins, including botulinum, anthrax, tentanus and ricin toxins. It is generally assumed that antibodies inactivate toxins by either preventing their ability to bind receptors on host cells or by promoting their clearance from circulation via Fc-receptor mediated uptake. In this study, we provide evidence that challenges this paradigm. Although ricin toxin uses its B-subunit (RTB) to bind to cells, the majority of antibodies elicited in response to ricin target its enzymatic A-subunit (RTA). We have recently produced a collection of ricin neutralizing monoclonal antibodies (mAbs) directed against RTA. Using two neutralizing anti-RTA mAbs called R70 and IB2 we showed that neither of them prevented ricin binding to cell surfaces, as measured by flow cytometry. R70 retained toxin neutralizing activity in a Vero cell cytotoxicity assay when added after ricin had attached to cell surfaces. Colocalization of both mAbs with ricin was detected intracellularly in Vero cells by confocal microscopy. Interestingly, in vitro assays that mimic specific steps in the intracellular trafficking of ricin demonstrated that IB2 affected the ability of protein disulfide isomerase to reduce the disulphide bond linking RTA to RTB. Furthermore IB2 interfered with RTA's capacity to arrest protein synthesis. In conclusion these results suggest that RTA-specific mAbs neutralize ricin intracellularly. These findings will have implications for toxin vaccine design as well as contribute to our fundamental understanding of how antibodies neutralize ricin as well as other medically relevant AB toxins.