RTA-specific monoclonal antibodies neutralize ricin toxin intracellularly (53.13)

Abstract

Abstract According to the current paradigm, antibodies neutralize protein toxins by either preventing toxin binding to cell surfaces or promoting toxin clearance via Fc-receptor mediated uptake. In the case of the biothreat agent ricin, although this toxin uses its B-subunit (RTB) to gain cell entry the majority of antibodies are produced against its enzymatic A-subunit (RTA). We have previously demonstrated using a collection of monoclonal antibodies (mAbs) against RTA that neutralizing activity and protection in a mouse model is associated with epitope specificity, and not mAb affinity or Ig subclass. We now put forth evidence to indicate that at least two RTA-specific mAbs, R70 and IB2, neutralize ricin intracellulary. Neither R70 nor IB2 prevented ricin binding to cell surfaces, as measured by flow cytometry. Moreover, R70 (and likely IB2) retained toxin neutralizing activity in a Vero cell assay when added after ricin had attached to cell surfaces. Using in vitro assays, we also found that R70 and IB2 affected the ability of protein disulfide isomerase to reduce the single disulphide bond linking RTA to RTB and interfered with RTA’s capacity to arrest protein synthesis. We are currently using a combination of techniques to define more precisely the mechanism by which these and other RTA-specific mAbs neutralize ricin.