A Biparatopic Intrabody Renders Vero Cells Impervious to Ricin Intoxication

Abstract

Expression of camelid-derived, single-domain antibodies (V_HHs) within the cytoplasm of mammalian cells as "intrabodies" has opened-up novel avenues for medical countermeasures against fast-acting biothreat agents. In this report, we describe a heterodimeric intrabody that renders Vero cells virtually impervious to ricin toxin (RT), a potent Category B ribosome-inactivating protein (RIP). The intrabody consists of two structurally defined V_HHs that target distinct epitopes on RT's enzymatic subunit (RTA): V9E1 targets RTA's P-stalk recruitment site, and V2A11 targets RTA's active site. Resistance to RT conferred by the biparatopic V_HH construct far exceeded that of either of the V_HHs alone and effectively inhibited all measurable RT-induced cytotoxicty in vitro. We propose that targeted delivery of bispecific intrabodies to lung tissues may represent a novel means to shield the airways from the effects of inhalational RT exposure.

Keywords: antibody; biodefense; intrabody; intracellular; neutralization; ribosome; single domain antibody; toxin.

Figure 1.. Intracellular neutralization of ricin by co-expressed neutralizing intrabodies.

V2A11 (green; top left), V9E1 (orange; top right), or a combination of both intrabodies (brown; bottom left) were transfected into Vero cells and treated with a range of ricin concentrations one day later. A superposition of all three is shown bottom right. Cell viabilities were analyzed as a percentage of control live cells (not treated with ricin) two days after intoxication, and compared to cell transfected with vehicle LNP controls (white).

Figure 2.. Intracellular expression of V2A11-V9E1 potently protects Vero cells from ricin intoxication.

Vero cells were transiently transfected with either V2A11 (green; top left), V9E1 (orange; top right), a cocktail of V2A11 and V9E1 (brown; bottom left), or the V2A11-V9E1 heterodimer (black; bottom right). One day after transfection, cells were treated with a range of ricin concentrations and analyzed for viability two days after ricin treatment. Cell viabilities were normalized to “live” control cells for each transfection condition, not treated with ricin, and compared to cells transfected with vehicle LNP controls (white).

Figure 3.. Vero cells stably expressing V2A11-V9E1 are extremely resistant to ricin.

Transfected cells were seeded in 96-well plates without selection media and treated with a range of ricin concentrations one day later in triplicate. Cell viabilities were determined two days following ricin treatment, normalized to “live” control cells for each intrabody condition. Un-transfected control Vero cells were grown in DMEM without G418 and tested simultaneously to generate standard ricin dose response curves. Cells were tested in cytotoxicity assays for ricin resistance at A) 4 weeks post-transfection, B) 11 weeks post-transfection, and C) 14- to 30-weeks post-transfection. Throughout the 14–30 weeks post-transfection, cells were continuously subjected to cytotoxicity assays as described above and results averaged. Significance for the 14- to 30-week assays was determined by two-way ANOVA with Sidak post-hoc test at each RTA concentration tested. Black asterisks represent significant differences between V2A11-V9E1-transfected and control Vero cells. Orange asterisks represent significant differences between V9E1 transfected and control cells. **** p<0.0001; *** p<0.001; ** p<0.005; * p<0.05.