Monoclonal antibodies against the ricin toxin B subunit: Evidence for extracellular and intracellular mechanisms of toxin neutralization. (53.2)

Abstract

Abstract Antibodies are remarkable in their capacity to inactivate even the most potent protein toxins, including anthrax, botulinum, diphtheria, and ricin. However, the underlying mechanisms by which this occurs are poorly understood. In this study, we have produced and characterized a collection of monoclonal antibodies (mAbs) directed against the B subunit (RTB) of ricin toxin. RTB plays two roles in ricin cytotoxicity; it promotes toxin attachment to host cell surfaces, and it mediates the intracellular trafficking of the toxin from the plasma membrane to the endoplasmic reticulum (ER). We screened ~2,000 RTB-specific murine B cell hybridomas, and identified 4 mAbs with toxin neutralizing activity. Two of these mAbs, SylH3 and 24B11, passively protected mice against a 5xLD50 dose of ricin, but only partially prevented ricin binding to cell surfaces, indicating that they must neutralize ricin by a mechanism other than simply interfering with receptor attachment. Using a modified Vero cell cytotoxicity assay we found that both SylH3 and 24B11 were able to completely neutralize ricin in vitro even after the toxin had bound to cell surfaces. These data reveal that SylH3 and 24B11 neutralize ricin downstream of attachment, perhaps by interfering with the ability of the toxin to traffic to the ER. We are currently using biochemical and microscopic techniques to localize where in the cell these mAbs interfere with ricin toxicity.