Evaluation of a Ricin Vaccine Candidate for Human Toxicity Using an In Vitro Vascular Leak Model

摘要

Inhaling lethal doses of ricin (RT) causes pulmonary edema and results in death. In lower concentrations, ricin minimally causes tissue damage to the respiratory tract and lungs, which can incapacitate the individuals exposed to this toxin. A genetically derived ricin A chain vaccine candidate (RTA 33/198) is available that lacks the cytotoxic N-glycosidase activity. RTA 33/198 was previously shown to be protective in animal studies against an aerosolized RT challenge. The current study evaluated if this ricin vaccine is safe or use in humans by using an in vitro vascular leak assay. Incubating human endothelial cells with the vaccine at concentrations ranging from 0.6 to 9 micrometers had no considerable toxic effects in measures of transendothelial electrical resistance for up to 24 hr. Cytotoxicity due to exposure of 9 microns ricin A chain in culture was apparent by changes in electrical resistance as early as 2 hr. Electron micrographs of endothelial cells exposed for 6 hr to 9 microns of the vaccine had reduced cytoxicity when compared to cells treated with the ricin holotoxin. Because the binding of ricin to the endothelial cell surface may suggest the occurrence of vascular leak, we performed flow cytometry of the endothelial cells exposed to either RTA, RTA 33/198 vaccine candidate, or the ricin holotoxin, to detect binding of the toxin to the cell. Our results indicated significant binding of the ricin holotoxin, and possibly a slight binding of ricin A chain. However, there was no binding by the ricin vaccine RTA 33/198. This human vascular leak model provides an adequate in vitro testing system to evaluate toxicities intrinsic to vaccines and can even be used for pre-testing therapeutics to biowarfare agents, reducing the need of animals in scientific research. In this specific study, the model successfully demonstrated the reduced toxicity of the lead ricin vaccine when compared to the toxicity caused by holotoxin and to ricin A chain.