Neutralizing Antibodies Against the Ricin Toxin Binding Subunit (RTB).

摘要

Ricin is a toxin that is naturally produced by the seeds of the castor bean plant Ricinus communis, and is part of a family of A-B toxins that includes Shiga, cholera, and anthrax toxins. The toxin consists of two subunits, RTA and RTB, which are linked by a disulfide bond. RTA is an RNA N-glycosidase that selectively targets and inactivates 28S ribosomal RNA, thereby arresting protein synthesis and leading to cell death. RTB is a galactose/ N-acetylgalactosamine-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Currently, there is no approved vaccine or therapeutics available against this Category B select agent and the details of toxin neutralization by monoclonal antibodies (mAbs) are still elusive.;In an effort to identify which regions on RTB are responsible for eliciting protective immunity to ricin, I generated and characterized a collection of RTB-specific murine mAbs. These mAbs were characterized based on their affinity for ricin and ability to neutralize ricin both in vitro and in vivo. I then identified their target epitope on RTB. I determined that the majority of RTB-specific mAbs are non-neutralizing and target the internal sub-domains 1β, 1γ, 2α and 2β of RTB. In contrast, neutralizing mAbs are relatively rare (<1% of total RTB-specific mAbs) and target the galactose binding sub-domains 1α and 2γ. These results suggest that there are a limited number of neutralizing epitopes present on RTB.;Furthermore, investigation of RTB-mAb interactions in vitro revealed that there are two classes of neutralizing RTB-specific mAbs; (i) ones that block ricin attachment to cell surface receptors (i.e mAb SylH3), and a novel class (ii) that neutralizes ricin intracellularly, but does not block attachment (i.e. mAb 24B11). I determined that 24B11 neutralized ricin intracellularly, by interfering with retrograde transport of ricin to the TGN. Finally, I investigated the mechanisms of ricin neutralization by mAbs in vivo, demonstrating that Fab fragments from SylH3 and 24B11 were sufficient to protect mice against a lethal systemic ricin challenge. These results suggest that neutralization of ricin in vivo, occurs independently of Fc Receptor-mediated clearance.