Sites of vulnerability on ricin B chain revealed through epitope mapping of toxin-neutralizing monoclonal antibodies

Abstract

Ricin toxin’s B subunit (RTB) is a multifunctional galactose (Gal)-/N-acetylgalactosamine (GalNac)-specific lectin that promotes uptake and intracellular trafficking of ricin’s ribosome-inactivating subunit (RTA) into mammalian cells. Structurally, RTB consists of two globular domains (RTB-D1, RTB-D2), each divided into three homologous sub-domains (α, β, γ). The two carbohydrate recognition domains (CRDs) are situated on opposite sides of RTB (sub-domains 1α and 2γ) and function non-cooperatively. Previous studies have revealed two distinct classes of toxin-neutralizing, anti-RTB monoclonal antibodies (mAbs). Type I mAbs, exemplified by SylH3, inhibit (~90%) toxin attachment to cell surfaces, while type II mAbs, epitomized by 24B11, interfere with intracellular toxin transport between the plasma membrane and the trans-Golgi network (TGN). Localizing the epitopes recognized by these two classes of mAbs has proven difficult, in part because of RTB’s duplicative structure. To circumvent this problem, RTB-D1 and RTB-D2 were expressed as pIII fusion proteins on the surface of filamentous phage M13 and subsequently used as “bait” in mAb capture assays. We found that SylH3 captured RTB-D1 (but not RTB-D2) in a dose-dependent manner, while 24B11 captured RTB-D2 (but not RTB-D1) in a dose-dependent manner. We confirmed these domain assignments by competition studies with an additional 8 RTB-specific mAbs along with a dozen a single chain antibodies (VHHs). Collectively, these results demonstrate that type I and type II mAbs segregate on the basis of domain specificity and suggest that RTB’s two domains may contribute to distinct steps in the intoxication pathway.

Structural Basis of Antibody-Mediated Inhibition of Ricin Toxin Attachment to Host Cells

“…6,12,33 Furthermore, evidence suggests that SylH3's epitope is localized in or in proximity to subdomain 1β, not subdomains 1α or 2γ. 23 With that in mind, it is possible that SylH3 has allosteric effects on RTB similar to those reported by mAbs against the bacterial mannose-specific adhesin, FimH. 34 We therefore embarked on a study to define the molecular interactions between RT and SylH3 and JB4.…”

“…23 Rather, SylH3 and JB4 were assigned to epitope cluster 5, which is situated in or immediately adjacent to subdomain 1β. 23 To elucidate SylH3 and JB4's epitopes at high resolution, we determined the X-ray crystal structures of SylH3 and JB4 Fab fragments in complex with RT at 2.9 and 3.3 Å, respectively (Figure 1; Table S1). The structures revealed that SylH3 and JB4 Fabs interact primarily with subdomain 1β in a nearly identical fashion.…”

“…Several lines of evidence, including competitive ELISAs with a panel of RTB-specific V H H's, indicate that SylH3 and JB4 recognize epitopes outside of RTB subdomains 1α and 2γ that are principally associated with Gal/GalNAc recognition. 23 Rather, SylH3 and JB4 were assigned to epitope cluster 5, which is situated in or immediately adjacent to subdomain 1β. 23 To elucidate SylH3 and JB4's epitopes at high resolution, we determined the X-ray crystal structures of SylH3 and JB4 Fab fragments in complex with RT at 2.9 and 3.3 Å, respectively (Figure 1; Table S1).…”

“…As a category B biothreat with the potential of being disseminated via aerosol, there is considerable interest in identifying antidotes to RT, including small molecule inhibitors and toxin-neutralizing monoclonal antibodies (MAbs). , Toward this end, we have identified two MAbs, PB10 and SylH3, that when combined protect mice against the deleterious effects of intranasal RT exposure. , PB10 is directed against an immunodominant epitope on RTA and interferes with retrograde transport of RT. − SylH3 is directed against RTB and blocks RT attachment to cell surfaces. − The combination of the two MAbs, one that works outside the cell and one that works inside, affords a degree of neutralizing activity not observed with other mAb combinations we have tested . Moreover, both PB10 and SylH3 retain full toxin neutralizing activity as Fab fragments, demonstrating that epitope specificity rather than Fc-mediated effector functions or toxin cross-linking is critical.…”

“…For many toxins such as Shiga, anthrax, and Clostridium difficile B (TcdB), inhibition of attachment to host cell receptors involves antibody occupancy or occlusion of receptor binding pockets. − This is unlikely to be the case with SylH3, as RTB’s two principal carbohydrate recognition domains (CRDs) are antigenically distinct and spaced too far apart (∼50 Å) to be obstructed by a single mAb or Fab. ,, Furthermore, evidence suggests that SylH3’s epitope is localized in or in proximity to subdomain 1β, not subdomains 1α or 2γ . With that in mind, it is possible that SylH3 has allosteric effects on RTB similar to those reported by mAbs against the bacterial mannose-specific adhesin, FimH …”