Neutralizing Monoclonal Antibodies against Disparate Epitopes on Ricin Toxin’s Enzymatic Subunit Interfere with Intracellular Toxin Transport

Abstract

Ricin is a member of the A-B family of bacterial and plant toxins that exploit retrograde trafficking to the Golgi apparatus and endoplasmic reticulum (ER) as a means to deliver their cytotoxic enzymatic subunits into the cytoplasm of mammalian cells. In this study we demonstrate that R70 and SyH7, two well-characterized monoclonal antibodies (mAbs) directed against distinct epitopes on the surface of ricin’s enzymatic subunit (RTA), interfere with toxin transport from the plasma membrane to the trans Golgi network. Toxin-mAb complexes formed on the cell surface delayed ricin’s egress from EEA-1+ and Rab7+ vesicles and enhanced toxin accumulation in LAMP-1+ vesicles, suggesting the complexes were destined for degradation in lysosomes. Three other RTA-specific neutralizing mAbs against different epitopes were similar to R70 and SyH7 in terms of their effects on ricin retrograde transport. We conclude that interference with toxin retrograde transport may be a hallmark of toxin-neutralizing antibodies directed against disparate epitopes on RTA.

High-Resolution Epitope Positioning of a Large Collection of Neutralizing and Nonneutralizing Single-Domain Antibodies on the Enzymatic and Binding Subunits of Ricin Toxin

“…Unfortunately, our current report does not address the mechanism(s) by which the supercluster II antibodies neutralize ricin. We can only speculate that they interfere with a region of the toxin that is required for retrograde transport from the plasma membrane to the ER and/or retrotranslocation from the ER to the cytoplasm (45)(46)(47)(48).…”

“…The second hot spot (as noted above) is located at the RTA-RTB interface, defined by RTA's ␣-helix F and the F-G loop and (possibly) RTB's subdomains 2␣ and/or 2␥ (16). At present, it is not clear why these regions render ricin vulnerable to antibody attack, although evidence is mounting that neutralization occurs intracellularly rather than extracellularly (13,(45)(46)(47).…”

High-Definition Mapping of Four Spatially Distinct Neutralizing Epitope Clusters on RiVax, a Candidate Ricin Toxin Subunit Vaccine

“…Legler and colleagues recently demonstrated, using V H Hs, a correlation between an antibody's impact on RTA unfolding dynamics and in vitro toxin-neutralizing activity, possibly due to events associated with ricin endocytosis or retrograde transport (39). Other reports have shown that anti-RTA MAbs reduced intracellular transport of ricin from the plasma membrane to the endoplasmic reticulum (ER) and even prevented protein-disulfide isomerase (PDI)-mediated dissociation of RTA from RTB in a cell-free assay (62)(63)(64). Whether these in vitro activities are relevant in vivo The strongly protected regions for each cluster are summed over all MAbs belonging to that cluster and shown mapped to the crystal structure of the ricin holotoxin (PDB ID: 2AAI) (14), with each cluster colored as indicated.…”

Toxicity of ricin A chain is reduced in mammalian cells by inhibiting its interaction with the ribosome

“…While a number of these have been shown to partially rescue mice from ricin intoxication (Hu et al , 2012; O’Hara et al , 2012; Sully et al , 2014), none have been tested in Phase I clinical trials to date. Several neutralizing antibodies have been shown to act by targeting ricin uptake and trafficking (Herrera et al , 2016; Yermakova et al , 2016). Interestingly, the ricin-neutralizing antibody 6C2 recognizes an epitope distinct from the catalytic active site of RTA and is more potent in its neutralizing ability relative to antibodies that bind the active site (Dai et al , 2011).…”