Spatial location of neutralizing and non-neutralizing B cell epitopes on domain 1 of ricin toxin’s binding subunit

Abstract

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.

Sensitivity of Kupffer cells and liver sinusoidal endothelial cells to ricin toxin and ricin toxin–Ab complexes

“…We next examined the capacity of ricin‐specific MAbs to protect KCs against ricin toxicity ex vivo. We have previously described a collection anti‐RTA and anti‐RTB MAbs with a range of neutralizing activities (IC 50 ) on Vero cells . The anti‐RTA MAbs are directed against 4 spatially distinct epitope clusters (I–IV).…”

“…The results of the ex vivo studies prompted us to examine the degree to which individual anti‐ricin MAbs are able to protect KCs in vivo. We chose PB10 (anti‐RTA) and SylH3 (anti‐RTB) for these studies as they represent 2 of our most potent classes of toxin‐neutralizing MAbs . Ricin was injected into mice by the i.p.…”

“…The MAbs were purified from hybridoma supernatants by Protein A chromatography at the Dana Farber Cancer Institute (DFCI) Monoclonal Antibody Core facility (Boston, MA) . The murine MAbs against RTA (SyH7, PA1, IB2, WECB2, JD4, and PB10) and RTB (MH3, SylH3, LC5, 24B11, 8B3, and 8A1) have been previously described . The V H Hs against RTA (JIVF5, V5E1, V1D3, and V13G5) and RTB (JIZB7, V5D1) have also been described, except for V11E10 (manuscript in preparation) …”

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

“…V5B1 was also unable to recognize R-ASF, presumably because its epitope is near the domain 1 Gal-specific binding element (5). We recently identified three additional MAbs specific for RTB's domain 1, although they were not included in this study (37).…”

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

“…Neutralizing antibodies are relatively rare, estimated at ϳ10% of the total ricin-specific pool. Complicating matters is the fact that there is often a disconnect between in vitro toxin-neutralizing activity and the capacity of a given MAb to neutralize ricin in an animal model (28,33). In other words, MAbs that have potent toxin-neutralizing activity in a cell-based assay may or may not passively protect mice from ricin intoxication.…”