A Collection of Single-Domain Antibodies that Crowd Ricin Toxin's Active Site.

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHs, which derive from 11 different B-cell lineages, were binned together based on competition ELISAs with IB2, a monoclonal antibody that defines a toxin-neutralizing hotspot ("cluster 3") located in close proximity to RTA's active site. HX-MS analysis revealed that the 21 VHs recognized four distinct epitope subclusters (3.

Fine-Specificity Epitope Analysis Identifies Contact Points on Ricin Toxin Recognized by Protective Monoclonal Antibodies.

Ricin is a fast-acting protein toxin classified by the Centers for Disease Control and Prevention as a biothreat agent. In this report, we describe five new mouse mAbs directed against an immunodominant region, so-called epitope cluster II, on the surface of ricin's ribosome-inactivating enzymatic subunit A (RTA). The five mAbs were tested alongside four previously described cluster II-specific mAbs for their capacity to passively protect mice against 10× LD ricin challenge by injection.

Contribution of an unusual CDR2 element of a single domain antibody in ricin toxin binding affinity and neutralizing activity.

Ricin toxin's enzymatic subunit (RTA) has been subjected to intensive B cell epitope mapping studies using a combination of competition ELISAs, hydrogen exchange-mass spectrometry and X-ray crystallography. Those studies identified four spatially distinct clusters (I-IV) of toxin-neutralizing epitopes on the surface of RTA. Here we describe A9, a new single domain camelid antibody (VHH) that was proposed to recognize a novel epitope on RTA that straddles clusters I and III.

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

RiVax is a promising recombinant ricin toxin A subunit (RTA) vaccine antigen that has been shown to be safe and immunogenic in humans and effective at protecting rhesus macaques against lethal-dose aerosolized toxin exposure. We previously used a panel of RTA-specific monoclonal antibodies (MAbs) to demonstrate, by competition enzyme-linked immunosorbent assay (ELISA), that RiVax elicits similar serum antibody profiles in humans and macaques. However, the MAb binding sites on RiVax have yet to be defined.

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

We previously produced a heavy-chain-only antibody (Ab) VH domain (VH)-displayed phage library from two alpacas that had been immunized with ricin toxoid and nontoxic mixtures of the enzymatic ricin toxin A subunit (RTA) and binding ricin toxin B subunit (RTB) (D. J. Vance, J.

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies.

In this report we investigated, within a group of closely related single domain camelid antibodies (V Hs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast-acting toxin and biothreat agent. The V1C7-like V Hs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin-neutralizing activities. Using the X-ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta-based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer.