Dataset Information

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

ABSTRACT: In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (VHHs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The VHHs, 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 VHHs recognized four distinct epitope subclusters (3.1-3.4). Sixteen of the 21 VHHs grouped within subcluster 3.1 and engage RTA α-helices C and G. Three VHHs grouped within subcluster 3.2, encompassing a-helices C and G, plus α-helix B. The single VHH in subcluster 3.3 engaged RTA α-helices B and G, while the epitope of the sole VHH defining subcluster 3.4 encompassed α-helices C and E, and β-strand h. Modeling these epitopes on the surface of RTA predicts that the 20 VHHs within subclusters 3.1-3.3 physically occlude RTA's active site cleft, while the single antibody in subcluster 3.4 associates on the active site's upper rim.

SUBMITTER: Angalakurthi SK

PROVIDER: S-EPMC6374049 | biostudies-literature | 2018 Dec

REPOSITORIES: biostudies-literature

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

Angalakurthi Siva Krishna SK Vance David J DJ Rong Yinghui Y Nguyen Chi My Thi CMT Rudolph Michael J MJ Volkin David D Middaugh C Russell CR Weis David D DD Mantis Nicholas J NJ

Antibodies (Basel, Switzerland) 20181217 4

In this report, we used hydrogen exchange-mass spectrometry (HX-MS) to identify the epitopes recognized by 21 single-domain camelid antibodies (V<sub>H</sub>Hs) directed against the ribosome-inactivating subunit (RTA) of ricin toxin, a biothreat agent of concern to military and public health authorities. The V<sub>H</sub>Hs, 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 (" ...[more]

PMID: 30775035

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Project description:Ricin, a member of the A-B family of ribosome-inactivating proteins, is classified as a Select Toxin by the Centers for Disease Control and Prevention because of its potential use as a biothreat agent. In an effort to engineer therapeutics for ricin, we recently produced a collection of alpaca-derived, heavy-chain only antibody VH domains (VHH or "nanobody") specific for ricin's enzymatic (RTA) and binding (RTB) subunits. We reported that one particular RTB-specific VHH, RTB-B7, when covalently linked via a peptide spacer to different RTA-specific VHHs, resulted in heterodimers like VHH D10/B7 that were capable of passively protecting mice against a lethal dose challenge with ricin. However, RTB-B7 itself, when mixed with ricin at a 1 ∶ 10 toxin:antibody ratio did not afford any protection in vivo, even though it had demonstrable toxin-neutralizing activity in vitro. To better define the specific attributes of antibodies associated with ricin neutralization in vitro and in vivo, we undertook a more thorough characterization of RTB-B7. We report that RTB-B7, even at 100-fold molar excess (toxin:antibody) was unable to alter the toxicity of ricin in a mouse model. On the other hand, in two well-established cytotoxicity assays, RTB-B7 neutralized ricin with a 50% inhibitory concentration (IC50) that was equivalent to that of 24B11, a well-characterized and potent RTB-specific murine monoclonal antibody. In fact, RTB-B7 and 24B11 were virtually identical when compared across a series of in vitro assays, including adherence to and neutralization of ricin after the toxin was pre-bound to cell surface receptors. RTB-B7 differed from both 24B11 and VHH D10/B7 in that it was relatively less effective at blocking ricin attachment to receptors on host cells and was not able to form high molecular weight toxin:antibody complexes in solution. Whether either of these activities is important in ricin toxin neutralizing activity in vivo remains to be determined.

Dataset Information

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

Publications

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

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