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

David J Vance; Jacqueline M Tremblay; Yinghui Rong; Siva Krishna Angalakurthi; David B Volkin; C Russell Middaugh; David D Weis; Charles B Shoemaker; Nicholas J Mantis

doi:10.1128/CVI.00236-17

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

Clin Vaccine Immunol. 2017 Dec 5;24(12):e00236-17. doi: 10.1128/CVI.00236-17. Print 2017 Dec.

Authors

David J Vance¹, Jacqueline M Tremblay², Yinghui Rong³, Siva Krishna Angalakurthi⁴, David B Volkin⁴, C Russell Middaugh⁴, David D Weis⁵, Charles B Shoemaker², Nicholas J Mantis^{1

6}

Affiliations

¹ Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, USA david.vance@health.ny.gov nicholas.mantis@health.ny.gov.
² Department of Infectious Disease and Global Health, Tufts Cummings School of Veterinary Medicine, North Grafton, Massachusetts, USA.
³ Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York, USA.
⁴ Department of Pharmaceutical Chemistry, Macromolecule and Vaccine Stabilization Center, University of Kansas, Lawrence, Kansas, USA.
⁵ Department of Chemistry, University of Kansas, Lawrence, Kansas, USA.
⁶ Department of Biomedical Sciences, University at Albany, SUNY, Albany, New York, USA.

Abstract

We previously produced a heavy-chain-only antibody (Ab) VH domain (V_HH)-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. M. Tremblay, N. J. Mantis, and C. B. Shoemaker, J Biol Chem 288:36538-36547, 2013, https://doi.org/10.1074/jbc.M113.519207). Initial and subsequent screens of that library by direct enzyme-linked immunosorbent assay (ELISA) yielded more than two dozen unique RTA- and RTB-specific V_HHs, including 10 whose structures were subsequently solved in complex with RTA. To generate a more complete antigenic map of ricin toxin and to define the epitopes associated with toxin-neutralizing activity, we subjected the V_HH-displayed phage library to additional "pannings" on both receptor-bound ricin and antibody-captured ricin. We now report the full-length DNA sequences, binding affinities, and neutralizing activities of 68 unique V_HHs: 31 against RTA, 33 against RTB, and 4 against ricin holotoxin. Epitope positioning was achieved through cross-competition ELISAs performed with a panel of monoclonal antibodies (MAbs) and verified, in some instances, with hydrogen-deuterium exchange mass spectrometry. The 68 V_HHs grouped into more than 20 different competition bins. The RTA-specific V_HHs with strong toxin-neutralizing activities were confined to bins that overlapped two previously identified neutralizing hot spots, termed clusters I and II. The four RTB-specific V_HHs with potent toxin-neutralizing activity grouped within three adjacent bins situated at the RTA-RTB interface near cluster II. These results provide important insights into epitope interrelationships on the surface of ricin and delineate regions of vulnerability that can be exploited for the purpose of vaccine and therapeutic development.

Keywords: antibody; biodefense; epitope; neutralizing; toxins; vaccines.

MeSH terms

Animals
Antibodies, Neutralizing / immunology*
Camelids, New World
Chemical Warfare Agents
Epitope Mapping*
Epitopes / immunology*
Protein Binding
Ricin / immunology*
Single-Domain Antibodies / immunology*

Substances

Antibodies, Neutralizing
Chemical Warfare Agents
Epitopes
Single-Domain Antibodies
Ricin

Grants and funding

HHSN272201400021C/AI/NIAID NIH HHS/United States