The Need for Continued Development of Ricin Countermeasures

Reisler, Ronald B.; Smith, Leonard A.

doi:10.1155/2012/149737

Cited by 44 publications

(44 citation statements)

References 27 publications

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“…Once within the ER, the disulfide bond that links RTA to RTB is reduced by protein disulfide isomerase and RTA is retrotranslocated (dislocated) into the cell cytoplasm, where it triggers programmed cell death (7,8). Ricin is classified as a category B biothreat agent by the Centers for Disease Control and Prevention (CDC) because of its potential, when delivered via aerosol, to induce severe lung inflammation and tissue damage (9,10). Concerns over ricin's possible use as a biothreat agent stem from the fact that the toxin is relatively easy to procure from its natural source, castor beans (Ricinus communis), as well as from its extreme toxicity following inhalation or injection (11).…”

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confidence: 99%

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

Vance

Tremblay

Rong

et al. 2017

Clin Vaccine Immunol

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We previously produced a heavy-chain-only antibody (Ab) VH domain (V H H)-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 RTAand RTB-specific V H Hs, 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 H Hdisplayed 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 H Hs: 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 H Hs grouped into more than 20 different competition bins. The RTA-specific V H Hs 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 H Hs 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 R icin is the prototype of the type II ribosome-inactivating protein (RIP) family of toxins (1). Ricin toxin A subunit (RTA) is an RNA N-glycosidase that catalyzes the hydrolysis of a conserved adenine residue within the sarcin/ricin loop (SRL) of 28S rRNA, resulting in ribosome arrest and apoptosis (2-4). Ricin toxin B subunit (RTB) is a galactose-and N-acetylgalactosamine (Gal/GalNAc)-specific lectin that promotes toxin entry into mammalian cells, including the epithelial cells that line the respiratory tract

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

Vance

Tremblay

Rong

et al. 2017

Clin Vaccine Immunol

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“…There remains a large demand in the public health community and military sectors for the development of prophylactics and therapeutics capable of neutralizing fast-acting plant and microbial toxins, especially those like botulinum neurotoxins, anthrax, and ricin toxin that have the potential to be used as biothreat agents (1)(2)(3)(4). Although a number of promising toxinneutralizing murine and humanized monoclonal antibodies (mAbs) are in the pipeline, the emergence of technologies surrounding the generation and expression of toxin-specific camelid heavy chain-only VH domains (VHHs) 3 has opened up new avenues for the rational design and delivery of antitoxin agents (5).…”

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confidence: 99%

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies

Herrera

Tremblay

Shoemaker

et al. 2015

Journal of Biological Chemistry

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Background: Bispecific heterodimeric camelid heavy chain-only antibodies (VHHs) have been shown to neutralize ricin toxin in vivo. Results: Heterodimeric VHHs promote ricin aggregation in solution and block toxin attachment to cell surfaces. Conclusion:The bispecific nature of the heterodimeric VHHs may be important for toxin neutralization in vivo. Significance: Understanding mechanisms of toxin neutralization will lead to the development of new antitoxin agents.

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“…Departments of Defense (DOD) and Health and Human Services (HHS) have ongoing initiatives to develop antibody-based products capable of providing passive protection against systemic and mucosal ricin exposure (16,17). In addition to protective efficacy, issues related to platform technology, scalability, and speed of manufacturing will ultimately dictate which product(s) will be pursued for advanced development and are consistent with the unique needs for biodefense.…”

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confidence: 99%

Chimeric Plantibody Passively Protects Mice against Aerosolized Ricin Challenge

Sully

Whaley

Bohorova

et al. 2014

Clin. Vaccine Immunol.

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e Recent incidents in the United States and abroad have heightened concerns about the use of ricin toxin as a bioterrorism agent. In this study, we produced, using a robust plant-based platform, four chimeric toxin-neutralizing monoclonal antibodies that were then evaluated for the ability to passively protect mice from a lethal-dose ricin challenge. The most effective antibody, c-PB10, was further evaluated in mice as a therapeutic following ricin exposure by injection and inhalation. Ricin toxin is a member of the medically important A-B family of plant and bacterial ribosome-inactivating proteins (RIPs) (1). In its mature form, ricin is a 65-kDa heterodimeric glycoprotein that is a natural constituent of the seeds of the castor plant (Ricinus communis). The 267-amino-acid A subunit (RTA) of ricin is an RNA N-glycosidase that depurinates a conserved adenosine residue within the so-called sarcin-ricin loop (SRL) of eukaryotic 28S rRNA, which is required for activation of the elongation factor EF-Tu (2, 3). RTA is linked via a single disulfide bond to ricin's B subunit (RTB), a 262-amino-acid lectin that is specific for glycoproteins and glycolipids terminating in galactose and Nacetylgalactosamine (Gal/GalNAc). In addition to its role in attachment, RTB also mediates the retrograde transport of ricin to the trans-Golgi network (TGN) and endoplasmic reticulum (ER), where RTA is ultimately delivered across the ER membrane and into the cytoplasm (4, 5). The extraordinary capacity of RTA to inactivate ribosomes (k cat , 1,500/min) makes ricin one of the most potent known RIPs (6, 7). For example, in rodents and nonhuman primates, the 50% lethal dose (LD 50 ) of ricin by injection is approximately 5 g/kg, while the LD 50 of ricin by inhalation is estimated to be as low as 3 g/kg (8). The respiratory mucosa is especially sensitive to ricin, as even trace amounts of toxin are known to elicit widespread necrosis in the airways and alveoli, peribronchovascular edema, mixed inflammatory cell infiltrates, and massive pulmonary alveolar flooding (9-15).The U.S. Departments of Defense (DOD) and Health and Human Services (HHS) have ongoing initiatives to develop antibody-based products capable of providing passive protection against systemic and mucosal ricin exposure (16,17). In addition to protective efficacy, issues related to platform technology, scalability, and speed of manufacturing will ultimately dictate which product(s) will be pursued for advanced development and are consistent with the unique needs for biodefense. In this regard, the Nicotiana benthamiana-based rapid antibody-manufacturing platform (RAMP) provides the potential for extremely fast and high-yield monoclonal antibody (MAb) production that has already proven to be applicable to biodefense (18-21). The technology entails mass infiltration of mature Nicotiana plants with an Agrobacterium tumefaciens suspension carrying T-DNA encoding viral replicons and results in high MAb recovery from original DNA constructs within days (22). The Nicotiana-based RAMP...

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The Need for Continued Development of Ricin Countermeasures

Cited by 44 publications

References 27 publications

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

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

Mechanisms of Ricin Toxin Neutralization Revealed through Engineered Homodimeric and Heterodimeric Camelid Antibodies

Chimeric Plantibody Passively Protects Mice against Aerosolized Ricin Challenge

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