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

doi:10.1128/cvi.00236-17

Cited by 35 publications

(77 citation statements)

References 52 publications

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“…To further examine the combinatorial effects of antibodies on ricin toxin neutralizing activity in the LSEC cytotoxicity assay, we turned to a collection of alpaca‐derived single domain antibodies (V H Hs) . V H Hs differ from conventional IgG MAbs in that they are monovalent and are devoid of Fc elements, thereby eliminating any contribution of aggregation and FcR involvement in the cytotoxicity assay.…”

Section: Resultsmentioning

confidence: 99%

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

Mooney

Torres-Vélez

Doering

et al. 2019

Journal of Leukocyte Biology

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Ricin toxin is a plant‐derived, ribosome‐inactivating protein that is rapidly cleared from circulation by Kupffer cells (KCs) and liver sinusoidal endothelial cells (LSECs)—with fatal consequences. Rather than being inactivated, ricin evades normal degradative pathways and kills both KCs and LSECs with remarkable efficiency. Uptake of ricin by these 2 specialized cell types in the liver occurs by 2 parallel routes: a “lactose‐sensitive” pathway mediated by ricin's galactose/N‐acetylgalactosamine‐specific lectin subunit (RTB), and a “mannose‐sensitive” pathway mediated by the mannose receptor (MR; CD206) or other C‐type lectins capable of recognizing the mannose‐side chains displayed on ricin's A (RTA) and B subunits. In this report, we investigated the capacity of a collection of ricin‐specific mouse MAb and camelid single‐domain (VHH) antibodies to protect KCs and LSECs from ricin‐induced killing. In the case of KCs, individual MAbs against RTA or RTB afforded near complete protection against ricin in ex vivo and in vivo challenge studies. In contrast, individual MAbs or VHHs afforded little (<40%) or even no protection to LSECs against ricin‐induced death. Complete protection of LSECs was only achieved with MAb or VHH cocktails, with the most effective mixtures targeting RTA and RTB simultaneously. Although the exact mechanisms of protection of LSECs remain unknown, evidence indicates that the Ab cocktails exert their effects on the mannose‐sensitive uptake pathway without the need for Fcγ receptor involvement. In addition to advancing our understanding of how toxins and small immune complexes are processed by KCs and LSECs, our study has important implications for the development of Ab‐based therapies designed to prevent or treat ricin exposure should the toxin be weaponized.

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Section: Resultsmentioning

confidence: 99%

“…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) …”

Section: Methodsmentioning

confidence: 99%

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

Mooney

Torres-Vélez

Doering

et al. 2019

Journal of Leukocyte Biology

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“…The nine toxin-neutralizing MAbs that were subjected to epitope mapping in this study (Table 1) were used in a study, reported in a companion paper, of competition ELISAs for the purpose of localizing the binding sites of a large collection of V H Hs (71). As part of that study, we reported the full-length DNA sequences, binding affinities, and neutralizing activities of 31 V H Hs against RTA, 33 against RTB, and 4 against ricin holotoxin.…”

Section: Discussionmentioning

confidence: 99%

“…In a recent study, we successfully adapted HX-MS for the purpose of identifying epitopes recognized by a family of RTA-specific single-domain alpaca-derived antibodies (V H Hs) (51). We also used HX-MS to assist in epitope refinement of a subset of the 68 V H Hs described in a companion paper (71). We now report the contact points on RiVax recognized by 10 MAbs in epitope clusters I to IV.…”

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confidence: 99%

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

Toth

Angalakurthi

Slyke

et al. 2017

Clin Vaccine Immunol

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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. In this study, we employed hydrogen exchange-mass spectrometry (HX-MS) to localize the epitopes on RiVax recognized by nine toxin-neutralizing MAbs and one nonneutralizing MAb. Based on strong protection from hydrogen exchange, the nine MAbs grouped into four spatially distinct epitope clusters (namely, clusters I to IV). Cluster I MAbs protected RiVax's ␣-helix B (residues 94 to 107), a protruding immunodominant secondary structure element known to be a target of potent toxin-neutralizing antibodies. Cluster II consisted of two subclusters located on the "back side" (relative to the active site pocket) of RiVax. One subcluster involved ␣-helix A (residues 14 to 24) and ␣-helices F-G (residues 184 to 207); the other encompassed ␤-strand d (residues 62 to 69) and parts of ␣-helices D-E (154 to 164) and the intervening loop. Cluster III involved ␣-helices C and G on the front side of RiVax, while cluster IV formed a sash from the front to back of RiVax, spanning strands b, c, and d (residues 35 to 59). Having a highresolution B cell epitope map of RiVax will enable the development and optimization of competitive serum profiling assays to examine vaccine-induced antibody responses across species.KEYWORDS antibody, biodefense, epitope, mass spectrometry, toxin, vaccine R icin is one of a small group of plant and bacterial toxins that are classified at the domestic and international levels as potential agents of bioterrorism (1, 2). Ricin is a product of castor beans (Ricinus communis), which are cultivated globally for their oils that are used in industrial lubricants, cosmetics, and biofuels. The toxin itself is an ϳ65-kDa glycoprotein that makes up ϳ5% of the dry weight of a castor bean. The toxin can be purified by relatively simple affinity chromatography (3, 4). On a cellular level, ricin exerts its cytotoxic effects through ribosome inactivation and triggering of programmed cell death (5). Ricin's binding subunit, ricin toxin B subunit (RTB), is a galactose/N-acetyl galactosamine (Gal/GalNAc)-specific lectin that promotes toxin entry into mammalian cells, while ricin's enzymatic subunit, RTA, is an RNA N-glycosidase (EC 3.2.2.22) that, when successfully delivered into the cytoplasm, cleaves the sarcin-ricin

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“…Clinical and Vaccine Immunology describe the continued mapping of the neutralizing epitopes within ricin toxin (RTX) and relate these studies to current research on the botulinum neurotoxins (BoNTs) (1,2). Our goal is to provide a perspective on how protein structure has facilitated development of vaccines and therapies against regulated select agent (SA) toxins.…”

Section: T His Commentary Addresses Studies By Mantis and Coworkers mentioning

confidence: 99%

Protein Structure Facilitates High-Resolution Immunological Mapping

Zuverink

Barbieri

2017

Clin Vaccine Immunol

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Select agents (SA) pose unique challenges for licensing vaccines and therapies. In the case of toxin-mediated diseases, HHS assigns guidelines for SA use, oversees vaccine and therapy development, and approves animal models and approaches to identify mechanisms for toxin neutralization. In this commentary, we discuss next-generation vaccines and therapies against ricin toxin and botulinum toxin, which are regulated SA toxins that utilize structure-based approaches for countermeasures to guide rapid response to future biothreats.KEYWORDS botulinum toxin, RTA, RVEc, RiVax, antibodies, mass spectrometry, ricin T his commentary addresses studies by Mantis and coworkers, who in this issue of Clinical and Vaccine Immunology describe the continued mapping of the neutralizing epitopes within ricin toxin (RTX) and relate these studies to current research on the botulinum neurotoxins (BoNTs) (1, 2). Our goal is to provide a perspective on how protein structure has facilitated development of vaccines and therapies against regulated select agent (SA) toxins. An overview of the properties of ricin toxin and botulinum neurotoxin is presented in Fig. 1. Ricin toxin and the botulinum neurotoxins are HHS and USDA Select Agents and Toxins (7 CFR part 331, 9 CFR part 121, and 42 CFR part 73).AB toxin structure/function. AB toxins contain modular domains with distinct functional activities (3). The A domain encodes an enzymatic activity which targets a host protein to modulate host cell physiology, often inactivating a biological process within cells, leading to cell death or occasionally enhancing the action of a cellular process. The B domain often binds a host receptor, such as a protein or glycolipid, via interactions that bind directly to a specific host receptor or that bind a carbohydrate present on a glycosylated host receptor. AB toxins are often synthesized as single-chain proteins in an inactive form and are converted to an active di-chain by proteolysis between the A and B domains, which remain connected by an interchain disulfide (4, 5).RTX is a category B bioterrorism toxigenic lectin enriched in seeds of the castor bean plant Ricinus communis. RTX is posttranslationally processed by host glycosylation and activated by cleavage between A and B domains (6, 7). The ricin toxin A domain (RTA) is a type II ribosome-inactivating protein with N-glycosidase activity, which depurinates 28S rRNA within the 60S host ribosome (8). Depurination stalls protein synthesis and induces a ribotoxic stress response (9). Ricin toxin B domain (RTB) contains at least two homologous lectin binding sites specific for galactose and binds galactose-containing lipids and glycoproteins (10). While binding of galactose results in nonproductive uptake of RTX, binding to mannose moieties on host mannose-containing receptors results in RTX retrograde trafficking to the endoplasmic reticulum, where RTA is delivered into the cytosol to target the ribosome (11). RTX exploits receptors on multiple cell types; therefore, the symptoms vary based ...

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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

Cited by 35 publications

References 52 publications

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

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

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

Protein Structure Facilitates High-Resolution Immunological Mapping

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