Background:We sought to engineer highly efficacious agents that neutralize ricin toxin. Results: We identified monomeric single-chain camelid V H domains (V H Hs) capable of neutralizing ricin in vitro and engineered heterodimeric V H Hs that neutralized ricin in vivo. Conclusion: Stepwise engineering of V H Hs resulted in highly potent ricin toxin-neutralizing antibodies. Significance: This study highlights the potential use of a V H H platform as a strategy for therapeutics against diverse biological toxins.

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

The B subunit (RTB) of ricin toxin is a galactose (Gal)−/N-acetylgalactosamine (GalNac)-specific lectin that mediates attachment, entry, and intracellular trafficking of ricin in host cells. Structurally, RTB consists of two globular domains with identical folding topologies. Domains 1 and 2 are each comprised of three homologous sub-domains (α, β, γ) that likely arose by gene duplication from a primordial carbohydrate recognition domain (CRD), although only sub-domains 1α and 2γ retain functional lectin activity. As part of our ongoing effort to generate a comprehensive B cell epitope map of ricin, we report the characterization of three new RTB-specific monoclonal antibodies (mAbs). All three mAbs, JB4, B/J F9 and C/M A2, were initially identified based on their abilities to neutralize ricin in a Vero cell cytotoxicty assay and to partially (or completely) block ricin attachment to cell surfaces. However, only JB4 proved capable of neutralizing ricin in a macrophage apoptosis assay and in imparting passive immunity to mice in a model of systemic intoxication. Using a combination of techniques, including competitive ELISAs, pepscan analysis, differential reactivity by Western blot, as well as affinity enrichment of phage displayed peptides, we tentatively localized the epitopes recognized by the non-neutralizing mAbs B/J F9 and C/M A2 to sub-domains 2α and 2β, respectively. Furthermore, we propose that the epitope recognized by JB4 is within sub-domain 2γ, adjacent to RTB’s high affinity Gal/GalNAc CRD. These data suggest that recognition of RTB’s sub-domains 1α and 2γ are critical determinants of antibody neutralizing activity and protective immunity to ricin.

The simultaneous binding of multiple ligands on one entity to multiple receptors on another can result in an affinity that is significantly greater than that for the binding of a single ligand to a single receptor. This concept of ''polyvalency'' can be used to design molecules that are potent inhibitors of toxins and pathogens. We describe the design of potent polyvalent inhibitors that neutralize anthrax toxin in vivo as well as our attempts to elucidate the relationship between inhibitor structure and activity. We also highlight promising future avenues for research in polyvalent drug design.

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

Ricin is a Select Agent Toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins (RIPs). In this study, we determined x-ray crystal structures of the enzymatic subunit of ricin (RTA) in complex with the antigen binding domains (VHH) of five unique single-chain monoclonal antibodies that differ in their respective toxin-neutralizing activities. None of the VHHs made direct contact with residues involved in RTA’s RNA N-glycosidase activity or induced notable allosteric changes in the toxin’s subunit. Rather, the five VHHs had overlapping structural epitopes on the surface of the toxin and differed in the degree to which they made contact with prominent structural elements in two folding domains of the RTA. In general, RTA interactions were influenced most by the VHH CDR3 elements, with the most potent neutralizing antibody having the shortest and most conformationally constrained CDR3. These structures provide unique insights into the mechanisms underlying toxin neutralization and provide critically important information required for the rational design of ricin toxin subunit vaccines.

Residues Y91-T116 of ricin toxin’s enzymatic subunit (RTA) constitute an immunodominant loop-helix-loop motif that is the target of two potent toxin neutralizing monoclonal antibodies (mAbs), PB10 and R70. To define the exact epitope(s) recognized by these mAbs, we affinity enriched from a phage-displayed peptide library 12 mers that bound one or both of these mAbs. We report that PB10 recognizes a distinct but overlapping epitope with R70, in which residues (Q98), E102, T105, and H106 are central to mAb recognition.

Ricin toxin’s binding subunit (RTB) is a galactose-/N-acetylgalactosamine (Gal/GalNac)-specific lectin that mediates uptake and intracellular trafficking of ricin within mammalian cells. Structurally, RTB consists of two globular domains, each divided into three homologous sub-domains (α, β, γ). In this report, we describe five new murine IgG monoclonal antibodies (mAbs) against RTB: MH3, 8A1, 8B3, LF1, and LC5. The mAbs have similar binding affinities (KD) for ricin holotoxin, but displayed a wide range of in vitro toxin-neutralizing activities. Competition ELISAs indicate that the two most potent toxin-neutralizing mAbs (MH3, 8A1), as well as one of the moderate toxin-neutralizing mAbs (LF1), recognize distinct epitopes near the low affinity Gal recognition domain in RTB subdomain 1α. Evaluated in a mouse model of systemic ricin challenge, all five mAbs afforded some benefit against intoxication, but only MH3 was protective. However, neither MH3 nor 24B11, another well-characterized mAb against RTB subdomain 1α, could passively protect mice against a mucosal (intranasal) ricin challenge. This is in contrast to SylH3, a previously characterized mAb directed against an epitope near RTB’s high affinity Gal/GalNac recognition element in sub-domain 2γ, which protected animals against systemic and mucosal ricin exposure. SylH3 was significantly more effective than MH3 and 24B11 at blocking ricin attachment to host cell receptors, suggesting that mucosal immunity to ricin is best imparted by antibodies that target RTB’s high affinity Gal/GalNac recognition element in subdomain 2γ, not the low affinity Gal recognition domain in subdomain 1α.