Structural Analysis of Toxin-Neutralizing,Single-Domain Antibodies that Bridge Ricin’s A-B Subunit Interface

Ricin toxin kills mammalian cells with notorious efficiency. The toxin’s B subunit (RTB) is a Gal/GalNAc-specific lectin that attaches to cell surfaces and promotes retrograde transport of ricin’s A subunit (RTA) to the trans Golgi network (TGN) and endoplasmic reticulum (ER). RTA is liberated from RTB in the ER and translocated into the cell cytoplasm, where it functions as a ribosome-inactivating protein. While antibodies against ricin’s individual subunits have been reported, we now describe seven alpaca-derived, single-domain antibodies (V_HHs) that span the RTA-RTB interface, including four Tier 1 V_HHs with IC₅₀ values <1 nM. Crystal structures of each V_HH bound to native ricin holotoxin revealed three different binding modes, based on contact with RTA’s F-G loop (mode 1), RTB’s subdomain 2γ (mode 2) or both (mode 3). V_HHs in modes 2 and 3 were highly effective at blocking ricin attachment to HeLa cells and immobilized asialofetuin, due to framework residues (FR3) that occupied the 2γ Gal/GalNAc-binding pocket and mimic ligand. The four Tier 1 V_HHs also interfered with intracellular functions of RTB, as they neutralized ricin in a post-attachment cytotoxicity assay (e.g., the toxin was bound to cell surfaces before antibody addition) and reduced the efficiency of toxin transport to the TGN. We conclude that the RTA-RTB interface is a target of potent toxin-neutralizing antibodies that interfere with both extracellular and intracellular events in ricin’s cytotoxic pathway.     

Single-domain antibodies neutralize ricin toxin intracellularly by blocking access to ribosomal P-stalk proteins

During ricin intoxication in mammalian cells, ricin''s enzymatic (RTA) and binding (RTB) subunits disassociate in the endoplasmic reticulum. RTA is then translocated into the cytoplasm where, by virtue of its ability to depurinate a conserved residue within the sarcin–ricin loop (SRL) of 28S rRNA, it functions as a ribosome-inactivating protein. It has been proposed that recruitment of RTA to the SRL is facilitated by ribosomal P-stalk proteins, whose C-terminal domains interact with a cavity on RTA normally masked by RTB; however, evidence that this interaction is critical for RTA activity within cells is lacking. Here, we characterized a collection of single-domain antibodies (V_HHs) whose epitopes overlap with the P-stalk binding pocket on RTA. The crystal structures of three such V_HHs (V9E1, V9F9, and V9B2) in complex with RTA revealed not only occlusion of the ribosomal P-stalk binding pocket but also structural mimicry of C-terminal domain peptides by complementarity-determining region 3. In vitro assays confirmed that these V_HHs block RTA–P-stalk peptide interactions and protect ribosomes from depurination. Moreover, when expressed as “intrabodies,” these V_HHs rendered cells resistant to ricin intoxication. One V_HH (V9F6), whose epitope was structurally determined to be immediately adjacent to the P-stalk binding pocket, was unable to neutralize ricin within cells or protect ribosomes from RTA in vitro. These findings are consistent with the recruitment of RTA to the SRL by ribosomal P-stalk proteins as a requisite event in ricin-induced ribosome inactivation.     

Using homology modeling to interrogate binding affinity in neutralization of ricin toxin by a family of single domain antibodies

In this report we investigated, within a group of closely related single domain camelid antibodies (V_HHs), the relationship between binding affinity and neutralizing activity as it pertains to ricin, a fast‐acting toxin and biothreat agent. The V1C7‐like V_HHs (V1C7, V2B9, V2E8, and V5C1) are similar in amino acid sequence, but differ in their binding affinities and toxin‐neutralizing activities. Using the X‐ray crystal structure of V1C7 in complex with ricin's enzymatic subunit (RTA) as a template, Rosetta‐based homology modeling coupled with energetic decomposition led us to predict that a single pairwise interaction between Arg29 on V5C1 and Glu67 on RTA was responsible for the difference in ricin toxin binding affinity between V1C7, a weak neutralizer, and V5C1, a moderate neutralizer. This prediction was borne out experimentally: substitution of Arg for Gly at position 29 enhanced V1C7's binding affinity for ricin, whereas the reverse (ie, Gly for Arg at position 29) diminished V5C1's binding affinity by >10 fold. As expected, the V5C1_R29G mutant was largely devoid of toxin‐neutralizing activity (TNA). However, the TNA of the V1C7_G29R mutant was not correspondingly improved, indicating that in the V1C7 family binding affinity alone does not account for differences in antibody function. V1C7 and V5C1, as well as their respective point mutants, recognized indistinguishable epitopes on RTA, at least at the level of sensitivity afforded by hydrogen‐deuterium mass spectrometry. The results of this study have implications for engineering therapeutic antibodies because they demonstrate that even subtle differences in epitope specificity can account for important differences in antibody function.     

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

Novel antibody constructs consisting of two or more different camelid heavy-chain only antibodies (VHHs) joined via peptide linkers have proven to have potent toxin-neutralizing activity in vivo against Shiga, botulinum, Clostridium difficile, anthrax, and ricin toxins. However, the mechanisms by which these so-called bispecific VHH heterodimers promote toxin neutralization remain poorly understood. In the current study we produced a new collection of ricin-specific VHH heterodimers, as well as VHH homodimers, and characterized them for their ability neutralize ricin in vitro and in vivo. We demonstrate that the VHH heterodimers, but not homodimers were able to completely protect mice against ricin challenge, even though the two classes of antibodies (heterodimers and homodimers) had virtually identical affinities for ricin holotoxin and similar IC₅₀ values in a Vero cell cytotoxicity assay. The VHH heterodimers did differ from the homodimers in their ability to promote toxin aggregation in solution, as revealed through analytical ultracentrifugation. Moreover, the VHH heterodimers that were most effective at promoting ricin aggregation in solution were also the most effective at blocking ricin attachment to cell surfaces. Collectively, these data suggest that heterodimeric VHH-based neutralizing agents may function through the formation of antibody-toxin complexes that are impaired in their ability to access host cell receptors.     

Structural analysis of nested neutralizing and non‐neutralizing B cell epitopes on ricin toxin's enzymatic subunit

In this report, we describe the X‐ray crystal structures of two single domain camelid antibodies (V_HH), F5 and F8, each in complex with ricin toxin's enzymatic subunit (RTA). F5 has potent toxin‐neutralizing activity, while F8 has weak neutralizing activity. F5 buried a total of 1760 Ų in complex with RTA and made contact with three prominent secondary structural elements: α‐helix B (Residues 98–106), β‐strand h (Residues 113–117), and the C‐terminus of α‐helix D (Residues 154–156). F8 buried 1103 Ų in complex with RTA that was centered primarily on β‐strand h. As such, the structural epitope of F8 is essentially nested within that of F5. All three of the F5 complementarity determining regions CDRs were involved in RTA contact, whereas F8 interactions were almost entirely mediated by CDR3, which essentially formed a seventh β‐strand within RTA's centrally located β‐sheet. A comparison of the two structures reported here to several previously reported (RTA‐V_HH) structures identifies putative contact sites on RTA, particularly α‐helix B, associated with potent toxin‐neutralizing activity. This information has implications for rational design of RTA‐based subunit vaccines for biodefense. Proteins 2016; 84:1162–1172. © 2016 Wiley Periodicals, Inc.     

Stability of isolated antibody-antigen complexes as a predictive tool for selecting toxin neutralizing antibodies

Ricin is an A-B ribosome inactivating protein (RIP) toxin composed of an A-chain subunit (RTA) that contains a catalytic N-glycosidase and a B-chain (RTB) lectin domain that binds cell surface glycans. Ricin exploits retrograde transport to enter into the Golgi and the endoplasmic reticulum, and then dislocates into the cytoplasm where it can reach its substrate, the rRNA. A subset of isolated antibodies (Abs) raised against the RTA subunit protect against ricin intoxication, and RTA-based vaccine immunogens have been shown to provide long-lasting protective immunity against the holotoxin. Anti-RTA Abs are unlikely to cross a membrane and reach the cytoplasm to inhibit the enzymatic activity of the A-chain. Moreover, there is not a strict correlation between the apparent binding affinity (K_a) of anti-RTA Abs and their ability to successfully neutralize ricin toxicity. Some anti-RTA antibodies are toxin-neutralizing, whereas others are not. We hypothesize that neutralizing anti-RTA Abs may interfere selectively with conformational change(s) or partial unfolding required for toxin internalization. To test this hypothesis, we measured the melting temperatures (T_m) of neutralizing single-domain Ab (sdAb)-antigen (Ag) complexes relative to the T_m of the free antigen (T_m-shift = T_m^complex – T_m^Ag), and observed increases in the T_m^complex of 9–20 degrees. In contrast, non-neutralizing sdAb-Ag complexes shifted the T_m^Complex by only 6–7 degrees. A strong linear correlation (r² = 0.992) was observed between the magnitude of the T_m-shift and the viability of living cells treated with the sdAb and ricin holotoxin. The T_m-shift of the sdAb-Ag complex provided a quantitative biophysical parameter that could be used to predict and rank-order the toxin-neutralizing activities of Abs. We determined the first structure of an sdAb-RTA1-33/44-198 complex, and examined other sdAb-RTA complexes. We found that neutralizing sdAb bound to regions involved in the early stages of unfolding. These Abs likely interfere with steps preceding or following endocytosis that require conformational changes. This method may have utility for the characterization or rapid screening of other Ab that act to prevent conformational changes or unfolding as part of their mechanism of action.     

Crystal Structures of Ricin Toxin's Enzymatic Subunit (RTA) in Complex with Neutralizing and Non-Neutralizing Single-Chain Antibodies

Ricin is a select agent toxin and a member of the RNA N-glycosidase family of medically important plant and bacterial ribosome-inactivating proteins. 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 (CDR, complementarity-determining region) 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.     

Arginine Residues on the Opposite Side of the Active Site Stimulate the Catalysis of Ribosome Depurination by Ricin A Chain by Interacting with the P-protein Stalk

Ricin inhibits protein synthesis by depurinating the α-sarcin/ricin loop (SRL). Ricin holotoxin does not inhibit translation unless the disulfide bond between the A (RTA) and B (RTB) subunits is reduced. Ricin holotoxin did not bind ribosomes or depurinate them but could depurinate free RNA. When RTA is separated from RTB, arginine residues located at the interface are exposed to the solvent. Because this positively charged region, but not the active site, is blocked by RTB, we mutated arginine residues at or near the interface of RTB to determine if they are critical for ribosome binding. These variants were structurally similar to wild type RTA but could not bind ribosomes. Their K_m values and catalytic rates (k_cat) for an SRL mimic RNA were similar to those of wild type, indicating that their activity was not altered. However, they showed an up to 5-fold increase in K_m and up to 38-fold decrease in k_cat toward ribosomes. These results suggest that the stalk binding stimulates the catalysis of ribosome depurination by RTA. The mutated arginines have side chains behind the active site cleft, indicating that the ribosome binding surface of RTA is on the opposite side of the surface that interacts with the SRL. We propose that stalk binding stimulates the catalysis of ribosome depurination by orienting the active site of RTA toward the SRL and thereby allows docking of the target adenine into the active site. This model may apply to the translation factors that interact with the stalk.     

Synthesis of a ricin toxin B subunit-rotavirus VP7 fusion protein in potato

A gene encoding the outer capsid glycoprotein (VP7) of simian rotavirus SA11, was genetically linked to the amino terminus of the ricin toxin B subunit (RTB) isolated from castor-oil plant (Ricinus communis) seeds. To assess fusion protein expression in plant cells, the VP7::RTB fussion gene was transferred into potato (Solanum tuberosum) cells by Agrobacterium tumefaciens-mediated transformation methods and transformed plants regenerated. The fusion gene was detected in transformed potato genomic DNA by polymerase chain reaction DNA amplification methods. Immunoblot analysis with anti-SA11 antiserum as the primary antibody verified the presence of VP7::RTB fusion protein in transformed potato tuber tissues. The plant-synthesized fusion protein bound RTB membrane receptors as measured by asialofetuin-enzyme-linked immunosorbent assay (ELISA). The ELISA results indicated that the VP7::RTB fusion protein was biologically active and made up approx 0.03% of total soluble transformed tuber protein. The biosynthesis of receptor binding VP7::RTB fusion protein in potato tissues demonstrates the feasibility of producing monomeric ricin toxin B subunit adjuvant-virus antigen fusion proteins in crop plants for enhanced immunity.     

Ricin B chain fragments expressed inEscherichia coli are able to bind free galactose in contrast to the full length polypeptide

Deleted forms of ricin B chain (RTB) containing only one of the two galactose binding sites were produced inE. coli and targeted to the periplasm by fusion to theompA orompF signal sequences. The proteins were then isolated from the periplasm and their sugar binding properties assessed. Previous studies investigating the properties of such proteins produced inXenopus laevis oocytes suggested that deleted forms of RTB, when not glycosylated, retain their ability to bind simple sugars, unlike the full-length unglycosylated proteins. When produced inE. coli however we found that only one, EB733, of a number of deleted forms of RTB closely related to those previously produced inXenopus laevis oocytes, bound to simple sugars. All of the deletion forms of RTB were found to bind in the asialofetuin binding assay; an assay which has been previously utilized to measure binding of lectins to the terminal galactose residues of glycoprotein oligosaccharides. However, in contrast to glycosylated RTB, binding of the deletion mutants could be competed to only a small degree or not at all with galactose. The only deletion mutant observed to bind to free galactose when produced inE. coli corresponded closely to the complete domain 2 of RTB. It is assumed that this mutant forms a stable structure similar to that of the C-terminal domain in the full-length protein. The structural integrity of EB733 was not only suggested by its sugar binding properties and solubility but also by its consistently higher level of expression and the absence of any apparent susceptibility toE. coli proteases.Abbreviations RTA ricin toxin A chain - RTB ricin toxin B chain - ER endoplasmic reticulum - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis - IPTG isopropyl -d-thiogalactopyranoside     

In Vivo Neutralization of α-Cobratoxin with High-Affinity Llama Single-Domain Antibodies (VHHs) and a VHH-Fc Antibody

Small recombinant antibody fragments (e.g. scFvs and V_HHs), which are highly tissue permeable, are being investigated for antivenom production as conventional antivenoms consisting of IgG or F(ab’)₂ antibody fragments do not effectively neutralize venom toxins located in deep tissues. However, antivenoms composed entirely of small antibody fragments may have poor therapeutic efficacy due to their short serum half-lives. To increase serum persistence and maintain tissue penetration, we prepared low and high molecular mass antivenom antibodies. Four llama V_HHs were isolated from an immune V_HH-displayed phage library and were shown to have high affinity, in the low nM range, for α-cobratoxin (α–Cbtx), the most lethal component of Naja kaouthia venom. Subsequently, our highest affinity V_HH (C2) was fused to a human Fc fragment to create a V_HH2-Fc antibody that would offer prolonged serum persistence. After in planta (Nicotiana benthamiana) expression and purification, we show that our V_HH2-Fc antibody retained high affinity binding to α–Cbtx. Mouse α–Cbtx challenge studies showed that our highest affinity V_HHs (C2 and C20) and the V_HH2-Fc antibody effectively neutralized lethality induced by α–Cbtx at an antibody:toxin molar ratio as low as ca. 0.75×:1. Further research towards the development of an antivenom therapeutic involving these anti-α-Cbtx V_HHs and V_HH2-Fc antibody molecules should involve testing them as a combination, to determine whether they maintain tissue penetration capability and low immunogenicity, and whether they exhibit improved serum persistence and therapeutic efficacy.     

蓖麻毒蛋白研究及应用进展(综述)

蓖麻毒蛋白(ricin)是一种核糖体失活蛋白,它由分子量分别为32KD和34KD的A、B两条链组成,具有很强的细胞毒性。本文综述蓖麻毒蛋白的结构和物理性质、毒性作用机理、制备及在医疗和生物农药方面的应用前景。     

Process development and cGMP manufacturing of a recombinant ricin vaccine: An effective and stable recombinant ricin a‐chain vaccine—RVEc™

Ricin is a potent toxin and a potential bioterrorism weapon with no specific countermeasures or vaccines available. The holotoxin is composed of two polypeptide chains linked by a single disulfide bond: the A‐chain (RTA), which is an N‐glycosidase enzyme, and the B‐chain (RTB), a lectin polypeptide that binds galactosyl moieties on the surface of the mammalian target cells. Previously (McHugh et al.), a recombinant truncated form of RTA (rRTA1‐33/44‐198 protein, herein denoted RVEa?) expressed in Escherichia coli using a codon‐optimized gene was shown to be non‐toxic, stable, and protective against a ricin challenge in mice. Here, we describe the process development and scale‐up at the 12 L fermentation scale, and the current Good Manufacturing Practice (cGMP)‐compliant production of RVEc? at the 40 L scale. The average yield of the final purified bulk RVEc? is approximately 16 g/kg of wet cell weight or 1.2 g/L of fermentation broth. The RVEc? was >99% pure by three HPLC methods and SDS‐PAGE. The intact mass and peptide mapping analysis of RVEc? confirmed the identity of the product and is consistent with the absence of posttranslational modifications. Potency assays demonstrated that RVEc? was immunoprotective against lethal ricin challenge and elicited neutralizing anti‐ricin antibodies in 95–100% of the vaccinated mice. Published 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011.     

Inhibition of ricin A-chain (RTA) catalytic activity by a viral genome-linked protein (VPg)

Ricin is a plant derived protein toxin produced by the castor bean plant (Ricinus communis). The Centers for Disease Control (CDC) classifies ricin as a Category B biological agent. Currently, there is neither an effective vaccine that can be used to protect against ricin exposure nor a therapeutic to reverse the effects once exposed. Here we quantitatively characterize interactions between catalytic ricin A-chain (RTA) and a viral genome-linked protein (VPg) from turnip mosaic virus (TuMV). VPg and its N-terminal truncated variant, VPg_1–110, bind to RTA and abolish ricin's catalytic depurination of 28S rRNA in vitro and in a cell-free rabbit reticulocyte translational system. RTA and VPg bind in a 1 to 1 stoichiometric ratio, and their binding affinity increases ten-fold as temperature elevates (5 °C to 37 °C). RTA-VPg binary complex formation is enthalpically driven and favored by entropy, resulting in an overall favorable energy, ΔG = −136.8 kJ/mol. Molecular modeling supports our experimental observations and predicts a major contribution of electrostatic interactions, suggesting an allosteric mechanism of downregulation of RTA activity through conformational changes in RTA structure, and/or disruption of binding with the ribosomal stalk. Fluorescence anisotropy studies show that heat affects the rate constant and the activation energy for the RTA-VPg complex, Ea = −62.1 kJ/mol. The thermodynamic and kinetic findings presented here are an initial lead study with promising results and provides a rational approach for synthesis of therapeutic peptides that successfully eliminate toxicity of ricin, and other cytotoxic RIPs.     

Sub-Domains of Ricin��s B Subunit as Targets of Toxin Neutralizing and Non-Neutralizing Monoclonal Antibodies

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.     

Neutralising antibodies against ricin toxin

The Centers for Disease Control and Prevention have listed the potential bioweapon ricin as a Category B Agent. Ricin is a so-called A/B toxin produced by plants and is one of the deadliest molecules known. It is easy to prepare and no curative treatment is available. An immunotherapeutic approach could be of interest to attenuate or neutralise the effects of the toxin. We sought to characterise neutralising monoclonal antibodies against ricin and to develop an effective therapy. For this purpose, mouse monoclonal antibodies (mAbs) were produced against the two chains of ricin toxin (RTA and RTB). Seven mAbs were selected for their capacity to neutralise the cytotoxic effects of ricin in vitro. Three of these, two anti-RTB (RB34 and RB37) and one anti-RTA (RA36), when used in combination improved neutralising capacity in vitro with an IC(50) of 31 ng/ml. Passive administration of association of these three mixed mAbs (4.7 μg) protected mice from intranasal challenges with ricin (5 LD(50)). Among those three antibodies, anti-RTB antibodies protected mice more efficiently than the anti-RTA antibody. The combination of the three antibodies protected mice up to 7.5 hours after ricin challenge. The strong in vivo neutralising capacity of this three mAbs combination makes it potentially useful for immunotherapeutic purposes in the case of ricin poisoning or possibly for prevention.     

Membrane destabilization by ricin

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.Abbreviations CF Carboxyfluorescein - DPhPC Diphytanoyl-phosphatidylcholine - DPA Dipicolinic acid - EDTA Ethylendiamine-tetracetate - ER Endoplasmic reticulum - ERAD ER-associated degradation - FRET Fluorescence-resonance energy transfer - GM₁ Monosialoganglioside - MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry - MES 2-Morpholino-ethanesulfonic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine) - PC Phosphatidylcholine - PE Phosphatidylethanolamine - PG Phosphatidylglycerol - Rh-PE N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine - RIP Ribosome-inactivating protein - RTA A-chain of ricin - RTB B-chain of ricin - TES N-[Tris-(hydroxymethyl)-methyl]-2-aminoethansulfonic acid - TOF Time-of-flight     

Prolonged Prophylactic Protection from Botulism with a Single Adenovirus Treatment Promoting Serum Expression of a VHH-Based Antitoxin Protein

Current therapies for most acute toxin exposures are limited to administration of polyclonal antitoxin serum. We have shown that VHH-based neutralizing agents (VNAs) consisting of two or more linked, toxin-neutralizing heavy-chain-only V_H domains (VHHs), each binding distinct epitopes, can potently protect animals from lethality in several intoxication models including Botulinum neurotoxin serotype A1 (BoNT/A1). Appending a 14 amino acid albumin binding peptide (ABP) to an anti-BoNT/A1 heterodimeric VNA (H7/B5) substantially improved serum stability and resulted in an effective VNA serum half-life of 1 to 2 days. A recombinant, replication-incompetent, adenoviral vector (Ad/VNA-BoNTA) was engineered that induces secretion of biologically active VNA, H7/B5/ABP (VNA-BoNTA), from transduced cells. Mice administered a single dose of Ad/VNA-BoNTA, or a different Ad/VNA, via different administration routes led to a wide range of VNA serum levels measured four days later; generally intravenous > intraperitoneal > intramuscular > subcutaneous. Ad/VNA-BoNTA treated mice were 100% protected from 10 LD₅₀ of BoNT/A1 for more than six weeks and protection positively correlated with serum levels of VNA-BoNTA exceeding about 5 ng/ml. Some mice developed antibodies that inhibited VNA binding to target but these mice displayed no evidence of kidney damage due to deposition of immune complexes. Mice were also successfully protected from 10 LD₅₀ BoNT/A1 when Ad/VNA-BoNTA was administered up to 1.5 hours post-intoxication, demonstrating rapid appearance of the protective VNA in serum following treatment. Genetic delivery of VNAs promises to be an effective method of providing prophylactic protection and/or acute treatments for many toxin-mediated diseases.     

Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).     

Algal chloroplast produced camelid VHH antitoxins are capable of neutralizing botulinum neurotoxin

We have produced three antitoxins consisting of the variable domains of camelid heavy chain‐only antibodies (V_HH) by expressing the genes in the chloroplast of green algae. These antitoxins accumulate as soluble proteins capable of binding and neutralizing botulinum neurotoxin. Furthermore, they accumulate at up to 5% total soluble protein, sufficient expression to easily produce these antitoxins at scale from algae. The genes for the three different antitoxins were transformed into Chlamydomonas reinhardtii chloroplasts and their products purified from algae lysates and assayed for in vitro biological activity using toxin protection assays. The produced antibody domains bind to botulinum neurotoxin serotype A (BoNT/A) with similar affinities as camelid antibodies produced in Escherichia coli, and they are similarly able to protect primary rat neurons from intoxication by BoNT/A. Furthermore, the camelid antibodies were produced in algae without the use of solubilization tags commonly employed in E. coli. These camelid antibody domains are potent antigen‐binding proteins and the heterodimer fusion protein containing two V_HH domains was capable of neutralizing BoNT/A at near equimolar concentrations with the toxin. Intact antibody domains were detected in the gastrointestinal (GI) tract of mice treated orally with antitoxin‐producing microalgae. These findings support the use of orally delivered antitoxins produced in green algae as a novel treatment for botulism.