Ricin Toxin

From the time Pharaohs first presided during the dawn of civilization until the present day, the castor bean plant, Ricinus communis, and its seeds have played a significant role in the advancement of science, industry, and war. From ancient times, the seeds have been a valuable source of oil used for light and medicinal purposes. Also co-existing in the seed is a toxin (ricin) that has been used in beneficial, and sometimes iniquitous, ways. This chapter provides a review of the history of ricin use (with background information on its source the castor plant and its seeds) that shows the impact that Ricinus communis has had upon many facets of civilization.

Ricin is a protein made of the castor bean plant, Ricinus communis, and found primarily in its seeds. Ricin accounts for about 5% of the proteins in the mature seeds and is one of the most poisonous naturally occurring substances. With the worldwide increase of castor bean production for biofuels and petrochemical replacements, it has been of increasing concern that ricin may become a major instrument of bioterrorism because of its heat stability, accessibility, and ease of production in massive quantities. To assure a safe food supply, it is necessary to have detection methods applicable to important food matrices. This review highlights detection of ricin in different matrices using methods ranging from classic animal bioassays to cutting edge molecular approaches.

Ricin is a highly toxic globular protein (lectin) found in castor (Ricinus communis) seeds. Inhaled ricin dust or intravenously injected ricin is ~1000 times more toxic than orally ingested ricin. Interactions between ricin, soil minerals, and other materials control the environmental fate of dispersed ricin. Ricin was sorbed by the common soil clay minerals, montmorillonite (~350 g/kg), illite (~50 g/kg), and kaolinite (10 g/kg). Ricin also effectively sorbed to other soil materials: ferrihydite, goethite, calcite, and Ca-humate, but sorption by activated carbon was minimal. Montmorillonite sorbed 5 times more ricin at pH 5 than at pH 10. Much of the ricin sorbed at pH 7 could be desorbed using pH 10 buffer solutions. X-ray diffraction patterns showed that ricin sorption expanded montmorillonite interlayers and shifted the basal spacing from ~ 11.8 Å to ~21.2 Å. Textiles, fruit, and vegetables sorbed ricin, but much less ricin was sorbed than by soil materials. Breakthrough curves for ricin leached through soil columns indicate that dissolved ricin is strongly sorbed by soils and most readily moves in sandy soils. Ricin readily moved in the dust from sandy soils and was concentrated in the fine dust particles. Ricin less readily moved in clayey soil dust and was less effectively concentrated in the fine dust. Experiments using PM10 (< 10 μm) and PM2.5 (< 2.5 μm) dust samplers demonstrated that significant quantities of respirable-size ricin particles were generated in fine soil dust. Dust control chemicals, particularly polyacrylamide, reduced the generation of fine ricin dust particles.

Commercial production of castor, as a source of highly valuable hydroxyl fatty acids, has been limited by both the real and perceived risks of commercial castor production in North America. Crop commodity groups, regulatory governmental agencies, and much of the general public may have reservations about the large scale production of an oilseed crop which produces a seed meal with high concentrations of the toxin ricin as an accidental contaminant in feed grains or human food commodities, and as a potential source of chemical weapons by terrorist organizations. A successful castor industry in North America has to provide assurance that castor production will not impact the quality of existing crop commodities or create public safety concerns. Both the genetic detoxification of castor seed and the development of vertically integrated production systems, to functionally isolate castor seed and its commercial products, are being developed by researchers in Texas and California.

Castor beans, from 10 different cultivars of Ricinus communis, were acquired from different commercial sources from three different countries. Ricin was extracted from 25 grams of beans from each cultivar. Resulting toxin yields varied from negligible to relatively high. Two methods of extraction were used, with one method yielding on average 7-fold more toxin than the other. Reducing SDS-PAGE showed that most of the ricin extractions had not only the A and B chains, but also additional bands of comparable molecular weights similar to those noted in the historical literature. Results from intra-peritoneal injection (i.p.) of mice, given the same amount of ricin extracted from different cultivars, varied in toxicity up to 10-fold. Regardless of the different sources of ricin, both polyclonal goat anti-ricin antisera and monoclonal mouse antibody (mD9) inhibited all of these, rescuing mice from 5 LD₅₀ of toxin when given via i.p. an hour later.

Ricin is a potent phytotoxin that presents a significant public health concern due to its potential use as a bioterrorism agent. Exposure to ricin results in local tissue necrosis and general organ failure leading to death within several days. Currently, there is no U.S. FDA-approved drug or vaccine against ricin poisoning. Because vaccination offers a practical prophylactic strategy to protect selected populations at risk of ricin exposure, there has been a great deal of interest in developing a safe and effective vaccine to protect humans, in particular soldiers and first responders. Generation of non-toxic derivatives of ricin or ricin A chain (RTA) for use as vaccines has been initially attempted by several groups using formalin treatment, chemical deglycosylation, or mutagenesis by substitution or insertion. Most of these efforts resulted in unstable protein products that aggregated in solution, had residual toxicity, or expressed poorly in recombinant form. At present, two leading recombinant RTA vaccine candidates, RiVax (University of Texas) and RVEc (USAMRIID), are in advanced development in clinical trials. This chapter reviews the efforts, challenges, and progress toward the development of ricin vaccines.

Antibodies provide the best defense against the effects of biological toxins. We have previously made a panel of 45 different murine monoclonal antibodies (mAbs) to ricin A chain, B chain, and determinants found on both chains. Comparative in vitro and in vivo studies have identified one anti-A chain mAb, designated RAC18, as the most effective at both in vitro neutralization and in vivo protection from ricin toxin. Here we describe experiments to improve the utility of this mAb. We have made a chimeric mouse/human Ab by grafting the murine V regions onto human IgG1/ constant regions, and show that it retained full in vitro and in vivo activity. We also used a novel approach to generate higher affinity Abs, based on our observation that hybridoma cell lines were resistant to ricin in proportion to the affinity of the Ab they produce. We induced AID-dependent V region mutagenesis while selecting cells in increasing concentrations of ricin. We were able to isolate cells that were 100X more resistant to ricin than the parental hybridomas, but the quality of the Ab was no different. Rather, cells had down-regulated the expression of cell surface structures that are bound by ricin. These results demonstrate a unique mechanism whereby cells become resistant to ricin’s lethal effects.

Development of anti-ricin protective monoclonal antibodies (mAbs) started in the early 1980s. Much progress has been made since then. Antibodies possess great potential for development as antidotes against toxins. These can be used either prophylactically to prevent, or therapeutically to treat, toxin-mediated intoxications in an emergency situation. Unlike many other therapeutic products, antibodies offer unique and high target specificity, and long half-life in serum. There are several mAbs which are currently in the discovery stage for medical countermeasures against ricin intoxication. This review summarizes these mAbs, including their anti-ricin mechanism, generation, and efficacies in vivo.

Murine monoclonal antibodies (mAbs) have great potentials as therapeutics developed for clinical applications. The major problem with these mAbs, however, is their immunogenicity due to their foreignness to humans. Humanization, a process to decease the content of murine residues in mAbs, can make it possible to reduce their immunogenicity for clinical uses. One of the humanization strategies, complementarity determining region (CDR)-grafting is well-established and popular, but it generally needs multiple design cycles, which are time-consuming. A CDR-grafting approach now described is based on a comprehensive analysis of the antibody sequence, and its three-dimensional structure from molecular modeling, to identify critical residues in the murine antibody of interest, which guides the completion of CDR-grafting to a human antibody template in a single cycle. The single-cycle structure-based method was used to create the first humanized (>94% human) anti-ricin monoclonal IgG antibody (results published and patented). The steps used in the successful creation of this antibody, hD9, are described to assist the researchers in their quest to develop humanized antibodies against threat agents.

A cell-based high throughput screening assay (HTS) was established to screen the Prestwick Chemical Library for candidates that acted against ricin. Of 1120 compounds screened, only 7 were identified as ricin inhibitors. Secondary screening with cell cultures identified only ethotoin as a dose-dependent inhibitor against ricin induced toxicity. Ethotoin was further evaluated in two in vivo studies. Study 1: When mice were given intra-peritoneal injections of 5×LD50 ricin (1 μg) pre-incubated with ethotoin (1 mg), all mice survived (monitored for 14 days). In contrast, control mice without ethotoin died within 2 days. Study 2: When mice were given 2×LD50 ricin (0.2 μg) by the intranasal route and then given ethotoin by multiple oral/gavage deliveries (at 1, 2, 4, 6, 8 and 10 hr after intoxication) 40% of the mice were alive at day 14. In contrast, all control mice (those that received sterile saline instead) died between days 3-9. Hence ethotoin, given by one route (oral) could rescue some mice from ricin given by another route (intra-nasal). Our findings suggest that ethotoin, although now an abandoned anti-convulsant drug, deserves further investigation and development as a potential antidote against the ricin biothreat.

A major difficulty in developing medical countermeasures against ricin (e.g. passive antibody therapy) is the need to work with authentic ricin, a Schedule 1 Chemical under the Chemical Weapons Convention. Twinstrand Therapeutics Inc. created a benign but antigenically intact variant of ricin by changing the genetic sequence of the A and B chain linker peptide (representing less than 4% of the gene) to block post-translational processing and activation. The toxoid protein, a single polypeptide chain rather than dimer, was produced in a Pichia pastoris expression system in accordance with Good Laboratory Practices (GLP). The toxoid was determined to be 1000-fold less toxic than authentic ricin by both in vitro and in vivo assays, allowing high immunogenic doses to be administered to test animals. Cangene Corporation oversaw a contract in which goats were inoculated with varying schedules and doses of the toxoid in accordance with GLP. DRDC Suffield Research Centre assessed the activity of the resultant goat antibodies in challenges involving the wild type toxin. The goat anti-toxoid antiserum was found to have high anti-ricin neutralizing activity. The collaboration described was supported by the Defence Research and Development Canada’s CBRNe Research and Technology Initiative (CRTI) project 02-0007TA (2003-2005). All partners contributed generous In-Kind support.

Ribosome Inactivating Proteins (RIPs) are toxins capable of specifically and irreversibly inhibiting protein synthesis in eukaryotic cells. The plant RIP prototype, ricin, is a vigorous toxin that can be easily extracted and purified from the castor plant Ricinus communis. This heterodimeric toxin consists of a catalytic subunit A linked to a galactose-binding lectin subunit B, which allows cell surface binding and toxin entry in most mammalian cells. Ricin exhibits potency in the picomolar range, thereby it is highly toxic at very low lethal doses. Due to its high systemic cytotoxicity, ease of production, and prevalence, ricin is considered as a possible weapon for warfare and terrorism attacks, and therefore included among the potential biological weapons by the United State Centre of Disease Control and Prevention. Since heating denatures the toxin, extracted castor oil has been widely used in the industry; for example, as twostroke engine oil or lubricant for aviation engine. However, castor oil has also detrimental effects causing violent diarrhea. Thus, even castor oil has been used as a convenient terror weapon under the Italian Fascist regime to convert “red” workers. Nowadays, more peaceful and beneficial medicinal uses have been created. Certainly, the most promising way is to exploit ricin as weapon to combat cancers. Novel molecules in which the toxin moiety of ricin was linked to selective tumour targeting domains have been generated for cancer therapy. The major recent advancements in this field will be discussed in this chapter.