A Monoclonal Immunoglobulin G Antibody Directed against an Immunodominant Linear Epitope on the Ricin A Chain Confers Systemic and Mucosal Immunity to Ricin

Due to the potential use of ricin and other fast-acting toxins as agents of bioterrorism, there is an urgent need for the development of safe and effective antitoxin vaccines. A candidate ricin subunit vaccine (RiVax) consisting of a recombinant attenuated enzymatic A chain (RTA) has been shown to elicit protective antitoxin antibodies in mice and rabbits and is currently being tested in phase I human clinical trials. However, evaluation of the efficacy of this vaccine for humans is difficult for a number of reasons, including the fact that the key neutralizing B-cell epitopes on RTA have not been fully defined. Castelletti and colleagues (Clin. Exp. Immunol. 136:365-372, 2004) recently identified a linear epitope on RTA, spanning residues L161 to I175, as a primary target of serum antibodies derived from humans who had been treated with ricin immunotoxin. While affinity-purified polyclonal IgG antibodies against this region of RTA were capable of neutralizing ricin in vitro, their capacity to confer protection against ricin challenge in vivo was not determined. In this report, we describe the production and characterization of GD12, a murine monoclonal IgG1 antibody specifically directed against residues 163 to 174 (TLARSFIICIQM) of RTA. GD12 bound ricin holotoxin with high affinity (K_D [dissociation constant], 2.9 × 10⁻⁹ M) and neutralized it with a 50% inhibitory concentration of ∼0.25 μg/ml, as determined by a Vero cell-based cytotoxicity assay. Passive administration of GD12 was sufficient to protect BALB/c mice against intraperitoneal and intragastric ricin challenges. These data are important in terms of vaccine development, since they firmly establish that preexisting serum antibodies directed against residues 161 to 175 on RTA are sufficient to confer both systemic and mucosal immunity to ricin. The potential of GD12 to serve as a therapeutic following ricin challenge was not explored in this study.Recent bioterrorism incidents in the United States and abroad have alerted public health officials to the need for vaccines against pathogens and toxins previously deemed to be of little concern (2, 25). The development and implementation of vaccines for biodefense and emerging infectious diseases are inherently challenging, because phase III clinical efficacy trials of candidate vaccines are generally not feasible or ethical. To address this issue, the Food and Drug Administration (FDA) has implemented the “two-animal rule,” which enables candidate vaccines to advance toward licensure based on efficacy studies performed with two or more relevant animal models (8, 49). For compliance with this FDA policy, the animal models must mimic the pathophysiology of human disease, and the defined end point(s) of the efficacy studies must correlate with the desired effects for humans. However, even well-established animal models cannot completely substitute for human studies. Therefore, whenever possible, specific correlates of protection against select agents and emerging infectious diseases should be established in humans, and surrogate assays should be developed that can be used to estimate immunity in vaccinated human populations (35).Ricin is a category B toxin, as classified by the Centers for Disease Control and Prevention (CDC). The toxin is naturally produced by the castor bean plant, Ricinus communis, which is cultivated on industrial levels around the world for the production of castor oil. Ricin constitutes up to 5% of the total dry weight of the castor bean and can be extracted from the mash through several simple enrichment steps. The toxin is a member of the so-called type II ribosome-inactivating proteins (RIPs); it consists of two subunits, RTA and RTB, each with a molecular mass of approximately 30,000 Da (31, 47). RTA is an RNA N-glycosidase whose substrate is a conserved adenine residue within the so-called sarcin/ricin loop of eukaryotic 28S rRNA. Ribosome progression is arrested upon cleavage of this residue by RTA (9). RTB is a bivalent lectin with specificity for glycoproteins and glycolipids containing β(1-3)-linked galactose and N-acetylgalactosamine residues (4). RTB mediates the attachment and internalization of ricin into host cells and facilitates retrograde transport of the toxin to the Golgi apparatus and endoplasmic reticulum (ER) (21, 38). Ricin in semipurified or purified form is extremely toxic to humans following injection, inhalation, or ingestion (3) and has been used as an agent of bioterrorism. Ricin was weaponized by the United States and other countries during World War II (24, 48); it has been used in assassinations; and it was recently uncovered in a number of government facilities, including a South Carolina postal facility, and packed in envelopes delivered to offices of the U.S. Senate (12, 40).Because of the toxicity of ricin and the ease of its preparation, public health officials and defense agencies have made a concerted effort to develop a vaccine against it that could be administered to emergency first responders and military personnel (25). Although formaldehyde-treated ricin toxoid (RT) preparations are effective at eliciting protective immunity in rodents, they are not being considered for use in humans, because of concerns about residual toxicity (11). Therefore, the current emphasis is on the development of attenuated subunit vaccines (6, 16, 32, 45, 52). One of the most promising candidates is a recombinant derivative of RTA containing two point mutations: one in the enzymatic active site (Y80A) and the other in a residue (V76M) involved in eliciting vascular leak syndrome (42-45, 52). This vaccine, known by the trade name RiVax, is safe and immunogenic in mice and rabbits and, when administered intramuscularly, elicits serum antitoxin IgG antibodies capable of protecting animals against a systemic ricin challenge of 10 50% lethal doses (LD₅₀s) (42, 44). RiVax has also been shown to elicit an antibody response capable of protecting mice against both intragastric (i.g.) and aerosol challenges (45). Based on these animal studies, a pilot phase I clinical trial of RiVax was undertaken in 2006 (52). The trial consisted of three groups of five healthy volunteers injected at monthly intervals with 10, 33, or 100 μg of the vaccine. The results of this study revealed that RiVax was well tolerated and resulted in dose-dependent seroconversion (52).While RiVax was deemed safe and immunogenic, evaluation of the efficacy of this vaccine in humans remains challenging. For example, in the pilot phase I clinical trial noted above, there was no observed correlation between serum anti-RTA IgG titers and in vitro ricin-neutralizing activity (52). Specifically, two individuals with virtually identical serum anti-RTA IgG levels (4.73 ± 0.019 μg/ml versus 4.36 ± 0.16 μg/ml) had toxin-neutralizing titers that differed by >10-fold (1.4 ± 0 versus 0.13 ± 0.02). These data suggest that the polyclonal response to RTA consists of a mixture of neutralizing and nonneutralizing antibodies and that the ratio of the two types of antibodies can differ from individual to individual. This interpretation is supported by work from Maddaloni and colleagues, who identified both potent neutralizing monoclonal antibodies (MAbs) (e.g., RAC18) and a number of MAbs that bound to RTA with high avidity but failed to neutralize ricin in vitro or in vivo (23, 37). In fact, one MAb, designated RAC23, actually enhanced ricin toxicity in vivo. These data indicate that distinct neutralizing and nonneutralizing B-cell epitopes exist on RTA.Castelletti and colleagues recently identified a linear B-cell epitope (L161 to I175) on RTA recognized by serum antibodies from 15 Hodgkin''s lymphoma patients who had received ricin immunotoxin therapy (7). In two of the serum samples tested, the majority of the antiricin specific antibodies were directed against this epitope. Affinity-purified serum antipeptide IgG was capable of neutralizing ricin in vitro, suggesting that this epitope is an important target of anti-RTA neutralizing antibodies in vivo. However, the possibility that the observed activity was due to a minor contaminating population of antibodies directed against a similar or closely situated epitope cannot be excluded. Moreover, Castelletti and colleagues did not examine whether antibodies directed against L161 to I175 were actually capable of neutralizing ricin in an animal model of ricin intoxication.In an effort to identify the epitopes on ricin that are the key targets of neutralizing antibodies in vivo with the goal of better understanding vaccine-induced immunity to ricin, we have produced and characterized a murine IgG1 MAb, referred to as GD12, that is specifically directed against the linear B-cell epitope on RTA described by Castelletti and colleagues as being immunodominant in humans. GD12 neutralized ricin with a 50% inhibitory concentration (IC₅₀) of ∼0.25 μg/ml, as determined by a Vero cell-based cytotoxicity assay. More importantly, passive administration of GD12 to mice was sufficient to protect the animals against both systemic (i.e., intraperitoneal [i.p.]) and mucosal (i.e., intragastric) ricin challenge, underscoring the importance of the epitope spanning residues 161 to 175 on RTA as a key target of neutralizing antibodies in vivo. However, because these studies focused on vaccine development, rather than on therapeutic applications of GD12, this MAb was not examined for the ability to rescue animals following toxin exposure.