INFECTION AND IMMUNITY, Jan. 2010, p. 552–561 Vol. 78, No. 10019-9567/10/$12.00 doi:10.1128/IAI.00796-09Copyright © 2010, American Society for Microbiology. All Rights Reserved.

A Monoclonal Immunoglobulin G Antibody Directed against anImmunodominant Linear Epitope on the Ricin A Chain

Confers Systemic and Mucosal Immunity to Ricin�

Lori M. Neal,1 Joanne O’Hara,2 Robert N. Brey III,3 and Nicholas J. Mantis1,2*Division of Infectious Disease, Wadsworth Center, New York State Department of Health, Albany, New York 122081; Department of

Biomedical Sciences, University at Albany School of Public Health, Albany, New York 122012; and Soligenix, Ewing, New Jersey 086283

Received 15 July 2009/Returned for modification 1 September 2009/Accepted 12 October 2009

Due to the potential use of ricin and other fast-acting toxins as agents of bioterrorism, there is an urgentneed 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 antitoxinantibodies 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 thatthe 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, asa 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 ricinin 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 specificallydirected against residues 163 to 174 (TLARSFIICIQM) of RTA. GD12 bound ricin holotoxin with high affinity(KD [dissociation constant], 2.9 � 10�9 M) and neutralized it with a 50% inhibitory concentration of �0.25�g/ml, as determined by a Vero cell-based cytotoxicity assay. Passive administration of GD12 was sufficient toprotect BALB/c mice against intraperitoneal and intragastric ricin challenges. These data are important interms of vaccine development, since they firmly establish that preexisting serum antibodies directed againstresidues 161 to 175 on RTA are sufficient to confer both systemic and mucosal immunity to ricin. The potentialof GD12 to serve as a therapeutic following ricin challenge was not explored in this study.

Recent bioterrorism incidents in the United States andabroad have alerted public health officials to the need forvaccines against pathogens and toxins previously deemed to beof little concern (2, 25). The development and implementationof vaccines for biodefense and emerging infectious diseases areinherently challenging, because phase III clinical efficacy trialsof candidate vaccines are generally not feasible or ethical. Toaddress this issue, the Food and Drug Administration (FDA)has implemented the “two-animal rule,” which enables candi-date vaccines to advance toward licensure based on efficacystudies performed with two or more relevant animal models (8,49). For compliance with this FDA policy, the animal modelsmust mimic the pathophysiology of human disease, and thedefined end point(s) of the efficacy studies must correlate withthe desired effects for humans. However, even well-establishedanimal models cannot completely substitute for human studies.Therefore, whenever possible, specific correlates of protectionagainst select agents and emerging infectious diseases shouldbe established in humans, and surrogate assays should be de-veloped that can be used to estimate immunity in vaccinatedhuman populations (35).

Ricin is a category B toxin, as classified by the Centers for

Disease Control and Prevention (CDC). The toxin is naturallyproduced by the castor bean plant, Ricinus communis, which iscultivated on industrial levels around the world for the pro-duction of castor oil. Ricin constitutes up to 5% of the total dryweight of the castor bean and can be extracted from the mashthrough several simple enrichment steps. The toxin is a mem-ber of the so-called type II ribosome-inactivating proteins(RIPs); it consists of two subunits, RTA and RTB, each with amolecular mass of approximately 30,000 Da (31, 47). RTA isan RNA N-glycosidase whose substrate is a conserved adenineresidue within the so-called sarcin/ricin loop of eukaryotic 28SrRNA. Ribosome progression is arrested upon cleavage of thisresidue by RTA (9). RTB is a bivalent lectin with specificity forglycoproteins and glycolipids containing �(1–3)-linked galac-tose and N-acetylgalactosamine residues (4). RTB mediatesthe attachment and internalization of ricin into host cells andfacilitates retrograde transport of the toxin to the Golgi appa-ratus and endoplasmic reticulum (ER) (21, 38). Ricin in semi-purified or purified form is extremely toxic to humans follow-ing injection, inhalation, or ingestion (3) and has been used asan agent of bioterrorism. Ricin was weaponized by the UnitedStates and other countries during World War II (24, 48); it hasbeen used in assassinations; and it was recently uncovered in anumber of government facilities, including a South Carolinapostal facility, and packed in envelopes delivered to offices ofthe U.S. Senate (12, 40).

Because of the toxicity of ricin and the ease of its prepara-tion, public health officials and defense agencies have made a

* Corresponding author. Mailing address: Division of InfectiousDisease, Wadsworth Center, 120 New Scotland Avenue, Albany, NY12208. Phone: (518) 473-7487. Fax: (518) 402-4773. E-mail: nmantis@wadsworth.org.

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concerted effort to develop a vaccine against it that could beadministered to emergency first responders and military per-sonnel (25). Although formaldehyde-treated ricin toxoid (RT)preparations are effective at eliciting protective immunity inrodents, they are not being considered for use in humans,because of concerns about residual toxicity (11). Therefore, thecurrent emphasis is on the development of attenuated subunitvaccines (6, 16, 32, 45, 52). One of the most promising candi-dates is a recombinant derivative of RTA containing two pointmutations: one in the enzymatic active site (Y80A) and theother in a residue (V76M) involved in eliciting vascular leaksyndrome (42–45, 52). This vaccine, known by the trade nameRiVax, is safe and immunogenic in mice and rabbits and, whenadministered intramuscularly, elicits serum antitoxin IgG an-tibodies capable of protecting animals against a systemic ricinchallenge of 10 50% lethal doses (LD50s) (42, 44). RiVax hasalso been shown to elicit an antibody response capable ofprotecting mice against both intragastric (i.g.) and aerosolchallenges (45). Based on these animal studies, a pilot phase Iclinical trial of RiVax was undertaken in 2006 (52). The trialconsisted of three groups of five healthy volunteers injected atmonthly intervals with 10, 33, or 100 �g of the vaccine. Theresults of this study revealed that RiVax was well tolerated andresulted in dose-dependent seroconversion (52).

While RiVax was deemed safe and immunogenic, evaluationof 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-RTAIgG titers and in vitro ricin-neutralizing activity (52). Specifi-cally, two individuals with virtually identical serum anti-RTAIgG levels (4.73 � 0.019 �g/ml versus 4.36 � 0.16 �g/ml) hadtoxin-neutralizing titers that differed by �10-fold (1.4 � 0versus 0.13 � 0.02). These data suggest that the polyclonalresponse to RTA consists of a mixture of neutralizing andnonneutralizing antibodies and that the ratio of the two typesof antibodies can differ from individual to individual. Thisinterpretation is supported by work from Maddaloni and col-leagues, who identified both potent neutralizing monoclonalantibodies (MAbs) (e.g., RAC18) and a number of MAbs thatbound to RTA with high avidity but failed to neutralize ricin invitro or in vivo (23, 37). In fact, one MAb, designated RAC23,actually enhanced ricin toxicity in vivo. These data indicate thatdistinct neutralizing and nonneutralizing B-cell epitopes existon RTA.

Castelletti and colleagues recently identified a linear B-cellepitope (L161 to I175) on RTA recognized by serum antibod-ies from 15 Hodgkin’s lymphoma patients who had receivedricin immunotoxin therapy (7). In two of the serum samplestested, the majority of the antiricin specific antibodies weredirected against this epitope. Affinity-purified serum antipep-tide IgG was capable of neutralizing ricin in vitro, suggestingthat this epitope is an important target of anti-RTA neutral-izing antibodies in vivo. However, the possibility that the ob-served activity was due to a minor contaminating population ofantibodies directed against a similar or closely situated epitopecannot be excluded. Moreover, Castelletti and colleagues didnot examine whether antibodies directed against L161 to I175were actually capable of neutralizing ricin in an animal modelof 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 betterunderstanding vaccine-induced immunity to ricin, we have pro-duced and characterized a murine IgG1 MAb, referred to asGD12, that is specifically directed against the linear B-cellepitope on RTA described by Castelletti and colleagues asbeing immunodominant in humans. GD12 neutralized ricinwith a 50% inhibitory concentration (IC50) of �0.25 �g/ml, asdetermined by a Vero cell-based cytotoxicity assay. More im-portantly, passive administration of GD12 to mice was suffi-cient to protect the animals against both systemic (i.e., intra-peritoneal [i.p.]) and mucosal (i.e., intragastric) ricin challenge,underscoring the importance of the epitope spanning residues161 to 175 on RTA as a key target of neutralizing antibodies invivo. However, because these studies focused on vaccine de-velopment, rather than on therapeutic applications of GD12,this MAb was not examined for the ability to rescue animalsfollowing toxin exposure.

MATERIALS AND METHODS

Chemicals, biological reagents, and cell lines. Ricin, RTA, and RTB werepurchased from Vector Laboratories (Burlingame, CA). The amount of ricintoxin maintained in the laboratory was less than 100 mg at any one time, andtherefore it was exempt from select-agent registration, as defined by the CDC.Ricin toxoid (RT) was produced by treatment of holotoxin with paraformalde-hyde (4%, vol/vol), as described previously (26). Ricin and RT were dialyzedagainst phosphate-buffered saline (PBS) at 4°C in 10,000-molecular-weight-cut-off Slide-A-Lyzer dialysis cassettes (Pierce, Rockford, IL) prior to use in cyto-toxicity and mouse challenge studies. Paraformaldehyde (16%) was purchasedfrom Electron Microscopy Sciences (Fort Washington, PA). General proteinelectrophoresis reagents, including Tween 20, molecular weight markers, andprecast polyacrylamide gels, were purchased from Bio-Rad (Torrance, CA).Goat serum was purchased from Gibco-Invitrogen (Carlsbad, CA). Unless notedspecifically, all other chemicals were obtained from the Sigma Company (St.Louis, MO). Vero cells, irradiated MRC-5 human lung fibroblast cells, and themurine myeloma cell line P3X63.Ag8.653 were purchased from the AmericanType Culture Collection (Manassas, VA). Cell culture media were prepared bythe Wadsworth Center medium facility. Cell lines and hybridomas were main-tained in a humidified incubator at 37°C under 5% CO2.

Mouse strains and animal housing. The studies described here utilized femaleBALB/c mice approximately 6 to 8 weeks of age, purchased from Taconic Labs(Hudson, NY). Animals were housed under conventional, specific-pathogen-freeconditions and were treated in compliance with the Wadsworth Center’s Insti-tutional Animal Care and Use Committee (IACUC) guidelines.

Production and identification of MAb GD12. Female BALB/c mice wereprimed with RT (250 �g) administered i.p. on day zero and were then boostedwith RT (100 �g) on days 10 and 20. On day 24, the animals were euthanized,and total splenocytes were fused with the myeloma cell line P3X63.Ag8.653 (10,26). Hybridomas were seeded into wells of 96-well microtiter plates containing alayer of irradiated MRC-5 feeder cells. Hybridomas were cultured in a 1:1mixture of NCTC (Sigma Co.) and RPMI media containing 10% fetal calf serumand penicillin-streptomycin, and occasionally supplemented with 1% Opti-MAb(Gibco-Invitrogen). Hybridoma GD12 was cloned by limiting dilution and wastransitioned to a serum-free, protein-free, antibiotic-free medium (CD Hybrid-oma; Gibco-Invitrogen). For cell cytotoxicity assays and mouse challenge studies,GD12 was prepared as a sterile-filtered, serum-free, protein-free hybridomasupernatant. MAbs R70 and TFTB-1 were prepared as described previously (27).

ELISAs and RTA peptide arrays. Nunc Maxisorb F96 microtiter plates(Thermo Fisher Scientific) were coated with ricin, RTA, RTB, or bovine serumalbumin (BSA) (0.1 �g/well) in PBS (pH 7.4) and were incubated overnight at4°C in a humidified chamber; they were then washed three times with PBS–Tween 20 (PBS-T; 0.05%, vol/vol) and were blocked with goat serum (2%,vol/vol, in PBS-T) for 1 h at room temperature before being probed with MAbsor hybridoma supernatants diluted in blocking solution. For the enzyme-linkedimmunosorbent assays (ELISAs), horseradish peroxidase (HRP)-labeled goatanti-mouse IgG-specific polyclonal antibodies (SouthernBiotech) were used asthe secondary reagents, and 3,3�,5,5�-tetramethylbenzidine (TMB; Kirkegaard &Perry Labs, Gaithersburg, MD) was used as the colorimetric detection substrate.

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ELISA plates were analyzed with a SpectroMax 250 spectrophotometer, withSoftmax Pro software (version 5.2; Molecular Devices, Sunnyvale, CA).

The RTA peptide array consisted of 44 12-mers, each overlapping its neigh-bors by 6 amino acids, collectively spanning the RTA sequence (Table 1). Therationale for using relatively short peptides for this study was based on our recentsuccess in identifying linear B-cell epitopes on anthrax toxin using a library ofpeptides of this length (15). The peptides were synthesized, unbound, in 96individual tubes, in a 96-well plate format, and were provided at a 2.5-�mol scale(1.8 mg per peptide, average) at �75% purity (New England Peptide, Gardner,MA). The peptides were solubilized in dimethyl sulfoxide, and aliquots werestored at �20°C. RTA peptide arrays were prepared by coating the wells ofmicrotiter plates with individual peptides (1 �g/well), followed by overnightincubation at 4°C. The plates were washed three times with 0.025% Tween 20 inPBS, blocked with 1% goat serum for 1 h, and then incubated with individualMAbs for 4 h, all at 4°C. The plates were developed as described above for theELISAs.

Western blot analysis. For denaturing conditions, ricin and RTA were dilutedinto Laemmli sample buffer containing 5% (vol/vol) �-mercaptoethanol; thenthey were boiled for 6 min prior to being subjected to sodium dodecyl sulfate(SDS)–12% polyacrylamide gel electrophoresis (PAGE). For nondenaturingconditions, ricin and RTA were simply diluted into Laemmli sample buffer(without �-mercaptoethanol) prior to SDS-PAGE. For Western blot analysis,proteins were transferred from the polyacrylamide gels to nitrocellulose mem-branes (pore size, 0.45 �m; Bio-Rad) by semidry electroelution. Nitrocellulosemembranes were blocked with 2% (wt/vol) goat serum in PBS-Tween and werethen incubated with GD12 (1 �g/ml) overnight at 4°C. Membranes were probedwith goat anti-mouse IgG conjugated to HRP (0.4 �g/ml) (SouthernBiotech),developed using an enhanced chemiluminescent detection (ECL) kit (Bio-Rad),and then exposed to Kodak X-Omat film (Thermo Fisher Scientific).

Vero cell cytotoxicity assays. Vero cells were cultured in Dulbecco’s modifiedEagle medium supplemented with 10% fetal calf serum and were maintained ina humidified incubator (37°C, 5% CO2). For cytotoxicity assays, the cells weretrypsinized, adjusted to approximately 0.5 � 105 to 1.0 � 105 cells per ml, seeded(100 �l/well) onto white 96-well plates (Corning), and allowed to adhere over-night. Vero cells were then treated with either ricin (10 ng/ml), ricin-MAbmixtures, or medium alone (negative control) for 2 h at 37°C. The cells werewashed to remove noninternalized toxin or toxin-MAb mixtures and were thenincubated for 40 h. Cell viability was assessed using CellTiter-Glo reagent (Pro-mega) according to the manufacturer’s instructions, except that the reagent wasdiluted 1:5 in PBS prior to use. Luminescence was measured with a SpectraMaxL luminometer, as indicated above. All treatments were performed in triplicate,and 100% viability was defined as the average value obtained from wells in whichcells were treated with medium only.

Systemic and mucosal ricin challenge studies. Systemic and mucosal ricinchallenge studies were conducted as described previously (23, 34, 53). For sys-temic challenge studies, female BALB/c mice were injected i.p. with ricin (50�g/kg) diluted in PBS (final volume, 0.4 ml). Thereafter, the animals wereallowed food and water ad libitum. Hypoglycemia was used as a surrogatemarker of intoxication (34). Blood (5 �l) was collected from the tail veins of theanimals at 18- to 24-h intervals. Blood glucose levels were measured with anAccu-Chek Aviva handheld blood glucose meter (Roche, Indianapolis, IN). Incompliance with the end point specified by the Wadsworth Center’s IACUC,mice were euthanized when they became overtly moribund and/or when bloodglucose levels fell below 25 mg/dl. For statistical purposes, readings at or belowthe meter’s limit of detection of �12 mg/dl were set to that value.

Mucosal challenge studies were conducted as previously described (53). Ricin(5 mg/kg) was diluted into PBS and administered i.g. to female BALB/c mice bymeans of a 22-gauge, 1.5-in blunt-end feeding needle (Popper Scientific, NewHyde Park, NY). Animals were fasted for �2 h prior to challenge and were thenprovided with food ad libitum �1 h after challenge. Twenty-four hours later, theanimals were euthanized by CO2 asphyxiation. Segments of the proximal smallintestine were immersed in ice-cold cell lysis buffer (Cell Signaling, Beverly, MA)supplemented with protease inhibitors and were then homogenized on ice usinga Tekmar Tissumizer (Thermo Fisher Scientific). Monocyte chemotactic protein1 (MCP-1) levels in intestinal homogenates were determined by the BD cyto-metric bead array (CBA) flex set (BD Biosciences, San Jose, CA), as describedpreviously (53).

Passive protection studies. For systemic challenge studies, individual MAbswere diluted into endotoxin-free PBS, and each MAb was administered in a finalvolume of 0.4 ml to BALB/c mice by i.p. injection. Mice were challenged withricin by i.p. injection 24 h later. For mucosal challenge studies, MAbs werepassively administered to mice by the “backpack tumor” method (5, 28, 33, 41).For the backpack tumor studies, �2 � 106 hybridoma cells secreting the desired

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MAb (e.g., GD12, TFTB-1) were implanted subcutaneously into the backs ofmice, using a 1-ml syringe and a 25-gauge needle. Approximately 10 days later,blood was collected from the mice by tail bleed, and serum MAb concentrationswere determined by ELISA. The animals were challenged with ricin 24 h later,as described above.

In vitro translation assays. Ricin (5 ng) was incubated with individual MAbs(50 to 300 ng) for 10 min at room temperature, and the mixture was then addedto a cell-free reticulocyte lysate mixture containing ribosomes, amino acids, andATP (Retic Lysate IVT; Ambion, Austin, TX). The cocktail was incubated at25°C for 20 min before the addition of uncapped in vitro-transcribed templateRNA (1 �g) encoding firefly luciferase (Luc) or Xenopus elongation factor 1(XeF-1), each obtained from Promega (Madison, WI). The xef-1 template wasused as a negative-control mRNA, since the translated XeF-1 protein is incapa-ble of cleaving the luciferin substrate. The samples (25 �l) were incubated inmicrocentrifuge tubes for �3 h in a 30°C water bath and were then transferredto white 96-well plates (Corning, Lowell, MA) before the addition of Bright-Gloluciferin substrate (Promega). Luminescence was measured with a SpectraMax Lluminometer interfaced with SoftMax Pro software (version 5.2; Molecular De-vices). All experiments were performed in triplicate.

Statistical analysis and other software. Statistical analysis was carried out withExcel 2003 (Microsoft, Redmond, WA) and SigmaStat, version 3.5 (Systat Soft-ware, San Jose, CA). Passive protection studies were analyzed by one-way anal-ysis of variance (ANOVA) and Tukey’s posthoc tests. All other data wereanalyzed using Student t tests, unless indicated otherwise. The open-sourcemolecular visualization software PyMol (DeLano Scientific LLC, Palo Alto, CA),accessed at www.pymol.org, was used for epitope modeling.

RESULTS

Identification of MAb GD12. We sought to produce a MAbdirected against the immunodominant linear epitope on RTAthat had been identified by Castelletti and colleagues as apotential target of neutralizing serum IgG antibodies in hu-mans who had received ricin immunotoxin therapy (7). Towardthis end, we immunized groups of BALB/c mice with RT and

screened their sera for antibodies capable of reacting with apeptide spanning RTA residues L163 to I174 (TLARSFIICIQM). The sera from the majority of RT-immunized animalsreacted with this peptide (data not shown), indicating that thisepitope is recognized by the BALB/c strain of mice. Sera fromRiVax-immunized animals also demonstrated reactivityagainst this peptide, although less strongly than the RT serumsamples (data not shown). B-cell hybridomas were thereforeproduced from the spleens of RT-immunized animals andwere then screened by ELISA for those secreting antibodiesreactive against both RTA and the L163-to-I174 peptide. Hy-bridoma GD12 was identified as producing an IgG1 havingthese properties and was therefore chosen for further study.

For confirmation of the epitope specificity of MAb GD12,a hybridoma supernatant was used to probe an RTA peptidearray consisting of 44 overlapping 12-mers that collectivelyspan the length of the RTA sequence (Table 1). GD12preferentially reacted with a peptide (“F11”) correspondingto residues L163 to I174 (Fig. 1). It should be noted thatdespite numerous attempts, synthesis of the original peptidedescribed by Castelletti and colleagues (i.e., L161 to I175)(7) proved unsuccessful, and we were therefore unable todetermine the reactivity of GD12 with this specific epitope.

For comparative purposes, MAb R70 was also subjected topeptide array analysis. R70 (also known as UNIVAX 70/138) isthe most potent neutralizing MAb identified to date and one ofthe few MAbs shown to be capable of protecting mice againsta lethal dose of ricin (20, 27). R70 is proposed to recognize alinear epitope within a 26-amino-acid loop-helix-loop motif(Y91 to T116) on RTA, although the precise epitope has not

FIG. 1. GD12 and R70 recognize distinct linear epitopes on RTA. (A and B) Molecular models of ricin depicting the toxin’s active site (red),the GD12 epitope (gold), and the R70 epitope (teal). (C and D) Reactivities of GD12 and R70 with an RTA peptide array. ELISA plates werecoated with 44 overlapping 12-mer peptides spanning the length of RTA (Table 1). The plates were probed with GD12 (C) or R70 (D). GD12bound preferentially to peptide F11 (gold), encompassing residues 163 to 174 (TLARSFIICIQM). GD12 bound less well to peptide “H1” (white),which spans residues 247 to 258 (ILIPIIALMVYR). The two peptides, F11 and H1, show no sequence similarity except for the centrally locateddoublet of isoleucine. R70 bound exclusively to peptide E12 (teal), spanning residues 97 to 108 (NQEDAEAITHLF). Both arrays were performedat least three independent times with similar results. The data shown are from one representative experiment. The reactivities (y axis) of the MAbsrefer to the values (optical densities at 450 nm [OD450]) minus the background obtained by the peptide array ELISA, as described in Materialsand Methods.

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been identified (19). Peptide array analysis revealed that R70bound exclusively to a single peptide (“E12”), correspondingto residues N97 to F108 (NQEDAEAITHLF) on RTA (Fig.1). The locations of the epitopes recognized by GD12 and R70were modeled on the structure of RTA using PyMol software.The resulting image revealed that the MAbs recognized spa-tially distinct, solvent-exposed regions on the tertiary structure

of ricin that are roughly equidistant from the active site of thetoxin (Fig. 1A and B).

Reactivities of GD12 with RTA and holotoxin. To furthervalidate the specificity of GD12, we examined the reactivity ofGD12 with ricin holotoxin, RTA, and RTB, and we then com-pared these profiles to those obtained previously with MAbR70 (27). As expected, GD12 bound to ricin holotoxin andRTA but not to RTB (Fig. 2A). This profile was similar to thatobserved for R70, except that GD12 reacted slightly less wellwith RTA than with holotoxin (Fig. 2A and B). By Westernblot analysis, GD12 recognized the reduced and nonreducedforms of RTA (Fig. 2C), a result consistent with the MAbbinding to a linear (or continuous) epitope on RTA. The actualaffinity of GD12 for ricin was determined by BIAcore analysis.This analysis revealed that GD12 had a dissociation constant(KD) for ricin of approximately 2.9 � 10�9 M (Table 2), whichis virtually identical to the value we reported for R70 (27).

GD12 neutralizes ricin in vitro. We used a Vero cell cyto-toxicity assay to assess the capacity of GD12 to neutralize ricinin vitro. Ricin (10 ng/ml) was incubated for 1 h with GD12 orR70 at a range of concentrations, and the mixture was thenapplied in triplicate to Vero cells grown in 96-well microtiterplates (see Materials and Methods). The viability of the Verocells was determined 40 h later. GD12 protected Vero cellsfrom the cytotoxic effects of ricin in a dose-dependent mannerand had an estimated IC50 of �0.25 �g/ml (Fig. 3). GD12 wasapproximately twice as effective at neutralizing ricin as R70,which had an IC50 of �0.5 �g/ml. These data demonstrate thatan antibody against the linear epitope encompassing L163 toI174 of RTA is capable of neutralizing ricin in vitro.

Because GD12 and R70 recognize different epitopes on

FIG. 2. Specificity of GD12 for RTA. (A and B) Reactivities ofGD12 (A) and R70 (B) with ricin, RTA, RTB, or BSA, as determinedby ELISA. Microtiter plates were coated with 0.1 �g/well of each of thetarget antigens. Each datum point represents the average value forthree replicate wells. (C) Reactivity of GD12 with RTA by Westernblot analysis. Ricin (lanes 1) or purified RTA (lanes 2) was subjectedto SDS-PAGE under either nondenaturing, nonreducing conditions(left) or denaturing and reducing conditions (right), transferred to anitrocellulose membrane, and probed with GD12. GD12 recognizedricin (filled arrowhead) and RTA (open arrowhead) under nonreduc-ing, nondenaturing conditions, and it also recognized RTA underreducing and denaturing conditions. Purified RTA characteristicallyruns as a doublet due to differing degrees of glycosylation.

TABLE 2. Affinity and epitope specificity of GD12a

MAbb KD (M) KA (M�1) ka (M�1 s�1) kd (s�1) Epitope RTA residues

GD12 2.9 � 10�9 3.5 � 108 2.3 � 104 6.8 � 10�5 TLARSFIICIQM 163–174R70c 3.2 � 10�9 1.1 � 105 3.5 � 10�4 NQEDAEAITHLF 97–108

a KA, equilibrium association constant; ka, association rate constant; kd, dissociation rate constant.b Both MAbs are of the IgG1 isotype.c Kinetic data are reproduced from reference 26.

FIG. 3. GD12 protects Vero cells from ricin intoxication. GD12 orR70 at the indicated concentrations was incubated with ricin (10 ng/ml) for 1 h, and then the mixture was applied in triplicate to Vero cellsseeded in 96-well plates. Cell viability was assessed 40 h later asdescribed in Materials and Methods. The data points represent aver-ages for at least three replicate wells. Error bars represent standarddeviations.

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RTA, we wanted to test whether a combination of the twoMAbs would be more effective than either of the individualMAbs singly at neutralizing ricin in vitro. To assess this, wetested equivalent concentrations of GD12, R70, and a 1:1 mix-ture of GD12 and R70 in a Vero cell cytotoxicity assay. Themixture of GD12 and R70 did not result in enhanced neutral-ization activity relative to the activities of the MAbs testedindividually (data not shown), indicating that GD12 and R70do not act additively or synergistically.

GD12 protects mice from systemic ricin intoxication. Weused an established mouse model of systemic ricin intoxicationto assess the capacity of GD12 to neutralize ricin in vivo (23,34). For these challenge studies, hypoglycemia was used as asurrogate marker of intoxication (34). MAb GD12 or R70 waspassively administered to female BALB/c mice (5 to 40 �g/animal) by i.p. injection. Twenty-four hours later, the animalswere challenged by i.p. injection with the equivalent of fivetimes the LD50 of ricin (50 �g/kg), and blood glucose levelswere subsequently assessed at 24-h intervals. Ricin-challengedcontrol mice experienced a dramatic decline in blood glucoselevels within 30 h and subsequently died or were euthanized(Table 3). Mice that received GD12 or R70 all survived ricinchallenge. At early time points (24 and 48 h), the mean bloodglucose levels of ricin-challenged, GD12- or R70-treated micewere not statistically different from each other. However, at 72to 77 h postchallenge, mice treated with low doses of R70 (5.0to 10 �g per animal) had blood glucose levels significantlylower than those of the control group (Table 3), suggesting thatonly partial protection, and/or delayed recovery, was achieved.In contrast, at the same time point, the mean blood glucoselevels of animals treated with GD12 at any of the four doseswere not statistically different from those of control animals,suggesting that GD12 confers full protection under these con-ditions.

GD12 confers intestinal immunity to ricin in an oral chal-lenge model. Ricin is highly toxic to mucosal tissues, includingboth the respiratory and intestinal tracts (45, 53). It was there-fore of interest to determine whether GD12 was sufficient toconfer mucosal immunity to this toxin. We have previouslyestablished an in vivo model of intestinal intoxication in whichmice are challenged by gavage with ricin toxin (5 mg/kg) and,24 h later, MCP-1 levels in the animals’ proximal small intes-tines are assessed as a surrogate marker of tissue damage andinflammation (53). While we speculated that secretory IgA(SIgA) is necessary to confer intestinal immunity to ricin (26,53), we have recently shown that serum antitoxin IgG antibod-ies are in fact sufficient to confer protection in this oral chal-lenge model (L. Neal, E. McCarthy, C. McGuinness, and N.Mantis, unpublished data). To assess the capacity of GD12 toconfer intestinal immunity to ricin, the MAb was passivelyadministered to mice by the “backpack tumor” method, andanimals were challenged with ricin 10 to 12 days later. Thebackpack tumor model is a widely used and well-establishedmethod to assess the efficacy of an individual MAb at confer-ring immunity to mucosal pathogens and enterotoxins (1, 5, 13,28, 33, 41). Thus, groups of mice were implanted with GD12 orTFTB-1 hybridoma “backpacks,” as described in Materials andMethods. TFTB-1 is an IgG1 MAb that binds ricin but lacksdemonstrable neutralizing activity (27). Approximately 10 dayslater, blood was collected from the mice by tail bleed, andserum MAb concentrations were determined by ELISA. Se-rum TFTB-1 concentrations in these animals ranged from 1.0to 216 �g/ml, whereas serum GD12 concentrations rangedfrom 1.7 to 1,022 �g/ml. Twenty-four hours following bloodcollection, the animals were challenged with ricin.

Prior to performing mucosal challenges, we first wanted toconfirm that administration of GD12 by the backpack tumormethod confers systemic immunity to ricin, as we observedwhen the MAb was administered by i.p. injection (Table 3).Therefore, groups of GD12 backpack mice were challengedwith the toxin by i.p. injection. GD12 hybridoma backpackmice showed no outward signs of ricin intoxication and hadvirtually normal blood glucose levels 48 to 54 h after ricinchallenge (Fig. 4A). In contrast, mice bearing TFTB-1 back-packs and injected with ricin showed severe signs of intoxica-tion (e.g., ruffled fur, hunched posture, lethargy, laboredbreathing) and experienced rapid and dramatic declines inblood glucose levels (from �100 mg/dl to 40 mg/dl) within24 h (Fig. 4A). These animals were indistinguishable from thecontrol, non-backpack-bearing animals that had been chal-lenged with ricin, and they were euthanized 30 h postchallenge.

To assess mucosal immunity to ricin, groups of control orhybridoma backpack-bearing mice were challenged with ricinby the i.g. route. In unchallenged animals, intestinal MCP-1levels were approximately 30 pg/ml, whereas the levels in theintestines of ricin-challenged control (data not shown) orTFTB-1-treated mice were greater than 100 pg/ml (Fig. 4B).MCP-1 levels in the intestinal homogenates of ricin-chal-lenged, GD12 hybridoma backpack-bearing mice were indis-tinguishable from those observed in control, unchallengedmice. These data demonstrate that GD12 is sufficient to confermucosal immunity to ricin, at least in a mouse i.g. challengemodel.

TABLE 3. GD12 protects mice against systemic ricin intoxication

MAb anddose

(�g/animal)

Blood glucose levels (mg/dl)a in mice at thefollowing time postchallenge:

No. ofanimals

surviving/no.tested0 h 24–30 h 48–54 h 72–76 h

GD1240 94 � 3.1 68 � 5.3 74 � 7.2 109 � 4.6 3/320 98 � 7.0 74 � 5.6 86 � 13 95 � 8.1 3/310 96 � 14 50 � 9.8 78 � 13 96 � 18 4/45 113 � 19 61 � 24 76 � 18 84 � 14 4/4

R7040 98 � 7.8 71 � 2.5 66 � 1.5 88 � 1.7 3/320 95 � 5.9 72 � 2.1 80 � 10 105 � 11 3/310 108 � 6.0 61 � 12 73 � 13 70 � 11b 3/35 97 � 12 48 � 9.5 55 � 16 69 � 5.9c 4/4

None 102 � 12 30 � 13 NAd NAd 0/8

Control 102 � 12e 8/8

a Values reported are means � standard deviations.b Significantly lower than the control value by ANOVA with Tukey’s posthoc

test (q 5.810; P 0.009).c Significantly lower than the control value by ANOVA with Tukey’s posthoc

test (q 6.642; P 0.002).d NA, not applicable; all animals were sacrificed prior to this time point.e Blood glucose levels in unchallenged control animals did not differ signifi-

cantly over the course of the experiment.

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GD12 interferes with the enzymatic activity of RTA. It hasbeen postulated that one mechanism by which anti-RTA MAbsmay neutralize ricin is by interfering with the capacity of thetoxin to arrest protein synthesis (23). We expected that bothGD12 and R70 might affect the activity of RTA, because bothMAbs bind epitopes located in close proximity to the active site(Fig. 1A and B). We used a cell-free in vitro translation assayto examine this hypothesis (see Materials and Methods). GD12or R70 was incubated with ricin holotoxin or RTA for 10 minat room temperature and was then added to a rabbit reticulo-cyte lysate mixture in which luciferase (luc) mRNA was pro-vided as the template. Luminescence was used as an indicatorof in vitro translation and protein synthesis. In control reac-tions in which luc mRNA was combined with the cell-free invitro translation mixture in the absence of ricin and MAbs,robust luciferase activity was detected (Fig. 5A). This activitywas dependent on luc mRNA, as evidenced by the lack ofdetectable luciferase activity when xef-1 mRNA was used as anirrelevant template control. The addition of ricin reduced luc-dependent luciferase activity by more than 5 log units, dem-onstrating the effect of the toxin on ribosome function. The

addition of GD12 inhibited the enzymatic activity of ricin in adose-dependent manner, although even at the highest concen-tration of MAb tested, only partial inhibition of activity wasobserved (Fig. 5A). R70 also interfered with the enzymaticactivity of ricin, but less effectively than GD12 (Fig. 5B). TwoMAbs directed against RTB, 24B11 and TFTB-1, had no affecton the enzymatic activity of the toxin (Fig. 5B). These datademonstrate that in an in vitro cell-free system, both GD12 andR70 can interfere, at least partially, with the enzymatic activityof ricin.

DISCUSSION

The development and licensure of vaccines for biodefense isinherently challenging, because human efficacy trials of candi-date vaccines are generally not feasible or ethical. Recognizingthis roadblock, the FDA has implemented the two-animal rule,by which candidate vaccine development can proceed based onefficacy studies of two or more relevant animal models. For ananimal model to be deemed relevant, the underlying patho-genesis and mechanisms of immunity in response to the select

FIG. 4. Mice bearing GD12 hybridoma backpack tumors are protected against the effects of both systemic and mucosal ricin exposure. Groupsof female BALB/c mice carrying GD12 or TFTB-1 hybridoma backpacks were challenged with ricin by the i.p. or i.g. route, as described inMaterials and Methods. (A) Intraperitoneal challenge. Ricin (50 �g/kg) was administered by i.p. injection to control, non-hybridoma-bearing mice(rightmost bar) or to mice bearing GD12 or TFTB-1 backpack tumors, as indicated. Blood glucose levels were assessed �48 h later. Each barrepresents the average (� standard deviation) for two independent experiments with a total of 4 to 12 mice per group. The asterisk indicates thatthe mean value is not significantly different from that for untreated control animals (P � 0.05). (B) Intragastric challenge. Ricin (5 mg/kg) wasadministered by gavage to mice bearing GD12 or TFTB-1 backpack tumors, as indicated. TFTB-1 is an IgG1 MAb that binds ricin but lacksdemonstrable neutralizing activity. The animals were euthanized 24 h later, and the small intestines were collected immediately for MCP-1 analysis,as described in Materials and Methods. Each bar represents the average value (� standard error of the mean) for a single experiment with a totalof 6 mice per group. The experiment was performed two independent times, with identical results. The asterisk indicates that the mean value isnot significantly different from that obtained for untreated control animals (P � 0.05).

FIG. 5. GD12 partially inhibits the enzymatic activity of ricin in vitro. (A) Ricin and GD12 (at the indicated concentrations) were incubated witha cell-free in vitro translation mixture before the addition of luciferase mRNA. Luciferase activity, reported in relative light units (RLUs), isrepresentative of protein translation. (B) GD12 and a panel of antiricin MAbs (400 ng each) were compared in the same assay. Each experimentwas performed in triplicate; error bars represent standard deviations.

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agent in question must be similar to those in humans. In ad-dition, the animal model(s) must take into account the possi-bility that toxin or pathogen exposure can occur by multipleroutes, such as injection, inhalation, and ingestion. In suchsituations, vaccines may need to elicit both systemic and mu-cosal immunity in order to be considered useful. Finally, when-ever possible, identification of the underlying correlates ofprotection, as well as the development of surrogate markers ofimmunity, should be validated in humans.

The fact that RTA has been under investigation for morethan 2 decades as a possible immunotoxin for cancer therapyhas yielded some basic information regarding the human im-mune response to ricin (7, 50). Most notable is the recentreport by Castelletti and colleagues, who identified an immu-nodominant linear B-cell epitope on RTA (residues 161 to175) that was recognized by serum antibodies from 15Hodgkin’s lymphoma patients who had received RTA immu-notoxin (7). While that study demonstrated that affinity-puri-fied antipeptide antibodies were capable of neutralizing ricin invitro, it did not determine whether the antibodies were capableof neutralizing ricin in vivo. This is not an academic point,given that Maddaloni and colleagues recently described severalMAbs against RTA that were highly effective at neutralizingricin in vitro yet afforded no protection in a mouse model ofricin challenge (23). In an effort to resolve this issue, we haveproduced and characterized GD12, a murine IgG1 MAb di-rected against virtually the same epitope on RTA as that de-scribed by Castelletti and colleagues. We found that GD12bound the holotoxin with high affinity and neutralized ricinwith an IC50 of �0.25 �g/ml, as determined by a Vero cell-based cytotoxicity assay. Most importantly, we found that thisMAb, when administered passively to mice, was sufficient toprotect the animals against both systemic (i.e., intraperitoneal)and mucosal (i.e., intragastric) ricin challenge. These data,when considered in the context of the work by Castelletti andcolleagues, establish the importance of residues 161 to 175 onRTA as a target of ricin-neutralizing antibodies in vitro and invivo.

RTA is composed of three distinct subdomains, two of whichare now known to be the targets of neutralizing MAbs (14).Subdomain 1 spans residues 1 to 117; subdomain 2 spansresidues 118 to 210; and subdomain spans 3 residues 211 to276. The epitope recognized by GD12 is localized entirelywithin subdomain 2. Specifically, the GD12 epitope maps to along �-helix that forms the core of the subdomain. R70, on theother hand, recognizes subdomain 1 (19), although prior to thisstudy the precise epitope recognized by this MAb was notknown. We determined by peptide array analysis that R70binds exclusively to a single 12-mer consisting of residues N97to F108 (NQEDAEAITHLF). This epitope, like that recog-nized by GD12, is situated within an �-helix (14). There isevidence to suggest that antibodies directed against subdomain3 may be poor neutralizers, or even potentiators, of ricin tox-icity. For example, Maddaloni and colleagues identified severalanti-RTA MAbs capable of enhancing the potency of ricin invitro and in vivo. One of these MAbs, RAC23, is postulated tobind residues within subdomain 3 (23). In addition, we haveidentified several MAbs directed against linear epitopes onsubdomain 3, and at least preliminarily, these MAbs appearunable to neutralize ricin in vitro (J. O’Hara and N. Mantis,

unpublished data). Systematic identification of the B-cellepitopes on RTA that elicit both neutralizing and nonneutral-izing antibodies may have immediate implications for vaccinedesign.

While this study clearly demonstrated the potential of GD12to neutralize ricin in vitro and in vivo, the mechanism by whichthis is achieved remains unresolved. The epitope recognized byGD12 is immediately adjacent to two residues (E177 andR180) on RTA known to be involved in rRNA catalysis (17).For this reason, we suspected that the association of GD12with RTA may interfere with the subunit’s enzymatic activity,possibly by blocking access to the rRNA substrate or by phys-ically distorting the active site. Indeed, GD12 was observed toreduce the capacity of RTA to inhibit protein synthesis in an invitro cell-free assay. However, the relevance of this observationto the capacity of GD12 to neutralize ricin in whole cells oranimals remains unclear. RTA interacts with its substrate onlyafter a circuitous journey of the holotoxin from the plasmamembrane. Specifically, following endocytosis, ricin undergoesvesicular retrograde transport to the ER. RTA and RTB dis-sociate within the ER, after which RTA is unfolded and deliv-ered across the ER membrane by the Sec61 complex, a processknown as retrotranslocation (21, 36, 39). If GD12 acts throughinterference with the enzymatic activity of RTA, then the MAbwould have to remain associated with RTA throughout theretrograde and retrotranslocation processes. Alternatively,based on several recent reports documenting the mechanismby which MAbs neutralize Shiga toxins (Stx) (18, 46), we spec-ulate that GD12 (and other anti-RTA MAbs) may interferewith the intracellular trafficking of ricin. For example, Krautz-Peterson and colleagues recently demonstrated that a MAbdirected against the A subunit of Stx1 blocked the retrogradetransport of the toxin into the Golgi apparatus and the endo-plasmic reticulum (18).

As a biothreat agent, ricin can potentially be disseminated asan aerosol, or it could be used to contaminate food and watersupplies. By either route, the toxin would first contact a mu-cosal surface in the host, an important consideration when aricin vaccine is being developed and evaluated. Studies of an-imal models have established that both the respiratory andintestinal epithelia are sensitive to ricin intoxication (45, 53). Inthe gut, protection of the intestinal epithelium from entero-toxins is a function generally ascribed to SIgA (22, 51). How-ever, we have shown in a separate study that serum antiricinIgG antibodies are themselves sufficient to protect the intesti-nal epithelium from ricin intoxication, at least in a mousemodel (L. Neal et al., unpublished). In the present study, wehave demonstrated, using the backpack tumor model, thatGD12 was sufficient to protect the intestinal mucosa from ricin.While the underlying mechanism by which serum IgG confersmucosal immunity to ricin remains to be elucidated, this find-ing has potentially important implications for vaccine delivery,in that it suggests that parenteral immunization with a ricinsubunit vaccine may be sufficient to elicit immunity in bothsystemic and mucosal compartments.

The results of a recent pilot phase I clinical trial of RiVaxunderscore the need to identify additional correlates of immu-nity to ricin (52). Of particular concern was the fact that totalanti-RTA titers, as determined by ELISA, were poor predic-tors of the ability of individual serum samples from RiVax-

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immunized patients to neutralize ricin. While antitoxin titers inthemselves provide a convenient means by which seroconver-sion can be assessed in a human cohort, they do not necessarilyprovide useful information regarding toxin-neutralizing activ-ity. The only means, at present, by which to assess neutralizingactivity is a cell-based cytotoxicity assay or passive protectionstudies of mice. Unfortunately, those types of assays are rela-tively insensitive and require significant amounts of serum. Wepropose, based in part on the results of the current study, thatit may be possible to predict an individual’s immune statusrelative to ricin from serum antibody reactivity against specificRTA-derived linear B-cell epitopes. For example, in prelimi-nary studies, we have observed a significant correlation be-tween serum antibody reactivity with three distinct linearepitopes of RTA (including L163 to I174) in mice and protec-tion against systemic lethal toxin challenge (J. O’Hara, L. Neal,and N. Mantis, unpublished data). Microarray profiling of se-rum antibodies is being applied to the development and eval-uation of effective diagnostics, therapeutics, and vaccines in theareas of cancer and emerging infectious diseases (29, 30). Wepropose that such technology is ideally suited to the field ofbiodefense, in which the ultimate licensure of vaccines must beachieved in the absence of clinical efficacy trials.

ACKNOWLEDGMENTS

We thank the following individuals at the Wadsworth Center forassistance with this project: Helen Johnson (Animal Pathology Core)for tissue processing, Jane Kasten-Jolly (Immunology Core) for BIA-core analysis, and Karen Chave (Protein Expression Core) for mono-clonal antibody purification.

This work was supported by grants from the National Institutes ofHealth (U01AI070624; to R.N.B.) and the Northeast Biodefense Cen-ter (U54-AI057158-Lipkin; to N.J.M.).

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