Toxin-Mediated Paracellular Transport of Antitoxin AntibodiesFacilitates Protection against Clostridium difficile Infection

Z. Zhang,a X. Chen,b L. D. Hernandez,a P. Lipari,a A. Flattery,a S.-C. Chen,a S. Kramer,a J. D. Polishook,a F. Racine,a H. Cape,a

C. P. Kelly,b A. G. Theriena

Merck Research Laboratories, Merck & Co., Inc., Kenilworth, New Jersey, USAa; Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School,Boston, Massachusetts, USAb

The exotoxins TcdA and TcdB are the major virulence factors of Clostridium difficile. Circulating neutralizing antitoxin anti-bodies are protective in C. difficile infection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlo-toxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralizetoxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in activeantibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevanthuman IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggest-ing that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in ham-sters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In an in vitro two-di-mensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelialcell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increasedupon addition of toxin to the apical side. Similar data were obtained with F(ab=)2 fragments, which lack an Fc domain, consistentwith FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damagecaused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mech-anism of humoral response-mediated protection against enteric pathogens.

The enteric pathogen Clostridium difficile is a Gram-positive,anaerobic, spore-forming bacterium. C. difficile infections

(CDI) cause diarrhea, pseudomembranous colitis, and in somesevere cases colonic rupture and death (1). In recent years, CDI-associated morbidity and mortality have increased significantly,and the disease poses a significant health care threat in the UnitedStates and globally (2).

The major virulence factors of C. difficile are the Rho-inacti-vating toxins A and B (TcdA and TcdB), which consist of largesingle-chain proteins with similar multidomain structures andfunctions (3–5). It is thought that both toxins bind to target mam-malian cells (typically gut epithelial cells) at least in part throughtheir C-terminal receptor-binding domains (combined repetitiveoligopeptide [CROP] domains) and become internalized via re-ceptor-mediated endocytosis (6, 7). Following internalization,acidification of the endosome leads to a conformational changewithin the toxins that results in translocation of the glucosyltrans-ferase domain (GTD) across the endosomal membrane and auto-cleavage via the cysteine protease domain (8–10). This releases theGTD into the cytoplasm, where it inactivates rho-type GTPase bycovalent glucosylation, resulting in disruption of the cytoskeleton,changes in cellular morphology, and eventually cell death (11, 12).The resulting disruption of the gut epithelial barrier leads to thesymptoms of the disease, which are exacerbated by toxin-medi-ated recruitment of a proinflammatory host immune response(3–5, 13).

Although standard-of-care antibiotic therapy with metronida-zole, vancomycin, or fidaxomicin is often effective in resolvingprimary cases of CDI, �25% of patients develop one or morerecurrent episode of CDI even after an initial cure (1, 14). Multiplelines of evidence suggest that adaptive humoral immune re-sponses against the C. difficile toxins are protective in both pri-

mary and recurrent CDI. Kyne et al. first showed that circulatinglevels of anti-TcdA IgG were positively correlated with a lower rateof primary CDI in colonized patients (15) and with a lower rate ofrecurrence among patients who had suffered a primary episode ofCDI (16). A more recent study has shown that anti-TcdB IgG levelsalso correlate with protection against CDI recurrence (17). Whilecorrelative in nature, these studies provided the impetus to test thehypothesis that antitoxin antibodies might be protective in CDI, andthis has now been demonstrated in multiple animal models (18–22).More significantly for human disease, passive immunotherapy withthe antitoxin neutralizing antibody combination consisting of actox-umab and bezlotoxumab (specific for TcdA and TcdB, respectively)has been shown to reduce CDI recurrence in human patients (23).The combination of actoxumab and bezlotoxumab, both fully hu-man IgG1 antibodies, is currently in phase III clinical developmentfor the prevention of recurrent CDI.

Despite the evidence that circulating antitoxin antibodies are

Received 26 August 2014 Returned for modification 12 September 2014Accepted 5 November 2014

Accepted manuscript posted online 10 November 2014

Citation Zhang Z, Chen X, Hernandez LD, Lipari P, Flattery A, Chen S-C, Kramer S,Polishook JD, Racine F, Cape H, Kelly CP, Therien AG. 2015. Toxin-mediatedparacellular transport of antitoxin antibodies facilitates protection againstClostridium difficile infection. Infect Immun 83:405–416. doi:10.1128/IAI.02550-14.

Editor: V. B. Young

Address correspondence to A. G. Therien, alex_therien@merck.com.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.02550-14.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.02550-14

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protective in CDI, the question of how systemic IgG antibodiesbind to and neutralize toxins presumably located largely in the gutlumen remains unanswered. Previous studies have shown that theneonatal immunoglobulin receptor FcRn mediates specific trans-port of IgG antibodies across the gut wall (24, 25) and plays a rolein antibody-mediated protection against infections of the gastro-intestinal tract in mice (26, 27). In this study, we use both in vitroand in vivo model systems to explore the mechanism throughwhich neutralizing antitoxin IgG antibodies cross the gut epithe-lium and neutralize C. difficile toxins located in the lumen of the gut,thereby protecting the host against CDI. We show that transepithelialneutralization depends on nonspecific paracellular transport of theantibodies rather than specific transcellular Fc-receptor-mediatedtransport mechanisms and that toxin-induced damage paradoxicallyfacilitates the transport of (and toxin neutralization by) antitoxin an-tibodies.

MATERIALS AND METHODSToxins and cells. Purified TcdA and TcdB from strain VPI 10463 werepurchased from Native Antigen (Upper Heyford, Oxfordshire, UnitedKingdom). T84 cells were cultured in Dulbecco’s modified Eagle medium(DMEM)-F12 medium (ATCC) supplemented with 5% fetal bovine se-rum (FBS) and penicillin-streptomycin (P/S). Caco-2 cells were culturedin Eagle’s minimal essential medium (EMEM) (ATCC) supplementedwith 10% FBS, nonessential amino acids, sodium-bicarbonate, and P/S.MDCK cells were cultured in DMEM (Life Technologies) supplementedwith 10% FBS plus P/S.

Mouse primary CDI model. The mouse antibiotic-associated CDImodel has previously been described (28). Briefly, 8-week-old femaleC57BL6 wild-type or FcRn�/�mice (purchased from Jackson Laboratory)were orally given an antibiotic mixture of kanamycin (40 mg/kg of bodyweight/day), gentamicin (3.5 mg/kg/day), colistin (4.2 mg/kg/day), met-ronidazole (21.5 mg/kg/day), and vancomycin (4.5 mg/kg/day) for 3 days,followed, 2 days later, by a single dose of clindamycin (10 mg/kg) intra-peritoneally 24 h prior to challenge with 1 � 105 CFU of C. difficile (strainVPI 10463) by gavage. Actoxumab and/or bezlotoxumab were adminis-tered intraperitoneally to mice, each at a dose of 250 �g, 1 day prior tochallenge with C. difficile. Statistical analysis was carried out using the logrank test with Bonferroni correction using the GraphPad Prism 6 softwareprogram (GraphPad Software, San Diego, CA).

Mouse recurrent CDI model. Mice were treated with oral vancomy-cin (50 mg/kg, administered by gavage) once daily for 5 days following C.difficile challenge. Actoxumab and bezlotoxumab in combination wereadministered intraperitoneally, each at a dose of 250 �g, 1 day prior tochallenge with C. difficile and again 1 day after discontinuing vancomycintherapy. Statistical significance was determined using the log rank testwith Bonferroni correction using the GraphPad Prism 6 software pro-gram (GraphPad Software, San Diego, CA).

Hamster CDI model. C. difficile B1 spores (generously provided by D.Gerding, Hines VA Hospital, Hines, IL) were prepared from confluentcultures grown anaerobically on agar plates for 12 days. Cells were washedfrom the surface of the agar in an aerobic environment using sterile ice-cold deionized water (diH2O) and then left on ice for 3 h. Spores wereplaced in microcentrifuge tubes and were separated from vegetative cellsby centrifugation at 13,000 rpm for 3 min at 4°C and then further purifiedby serial washes in ice-cold diH2O and repeat centrifugation. Spores wereresuspended in diH2O and quantified by anaerobically incubating ali-quots on Clostridium difficile selective agar (CDSA) supplemented with0.12 ml 10% sodium taurocholate in sterile water, and aliquots werestored at �80°C. Male golden Syrian hamsters, approximately 100 g inweight (Charles River Laboratories), were preconditioned for C. difficilesusceptibility by oral administration of clindamycin at 30 mg/kg 5 daysprior to infectious spore challenge. On day 0, hamsters were infected witha saline suspension containing �50 spores of toxigenic C. difficile strain

B1. Hamsters were divided into 3 treatment groups of 6 animals each, plus1 group of infected controls (n � 2). Treatment groups included actox-umab-bezlotoxumab at 50 mg/kg each subcutaneously (s.c.) once daily(q.d.) for 4 days beginning 5 h after infectious challenge, nonspecific hu-man IgG (huIgG) (Equitech-Bio., Inc.) administered at 2 g/kg s.c. q.d. for4 days beginning 4 h after infectious challenge, or a combination of thetwo above treatment regimens (actoxumab-bezlotoxumab plus huIgG).Hamsters were monitored at least twice daily for morbidity, mortality,and signs of disease, including diarrhea (“wet tail”), body weight loss,lethargy, hunched posture, or distended abdomen. Animals were eutha-nized if judged to be in a moribund state or if weight loss exceeded 20%.Statistical significance was determined using the log rank test with Bon-ferroni correction using the GraphPad Prism 6 software program (Graph-Pad Software, San Diego, CA).

Tissue distribution of actoxumab-bezlotoxumab in hamsters. Malegolden Syrian hamsters were preconditioned with clindamycin on day �5relative to infectious spore challenge, as described above. On day 0, ham-sters were divided into 2 groups, one left untreated and the second in-fected with �50 spores of C. difficile strain B1. At 5 h after spore challenge,infected and uninfected hamsters received a single s.c. dose of actoxumab-bezlotoxumab at 50 mg/kg each. Hamsters were euthanized at time pointsafter administration of antibodies, and the gastrointestinal tract (GI) fromthe stomach to the rectum was collected. The GI tract was sectioned intoduodenum, jejunum, ileum, cecum, and ascending colon. Cecum con-tents were collected undiluted, and contents of other GI sections wereobtained from the lumen by flushing each section with 1 ml sterile phos-phate-buffered saline (PBS). GI tissue was then thoroughly rinsed withsterile PBS, weighed, and homogenized in sterile PBS containing 10%glycerol (5 ml for cecum and 1.5 ml for other GI sections). The concen-tration of human IgG in each tissue or lumenal contents was determinedusing a human IgG quantitation enzyme-linked immunosorbent assay(ELISA) (Bethyl Laboratories). Statistical significance was determined byfirst converting the data to the log of the antibody concentration, carryingout unpaired, two-tailed t tests comparing healthy versus CDI hamsters,and applying the Bonferroni correction for multiple comparisons.

Immunohistochemistry. Hamsters were preconditioned with clinda-mycin, infected (or not) with C. difficile strain B1 spores as describedabove, and dosed with actoxumab-bezlotoxumab (50 mg/kg of each an-tibody) subcutaneously, once 5 h after spore challenge and a second time24 h later. Cecum tissues were collected 24 h after the second dose ofactoxumab-bezlotoxumab or vehicle from healthy untreated hamsters(unchallenged and dosed with vehicle), healthy treated hamsters (unchal-lenged with C. difficile spores and dosed with actoxumab-bezlotoxumab),or diseased treated hamsters (challenged with C. difficile spores and dosedwith actoxumab-bezlotoxumab), fixed with 10% neutral buffered forma-lin for 22 h, and subsequently processed for formalin-fixed paraffin-em-bedded sections. Localization of injected actoxumab-bezlotoxumab wascarried out using a goat anti-human IgG antibody from Jackson Immuno-Research (West Grove, PA), followed by sequential incubation with a goathorseradish peroxidase (HRP) polymer and diaminobenzidine in a Super-Picture polymer detection kit (Invitrogen, Grand Island, NY). Tissue sectionswere counterstained with hematoxylin (Biocare Medical, Concord, CA) forstructural analysis.

Generation of F(ab=)2 fragments of actoxumab and bezlotoxumab.F(ab=)2 fragments of actoxumab and bezlotoxumab were generated usingthe Pierce F(ab=)2 preparation kit (Thermo Scientific, Waltham, MA,USA) according to the manufacturer’s instructions. Fc fragments wereremoved from F(ab=)2 by gel filtration chromatography using a Superdex-S200 column (GE Healthcare Life Sciences, Piscataway, NJ, USA) pre-equilibrated with PBS. Five-milliliter fractions were collected, and thepeaks corresponding to the F(ab=)2 fragment were pooled. F(ab=)2 puritywas ascertained under reducing and nonreducing conditions by SDS-PAGE, using 4 to 12% Novex Tris-glycine gels (Life Technologies, GrandIsland, NY, USA), followed by Coomassie brilliant blue staining. No con-

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tamination of samples by uncleaved IgG was detectable (see Fig. S4A andB in the supplemental material).

Two-dimensional cell culture and TER measurements. To establishthe two-dimensional culture system, cells were seeded on the well insert(HTS 24-multiwell insert, 351181; BD Falcon, Bedford, MA, USA) at (0.5to 1.0) � 105 cells per well, with 250 �l medium in the apical chamber and800 �l medium in the basolateral chamber. Cells were cultured for �14days at 37°C (in 5% CO2) to ensure full differentiation and confluence, asassessed by transepithelial electrical resistance (TER) measurements pla-teauing at �600 � · cm2 (Caco-2) or �1,000 � · cm2 (T84), measuredusing the epithelial volt-ohm meter Millicell ERS-2 (EMD Millipore, Bil-lerica, MA, USA). For fast-growing MDCK cells, maximal TER (plateau-ing at �600 � · cm2) was achieved 1 to 2 days after seeding. For assess-ments of toxin neutralization, actoxumab or bezlotoxumab (or F(ab=)2

fragments thereof) were added to a final concentration of 50 �g/ml to theapical chamber or 100 �g/ml to the basolateral chamber, either 18 h be-fore or immediately before addition of toxin to the apical chamber (see thefigure legends). TER measurements were obtained at different time pointseither immediately before (t � 0 h) or at various time points after additionof various concentrations of TcdA or TcdB to either the apical or basolat-eral chamber. TER measurements were normalized to values obtained inthe absence of toxin at each time point to account for minor time-depen-dent variability. Ten-microliter samples were taken from the apical cham-ber either immediately before addition of the toxin (t � 0 h) or at varioustime points after addition of toxin (see the figure legends) for quantitationof the antibody concentration. Fifty-microliter samples were taken fromthe basolateral chamber 48 h after addition of toxin for quantitation of thetoxin concentration (see below). Statistical significance was determinedby matched two-way analysis of variance (ANOVA) with Tukey’s multi-ple-comparison test using the GraphPad Prism 6 software program(GraphPad Software, San Diego, CA).

ELISAs. ELISAs were carried out according to standard methodology.Briefly, high-protein-binding ELISA plates (Fisher Scientific, Waltham,MA, USA) were coated with capture antibody [chicken anti-humanF(ab=)2 secondary antibody, SA1-72043; Fisher] at 10 �g/ml and blockedusing blocking buffer (Fisher Scientific). Samples isolated from the apicalchamber of the two-dimensional culture system (see above) were dilutedin blocking buffer and added to wells, with purified actoxumab, bezlotox-umab, or F(ab=)2 fragments thereof used as standards. Wells were washed,and HRP-linked secondary antibody (300 ng/ml; Fisher Scientific) inblocking buffer was added for detection of bound antibodies. Followingextensive washing, HRP substrate solution (Fisher Scientific) was addedand the luminescent signal was read on a SpectraMax M4 instrument.Quantification of toxins in the basolateral chamber was carried out byELISA using a kit from tgcBIOMICS (Bingen, Germany) and followingthe manufacturer’s instructions. Statistical significance was determinedby matched two-way ANOVA with Tukey’s multiple-comparison test us-ing GraphPad Prism 6 software (GraphPad Software, San Diego, CA).

Ethics statement. All procedures with animals were performed in ac-cordance with the highest standards for the humane handling, care, andtreatment of research animals and adhered to the National ResearchCouncil’s Guide for Care and Use of Laboratory Animals, 8th ed. Mousestudies were approved by the Beth Israel Deaconess Medical Center(BIDMC) Institutional Animal Care and Use Committee (protocol num-ber 101-2013). Hamsters studies were approved by the Merck & Co., Inc.(in Kenilworth, NJ) Institutional Animal Care and Use Committee (pro-tocol number 0280-12).

RESULTSRole of Fc-mediated transport in protection against C. difficileinfection. The neonatal Fc receptor FcRn has previously been im-plicated in transport of IgG from the systemic to the lumenal sideof the gut wall (24, 25, 29) and has been shown to play a role inIgG-mediated protection against some forms of bacterial infec-tions (26, 27). To assess whether these observations extend to C.

difficile infections, we evaluated the human IgG1 antibody com-bination actoxumab-bezlotoxumab using a murine CDI modelcomparing FcRn knockout mice with wild-type (WT) littermates.Since efficacy of actoxumab-bezlotoxumab had not previouslybeen demonstrated in mice, we first administered actoxumab, be-zlotoxumab, or a combination of both antibodies intraperitone-ally to WT mice 24 h before challenge with the toxigenic C. difficilestrain VPI 10463. The antibody combination provided excellentprotection against mortality in this model, while the individualantibodies alone were only partially protective (Fig. 1A). Further-more, in both primary (Fig. 1B) and recurrent (Fig. 1C) CDI par-adigms (see Materials and Methods), a single administration ofactoxumab-bezlotoxumab increased survival to the same extentin WT mice and in FcRn-null mice, demonstrating that the FcRnreceptor does not play a significant role in antibody-mediatedprotection in this murine model. These data, however, do not ruleout the possibility that other Fc-mediated transport mechanismsmay be involved in the efficacy of actoxumab-bezlotoxumab. Toaddress this, we used the gold standard Syrian hamster model ofCDI, in which actoxumab-bezlotoxumab has previously beendemonstrated to be protective (18). Since targeted genetic dele-tion is not possible in hamsters, and to cover any and all possibleFc-mediated activities of actoxumab-bezlotoxumab, we codosedthe antibody combination in a therapeutic paradigm (see Materi-als and Methods) with or without a 40-fold excess of irrelevanthuman IgG (which contains �63% IgG1; data not shown), rea-soning that such antibodies should compete with actoxumab-be-zlotoxumab for binding to Fc receptors. As shown in Fig. 1D,codosing with excess human IgG had no significant impact onactoxumab-bezlotoxumab-mediated protection, suggesting thatsuch protection is independent of Fc-mediated transport mecha-nisms.

Tissue distribution of actoxumab-bezlotoxumab in thehamster CDI model. To gain insight into whether and to whatextent systemic antibodies reach the site of infection in thelumen of the gut, we characterized the intestinal tissue distri-bution of systemically administered actoxumab and bezlotox-umab in hamsters. Hamsters challenged with or without a tox-igenic strain of C. difficile (strain B1 [18]) were injected with asingle dose of actoxumab-bezlotoxumab at 50 mg/kg. Samples ofintestinal tissues were collected from both infected and uninfectedanimals at various time points after challenge, and levels of humanIgG were measured by ELISA in lumenal contents and in thewashed whole-gut tissues. While no significant differences were ob-served in actoxumab-bezlotoxumab levels within the intestinal tis-sues between infected and uninfected hamsters (Fig. 2A), levels ofactoxumab-bezlotoxumab in the GI lumen were significantly higherin infected hamsters, in particular in the cecum, where antibody levelswere nearly undetectable in healthy animals (Fig. 2B; see also Fig. S1in the supplemental material). Increased antibody transport acrossthe gut wall in diseased hamsters was confirmed at the cellular levelusing immunohistochemistry (Fig. 2C; see also Fig. S1C). In healthyhamsters, antibodies are located primarily in the subepithelial space(including the lamina propria) of the mucosa, with no staining in theepithelial layer, which acts as a barrier preventing leakage of antibod-ies into the gut lumen. Conversely, in hamsters infected with C. dif-ficile, the actoxumab-bezlotoxumab signal is present throughoutthe mucosa, including parts of the epithelial layer, which exhibitssignificant damage/sloughing, allowing antibodies to enter the in-testinal lumen. Together, these data show that transport of sys-

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temic antibodies to the gut lumen is significantly facilitated bydamage to the gut epithelium mediated by C. difficile toxins.

Transepithelial toxin neutralization by antibodies in a two-dimensional culture system. To better understand the protectiverole of circulating antitoxin antibodies against toxins in the gutlumen, we used a two-dimensional cell culture system whereinapical and basolateral compartments are separated by a singlemonolayer of differentiated epithelial cells (30–33). The systemmimics the polarized nature of the intact intestinal mucosal epi-thelium which separates the gut lumen (apical side) from the sub-epithelial/systemic space (basolateral side). The integrity of theepithelial layer is monitored by measuring the transepithelial elec-trical resistance (TER); a drop in TER indicates that the integrityof the epithelial monolayer has been compromised (31).

For studies aimed at understanding how neutralizing antibod-ies present on the basolateral/systemic side can neutralize toxin onthe apical/lumenal side, we first demonstrated that TcdA andTcdB added to the apical chamber cause significant time- andconcentration-dependent decreases in TER in the colonic epithe-lial cell line, Caco-2 (Fig. 3A and B). To confirm that the epithelialmonolayer was fully differentiated and polarized, we replicatedthe previously published observation (33) that colonic epithelialcells are more sensitive to TcdB applied to the basolateral side thanto that applied to the apical side, whereas sensitivity to TcdA iscomparable on the two sides (see Fig. S2 in the supplemental ma-terial). The effects of the neutralizing antibodies actoxumab and

bezlotoxumab on toxin-induced damage are shown in Fig. 3C, D,E, and F for Caco-2 cells (see also Fig. S3 for T84 cells). Whenapplied to the same side (apical) as the toxins, the antibodies showa strong neutralizing effect on the toxins, as shown by rightwardsshifts in the concentration-response curves of the toxins (shown24 h after addition of toxin) (Fig. 3E and F). Significant, thoughsmaller, shifts are also observed when antibodies are added to thebasolateral/systemic side of the epithelium, approximating theprotection afforded by systemically circulating neutralizing anti-bodies in the context of CDI. To assess the potential role of Fc-mediated transport in transepithelial neutralization, we generatedF(ab=)2 fragments of actoxumab and bezlotoxumab, which lack anFc region. The F(ab=)2 fragments, which were first shown to bedevoid of contamination by uncleaved antibody and fully neutral-izing in a toxin-induced cell death assay (34) (see Fig. S4), neu-tralized the effects of toxins at least as efficiently as intact antibod-ies (Fig. 3E and F), demonstrating that the Fc regions of IgGmolecules on the basolateral/systemic side of the epithelial layerare not necessary for transepithelial toxin neutralization. Support-ing this notion, addition of excess irrelevant human IgG to thebasolateral chamber had no impact on the transepithelial neutral-ization of toxin (see Fig. S5).

Mechanism of antibody transport across the epithelialmonolayer. Neutralization of apical toxin by basolateral antibod-ies strongly suggests that antibodies are transported to the apicalside. To confirm this, we measured the extent to which antibody

FIG 1 Role of FcRn and other Fc receptors in protection against CDI. (A) Kaplan-Meier survival curves of mice (n � 15 per group) infected with C. difficile andtreated with vehicle (dashed blue line) or with 250 �g actoxumab alone (dotted blue line), bezlotoxumab alone (thin blue line), or both antibodies together (thickblue line). (B) Survival curves of wild-type (dark blue lines) and FcRn knockout (light-blue lines) mice (n � 10 per group) infected with C. difficile in the primaryinfection mode and treated with vehicle (dashed lines) or 250 �g actoxumab-bezlotoxumab (solid lines). (C) As in panel B but in the recurrent infection mode(n � 10 per group for WT mice; n � 12 for FcRn knockout mice). (D) Survival curves of Syrian hamsters infected with C. difficile and treated with vehicle (blackline; n � 2), 2 g/kg human IgG (red line; n � 6), 50 mg/kg actoxumab-bezlotoxumab (blue line; n � 6), or 50 mg/kg actoxumab-bezlotoxumab with 2 g/kg humanIgG (purple line; n � 6). �, P � 0.02; ��, P � 0.001; ���, P � 0.0001 (compared to results for corresponding vehicle groups).

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applied to the basolateral chamber translocates to the apicalchamber in the presence and absence of toxin on the apical side.Similar to the TER assays described above, 100 �g/ml antibodywas added to the basolateral side of a confluent monolayer ofCaco-2 cells 18 h prior to addition of buffer or toxin at differentconcentrations, and the antibody concentration in the apicalchamber was measured by ELISA at different time points. Asshown in Fig. 4A and B (see also Fig. S6A and B in the supplemen-tal material for T84 cells), transport of actoxumab and bezlotox-umab into the apical chamber is minimal 18 h after addition ofantibodies to the basolateral chamber (at t � 0 h with respect tothe time of toxin addition). Transport of antibodies into the apicalchamber increases with time in the presence of toxin, and thiseffect is dependent on the concentration of toxin added to theapical chamber. Indeed, while the concentration of antibodies onthe apical side is �0.002% of that added to the basolateral cham-ber at 48 h (66 h after addition of antibodies) in the absence oftoxin, this value reaches 40% and 20% in the presence of 64ng/ml TcdA or 256 ng/ml TcdB, respectively. The dependence ofantibody transport on toxin is presumed to result from toxin-dependent disruptions in the epithelial barrier function, leading

to increased nonspecific paracellular leakage of antibodies to theapical side, analogous to the high levels of antibodies observed inC. difficile-infected hamsters versus those in healthy hamsters (Fig.2). Importantly, transport of F(ab=)2 fragments of actoxumab andbezlotoxumab was at least as high as that of intact antibody in thepresence and absence of toxin (Fig. 4C and D), confirming thatFc-dependent transport mechanisms, such as FcRn, are not in-volved in antibody transport in this system. Indeed, concentra-tions of F(ab=)2 were consistently higher in the apical chamber,particularly at high toxin concentrations. This may be due to thesmaller size of F(ab=)2 fragments allowing them to leak throughparacellular gaps more efficiently and/or a slightly higher molarconcentration of F(ab=)2 added to the basolateral chamber than ofintact antibodies (since 100 �g/ml each of intact antibodies andF(ab=)2 fragments were used in these experiments, and the molec-ular mass of F(ab=)2 is �1.5-fold lower than that of intact anti-body).

Antibody-induced recovery of epithelial monolayer follow-ing toxin-induced damage. In order to better understand howtoxin neutralization might protect the gut epithelium in thecontext of the gut wall (where damage is repaired much faster

FIG 2 Tissue distribution of actoxumab-bezlotoxumab in hamsters. (A) Levels of actoxumab-bezlotoxumab in homogenized whole intestinal tissuesfrom healthy (white bars; n � 4) and C. difficile-infected (black bars; n � 3) hamsters, harvested 2 days after challenge. Values shown are means SD. Nosignificant differences in the levels of actoxumab-bezlotoxumab exist when comparing healthy to C. difficile-infected hamsters across all tissues. hIgG, humanIgG; Duod, duodenum; Jej, jejunum; Ile, ileum; Col, colon; Cec, cecum. (B) Levels of actoxumab-bezlotoxumab in the lumenal contents of various intestinalsegments from healthy (white bars; n � 4) and C. difficile-infected (black bars; n � 3) hamsters, harvested 2 days after challenge. Values shown are means SD.�, P � 0.01 (compared to results for corresponding lumenal contents in healthy hamsters). Representative experiments from 2 independent experiments areshown. (C) Localization of actoxumab-bezlotoxumab in the cecum wall of vehicle-injected healthy hamsters (“Control”), healthy hamsters injected withactoxumab-bezlotoxumab (“Healthy”), and infected hamsters injected with actoxumab-bezlotoxumab (“CDI”) (for additional pictures, see Fig. S1 in thesupplemental material), as determined by immunohistochemistry, harvested 2 days after challenge. Insets show representative epithelial layers of healthy andCDI hamsters at higher magnification. LP, lamina propria; E, epithelium; L, lumen; S, submucosa. Representative panels from at least 3 independent experimentsare shown.

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than in isolated epithelial cells owing to the presence of othercells and growth factors in the intact gut wall [35]), we assessedtransepithelial neutralization and transport in the two-dimen-sional culture assay using MDCK cells, which proliferate at amuch higher rate in vitro than gut epithelial cells. Similar toresults with Caco-2 and T84 cells (Fig. 3; see also Fig. S3 in thesupplemental material), we observed a robust TcdA-dependentdecrease in TER and significant neutralization of apical TcdAby basolateral actoxumab (Fig. 5A and B). Interestingly, theprotection afforded by actoxumab was biphasic, with a signif-

icant yet incomplete effect at 6 h and complete neutralization (exceptfor the highest concentration of TcdA) at 24 h. We confirmed thattoxin is indeed fully neutralized at 24 h by transferring the contents ofthe apical chambers at this time point (TcdA concentrations up to 64ng/ml) to intact MDCK and Caco-2 monolayers and showing limitedeffects on TER after 24 h (data not shown). These data are consistentwith the notion that toxin on the apical side must first cause damageto the epithelial layer (shown by a drop in TER at 6 h) in order for theantibody on the basolateral side to fully neutralize apical toxin.

In parallel with these TER measurements, we assessed trans-

FIG 3 Effects of apical toxin on TER and neutralization by actoxumab-bezlotoxumab in Caco-2 cells. (A) Time-dependent effects on TER of TcdA addedto the apical chamber (circles, 0.1 ng/ml TcdA; squares, 0.3 ng/ml; triangles, 1 ng/ml; inverted triangles, 3 ng/ml; diamonds, 10 ng/ml). The dashed boxhighlights the time point shown in panel E. All curves are significantly different from each other at at least at one time point (P � 0.0001), except for 1ng/ml versus 3 ng/ml (P 0.05 at all time points). (B) Time-dependent effects on TER of TcdB added to the apical chamber (circles, 1 ng/ml TcdB;squares, 3 ng/ml; triangles, 10 ng/ml; inverted triangles, 30 ng/ml; diamonds, 100 ng/ml). The dashed box highlights the time point shown in panel F. Allcurves are significantly different from each other at at least at one time point (P � 0.0005), except for 30 ng/ml versus 100 ng/ml (P 0.05 at all timepoints). (C) Like panel A but in the presence of 100 �g/ml actoxumab in the basolateral chamber. All curves are significantly different from each other atleast at one time point (P � 0.0005). (D) Same as panel B but in the presence of 100 �g/ml bezlotoxumab in the basolateral chamber. All curves aresignificantly different from each other at at least at one time point (P � 0.0005 except for 3 ng/ml versus 10 ng/ml: P � 0.014 at 48 h only), except for 1ng/ml versus 3 ng/ml (P 0.05 at all time points). (E) Concentration-dependent effects of TcdA on TER measured 24 h after addition of toxin to the apicalchamber in the absence (circles) or presence of 100 �g/ml actoxumab added to the apical chamber (squares) or to the basolateral chamber (triangles) orin the presence of 100 �g/ml F(ab=)2 fragments of actoxumab added to the basolateral chamber (inverted triangles). (F) Concentration-dependent effectsof TcdB on TER measured 24 h after addition of toxin to the apical chamber in the absence (circles) or presence of 100 �g/ml bezlotoxumab added to theapical chamber (squares) or to the basolateral chamber (triangles), or in the presence of 100 �g/ml F(ab=)2 fragments of bezlotoxumab added to thebasolateral chamber (inverted triangles). �, P � 0.05; ��, P � 0.001, versus results with no antibody added. In all cases, antibodies were added 18 h priorto addition of toxin. Values are means SD from at least 4 independent experiments.

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port of antibody to the apical side 24 h after addition of variousconcentrations of toxin (Fig. 5C; see also Fig. S6C in the supple-mental material). Surprisingly, the apical concentration of actox-umab was �20% of the amount added to the basolateral side, evenat the top TcdA concentration of 256 ng/ml. Conversely, when theantibody applied to the basolateral chamber was bezlotoxumab,which does not neutralize TcdA (data not shown), antibody con-centrations were up to 8-fold higher than with actoxumab. Ananalogous result was obtained when TcdB was added to the apicalchamber; transport of the nonneutralizing antibody actoxumabwas up to 60-fold higher than that of the neutralizing antibodybezlotoxumab (Fig. 5D). This suggests that neutralizing antibod-ies limit their own transport across the epithelium by halting andreversing toxin-induced damage on the apical side, thereby pre-venting further antibody transport.

Taken together, the data for MDCK cells suggest that toxin-induced damage to the epithelium results in paracellular diffu-sion of antibodies from the basolateral side to the apical side,leading to transepithelial neutralization of the toxin, restora-tion of the epithelial layer, and inhibition of further antibodytransport. These observations also apply to the intestinal epi-thelial cell line Caco-2, albeit more modestly (see Fig. S7 in thesupplemental material). Since Caco-2 cells proliferate at alower rate than MDCK cells, recovery of the epithelial mono-layer occurs 120 h (rather than 24 h) after addition of toxin,and transport of the neutralizing antibody compared to that of

the nonneutralizing antibody is less dramatically depressedthan with MDCK cells.

Toxin-induced toxin transport across the epithelial mono-layer. Since toxin-dependent transport of antibodies across theepithelial monolayer appears to be nonspecific, we posited thattoxin may facilitate its own transport in the opposite direction.Indeed, the concentrations of TcdA and TcdB in the basolateralchamber 48 h after addition of the toxins to the apical chamberincrease in a toxin-dependent manner (Fig. 6), consistent withprevious data (30). The increase is observed whether the data areexpressed as absolute toxin concentrations or as a percentage ofthe toxin concentration applied to the apical chamber, showingthat toxin transport is at least partly due to toxin-dependent dis-ruption of the epithelial cell layer and is not solely driven by theconcentration gradient. Notably, translocation of toxin from theapical to the basolateral side is less efficient than transport of an-tibodies from the basolateral to the apical side, likely due to themuch larger size of the toxins.

Non-toxin-induced paracellular transport of antibodies andcontribution to transepithelial toxin neutralization. While thedata described herein demonstrate that neutralization of apicaltoxin by basolateral antibodies is largely dependent on toxin-in-duced damage leading to paracellular diffusion of antibodies tothe apical side, they do not rule out the possibility that basal trans-port of antibodies even in the absence of toxin also contributes totoxin neutralization; low but significant levels of antibodies are

FIG 4 Transport of actoxumab and bezlotoxumab from the basolateral to the apical side of Caco-2 monolayers. (A) Concentrations of actoxumab in the apicalchamber at various times after addition of increasing concentrations of TcdA (as indicated, in ng/ml) to the apical chamber. (B) Concentrations of bezlotoxumabin the apical chamber at various times after addition of increasing concentrations of TcdB (as indicated, in ng/ml) to the apical chamber. �, P � 0.05; ��, P � 0.001(compared to results for all other toxin concentrations at the same time point); #, P � 0.05; ##, P � 0.001 (compared to all other time points at the same toxinconcentration). (C) Time-dependent accumulation of actoxumab (circles and triangles) or F(ab=)2 fragments thereof (squares and diamonds) in the apicalchamber after addition of buffer (circles and squares) or 64 ng/ml TcdA (triangles and diamonds) to the apical chamber. (D) Time-dependent accumulation ofbezlotoxumab (circles and triangles) or F(ab=)2 fragments thereof (squares and diamonds) in the apical chamber after addition of buffer (circles and squares) or256 ng/ml TcdB (triangles and diamonds) to the apical chamber. ��, P � 0.0001; �, P � 0.01 (compared to results in the absence of toxin); ##, P � 0.0001; #, P� 0.005 (compared to results for F(ab=)2 in the presence of toxin). In all cases, antibodies were added to the basolateral chamber 18 h prior to addition of toxin.Values are means SD from at least two independent experiments.

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indeed detectable on the apical side 18 h after their addition to thebasolateral chamber (at t � 0 h in relation to toxin addition) (Fig.4 and 5; see also Fig. S6 in the supplemental material). To gaininsight into whether basal (non-toxin-dependent) transport ofantibodies contributes to toxin neutralization once toxins areadded to the apical side, we compared the transepithelial neutral-ization of TcdA by actoxumab after adding the antibody to thebasolateral side either 18 h prior to addition of toxin to the apical

side (as was done for all other experiments described in this study)or immediately prior to addition of the toxins. As shown in Fig. 7,transepithelial neutralization is more pronounced when antibod-ies are added 18 h prior to addition of toxins, indicating that theminimal transport that occurs across the intact epithelial mono-layer does contribute significantly to protection. Differences in thelevel of protection when comparing addition of antibodies added18 h before toxin addition versus immediately before toxin addi-

FIG 5 Toxin-induced transepithelial toxin neutralization and transport of basolateral antibodies in MDCK cells. (A) Time-dependent effects on TER of TcdAadded to the apical chamber (circles, 1 ng/ml TcdA; squares, 4 ng/ml; triangles, 16 ng/ml; inverted triangles, 64 ng/ml; diamonds, 256 ng/ml). (B) Like panel Abut with 100 �g/ml actoxumab added to the basolateral chamber. � (P � 0.05), �� (P � 0.01), and ��� (P � 0.001) indicate values significantly lower than thosefor both the 0-h and 24-h time points. Values are means SD from 4 independent experiments. (C) Concentrations of actoxumab (green bars) or bezlotoxumab(blue bars) in the apical chamber 42 h after addition of the antibodies individually to the basolateral chamber and 24 h after addition of various concentrations(x axis) of TcdA to the apical chamber. ���, P � 0.0001 compared to results for actoxumab at the same toxin concentration; #, P � 0.05 compared to results forother toxin concentrations with bezlotoxumab, except for 16 ng/ml (P 0.05); ##, P � 0.01 compared to results for other toxin concentrations with bezlotox-umab; @, P � 0.05 compared to results for other toxin concentrations with actoxumab, except for 64 ng/ml (P 0.05). (D) Like panel C but with TcdB addedto the apical chamber. ���, P � 0.0001 compared to results for bezlotoxumab at 256 ng/ml TcdB; ###, P � 0.0001 compared to results for all other toxinconcentrations with actoxumab. In all cases, antibodies were added to the basolateral chamber 18 h prior to addition of toxin. Values are means SD from 4independent experiments.

FIG 6 Toxin-dependent transport of toxin across Caco-2 monolayers. (A) Concentrations of TcdA in the basolateral chamber 48 h after addition of the toxinto the apical chamber at various concentrations (as indicated on the x axis). Data are expressed as absolute concentrations (light green, left y axis) and aspercentages of the TcdA concentration added to the apical chamber (dark green, right y axis). �, P � 0.05 compared to results for 4 ng/ml TcdA. (B)Concentrations of TcdB in the basolateral chamber 48 h after addition of the toxin to the apical chamber at various concentrations (as indicated on the x axis).Data are expressed as absolute concentrations (light blue, left y axis) and as percentages of the TcdB concentration added to the apical chamber (dark blue, righty axis). �, P � 0.05 compared to results for 16 ng/ml TcdB; ��, P � 0.0001 compared to results for 16 and 64 ng/ml TcdB. BLQ, below the limit of quantitation.All values are means SD from five independent experiments.

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tion are observed for both actoxumab (Fig. 7A) and bezlotoxumab(Fig. 7C) and are larger when assessed 24 h compared to 48 h afteraddition of the toxin (Fig. 7B and D). The non-toxin-dependentprotection is likely due to paracellular rather than transcellulartransport, since similar data were obtained using F(ab=)2 frag-ments of actoxumab (data not shown).

DISCUSSION

Systemic antibodies are thought to play a significant role inprotecting the host from infections by enteric pathogens (36).In the case of Clostridium difficile infections, studies by Kyne andcoworkers first showed a correlation between levels of circulatingantitoxin antibodies and protection from primary and recurrentinfections (15, 16). Antitoxin antibodies, whether systemically ad-ministered or induced through vaccination, have been shown toprotect against CDI in multiple animal models (18–22) and in theclinic (23). However, the question of how systemic antibodiesneutralize bacterial toxins in the intestinal lumen has remainedunanswered. In this study, we have shown that antibody transportand efficient toxin neutralization are largely dependent on para-cellular transport across the gut epithelium, rather than specifictranscellular transport mechanisms involving the Fc domains ofthe antibodies, and that the damage induced by the toxins them-selves facilitates the process of transepithelial neutralization.

Multiple studies have shown that the neonatal Fc receptor(FcRn) is involved in transport of IgG molecules across the gutepithelium (reviewed in reference 37). This receptor is known totransport antibodies from the lumenal to the systemic side of thegut and is thought to function as a facilitator of antibody absorp-

tion from maternal milk in suckling infants. However, transportin the opposite direction (i.e., from the systemic side to the GIlumen) has also been observed (24, 25, 29) and has been proposedas the mechanism of IgG-mediated protection against Helicobac-ter (26) and Citrobacter (27) infections in mice. In the currentstudy, protection from CDI by the antitoxin antibody combina-tion actoxumab-bezlotoxumab was found to be equivalent inFcRn knockout mice and in WT mice (Fig. 1B and C), suggestingthat FcRn is not involved in antibody-mediated protection againstC. difficile in this model. One important caveat to this experimentis the fact that FcRn expression in mice decreases rapidly followingweaning, whereas high expression is maintained throughoutadulthood in humans (37). Conclusions regarding the role ofFcRn in murine CDI therefore cannot necessarily be applied tohuman disease based on this experiment alone. Nevertheless, sev-eral additional lines of evidence are consistent with the notionthat, in the context of CDI, transport of antitoxin IgG antibodiesto the gut lumen is not mediated by FcRn or by any other Fc-mediated transport mechanism. Indeed, excess irrelevant humanIgG, which should compete with the antitoxin antibodies forbinding to any and all Fc receptors, has no impact on protec-tion against CDI in the hamster model (Fig. 1D) and on trans-epithelial neutralization in the two-dimensional culture assay(see Fig. S5 in the supplemental material). Consistent with this,transepithelial toxin neutralization by (and transport of)F(ab=)2 fragments of actoxumab and bezlotoxumab is compa-rable to that by intact versions of the antibodies in the TERassay (Fig. 3 and 4). Finally, toxin-induced damage accountsfor the majority of antibody transport to the apical chamber (in

FIG 7 Non-toxin-mediated transport of actoxumab and impact on transepithelial neutralization of TcdA. (A) Effect of apical TcdA on TER 24 h after additionof toxin, in the absence (circles) or presence of 100 �g/ml actoxumab added to the basolateral chamber either 18 h (squares) or immediately (triangles) beforeaddition of TcdA. (B) Like panel A but 48 h after addition of toxin. (C) Effect of apical TcdB on TER 24 h after addition of toxin, in the absence (circles) or in thepresence of 100 �g/ml bezlotoxumab added to the basolateral chamber either 18 h (squares) or immediately (triangles) before addition of TcdB. (D) Like panelC but 48 h after addition of toxin. �, P � 0.05; ��, P � 0.01; ���, P � 0.001 (compared to absence of antibody); #, P � 0.05; ##, P � 0.01; ###, P � 0.001 (comparedto antibody added immediately before toxin). All values are means SD from two independent experiments.

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the TER assay) (Fig. 4 and 5; see also Fig. S6) or to the gutlumen (in infected versus healthy hamsters) (Fig. 2), presum-ably through disruption of the tight junctions that normallylimit paracellular transport of molecules across the epithelium.Overall, our data preclude a role of specific Fc-dependenttransport mechanisms in protection against CDI, analogous torecent data from Johnston et al. (38) showing that transport ofIgA via the polymeric immunoglobulin receptor (pIgR) is alsonot necessary for protection against CDI in mice.

The concept that toxin-induced damage enables efficienttransepithelial neutralization of that same toxin by antibodies onthe opposite side of the epithelium may at first glance appearcounterintuitive; after all, if damage to the gut lining is required inorder for systemic antibodies to neutralize the toxin, how can suchantibodies prevent disease in the first place? The answer may lie inthe notion that antibody transport and epithelial damage impacteach other in ways that render them self-limiting. Thus, as toxin-induced damage increases, more antibody is allowed to translo-cate to the gut lumen, leading to neutralization of the toxin, re-covery of the epithelium, and inhibition of further antibodytransport. Lower antibody transport, in turn, decreases toxin neu-tralization, leading to increased damage to the gut lining. In thisparadigm, the extent of epithelial disruption required to achievesufficient antibody transport for toxin neutralization determinesthe severity of CDI symptoms or indeed whether symptoms occurat all. Our data show that this “tipping point” depends on multiplefactors, including the concentrations of toxin and antibody, thesensitivity of the epithelial cells to toxins on the apical side, and therate of epithelial cell proliferation (which will affect the efficiencyof epithelial healing). Illustrating this idea, transepithelial neutral-ization of TcdB by bezlotoxumab is more efficient than neutral-ization of TcdA by actoxumab in Caco-2 (Fig. 3), T84 (see Fig. S3in the supplemental material), and MDCK cells (data not shown),possibly because these cells are less sensitive to TcdB on the apicalside (see Fig. S2). A more efficient neutralization is associated witha lower overall level of bezlotoxumab transport across the epithe-lium (Fig. 4; see also Fig. S6). For TcdB and bezlotoxumab, there-fore, the tipping point is skewed toward limited epithelial damageand antibody transport. For TcdA and actoxumab, the tippingpoint is generally shifted toward a higher level of epithelial damageand antibody transport, although under conditions where healingof the epithelium is rapid (as with MDCK cells) (Fig. 5), any sig-nificant and/or lasting effects of toxin are quickly reversed by thetransported antibody. Given the high rate at which the intestinalbarrier function is reestablished following injury in vivo (35), itmay well be that efficient neutralization of toxins in the gut lumenoccurs with minimal (i.e., asymptomatic) damage to the epithe-lium.

The aforementioned tipping point dictating the levels of epi-thelial damage and antibody transport under specific conditions isalso predicted to be impacted by how much, if any, antibodytransport occurs via paracellular leakage in the absence of toxin.Low levels of antibodies were indeed detected in vitro in the apicalchamber prior to addition of toxin, following 18 h of incubationwith antibodies on the basolateral side (Fig. 4 and 5; see also Fig. S6and S7 in the supplemental material), and in vivo in the cecumcontents of uninfected hamsters (Fig. 2; see also Fig. S1). Further-more, basal antibody transport was found to contribute somewhatto transepithelial toxin neutralization (Fig. 7) and under someconditions may be sufficient for at least partial neutralization. For

example, significant reversal of the toxin-induced effects on TERwas observed at low toxin concentrations (Fig. 3) even in the ab-sence of significant toxin-dependent antibody transport (Fig. 4Aand B). The latter observation, however, may be misleading, sincethe concentration of antibodies in the entire apical chamber doesnot necessarily accurately reflect the local concentration of anti-bodies at or near the epithelial cell surface or indeed within theintercellular space between colonocytes. Overall, our data indicatethat complete neutralization of apical toxin, at least at higher toxinconcentrations, requires prior disruption of the epithelial cell re-sistance by toxin, as evidenced by the biphasic nature of the trans-epithelial neutralization in MDCK cells (Fig. 5) and Caco-2 cells(see Fig. S7).

Our data lead us to propose a model of antibody transportacross the gut wall that accounts for both the previously de-scribed correlation between circulating antitoxin IgG and pro-tection against CDI and the demonstration that systemic ad-ministration of antitoxin IgG against the C. difficile toxins isprotective in animal models and in humans. According to thismodel (Fig. 8), antitoxin immunoglobulins are present at a highconcentration on the systemic side of the gut epithelium, withlimited but significant paracellular leakage to the gut lumen (Fig.8, I). Upon colonization of the gut with a toxigenic strain of C.difficile, toxin begins to accumulate on the apical/lumenal side ofthe gut, where some neutralization of the toxin may occur due tobasal antibody transport (Fig. 8, II). Toxin-induced disruption ofthe epithelium causes increased transport of antibodies to the lu-menal side and possibly of toxin to the systemic side, leading toefficient neutralization of the toxin; at this point, significant levelsof antibodies may be detectable on the lumenal/apical side (Fig. 8,III). Disruption of the epithelial monolayer eventually reaches atipping point, when enough antibody is transported to preventfurther damage and allow the epithelium to recover and heal. Af-ter the antibody/toxin complexes are cleared from the lumen, thegut wall returns to a basal state with a high concentration of anti-bodies on the basolateral side (I). As discussed above, the tippingpoint may occur prior to the infliction of significant damage onthe gut wall, in which case CDI symptoms might never arise(asymptomatic colonization). In the case of antitoxin-dependentprevention of recurrent disease, the patient suffering from CDIwould have preexisting gut wall lesions and the cycle would start instate III of the model; clearance of the infection by standard-of-care antibiotics would lead to state I, and subsequent episodesof CDI (i.e., recurrent CDI) might be prevented as describedabove.

This model reveals a heretofore underappreciated advantage ofsystemic versus mucosal antibodies in protection against C. diffi-cile. The long half-life of antibodies in circulation allows for aconstant and long-lasting source of neutralizing activity on thebasolateral/systemic side of the gut epithelium. Furthermore,since toxins can induce their own transport to the basolateral/systemic side (Fig. 6 and reference 30), perhaps allowing them toenter the circulation and reach other organs (39, 40), circulatingneutralizing antibodies should prevent systemic effects, if any, ofC. difficile toxins. Whether these advantages will translate to sys-temically administered antibodies, such as actoxumab-bezlotox-umab, versus orally delivered neutralizing antibodies remains tobe confirmed.

We have demonstrated in this study that systemic anti-C. dif-ficile toxin IgG antibodies translocate to the gut lumen via non-

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specific paracellular transport, facilitated by a toxin-induced dis-ruption in the integrity of the intestinal epithelium. The dataprovide insight into the mechanism of action of circulating anti-toxin antibodies, which have been shown to be protective in ani-mal models of CDI and in human disease. Since disruption of theintestinal epithelial barrier function is a well-recognized conse-quence of infections by various enteric pathogens (41), it will beinteresting to see whether this mechanism of antibody transportplays a role in protection against other pathogens or is specific toClostridium difficile.

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Zhang et al.

416 iai.asm.org January 2015 Volume 83 Number 1Infection and Immunity

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Correction for Zhang et al., Toxin-Mediated Paracellular Transport ofAntitoxin Antibodies Facilitates Protection against Clostridium difficileInfection

Z. Zhang,a X. Chen,b L. D. Hernandez,a P. Lipari,a A. Flattery,a S.-C. Chen,a S. Kramer,a J. D. Polishook,a F. Racine,a H. Cape,a

C. P. Kelly,b A. G. Theriena

Merck Research Laboratories, Merck & Co., Inc., Kenilworth, New Jersey, USAa; Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School,Boston, Massachusetts, USAb

Volume 83, no. 1, p. 405– 416, 2015. Page 408: Figure 1 should appear as shown below.

Citation Zhang Z, Chen X, Hernandez LD, Lipari P, Flattery A, Chen S-C, Kramer S,Polishook JD, Racine F, Cape H, Kelly CP, Therien AG. 2015. Correction for Zhang etal., Toxin-mediated paracellular transport of antitoxin antibodies facilitatesprotection against Clostridium difficile infection. Infect Immun 83:4899.doi:10.1128/IAI.01214-15.

Copyright © 2015, American Society for Microbiology. All Rights Reserved.

FIG 1

AUTHOR CORRECTION

December 2015 Volume 83 Number 12 iai.asm.org 4899Infection and Immunity