Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab Zhiyong Yang,a Jeremy Ramsey,a Therwa Hamza,a Yongrong Zhang,a Shan Li,a Harris G. Yfantis,b Dong Lee,b Lorraine D. Hernandez,c Wolfgang Seghezzi,d Jamie M. Furneisen,d Nicole M. Davis,d Alex G. Therien,c Hanping Fenga

Clostridium difficile infection (CDI) represents the most prevalent cause of antibiotic-associated gastrointestinal infections in health care facilities in the developed world. Disease symptoms are caused by the two homologous exotoxins, TcdA and TcdB. Standard therapy for CDI involves administration of antibiotics that are associated with a high rate of disease recurrence, highlighting the need for novel treatment paradigms that target the toxins rather than the organism itself. A combination of human monoclonal antibodies, actoxumab and bezlotoxumab, directed against TcdA and TcdB, respectively, has been shown to decrease the rate of recurrence in patients treated with standard-of-care antibiotics. However, the exact mechanism of antibodymediated protection is poorly understood. In this study, we show that the antitoxin antibodies are protective in multiple murine models of CDI, including systemic and local (gut) toxin challenge models, as well as primary and recurrent models of infection in mice. Systemically administered actoxumab-bezlotoxumab prevents both the damage to the gut wall and the inflammatory response, which are associated with C. difficile in these models, including in mice challenged with a strain of the hypervirulent ribotype 027. Furthermore, mutant antibodies (N297Q) that do not bind to Fc␥ receptors provide a level of protection similar to that of wild-type antibodies, demonstrating that the mechanism of protection is through direct neutralization of the toxins and does not involve host effector functions. These data provide a mechanistic basis for the prevention of recurrent disease observed in CDI patients in clinical trials.

C

lostridium difficile is an anaerobic, spore-forming, Gram-positive bacterium that causes infections in the lumen of the colon and is the most frequent cause of nosocomial diarrhea in the developed world (1, 2). C. difficile infections (CDI) contribute to thousands of deaths and are associated with over $1 billion in health care-related costs in the United States each year (3–5). The symptoms of CDI range from asymptomatic carriage or mild diarrhea to fatal pseudomembranous colitis, colonic rupture, and death (6, 7). The disease occurs mainly in patients undergoing (or who have recently undergone) a course of broad-spectrum antibiotics; in such patients, composition of the gut microbiota is altered, disrupting the body’s natural defense against C. difficile infections. Clinical management of CDI consists of discontinuation of the offending antibiotic and treatment with either metronidazole, vancomycin, or the newly approved fidaxomicin (8). A major concern with CDI is that even when treatment of a primary infection is successful, 20 to 30% of patients experience a recurrence of the disease within days or weeks of symptom resolution. Disease recurrence results from continued disruption of the gut microbiota by standard-of-care antibiotics (9) combined with persistence of resistant C. difficile spores (relapse) or reacquisition of new spores from the environment (reinfection) (10, 11). Multiple recurrences often occur, as repeated antibiotic use prevents the gut microbiota from reestablishing itself, allowing C. difficile spores to germinate and reinfect the gut as soon as antibiotic use is discontinued (12). These challenges highlight the need for nonantibiotic therapies for CDI that may spare the intestinal microbiota and thus be associated with lower rates of recurrence. The symptoms of CDI are caused by two homologous exotoxins, TcdA and TcdB, expressed by pathogenic strains of C. difficile (13). The toxins target the epithelial cells of the gut lining by binding to unknown receptors at the cell surface, entering the cells via

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endocytosis and inactivating Rho-type GTPases through covalent glucosylation. Inactivation of these enzymes leads to dysregulation of the actin cytoskeleton and loss of tight junction integrity (6, 13), as well as to the release of proinflammatory factors such as interleukin 8 (IL-8) (14, 15). The resulting increase in gut wall permeability and acute proinflammatory response leads to diarrhea and, if left unchecked, to the more severe symptoms of CDI. Interestingly, recently emerging hypervirulent strains of C. difficile, including the common NAP1/BI/027 variant which has been linked to higher rates of recurrence (11, 16, 17), overexpress both TcdA and TcdB (18, 19). Targeting the toxins of C. difficile thus represents a novel antibiotic-sparing approach to CDI therapy. The notion that targeting the toxins of C. difficile may be beneficial in CDI is supported by multiple studies in animal models

Received 7 November 2014 Returned for modification 20 November 2014 Accepted 1 December 2014 Accepted manuscript posted online 8 December 2014 Citation Yang Z, Ramsey J, Hamza T, Zhang Y, Li S, Yfantis HG, Lee D, Hernandez LD, Seghezzi W, Furneisen JM, Davis NM, Therien AG, Feng H. 2015. Mechanisms of protection against Clostridium difficile infection by the monoclonal antitoxin antibodies actoxumab and bezlotoxumab. Infect Immun 83:822–831. doi:10.1128/IAI.02897-14. Editor: V. B. Young Address correspondence to Alex G. Therien, [email protected], or Hanping Feng, [email protected]. Z.Y. and J.R. contributed equally to this work. Supplemental material for this article may be found at http://dx.doi.org/10.1128 /IAI.02897-14. Copyright © 2015, American Society for Microbiology. All Rights Reserved. doi:10.1128/IAI.02897-14

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Department of Microbial Pathogenesis, University of Maryland Dental School, Baltimore, Maryland, USAa; Department of Pathology and Laboratory Medicine, VAMHCS, University of Maryland School of Medicine, Baltimore, Maryland, USAb; Merck & Co., Inc., Kenilworth, New Jersey, USAc; Merck & Co., Inc., Palo Alto, California, USAd

Antitoxin Monoclonal Antibodies Protect against CDI

MATERIALS AND METHODS Mice. Six- to eight-week-old CD1 and C57BL/6 mice were purchased from Harlan Laboratories (Maryland, USA). All mice were housed in dedicated pathogen-free facilities in groups of 5 mice per cage under the same conditions. Food, water, bedding, and cages were autoclaved. All procedures involving mice were conducted under protocols approved by the Institutional Animal Care and Use Committees at the University of Maryland and at Merck & Co., Inc. Systemic toxicity assay. Six- to eight-week-old CD1 mice were treated intraperitoneally (i.p.) with anti-TcdA (actoxumab) and anti-TcdB (bezlotoxumab) mixed together at doses of 3, 30, and 300 ␮g/mouse (⬃0.1, 1, and 10 mg/kg of body weight) of each antibody. Phosphate-buffered saline (PBS) was used as a vehicle control. One hour posttreatment, mice were challenged i.p. with a mixture of purified TcdA and TcdB at a final concentration of 25 ng of each toxin (from strain VPI 10463 [38]). This toxin dose was previously determined to be the minimally lethal dose. Mice were closely monitored for signs of systemic disease, and those that became moribund were sacrificed. Ileal loop model. CD1 mice were injected i.p. with either a mixture of 50 mg/kg actoxumab and 50 mg/kg bezlotoxumab (final concentration of 100 mg/kg antibody) or PBS as a vehicle control. Mice were rested for 24 h with fasting and anesthetized with 250 mg/kg avertin (0.5 g 2,2,2-tribromoethanol and 1 ml amylene hydrate in 40 ml distilled water) administered i.p. A midline laparotomy was performed by making a 1-cm incision in the abdomen and isolating the ileum. An approximately 2- to 3-cm section of ileum was ligated at each end with suture silk to separate it from the rest of the intestinal tract. The loops were inoculated with 100 ␮l of either a mixture of TcdA and TcdB (2.5 ␮g each toxin, from strain VPI

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10463 [38], previously determined to be the lowest dose to cause full effects) or PBS as a vehicle control. The ileum was returned to the abdomen, and the incision was closed with suture silk. The mice were allowed to recover after surgery and were sacrificed 4 h later. The ligated ileal loop was removed, and its weight and length were determined. Sections of the ileal loops were also collected for histological examination. Murine C. difficile infection model. Mouse infection was performed as described previously (39, 40), and schematic diagrams of the various challenge paradigms are shown in Fig. S1 in the supplemental material. C57BL/6 mice were treated with an antibiotic cocktail for 3 days, followed by normal water for 3 additional days before C. difficile spore challenge. Mice were i.p. injected with 10 mg/kg clindamycin 24 h prior to spore challenge. Pilot experiments were first carried out to show that a challenge with 105 C. difficile UK1 spores results in the most reproducible and robust generation of symptoms. For antitoxin treatment, mice were injected i.p. with vehicle (PBS) or with different doses of actoxumab-bezlotoxumab either 24 h prior to (prophylactic paradigm; see Fig. S1A in the supplemental material) or 24 h after (therapeutic paradigm; see Fig. S1B) orogastric inoculation with C. difficile spores. For recurrent CDI (40) (see Fig. S1C), surviving mice from the primary infection were treated 7 days after the initial spore challenge with antibiotic-water containing dexamethasone (0.1 mg/ml) for 3 days, followed by antibiotic-free water containing added dexamethasone for 3 additional days, and then injected i.p. with clindamycin 1 day prior to a second orogastric spore challenge with 105 C. difficile UK1 spores. For the experiment in which mice were treated a second time with actoxumab-bezlotoxumab 24 h prior to spore challenge (see Fig. 4C and D), mice were rested for 14 days following the primary challenge (see Fig. S1D). Mice were monitored postchallenge for weight changes and CDI symptoms. Any mice demonstrating severe CDI symptoms or those that lost ⬎20% body weight were considered moribund and were euthanized. Tissue sampling, histology, and damage scoring. Sections of the ileum (ileal loop model) or cecum (spore challenge model) of mice were collected, fixed in 10% formalin, and stained with hematoxylin and eosin by the EM/Histology Lab, Department of Pathology, University of Maryland Baltimore. Overall damage was analyzed by histologists blinded to the identity of each sample. Damage scores were graded based on five criteria, each on a scale of 0 to 3, and added together to generate a score with a maximum value of 15. The criteria were inflammatory cell infiltration and inflammation, mucosal hypertrophy and thickness, vascular congestion and exudates, epithelial cell and architectural disruption, and submucosal edema. The number of neutrophils and apoptotic bodies were also counted over six fields of view at ⫻200 magnification and were also scored blindly. Cell rounding toxin neutralization assay. Vero cells (African green monkey kidney epithelial cells; ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA) with 10% fetal bovine serum, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, and 40 ␮g/ml streptomycin sulfate. Cell rounding assays were carried out as previously described (38, 39, 41). Serum from mice treated with vehicle or with actoxumab-bezlotoxumab was collected at various time points following spore challenge and diluted 1:30 in cell culture medium. Diluted sera were added to Vero cells, and the extent of cell rounding was assessed using a phase-contrast microscope after 16 h of exposure to the serum. Cell death toxin neutralization assay. To measure antibody-mediated toxin neutralization, Vero cells were seeded at 2,000 cells/well in 96-well dishes and incubated overnight. Purified TcdA (2 ng/ml) or TcdB (10 pg/ml) was combined with serially diluted wild-type (WT) anti-TcdA (actoxumab), anti-TcdB (bezlotoxumab), or N297Q mutant antibodies for 2 h at 37°C and then added to Vero cells. After 24 h, the medium was aspirated and plates were washed 2 times with phosphate-buffered saline. A total of 200 ␮l/well of complete medium was added, and plates were incubated for an additional 48 h before medium was removed and cells were fixed with 100 ␮l/well of cold 10% trichloroacetic acid (TCA). After

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wherein passive or active immunization against the toxins has been shown to be highly protective (20–25). A recent report from this laboratory showed that a novel multivalent toxin-neutralizing antibody reverses fulminant CDI in mice when the antibody is given after disease symptoms have developed (26). Evidence that toxin blockade may also be protective in human patients originates from studies showing that high titers of antitoxin antibodies correlate with lower rates of primary and recurrent CDI in humans (27–31). Furthermore, intravenous immunoglobulin treatment is sometimes used to treat severe CDI under the assumption that such immunoglobulin preparations contain significant levels of antitoxin antibodies (32–36). These data clearly demonstrate that administration of neutralizing antitoxin antibodies is a viable approach to the treatment and prevention of CDI. Two particularly appealing features of this approach are that blocking the toxins should not have an impact on the normal gut flora and should not engender resistance emergence since the pathogen itself is not targeted. Actoxumab and bezlotoxumab are two human monoclonal antibodies that bind to and neutralize TcdA and TcdB, respectively (20). A combination of the antibodies (referred to herein as actoxumab-bezlotoxumab) is currently in phase III clinical trials for the prevention of recurrent CDI. When administered concurrently with the standard of care antibiotics vancomycin and metronidazole, actoxumab-bezlotoxumab caused a 73% decrease in recurrence rates in phase II clinical trials (37). Despite these findings, the cellular and physiological mechanisms through which actoxumab and bezlotoxumab protect against disease are poorly understood. In this study, we assess the efficacy of actoxumab and bezlotoxumab in murine models of CDI and demonstrate that (i) the antibodies prevent both epithelial damage and inflammatory disease and (ii) host effector functions are not involved in protection.

Yang et al.

RESULTS

Actoxumab-bezlotoxumab protects mice in C. difficile toxin challenge models. As a first step toward evaluating the ability of actoxumab-bezlotoxumab to protect against the C. difficile toxins in mice, we utilized a mouse model of systemic toxin challenge wherein mice were dosed with or without actoxumab-bezlotoxumab (0.1, 1, or 10 mg/kg) an hour before toxin administration. All vehicle-treated mice challenged with a mixture of TcdA and TcdB (25 ng of each toxin) died within 18 h, whereas pretreatment of mice with actoxumab-bezlotoxumab significantly protected the mice in a dose-dependent manner (Fig. 1A). These results demonstrate that actoxumab-bezlotoxumab neutralizes the toxins of C. difficile in vivo. Since CDI is largely a disease of the gut lumen, we utilized the ileal loop model to assess the effects of toxins administered directly into the gut lumen. Vehicle or toxin (2.5 ␮g each of TcdA and TcdB) was injected into isolated ileal loops of mice that had been dosed with vehicle or with actoxumab-bezlotoxumab 24 h earlier, and the effects of toxin on fluid accumulation within the ligated ileal loop was assessed 4 h later. Fluid accumulation in toxin-challenged mice (109.4 mg/cm) was significantly reduced in mice treated with actoxumab-bezlotoxumab (62.8 mg/cm), although the reduction was not complete (35.7 mg/cm or 38.9 mg/cm for vehicle-challenged mice treated with or without actoxumab-bezlotoxumab, respectively) (Fig. 1B). To assess the effect of actoxumab-bezlotoxumab on toxin-induced damage and inflammation, sections of the ileal walls of mice from each treatment group were examined histologically. As shown in Fig. 1C (quantified in Fig. 1D, E, and F), control mice treated with vehicle or with actoxumab-bezlotoxumab showed normal villous architec-

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ture with no evidence of inflammation or epithelial damage. Conversely, untreated toxin-challenged mice showed marked neutrophil infiltration, epithelial cell damage, erosion of the epithelium, loss of villous architecture, and edema. Treatment with actoxumab-bezlotoxumab was protective against damage and inflammation; although the decrease in overall damage score did not rise to the level of significance (Fig. 1D), effects on the epithelium were largely negated (Fig. 1C), and neutrophil infiltration was largely prevented by antibody treatment (Fig. 1E). Measurements of apoptotic cells within the gut wall were statistically indistinguishable across all treatment groups. The data overall indicate that actoxumab-bezlotoxumab protects against inflammation and damage associated with TcdA and TcdB in this acute and severe model of CDI. Actoxumab-bezlotoxumab treatment is protective against primary CDI in a spore challenge model. Actoxumab-bezlotoxumab has previously been shown to be effective in hamster and piglet spore challenge models of CDI (20, 24), but damage and inflammatory components of the disease have not been extensively studied. We therefore evaluated the efficacy of the antibodies in a mouse model of primary CDI (42) with the intent of understanding the mechanisms of protection and of assessing whether the effector functions of the antibodies are involved in efficacy. To address the latter question, we generated versions of actoxumab and bezlotoxumab that contain the N297Q mutation, rendering them incapable of binding to Fc␥R (data not shown) and of engendering effector functions of the host (43). These mutant antibodies neutralize the toxins with similar potency as the WT antibodies (see Fig. S2 in the supplemental material). We assessed the ability of actoxumab-bezlotoxumab to protect against CDI in a previously described model in mice challenged with the epidemic ribotype 027 strain UK1 (40). Mice treated with 10 mg/kg actoxumab-bezlotoxumab antibodies prophylactically 24 h before spore challenge (see Fig. S1A in the supplemental material) showed significantly reduced weight loss and mortality (Fig. 2A and B) throughout the time course of the experiment. Treatment doses of 2, 10, and 50 mg/kg had previously been evaluated in a pilot study, and 10 mg/kg was found to be the lowest fully efficacious dose (see Fig. S3 in the supplemental material). Importantly, the N297Q mutants of actoxumab and bezlotoxumab provided a level of protection similar to that of WT antibodies, demonstrating that effector functions of antibodies (which are dependent on engagement of Fc␥ receptors) are not required for efficacy (Fig. 2A and B). In a separate experiment, the efficacy of actoxumab-bezlotoxumab was also evaluated following injection of the antibodies 24 h after spore challenge (see Fig. S1B in the supplemental material). In this therapeutic paradigm, actoxumab-bezlotoxumab, compared to untreated mice, significantly prevented weight loss and mortality (Fig. 2C and D), although the effect on weight loss was not as robust as in the prophylactic paradigm even at a high dose of 50 mg/kg. To gain insight into the mechanism through which actoxumab and bezlotoxumab protect against morbidity (weight loss) and mortality in this model, we determined the extent of damage and inflammation in the intestines of mice 48 h after spore challenge in the prophylactic paradigm of the model (i.e., 10 mg/kg WT and N297Q mutant actoxumab-bezlotoxumab administered 24 h prior to spore challenge). The intestines of untreated infected mice showed significant signs of hemorrhaging and swelling due to fluid accumulation and also contained diffused and unformed

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60 min at 4°C, the TCA was removed and plates were washed 4 times with distilled water. A total of 100 ␮l/well of 100 ␮g/ml sulforhodamine B (Sigma, St. Louis, MO) in 10% acetic acid was added. Plates were incubated for 15 min at room temperature (RT) and then washed 4 times with 10% acetic acid and air dried. A total of 150 ␮l/well of 10 mM Tris was added, and plates were incubated with shaking at room temperature for 10 min. Plates were read in a SpectraMax plate reader (Molecular Biosystems) at an absorbance wavelength of 570 nm. Percent cell survival was calculated by comparing wells of treated and untreated cells. Binding of wild type and N297Q mutants of actoxumab and bezlotoxumab to Fc␥R. Kinetic binding activities of actoxumab, bezlotoxumab, and N297Q mutants (all IgG1) were evaluated against recombinant human Fc␥RI/CD64 (R&D Systems, Inc., Minneapolis, MN), hFc␥RIIIA/ CD16A (V) (R&D Systems, Inc.), hFc␥RIIA/CD32a, hFc␥RIIB/CD32b, and hFc␥RIIIA/CD16a (F), as well as murine Fc␥ RI/CD64 and mFcg RIIIA/ B/CD16, by surface plasmon resonance using a Biacore T200 system (Biacore, GE Healthcare, Piscataway, NJ). Approximately 5,000 resonance units of goat F(ab=)2 anti-human kappa (k chain specific) (Southern Biotech, Birmingham, AL) was immobilized via amine coupling chemistry onto a series S CM5 sensor chip (GE Healthcare). HBS-EP⫹ buffer (GE Healthcare) was used as the running buffer with a flow rate of 30 ␮l/min. Antibodies were injected separately at 1 ␮g/ml for a capture level of 200 RU. Various concentrations of Fc␥ receptors were injected over each antibody surface at 40 ␮l/min. Following each injection cycle, the sensor chip surface was regenerated using 10 mM glycine (pH 1.5) solution followed by an injection of 12.5 mM NaOH solution at a flow rate of 45 ␮l/min. Background subtraction binding sensorgrams were used for analyzing the rate constant of association (kon) and dissociation (koff) and the equilibrium dissociation constant (KD). The resulting data sets were fitted with either a 1:1 Langmuir binding model (for human and murine FcgRI) or a steady-state affinity model (for other receptors) using the Biacore T200 Evaluation Software (version 2.0).

Antitoxin Monoclonal Antibodies Protect against CDI

Downloaded from http://iai.asm.org/ on March 7, 2015 by UNIV OF CALIF-SAN FRANCISCO FIG 1 Actoxumab-bezlotoxumab protects against systemic and intestinal toxin challenge in mice. (A) Survival curve of CD1 mice treated with vehicle (blue line) or with 0.1 (light-pink line), 1 (pink line), and 10 (red line) mg/kg actoxumab-bezlotoxumab and systemically challenged 1 h later with TcdA and TcdB (25 ng each). Survival of mice was monitored over time. *, P ⫽ 0.05; **, P ⬍ 0.01, compared to the vehicle-treated mice, as determined by a log-rank/Mantel-Cox test. (B) Fluid accumulation in ileal loops after inoculation with vehicle (Veh) or C. difficile toxins (2.5 ␮g each; Tx) in mice treated with actoxumab-bezlotoxumab (A/B) or PBS, calculated as the ratio of loop weight (in mg) to loop length (in cm). *, P ⬍ 0.01 by one-way analysis of variance (ANOVA) with Dunnett’s posttest. (C) Histology sections from cecal wall of ileal loops taken from each group for panel B. (D to F) Histological analysis of ileal loop sections taken from each group in panel B. Damage score (D), average number of neutrophils (E), and average number of apoptotic cells (F) were assessed based on criteria listed in Materials and Methods. *, P ⬍ 0.05, compared to PBS-Tx group, as assessed by one-way ANOVA with Dunnett’s posttest.

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circles), with 10 mg/kg actoxumab-bezlotoxumab (red circles), or with 10 mg/kg N297Q mutant actoxumab-bezlotoxumab (green circles) 24 h prior to challenge with C. difficile spores. Each point is the mean ⫾ standard deviation (SD) from 6 to 15 mice. *, P ⬍ 0.05; **, P ⬍ 0.001, compared to vehicle-treated mice as assessed by two-way ANOVA with Tukey’s posttest. (B) Survival of mice treated and challenged as described for panel A (blue line, vehicle; red line, actoxumabbezlotoxumab; green line, N297Q actoxumab-bezlotoxumab). *, P ⬍ 0.02, compared to vehicle-treated mice, as determined by a log-rank/Mantel-Cox test. (C) Weights of mice treated with vehicle (blue circles), with 50 mg/kg actoxumab-bezlotoxumab (red circles), or with 5 mg/kg actoxumab-bezlotoxumab (pink circles) 24 h postchallenge with C. difficile spores. Each point is the mean ⫾ SD from 5 to 10 mice, except for vehicle-treated mice on days 3 and 5 (only one mouse remaining). *, P ⬍ 0.01; **, P ⬍ 0.0001, compared to vehicle-treated mice as assessed by two-way ANOVA with Tukey’s posttest. (D) Survival of mice treated and challenged as described in panel C (blue line, vehicle; pink line, 5 mg/kg actoxumab-bezlotoxumab; red line, 50 mg/kg actoxumab-bezlotoxumab). *, P ⬍ 0.01; **, P ⬍ 0.001, compared to vehicle-treated mice, as determined by a log-rank/Mantel-Cox test.

feces (Fig. 3A). Conversely, the intestines of actoxumab-bezlotoxumab-treated mice were unremarkable, with no discernible swelling or hemorrhaging and fully formed fecal pellets. Histological examination of the ceca revealed marked neutrophil infiltration within the lamina propria of untreated infected mice, along with significant damage to (and sloughing of) the epithelium, enterocyte necrosis, and presence of apoptotic cells (Fig. 3B to E). Treatment with 10 mg/kg wild-type and N297Q actoxumab-bezlotoxumab resulted in significant reduction in damage to the epithelium and other structures of the gut wall (Fig. 3B), consistent with a significant decrease in overall damage score (Fig. 3C) and in numbers of infiltrated neutrophils (Fig. 3D) and apoptotic cells (Fig. 3E), although the effects on neutrophils failed to reach statistical significance. We have previously demonstrated in animal CDI models that the leakage of the toxins into the circulation causes severe and systemic disease (44). To determine the extent of toxin leakage from the lumen of the intestines to the circulation in the mouse model and, by extension, to evaluate the ability of actoxumabbezlotoxumab to prevent this leakage, serum samples from infected mice were tested for their ability to induce rounding of Vero cells as described previously (44, 45). Significant rounding of Vero cells was observed on days 1 through 4 postinfection in untreated mice, whereas samples from mice treated with WT and N297Q actoxumab-bezlotoxumab were largely devoid of cell rounding activity throughout this period with one or two exceptions

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(Fig. 3F). This observation indicates either that toxins do not leak into the circulation due to antibody-mediated protection against damage to the gut wall (Fig. 3B and C) or that toxin is present systemically but is fully neutralized by circulating antibodies. In either case, these data are consistent with the previous observation that actoxumab-bezlotoxumab protects against systemic disease in CDI (24). Taken together, these data demonstrate that actoxumab-bezlotoxumab treatment has profound protective effects on the toxin-induced intestinal wall damage and on the inflammatory responses that are typical of this model and that this protection prevents not only the local (intestinal) effects of toxins but also any potential systemic effects. Actoxumab-bezlotoxumab treatment is protective against recurrent CDI in a spore challenge model. The combination of actoxumab and bezlotoxumab, when administered during a primary CDI, has been shown to be efficacious in preventing recurrent CDI clinically (37). We therefore assessed the ability of the antibodies to prevent recurrence in a mouse model of recurrent CDI (40). In this model, actoxumab-bezlotoxumab-treated mice that have been subjected to, and survived, a primary infection (prophylactic paradigm; Fig. 2A and B) were allowed to recover for 7 days following the first spore challenge and sensitized with a second round of antibiotics (see Fig. S1C in the supplemental material and Materials and Methods). This second antibiotic treatment caused some of the mice in the vehicle-injected group, but not in the antibody groups, to succumb to disease following

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FIG 2 Actoxumab-bezlotoxumab is protective in both prophylactic and therapeutic paradigms of primary CDI. (A) Weights of mice treated with vehicle (blue

Antitoxin Monoclonal Antibodies Protect against CDI

actoxumab-bezlotoxumab (WT) collected 48 h following spore challenge. Pictures are representative of three animals per group. (B) Hematoxylin-eosin-stained histology sections from the ceca of spore-challenged mice treated with PBS or with 10 mg/kg WT or N297Q mutant (N297Q) actoxumab-bezlotoxumab, collected 48 h following spore challenge. (C to E) Histological analysis of ileal loop sections taken from each group in panel B. Damage score (C), average number of neutrophils (D), and average number of apoptotic cells (E) were assessed based on criteria listed in Materials and Methods. *, P ⬍ 0.05, compared to the PBS group, as assessed by one-way ANOVA with Dunnett’s posttest. (F) Toxin-dependent cell rounding in sera of mice treated with PBS or with WT or N297Q mutant actoxumab-bezlotoxumab. Cell rounding in Vero cells incubated with serum samples from mice on days 1 (red circles), 2 (green circles), 3 (pink triangles), 4 (blue inverted triangles), and 6 (brown diamonds) following spore challenge was assessed as described in Materials and Methods.

antibiotic treatment (starting 2 days before rechallenge; see Fig. 4B), presumably due to the reemergence of C. difficile from spores remaining from the primary challenge (relapse). Surviving mice were rechallenged with C. difficile spores 24 h after clindamycin administration (13 days total after the first challenge) to simulate

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a reinfection, and body weight and survival were monitored. In this paradigm, a single 10-mg/kg dose of actoxumab-bezlotoxumab (WT and N297Q mutants) administered prior to the primary infection failed to protect against the decrease in body weight observed following the second challenge (Fig. 4A), al-

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FIG 3 Actoxumab-bezlotoxumab protects against damage and inflammation in primary CDI. (A) Intestines of mice treated with either PBS or 10 mg/kg

Yang et al.

though mouse survival was increased with antibody treatment (P ⫽ 0.16 for WT and P ⫽ 0.03 for N297Q) (Fig. 4B). Since antibodies had been administered a full 14 days prior to the second spore challenge in this experiment (see Fig. S1C), we hypothesized that the weaker level of protection observed in the recurrence model than the prophylactic and therapeutic primary infection models (Fig. 2) was due to a poor pharmacokinetic profile of the human antibodies in the mouse. Indeed, the rate of clearance of actoxumab-bezlotoxumab is much higher, and the concentration of antibodies at 48 h postinjection is much lower, in mice administered a 10-mg/kg dose than in humans administered the same dose (half-life of 6 to 7 compared to 22 to 26 days, and serum concentrations of ⬃50 compared to ⬃300 ␮g/ml 48 h postadministration, in mice and humans, respectively) (see Fig. S4 in the supplemental material) (37). As a result, antibody levels in the context of the recurrence model are much lower than during the primary infection. To mitigate against the poor pharmacokinetic profile of the antibodies in mice, and to better mimic the situation in human patients where high levels of circulating antibodies persist for weeks to months (37), we carried out an additional recurrence experiment wherein the mice were allowed to rest for a longer period (14 days) following primary challenge (to minimize the chance of spontaneous relapse observed with only 7 days of

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rest) and were administered a second dose of actoxumab-bezlotoxumab 24 h prior to the reinfection (see Fig. S1D). The second dose of antibodies protected mice against both weight loss (Fig. 4C) and death (Fig. 4D), even at a low dose of 2 mg/kg, demonstrating that the poor efficacy observed in the absence of a second dose was indeed due to low antibody levels. Overall, therefore, actoxumab-bezlotoxumab prevents CDI recurrence in mice when administered prior to the recurrent episode, similar to results obtained in human patients (37). DISCUSSION

Although current antibiotic treatments are largely successful in curing initial episodes of CDI, the morbidity, mortality, and rate of recurrence are increasing, in part due to the emergence of toxinhyperproducing strains such as NAP1/BI/027. The need for new therapeutic options that inhibit or neutralize the bacterial toxins is therefore critical. One approach currently in development involves administration of a combination of human monoclonal antitoxin antibodies, actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively (20, 37). These antibodies are protective against CDI in hamsters (20) and piglets (24) and have shown promising results against recurrent CDI when tested in humans (37). The ability of actoxumab-bezlotox-

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FIG 4 Actoxumab-bezlotoxumab is protective in recurrent CDI. (A) Weights of mice that have undergone a primary challenge with C. difficile spores and have been allowed to recover for 13 days before rechallenge with C. difficile spores (see Fig. S1C in the supplemental material). Day 0 represents the day of the rechallenge. Mice were treated with vehicle (blue circles), with 10 mg/kg actoxumab-bezlotoxumab (red circles), or with 10 mg/kg N297Q mutant actoxumabbezlotoxumab (green circles), 14 days prior to the rechallenge (24 h prior to the primary challenge). Each point is the mean ⫾ SD from 7 to 12 mice. (B) Survival of mice treated and challenged as described for panel A (blue line, vehicle; red line, actoxumab-bezlotoxumab; green line, N297Q actoxumab-bezlotoxumab). *, P ⬍ 0.05, compared to vehicle-treated mice, as determined by a log-rank/Mantel-Cox test. Day 0 represents the day of rechallenge. (C) Weights of mice that have undergone a primary challenge with C. difficile spores and have been allowed to recover for 20 days before being rechallenged with C. difficile spores (see Fig. S1D in the supplemental material). Day 0 represents the day of the rechallenge. Mice were treated with vehicle (blue circles), with 2 mg/kg actoxumab-bezlotoxumab (light-pink circles), with 10 mg/kg actoxumab-bezlotoxumab (pink circles), or with 50 mg/kg actoxumab-bezlotoxumab (red circles) 21 days prior to the rechallenge (24 h prior to the primary challenge) and again 24 h prior to the rechallenge. Each point is the mean ⫾ SD from 4 to 10 mice. *, P ⬍ 0.05; **, P ⬍ 0.001, compared to vehicle-treated mice, as assessed by two-way ANOVA with Tukey’s posttest. (D) Survival of mice treated and challenged as described in panel C (blue line, vehicle; light-pink line, 2 mg/kg actoxumab-bezlotoxumab; pink line, 10 mg/kg actoxumab-bezlotoxumab; red line, 50 mg/kg actoxumab-bezlotoxumab).

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actoxumab-bezlotoxumab to prevent damage and inflammation in the gut wall in the ileal loop and spore challenge models. Treatment with the antibodies reduced or prevented intestinal wall damage, including disruption of the epithelium and recruitment of neutrophils, in both models (Fig. 1C and D and 3B and C), although the protection failed to reach the level of significance in the ileal loop model when looking at overall damage score (Fig. 1D). These data are consistent with previously observed in vitro neutralization of toxin-induced cytopathic and cytotoxic effects on mammalian cells, including gut epithelial cells, by actoxumab and bezlotoxumab (20, 46, 47). In addition to their protective effects against epithelial damage, the antibodies significantly decrease neutrophil recruitment to the gut wall in the ileal loop model (with a nonsignificant trend toward a decrease in the C. difficile challenge model; see Fig. 1E and 3D), consistent with previous in vitro data obtained by Koon and coworkers showing that actoxumab and bezlotoxumab prevent toxin-induced release of proinflammatory cytokines in peripheral blood mononuclear cells and human colonic explants (46). The protective effects of actoxumab-bezlotoxumab in the spore challenge model not only results in decreased damage and inflammation at the site of infection (i.e., the gut) but may also prevent systemic dissemination of active toxins from the gut lumen (Fig. 3F), which can occur in severe models of CDI (44). Actoxumab and bezlotoxumab bind to the receptor binding or combined repetitive oligopeptide (CROP) domains of TcdA and TcdB, respectively (20), and we have recently demonstrated that bezlotoxumab binds to regions of the CROP that overlap the putative receptor binding pockets and prevent binding of TcdB to mammalian cells (47). Despite this demonstration that bezlotoxumab directly neutralizes TcdB, the possibility exists that the antibodies may protect mice from severe inflammation at least partially through the recruitment of effector functions in host immune cells via the Fc␥ receptors. In testing mutant forms of actoxumab and bezlotoxumab in which a single amino acid change (N297Q) renders the antibodies unable to bind to Fc␥ receptors and to induce effector cell activity (43), we found no significant differences in protection compared to wild-type actoxumab-bezlotoxumab (Fig. 3 and 4). These results suggest that actoxumab-bezlotoxumab does not ameliorate symptoms through Fc␥ receptor-mediated effector functions but rather solely via direct neutralization of the toxins. This is in line with a recent study from our group showing that systemic administered neutralizing VHH antibodies devoid of an Fc fragment protect mice from fulminant CDI (26). The overall results from this study indicate that treatment of mice with antitoxin antibodies significantly protects against the morbidity and mortality associated with CDI induced by both historical (VPI 10463, in the case of the toxin challenge models) and hypervirulent (NAP1/BI/027) strains of C. difficile. The mechanism of protection is through direct neutralization of the toxins and prevention of toxin-induced epithelial damage and subsequent inflammatory response and does not appear to involve host effector functions. The antibodies are beneficial both for the prevention and treatment of primary infections and for the prevention of recurrent CDI. The intended clinical use of actoxumabbezlotoxumab is for the prevention of recurrent disease when dosed in conjunction with standard-of-care antibiotics, since the antibodies alone are not predicted to have an impact on C. difficile bacterial burden. Our results suggest that preventing the epithelial

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umab to protect against the clinically common hypervirulent strain NAP1/BI/027 is still largely unknown, as are the physiological mechanisms through which the antibodies protect. In this study, we used various mouse models of CDI to assess the protection afforded by these antibodies and to better understand specific aspects of disease that are prevented by the antibodies. Additional questions addressed included whether actoxumab-bezlotoxumab is efficacious against a strain of ribotype 027 and whether effector functions, in addition to direct neutralization of toxins, play a role in protection. Actoxumab and bezlotoxumab neutralize their respective toxins in vivo, as shown by increased survival of mice after systemic challenge with C. difficile toxins (Fig. 1A) and reduced fluid accumulation in the ileal loop model (Fig. 1B). These data are consistent with previous results obtained by Babcock et al. in hamsters (20) but provide additional data about the specific mechanism of protection, as discussed below. Given the artificially acute and severe nature of these aforementioned models, we assessed actoxumab-bezlotoxumab in a more traditional model of CDI, the previously described intragastric spore challenge mouse model (40, 42). Using this model, we showed that actoxumab-bezlotoxumab antibodies are protective against a hypervirulent strain (UK1, a ribotype 027 strain) of C. difficile in both prophylactic and therapeutic treatment paradigms (Fig. 2). Actoxumab-bezlotoxumab significantly protected mice from morbidity (weight loss) and mortality associated with CDI. The antibody combination was also protective in a mouse recurrent CDI model (Fig. 4), a paradigm which simulates its intended use in human patients, where actoxumab and bezlotoxumab will be administered concurrently with standard-of-care antibiotics during a primary infection to prevent a recurrent episode (37). Importantly, the level of protection afforded by the antibodies depends on systemic antibody concentrations since a single dose administered 14 days prior to the recurrent episode in mice failed to significantly impact the loss in body weight associated with the second spore challenge (Fig. 4A). Given the short half-life of the antibodies in mice (⬃6 days), this is not entirely surprising, although it is noteworthy that even at serum concentrations of ⬃15 ␮g/ml (see Fig. S4 in the supplemental material), mortality was lower in antibody-treated mice than in the vehicle group (Fig. 4B). When mice were dosed a second time with actoxumab-bezlotoxumab 24 h before the second challenge, clear protection against both weight loss and mortality was observed (Fig. 4C and D), demonstrating that antibody serum levels are indeed a key determinant of protection. To gauge the predictive value of these data in terms of the clinical efficacy of actoxumab-bezlotoxumab, one must take into account the fact that the half-life of actoxumab-bezlotoxumab is at least 4-fold longer in humans than in mice (see Fig. S4 in the supplemental material) (37). Indeed, serum levels of actoxumab and bezlotoxumab remain above 15 ␮g/ml (at which concentration partial protection was observed in mice, as discussed above) for at least 56 days following antibody administration (37). Therefore, based on the mouse data, the antibodies are predicted to be protective for several weeks postadministration in human patients, consistent with the results of a phase 2 study assessing the ability of the antibodies to prevent recurrent episodes of CDI (37). The symptoms of CDI are due primarily to direct damage to the gut wall epithelium as well as to the induction of a local proinflammatory response that involves the recruitment of neutrophils to the site of damage. We therefore assessed the ability of

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damage and inflammation that underlie the symptoms of CDI is sufficient for protection against morbidity and mortality, at least in the mouse models described herein.

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This work was supported by awards R01AI088748, R01DK084509, R56AI99458, and U19AI109776 funded from the National Institute of Allergy and Infectious Diseases and National Institute of Diabetes and Digestive and Kidney Diseases at the National Institutes of Health (NIH) and contracts LKR-107608 and LKR-128468 from Merck & Co., Inc.

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