Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

Orth, Peter; Xiao, Li; Hernandez, Lorraine D.; Reichert, Paul; Sheth, Payal R.; Beaumont, Maribel; Yang, Xiaoyu; Murgolo, Nicholas; Ermakov, Grigori; DiNunzio, Edward; Racine, Fred; Karczewski, Jakub; Secore, Susan; Ingram, Richard; Mayhood, Todd; Strickland, Corey; Therien, Alex G.

doi:10.1074/jbc.m114.560748

Cited by 118 publications

(131 citation statements)

References 44 publications

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“…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.…”

Section: Discussionsupporting

confidence: 81%

“…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.…”

Section: Discussionmentioning

confidence: 99%

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Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab

et al. 2015

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dClostridium 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. Clostridium 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 (rein...

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Section: Discussionsupporting

confidence: 81%

Section: Discussionmentioning

confidence: 99%

Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab

et al. 2015

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“…Actoxumab (MK-3415, GS-CDA1, and MDX-066) and bezlotoxumab (MK-6072, MBL-CDB1, and MDX-1388) are human monoclonal antibodies that target TcdA and TcdB, respectively (6). We showed previously that bezlotoxumab binds to two separate sites in the CROP domain of TcdB, partially overlapping with putative receptor binding pockets and blocking binding of the toxin to host cells (8); actoxumab, which binds to the CROP domain of TcdA (6), is presumed to work in a similar manner. In a relatively small phase II study that enrolled 200 patients in the United States and Canada, the actoxumab-bezlotoxumab combination, when given with standard-of-care therapy, reduced the rate of CDI recurrence, compared to placebo (7% and 25% recurrence, respectively) (7).…”

mentioning

confidence: 99%

Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted by In Vitro Neutralization and Epitope Modeling

Hernandez

Racine

Xiao

et al. 2015

Antimicrob Agents Chemother

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Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea and primarily involve two exotoxins, TcdA and TcdB. Actoxumab and bezlotoxumab are human monoclonal antibodies that neutralize the cytotoxic/cytopathic effects of TcdA and TcdB, respectively. In a phase II clinical study, the actoxumab-bezlotoxumab combination reduced the rate of CDI recurrence in patients who were also treated with standard-of-care antibiotics. However, it is not known whether the antibody combination will be effective against a broad range of C. difficile strains. As a first step toward addressing this, we tested the ability of actoxumab and bezlotoxumab to neutralize the activities of toxins from a number of clinically relevant and geographically diverse strains of C. difficile. Neutralization potencies, as measured in a cell growth/survival assay with purified toxins from various C. difficile strains, correlated well with antibody/toxin binding affinities. Actoxumab and bezlotoxumab neutralized toxins from culture supernatants of all clinical isolates tested, including multiple isolates of the BI/NAP1/027 and BK/NAP7/078 strains, at antibody concentrations well below plasma levels observed in humans. We compared the bezlotoxumab epitopes in the TcdB receptor binding domain across known TcdB sequences and found that key substitutions within the bezlotoxumab epitopes correlated with the relative differences in potencies of bezlotoxumab against TcdB of some strains, including ribotypes 027 and 078. Combined with in vitro neutralization data, epitope modeling will enhance our ability to predict the coverage of new and emerging strains by actoxumab-bezlotoxumab in the clinic. Infection with the Gram-positive, spore-forming, anaerobic bacterium Clostridium difficile is the leading cause of hospital-acquired infectious diarrhea in the developed world and can have potentially life-threatening effects. In the United States, approximately 14,000 deaths per year are attributed to C. difficile infections (CDIs), with an additional 250,000 patients per year requiring hospitalization or an increased length of hospital stay due to infection. As a result, it is estimated that more than $1 billion per year are spent in excess medical costs for treatment of CDIs in the United States (1, 2).C. difficile is transmitted by spores through the fecal-oral route, often in a hospital or health care facility setting. Treatment with broad-spectrum antibiotics, which suppress the normal gut flora, is the primary risk factor for development of CDIs. In the absence of bacterial competition, C. difficile is able to thrive and to colonize the large intestine, leading to symptoms that can include mild to severe diarrhea, fever, pseudomembranous colitis, and toxic megacolon (2). While primary CDIs are generally successfully treated with the current standard-of-care antibiotics vancomycin, metronidazole, and most recently fidaxomicin, over the past decade there has been an increase in antibiotic-resistant and socalled hypervi...

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“…Additionally, normal metabolismrelated fluctuations in ATP levels that are unrelated to cell viability can further affect the assay readout. We developed a more robust colorimetric assay that measures cellular protein content as a surrogate of cell growth and survival [14]. The sulforhodamine B (SRB) assay was used to determine the cytotoxic effects of purified C. difficile toxins of the reference strain VPI 10463 (ribotype 087) and from strains of ribotypes from the so-called hyper-virulent ribotypes 027 and 078.…”

Section: Toxin-induced Cytotoxicity (Step 5 Inmentioning

confidence: 99%

“…In this chapter, we describe multiple quantitative cell-based assays that were newly developed, or adapted and optimized from previous reports, and used to interrogate the effect of the C. difficile toxins on epithelial cells. The assays are validated using the highly specific and potent antitoxin antibodies, actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively [13][14][15].…”

Section: Introductionmentioning

confidence: 99%

Assays for Measuring C. difficile Toxin Activity and Inhibition in Mammalian Cells

Cox¹,

Hernandez²,

Gupta³

et al. 2017

Clostridium Difficile - A Comprehensive Overview

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Clostridium difficile infections (CDIs) are the leading cause of hospital-acquired infectious diarrhea. The symptoms of CDI are caused by two exotoxins, TcdA and TcdB, which are structurally and functionally highly homologous. Both toxins bind to specific receptors on mammalian cells, are internalized through endocytosis, translocate to the cytoplasm, and inactivate Rho-type GTPases via covalent glucosylation. This leads to downstream events that include morphological changes and disruption of epithelial tight junctions, release of pro-inflammatory mediators, and cell death. Assays used to assess the effects of toxins on cells have historically relied on evaluation of cell rounding or quantitation of ATP levels to estimate cell death-assays which can be qualitative and variable. In this chapter, several assays are described that robustly and quantitatively measure early and late toxin-dependent events in cells, including (i) toxin binding, (ii) Rac1 glucosylation, (iii) changes in cellular morphology (measured as dynamic mass redistribution), (iv) loss of epithelial integrity (measured as transepithelial electrical resistance), and (v) cell death (measured as total cellular protein using a colorimetric assay). The assays were validated using the highly specific monoclonal antitoxin antibodies, actoxumab and bezlotoxumab, which neutralize TcdA and TcdB, respectively.

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Mechanism of Action and Epitopes of Clostridium difficile Toxin B-neutralizing Antibody Bezlotoxumab Revealed by X-ray Crystallography

Cited by 118 publications

References 44 publications

Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab

Mechanisms of Protection against Clostridium difficile Infection by the Monoclonal Antitoxin Antibodies Actoxumab and Bezlotoxumab

Broad Coverage of Genetically Diverse Strains of Clostridium difficile by Actoxumab and Bezlotoxumab Predicted by In Vitro Neutralization and Epitope Modeling

Assays for Measuring C. difficile Toxin Activity and Inhibition in Mammalian Cells

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