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

Abstract

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...

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

“…Both antibodies fully neutralized the effects of their respective toxins from all ribotypes tested (Figure 7C and D). However, the neutralization potencies of both antibodies for toxins of ribotypes 027 and 078 were significantly lower than toxins of ribotype 087, similar to data obtained in the Rac1 glucosylation assay above (Section 2.3) and consistent with previous data in the SRB assay [15].…”

“…Notably, the potency of actoxumab and bezlotoxumab on their respective toxins was lower for toxins of ribotype 027 and 078 compared to ribotype 087. This is consistent with the lower affinities of the antibodies against toxins of these ribotypes, as previously described by Hernandez et al [15]. Figure 1)…”

“…To confirm this finding, full concentration-response curves of actoxumab and bezlotoxumab were generated against dilutions of intact or immunodepleted supernatants associated with ~90% reduction in cell viability (EC 90 ); actoxumab neutralized the cytotoxic activity of immunodepleted supernatants, whereas bezlotoxumab neutralized the cytotoxic activity of intact supernatants, and no cross-neutralization was observed (Figure 8C and D). This approach has been used successfully to assess the activities of actoxumab and bezlotoxumab on TcdA and TcdB of dozens of clinical isolates of C. difficile, covering 18 distinct ribotypes (seven toxinotypes) [15]. …”

“…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].…”

Exposure of Neutralizing Epitopes in the Carboxyl-terminal Domain of TcdB Is Altered by a Proximal Hypervariable Region

“…Based on this assessment, we can reasonably conclude that bezlotoxumab should interact with target epitopes both in TcdB 027 and TcdB 012 , yet the neutralization experiments suggest that TcdB 027 is less sensitive to the antibody (30). More recently, Hernandez et al (33) performed a comprehensive analysis of bezlotoxumab reactivity with TcdB from several ribotypes and found that subtle sequence variations do indeed affect the EC 50 , but this can be overcome by reasonable increases in the amounts of antibody applied to the toxin. The extent to which intramolecular or intermolecular interactions shield these and other specific neutralizing epitopes has yet to be determined.…”