Development and Optimization of a Novel Assay To Measure Neutralizing Antibodies against Clostridium difficile Toxins

Abstract

e Clostridium difficile produces two major virulence toxins, toxin A (TcdA) and toxin B (TcdB). Antitoxin antibodies, especially neutralizing antibodies, have been shown to be associated with a lower incidence of C. difficile infection (CDI) recurrence, and antibody levels are predictive of asymptomatic colonization. The development of an assay to detect the presence of neutralizing antibodies in animal and human sera for the evaluation of vaccine efficacy is highly desired. We have developed such an assay, which allows for the quantification of the effect of toxins on eukaryotic cells in an automated manner. We describe here the optimization of this assay to measure toxin potency as well as neutralizing antibody (NAb) activity against C. difficile toxins using a design-of-experiment (DOE) methodology. Toxin concentration and source, cell seeding density, and serum-toxin preincubation time were optimized in the assay using Vero cells. The assay was shown to be robust and to produce linear results across a range of antibody concentrations. It can be used to quantify neutralizing antibodies in sera of monkeys and hamsters immunized with C. difficile toxoid vaccines. This assay was shown to correlate strongly with traditional assays which rely on laborintensive methods of determining neutralizing antibody titers by visual microscopic inspection of intoxicated-cell monolayers. This assay has utility for the selection and optimization of C. difficile vaccine candidates.

Caspase Activation as a Versatile Assay Platform for Detection of Cytotoxic Bacterial Toxins

“…Detection of filamentous actin (F-actin) polymerization was employed as a means to correlate the results with the caspase activation assay data. The F-actin assay was described in detail by Xie et al (29). Briefly, Vero cell suspensions were added to 384-well plates and incubated overnight at 37°C in 5% CO 2 .…”

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

“…This method is plagued with low signal to noise ratios and variability due to substantial ATP levels remaining in cells that are not yet dead and still undergoing morphological changes due to intoxication [13]. Additionally, normal metabolismrelated fluctuations in ATP levels that are unrelated to cell viability can further affect the assay readout.…”

“…Historically, studying the effects of TcdA and TcdB on mammalian cells has been hampered by time-consuming and subjective assays that rely, for example, on visualization of cells to assess cell rounding or on the variable quantitation of ATP levels to measure cell death [13]. Thus, there is a scarcity of robust quantitative assays that measure the various cellular events associated with the intoxication cascade, making it difficult to evaluate new toxin-directed agents.…”

“…As a result, up to 25% or more patients successfully cured of an initial episode of CDI with these antibiotics suffer a recurrent episode within days to weeks. To address this, recent approaches to CDI treatment, including vaccines and monoclonal antibodies, have focused on neutralizing the effects of TcdA and TcdB, specifically, rather than the organisms itself [11][12][13]. Foremost among these novel therapies is bezlotoxumab, the anti-TcdB antibody recently approved by the Food and Drug Administration for reducing recurrence of CDI in patients 18 years of age or older who are receiving antibacterial drug treatment of CDI and are at a high risk for CDI recurrence.…”

“…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].…”