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.

e Clostridium difficile strains producing binary toxin, in addition to toxin A (TcdA) and toxin B (TcdB), have been associated with more severe disease and increased recurrence of C. difficile infection in recent outbreaks. Binary toxin comprises two subunits (CDTa and CDTb) and catalyzes the ADP-ribosylation of globular actin (G-actin), which leads to the depolymerization of filamentous actin (F-actin) filaments. A robust assay is highly desirable for detecting the cytotoxic effect of the toxin and the presence of neutralizing antibodies in animal and human sera to evaluate vaccine efficacy. We describe here the optimization, using design-of-experiment (DOE) methodology, of a high-throughput assay to measure the toxin potency and neutralizing antibodies (NAb) against binary toxin. Vero cells were chosen from a panel of cells screened for sensitivity and specificity. We have successfully optimized the CDTa-to-CDTb molar ratio, toxin concentration, cell-seeding density, and sera-toxin preincubation time in the NAb assay using DOE methodology. This assay is robust, produces linear results across serial dilutions of hyperimmune serum, and can be used to quantify neutralizing antibodies in sera from hamsters and monkeys immunized with C. difficile binary toxin-containing vaccines. The assay will be useful for C. difficile diagnosis, for epidemiology studies, and for selecting and optimizing vaccine candidates. Clostridium difficile is a Gram-positive bacterium and the primary cause of hospital-acquired diarrhea. C. difficile infection (CDI) occurs when antibiotic treatment disrupts the normal bacterial flora of the intestine, which allows for the colonization of C. difficile bacteria. CDI is characterized by symptoms that range from mild diarrhea to severe and often life-threatening colitis. The incidence of more severe CDI has been increasing in recent years due to the emergence of epidemic hypervirulent strains (1, 2).Two glucosylating toxins, TcdA and TcdB, are considered the main virulence factors of C. difficile. These two toxins catalyze the glucosylation of proteins RhoA, Rac, and Cdc42, which leads to the disruption of actin filaments and collapse of the cell cytoskeleton, disruption of other signaling pathways, and eventually, cell death (3-5). In addition to TcdA and TcdB, hypervirulent strains have been found to express a binary toxin referred to as C. difficile transferase (CDT). Much of what we know of CDT was learned through a comparison to related binary toxins found in other clostridial species, such as Clostridium botulinum C2 toxin and Clostridium perfringens iota toxin (6-8). Binary toxins are characterized as having an enzymatically active "A" component that causes ADP-ribosylation of globular actin (G-actin) and a cell binding and translocation "B" component. For C. difficile, CDTa and CDTb are transcribed monocistronically but translated as two separate proteins. The proposed mechanism of action for the C. difficile binary toxin has been described in the literature (7). Briefly, the precursor C...

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Clostridium difficile infections (CDI) are caused by colonization and growth of toxigenic strains of C. difficile in individuals whose intestinal microbiota has been perturbed, in most cases following antimicrobial therapy. Determination of the protective commensal gut community members could inform the development of treatments for CDI. Here, we utilized the lethal enterocolitis model in Syrian golden hamsters to analyze the microbiota disruption and recovery along a 20-day period following a single dose of clindamycin on day 0, inducing in vivo susceptibility to C. difficile infection. To determine susceptibility in vitro, spores of strain VPI 10463 were cultured with and without soluble hamster fecal filtrates and growth was quantified by quantitative PCR and toxin immunoassay. Fecal microbial population changes over time were tracked by 16S ribosomal RNA gene analysis via V4 sequencing and the PhyloChip assay. C. difficile culture growth and toxin production were inhibited by the presence of fecal extracts from untreated hamsters but not extracts collected 5 days post-administration of clindamycin. In vitro inhibition was re-established by day 15, which correlated with resistance of animals to lethal challenge. A substantial fecal microbiota shift in hamsters treated with antibiotics was observed, marked by significant changes across multiple phyla including Bacteroidetes and Proteobacteria. An incomplete return towards the baseline microbiome occurred by day 15 correlating with the inhibition of C. difficile growth in vitro and in vivo. These data suggest that soluble factors produced by the gut microbiota may be responsible for the suppression of C. difficile growth and toxin production.