Development of a Novel Vaccine Containing Binary Toxin for the Prevention of Clostridium difficile Disease with Enhanced Efficacy against NAP1 Strains.

Clostridium difficile infections (CDI) are a leading cause of nosocomial diarrhea in the developed world. The main virulence factors of the bacterium are the large clostridial toxins (LCTs), TcdA and TcdB, which are largely responsible for the symptoms of the disease. Recent outbreaks of CDI have been associated with the emergence of hypervirulent strains, such as NAP1/BI/027, many strains of which also produce a third toxin, binary toxin (CDTa and CDTb). These hypervirulent strains have been associated with increased morbidity and higher mortality. Here we present pre-clinical data describing a novel tetravalent vaccine composed of attenuated forms of TcdA, TcdB and binary toxin components CDTa and CDTb. We demonstrate, using the Syrian golden hamster model of CDI, that the inclusion of binary toxin components CDTa and CDTb significantly improves the efficacy of the vaccine against challenge with NAP1 strains in comparison to vaccines containing only TcdA and TcdB antigens, while providing comparable efficacy against challenge with the prototypic, non-epidemic strain VPI10463. This combination vaccine elicits high neutralizing antibody titers against TcdA, TcdB and binary toxin in both hamsters and rhesus macaques. Finally we present data that binary toxin alone can act as a virulence factor in animal models. Taken together, these data strongly support the inclusion of binary toxin in a vaccine against CDI to provide enhanced protection from epidemic strains of C. difficile.

Development and optimization of a high-throughput assay to measure neutralizing antibodies against Clostridium difficile binary toxin.

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.

Establishment of higher passage PER.C6 cells for adenovirus manufacture.

PER.C6 cells, an industrially relevant cell line for adenovirus manufacture, were extensively passaged in serum-free suspension cell culture to better adapt them to process conditions. The changes in cell physiology that occurred during this passaging were characterized by investigating cell growth, cell size, metabolism, and cultivation of replication-deficient adenovirus. The changes in cell physiology occurred gradually as the population doubling level, the number of times the cell population had doubled, increased. Higher passage PER.C6 (HP PER.C6) proliferated at a specific growth rate of 0.043 h(-1), 2-fold faster than lower passage PER.C6, and were capable of proliferation from lower inoculation cell densities. HP PER.C6 cell volume was 16% greater, and cellular yields on glucose, lactate, oxygen, and amino acids were greater as well. In batch cultures, HP PER.C6 cells volumetrically produced 3-fold more adenovirus, confirmed with three different constructs. The increase in productivity was also seen on a cell-specific basis. Although HP PER.C6 were more sensitive to the "cell density effect", requiring lower infection cell densities for optimal specific productivity, they proliferated more after infection than lower passage PER.C6, increasing the number of cells available for virus production. The extensive passaging established HP PER.C6 cells with several desirable attributes for adenovirus manufacture.