The development of physiologically relevant assays at early stages of the screening cascade can reduce attrition further downstream by ensuring target activity in an appropriate biological context with respect to disease. We set out to design and implement just such an assay cascade to identify and characterise antibodies capable of neutralising Clostridium difficile enterotoxins.
C. difficile is the most common cause of hospital infection and in the US incidence is estimated as 478,000 cases p.a. with up to $3.8bn of healthcare costs. The two major exotoxins, TcdA and TcdB, have been established as the major pathogenicity determinants of C. difficile in a large number of in vitro and in vivo studies.
The standard bioassay of toxin activity measures viability of the Vero monkey kidney cell line. We developed several assays using the human cell line, Caco-2. This gut epithelial cell line is derived from a pertinent human organ and cell-type with respect to disease, and has the advantage of being suitable for in vitro differentiation to a polarised morphology. An array of assays using this cell line was used to inform our selection process. In this poster we show that Caco-2 cell proliferation is inhibited by both TcdA and TcdB and that this activity is potently neutralised by several toxin-binding antibodies. We will also show data from a differentiated, polarised Caco-2 cell assay, established using Matrigel-coated Boyden -chamber plates, where toxin activity was determined by measurement of tight-junction integrity using electrical resistance. Loss of tight junction integrity is associated with diarrhoea in patients. Assays were also developed to measure other functional and morphological parameters to better define antibody activity. Use of this screening cascade allowed antibodies to be selected on their ability to neutralise key toxins in assays using disease-relevant biology in a differentiated gut epithelial cell monolayer. Antibodies selected in these assays have shown good in vivo activity. In conclusion, we have developed a series of assays allowing the selection of C. difficile¬ toxin-neutralising antibodies in a disease-relevant setting.