Isolation by phage display of recombinant antibodies able to block adherence of Escherichia coli mediated by the K99 colonisation factor

Abstract

K99 fimbriae are important for intestinal colonisation by bovine strains of enterotoxigenic Escherichia coli. The mode of action of this colonisation factor is well understood and specific immune responses are protective. K99 was therefore chosen for this study as a model to test if antibodies with anti-adhesion activity could be isolated from recombinant libraries using phage display techniques. Potentially, this strategy could be used to understand better the action of bacterial colonisation factors and aid the design of therapies (e.g. vaccines, purified protein products or bacteria bearing colonisation-blocking antibodies) to inhibit bacterial adherence. The major fimbrial subunit from K99, FanC, was purified from a clinical E. coli isolate. The protein was coated to plastic immunotubes and used as a target for selection of antibodies from the Tomlinson I and J libraries of single chain (scFv) antibodies. Clones able to recognise K99 were isolated by iterative rounds of binding, elution and amplification. scFv antibodies chosen from the resulting panel were purified and their specificity confirmed by ELISA. Pre-incubation of several scFvs with bacteria expressing K99 fimbriae inhibited the agglutination of erythrocytes. Further investigation by microscopy confirmed that when E. coli expressing K99 were exposed to scFv antibodies, the binding of bacteria to erythrocytes was blocked with high efficiency. The study showed that recombinant antibodies were able to block the action of a bacterial colonisation factor and hence that phage display techniques might be applied to the identification of less well-characterised virulence factors and the analysis of their structure and function.