Moreland, NBaker, EProft, APTRaynes, Jeremy2019-02-262019http://hdl.handle.net/2292/45256Streptococcus pyogenes is a globally important pathogen causing a broad range of human disease and significant morbidity and mortality. No vaccines for S. pyogenes are currently available, but vaccine candidates based on the T antigen, which forms the backbone of the S. pyogenes pilus, are in pre-clinical development. Pilus proteins have previously been shown to have protective properties and exhibit relatively low antigenic variation. This project aimed to investigate antibody-T antigen interactions and provide 3-dimensional structural data of protective epitopes to inform structure-led, vaccine design. Large Fab-based antibody phage-display libraries were generated from mice vaccinated with the T18.1 pilus and were biopanned to identify individual Fab that bound to T18.1. The strainspecificity and cross-reactivity of the isolated Fab were determined using a panel of T antigens that covers all of the major circulating strain types. The binding affinity of the Fab to T18.1 as a monomer and in the polymerised pilus was assessed. Biopanning identified 20 unique Fab that bound to T18.1 with high affinity. Four promising Fab were selected for further characterisation. Three of these were T18-specific Fab while one also cross-reacted with T3.2. The epitopes of the four selected Fab were mapped using Fab-T18.1 co-crystallography and peptide tiling. The atomic structures of T18.1 in isolation and in complex with one of the Fab were determined by x-ray crystallography. The co-crystal and peptide tiling data mapped the epitopes of all four antibodies to a single 35-residue region on the N-domain of T18.1. This region was also targeted by antibodies in human and animal sera. Competition ELISAs and flow cytometry identified multiple epitopes within this region and indicated that they have varying accessibility in the polymerised pilus.Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. Previously published items are made available in accordance with the copyright policy of the publisher.https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htmhttp://creativecommons.org/licenses/by-nc-sa/3.0/nz/ThesisCopyright: The authorhttp://purl.org/eprint/accessRights/OpenAccessQ112200891