Generating Monoclonal Antibodies against Group A Streptococcal M6 Protein using Phage Display

Abstract

Group A streptococcus (GAS, Streptococcus pyogenes) is a human-specific pathogen, responsible for more than 500,000 deaths annually. Acute rheumatic fever (ARF) is a serious autoimmune condition that can develop after a GAS infection. The rates of ARF in Maori and Pacific populations in New Zealand are among the highest in the developed world. GAS M protein is a major virulence factor that extends from the cell surface as a coiled-coil homodimer. It comprises an N-terminal hypervariable region (HVR) and conserved C-repeat region, with both regions being developed as vaccines. TheMprotein has also been implicated in generating cross-reactive antibodies that contribute to ARF. Despite the importance of M protein in both vaccine and disease, there is a lack of understanding of M protein immunogenicity at the molecular level. In this project, a rabbit was vaccinated with M protein from the clinically important M6 strain. The serum derived from the rabbit was characterised and showed high titre antibodies to the full length M protein as well as the HVR and C-repeat regions. Phage display technology was utilised to generate a Fab-phage library from the mRNA extracted from the rabbit bone marrow and spleen. This incorporated the development of a new, optimised PCR protocol for the synthesis of rabbit Fab-phage libraries with a next-generation polymerase. The library was subjected to bio-panning against the full-length M6 protein and the M6 HVR peptide. Despite no M6-specific antibodies being identified in this study, a number of key learnings were made with respect to M protein for future phage display studies. Firstly, non-specific attachment of biotin to lysine residues of M6 blocks important epitopes in the protein. Second, immobilisation of M protein onto plastic exposes hydrophobic residues and results in the selection of unwanted non-specific Fab-phage clones. Finally, HVR antibodies are rare and difficult to select for by phage display. Future attempts to identifyMprotein specific antibodies by phage display should include the use of a specific C-terminal tag (such as an AviTag) on the full-length M protein to enable the preservation of native protein structure and major epitopes in a solution panning protocol.