Exploring Monoclonal Antibody Action Against the Group A Streptococcal M Protein

Abstract: Group A Streptococcus (GAS) is a significant human pathogen that has developed multipleimmune evasion mechanisms to counter the host immune response. One of these mechanisms involvesthe production of the M protein, which, amongst other things, acts as an anti-phagocytic factor and canbind host proteins. Another is the ability of M protein to bind a human protein called fibronectin (Fn). Thisprotein plays a key role in a number of physiological processes and can be used by GAS to evade theimmune system. In this PhD dissertation, we aimed to assess the binding efficacy and function ofmonoclonal antibodies targeting the GAS M protein.In the first paper we start by developing a robust method to assess phagocytosis. This method highlightsthe importance of factors such as volume, time, and the ratio of phagocyte to prey on the phagocyticprocess. It has allowed us to, henceforth, attain precise, high quality phagocytosis data and has been amajor driving force for other projects within the lab – especially the three other papers included in thisthesis.In the second paper we discovered a novel form of antibody binding whereby a monoclonal binds theGAS M protein in a bivalent dual-Fab cis mode. This means that both Fab arms of the Ab bind to distinctepitopes on the target molecule simultaneously. Even so this antibody bound to a region of the M proteinassociated with non-opsonic antibodies we found that this Ab could enhance phagocytosis suggestingthat this novel binding form can circumvent the M protein's anti-phagocytic properties.In the third paper we investigated the M protein’s ability to bind fibronectin. While this function wasdescribed in previous studies, we found it could only do so with very low affinity. We found that the bindingof antibodies from the blood of donors who had recently recovered from a severe GAS infection couldgreatly enhance this fibronectin binding. We show that same occurs with certain anti-M monoclonals andthat this mechanism leads to a reduction in opsonophagocytosis. Moreover we find that Ab flexibility mayplay a role and that Ab Fc domains are a crucial factor in mechanism.In the fourth paper we further explore this anti-phagocytic effect. Here we assess the effects of varyingconcentrations of Fn since this can differ greatly within the human body. We found that both very low andhigh concentrations of Fn, corresponding with the nasopharyngeal niche and blood respectively, led to asubstantial reduction in phagocytosis. We moreover found that this reduction in phagocytosis is likelylinked to a modulation of integrins. Overall, this work provides insights into immune evasion mechanismsdeveloped by GAS and highlights how this remarkable pathogen always seems to be one step ahead ofus.