Final Report Summary - SURFACE (Gram positive Surface proteins in immune evasion) This project aimed to develop a new Staphylococcus aureus (S. aureus) vaccine by targeting surface-exposed immune evasion molecules. Instead, the work of the fellow (van Sorge) on vaccines targeting surface-exposed molecules of Streptococcus pyogenes (S. pyogenes, or Group A Streptococcus, GAS) was continued, since this project was yielding promising results. S. aureus and GAS share many of the same challenges when it comes to human health: both are preeminent human pathogens without the availability of a safe and efficacious vaccine. An extra complication with GAS vaccines is the potential risk of autoreactivity of vaccine-induced antibodies. These antibodies recognize similar structures on human tissue, resulting in the post-infectious immune-mediated disease rheumatic fever, which is a major cause of chronic heart disease and mortality in many parts of the developing world. Our research focused on the surface-expressed polysaccharide Group A carbohydrate (GAC), which defines GAS as a species and constitutes approximately half of the cell wall by weight. This antigen has been shown previously to be effective as an universal GAS vaccine. However, theoretical concerns regarding crossreactivity of antibodies that recognize a specific GAC epitope against human tissues have been raised. In addition, no specific biological function has been attributed to this conserved and abundant antigen. We therefore hypothesized that a modified version of this molecule, lacking this autoreactive epitope, might be a safe vaccine antigen. To this end, the fellow first identified the genes responsible for GAS biosynthesis (Objective 1) by mining sequenced GAS genomes. A 12-gene operon was identified and probed by integrational mutagenesis scan. Several knockout bacteria expressed modified GAC structures on the GAS cell surface. In with objective 2, these knockout bacteria were tested in in vitro assays that mimic specific steps of human disease, including killing in human whole blood and by isolated human neutrophils, complement deposition, resistance to reactive oxygen species and antimicrobial peptides (Objective 2). A specific mutant, ΔgacI, was attenuated in human whole blood and neutrophil killing, due to hypersusceptibility to human antimicrobial peptide LL-37 and platelet-derive antimicrobials. As per objective 3, virulence of wild-type and knockout bacteria was compared in two in vivo animal models. In line with the in vitro results, knockout bacteria caused reduced mortality and morbidity compared to wild-type bacteria in both models. Reduced virulence was associated with lower number of recovered bacteria, suggesting that knockout bacteria are more susceptible to immune clearance. Finally, we tested the capacity of the modified carbohydrate to be an effective vaccine antigen (Objective 4). The modified GAC were conjugated to a carrier protein and used to immunize rabbits to raise specific antibodies. The specific antiserum was highly reactive against the immunizing antigen as well as the native GAC from wild-type bacteria. Moreover, the antiserum enhanced neutrophil-mediated killing of different GAS serotypes as well as provided protection against lethal infection in mice after passive transfer. This finding is of great interest since this modified antigen would provide broad coverage against all (> 150) serotypes without the risk of inducing autoimmunity in vaccinees. Our results demonstrate how genetic and functional insight into cell wall-associated structures has implications for rational universal vaccine design. This work was recently accepted in the peer-reviewed journal Cell Host and Microbe and the invention is covered by international patent PCT/US12/049604 (the fellow is co-inventor). Finally, this work enabled the fellow to secure a prestigious personal VIDI grant from the Dutch Scientific Organization (€800,000, 5 years) and a permanent position as assistant professor at our Department.