Novel Determinants of Streptococcus Pyogenes Virulence and Protective Immunity in the Primate Oropharynx: A Genome-wide Strategy

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PROJECT SUMMARY The human bacterial pathogen Streptococcus pyogenes (group A streptococcus, GAS) causes more than 600 million cases of pharyngitis annually worldwide. Despite 100 years of effort, this organism has successfully defied attempts to create a vaccine that protects humans from pharyngitis and invasive infections. We believe a new strategy is warranted and necessary. The proposed research seeks to discover, on a genome-wide scale using a strategy not previously applied to GAS vaccinology, novel GAS genes contributing to pharyngitis, and exploit this information for vaccine research efforts. It is our thesis that this knowledge gap has severely limited the ability to fully understand the virulence determinants at work in the upper respiratory tract (URT) and create a protective GAS vaccine. We will address these issues by combining our recent successes applying transposon- directed insertion site sequencing (TraDIS) technology to GAS in several settings, together with our 19 years of productively using a cynomolgus macaque (nonhuman primate, NHP) model of pharyngitis, in vaccinology research. We will first use TraDIS to conduct in vivo genome-wide screens to systematically identify serotype M89 GAS genes required for colonization, clinical disease, and persistence in the URT of NHPs. These data will complement and enrich analogous information we have already generated for serotype M1 and M28 GAS. Our central hypothesis is that defining the GAS genes that contribute to fitness in the NHP URT will significantly improve our understanding of the molecular processes occurring in this niche, thereby filling a massive knowledge gap and leading to new strategies for creating a vaccine that protects against GAS pharyngitis. We propose the following three specific aims: Specific Aim 1: Exploit TraDIS for genome- wide identification of serotype M89 GAS genes required for colonization, acute clinical disease, and persistence (i.e., fitness) in the URT of NHPs. Specific Aim 2: Use our combined M1, M28, and M89 TraDIS URT screen data to generate isogenic gene-deletion mutant strains and validate the importance of additional selected candidate genes in causing GAS pharyngitis in NHPs. Specific Aim 3: Determine if vaccination of NHPs with proteins encoded by genes identified in Specific Aims 1 and 2 confers protective immunity against experimental pharyngitis caused by homologous (serotype M1) and heterologous (serotype M28) M protein serotypes of GAS. The proposed line of research will exploit and significantly expand studies funded by an R21 grant, and our innovative and successful application of TraDIS to GAS, our long history of GAS pharyngitis and vaccinology studies using NHPs, our analyses of human specimens, and our contributions to understanding the basic biology of GAS molecular pathogenesis and genomics. In essence, this research represents a new way forward for GAS vaccine efforts. Important to note, an analogous strategy has been used to identify four proteins present in a new vaccine that protects horses against “strangles,” a severe URT disease caused by the closely related pathogen, Streptococcus equi.