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

项目总结 人类致病菌化脓性链球菌(GAS)引起600多人 全世界每年有数百万例咽炎病例。尽管经过了100年的努力，这种生物还是成功地挑战了 试图创造一种疫苗，保护人类免受咽炎和侵入性感染。我们相信一个新的 战略是有根据的，也是必要的。这项拟议的研究试图在全基因组范围内利用 以前没有应用于气体疫苗学的策略，导致咽炎的新气体基因，以及开发 这些信息用于疫苗研究工作。我们的论点是，这种知识差距严重限制了 能够充分了解在上呼吸道(URT)中起作用的毒力决定因素，并创建 保护性气体疫苗。我们将结合我们最近应用转座子的成功来解决这些问题- 定向插入位点测序(Tradis)技术在几个环境中的应用，以及我们19年的 在疫苗学中有效地使用食蟹猴(非人灵长类，NHP)咽炎模型 研究。我们将首先使用TRADIS进行活体全基因组筛查，以系统地鉴定血清型 非霍奇金淋巴瘤的定植、临床疾病和持续存在所需的M89 GAS基因。这些数据将 补充和丰富我们已经为M1型和M28型GAS生成的类似信息。我们的 中心假设是，在NHP城市轨道交通中定义有助于健身的GAS基因将 极大地提高了我们对这一利基领域中发生的分子过程的理解，从而 填补了巨大的知识空白，并导致了创造预防流感疫苗的新策略 气性咽炎。我们提出了以下三个具体目标：具体目标1：开发基因组交易-- 广泛鉴定定植、急性临床疾病和持久性所需的M89型GAS基因 (即，适合度)在城市轨道交通中。具体目标2：使用我们的组合M1、M28和M89 Tradis URT屏幕 产生等基因缺失突变株的数据和验证额外选择的重要性 致气性咽炎的候选基因。具体目标3：确定是否接种非传染性疾病疫苗 由特定AIMS 1和2中确定的基因编码的蛋白质对实验动物具有保护性免疫力 由同源(M1型)和异型(M28型)M蛋白血清型GAS引起的咽炎。 建议的研究项目将利用并显著扩展由R21拨款资助的研究，以及我们的 创新和成功地将Tradis应用于GAS，我们在气性咽炎和疫苗方面的悠久历史 使用NHPS的研究，我们对人体样本的分析，以及我们对理解基础生物学的贡献 GAS分子发病机制和基因组学的研究。从本质上讲，这项研究代表了天然气的一种新的发展方向 疫苗努力。值得注意的是，一种类似的策略已经被用来鉴定存在的四种蛋白质 在一种新的疫苗中，保护马匹免受“勒死”，一种严重的城市轨道交通疾病引起的密切 相关病原体，马链球菌。

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.