Project 2: Regulation of Rotavirus Host Range, Neutralization, and M cell Interactions in Enteric Biomimetics

项目概要/摘要（项目2） 轮状病毒（RV）感染每年导致20万儿童死亡。轮状病毒是无包膜的三层病毒， dsRNA基因组由11个独立片段组成的二十面体病毒。房车无处不在， 具有传染性，并在包括人类在内的大多数哺乳动物物种的幼仔中引起严重的腹泻疾病。RV 仍然是婴儿和四岁以下幼儿急性危及生命的腹泻的主要原因。 RV也可以折磨老年人，免疫功能低下和健康的成年人，但疾病通常不太严重。 尽管有几种安全有效的疫苗，但RV导致1.14亿次腹泻，24 240万人次门诊就诊，240万人次住院，约20万名幼儿死亡 每年。目前获得许可的疫苗在欠发达国家的大部分地区的效力有限（<60%）。 世界第三世界缺乏高效的RV疫苗，至少部分原因是由于不完整的 了解RV与肠道免疫系统和肠道的相互作用。利用原发性小肠类肠， 我们先前在调节RV宿主范围限制的机制上取得了一些开创性的发现 (HRR)中和，并在肠道中扩散。我们的目标是在这些成功的基础上， RV生物学、发病机制和免疫的关键特征。我们将利用最新的技术 这些进展包括：i）使用人供体来源的小肠类肠体来模拟人RV感染， ii）开发有效的反向遗传学（RG）系统以修饰RV基因组;以及iii） 开发一种分化回肠类器官中功能性微折叠（M）细胞的系统。该项目具有 三个目标： 1)我们将使用我们优化的RG系统来产生人类和选择之间的靶向遗传参考物 动物RV并检查它们的复制和抑制人类类器官中先天免疫反应的能力。 我们希望能够明确人类RV HRR的遗传基础，这将有助于指导合理的设计 第三代RV疫苗2)我们将进行竞争阻断分析和晶体学分析， 检查mAb/RV蛋白相互作用，以阐明人RV中和的结构基础， 人肠道，并使用新的全基因组CRISPR-Cas9筛选方法来鉴定RV特异性结合 肠上皮细胞表面的依赖因子。3)我们将使用遗传工具（例如，CRISPR-Cas9缺失 STAT 1）或IFN信号传导的药理学抑制（即，ruxolitinib），以确定是否先天性致残 免疫性使M细胞对RV感染敏感。最后，我们将研究M细胞是否起作用， 通过建立基底外侧细胞表面通路，是广泛上皮细胞感染的关键进入通道。这些 RV研究和发现也应该与其他肠道病原体和非感染性肠道病原体广泛相关。 炎症性疾病也是如此。

PROJECT SUMMARY/ABSTRACT (Project 2) Rotavirus (RV) infection kills 200,000 children annually. Rotaviruses are non-enveloped, triple-layered, icosahedral viruses with dsRNA genomes composed of 11 separate segments. RVs are ubiquitous, highly infectious and cause severe diarrheal diseases in the young of most mammalian species including humans. RV remains the primary cause of acute life-threatening diarrhea in infants and young children under the age of four. RVs can also afflict elderly, immunocompromised, and healthy adults but disease is generally less severe. Despite the availability of several safe and effective vaccines, RV causes 114 million diarrhea episodes, 24 million outpatient visits, 2.4 million hospitalizations, and approximately 200,000 deaths in young children annually. Currently available licensed vaccines have limited efficacy (<60%) in most parts of the less-developed world. The lack of highly effective RV vaccines for the third world is due, at least in part, to an incomplete understanding of RV interactions with the enteric immune system and gut. Using primary small bowel enteroids, we previously made several seminal discoveries on the mechanisms that regulate RV host range restriction (HRR), neutralization, and spread across the intestine. Our goal is to build on these successes to understand key features of RV biology, pathogenesis, and immunity. We will take advantage of recent technological advances including: i) use of human donor-derived small bowel enteroids to model human RV infection in and through the gut; ii) development of an efficient reverse genetics (RG) system to modify RV genomes; and iii) development of a system to differentiate functional microfold (M) cells in ileum organoids. This project has the following three Aims: 1) We will use our optimized RG system to generate targeted genetic reassortants between human and select animal RVs and examine their replication and abilities to inhibit innate immune responses in human organoids. We expect to identify the genetic basis of human RV HRR, which will prove useful to guide the rational design of third-generation RV vaccines. 2) We will perform competition blocking analysis and crystallographic examination of mAb/RV protein interactions to elucidate the structural basis of human RV neutralization in the human gut and use a novel genome-wide CRISPR-Cas9 screening approach to identify the RV-specific binding dependency factors on the enterocyte surface. 3) We will employ genetic tools (e.g., CRISPR-Cas9 deletion of STAT1) or pharmacological inhibition of IFN signaling (i.e., ruxolitinib) to determine whether disabling innate immunity renders M cells susceptible to RV infection. Finally, we will examine whether the M cells function as critical entry conduits to widespread epithelial cell infection via creating basolateral cell surface access. These RV studies and findings should also be broadly relevant to other enteric pathogens and non-infectious intestinal inflammatory diseases as well.