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

项目摘要/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， 二十面体病毒，dsRNA基因组由11个独立片段组成。房车无处不在，高度 在包括人类在内的大多数哺乳动物物种的幼体中具有传染性，并导致严重的腹泻疾病。房车 仍然是四岁以下婴幼儿急性危及生命的腹泻的主要原因。 房车也可以折磨老年人、免疫功能低下的和健康的成年人，但疾病通常不那么严重。 尽管有几种安全有效的疫苗可用，轮状病毒仍导致1.14亿起腹泻事件，24 门诊就诊人数为100万人次，住院人数为240万人次，幼儿死亡人数约为20万人 每年一次。目前获得许可的疫苗在欠发达地区的大部分地区效力有限(60%)。 世界。第三世界缺乏高效的轮状病毒疫苗，至少在一定程度上是由于 了解轮状病毒与肠道免疫系统和肠道的相互作用。使用初级小肠肠样病变， 我们之前在调节RV宿主范围限制的机制上有了几个开创性的发现 (HRR)，中和，并扩散到整个肠道。我们的目标是在这些成功的基础上进一步了解 轮状病毒生物学、致病机制和免疫的主要特征。我们将利用最新的技术 进展包括：i)使用人类供体来源的小肠小肠腺来模拟人类轮状病毒感染。 通过肠道；ii)开发高效的反向遗传学(RG)系统来修改RV基因组；以及III) 回肠器官中功能微折叠(M)细胞分化系统的建立。这个项目有 以下三个目标： 1)我们将使用我们优化的RG系统在人类和SELECT之间产生有针对性的基因重排 动物RV，并检测它们的复制和在人类器官中抑制先天免疫反应的能力。 我们希望确定人类RV HRR的遗传基础，这将有助于指导合理的设计 第三代房车疫苗。2)我们将进行竞争阻断分析和晶体分析 检测mAb/RV蛋白相互作用以阐明人RV中和的结构基础 并使用一种新的全基因组CRISPR-Cas9筛选方法来鉴定RV的特异性结合 肠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 STAT1)或对干扰素信号的药物抑制(即Ruxolitinib)，以确定是否先天禁用 免疫使M细胞对RV感染易感。最后，我们将检查M细胞是否具有 通过建立基底侧细胞表面通路，关键进入通路是广泛的上皮细胞感染的通道。这些 轮状病毒的研究和发现也应该与其他肠道病原体和非传染性肠道广泛相关。 还有炎症性疾病。

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.