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

项目2：肠道仿生学中轮状病毒宿主范围、中和和M细胞相互作用的调节

• 批准号: 10614394
• 负责人: Harry Bernard Greenberg
• 金额: $ 15.79万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-03-01 至 2026-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10614394
• 关键词: Acute; Adult; Age; Air; Animals; Antigen Presentation; Apical; Binding; Biology; Biomimetics; Biopsy; CRISPR screen; CRISPR/Cas technology; Cell Communication; Cell physiology; Cell surface; Cells; Cessation of life; Child; Colon; Communication; Coronavirus; Coronavirus Infections; Crystallography; Data; Dependence; Development; Diarrhea; Disease; Double-Stranded RNA; Elderly; Enteral; Enterocytes; Epithelial Cells; Epithelial Receptor Cell; Epithelium; Escape Mutant; Generations; Genes; Genetic; Genetic Determinism; Genetic Variation; Genome; Goals; HT29 Cells; Hospitalization; Human; Immune; Immune response; Immune system; Immunity; Immunocompromised Host; In Vitro; Infant; Infection; Inflammatory Bowel Diseases; Information Dissemination; Innate Immune Response; Interferons; Intestines; Knock-out; Libraries; Licensing; Life; Liquid substance; M cell; Mediating; Modeling; Monoclonal Antibodies; Morphology; Mucous Membrane; Mus; Natural Immunity; Occupational activity of managing finances; Organoids; Outpatients; Pathogenesis; Peyer' s Patches; Population; Predisposition; Process; Production; Productivity; Proteins; Recombinants; Recovery; Regulation; Research; Role; Rotavirus; Rotavirus Infections; Rotavirus Vaccines; SH2D3A gene; STAT1 gene; Salmonella typhi; Seminal; Signal Transduction; Site; Small Intestines; Surface; System; Technology; Vaccines; Viral; Viral Pathogenesis; Virus; Visit; Work; attenuation; cytokine; diarrheal disease; enteric infection; enteric pathogen; genome-wide; human model; human monoclonal antibodies; ileum; interest; intestinal epithelium; method development; microbial; mouse model; neutralizing monoclonal antibodies; novel; pathogen; pharmacologic; prevent; programs; rational design; respiratory virus; response; reverse genetics; single-cell RNA sequencing; success; suckling; synergism; tool; transcriptome; transcriptome sequencing; transcytosis

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.

项目概要/摘要（项目 2） 轮状病毒 (RV) 感染每年导致 200,000 名儿童死亡。轮状病毒是无包膜的三层病毒， 具有由 11 个独立片段组成的 dsRNA 基因组的二十面体病毒。房车无处不在，高度 在包括人类在内的大多数哺乳动物的幼年中具有传染性并引起严重的腹泻病。房车 仍然是四岁以下婴幼儿急性危及生命腹泻的主要原因。 RV 也会影响老年人、免疫功能低下和健康的成年人，但疾病通常不太严重。 尽管有多种安全有效的疫苗可供使用，RV 仍导致 1.14 亿次腹泻发作，24 100 万人次门诊就诊，240 万人次住院治疗，约 20 万名幼儿死亡 每年。目前可用的许可疫苗在大多数欠发达地区的功效有限（<60%） 世界。第三世界缺乏高效的 RV 疫苗至少部分是由于不完整的研究 了解 RV 与肠道免疫系统和肠道的相互作用。使用初级小肠肠类， 我们之前在调节 RV 宿主范围限制的机制方面取得了几项开创性的发现 (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了解 RV 生物学、发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肠类来模拟人类 RV 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；和三） 开发一个系统来区分回肠类器官中的功能性微折叠（M）细胞。该项目有 以下三个目标： 1) 我们将使用我们优化的 RG 系统在人类和选择之间生成有针对性的基因重配 动物 RV 并检查它们的复制和抑制人类类器官先天免疫反应的能力。 我们期望确定人类 RV HRR 的遗传基础，这将有助于指导合理设计 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 人类肠道并使用新型全基因组 CRISPR-Cas9 筛选方法来识别 RV 特异性结合 肠上皮细胞表面的依赖性因素。 3) 我们将采用遗传工具（例如，CRISPR-Cas9 删除 STAT1）或 IFN 信号传导的药物抑制（即鲁索替尼）以确定是否禁用先天性 免疫力使 M 细胞容易受到 RV 感染。最后，我们将检查 M 细胞是否发挥作用 通过创建基底外侧细胞表面通道，成为广泛上皮细胞感染的关键进入通道。这些 RV 研究和发现也应与其他肠道病原体和非感染性肠道广泛相关 炎症性疾病也是如此。