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: 10392441
• 负责人: Harry Bernard Greenberg
• 金额: $ 29.3万
• 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/10392441
• 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; Communication; Coronavirus; Coronavirus Infections; Data; Dependence; Development; Diarrhea; Disease; Double-Stranded RNA; Elderly; Enteral; Enterocytes; Epithelial; Epithelial Cells; Epithelial Receptor Cell; 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; Life; Liquid substance; Mediating; Modeling; Monoclonal Antibodies; Morphology; Mucous Membrane; Mus; Natural Immunity; Occupational activity of managing finances; Organoids; Outpatients; Pathogenesis; Peyer' s Patches; Pharmacology; Population; Process; Production; 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 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; prevent; programs; rational design; respiratory virus; response; reverse genetics; single-cell RNA sequencing; success; suckling; synergism; tool; transcriptome; transcriptome sequencing; transcytosis; 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; Communication; Coronavirus; Coronavirus Infections; Data; Dependence; Development; Diarrhea; Disease; Double-Stranded RNA; Elderly; Enteral; Enterocytes; Epithelial; Epithelial Cells; Epithelial Receptor Cell; 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; Life; Liquid substance; Mediating; Modeling; Monoclonal Antibodies; Morphology; Mucous Membrane; Mus; Natural Immunity; Occupational activity of managing finances; Organoids; Outpatients; Pathogenesis; Peyer' s Patches; Pharmacology; Population; Process; Production; 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 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; 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.

项目摘要/摘要（项目2） 轮状病毒（RV）感染每年杀死200,000名儿童。轮状病毒是非发育的，三层的， 二十面体病毒，由dsRNA基因组由11个单独的段组成。 RV无处不在，高度 包括人类在内的大多数哺乳动物物种的年轻人中，传染性并引起严重的腹泻疾病。 RV 仍然是四岁以下的婴儿和幼儿急性威胁生命的腹泻的主要原因。 RVS还可能折磨老年人，免疫功能低下和健康的成年人，但疾病通常不那么严重。 尽管有几种安全有效的疫苗可用，但RV会导致1,400万次腹泻发作，24个发作 百万门诊就诊，240万个住院治疗以及大约200,000次死亡的幼儿 每年。目前可用的许可疫苗在大多数欠发达的部分的疗效有限（<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 了解RV与肠道免疫系统和肠道的相互作用。使用主要的小肠肠肠to， 我们之前就调节RV主机限制的机制进行了一些开创性发现 （HRR），中和并散布在肠道上。我们的目标是基于这些成功来理解 RV生物学，发病机理和免疫力的关键特征。我们将利用最近的技术 进步包括：i）使用人类供体衍生的小肠肠todoids对人的RV感染进行建模和 通过肠道； ii）开发有效的反向遗传学（RG）系统以修饰RV基因组；和iii） 开发系统，以区分回肠器官中的功能性微量（M）细胞。这个项目有 以下三个目标： 1）我们将使用优化的RG系统在人与选择之间生成有针对性的遗传改良剂 动物RV并检查它们的复制和能力，以抑制人类器官中的先天免疫反应。 我们期望确定人RV HRR的遗传基础，这将被证明有助于指导理性设计 第三代RV疫苗。 2）我们将执行竞争阻塞分析和晶体学 检查MAB/RV蛋白相互作用，以阐明人类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 肠肠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）或药理学抑制IFN信号传导（即ruxolitinib），以确定是否禁用先天性 免疫使M细胞易受RV感染。最后，我们将检查M细胞是否起作用 通过创建基底外侧细胞表面访问，关键进入导管可广泛地上皮细胞感染。这些 RV研究和发现也应与其他肠道病原体和非感染肠道广泛相关 炎症性疾病也是如此。