Human Intestinal Enteroids as Ex Vivo Models of Human Rotavirus Infection

项目摘要 轮状病毒（RV）是5岁以下儿童威胁生命的腹泻病的主要原因， 每年导致近50万人死亡，尽管使用了两种获得许可的RV疫苗。虽然RV 虽然疫苗是可以获得的，但在最需要疫苗的国家，这些疫苗并没有发挥最佳作用。人类 轮状病毒（HRV）在培养的细胞中生长不良，不感染小鼠，小鼠只对动物敏感。 轮状病毒感染我们不了解HRV感染如何导致危及生命的疾病， 来自现有动物模型的知识完全适用于HRV。我们率先建立了人类 小肠类肠体（HIEs）作为新型离体模型“微肠”系统，以检查宿主-病原体相互作用 和病理生理学。HIE是自组织的、三维生理活性的多细胞培养物， 模仿肠道的有序结构。我们发现HRV毒株在HIE中复制得非常好 模仿在人体中观察到的宿主宽容性，从而建立了一个研究HRV感染的新模型 和发病机制。我们建议研究直接检查病理生理学和遗传学机制， 控制HRV感染和生长。最近发现，HRV毒株使用的细胞受体是 组织相容性抗原（HBGAs）的发现导致了对宿主遗传影响的思考范式转变 为轮状病毒的人畜共患病传播提供了新的视角。独特的差异， HBGA表达可以解释非洲不寻常的HRV毒株的高流行率和低疫苗接种率。 在不同种族人群中观察到的疗效。我们假设类肠病毒的HRV感染是遗传性的， 限制性和允许性感染导致新的细胞内和细胞外先天性信号传导和应答 HRV发病机制和病理生理学的基础途径。此外，HRV感染或 疫苗接种可以由有益微生物调节。我们建议在目标1中进行研究，以确定 遗传控制的易感性调节因子（HBGAs）和HIE中不同的先天反应调节HRV 感染和病理生理学（核心B和C）。目标2中的研究将确定西格列汀或 病原体对HRV或疫苗感染的影响。这些研究将阐明菌株的分子基础- 在不同种族群体的儿童中观察到的特定宿主范围限制， 疫苗特性。此外，评价改良的HIE培养物， 益生菌或其他病原体（核心B、C和项目2）在新的定制生物材料平台（ 项目3）将导致更好地了解上皮细胞对病毒感染的反应是如何调节的。 总的来说，这项工作将提供一个新的理解轮状病毒的病理生理学和随后的 人类的关键适应性反应，引发免疫和疾病。

PROJECT SUMMARY Rotavirus (RV) is the leading cause of life-threatening diarrheal disease in children under the age of 5, and results in nearly half a million deaths annually, despite the use of two licensed RV vaccines. Although RV vaccines are available, they do not work optimally in countries where they are needed most. Human rotaviruses (HRVs) grow poorly in cultured cells and do not infect mice, which are only susceptible to animal rotavirus infection. We do not understand how HRV infections cause life-threatening disease and question if knowledge from existing animal models is fully applicable to HRVs. We pioneered establishing human intestinal enteroids (HIEs) as novel ex vivo model “mini-gut” systems to examine host-pathogen interactions and pathophysiology. HIEs are self-organizing, 3-dimensional physiologically active, multicellular cultures that mimic the ordered structure of the intestine. We have found that HRV strains replicate extremely well in HIEs mimicking the host permissiveness observed in people, thus establishing a new model to study HRV infections and pathogenesis. We propose studies to directly examine mechanisms of pathophysiology and genetic control of HRV infection and growth. The recent discovery that cellular receptors used by HRV strains are histoblood group antigens (HBGAs) resulted in a paradigm shift in thinking about the host's genetic influence on RV infection and provides a new perspective on zoonotic transmission of rotaviruses. Unique differences in HBGA expression may explain the higher prevalence of unusual HRV strains in Africa and lower vaccine efficacy seen in distinct ethnic populations. We hypothesize that HRV infection of enteroids is genetically restricted and permissive infection leads to novel intracellular and extracellular innate signaling and response pathways that underlie HRV pathogenesis and pathophysiology. In addition, the outcome of HRV infection or vaccination can be modulated by beneficial microbes. We propose studies in Aim 1 to determine whether genetically-controlled modulators of susceptibility (HBGAs) and distinct innate responses in HIEs regulate HRV infection and pathophysiology (with Cores B and C). Studies in Aim 2 will establish the effect of commensal or pathogenic organisms on HRV or vaccine infection. These studies will elucidate the molecular basis for strain- specific host range restriction observed in children of different ethnic groups and be relevant to understanding vaccine properties. Furthermore, evaluation of modified HIE cultures that house human commensals, probiotics or other pathogens (with Cores B, C and Project 2) in new tailored biomaterial platforms (with Project 3) will lead to a better understanding of how epithelial responses to a viral infection are modulated. Collectively, this work will provide a new understanding of rotavirus pathophysiology and the subsequent critical adaptive responses in humans that trigger immunity and disease.