Human Gastrointestinal Biomimetics for Enteric Viral Infections

项目摘要 胃肠炎（GE）是全球死亡的主要原因之一。我们的研究重点是人类轮状病毒 (HRV)和人类诺如病毒（HuNoV），这是全球病毒性GE的两个主要原因， 每年死亡。这两种病毒都没有抗病毒药物，也没有针对HuNoV的疫苗。虽然疫苗 在高收入国家（84 - 90%），HRV的有效性仍然不理想（45 - 90%）， 57%）在疾病负担最大的低收入环境中。经济上，HuNoV感染导致 每年直接医疗成本超过40亿美元，社会成本超过600亿美元。这些数据强调 需要继续投资于克服粘膜肠道疾病的研究。 这两种人GI病毒都不感染小鼠;此外，HRV在培养的细胞中复制较差，并且HuNoV在培养的细胞中不存在。 近50年来一直无法耕种使用组织干细胞衍生的人肠类器官（HIO）培养物 作为这些人类胃肠道病原体的复制模型，我们取得了一些显著的基础性发现。关键 研究结果包括：两种人类病毒在至少两种不同的肠细胞类型（肠上皮细胞和 在小肠中，HRVs（内分泌细胞）也在结肠中复制。每一种病毒都能通过基因 编码组织血抗原（HBGAs），但这些聚糖在感染中发挥不同的作用。HBGA表达不 不限制感染，但与严重的HRV疾病相关，而感染HuNoV需要它。 与HBGA的相互作用对于两种病毒都是菌株依赖性的。每一种病毒通过一种 不同的途径，其中HRV感染基底外侧，HuNoV感染顶部。感染的HIO培养物产生一种 在囊泡中释放的新形式的HRV表现出与标准细胞培养衍生病毒不同的特性。 这两种病毒都诱导了III型干扰素（IFN）的主要上皮先天性应答;令人惊讶的是， 不限制病毒复制，提示III型IFN可能具有抗病毒以外的其他功能。HIO允许 多个HuNoV菌株和胆汁的培养是复制的关键因素。 尽管这些先前使用仅上皮HIO的研究提供了关于HRV和HuNoV的新知识， 感染，我们仍然缺乏对导致感染的病理生理学和宿主反应的全面了解。 危及生命的疾病。需要继续开发"人类迷你肠道"模型， 与发病机制相关的人-胃肠道病毒相互作用和改善对病毒感染的粘膜免疫应答。 利用复杂的仿生培养，我们提出了两个生物学问题的答案，基础和临床 相关性：在HRV和HuNoV感染期间，什么机制介导严重GI疾病（目标1），以及 微生物-微生物相互作用在病毒性GE的病理生理学中的作用是什么（目标2）？的交互 通过项目2和3以及我们的两个科学核心，我们预测我们的研究将取得进展，并使人类能够 器官仿生学作为理解人类粘膜感染的转化模型，并作为桥梁 在临床前动物模型和首次人体临床试验之间。

PROJECT SUMMARY Gastroenteritis (GE) is among the leading causes of mortality globally. Our research focuses on human rotavirus (HRV) and human norovirus (HuNoV), the two leading causes of viral GE worldwide causing over 320,000 deaths annually. No antivirals are available for either virus and there is no vaccine for HuNoVs. While vaccines to HRV are available and are effective in high-income countries (84-90%), the efficacy remains suboptimal (45- 57%) in low-income settings where the burden of disease is greatest. Economically, HuNoV infections result in over $4 billion in direct healthcare costs and over $60 billion in societal costs each year. These data underscore the need for continued investment in studies to overcome mucosal enteric disease. Both these human GI viruses do not infect mice; further, HRV replicates poorly in cultured cells, and HuNoV was noncultivatable for over almost 50 years. Using tissue stem cell-derived human intestinal organoid (HIO) cultures as a replication model for these human GI pathogens, we made some remarkable fundamental discoveries. Key findings include: both human viruses replicate in at least two distinct intestinal cell types (enterocytes and enterendocrine cells) in the small intestine, and HRVs also replicate in the colon. Each virus binds to genetically encoded histo-blood antigens (HBGAs) but these glycans play different roles in infection. HBGA expression does not restrict infection but correlates with severe HRV disease while it is required for infection with HuNoV. Interactions with HBGA are strain-dependent for both viruses. Each virus infects the polarized epithelium by a different route with HRVs infecting basolaterally and HuNoVs infecting apically. Infected HIO cultures produce a new form of HRV released in vesicles that exhibit different properties from standard cell-culture derived virus. Both viruses induce a predominant epithelial innate response of type III interferon (IFN); surprisingly this does not restrict virus replication suggesting type III IFN may have other functions than being antiviral. HIOs allow cultivation of multiple HuNoV strains and bile is a critical factor for replication. Although these previous studies using epithelial-only HIOs provide new knowledge on HRV and HuNoV infections, we still lack a comprehensive understanding of the pathophysiology and host responses that lead to life-threatening disease. Continued development of “human mini-gut” models is required to fully understand human-GI virus interactions linked to pathogenesis and improve mucosal immune responses to viral infections. Using complex biomimetic cultures, we propose to answer two biological questions of fundamental and clinical relevance: What mechanisms mediate severe GI disease during infection with HRV and HuNoV (Aim 1), and what is the role of microbe-microbe interactions in the pathophysiology of viral GE (Aim 2)? Through interactions with Projects 2 and 3 and our two Scientific Cores, we predict our studies will advance and enable human organ biomimetics as translational models to understand human mucosal infections and to serve as a bridge between preclinical animal models and first-in-human clinical trials.