Development of a human norovirus cell culture model

 描述（由申请方提供）：人诺如病毒（HuNoV）是全球非细菌性胃肠炎的主要原因，导致显著的死亡率、发病率和经济损失。 成本然而，没有针对HuNoV的有效疫苗或抗病毒药物。这在很大程度上是由于无法在实验室中培养HuNoV。由于缺乏HuNoV细胞培养模型，科学家们无法研究HuNoV生物学的基本方面，识别治疗靶点，并且抑制了快速识别抗病毒候选药物的能力。因此，迫切需要开发用于HuNoV的可再现的组织培养系统。长期目标是阐明HuNoV-宿主相互作用的机制，并制定有效的HuNoV疾病预防和控制策略。该项目的目的是确定在培养物中可再现地支持HuNoV感染的细胞类型。核心假设是HuNoV单独感染肠免疫细胞或与肠上皮细胞共培养。这 假设是基于我们在新开发的HuNoV小鼠模型中的数据，表明HuNoV感染巨噬细胞。此外，其他人的工作表明，HuNoV感染黑猩猩肠道固有层中的B细胞和树突状细胞，HuNoV病毒样颗粒结合到 肠固有层细胞和人十二指肠活检标本的组织切片对HuNoV抗原染色呈阳性。长期以来，人们一直认为HuNoV感染肠上皮细胞，但肠免疫细胞的感染将为在上皮细胞中培养HuNoV的失败尝试提供解释。将通过追求两个具体目标来测试该假设：1）确定HuNoV是否在人肠免疫细胞中复制，以及2）确定在与3D原代人肠微型器官共培养后HuNoV是否在免疫细胞中复制。在第一个目标中，我们将直接感染来自人小肠组织的肠道免疫细胞、巨噬细胞或来源于人多能干细胞的树突状细胞和已建立的人免疫细胞系。在第二个目标中，原代和干细胞衍生的免疫细胞将首先在感染前与人肠道微型器官共培养。这种方法 在概念上是创新的，因为研究测试了一个范式转变的假设，即，HuNoV在免疫细胞中复制，而不是在肠道中的肠细胞中复制。此外，原代人免疫细胞/上皮细胞共培养的发展代表了人类肠道微器官系统的技术创新，并将为研究肠道感染提供新的模型。所提出的研究是进一步重要的，因为预期其结果是开发用于HuNoV的可再现的组织培养模型。这样的发展将克服该领域的一个主要障碍，该障碍阻碍了过去40年的HuNoV研究。最终，这种技术突破将为HuNoV研究在基础和转化水平上的未来进展提供基础。

 DESCRIPTION (provided by applicant): Human noroviruses (HuNoVs) are the major cause of non-bacterial gastroenteritis worldwide, resulting in significant mortality, morbidity and economic cost. However, no effective vaccines or antivirals exist for HuNoV. This is in large part due to the inability to culture HuNoV in the laboratory. This lack of a HuNoV cell culture model has prevented scientists from studying fundamental aspects of HuNoV biology, identifying therapeutic targets, and it has stifled the ability to rapidly identify antiviral drug candidates. hus, there is a critical need to develop a reproducible tissue culture system for HuNoV. The long-term goal is to elucidate mechanisms of HuNoV-host interactions and develop effective HuNoV disease prevention and control strategies. The objective of this project is to determine a cell type that reproducibly supports HuNoV infection in culture. The central hypothesis is that HuNoVs infect intestinal immune cells alone or in co-culture with intestinal epithelial cells. This hypothesis is based on our data in a newly developed HuNoV mouse model demonstrating that HuNoV infects macrophages. In addition, work by others shows that HuNoV infects B cells and dendritic cells in the intestinal lamina propria of chimpanzees, HuNoV virus-like particles bind to intestinal lamina propria cells, and tissue sections of human duodenal biopsy specimens stain positive for HuNoV antigen. It has long been presumed that HuNoV infects intestinal epithelial cells, but infection of intestinal immune cells would provide an explanation for the failed attempt to culture HuNoV in epithelial cells. The hypothesis will be tested by pursuing two specific aims: 1) Determine whether HuNoV replicates in human intestinal immune cells, and 2) Determine whether HuNoV replicates in immune cells following co-culture with 3D primary human intestinal mini-organs. In the first aim, we will directly infect intestinal immune cells from human small intestinal tissue, macrophages or dendritic cells derived from human pluripotent stem cells and established human immune cell lines. In the second aim, primary and stem cell-derived immune cells will first be co-cultured with human intestinal mini-organs prior to infection. This approach is innovative conceptually because the research tests a paradigm-shifting hypothesis, i.e., that HuNoVs replicate in immune cells instead of enterocytes in the intestine. In addition, the development of primary human immune cell/epithelial co-cultures represents a technical innovation of the human intestinal mini-organ system and will provide a new model for studying enteric infections. The proposed research is further significant because it is expected to result i the development of a reproducible tissue culture model for HuNoVs. Such a development would overcome a major barrier in the field, which has hampered HuNoV research for the past 40 years. Ultimately, such technical break-through will provide the basis for future advances in HuNoV research both at the basic and translational level.