Rotavirus: Studies of the Effectors and Mechanisms of Heterotypic Humoral Immunity in Human Intestinal Epithelial Cells

轮状病毒（RV）是全球幼儿严重腹泻的最重要原因。 这些病毒也会引起健康成人、老年人和免疫系统疾病。 暴露了RV主要在小肠的成熟绒毛尖端细胞中复制。房车感染最多 在宿主特异性方式感染哺乳动物;一般来说，在一种哺乳动物中引起感染的RV菌株不 在另一个物种中引起疾病。RV自然感染和减毒活疫苗有效接种 诱导异型（以及同型）保护症状的再感染，尽管存在 在循环的RV菌株中具有很大的血清型多样性。B细胞和RV特异性抗体介导了这一过程 对症状性再感染的免疫力。无论是体液异型免疫的RV靶点， 导致人的广泛交叉反应性免疫的机制目前已被充分理解。到 为了解决这两个未知数，我们建议研究人肠源性RV单克隆抗体库， 具有以下特异性目的的抗体（mAbs）：目的1）鉴定异型和 - 轮状病毒病毒粒子上的在蛋白质、表位处的同型中和人抗体， 结构层次。我们将扩大和扩展我们的结合和传统血清学的初步研究， 人精氨酸衍生的抗RV mAb的中和表型。表型分析将包括新型 使用原代人肠上皮细胞（IEC）小肠类肠细胞的中和测定。我们 将进一步扩展这些研究，以确定在原子水平上，这些结构之间的相互作用， 功能重要的mAb和三种靶RV表面蛋白。 目的2）确定抗RV蛋白VP 8 *、VP 5 * 和VP 7的人源单克隆抗体的作用机制 在新型人小肠类肠培养系统中中和RV感染。我们将确定 这些mAb使用多种传统的和新的方法抑制（中和）RV复制的机制 评估mAb抑制RV与人IEC结合的能力、抑制细胞增殖的能力、 进入，并抑制病毒脱壳。 目的3）使用最有效抑制人轮状病毒与人轮状病毒结合的人单克隆抗体， 肠上皮细胞，以表征人肠细胞上的RV细胞表面受体。我们 将利用许多靶向RV VP 8 * 的人mAb有效中和人RV的新观察结果 在人肠样培养物和HT-29细胞中，但在MA 104细胞中没有。这一发现表明不同的RV 人IEC与MA 104猴肾细胞中的受体使用。我们将使用几种基因和生化技术 推定RV受体的特征，以更好地表征/鉴定人RV利用的受体 最初与人类IEC结合。 目标4）。使用针对VP 8 *、VP 5 * 和/或VP 7的RV特异性中和mAb的选择来设计一种新的中和抗体。 基于竞争的血清学检测可以更好地预测体液免疫的水平和有效性 与目前的中和和/或伊加固相免疫测定相比，我们将使用选定的 RV中和mAb，以开发一种简单但具有预测性的血清学竞争抑制试验， 在天然感染和/或RV疫苗接种的个体中对RV的表位特异性抗体应答。然后我们将 将来自感染和/或接种个体的血清的竞争抑制水平与先前的 从相同个体获得中和和/或RV结合滴度，以鉴定那些单一或组合的 竞争结合活性提供与中和滴度最佳相关性的mAb和/或 保护状态免受随后的RV疾病。

Rotaviruses (RVs) are the most important cause of severe diarrhea in young children worldwide. These viruses also cause diarrheal disease in healthy adults, the elderly, and the immune compromised. RV replicates primarily in the mature villous tip cells of the small intestine. RVs infect most mammals in a host-specific fashion; in general, RV strains that cause infection in one mammal do not cause disease in another species. Natural RV infection and live attenuated vaccination effectively induces heterotypic (as well as homotypic) protection from symptomatic re-infection despite the existence of great serotype diversity among circulating RV strains. B cells and RV-specific antibodies mediate this immunity to symptomatic re-infection. Neither the RV target of humoral heterotypic immunity nor the mechanism that results in broadly cross-reactive immunity in people are currently well understood. To address these two unknowns, we propose to study a library of human intestine origin RV monoclonal antibodies (mAbs) with the following Specific Aims: Aim 1) Identify the targets of heterotypic and homotypic neutralizing human antibodies on the rotavirus virion at the protein, epitope, and structural levels. We will expand and extend our initial studies of the binding and traditional serologic neutralization phenotypes of human intestine-derived anti RV mAbs. Phenotyping will include novel neutralization assays employing primary human intestinal epithelial cell (IECs) small bowel enteroids. We will further extend these studies to identify, at the atomic level, the structural interaction between these functionally important mAbs and the three target RV surfaces proteins. Aim 2) Determine the mechanisms by which human mAbs to RV proteins VP8*, VP5*, and VP7 neutralize RV infection in a novel human small intestinal enteroid culture system. We will identify the mechanism by which these mAbs suppress (neutralize) RV replication using a variety of traditional and novel technical approaches to assess the ability of the mAbs to inhibit RV binding to human IECs, to inhibit cell entry, and to inhibit viral decapsidation. Aim 3) Use the human mAbs that most efficiently inhibit the binding of human rotaviruses to human intestinal epithelial cells to characterize the RV cell surface receptor on human intestinal cells. We will exploit the novel observation that many human mAbs targeting RV VP8* efficiently neutralize human RVs in human enteroid cultures and in HT-29 cells but not in MA104 cells. This finding suggests different RV receptor usage in human IECs vs. MA104 monkey kidney cells. We will use several genetic and biochemical characteristics of the putative RV receptor to better characterize/ identify the receptor that human RVs utilize to initially bind to human IECs. Aim 4). Use a selection of RV-specific neutralizing mAbs to VP8*, VP5*, and/or VP7 to design a competition-based serologic assay to better predict the level and effectiveness of humoral immunity to RV in people than current neutralization and/or IgA solid phase immunoassays. We will use selected RV neutralizing mAbs to develop a simple but predictive serological competition inhibition assay to measure epitope specific antibody responses to RV in naturally infected and/or RV vaccinated individuals. We will then correlate the level of competition inhibition of sera from infected and/or vaccinated individuals with previously obtained neutralization and /or RV binding titers from the same individuals to identify those single or combined mAbs for which competition binding activity provide the best correlate with neutralization titers and/or protected status from subsequent RV illness.