Pathogenesis and Antivirals for Noroviruses

项目总结：项目1 计划1的重点是增加我们对诺沃克病毒感染的发病机制的了解，并 制定可应用于受感染人群和高危人群的治疗和预防战略。人类 诺如病毒是美国流行性和散发性胃肠炎的最常见原因， 造成重大的健康和经济影响，但我们目前只有非具体措施来 预防和治疗感染。我们先前的研究表明，血清抗体阻断的人 病毒颗粒与可能的多糖附着因子结合可降低疾病和感染的风险 在接触病毒后，在感染后，这种封闭抗体可以产生对抗异种病毒 菌株。然而，疫苗开发的一个主要潜在障碍是显著的遗传和抗原性。 发生的变异。免疫功能低下患者的慢性感染被认为是 新出现变种的来源。我们假设，儿童和老年人的感染是另一种 病毒变种的来源。在具体目标1中，我们将确定出现变异的频率是否为 与具有免疫能力的成年人相比，儿童或老年人的发病率更高，这些变异的频率 将与在免疫受损人群中观察到的结果形成对比。遗传基因的潜在影响 VP1结构的变化(例如，在组织母细胞组抗原结合特异性中)将在 与项目3合作。该项目中收集的病毒株还将用于 项目2.在具体目标2中，我们将进行研究，以更好地了解体液免疫反应 感染后诱导的。我们将检验这样一种假设，即抗体反应的产生 与感染毒株不同的基因型别在具有免疫能力的成年人中比在儿童中更常见。 通过克隆人的重链可变区和轻链可变区，将获得人源单抗 在急性感染期间产生的浆母细胞，并与免疫球蛋白结合表达 恒定域。抗体的反应性和抗体阻断病毒-糖链相互作用的能力 将确定对同型和异型菌株的抵抗力。阻断病毒-葡聚糖相互作用的抗体 因为大量的基因型别可能在高危人群的预防中有使用价值。在具体目标3中，我们 将采用包括基于结构的抑制剂设计，合成药物化学， 结构活性关系分析，以及开发和表征抗病毒药物的毒理学研究 以病毒蛋白酶为靶标。将获得在这一目标中开发的关键抑制剂的结构信息 与项目3合作。在AIMS 2和3中开发的单抗和抗病毒药物将 对项目2中开发的RNA表达和复制系统的活性进行了评估。总体而言，这些 研究旨在了解和评估年龄和免疫状态对病毒变异的重要性。 以及关于潜在体液免疫的发展和开发可用于预防或 治疗诺沃克病毒感染。

PROJECT SUMMARY: Project 1 The focus of Project 1 is to increase our understanding of the pathogenesis of norovirus infection and to develop treatment and prevention strategies that can be applied to infected and at risk populations. Human noroviruses are the most common cause of epidemic and sporadic gastroenteritis in the United States, causing both significant health and economic impact, but we currently have only non-specific measures to prevent and treat infection. Our prior research has shown that persons with serum antibody that blocks binding of viral particles to putative glycan attachment factors have decreased risks of illness and infection after exposure to virus, and that following infection such blocking antibodies can develop against heterologous strains. Nevertheless, a major potential barrier to vaccine development is the significant genetic and antigenic variation that occurs. Chronic infection among immunocompromised patients has been proposed as one source of emergent variants. We hypothesize that infections in children and elderly individuals are another source of viral variants. In Specific Aim 1 we will determine whether the frequency of emergent variants is greater in children or the elderly compared to immunocompetent adults, and the frequency of these variants will be contrasted with that observed in an immunocompromised population. The potential impact of genetic changes on VP1 structures (e.g., in histoblood group antigen binding specificities) will be assessed in collaboration with Project 3. Viral strains collected in this project will also be used in cultivation studies in Project 2. In Specific Aim 2, we will perform studies to better understand the humoral immune response induced following infection. We will test the hypothesis that the generation of antibody responses to genotypes distinct from the infecting strain is more common in immunocompetent adults than in children. Human monoclonal antibodies will be made by cloning the heavy and light chain variable domains from plasmablasts generated during acute infection, and expressing these in combination with immunoglobulin constant domains. Antibody reactivity and the ability of the antibodies to block virus-glycan interactions against homotypic and heterotypic strains will be determined. Antibodies that block virus-glycan interactions for a large number of genotypes may have value for use in prophylaxis of risk groups. In Specific Aim 3, we will use an integrated strategy including structure-based inhibitor design, synthetic medicinal chemistry, structure activity relationship analysis, and toxicological studies to develop and characterize antivirals targeting the viral protease. Structural information about key inhibitors developed in this aim will be obtained in collaboration with Project 3. Monoclonal antibodies and antiviral agents developed in Aims 2 and 3 will be evaluated for activity in RNA expression and replication systems developed in Project 2. Overall, these studies are designed to understand and evaluate the importance of age and immune status on virus variation and on the development of potential humoral immunity and to develop reagents that can be used to prevent or treat norovirus infection.