Rotavirus Outer Capsid Functions in Neutralization

项目总结 轮状病毒是婴幼儿腹泻发病率和死亡率的重要原因，尤其是在 发展中国家。轮状病毒外壳由VP4和VP7蛋白组成，这两种蛋白有助于病毒 附着和进入，是中和抗体的主要目标。轮状病毒的种类很窄 以及细胞嗜性，人类轮状病毒在大多数细胞系和动物模型中生长不良。这种表型， VP4对此有重大贡献，阻碍了人类轮状病毒分子生物学和疫苗的研究 发展。在引入轮状病毒疫苗后，严重的 轮状病毒病，但同时出现和传播以前罕见的轮状病毒血清型和 常见血清型的持续进化。相关的循环轮状病毒血清型的不同模式 在一个地理区域实施的特定疫苗的报告表明，疫苗- 介导的血清型选择。虽然当代和新兴人类轮状病毒的抗原多样性 表明由当前疫苗引发的抗体可能不等同地中和它们，但中和还没有 经过了经验的检验。以质粒为基础的猿类反向遗传系统的研究进展 轮状病毒和利用人类肠道培养人类轮状病毒提供了新的机会 阐明轮状病毒外壳抗原和疫苗引发的抗体之间相互作用的结果。 检验外衣壳抗原多样性、疫苗和细胞类型影响血清的假设 为了中和人轮状病毒，我们提出了两个整合的Subaim。在苏巴伊姆1号，我们将设计 嵌合的猿猴轮状病毒与当代和新出现的致病病毒的外壳基因 使用反向遗传学的人类菌株或疫苗菌株。我们将量化血清抗体的能力 用不同轮状病毒疫苗在兔或婴儿体内诱导结合和中和工程病毒 标准化验，以便与以前的研究进行比较。通过将人类G型或P型分离在另一种 这些研究将为深入了解VP7和VP4的抗原特异性功能提供依据。 中和。在Subaim 2中，我们将在人类细胞系和 使用一组嵌合轮状病毒和差异疫苗接种兔和婴儿的人肠道类 塞拉。这些研究将提供有关血清抗体反应的生物学相关见解。 不同的疫苗和VP4和VP7在中和中的作用，这可能支持不同的模型 基于动物轮状病毒或动物细胞系实验而提出的。总而言之，这些研究将揭示 轮状病毒外衣壳抗原的中和作用及其广度和差异 疫苗诱导的血清抗体反应的特异性，提供了对免疫压力的洞察 影响轮状病毒种群动态，并使改进的轮状病毒中和试验的发展成为可能 以及测试对目前在全球流行的轮状病毒的免疫反应的平台。

PROJECT SUMMARY Rotavirus is an important cause of diarrheal morbidity and mortality in infants and children, particularly in developing countries. The rotavirus outer capsid is composed of VP4 and VP7 proteins, which facilitate viral attachment and entry and are primary targets of neutralizing antibodies. Rotaviruses exhibit narrow species and cell tropism, with human rotaviruses growing poorly in most cell lines and animal models. This phenotype, to which VP4 contributes significantly, has hampered studies of human rotavirus molecular biology and vaccine development. Following rotavirus vaccine introduction, there has been a significant decrease in severe rotavirus disease but concurrent emergence and spread of previously uncommon rotavirus serotypes and continued evolution of common serotypes. Differential patterns of circulating rotavirus serotypes that correlate with the specific vaccine implemented in a geographic region have been reported, suggesting vaccine- mediated serotype selection. While the antigenic diversity of contemporary and emerging human rotaviruses suggests antibodies elicited by current vaccines may neutralize them inequivalently, neutralization has not yet been empirically tested. The recent development of a plasmid-based reverse genetics system for a simian rotavirus and the use of human intestinal enteroids to culture human rotaviruses present new opportunities to elucidate outcomes of interactions between rotavirus outer-capsid antigens and vaccine-elicited antibodies. To test the hypothesis that that outer-capsid antigenic diversity, vaccine, and cell type influence serum neutralization of human rotaviruses, we propose two integrated Subaims. In Subaim 1, we will engineer chimeric simian rotaviruses incorporating outer-capsid genes from contemporary and emerging pathogenic human strains or vaccine strains using reverse genetics. We will quantify the capacity of serum antibodies elicited in rabbits or infants by different rotavirus vaccines to bind and neutralize the engineered viruses using standard assays, to permit comparison with previous studies. By isolating a human G or P type in an otherwise isogenic background, these studies will provide insight into antigen-specific functions of VP7 and VP4 in neutralization. In Subaim 2, we will perform replication and neutralization assays in human cell lines and human intestinal enteroids, using a panel of chimeric rotaviruses and differentially vaccinated rabbit and infant sera. These studies will provide biologically relevant insights into serum antibody responses elicited by different vaccines and roles of VP4 and VP7 in neutralization, which may support models that differ from those proposed based on animal rotaviruses or experiments in animal cell lines. Together, these studies will reveal individual rotavirus outer-capsid antigen functions in neutralization and differences in the breadth and specificity of vaccine-elicited serum antibody responses, provide insight into immunological pressures influencing rotavirus population dynamics, and enable development of improved rotavirus neutralization assays and platforms to test immune responses to current globally circulating rotaviruses.