Mechanism of alphavirus packaging: designing of pseudoinfectious viruses

描述(申请人提供)：Togaviridae家族中的甲型病毒属含有广泛分布的各种重要的人类和动物病原体。委内瑞拉(VEEV)、东部(EEEV)和西部(WeEV)马脑炎病毒在美国代表着严重的公共卫生威胁，可被生物恐怖分子应用。然而，迄今为止，既没有开发出有效的抗病毒疫苗，也没有开发出安全有效的疫苗来预防这些感染。现有的实验性疫苗要么效果不佳，要么在人体上表现出非常强烈的不良反应。我们最近对甲型病毒RNA包装的分子机制的研究使我们了解了甲型病毒RNA特异性包装信号的结构和功能，该信号存在于其他脑源性甲型病毒的基因组中，并被同源蛋白和衣壳蛋白识别。这些数据将被应用于拟议的研究，以开发从根本上设计活的甲型病毒变异株的新策略，该变异株具有不可逆的、高度减毒的表型，但产生病毒特异性抗原，这是诱导灭菌免疫所必需的，与wt病毒一样有效。通过将多个合理设计的突变引入衣壳蛋白或新发现的通用rna包装信号，我们将开发保持高水平rna复制的甲型病毒，并产生主要释放的结构蛋白(策略1)或 以非传染性的、无基因组的病毒样颗粒的形式独占(策略2)，因此， 体内不会出现病毒血症。这些病毒将能够诱导细胞免疫反应和高水平中和抗体的平衡组合。 与公共卫生相关：该研究提案旨在从根本上开发设计甲型病毒活疫苗的新策略，该疫苗表现出不可逆转、高度减毒的表型，但产生病毒特异性抗原，这是诱导灭菌免疫所必需的，与wt病毒一样有效。

DESCRIPTION (provided by applicant): The Alphavirus genus in the Togaviridae family contains a variety of widely distributed important human and animal pathogens. Venezuelan (VEEV), eastern (EEEV) and western (WEEV) equine encephalitis viruses represent a serious public health threat in the US and can be applied by bioterrorists. However, to date, neither effective antiviral nor safe and efficient vaccines have been developed for preventing any of these infections. The existing experimental vaccines are either of poor efficacies or demonstrate very strong adverse reactions in humans. Our recent studies of the molecular mechanism of alphavirus RNA packaging led to understanding of structure and function of the alphavirus RNA-specific packaging signal, which is present in the genomes of other encephalitogenic alphaviruses and recognized by both homologous and capsid proteins. These data will be applied in the proposed research to develop fundamentally new strategies of designing live alphavirus variants possessing irreversible, highly attenuated phenotypes, but producing virus-specific antigens, required for induction of sterilizing immunity as efficiently as a wt virus. By introducing multiple, rationally designed mutations into capsid protein or newly identified universal RNA packaging signal, we will develop alphaviruses that retain high levels of RNA replication, and production of structural proteins which are released either mostly (strategy 1) or exclusively (strategy 2) in the form of non-infectious, genome-free virus-like particles and, thus, develop no viremia in vivo. These viruses will be capable of inducing a balanced combination of cellular immune response and high levels of neutralizing antibodies. PUBLIC HEALTH RELEVANCE: The research proposal is aimed to develop fundamentally new strategies of designing live alphavirus vaccines demonstrating irreversible, highly attenuated phenotype, but producing virus-specific antigens, required for induction of sterilizing immunity as efficiently as wt viruses.