Rational Design of live-attenuated vaccines for flaviviruses

项目概要： 建议研究的目的是为合理设计开发广泛有效的衰减策略 抗黄病毒减毒活疫苗（LAV）。致病性黄病毒引起严重的人类疾病 如出血热和脑炎。使用两种传统疫苗，黄热病病毒（YFV）17 D和 日本脑炎病毒SA 14 -14-2，已经证明了免疫接种如何成为一种有效的策略， 疾病控制。然而，用于开发这些LAV的经验方法已被证明是无效的 在生产其他黄病毒的候选LAV中，因此需要合理疫苗设计的新策略。 以前，黄病毒LAV的合理设计是基于引入突变， 在两种传统疫苗和其他减毒突变体中观察到减毒表型。但这并没有产生 广泛有效的减毒概念，因为诱变靶缺乏保守序列或 与不同的宿主分子相互作用LAV设计的一个主要挑战是该领域对以下方面的知识有限： 黄病毒毒力机制包含共有序列的靶基因，对基因组的功能重要， 不同黄病毒的毒力尚待鉴定。在这项研究中，PI将开发两个衰减 通过干扰黄病毒包膜（E）蛋白的结构域间运动的策略。域间 E蛋白的运动是所有黄病毒中普遍保守的机制，并且对于病毒膜至关重要。 融合和病毒体装配;它们由两组结构域间的高度保守序列控制 多肽，包膜蛋白结构域I（EDI）-包膜蛋白结构域II（EDII）铰链和EDI -包膜蛋白结构域II（EDII）铰链。 蛋白质结构域III（EDIII）接头。核心假设是，结构域间的保守残基 肽对于黄病毒的毒力具有重要的功能，无论它们的组织嗜性和疾病 发病机制为了去除两个结构域间肽中的毒力决定簇，将进行诱变分析。 在以下两个具体目标中进行：在目标1中，将高度保守的疏水残基突变为 干扰有助于EDI-EDII铰链结构和功能的疏水相互作用;在目的2中， EDI-EDIII接头的结构和功能将通过去除功能上重要的侧链而被破坏 保守残基和插入额外的甘氨酸/脯氨酸残基，以增加肽的灵活性。广泛 将通过将选择的突变工程化到全长 两种模式黄病毒，西尼罗河病毒和YFV的互补DNA感染性克隆，然后 证明在各自的小鼠模型中丧失了毒力。拟议研究的完成将导致 关于EDI-EDII铰链和EDI-EDIII接头的功能重要性的知识的进步 黄病毒毒力的结构域间区域。这项研究的结果预计将提供广泛的基础， 病原性黄病毒的有效减毒策略，并促进未来候选疫苗的合理设计 黄病毒LAVs

Project summary: The objective of the proposed study is to develop broadly effective attenuation strategies for the rational design of live-attenuated vaccines (LAVs) against flaviviruses. Pathogenic flaviviruses cause severe human diseases such as hemorrhagic fever and encephalitis. The use of two legacy vaccines, yellow fever virus (YFV) 17D and Japanese encephalitis virus SA14-14-2, has demonstrated how immunization can be an efficient strategy for disease control. However, the empirical approach used for the development of these LAVs has proven ineffective in producing candidate LAVs for other flaviviruses, thereby demanding new strategies for rational vaccine design. Previously, the rational design of flavivirus LAVs was based on the introduction of mutations that lead to attenuated phenotypes observed in the two legacy vaccines and other attenuated mutants. This failed to produce broadly effective attenuation concepts, because the mutagenesis targets lacked conserved sequences or interacted with diverse host molecules. A major challenge in designing LAVs is the field's limited knowledge of flavivirus virulence mechanisms. Target genes that contain consensus sequences, functionally important for the virulence of different flaviviruses, are yet to be identified. In this study, the PI will develop two attenuation strategies by interfering with the interdomain movements of flavivirus envelope (E) proteins. Interdomain movements of E proteins are universally conserved mechanisms in all flaviviruses and critical for viral membrane fusion and virion assembly; they are controlled by highly conserved sequences in two sets of interdomain peptides, the envelope protein domain I (EDI) – envelope protein domain II (EDII) hinge and the EDI – envelope protein domain III (EDIII) linker. The central hypothesis is that conserved residues in the interdomain peptides are functionally important for flavivirus virulence, regardless of their tissue tropism and disease pathogenesis. To remove virulence determinants in the two interdomain peptides, mutagenesis analyses will be conducted in the following two specific aims: In Aim 1, highly conserved hydrophobic residues will be mutated to interfere with the hydrophobic interactions that contribute to EDI-EDII hinge structure and functions; in Aim 2, the structures and functions of EDI-EDIII linker will be disrupted by removing functionally important side-chains of conserved residues and inserting additional glycine/prolin residues to increase peptide flexibility. Broadly effective attenuation strategies will be developed by engineering selected mutations into the full-length complementary DNA infectious clones of two model flaviviruses, West Nile virus and YFV, and then demonstrating the loss of virulence in respective mouse models. The completion of the proposed study will lead to an advancement in knowledge regarding the functional importance of the EDI-EDII hinge and EDI-EDIII linker interdomain regions for flavivirus virulence. The results of this study are expected to provide the basis for broadly effective attenuation strategies for pathogenic flaviviruses and facilitate the rational design of future candidate flavivirus LAVs.