Rational Development of Candidate Attenuated Flavivirus Vaccines

描述（由申请人提供）：黄病毒含有许多具有公共卫生重要性的病原体，包括登革热病毒（DENV）和西尼罗河病毒（WNV），我们没有获得许可的疫苗。减毒活疫苗可诱导持久的保护性免疫，是控制黄病毒疾病的重要策略之一，在控制黄热病和日本脑炎方面取得了巨大成功。非结构蛋白（NS）与逃避宿主先天免疫有关。其中，NS4B蛋白在黄病毒之间具有广泛的同源性，其n端区域编码干扰素（IFN）的主要拮抗功能。因此，我们将重点放在NS4B上，以确定可用于合理减毒候选黄病毒活疫苗的稳定突变。我们使用西尼罗河病毒作为模型，并确定了两个高度减毒的西尼罗河病毒NS4B突变体。C102S突变体在黄病毒保守的NS4B蛋白中心疏水区域有一个取代，该区域已知对黄病毒的毒力表型有贡献，并且在YFV、JEV和DENV的减毒衍生物中含有突变。尽管NS4B-C102S突变体对小鼠的神经侵袭性和神经毒力高度减弱，并在小鼠中赋予保护性免疫，但它很容易逆转。它本身不是一种理想的候选疫苗。另一个减弱的西尼罗河病毒突变体在NS4B蛋白n端高度保守的P38残基上有一个替换。它对小鼠神经侵袭性有高度减毒，高度免疫原性，突变体的逆转尚未确定。我们最近发表的研究表明，与野生型（WT） NY99菌株相比，NS4B-P38G突变体在小鼠体内诱导了更高的先天细胞因子和记忆T细胞反应，免疫后的小鼠都能免受WT WNV的攻击。潜在的免疫机制尚不清楚。我们假设，与WT NY99相比，WNV NS4B蛋白中IFN拮抗区域的P38G替代或与保守的中心疏水区域突变结合，将赋予其减毒表型和诱导更高保护性免疫的能力，这可能作为开发潜在黄病毒活疫苗候选物的良好模型。我们将首先阐明WNV NS4B-P38G突变体比WT NY99触发更高保护性免疫的免疫机制。接下来，我们将确定加入NS4B-C102S突变是否会进一步增强和稳定WNV NS4B-P38G突变体的减毒表型。最后，我们将描述小鼠和人类细胞对WNV NS4B- p38g /C102S双突变株感染的免疫反应，并评估WNV NS4B突变株在小鼠免疫后的致命WNV攻击后的保护作用。研究WNV NS4B蛋白高度保守编码区稳定突变的特征，探讨WNV NS4B突变体诱导更高保护性免疫的机制，可作为合理开发其他有效的黄病毒减毒活疫苗的范例。

DESCRIPTION (provided by applicant): The flaviviruses contain many pathogens of public health importance, including dengue virus (DENV) and West Nile virus (WNV) for which we have no licensed vaccines. Live attenuated vaccines, which induce durable protective immunity, are one of the important strategies to control flavivirus diseases and have been very successful at controlling yellow fever (YF) and Japanese encephalitis (JE). The nonstructural (NS) proteins are associated with evasion of host innate immunity. Among them, NS4B protein has extensive homology between flaviviruses and the N-terminal region has been shown to encode a major interferon (IFN) antagonism function. Thus, we focus on NS4B to identify stable mutations that can be used to rationally attenuate candidate live flavivirus vaccines. We are using WNV as a model and have identified two highly attenuated WNV NS4B mutants. The C102S mutant has a substitution in the flavivirus conserved central hydrophobic domain of NS4B protein, which is known to contribute to the virulence phenotype of flaviviruses, and contains mutations in the attenuated derivatives of YFV, JEV and DENV. Although the NS4B-C102S mutant was highly attenuated for mouse neuroinvasiveness and neurovirulence, and conferred protective immunity in mice, it was prone to reversion. It is not an ideal candidate vaccine by itself. Another attenuated WNV mutant has a substitution in the highly conserved P38 residue in the N-terminal region of the NS4B protein. It is highly attenuated for mouse neuroinvasiveness, highly immunogenic, reversion of the mutant has not been identified. Our recent published work showed that the NS4B-P38G mutant induced higher innate cytokine and memory T cell responses in mice than the wild-type (WT) NY99 strain, and immunized mice were all protected from WT WNV challenge. The underlying immune mechanisms are not clearly understood. We hypothesize that the P38G substitution in the IFN antagonism region or in combination with mutation(s) in the conserved central hydrophobic region of WNV NS4B protein will confer an attenuated phenotype and the ability to induce higher protective immunity than WT NY99, and this may serve as an excellent model to develop potential live flavivirus vaccine candidate(s). We will first elucidate the immune mechanisms by which WNV NS4B-P38G mutant triggers higher protective immunity than WT NY99. We will next determine whether incorporating the NS4B-C102S mutation will further enhance and stabilize the attenuated phenotype of WNV NS4B-P38G mutant. Lastly, we will characterize immune responses to WNV NS4B-P38G/C102S double mutant infection in mouse and human cells and evaluate the protective effects of WNV NS4B mutant strains after immunization of mice followed by lethal WT WNV challenge. Characterization of stable mutations in the highly conserved coding regions of WNV NS4B protein and investigation the mechanism by which WNV NS4B mutant induces higher protective immunity can be utilized as a paradigm to aid in the rational development of other efficacious live attenuated flavivirus vaccines.