Rational Development of Candidate Attenuated Flavivirus Vaccines

描述(申请人提供)：黄病毒含有许多对公共卫生重要的病原体，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。减毒活疫苗能诱导持久的保护性免疫，是控制黄病毒病的重要策略之一，在控制黄热病和流行性乙型脑炎方面取得了很好的效果。非结构蛋白(NS)与逃避宿主天然免疫有关。其中，NS4B蛋白在黄病毒之间具有广泛的同源性，N-末端已被证明编码一个主要的干扰素拮抗功能。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。我们正在使用西尼罗河病毒作为一个模型，并已经鉴定出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蛋白的中心疏水结构域有一个替换，该结构域与黄病毒的毒力表型有关，并包含YFV、JEV和DENV的减毒衍生品的突变。虽然NS4B-C102S突变体对小鼠的神经侵袭力和神经毒力具有高度的减毒作用，并对小鼠产生保护性免疫，但它容易逆转。它本身并不是一个理想的候选疫苗。另一个减毒的西尼罗河病毒突变体在NS4B蛋白的N端区域的高度保守的P38残基上有一个替换。它是高度减毒的小鼠神经侵袭性，高度免疫原性，突变体的逆转还没有被鉴定。我们最近发表的工作表明，NS4B-P38G突变株在小鼠体内诱导了比野生型(WT)NY99株更高的固有细胞因子和记忆T细胞应答，免疫小鼠都能抵抗WT WNV攻击。其潜在的免疫机制尚不清楚。我们推测，WNV NS4B蛋白干扰素拮抗区的P38G替换或与保守的中心疏水区突变(S)相结合，将产生比WT NY99更强的表型和更高的保护性免疫，这可能是开发潜在的黄病毒活疫苗候选株(S)的良好模型。我们将首先阐明WNV NS4B-P38G突变株触发比WT NY99更高的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突变株的减毒表型。最后，我们将对WNV NS4B-P38G/C102S双突变株在小鼠和人细胞中的免疫应答进行表征，并评价WNV NS4B突变株在小鼠免疫后对WT WNV致死攻击的保护作用。对WNV NS4B蛋白高度保守编码区的稳定突变进行鉴定，并研究WNV NS4B突变株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.