Rational Development of Candidate Attenuated Flavivirus Vaccines

描述（由申请方提供）：黄病毒含有许多对公共卫生具有重要意义的病原体，包括登革病毒（DENV）和西尼罗河病毒（WNV），我们没有许可的疫苗。减毒活疫苗可诱导持久的保护性免疫，是控制黄病毒病的重要策略之一，在控制黄热病（YF）和日本脑炎（JE）方面非常成功。非结构蛋白（NS）与宿主天然免疫的逃避有关。其中，NS 4 B蛋白在黄病毒之间具有广泛的同源性，并且已显示N-末端区域编码主要的干扰素（IFN）拮抗功能。因此，我们专注于NS 4 B以鉴定可用于合理减毒候选活黄病毒疫苗的稳定突变。我们正在使用WNV作为模型，并已确定了两个高度减毒的WNV NS 4 B突变体。C102 S突变体在黄病毒NS 4 B蛋白的保守中心疏水结构域中具有取代，已知其有助于黄病毒的毒力表型，并且包含YFV、JEV和DENV的减毒衍生物中的突变。尽管NS 4 B-C102 S突变体对小鼠神经侵袭性和神经毒力高度减毒，并在小鼠中赋予保护性免疫，但它易于回复。它本身并不是一种理想的候选疫苗。另一个减毒的WNV突变体在NS 4 B蛋白的N-末端区域的高度保守的P38残基中具有取代。它对小鼠神经侵袭性高度减毒，具有高度免疫原性，突变体的逆转尚未确定。我们最近发表的工作表明，NS 4 B-P38 G突变体在小鼠中诱导的先天性细胞因子和记忆性T细胞应答高于野生型（WT）NY 99株，并且免疫小鼠均免受WT WNV攻击。潜在的免疫机制还不清楚。我们假设IFN拮抗区域中的P38 G取代或与WNV NS 4 B蛋白的保守中心疏水区域中的突变组合将赋予减毒表型和诱导比WT NY 99更高的保护性免疫的能力，并且这可以用作开发潜在的活黄病毒疫苗候选物的极好模型。我们将首先阐明WNV NS 4 B-P38 G突变体触发比WT NY 99更高的保护性免疫的免疫机制。我们接下来将确定掺入NS 4 B-C102 S突变是否将进一步增强和稳定WNV NS 4 B-P38 G突变体的减毒表型。最后，我们将在小鼠和人细胞中表征对WNV NS 4 B-P38 G/C102 S双突变体感染的免疫应答，并评估WNV NS 4 B突变株在小鼠免疫后随后进行致死性WT WNV攻击的保护作用。对WNV NS 4 B蛋白高度保守的编码区的稳定突变进行表征并研究WNV NS 4 B突变体诱导更高保护性免疫的机制可以作为一个范例，以帮助合理开发其他有效的减毒活黄病毒疫苗。

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.