IRES新型黄热减毒活疫苗的构建及其减毒机制研究

Yellow fever virus (YFV) is an important member among the mosquito-borne flaviviruses, which leads to the high mortality yellow fever disease in infected humans and poses a great threat to the public health. The risks of yellow fever expansion around the world are increasing in recent years. The 17D live attenuated vaccine has made great contributions to the control and prevention of the infections of yellow fever virus. However, residual virulence factors still remain within the 17D vaccine, limiting its application in the newborn and immunocompromised populations; moreover, the 17D vaccine retains the ability to infect mosquito vectors, which leads to potential risks that the recombination occurs between the 17D and wild type strain in the mosquitoes. For these reasons, the improvement of the 17D, and the development of new generation of yellow fever vaccines are in great need. In this project, the internal ribosome entry site (IRES) from the encephalomyocarditis virus (EMCV) will be inserted into different positions of the 17D genome by reverse genetics approaches, and a series of vaccine candidates in which the translation of different viral proteins are directed by the IRES element will be generated, and then the replication characteristics, host tropism and genetic stability of the vaccine candidates will be evaluated. Based on these results, the attenuation phenotype of the recombinant vaccine strains will be accessed by using the suckling mice and innate-immunity-deficient mice models. Next, the immunogenicity and protection efficacy of the vaccine candidates will be tested in both immunocompetent and immunocompromised mice models. In the meanwhile, the molecular mechanism about IRES-directed attenuation will be illuminated by a series of experiments, including high-throughput transcriptome sequencing, knockdown and over-expression of interferon-simulated genes, as well as viral mini-genome and replicon assay. The conduction of this project will not only generate safer yellow fever vaccine candidates with good efficacy and without transmission potency, but also provide theoretical basis for the rational design of other viral vaccines.

黄热病毒是一种严重威胁人类健康的蚊媒黄病毒，近年来，其在全球扩散的风险日益升高。17D减毒活疫苗对防控黄热病毒感染发挥了关键作用，然而17D存在减毒不彻底的缺点，使其应用受到一定限制；且17D仍能感染蚊虫，带来潜在的安全风险。因此，对17D加以改进以研制新型黄热疫苗极为重要。本项目通过反向遗传学操作将内部核糖体进入位点（IRES）引入17D基因组不同位置，以获得由IRES元件介导翻译的重组疫苗候选株，并评价其复制特征、宿主嗜性与遗传稳定性。在此基础上，利用不同小鼠模型分析其减毒特征，并进一步在动物模型中评价其免疫原性与免疫保护效果。同时，通过转录组测序、干扰素刺激基因沉默与过表达试验、病毒微型基因组及复制子试验等一系列技术手段，对IRES元件介导的减毒机制进行深入研究。本项目的实施不仅有望获得消除感染蚊媒能力、更为安全高效的新型黄热疫苗候选株，也可为其它病毒疫苗的研制提供理论依据。

黄热病毒感染人类可引起高致死率的黄热病。尽管黄热减毒活疫苗17D具有良好的安全性和保护效果，该减毒株仍存在一定的毒力残留，且具有感染蚊虫的可能性。基于脑心肌炎病毒（EMCV）内部核糖体进入位点（IRES）活性的种属特异性，我们构建了由该元件介导翻译的17D-IRES重组疫苗候选株。17D-IRES与原型株17D在哺乳动物细胞中的增殖能力相近，但在蚊虫细胞系中，17D-IRES不能增殖。与17D相比，17D-IRES在A129和BALB/c小鼠模型中具有更低的神经毒力，在AG6小鼠模型中亦表现为播散能力和致病性减弱。体外试验表明，17D-IRES对I型干扰素的敏感性增强。随后，我们证实17D-IRES接种后可使小鼠产生特异性体液免疫和细胞免疫，并可产生有效的免疫保护。为构建可用于黄热病毒致病性研究和疫苗、药物评价的便利动物模型，我们首先构建了表达不同报告基因的重组17D疫苗株。实验结果表明，表达NanoLuc萤光素酶或者海肾萤光素酶基因的重组报告病毒均可用于抗病毒化合物的体外评价。乳鼠神经毒力测定等体内外试验表明，表达萤光素酶报告基因的NLuc-YF17D与原型株17D具有相似的生物学特性与致病性。通过生物发光成像技术，我们实现了对NLuc-YF17D在感染小鼠体内复制的准确便捷监测。在此基础上，我们进一步鉴定了NLuc-YF17D对不同周龄和免疫状态小鼠的致病性。值得指出的是，生物发光成像技术显示，将NLuc-YF17D经腹腔途径感染两周龄A129小鼠后，病毒可迅速增殖并播散至各内脏器官和中枢神经系统，从而使小鼠发病并死亡。因此两周龄A129小鼠可作为研究黄热病毒减毒机制以及黄热疫苗相关嗜内脏疾病和黄热疫苗相关嗜神经疾病的发生机制的动物模型。综上，本项目的开展对黄热疫苗的进一步优化设计及黄热病毒致病机制研究起到了重要的促进作用。