MicroRNA介导肺组织特异性流感病毒减毒株的构建及免疫效果研究

Vaccination is the most efficient method to control influenza pandemics. Administration of live attenuated influenza vaccine(LAIV) has been associated with durable immunity and significant protection. Nowaday, LAIV is developed on cold-adapted temperature-sensitive phenotype. The production of LAIV is based on the classic reassortment methodology, which is a complex and time consuming technology. MiRNAs have tissue-specific localizations and post-transcriptionally regulate mumerous cellular processes. In addition to these characteristics, endogenous cellular miRNAs can also play a role in viral replication. Some researches about engineering miRNA-responsive viruses have emerged. We intend to develop a complementary attenuation strategy for influenza virus using miRNA-mediated gene silencing. In our preliminary experiments, let-7b has been choosed basing on its high expression in lung. PB1 segment of wild-type H1N1(WT-H1N1) has been mutated to contain miRNA target elements(miRT). Recombinant virus(miRT-H1N1) has been constructed using reverse genetic. Compared to the WT-H1N1, miRT-H1N1 caused few cytopathic effects when infecting human bronchial cells. We intend to further investegate the virulence of miRT-H1N1 and evaluate its immunogenicity and immuno protection. We also try to use miRT-H1N1 as the master donor viruses with its attenuated properties in order to express the hemagglutinin(HA) and neuraminidase(NA) surface glycoproteins of the other wild-type strains. The new recombinant virus would been evaluated as new vaccine strains against it wild type virus. Lastly, as the mechanism is distinct from temperature-based attenuation, this technology could be used as a means of increasing vaccine safety and extending vaccine application population.

疫苗接种是人类预防流行性感冒大流行的主要手段。流感减毒活疫苗能够诱导宿主产生广泛而持久的免疫保护，但当前基于经典重组技术制备的低温适应减毒活疫苗存在制备技术复杂、耗时长等缺点。MiRNA具有组织特异性表达及转录后调节作用，利用这两个特性构建组织特异性病毒减毒株已在多种病毒中探讨，而构建miRNA介导流感病毒减毒株是一种新思路。项目组前期工作中，选用肺组织高表达miRNA（let-7b），通过碱基突变构建编码区内含let-7b结合靶位的PB1片段，利用反向遗传学方法得到含let-7b靶位的重组流感病毒，并在气道上皮细胞感染实验中发现其致细胞病变能力较野毒株明显减弱。本项目拟进一步在细胞及动物水平研究该重组毒株的毒力变化，阐明其宿主免疫原性和攻毒保护效应，探究利用该重组病毒投送异源流感株表面抗原的功能，明确miRNA介导的流感病毒减毒株应用价值，从而为流感病毒减毒活疫苗的研制提供了新思路。

疫苗接种是人类预防流行性感冒大流行的有效手段，减毒活疫苗能够诱导宿主产生广泛而持久的免疫保护。当前基于经典重组技术制备的低温适应减毒活疫苗存在制备技术复杂、耗时长等缺点。MiRNA具有组织特异性表达及转录后调节作用，利用这两个特性构建组织特异性病毒减毒株已在多种病毒中探讨，而构建miRNA介导流感病毒减毒株是一种新思路。课题采用miRNA介导的基因沉默机制设计一种全新的病毒减毒株。利用miRNA的调节机制抑制病毒的复制，从而降低病毒在重要器官和组织毒力。我们选择将miR-let-7b结合靶位设计在流感病毒的RNA聚合酶复合体PB1片段编码序列上。将miR-let-7b靶序列分别与流感毒株（A/Nanjing/NJU-108/2009 H1N1）的PA、PB1和PB2的开放阅读框序列比对，最终选择PB1片段最优序列（位置83bp-107bp）。发现病毒在表达相应miRNA的人支气管上皮细胞中（human bronchial epithelial cell line，HBE）复制受限，毒力下降，从而产生减毒表型。而在没有表达相应miRNA的293T细胞（human embryonic kidney cells，HEK293T）中保持复制能力和毒力。另外，减毒株在不同细胞组织中差异分布，避免全身组织的扩增和验证反应。分子水平实验证实了miR-let-7b特异作用于miRT-H1N1（miRNA recognition element H1N1，miRT-H1N1）株PB1片段，抑制miRT-H1N1的复制。体内小鼠研究证明miRNA-let-7b介导的甲型流感减毒株( miRT-H1N1 )在小鼠体内是安全有效的。感染MiRT-H1N1的小鼠体重下降不明显，生存率为100%，即使接种最高剂量（LD50>107 TCID50）也不会引起小鼠死亡；肺脏组织中miRT-H1N1病毒复制明显受限，病毒载量与wt-H1N1和scbi-H1N1相比下降超过100倍。感染miRT-H1N1的小鼠肺脏组织损伤最轻，促炎因子和趋化因子表达水平显著下降，炎症反应显著减轻。攻毒保护实验发现，经鼻腔接种免疫后，小鼠在致死剂量WT-H1N1感染时受到显著的免疫保护。课题验证了一种全新的减毒机制，为将来流感病毒快速制备提供技术储备，对流感病毒新型减毒活疫苗的探讨研究对流感的防治具有重要的意义。