利用内源性microRNA的特性对修饰型柯萨奇B3病毒感染组织趋向性及心肌致病性的可控性研究

One of the major drawbacks in the development of live attenuated coxsackievirus B group(CVB) vaccine is the fact that attenuated vaccine is reversion to a virulent phenotype and pathogenic damage occurs again. Aiming at the problem of instability in CVB genome, a rational strategy will be developed in this study for viral attenuation by exploiting the regulatory capacity of microRNA (miRNA)-mediated gene-silencing system and tissue-specific distribution of cellular miRNAs. Since high sequence complementarity between the miRNA and its target sequence is essential for suppression of mRNA translation, and catalytic mRNA degradation in the tissue-specific regulation of gene expression patterns, endogenous muscle- specific miRNA-complementary target sequences (miRNAs-T) will be inserted into CVB3 genome in our research. These recombinant viruses will not replicate in cells expressing muscle-specific miRNAs and therefore do not cause myocardial damage, meanwhile the recombinant viruses will still propagate robustly in a limited number of tissues, so as to elicit a complex and long-lasting immune response. The study mainly includes: To construct recombinant CVB3 which include target sequences bearing perfect sequence complementarity to cardiomyocyte- specific miRNAs ;The replication ability of viruses containing these sequences will be evaluated in different cultured cells which express or do not express cognate miRNAs to inserted targets; To determine the mechanism and feasibility of the potent inhibition of viral replication executed by microRNA- based gene-silencing system; To observe the tissue tropism of miRNA-modified CVB3 and estimate state of pathogenic pathology in mice vaccinated miRNA-modified CVB3; Next to ascertain whether engineered miRNA-modified CVB3 can elicit protective immunity against challenge with a wild-type pathogenic strain of coxsackievirus. This research may contribute a useful experimental data to develop a complementary attenuation strategy for coxsackievirus.

针对柯萨奇B3减毒活疫苗在研制过程中存在着不稳定性易发生致病表型回复造成致病性损伤的问题，研究拟利用内源性miRNA基因沉默系统调控基因表达的能力和特异性分布的特点，在CVB3基因组序列中插入心肌骨骼肌内特定miRNA的互补序列(miRNAs-T)，限制病毒在心肌细胞内复制，以此控制病毒对心肌骨骼肌的致病性，同时不改变病毒在其他组织中复制而诱导机体产生抗病毒免疫。研究包括：构建含有心肌特异性miRNA互补链的CVB3感染性克隆，在不表达和表达相应miRNA的细胞系中观察miRNAs-T修饰病毒复制能力，评估内源性miRNA对修饰病毒的细胞亲嗜性限制状况；确证内源性miRNA系统介导病毒复制抑制机理；同时在实验动物中观察和评价心肌内源性miRNA系统对修饰型CVB3致病病理的影响、心肌限制型修饰病毒诱发机体的免疫状况和抗CVB3再感染能力。研究结果将对探索新的安全有效的减毒途提供实验基础。

本项目为非连续资助类项目，因此在一年内顺利完成了任务书中计划的内容，主要研究结果包括： .1、获得含有心肌特异性microRNAs (miRNAs,miRs) 互补序列的修饰病毒毒株。加入内源性的miR-206（CVB-miR206）、miR-133/206/133 （CVB-miR133/206/133）互补序列以及插入无关序列的阴性对照(CVB-miR39*1)修饰病毒株与CVB3原病毒一样保持了对HeLa细胞的感染复制能力。同时经多次传代后证明修饰病毒能保持序列的稳定性。.2、通过细胞学研究证实心肌内源性miRNA能够抑制修饰病毒对心肌细胞的趋向性。使用相同MOI，修饰病毒在感染TE671细胞第24、48和72h，与CVB3相比未出现明显致细胞病变作用，在48h和72h后miRNAs修饰病毒感染组细胞增殖率比CVB3明显高（p<0.05），与细胞对照组相比没有明显差异。证明肌肉细胞内的miRNA-133及miRNA-206对修饰性病毒的复制起到明显抑制作用。而这些病毒仍保持在HeLa细胞（无内源性miR133和miR206）中的复制。.3、动物实验表明在相同剂量下与CVB3相比较，miRNAs修饰病毒有明显降低小鼠病死率的作用（p<0.05）。病理结果证实：CVB3感染后小鼠的心肌组织从第3天开始到第7天陆续呈现大量炎性细胞浸润，细胞坏死等典型心肌损伤病理变化。而感染miRNAs修饰病毒的小鼠心肌仅在第三天出现轻微水肿和出血后，然后逐步好转到第7天基本恢复正常。病毒核酸检测发现：CVB-miR133/206/133感染的小鼠心脏中的病毒核酸量明显低于CVB3的病毒核酸量，相差约350倍。表明心肌中内源性miR133或miR206识别到含有互补链的修饰性病毒时，开始抑制的病毒复制，并逐步降解病毒的基因组因而降低心肌细胞的病毒量，起到对心肌的保护作用。.4、研究证实修饰病毒同样可诱发机体的免疫产生针对CVB3的中和抗体，CVB-miR206免疫接种小鼠四周后血清中和抗体效价平均可达716，CVB-miR133/206/133可达到868,而对照组CVB3病毒免疫血清的中和抗体效价为886。用修饰型病毒免疫一个月后再用原始野生型CVB3感染小鼠，与对照相比较其死亡率降低。本研究初步验证和确定了修饰型病毒作为减毒活疫苗新途径的可行性和有效性。