马传染性贫血病毒弱毒疫苗株和亲本强毒株的体内进化及其与免疫应答关系研究

Live attenuated vaccine is considered the only type of vaccine that induces protective immunity to the infections of lentiviruses. The Chinese attenuated vaccine against equine infectious anemia virus (EIAV) is the first vaccine that successfully applied country-widely to control a disease coursed by the infection of a lentivirus. Although live attenuated lentiviral vaccines are no longer being accepted for practical using due to the concerning of safety, the hidden mechanism, by which the attenuated lentiviral vaccines effectively elicit protective immunity, will provide valuable references to studies on the immunity to and vaccine development of lentivirus. Our preliminary studies identified that there were significant differences in viral genome sequences and compositions between the EIAV vaccine strains and their parental pathogenic strains. Unique evolution patterns were observed in genomes, especially in the gene of the envelope surface glycoprote in gp90, of these two types of viruses when these viruses were inoculated to the hosts. In this proposed study, a systematic investigation will be performed to analyze the genomic evolution patterns and immune response levels of the EIAV vaccine and pathogenic strains, compare the differences in these viral behaviors between viral strains, and verify the correlation of the viral genomic evolutions to immune responses of the hosts. The goal of this proposal is to identify the correlations of the infectious status and genomic evolution patterns of the EIAV attenuated vaccine to the immune protection induced by the vaccine. By this approach, the safety and efficacy of live attenuated lentiviral vaccines will be examined for a better understanding of the lentiviral infection and corresponding immune responses. These efforts will improve the development of vaccines that effectively prevent the infection of lentiviruses.

研究发现，只有减毒活疫苗才能诱导对慢病毒感染的有效保护。马传染性贫血病毒（EIAV）中国弱毒疫苗是唯一成功大规模应用的慢病毒疫苗。虽因安全因素，慢病毒活疫苗已难以实际应用，但其诱导免疫保护的机制，可为慢病毒免疫研究和疫苗研发提供借鉴。前期研究发现，EIAV弱毒疫苗株与亲本强毒株的基因组之间存在明显差异，接种宿主后更表现出各自的独特体内进化，特别是囊膜表面糖蛋白 gp90 基因。由于二者诱导机体免疫应答能力显著不同，提示病毒的体内进化与疫苗的保护作用密切相关。本项目将通过系统检测疫苗株和强毒株在接种马体内的基因组进化规律和诱导宿主免疫应答特点，比较不同毒株间差异，综合分析病毒基因组变异与诱导免疫应答的关系。本研究的最终目标是，确定 EIAV 弱毒疫苗在体内基因组进化特征与免疫保护的关系，从分子生物学角度探讨弱毒疫苗的安全性和有效性，加深对慢病毒感染和免疫的理解，促进疫苗研发。

EIAV弱毒疫苗作为成功应用的慢病毒疫苗，对该疫苗在免疫马匹后病毒复制与感染状态的研究不仅关系疫苗的生物安全评价，也是研究疫苗诱导免疫保护的基础。与亲本毒株相比，弱毒疫苗在各个基因都有稳定的变异位点，EIAV疫苗在免疫马匹6个月后才能诱导出坚强的免疫保护反应。本项目系统研究了疫苗株和强毒株在接种马体内病毒基因组主要变异区的进化特征及其诱导中和抗体动态变化规律，并分析了Env变异对中和抗体体敏感性的影响，为揭示EIAV弱毒疫苗诱导免疫保护机制提供参考。. 完成了EIAV弱毒疫苗致弱过程中病毒基因组进化规律研究。发现伴随着病毒毒力的减弱，在基因组的各个区域均出现了一系列稳定变异，统计分析显示绝大部分变异位点在体外适应病毒和体内强毒株、致病病毒和弱毒株之间突变频率差异显著。. 完成了强毒株EIAVLN40感染实验，检测了强毒株LTR、S2和gp90基因在体内变异特征。发现在S2进化过程中有19.1%以上的氨基酸出现稳定替换，有7个变异位点呈现明显正选择压力。进化分析显示，所有发病点的样本基因序列高度同源，而感染早期的样本与疫苗株相似。统计分析表明S2的变异与发病状态和感染阶段相关。中和抗体检测发现，强毒株感染马只能产生较低的中和抗体。. 完成了EIAV弱毒疫苗免疫实验及其攻毒保护实验，检测了疫苗毒LTR、S2和gp90基因在体内进化特征。发现EIAV弱毒疫苗在体内维持低水平复制，并持续进化，病毒抗原基因gp90的多样性得到了进一步的丰富。中和抗体检测发现，疫苗免疫马在免疫后3个月产生较高的中和抗体，而且免疫保护马的中和抗体水平均高于未保护马。对EIAV强毒和疫苗免疫马的外周血单个核细胞（PBMC）、肺脏和脾脏进行了多种重要细胞因子的检测发现，EIAV强毒和疫苗毒感染马体后都激活了细胞因子应答，但是二者诱导细胞因子上调程度具有显著差异。. 完成了不同Env变异体对中和抗体敏感性研究，发现Env糖基化位点变化影响病毒对中和抗体的敏感性。. 发表SCI文章5篇，中文核心期刊2篇；培养博士研究生3名，硕士研究生2名。