伪狂犬病病毒变异株逃逸抗体介导的免疫保护的机制

The outbreaks of pseudorabies (PR) caused by the pseudorabies virus (PRV) variant occurred in Bartha-K61-vaccinated swine herds in many regions of China since 2011. The PRV variant strain (TJ strain) showed unique variations in the ectodomains and other regions of gC and gI/gE genes compared with the classical PRV strain (SC strain). The glycoprotein gE binds the IgG Fc domain and interferes with C1q-binding and antibody-dependent cellular cytotoxicity (ADCC). The glycoprotein gC binds to the complement component C3 and its activation products, C3b and C3c, and inhibits the pathways of complement activation. Thus, we hypothesize that these variations may affect the gC-mediated C3-binding and gE-mediated IgG Fc-binding, and affects the antibody-mediated immune protection. Therefore, in this proposal, firstly, we aim to generate a number of chimeric viruses exchanging the gC and gI/gE genes using the Fosmid libraries of PRV TJ and SC strains. Next, the effects of the gI/gE variations on antibody bipolar bridging will be analyzed using erythrocyte-binding assay; the effects of the gC and gI/gE variations on complement-mediated lysis and ADCC will be evaluated using cytotoxicity assay and ADCC reporter gene bioassay. Then the effects of the gC and gI/gE variations on the gC-mediated C3-binding and gE-mediated IgG Fc-binding will be analyzed using flow cytometry assay. A panel of PRV mutants will be generated to identify the critical mutation site(s) of gC and gI/gE proteins that affect its binding with the IgG Fc domain or C3. Finally, the virulence of the chimeric viruses and parental viruses will be compared to evaluate the effects of the gC and gI/gE variations on antibody-mediated immune protection. From this project we expect to reveal the molecular mechanisms how the PRV variant escapes from the antibody-mediated immune protection. Furthermore, this project will provide new insights into the prevention and control of pseudorabies.

伪狂犬病病毒（PRV）变异株能够逃逸Bartha-K61株疫苗的免疫保护。与PRV经典毒株（如SC株）相比，变异株（如TJ株）的gC和gI/gE胞外区等区域均出现特征性变异。据报道gC和gI/gE蛋白可以与补体C3b或IgG Fc片段结合使病毒逃逸抗体介导的免疫保护。我们推测，gC和gI/gE变异可能是变异株逃逸疫苗保护的原因之一。本项目首先利用PRV Fosmid平台构建TJ与SC株gC和gI/gE互换的嵌合病毒。然后，通过红细胞结合试验和细胞毒性试验评价gC和gI/gE变异对抗体介导的免疫保护的影响；流式细胞术试验评价变异对gI/gE与IgG Fc片段、gC与C3b结合的影响；通过构建突变体病毒确定影响结合的关键氨基酸位点。最后，动物实验检测gC和gI/gE变异对抗体介导的免疫保护的影响。本项目将揭示PRV变异株逃逸抗体介导的免疫保护的分子机制，为PRV疫苗及抗病毒药物开发提供新思路。

伪狂犬病（Pseudorabies, PR）是由伪狂犬病病毒（Pseudorabies virus, PRV ）引起的一种急性传染病。PRV基因组十分庞大，全长约143 kb，对其进行改造和修饰比较困难。本研究构建了PRV变异株（PRV-TJ）的基因组Fosmid文库。筛选出基因组插入片段大小为30-45 kb的19个粘 粒用于后续研究。选取10组粘粒组合用于拯救病毒。将10种粘粒组合分别转染Vero细胞 ，其中有5组组合拯救病毒成功。与PRV经典毒株（如 SC株）相比，变异株（如TJ株）的gC和gI/gE胞外区等区域均出现特征性变异。其中gC蛋白的变异尤为显著。我们利用实验室已有的PRV TJ和PRV SC株的fosmid系统，分别构建了gC基因替换的嵌合重组病毒rPRVTJ-SCgC和rPRVSC-TJgC。重组嵌合病毒rPRVTJ-SCgC和rPRVSC-TJgC以及亲本毒感染PK-15细胞，发现PRV TJ和rPRVSC-TJgC对PK15细胞的吸附效率显著高于rPRVTJ-SCgC和PRV SC株，说明TJ-gC蛋白能提高病毒的吸附效率。为进一步证实这一结果，分别构建TJ-gC和SC-gC蛋白His标签的表达细胞系，经镍柱亲和纯化后获得高纯度的gC蛋白，利用表面等离子共振技术(SPR)分析TJ-gC和SC-gC蛋白与受体分子硫酸乙酰肝素亲和力，结果显示SC-gC的KD值是TJ-gC的5倍，即TJ-gC蛋白与硫酸乙酰肝素的亲和力高于SC-gC蛋白。本研究发现和解析了PRV TJ株gC蛋白变异影响了病毒吸附能力，这将为伪狂犬病病毒（PRV）变异株能够逃逸Bartha-K61株疫苗免疫保护的机制研究奠定基础。