猪瘟病毒流行毒株逃逸C株疫苗诱导的中和抗体的分子机制

Recently, outbreaks of classical swine fever (CSF) have occurred in a large number of C-strain-vaccinated swine farms in many regions of China. We have reported that the emerging subgenotype 2.1b classical swine fever virus (CSFV) is responsible for the outbreaks. The new subgenotype isolates were clustered into an independent branch, which is distant from subgenotype 1.1 C-strain and Shimen strain and early isolates of subgenotype 2.1b in the phylogenetic tree. In addition, C-strain could provide clinical but not pathological or virological protection against the new subgenotype 2.1b isolates. Considering the critical role of the E2 protein in eliciting neutralizing antibodies and the sequence differences in E2 neutralizing antigenic domains between the new isolates of subgenotypes 2.1b and 1.1 CSFV, we hypothesize that the genetic variations might be responsible for the immune escape from neutralizing antibodies-mediated protection induced by C-strain against the new subgenotype 2.1b isolates. In this proposal, we will: 1) generate a series of chimeric viruses harboring the different neutralizing antigenic regions and sites of the E2 protein of the new subgenotype 2.1b strain based on the C-strain backbone; 2) assess the antigenic differences between the chimeric and parental viruses by serum neutralization test and surface plasmon resonance aasay, and resolve the three-dimensional structure of the E2 protein using X-ray diffraction to map the key antigenic region(s) and site(s) responsible for neutralizing antibodies-mediated protection; 3) evaluate the protection against the epidemic CSFV strains conferred by chimeric viruses or C strain. This project is expected to elucidate the molecular mechanisms of immune escape from neutralizing antibodies-mediated protection against the epidemic CSFV strain emerging in China, and provide scientific data for updating control strategies for CSF.

近年来我国多地C株疫苗免疫猪场爆发猪瘟。我们研究证实，猪瘟病毒（CSFV）优势流行毒株为2.1b基因亚型并不断演化，且不能被C株（1.1亚型）诱导的抗体有效中和。鉴于E2蛋白是CSFV主要免疫保护性抗原，且流行毒株与C株和石门株在E2中和抗原区存在20多处氨基酸的差异，我们推测，这些差异可能是其不能被C株有效保护的主要原因。基于此，本项目拟以C株为骨架，构建一系列替换流行毒株E2不同抗原区和差异氨基酸的突变体病毒，通过血清交叉中和试验、同源建模和表面等离子体共振等技术定位流行毒株逃逸C株诱导的中和抗体的关键抗原区和氨基酸位点；然后通过X射线晶体衍射解析其逃逸中和抗体的结构基础；最后通过评价突变体病毒对流行毒株的免疫保护效力，确定E2关键抗原区和氨基酸在流行毒株逃逸疫苗免疫保护中的作用。本研究可望揭示CSFV流行毒株逃逸中和抗体介导的免疫保护的分子机制，为猪瘟防控提供科学依据。

猪瘟（CSF）是由猪瘟病毒（CSFV）感染引起的一种猪烈性传染病。数十年来，我国通过大规模强制接种猪瘟兔化弱毒疫苗（C株）有效控制了猪瘟，但近年来我国多地C株疫苗免疫猪场爆发猪瘟。项目组前期研究证实，猪瘟病毒优势流行毒株为2.1基因亚型并不断演化，且与C株（1.1亚型）遗传关系较远。鉴于E2蛋白是猪瘟病毒最主要的免疫保护性抗原，且流行毒株与疫苗C株和强毒石门株在E2中和抗原区存在多处氨基酸的差异，我们推测，这些差异可能是其不能被C株有效保护的主要原因。基于此，本项目进一步开展了猪瘟流行病学调查，对近年我国猪瘟的流行本底进行了监测，结果显示我国当前流行毒株为2.1b或2.1d基因亚型，其中2.1d基因亚型占主导。而且，E2和Erns显示出多处氨基酸位点的独特变异，包括Erns抗原区（AR1、AR2和AR3）和E2抗原区（B/C和A/D），尤其是在E2的中和表位区。另外，与早前基因1型强毒石门株相比，当前流行毒株表现出对猪瘟兔化弱毒疫苗C株免疫抗体的逃逸能力。同时在E2和Erns的抗原区鉴定出潜在的正选择压力位点，其与抗体结合亲和力相关。还预测出猪瘟疫苗C株和流行野毒株之间的重组事件。以上结果表明流行毒株表现出分子变异和抗原性变化，并确定了流行毒株逃逸 C株诱导的中和抗体的关键抗原区和氨基酸位点，免疫相关位点的变化尤其是B/C抗原区的变异，与病毒逃逸相关，为当前疫苗逃逸毒株的出现提供了理论证据，也提示适时更新猪瘟防控策略的必要性。另外，为助力猪瘟流行病学监测和防控，研制了猪瘟病毒ELISA抗体检测试剂盒并通过新兽药注册复核检验和复审，并实现转化。本项目初步解析了猪瘟病毒流行毒株逃逸中和抗体介导的免疫保护的分子机制，为猪瘟防控提供科学依据。