ORF2-6嵌合型猪繁殖与呼吸综合征病毒诱导广谱中和抗体的作用与机制

Current porcine reproductive and respiratory syndrome (PRRS) vaccines cannot provide sufficient cross-protection, therefore, it's of great significance to study how to induce PRRS virus (PRRSV) cross-protection. Neutralizing antibody is an important component of anti-PRRSV immunity. Our previous studies showed that a portion of PRRSV vaccinated and challenged pigs induced broadly neutralizing antibodies (bnAbs), and the broadly neutralizing epitopes are closely related to envelope-associated proteins encoded by ORFs 2-6 genes. Previous studies from other groups showed that the diversity of viral envelope proteins is critical for inducing bnAbs. Therefore, in this study, we will firstly construct chimeric viruses containing ORFs 2-6 from distinct sub-genotypes of PRRSV strains using reverse genetic system. The constructs of ORFs 2-6 chimeric PRRSV will be used to inoculate piglets to explore the effects of ORFs 2-6 diversity on the induction of PRRSV bnAbs. Secondly, we will propagate PRRSV in vitro with the presence of homologous nAbs or bnAbs to obtain PRRSV mutants that are resistant to distinct nAbs. And the ORFs 2-6 of the mutants will be sequenced to select for potential action sites of bnAbs. At last, we will construct chimeric viruses containing the above mutation sites using site-directed mutagenesis and reverse genetic system. The neutralizing activities of homologous nAbs and bnAbs on the mutants will be determined to identify the key sites of broadly neutralizing epitopes. The diversity of the key sites will also be analyzed by sequence alignment. This study explores the effects and mechanisms of ORFs 2-6 chimeric PRRSV on the induction of bnAbs via in vivo and in vitro experiments, which will provide fundamental knowledge for the development of a PRRS vaccine that could induce bnAbs.

猪繁殖与呼吸综合征（PRRS）疫苗交叉保护性差，研究如何诱导PRRS病毒（PRRSV）交叉保护有重要意义。中和抗体是抗PRRSV免疫的重要成分。前期研究显示部分PRRSV免疫攻毒猪可诱导广谱中和抗体，广谱中和表位可能与ORF2-6基因编码的囊膜蛋白密切相关。前人研究表明病毒囊膜蛋白多样性是诱导广谱中和抗体的重要条件。因此，本研究首先利用反向遗传操作构建含不同亚型毒株ORF2-6的嵌合病毒，并使用ORF2-6嵌合型PRRSV进行猪体接种，以明晰ORF2-6多样性在诱导PRRSV广谱中和抗体中的作用。其次通过同源/广谱中和抗体与PRRSV体外共培养，获得不同中和抗体耐受性突变株并测定其ORF2-6，以筛选广谱中和抗体潜在作用位点。最后再构建含相应位点突变病毒并测定不同中和抗体对其中和活性，以判定广谱中和表位关键位点及其多样性。本研究结果将为后续研制可诱导广谱中和抗体的PRRS疫苗提供理论基础。

猪繁殖与呼吸综合征病毒（PRRSV）遗传多样性高，但现有PRRS疫苗缺乏交叉保护。因此，研究诱导PRRSV交叉保护的策略与机制具有重要意义。中和抗体是抗PRRSV免疫的重要成分，部分PRRSV感染猪可产生广谱中和抗体。研究表明PRRSV广谱中和表位与ORF2-6基因编码的囊膜蛋白密切相关，但如何诱导PRRSV广谱中和抗体仍不明确。本项目首先以HP-PRRSV天然弱毒株感染性克隆为框架，成功构建拯救了含PRRSV2 ORF2-6基因共有序列的嵌合病毒（rJS-ORF2-6-CON）。其次通过动物接种与攻毒试验证实该嵌合病毒可以诱导广谱中和抗体并为猪体提供抵抗NADC30-like PRRSV2异源强毒的交叉保护作用。而商品化疫苗接种只能诱导同源中和抗体且无法为猪体提供足够的交叉保护。进一步分析表明rJS-ORF2-6-CON嵌合病毒与商品化疫苗诱导PRRSV特异性IFN-γ分泌型T细胞和IFN-γ水平相当，提示广谱中和抗体在提供交叉保护中发挥了关键作用。然后通过构建分别含有小囊膜蛋白或者主要囊膜蛋白编码基因共有序列的嵌合病毒（rJS-ORF2-4-CON和rJS-ORF5-6-CON），并进行动物接种与攻毒试验。结果表明这两株嵌合病毒均无法提供类似于rJS-ORF2-6-CON的良好交叉保护。提示小囊膜蛋白和主要囊膜蛋白在诱导异源中和抗体和提供交叉保护中很可能起协同作用。本项目研究成果可为后续进一步研制可诱导广谱中和抗体的广谱高效PRRS疫苗提供理论基础和技术保障。