麻疹病毒P蛋白特异IgA抗体的胞内双重抗病毒分子机制

The immunoglobulin A (IgA) provides the initial specific immunological protection against most viral infections in relation to its transportation through mucosal epithelial cells. IgA is the most important effector in mucosal antiviral front line because most viruses enter the body through mucosal surfaces, replicates within epithelial cells, and then spreads to surrounding cells and tissues. Our previous and recent works demonstrated that IgA antibody has multiple antiviral functions in relation to epithelium, the viral internal matrix protein(M)-specific IgA antibody can indeed inhibit measles virus replication within epithelial cell efficiently. However, the detailed molecular mechanism is still unknown. The present project will focus on investigating the intracellular inhibition of virus replication by IgA antibody against viral non-surface phosphoprotein (P). Based on the P protein as an indispensable cofactor of viral RNA-dependent RNA polymerase (RdRp) and an antagonist against cellular interferon pathway during early viral infection, we will try to elucidate dual molecular mechanisms of intracellular antiviral function of IgA antibody against P protein by addressing following two questions from both viral and cellular tiers: First, how IgA specifically targets viral P protein to inhibit viral genome replication, gene expression and viral assembly? Second, whether and how IgA can disable the viral ability of inhibiting the host cellular interferon pathway, and maintain cellular innate immune activity against viral infection? The answers of these questions will advance our understanding of the IgA antiviral mechanisms that will be benefit to the design of new antiviral vaccine, antibody drug and more efficient antiviral strategies.

IgA抗体通过黏膜上皮细胞转运到黏膜表面，为机体提供黏膜局部特异免疫保护，是机体抗病毒防线的重要效应因子。我们的早期工作证明了IgA抗体抗病毒功能的多种方式，近年又发现了麻疹病毒基质蛋白M特异的IgA抗体可有效抑制细胞内病毒复制，确证了IgA细胞内中和病毒的独特作用，但相关细胞内中和的分子机制不详。本申请拟以麻疹病毒磷蛋白P为靶标，重点研究病毒非表面蛋白特异IgA抗体在细胞内对病毒复制的抑制效应。根据P蛋白是病毒复制必须的RdRp辅助因子，且在病毒感染早期作用于细胞干扰素通路抑制细胞固有免疫抗病毒效能，本研究将从病毒和细胞两个层次探索P蛋白特异IgA抗体胞内双重抗病毒分子机制。一，分析IgA抗体如何靶向控制病毒基因组复制、基因表达和组装；二、阐明IgA抗体能否以及如何拮抗病毒对干扰素通路的抑制，从而维护细胞固有免疫抗病毒的能力。本项目将为新型抗病毒疫苗和抗体药物设计提供理论依据和全新方案。

免疫球蛋白A（IgA）具有通过转胞吞作用(transcytosis)贯穿上皮细胞转运到黏膜腔面的能力，因此有机会在病毒复制过程中与所有病毒成分相互作用，从而发挥细胞内中和病毒抑制病毒复制的能力。我们的前期研究表明，IgA抗体具有多种黏膜上皮细胞内相关的抗病毒功能，例如，病毒基质蛋白（M）特异性IgA抗体确实能有效抑制上皮细胞内的麻疹病毒（MV）复制。本项目进一步研究了MV非表面磷蛋白（P）特异的IgA抗体在细胞内抑制病毒复制的效应和机制。我们筛选并得到了两株鼠单克隆IgA抗体，anti-P 1D11-IgA和anti-P 7F1-IgA。采用trans-well细胞培养和MV感染系统，我们证明，anti-P 1D11-IgA能够在MV感染的极化Vero-pIgR细胞中与P相互作用并抑制病毒复制。揭示了IgA能够通过与病毒P蛋白胞内的相互作用，阻断病毒P-N复合体的形成和RdRp的功能，从而抑制病毒复制的机制。另一方面，我们发现anti-P 7F1-IgA只能在Caco-2细胞中抑制MV的复制，而不能在干扰素缺陷的Vero-IgR细胞中抑制病毒复制。进一步研究表明，在MV感染的Caco-2细胞中，7F1-IgA的转运能增强干扰素-β（IFN-β）的产生和IFN刺激基因的表达，使Caco-2细胞具有更高的抗病毒效力。7F1-IgA能够在MV 感染的 Caco-2细胞内特异地与P相互作用，拮抗P对JAK1和STAT1磷酸化的抑制，从而保证细胞JAK1和STAT1能够及时有效地磷酸化，并高效迁移入核，有效激活I型干扰素通路。因此，我们揭示了P特异的IgA抗体可以拮抗病毒逃逸I型干扰素通路，保证上皮细胞有效的先天免疫抗病毒活性。总之，这些发现揭示了IgA可以通过多种机制针对病毒关键的保守性靶点发挥独特的抗病毒效应。本项目研究结果拓宽了我们对病毒免疫靶标及IgA独特抗病毒机制的认识，为新型抗病毒疫苗和抗体药物设计提供了新的靶点和策略。