Immune Response to Respiratory Syncytial Virus

摘要：呼吸道合胞病毒是全世界幼儿下呼吸道疾病最常见的病因。已知该病毒的F和G糖蛋白是保护性免疫反应的主要靶点，然而，在自然感染后，免疫是不完整的，尽管具有高水平的中和抗体，但仍会发生同源或异源亚型病毒的重复感染。此外，中和细胞外病毒的抗体可能无法抑制感染细胞的融合，从而导致细胞间传播。这些发现表明，感染后的B细胞反应可能缺乏阻断病毒受体相互作用的抗体。为了更好地了解感染所需的病毒-细胞相互作用，研究人员开始更完整地定义RSV使用的受体。采用肝素琼脂糖亲和层析法评价病毒蛋白与细胞表面糖胺聚糖的结合。利用整个病毒粒子、感染细胞裂解物、纯化包膜糖蛋白和表达RSV F或G糖蛋白的痘苗重组病毒裂解物进行的研究表明，F和G都能与肝素结合。在此之前，我们使用重叠的合成F肽确定了负责肝素结合的线性F序列。这些肽在肝素琼脂糖亲和层析中结合的能力，与Vero， Hep-2和A549细胞结合的能力，阻断病毒附着的能力以及抑制亚群A和B株细胞感染的能力进行了评估。可溶性肝素和牛肺肝素特异性抑制细胞结合。在过去的一年中，我们还使用FACS分析比较了肽与未经处理的细胞或经GAG裂解酶处理的细胞的结合。酶处理后，结合F和G肝素结合结构域肽的细胞百分比下降，荧光强度中位数下降，表明F和G肝素结合结构域肽的结合被取消。对Vero细胞的竞争性结合研究表明，纯化后的病毒可以阻断F和G肝素结合区域肽的结合。相反，没有看到相互竞争，多余的肽不会阻止纯化病毒粒子的结合。综上所述，这些研究表明线性F和G肝素结合域肽特异性地附着在细胞表面的糖胺聚糖上。竞争性结合研究还表明，RSV与细胞表面分子而非GAGS结合，肝素结合是附着后事件，或者病毒粒子与细胞GAGS结合的渴望度可能大于单独的肽结合。

Summary: Respiratory syncytial virus is the most common cause of lower respiratory tract disease in young children worldwide. The F and G glycoproteins of this virus are known to be the major targets of a protective immune response, however, following natural infection, immunity is incomplete and repeated infections with viruses of homologous or heterologous subtypes occur despite high levels of neutralizing antibody. In addition, antibodies that neutralize extracellular virus may not be able to inhibit fusion of infected cells, allowing cell-to-cell spread. These findings suggest that B cell responses seen following infection may be deficient in antibodies that block virus-receptor interactions. In order to better understand virus-cell interactions required for infection, studies were initiated to more completely define the receptor(s) used by RSV. The binding of viral proteins to cell surface glycosaminoclycans was evaluated using heparin agarose affinity chromatography. Studies using whole virions,infected cell lysates, purified envelope glycoproteins, and lysates of vaccinia recombinant viruses expressing individual RSV F or G glycoprotein, showed that both F and G bind heparin. Previously, we identified linear F sequences responsible for heparin binding using overlapping, synthetic F peptides. These peptides were evaluated for their ability to bind in heparin agarose affinity chromatography, their ability to bind to Vero, Hep-2 and A549 cells, ability to block virus attachment and capacity to inhibit infection of cells with subgroup A and B strains. Binding to cells was specifically inhibited by soluble heparin and bovine lung heparin. During the past year we have also compared binding of peptides to untreated cells or to cells treated with GAG lysases using FACS analysis. Binding of F and G heparin-binding domain peptides was abrogated following enzyme treatment as indicated by both a decrease in the percentage of cells binding each peptide and a decrease in the median flourescent intensity. Competitive binding studies on Vero cells showed that purified virus could block binding of F and G heparin binding domain peptides. In contrast, reciprocal competition was not seen and peptide in excess did not block binding of purified virions. Taken together, these studies suggest that linear F and G heparin binding domain peptides attach specifically to cell surface glycosaminoglycans. Competive binding studies also suggested that RSV binds to cell surface molecules other than GAGS, that heparin binding is a post-attachment event or that avidity of virion binding to cellular GAGS may be greater than peptide binding alone.