Vaccine Efficacy: Characterization of the Protective Im

摘要：呼吸道合胞病毒是全球幼儿下呼吸道疾病的最常见原因。 已知该病毒的F和G糖蛋白是保护性免疫应答的主要靶标，然而，在自然感染后，免疫是不完全的，并且尽管中和抗体水平高，但仍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，从而允许细胞间传播。 这些发现表明，感染后观察到的B细胞反应可能缺乏阻断特异性病毒-受体相互作用的抗体。 为了更好地理解感染所需的病毒-细胞相互作用，开始了研究以更完整地定义RSV所使用的受体。使用肝素琼脂糖亲和层析法评估病毒蛋白与细胞表面糖胺聚糖的结合。使用完整病毒体、感染细胞裂解物、纯化包膜糖蛋白和表达单个RSV F或G糖蛋白的牛痘重组病毒裂解物的研究表明，F和G均结合肝素。此外，感染的细胞而不是病毒体表达膜相关糖蛋白F-G-SH的寡聚复合物，其结合亲和力比单独的F或G更高。 在过去的一年中，我们确定了几个线性的F序列负责肝素结合使用重叠，合成的F肽。评价这些肽在肝素琼脂糖亲和层析中的结合能力，它们与Vero、Hep-2和A549细胞结合的能力，阻断病毒附着的能力和抑制亚群A和B菌株感染细胞的能力。可溶性肝素和牛肺肝素特异性抑制与细胞的结合。在过去的一年中，我们还使用FACS分析比较了肽与未处理的细胞或用GAG裂解酶处理的细胞的结合。 酶处理后，F和G肝素结合结构域肽的结合被消除，如结合每种肽的细胞百分比降低和中值荧光强度降低所示。与硫酸肝素缺陷突变CHO细胞系的结合相对于亲本细胞减少，并且在氯酸盐处理后也减少。 在Vero细胞上的竞争结合研究表明，纯化的病毒阻断F和G肝素结合域肽的结合。相反，没有看到相互竞争，过量的肽不会阻断纯化的病毒体的结合。总之，这些研究表明，线性F和G肝素结合结构域肽特异性地附着于细胞表面糖胺聚糖，并且结合促进感染。竞争性结合研究还表明，RSV结合细胞表面分子而不是GAGS，F-肝素结合是附着后事件，病毒体结合细胞GAGS的亲合力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 specific 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. Furthermore, infected cells but not virions expressed and oligomeric complex of membrane associated glycoproteins F-G-SH that bound with higher affinity than either F or G alone. Over the past year we identified several 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. Binding to heparin sulfate deficient mutant CHO cell lines was decreased relative to the parental cells and was also diminished following chlorate treatment. Competitive binding studies on Vero cells showed that purified virus blocked 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 and that binding facilitates infection. Competitive binding studies also suggested that RSV binds to cell surface molecules other than GAGS, that F-heparin binding is a post-attachment event and that avidity of virion binding to cellular GAGS may be greater than peptide binding alone.