VACCINE EFFICACY--CHARACTERIZATION OF IMMUNITY TO RSV

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靶点;然而，在自然感染后，免疫是不完全的。 尽管中和抗体水平很高，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融合，从而允许病毒在细胞间传播。 这些发现表明，感染后观察到的B细胞反应可能缺乏阻止病毒-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，开始了研究以更完整地定义RSV使用的受体。使用肝素琼脂糖亲和层析评价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。RSV/B cp 52株表达F糖蛋白，但缺乏G和SH，用于显示F-肝素结合不依赖于G。此外，在肝素、牛肺肝素和脱N硫酸化肝素存在下，Vero细胞的cp 52感染减少，并且这种抑制是剂量依赖性的。这些结果表明RSV-F-硫酸肝素相互作用在病毒生物学中很重要。 我们确定了线性F序列负责肝素结合使用重叠的F肽。评价这些肽在肝素琼脂糖亲和层析中结合的能力，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A和B菌株以及菌株cp 52感染的能力。可溶性肝素和牛肺肝素特异性抑制与细胞的结合，表明这种相互作用对细胞表面糖胺聚糖具有特异性。这些研究鉴定了RSV亚组F内结合肝素的线性序列。 该实验室的工作还表明，RSV F、G和SH在感染细胞表面的复合物中表达。有趣的是，F-G-SH复合物对肝素的亲和力大于单独的F或G糖蛋白的亲和力。 这些结果可以部分解释当肝素和硫酸肝素衍生物用于抑制RSV感染时观察到的不完全抑制。 此外，我们与OBRR的同事合作，确定了主要的人中和抗体应答是同种型IgG 1，并鉴定了RSV F和G上可被该人抗体亚类识别的线性结构域。

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 targets of a protective immune response; however, following natural infection, immunity is incomplete. Repeated infections with viruses of the 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, allowing cell-to-cell spread of virus. These findings suggest that B cell responses seen following infection may be deficient in antibodies that prevent virus- receptor interactions. In order to better understand the 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. Strain RSV/B cp 52, which expresses F glycoprotein but lacks G and SH, was used to show that F-heparin binding was independent of G. In addition, cp52 infection of Vero cells was diminished in the presence of heparin, Bovine lung heparin and de-N sulfated heparin and this inhibition was dose-dependent. These results indicate that RSV-F-heparin sulfate interactions are important in the biology of the virus. We identified linear F sequences responsible for heparin binding using overlapping 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 with A and B strains as well as strain cp52. Binding to cells was specifically inhibited by soluble heparin and bovine lung heparin, suggesting that the interaction was specific for cell surface glycosaminoglycans. These studies identified a linear sequence within RSV subgroup F that bound heparin. Work in this laboratory has also shown that RSV F,G and SH are expressed in a complex on the surface of infected cells. Interestingly, the affinity of the F-G-SH complex for heparin is greater than the affinity of F or G glycoproteins alone. These results may explain, in part, the incomplete inhibition observed when heparin and heparin sulfate derivatives are used to inhibit RSV infection. In addition, working with colleagues in OBRR, we have determined that the dominant human neutralizing antibody response is isotype IgG1 and identified linear domains on RSV F and G recognized by this subclass of human antibody.