VACCINE EFFICACY: CHARACTERIZATION OF THE PROTECTIVE IMMUNE RESPONSE TO RESPIRAT

呼吸道合胞病毒是全世界幼儿下呼吸道疾病的最常见病因。已知该病毒的F和G糖蛋白是保护性免疫反应的目标；然而，在自然感染后，免疫是不完整的。尽管有高水平的中和抗体，同源或异源亚型病毒的重复感染仍会发生。此外，中和细胞外病毒的抗体可能无法抑制融合，从而使病毒在细胞间传播。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，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。利用整个病毒粒子和感染细胞裂解物进行的研究表明，F糖蛋白和G糖蛋白都能与肝素结合。利用表达F糖蛋白但缺乏G和SH的RSV/B cp52菌株，证明F-肝素与G的结合不依赖于G。此外，肝素、牛肺肝素和去n硫酸肝素存在时，cp52对Vero细胞的感染减弱，且这种抑制作用呈剂量依赖性。这些结果表明，rsv - f -硫酸肝素相互作用在病毒的生物学中很重要。为了鉴定负责肝素结合的线性F序列，在肝素琼脂糖亲和层析中评估了重叠F肽的结合能力。这些研究确定了RSV亚群a - F与肝素结合的线性序列。我们计划评估这些肽与Vero、Hep-2和A549细胞结合的能力，并评估这些肽限制A2和cp52等菌株感染的能力。该实验室的工作还表明，RSV F、G和SH在感染细胞表面以复合物的形式表达。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。这些结果可能部分解释了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，与OBRR的同事合作，我们已经确定了主要的人类中和抗体反应是IgG1同型。人IgG1与整个病毒粒子反应，与感染细胞表面表达的F和G以及纯化的RSV F和G结合，目前正在研究确定RSV F和G上被人中和抗体识别的线性和构象位点。

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 and infected cell lysates showed that both F and G glycoproteins bind to 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. In order to identify linear F sequences responsible for heparin binding, overlapping F peptides were evaluated for their ability to bind during heparin agarose affinity chromatography. These studies identified a linear sequence on RSV subgroup A F that bound to heparin. We plan to evaluate the ability of these peptides to bind to Vero, Hep-2 and A549 cells and assess the ability of the peptides to limit infection with strains such as A2 and cp52. 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 of the IgG1 isotype. Human IgG1 reacts with whole virions and binds to F and G expressed on the surface of infected cells as well as purified RSV F and G. Studies are in progress to determine linear and conformational sites on RSV F and G recognized by human neutralizing antibody.