The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity
The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity
复制标题
DOI：
10.1371/journal.pntd.0000739
复制
发表时间：
2010-07-01
影响因子：
3.8
通讯作者：
Blair, Carol D.
中科院分区：
文献类型：
--
作者：
Hunt, Ann R.;Frederickson, Shana;Blair, Carol D.
背景资料：委内瑞拉马脑炎病毒（VEEV）是导致南美洲和中美洲以及美国发生的VEE流行病的原因。VEEV包膜含有两种糖蛋白E1（介导细胞膜融合）和E2（结合受体和elevinavirus中和抗体）。以前，我们构建了E1和E2表位地图使用鼠单克隆抗体（单克隆抗体）。六个E2表位（E2（c，d，e，f，g，h））结合VEEV中和抗体并定位于氨基酸（aa）182-207。关于VEEV的人抗体库或与人病毒中和抗体接合的表位还不清楚。VEE没有特异性治疗;然而，病毒中和mAb是通过外周或气雾剂途径用VEEV激发的小鼠的有效保护剂和治疗剂。因此，完全人单克隆抗体（hMAbs）的病毒中和活性，应是有用的预防或临床治疗的人VEE。方法：我们使用噬菌体展示分离VEEV特异性hFab从人骨髓供体。通过测序、特异性测试、使用间接ELISA的VEEV亚型交叉反应性和体外病毒中和能力对这些hFab进行了表征。一个E2-特异性中和hFAb，F5 n，转化为IgG，其结合位点进行了鉴定，使用竞争性ELISA与单克隆抗体，并通过制备和测序抗体中和逃逸variants.Findings：使用11 VEEV-反应hFab，我们构建了第一个人的甲病毒表面蛋白E1和E2的表位图。我们确定了一个重要的中和相关表位独特的人类免疫反应，E2 aa 115 -119。使用9埃分辨率的辛德毕斯病毒E2蛋白的冷冻电子显微镜图，我们显示了这个人VEEV epitope.Conclusions的可能的表面位置：的VEEV中和能力的hMAb F5 nIgG是类似的人源化mMAb Hy 4 IgG所表现出的。Hy 4 IgG已显示在预防和治疗上限制小鼠中的VEEV感染。给予结合不同E2表位的F5 n和Hy 4 IgG的混合物可以为VEEV提供增强的预防或免疫治疗，同时降低体内产生可能有害的病毒中和逃逸变体的可能性。
Background: Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2(c,d,e,f,g,h)) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.Methods: We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.Findings: Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 angstrom resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.Conclusions: The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.