Department of Microbiology and Immunology, University of North Carolina School of Medicine, Chapel Hill, NC, USA.
Department of Epidemiology, University of North Carolina School of Public Health, Chapel Hill, NC, USA.
Sci Rep. 2017 Jul 3;7(1):4524. doi: 10.1038/s41598-017-04767-6.
Zika virus (ZIKV) and the 4 dengue virus (DENV) serotypes are mosquito-borne Flaviviruses that are associated with severe neuronal and hemorrhagic syndromes. The mature flavivirus infectious virion has 90 envelope (E) protein homo-dimers that pack tightly to form a smooth protein coat with icosahedral symmetry. Human antibodies that strongly neutralize ZIKV and DENVs recognize complex quaternary structure epitopes displayed on E-homo-dimers and higher order structures. The ZIKV and DENV E protein expressed as a soluble protein is mainly a monomer that does not display quaternary epitopes, which may explain the modest success with soluble recombinant E (sRecE) as a vaccine and diagnostic antigen. New strategies are needed to design recombinant immunogens that display these critical immune targets. Here we present two novel methods for building or stabilizing in vitro E-protein homo-dimers that display quaternary epitopes. In the first approach we immobilize sRecE to enable subsequent dimer generation. As an alternate method, we describe the use of human mAbs to stabilize homo-dimers in solution. The ability to produce recombinant E protein dimers displaying quaternary structure epitopes is an important advance with applications in flavivirus diagnostics and vaccine development.
Zika 病毒 (ZIKV) 和 4 种登革热病毒 (DENV) 血清型都是蚊媒传播的黄病毒，与严重的神经元和出血综合征有关。成熟的黄病毒感染性病毒粒子有 90 个包膜 (E) 蛋白同源二聚体，紧密包装形成具有二十面体对称性的光滑蛋白衣壳。强烈中和 ZIKV 和 DENVs 的人抗体识别 E-同源二聚体和更高阶结构上展示的复杂四级结构表位。作为可溶性蛋白表达的 ZIKV 和 DENV E 蛋白主要是单体，不显示四级表位，这可能解释了可溶性重组 E (sRecE) 作为疫苗和诊断抗原的效果并不理想。需要新的策略来设计展示这些关键免疫靶标的重组免疫原。在这里，我们提出了两种构建或稳定体外 E 蛋白同源二聚体以显示四级表位的新方法。在第一种方法中，我们将 sRecE 固定化以实现随后的二聚体生成。作为替代方法，我们描述了使用人单克隆抗体在溶液中稳定同源二聚体的方法。能够产生展示四级结构表位的重组 E 蛋白二聚体是在黄病毒诊断和疫苗开发中有应用的重要进展。
