Cryo-Electron Microscopy Study of Insect Cell-Expressed Enterovirus 71 and Coxsackievirus A16 Virus-Like Particles Provides a Structural Basis for Vaccine Development

Enterovirus 71 (EV71) and coxsackievirus A16 (CA16) are the two most common etiological agents responsible for the epidemics of hand, foot, and mouth disease (HFMD), a childhood illness with occasional severe neurological complications. A number of vaccine candidates against EV71 or CA16 have been reported; however, no vaccine is currently available for clinical use. Here, we generated a secreted version of EV71 and CA16 virus-like particles (VLPs) using a baculovirus-insect cell expression system and reconstructed the three-dimensional (3D) structures of both VLPs by cryo-electron microscopy (cryo-EM) single-particle analysis at 5.2-angstrom and 5.5-angstrom resolutions, respectively. The reconstruction results showed that the cryo-EM structures of EV71 and CA16 VLPs highly resemble the recently published crystal structures for EV71 natural empty particles and CA16 135S-like expanded particles, respectively. Our cryo-EM analysis also revealed that the majority of previously identified linear neutralizing epitopes are well preserved on the surface of EV71 and CA16 VLPs. In addition, both VLPs were able to induce efficiently neutralizing antibodies against various strains of EV71 and CA16 viruses in mouse immunization. These studies provide a structural basis for the development of insect cell-expressed VLP vaccines and for a potential bivalent VLP vaccine against both EV71-and CA16-associated HFMD.

肠道病毒71型（EV71）和柯萨奇病毒A16型（CA16）是引起手足口病（HFMD）流行的两种最常见病原体，手足口病是一种儿童疾病，偶尔会出现严重的神经系统并发症。已有许多针对EV71或CA16的候选疫苗被报道；然而，目前尚无疫苗可用于临床。在此，我们利用杆状病毒 - 昆虫细胞表达系统制备了分泌型的EV71和CA16病毒样颗粒（VLPs），并通过冷冻电镜（cryo - EM）单颗粒分析分别以5.2埃和5.5埃的分辨率重构了这两种VLPs的三维（3D）结构。重构结果显示，EV71和CA16的VLPs的冷冻电镜结构分别与最近发表的EV71天然空颗粒和CA16的135S样膨胀颗粒的晶体结构高度相似。我们的冷冻电镜分析还表明，大多数先前确定的线性中和表位在EV71和CA16的VLPs表面保存良好。此外，在小鼠免疫中，这两种VLPs都能有效诱导针对各种EV71和CA16病毒株的中和抗体。这些研究为开发昆虫细胞表达的VLP疫苗以及针对与EV71和CA16相关的手足口病的潜在二价VLP疫苗提供了结构基础。