Structure, function, and antigenicity of emerging henipavirus surface glycoproteins

摘要 亨尼帕病毒(HNV)属，以最早发现的两个成员亨德拉病毒(HEV)和尼帕病毒(Nipah)命名 (NIV)，是一组不断扩大的人畜共患病病毒，曾多次暴发，病死率很高 达到75%。异常广泛的物种趋向性和不同的传播途径使HNV有可能 潜在的未来大流行。HEV和NIV都被归类为生物安全级别4(BSL-4) 跨界药物和NIAID C类优先病原体。年报告了六种新出现的HNV 近年来，与新城疫和戊型肝炎病毒的抗原性交叉反应有限。糖蛋白F和G是两种 仅HNV表面的尖峰信号，通过G糖蛋白介导的受体协调病毒进入过程 病毒与宿主细胞之间附着后F糖蛋白介导的膜融合。G和 F蛋白是HNV中和抗体的靶标。疫苗和单抗(MAb) 针对这两种HNV糖蛋白的对策研究迫在眉睫 为潜在的HNV溢出事件做好准备的重要性。HNV G糖蛋白的胞外区是一个同源的 四聚体，每个原始体由一个球形头部和一个柄区域组成。亚稳态四聚体 构象限制了以前G蛋白的结构特征仅限于单体头部， 这是全面了解HNV G蛋白介导的机制的主要障碍 进入和抗体识别。HNV F糖蛋白三聚体从输注前过渡到融合后 病毒进入过程中的构象。可以较好地识别亚稳态预融合体构象 通过中和抗体，从而成为疫苗设计和治疗开发的一个有希望的目标。而当 HEV和NIV的糖蛋白都得到了广泛的研究，新出现的HNV的糖蛋白， 它们在基因和抗原性上与两种典型的HNV截然不同，在 他们自己，还没有得到很好的调查。我们用合理的设计创造了溶解的、稳定的、 来自几种新出现的HNV的寡聚糖蛋白胞外结构域，包括两个在系统发育上相距较远的 蝙蝠携带的HNV，CedPV和AngV，以促进结构、功能和抗原性鉴定。我们 建议使用这些糖蛋白的结构和基于结构的功能分析来辅助描述 HNV糖蛋白介导的进入功能。我们还将确定这些HNV糖蛋白的抗原性 用中和抗体，包括常规的小鼠抗体和骆驼单域抗体，以 从结构和功能上研究它们的中和表位及其作用机制。联合抗原性 结果将给出一个全面的G和F糖蛋白易损部位的定义 新兴的HNV。总而言之，从这个项目得出的结果将为HNV进入机制提供见解，如 并为未来设计基于包膜糖蛋白的疫苗和免疫治疗提供信息 新兴的HNV。

Abstract Henipavirus (HNV) genus, named after the first two identified members, Hendra virus (HeV) and Nipah virus (NiV), is a group of expanding zoonotic viruses that have caused repeated outbreaks with case fatality rate reaching 75%. The exceptional broad species tropism and various transmission routes make HNV a risk of potential future pandemics. Both HeV and NiV have been categorized as Biological Safety Level-4 (BSL-4) transboundary agents, and NIAID Category C Priority Pathogens. There are six emerging HNVs reported in recent years, showing limited antigenic cross-reactivity with NiV and HeV. Glycoproteins F and G are the two only spikes on the HNV surface, which coordinate the viral entry process via G glycoprotein-mediated receptor attachment followed by the F glycoprotein-mediated membrane fusion between virus and host cell. Both G and F proteins are the targets of HNV-neutralizing antibodies. Vaccine and monoclonal antibody (mAb) countermeasure development focusing on these two HNV glycoproteins are now of critical and urgent importance to prepare for potential HNV spillover events. The ectodomain of HNV G glycoprotein is a homo- tetramer with each protomer composed of a globular head and a stalk region. The metastable tetrameric conformation has restricted the previous structural characterization of G protein to the monomeric head only, which is a major obstacle for a comprehensive understanding of the G protein-mediated mechanisms of HNV entry and antibody recognition. HNV F glycoprotein trimer transits from the prefusion to the post-fusion conformation during viral entry process. The metastable prefusion conformation can be preferably recognized by neutralizing antibodies, whereby a promising target for vaccine design and therapeutic development. While the glycoproteins of both HeV and NiV have been extensively studied, the glycoproteins of the emerging HNVs, which are genetically and antigenically distinct from the two prototypic HNVs, also extremely diverse among themselves, have not been well investigated. We have used rational designs to created soluble, stabilized, oligomeric glycoprotein ectodomain constructs from several emerging HNVs, including two phylogenically distant bat-borne HNVs, CedPV and AngV, to facilitate structural, functional and antigenicity characterization. We propose to use structural and structure-based functional analyses of these glycoproteins to assist delineation of the function of HNV glycoprotein-mediated entry. We will also define the antigenicity of these HNV glycoproteins with neutralizing antibodies, including conventional murine antibodies and camelid single-domain antibodies, to investigate their neutralization epitopes and mechanisms structurally and functionally. The combined antigenicity results will render a comprehensive definition of sites of vulnerability on both G and F glycoproteins of the emerging HNVs. Collectively, findings derived from this project will provide insights to HNV entry mechanism, as well as inform future work on designing envelope glycoprotein-based vaccine and immunotherapeutic against emerging HNVs.