HIV neutralization and mechanisms of cellular entry

了解三聚体表面糖蛋白的分子结构和描述传播机制是设计有效的免疫原和治疗剂以对抗艾滋病毒/艾滋病、流感、埃博拉和相关包膜病毒的核心。这些被包裹的病毒中的大多数都有相似的病毒进入机制，因此，对这些病毒的结构研究可能为所有这三种病毒的疫苗设计提供洞察力。在过去的一年里，我们继续在实现这些目标方面取得重大进展。在我们关于HIV的工作中，我们继续改进断层扫描方法，以更高的分辨率原位确定病毒表面包膜糖蛋白尖峰的3D结构。我们最令人兴奋的新结果来自于比较利用CXCR4或CCR5共受体的病毒株之间的结构差异，这些病毒株要么是原始株，要么是实验室适应株。这些结果正在准备出版。我们去年取得的重要进展之一是在广泛中和流感抗体方面取得了进展。与流感有关的儿童死亡中，相当大一部分是由于感染B型流感病毒，这种病毒作为两种不同的抗原谱在人类群体中共同传播，由免疫优势受体结合蛋白血凝素定义。虽然已经描述了针对B型流感病毒血凝素的广泛交叉反应的保护性单抗，但尚未有针对第二丰富的病毒糖蛋白神经氨酸酶的报道。在《自然微生物学》上发表的一篇论文中，我们分析了一组五种小鼠抗神经氨酸酶的单抗，这些抗体表现出广泛的结合、神经氨酸酶抑制、体外抗体依赖的细胞介导的细胞毒性和体内对B型流感病毒的保护作用，这些病毒既属于血凝素谱系，又跨越了70多年的抗原漂移。对两个神经氨酸酶-抗体复合体的电子显微镜分析表明，保守的神经氨酸酶表位位于分子的头部，它们与酶的活性部位不同。在小鼠模型中，一种治疗性剂量的抗体1F2比目前的治疗标准奥司他韦更具保护作用，奥司他韦每天两次，连续六天。埃博拉病毒是一种新出现的病原体，已成为疫苗和治疗开发的关键目标。埃博拉病毒在成熟的病毒粒子表面显示出单一复合体--包膜糖蛋白的多个副本。我们的结构研究涉及使用ZMapp抗体鸡尾酒，它由c2G4、c4G7和c13C6抗体组成，与来自西非2014分离株的天然全长埃博拉包膜糖蛋白结合，嵌入丝状病毒样颗粒中。这些研究表明，这些抗体中的每一种都与埃博拉糖蛋白尖峰的不同区域结合，并为病毒进入机制提供了新的见解，并为阻止这一步骤提供了策略。

Knowledge of the molecular structure of trimeric surface glycoproteins and delineating the mechanisms of transmission are central to the design of effective immunogens and therapeutic agents to combat HIV/AIDS, influenza, Ebola and related enveloped viruses. Most of these enveloped viruses share similar mechanisms for viral entry, and as such, structural studies of these viruses may offer insight towards vaccine design for all three of these viruses. We have continued to make significant progress towards these goals over the last year. In our work on HIV, we continue to refine tomographic methods to determine the 3D structure of envelope glycoprotein spikes in situ on the virus surface at pregoressively higher resolutions. Our most exciting new results are from comparing the differences in structure between viral strains that utilize CXCR4 or CCR5 co-receptors, and are either primary or lab-adapted strains. These results are being prepared for publication. One of the important advances we made last year is progress towards broadly neutralizing antibodies against influenza. A substantial proportion of influenza-related childhood deaths are due to infection with influenza B viruses, which co-circulate in the human population as two antigenically distinct lineages defined by the immunodominant receptor binding protein, haemagglutinin. While broadly cross-reactive, protective monoclonal antibodies against the haemagglutinin of influenza B viruses have been described, none targeting the neuraminidase, the second most abundant viral glycoprotein, have been reported. In a paper published in Nature Microbiology, we analyzed a panel of five murine anti-neuraminidase monoclonal antibodies that demonstrate broad binding, neuraminidase inhibition, in vitro antibody-dependent cell-mediated cytotoxicity and in vivo protection against influenza B viruses belonging to both haemagglutinin lineages and spanning over 70 years of antigenic drift. Electron microscopic analysis of two neuraminidase-antibody complexes shows that the conserved neuraminidase epitopes are located on the head of the molecule and that they are distinct from the enzymatic active site. In the mouse model, one therapeutic dose of antibody 1F2 was more protective than the current standard of treatment, oseltamivir, given twice daily for six days. The Ebola virus is an emerging pathogen that has become a critical target for vaccine and therapeutic development. Ebola displays many copies of a single complex, the envelope glycoprotein, on the surface of mature virions. Our structural studies have involved using the Zmapp antibody cocktail, which is composed of the c2G4, c4G7 and c13C6 antibodies, bound to native, full-length Ebola envelope glycoprotein from the West African 2014 isolate embedded in filamentous viral-like particles. These studies have revealed that each of these antibodies binds to different regions of the Ebola glycoprotein spikes, and are providing new insights into mechanisms of viral entry and inform strategies for blocking this step.