Targeting glycoprotein (G) domain-III for pan-lyssavirus nanobody therapeutics

摘要 致命性狂犬病脑炎是由人畜共患病溶酶病毒属的成员引起的。高潮 遗传多样性将溶病病毒成员分成三个系统群，而跨系统群(尤其是 种子组2和3)当前暴露后预防(PEP)或 抗体疗法。在全球范围内实现更广泛有效的狂犬病对策，并 为新的溶血病毒的出现做好准备，针对所有系统群的有效治疗药物是 这是必要的。溶酶病毒G糖蛋白是病毒粒子表面唯一的抗体靶点，它采用了截然不同的 与其他III类病毒融合机制一样，融合前和融合后状态之间的构象。领域-III，共 溶酶病毒G糖蛋白在天然状态下包含一个大的中和抗体可及的表面。 结构域III上的序列分析显示，所有系统群中都有几个保守的斑块，这可能 有可能成为泛溶血病毒中和剂的靶位。抗原特异性纳米抗体已经被 被认为是治疗各种传染病和非传染性疾病的有前途的治疗剂， 它们在结合常规方法无法达到的紧凑和隐藏的表位方面具有优势 抗体。我们假设，通过将免疫识别集中在溶酶病毒的G结构域-III上， 根据表位，我们可以识别泛溶血病毒中和纳米抗体，用于免疫治疗。在这项研究中，我们 提出两个具体目标：(1)使用基于结构和抗体引导的方法来设计和表征 溶酶病毒糖蛋白结构域-III的抗原性，该结构域是几个广泛靶向的病毒脆弱部位 中和抗体，用于揭示抗体中和机制；(2)鉴定和表征 泛溶酶病毒G结构域免疫识别技术中和治疗用纳米抗体 III保守表位。

Abstract The lethal rabies encephalitis diseases are caused by members of the zoonotic lyssavirus genus. The high genetic diversity separates lyssavirus members into three phylogroups, while cross-phylogroup (especially to phylogroups 2 and 3) protection has not been established by current post-exposure prophylaxis (PEP) or antibody therapeutics. To achieve more broadly effective countermeasures to rabies disease worldwide and to prepare for the emergence of new lyssaviruses, effective therapeutic agents against all phylogroups are necessary. Lyssavirus G glycoprotein is the sole antibody target on the virion surface, which adopt distinct conformations between pre- and post- fusion states as other class-III viral fusion machineries. Domain-III of lyssavirus G glycoprotein encompasses a large neutralizing antibody-accessible surface in the native state. Sequence analysis on domain-III revealed several conserved patches across all phylogroups, which could potentially serve as target epitopes for pan-lyssavirus neutralizers. Antigen-specific nanobodies have been considered as promising therapeutic agents against various infectious diseases and non-infectious diseases, which have the advantage in binding compact and hidden epitopes that are out of reach for conventional antibodies. We hypothesized that by focusing immune recognition on the lyssavirus G domain-III conserved epitopes, we can identify pan-lyssavirus neutralizing nanobodies for immunotherapeutics. In this study, we propose two specific aims: (1) to use a structure-based and antibody-guided approach to design and characterize the antigenicity of lyssavirus glycoprotein domain-III, a site of viral vulnerability targeted by several broadly neutralizing antibodies, for revelation of the antibody neutralization mechanism; (2) to identify and characterize pan-lyssavirus neutralizing nanobodies for therapeutics by focusing immune recognition on lyssavirus G domain III conserved epitopes.