Eliciting antibodies to probe native trimeric gp41 for HIV vaccine design

目前的HIV候选疫苗缺少的一个关键组成部分是引发广泛中和抗体的能力。在拟议的研究中，我们将疫苗设计、下一代测序（NGS）生物信息学、结构生物学和机制研究结合起来，克服HIV疫苗开发中的一个重要障碍，即激发识别gp41天然构象的抗体。gp41的膜近端外区（MPER）是一个特别理想的疫苗靶点，因为它被许多广泛中和的抗体识别，其中最有效的是10E8。然而，抗体如何被诱导到MPER实际上是未知的。同时，初步数据表明10E8表位涉及三聚包膜突亚基之间的相互作用。MPER在其天然构象中的结构和功能也不完全清楚。我们建议用三聚体MPER在稳定的天然尖峰、脂质体和纳米颗粒上免疫小鼠。我们将从免疫小鼠和非洲供体中鉴定与10E8重叠表位的新型MPER抗体。我们将利用NGS/生物信息学分析深入探测小鼠和人类的B细胞谱，以确定mper特异性抗体的种系v区。我们将尝试通过抗原修饰来操纵小鼠MPER特异性B细胞反应。在合作中，我们将研究10E8络合物中稳定包络峰的EM结构，同时使用一系列测定来确定10E8的中和机制。

DESCRIPTION: A crucial missing component of current HIV vaccine candidates is an ability to elicit broadly neutralizing antibodies. In the proposed studies we combine vaccine design, next generation sequencing (NGS) bioinformatics, structural biology and mechanistic studies to overcome an important roadblock in HIV vaccine development, that of eliciting antibodies that recognize native conformations of gp41. The membrane proximal external region (MPER) of gp41 is a particularly desirable vaccine target as it is recognized by numerous broadly neutralizing antibodies, the most potent of which is 10E8. However, how antibodies are elicited to the MPER is virtually unknown. Meanwhile, preliminary data suggest that the 10E8 epitope involves interactions between subunits of the trimeric envelope spike. The structure and function of the MPER in its native conformations is also incompletely understood. We propose to immunize mice with three trimeric presentations of the MPER on stabilized native spikes, on liposomes and on nanoparticles. We will identify novel MPER antibodies to epitopes overlapping with 10E8 from immunized mice and from African donors. We will use NGS/bioinformatics analyses to deeply probe the B cell repertoires of the mice and humans to identify germline V-regions of MPER-specific antibodies. We will attempt to manipulate MPER specific B cell responses in mice through antigen modification. In collaboration we will study the EM structure of stabilized envelope spikes in complex with 10E8, while using a battery of assays to define the mechanism of neutralization by 10E8.