Rational design and B cell responses of HIV epitope vaccines

项目摘要 许多广谱中和抗体(BNAbs)识别包膜糖蛋白上结构上不连续的部位。 人类免疫缺陷病毒1型(HIV-1)的(Env)，如CD4结合位点(CD4bs)， 可变区1和2(V1V2)，可变区3(V3)底部的N332超位，以及膜- 近端外区(MPER)。结晶学和电子显微镜(EM)揭示了这些 BNAbs与单个表位、工程环境结构域和gp140三聚体相互作用，提供了一个合理的基础 用于疫苗设计。这种支架方法被用来设计新的抗原，希望能激发出表位-- 特异性的bNAb样B细胞反应。在这个R01应用程序中，我们将把最新的结构发现与 蛋白质设计、B细胞工程和转基因小鼠以及下一代测序方面的新技术 (NGS)，以开发和评估三个bNAb表位的以表位为重点的疫苗候选。 我们R01提案的具体目标(SA)是：(1)为以下三个部位设计以表位为重点的免疫原 HIV-1的脆弱性。我们假设V3碱基的N332超位、三聚体V1V2顶点和MPER 可以通过结合bNAb构象的支架和纳米颗粒来呈现。在初步研究中，我们 基于表位支架、颗粒展示、 和FC演示。我们已经对阳性的抗原子集进行了结构和抗原分析 结果，并将筛选整个抗原小组，以便于合理选择；(2)评估以表位为重点的 表达bNAb的B细胞系和敲入小鼠中的免疫原。我们假设成功地设计了 表位集中的免疫原可以激活表达bNAb的工程化B细胞并在 BNab敲入鼠。此前，我们已经建立了表达CD4bs-、V1V2-和N332-的小鼠B细胞系 特异性bNAbs和B12敲入小鼠，用于评价合理设计的HIV-1免疫原。 我们将建立表达PGT145、PGT121/128和10E8的B细胞系和敲入小鼠，并使用这些细胞 用于评估目标1中选择的以表位为中心的免疫原的工具；(3)用于评估三聚体启动/表位增强的工具 NHP中的策略和B细胞反应。我们假设gp140三聚体将引起不同表位的nabs。 用集中于表位的免疫原进行的连续增强将引导B细胞对目标表位的反应。在……里面 我们的初步研究，我们已经测试了对选定的N332抗原的表位聚焦效应和中和 在C57BL/6小鼠体内。此前，我们已经对B细胞对HIV-1 gp140的反应进行了纵向研究- 非人灵长类(NHP)中的三聚体。在这里，我们将首先测试三聚体-质点/表位-增强策略在 然后评估免疫原性和B细胞在NHP中的反应。除了……之外 在血清学检测中，我们将使用抗体NGS来监测暂时性B细胞反应。我们建议的研究 从而构成了一个创新和实用的研究项目，以开发以表位为重点的艾滋病毒-1疫苗。

Project Summary Many broadly neutralizing antibodies (bNAbs) recognize structurally discrete sites on the envelope glycoprotein (Env) of human immunodeficiency virus type-1 (HIV-1), such as the CD4-binding site (CD4bs), the strand C of variable regions 1 and 2 (V1V2), the N332 supersite at the base of variable region 3 (V3), and the membrane- proximal external region (MPER). Crystallography and electron microscopy (EM) have revealed how these bNAbs interact with individual epitopes, engineered Env domains, and gp140 trimers, providing a rational basis for vaccine design. The scaffolding method has been used to design novel antigens in hope to elicit epitope- specific, bNAb-like B cell responses. In this R01 application, we will combine the latest structural findings with novel technologies in protein design, B cell engineering and knock-in mouse, and next-generation sequencing (NGS) of B cell repertoire to develop and assess epitope-focused vaccine candidates for three bNAb epitopes. The specific aims (SAs) of our R01 proposal are: (1) to design epitope-focused immunogens for three sites of HIV-1 vulnerability. We hypothesize that the N332 supersite of V3 base, the trimeric V1V2 apex, and MPER can be presented by scaffolds and nanoparticles in their bNAb-bound conformations. In a preliminary study, we have designed a panel of antigens based on various principles such as epitope scaffolding, particulate display, and Fc presentation. We have performed structural and antigenic profiling for a subset of antigens with positive results, and will screen the whole panel of antigens to facilitate rational selection; (2) to assess epitope-focused immunogens in bNAb-presenting B cell lines and knock-in mice. We hypothesize that successfully designed epitope-focused immunogens can activate engineered bNAb-expressing B cells and elicit robust responses in bNAb knock-in mice. Previously, we have developed mouse B cell lines expressing CD4bs-, V1V2- and N332- specific bNAbs and b12 knock-in mouse, which were used to assess rationally designed HIV-1 immunogens. We will develop PGT145-, PGT121/128-, and 10E8-expressing B cell lines and knock-in mice and use these tools to assess epitope-focused immunogens selected in Aim 1; (3) to assess the trimer-prime/epitope-boost strategy and B cell responses in NHPs. We hypothesize that a gp140 trimer will elicit NAbs to diverse epitopes and sequential boosts with epitope-focused immunogens will direct B cell responses to the target epitopes. In our preliminary study, we have tested the epitope-focusing effect and neutralization for selected N332 antigens in C57BL/6 mice. Previously, we have conducted a longitudinal study of B cell responses to an HIV-1 gp140- foldon trimer in non-human primates (NHPs). Here, we will first test the trimer-prime/epitope-boost strategy in mice for different epitopes and then assess the immunogenicity and B cell responses in NHPs. In addition to serological assays, we will use antibody NGS to monitor the temporal B cell responses. Our proposed studies thus constitute an innovative and practical research project to develop epitope-focused HIV-1 vaccines.