Rational design and B cell responses of HIV epitope vaccines

项目摘要 许多广泛中和抗体（bNAb）识别包膜糖蛋白上结构上的离散位点， (Env)人免疫缺陷病毒1型（HIV-1）的C链，如CD 4结合位点（CD 4 bs）， 可变区1和2（V1 V2）、位于可变区3（V3）基部的N332超位点和膜- 近端外部区域（MPER）。晶体学和电子显微镜（EM）揭示了这些 bNAb与单个表位、工程化Env结构域和gp 140三聚体相互作用，提供了合理的基础 用于疫苗设计。支架方法已被用于设计新的抗原，希望能引出表位- 特异性bNAb样B细胞应答。在此R 01应用程序中，我们将结合联合收割机的最新结构发现， 蛋白质设计、B细胞工程和基因敲入小鼠以及下一代测序方面的新技术 (NGS)的B细胞库，以开发和评估针对三个bNA B表位的表位聚焦疫苗候选物。 我们的R 01提案的具体目标（SA）是：（1）针对以下三个位点设计表位聚焦的免疫原： HIV-1的脆弱性。我们假设N332位于V3碱基的超位点，三聚体V1 V2顶点，MPER 可以通过支架和纳米颗粒以其bNAb结合的构象呈现。在初步研究中，我们 已经基于各种原理设计了一组抗原，例如表位支架，颗粒展示， Fc介绍我们已经对一个抗原子集进行了结构和抗原分析， 结果，并将筛选整个面板的抗原，以促进合理的选择;（2）评估表位集中 bNAb呈递B细胞系和敲入小鼠中的免疫原。我们假设成功设计的 表位聚焦的免疫原可以激活工程化的表达bNAb的B细胞， bNAb基因敲入小鼠。以前，我们已经开发了表达CD 4 bs-、V1 V2-和N332-的小鼠B细胞系， 特异性bNAbs和b12基因敲入小鼠，用于评估合理设计的HIV-1免疫原。 我们将开发表达PGT 145、PGT 121/128和10 E8的B细胞系和基因敲入小鼠，并使用这些细胞系和基因敲入小鼠。 评估目标1中选择的表位聚焦免疫原的工具;（3）评估三聚体-初免/表位-加强 策略和B细胞应答。我们假设gp 140三聚体将引发针对不同表位的NAb 用表位聚焦的免疫原连续加强将引导B细胞应答靶表位。在 我们的初步研究中，我们测试了选定N332抗原的表位聚焦效应和中和作用 C57 BL/6小鼠。以前，我们已经进行了一项纵向研究，研究B细胞对HIV-1 gp 140的反应， 非人灵长类动物（NHP）中的折叠子三聚体。在这里，我们将首先在以下中测试三聚体-引发/表位-加强策略： 小鼠的不同表位，然后评估NHP中的免疫原性和B细胞应答。除了 血清学检测，我们将使用抗体NGS来监测时间B细胞反应。我们建议的研究 从而构成了一个创新的和实用的研究项目，以开发针对表位的HIV-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.