Env-Specific B Cell Repertoire Responses to NFL Trimer vaccines

项目摘要 广泛中和抗体（bnAb）识别大肠杆菌的包膜糖蛋白（Env）上的保守表位。 人类免疫缺陷病毒1型（HIV-1）。BG 505 SOSIP.664 gp 140三聚体的原子结构 为基于Env的HIV-1疫苗设计提供了合理的框架。怀亚特集团最近开发了 不依赖于切割的天然柔性连接（NFL）三聚体，其为基于Env的 疫苗设计疫苗递送是在开发良好折叠的三聚体中尚未解决的关键问题， 疫苗产品。在最近的一篇综述中，Schiller等人提出了一种针对HIV-1的病毒样颗粒（VLP）方法 疫苗运送其他递送系统包括mRNA和病毒载体。对于NFL三聚体，它仍然是 目前还不清楚哪种传递系统将产生最强大的免疫反应，以保护免受艾滋病毒感染， 1例感染。我们假设，B细胞反应的定量读数将有助于合理评估 候选疫苗，因为对于有效的HIV-1疫苗， 疫苗下一代测序（NGS）已被广泛用于研究基因组的多样性和成熟度。 bnAbs。我们已经开发了一系列新的NGS技术，用于分析动态B细胞反应， 自然感染、动物免疫和人类疫苗试验。在这个HIVRAD应用程序的项目3中，我们 将研究Env特异性B细胞对基于NFL三聚体和不同递送的四种疫苗的反应 系统在各种动物模型-近交系小鼠，VelocImmune小鼠，兔和非人灵长类动物（NHP）。 在目标1中，我们将对近交系小鼠、VelocImmune小鼠和免疫兔的B细胞反应进行深度测序 四种疫苗我们将描述基本的剧目性质，如种系基因的使用，程度， SHM和CDR 3长度，并鉴定与每种疫苗相关的独特库模式。为每个 在动物模型中，我们将比较B细胞对四种疫苗的反应。我们将比较B细胞反应， 人类-小鼠和人类疫苗试验。在目标2中，我们将对免疫的NHP中的B细胞反应进行深度测序 四种疫苗同样，我们将描述基本的剧目属性，并确定独特的模式 与NHP模型中的四种疫苗相关。我们将比较B细胞对四种疫苗的反应， 比较NHP和人类疫苗试验中的B细胞反应。在目标3中，我们将研究 Env特异性B细胞谱系的途径。我们将追踪单克隆抗体的谱系 通过单B细胞分选（项目2，Wyatt）和基于微流体的单细胞 分析（项目3，朱）在NGS剧目。我们将研究疫苗是否引发中和mAb 类似于已知的bnAb及其发育途径。项目3，连同项目1（RNA和VSV 载体），项目2（NFL三聚体，VLP和血清Ab分析），以及两个核心的体内研究和基因 表达谱分析，将构成一个全面的HIVRAD计划，以有效的HIV-1疫苗。

Project Summary Broadly neutralizing antibodies (bnAbs) recognize conserved epitopes on the envelope glycoprotein (Env) of the human immunodeficiency virus type-1 (HIV-1). Atomic structures of the BG505 SOSIP.664 gp140 trimer have provided a rational framework for Env-based HIV-1 vaccine design. The Wyatt group recently developed a cleavage-independent, native flexibly linked (NFL) trimer, which offers a promising alternative for Env-based vaccine design. Vaccine delivery is a critical issue yet to be addressed in developing well-folded trimers toward vaccine products. In a recent review, Schiller et al. promoted a virus-like particle (VLP) approach for HIV-1 vaccine delivery. Other delivery systems include mRNA and virus vectors. For the NFL trimer, it remains unclear which delivery system will produce the most robust immune response to enable protection against HIV- 1 infection. We hypothesize that a quantitative readout of B-cell response will facilitate rational evaluation of vaccine candidates since a robust cross-neutralizing antibody response is expected for an effective HIV-1 vaccine. Next-generation sequencing (NGS) has been widely used to study the diversity and maturation of bnAbs. We have developed a series of novel NGS technologies for the analysis of dynamic B-cell responses in natural infection, animal immunization, and human vaccine trials. In Project 3 of this HIVRAD application, we will investigate the Env-specific B-cell response to four vaccines based on the NFL trimer and different delivery systems in various animal models – inbred mice, VelocImmune mice, rabbits and non-human primates (NHPs). In Aim 1, we will deep sequence the B-cell response of inbred mice, VelocImmune mice, and rabbits immunized with four vaccines. We will characterize the basic repertoire properties such as germline gene usage, degree of SHM, and CDR3 length and identify distinctive repertoire patterns associated with each vaccine. For each animal model, we will compare the B-cell responses to four vaccines. We will compare the B-cell responses in hu-mice and human vaccine trials. In Aim 2, we will deep sequence the B-cell response in NHPs immunized with four vaccines. Similarly, we will characterize the basic repertoire properties and identify distinctive patterns associated with four vaccines in the NHP model. We will compare the B-cell responses to four vaccines and compare the B-cell responses in NHPs and human vaccine trials. In Aim 3, we will study the developmental pathways of Env-specific B-cell lineages elicited by four vaccines. We will trace the lineages of monoclonal antibodies (mAbs) identified by single B-cell sorting (Project 2, Wyatt) and microfluidics-based single-cell analysis (Project 3, Zhu) in NGS repertoires. We will investigate whether the vaccine-elicited neutralizing mAbs resemble known bnAbs and their developmental pathways. Project 3, together with Project 1 (RNA and VSV vector), Project 2 (NFL trimer, VLP, and serum Ab analysis), and two Cores on in-vivo study and gene expression profiling, will constitute a comprehensive HIVRAD program towards an effective HIV-1 vaccine.