Env-Specific B Cell Repertoire Responses to NFL Trimer vaccines

项目摘要 广谱中和抗体(BNAbs)识别猪瘟病毒包膜糖蛋白(Env)上的保守表位 人类免疫缺陷病毒1型(HIV-1)。BG505 SOSIP.664 gp140三聚体的原子结构 为基于环境病毒的HIV-1疫苗设计提供了一个合理的框架。怀亚特集团最近开发了 一种裂解不依赖、天然灵活连接(NFL)的三聚体，为基于环境的病毒提供了一种有前途的替代方案 疫苗设计。疫苗递送是开发折叠良好的三聚体的关键问题，以实现 疫苗产品。在最近的一篇综述中，席勒等人。推广针对HIV-1的病毒样颗粒(VLP)方法 疫苗投放。其他递送系统包括信使核糖核酸和病毒载体。对于NFL三聚体来说，它仍然是 不清楚哪种递送系统将产生最强大的免疫反应，以实现对艾滋病毒的保护- 感染1例。我们假设，B细胞反应的定量读数将有助于理性评估 候选疫苗，因为有效的HIV-1需要强大的交叉中和抗体反应 疫苗。下一代测序技术(NGS)已被广泛应用于研究生物多样性和成熟度 BNAbs。我们开发了一系列新的NGS技术来分析动态B细胞反应 自然感染、动物免疫和人类疫苗试验。在此HIVRAD应用程序的项目3中，我们 将研究基于NFL三聚体和不同递送方式的四种疫苗对Env特异性B细胞的反应 在各种动物模型--近亲交配小鼠、速度免疫小鼠、兔子和非人灵长类动物(NHP)中的系统。 在目标1中，我们将对近交系小鼠、速度免疫小鼠和免疫兔子的B细胞反应进行深度排序 用了四种疫苗。我们将表征基本的曲目属性，如生殖系基因的使用，程度 SHM和CDR3长度，并识别与每种疫苗相关的独特的谱系模式。对于每个 动物模型，我们将比较四种疫苗对B细胞的反应。我们将比较B细胞的反应 人-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 用了四种疫苗。类似地，我们将确定基本曲目的属性并确定不同的模式 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和 比较NHP和人类疫苗试验中的B细胞反应。在目标3中，我们将研究发展的 四种疫苗诱导的Env特异性B细胞谱系的途径。我们将追踪单克隆体的血统 通过单个B细胞分选(项目2，怀亚特)和基于微流体的单细胞鉴定的抗体(MAbb) 分析(项目3，朱)在NGS剧目中。我们将调查疫苗引发的中和单抗是否 与已知的bNAbs及其发育途径相似。项目3和项目1(RNA和VSV 载体)、项目2(NFL三聚体、VLP和血清抗体分析)，以及体内研究和基因研究的两个核心 表达谱分析，将构成一个全面的HIVRAD计划，以获得有效的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.