Memory B Cell Isolation and Antibody Characterization

HIV-1疫苗开发的主要挑战之一是引发广泛中和抗体 （bNAbs）抗HIV Env.最近从HIV感染者中分离出的几种bNAb表明， 人B细胞库可以产生靶向保守Env区的bNAb。不过有 关于如何在慢性HIV-1感染期间引起广泛反应， 感染，以及这些与Env引起的更有限的反应相比如何 预防针为了填补这一信息空白，我们建议定义和改进中和的启发 抗体对HIV-1 Env中功能最保守和最易接近的元件的反应 复合物，包括受体CD 4结合位点（CD 4 bs）。以前，我们开发了一个 表位特异性B细胞分选和RT-PCR分析HIV感染者记忆B细胞区室 个体，这导致分离有效的CD 4 bs和bNAb，VRC 01，其中和>90% 传播的主要病毒。我们采用了这种表位特异性记忆B细胞分选策略，以扩展我们的 分析以了解Env免疫原接种非人期间和之后的Env B细胞应答 灵长类动物（NHP）。因此，本项目3的目的总结如下。在目标1中， 开发CD 4 b特异性记忆B细胞分离和单克隆抗体 从Env免疫原接种的NHP动物中克隆并设计新的和新颖的bNAb特异性探针 隔离在目标2中，我们将用最新技术表征Env特异性单克隆抗体的特异性 结合和表征分析。在目标3中，我们将研究角色，组成和演变 Env特异性IgM记忆B细胞区室的通路，记忆B细胞的一个亚群，最近 已显示在长期B细胞应答中起重要作用。

One of the major challenges of HlV-1 vaccine development is the elicitation of broadly neutralizing antibodies (bNAbs) against HIV Env. The recent isolation of several bNAbs from HIV-infected individuals demonstrates that the human B cell repertoire can generate bNAbs targeting the conserved Env region. However, there is still a tremendous knowledge gap regarding how the broad responses are elicited during chronic HIV-1 infection and, additionally, how these compare to the much more limited responses elicited by Env vaccination. To fill this information gap, we propose to define and improve the elicitation of neutralizing antibody responses toward the most functionally conserved and accessible element ofthe HIV-1 Env complex, including the receptor CD4 binding site (CD4bs). Previously, we developed a multicolor Env epitope-specific B cell sorting and RT-PCR strategy to analyze the memory B cell compartment of HIVinfected individuals, which led to the isolation of CD4bs potent and bNAb, VRCOl, which neutralizes >90% of circulating primary viruses. We adapted this epitope-specific memory B cell sorting strategy to extend our analyses to gain insight of Env B cell response during and following Env immunogen vaccination into nonhuman primates (NHPs). Accordingly, the aims of this Project 3 are summarized as follows. In Aim 1 we will develop envelope-specific, including CD4bs-specific memory B cell isolation and monoclonal antibody cloning from Env immunogen vaccinated NHP animals and design new and novel probes specific for bNAb isolation. In Aim 2, we will characterize the Env-specific monoclonal antibody specificities with state ofthe art binding and characterization assays. In Aim 3, we will investigate the role, composition, and evolution pathway ofthe Env-specific IgM memory B cell compartment, a subset of memory B cells, which recently has been shown to play an important role in the long term B cell response.