Memory B Cell Isolation and Antibody Characterization

HLV-1疫苗开发的主要挑战之一是诱导广谱中和抗体 (BNAbs)抗HIV Env.最近从HIV感染者身上分离出的几种bNAb证明 人类B细胞谱系可以产生针对保守的Env区的bNAb。然而，还有 关于慢性HIV-1是如何引起广泛反应的，仍然存在巨大的知识差距 感染，以及与环境引起的更有限的响应相比如何 接种疫苗。为了填补这一信息空白，我们建议定义和改进中和启发式 对HIV-1环境中功能最保守和最容易接近的元件的抗体应答 复合体，包括受体的CD4结合位点(CD4bs)。在此之前，我们开发了一个多色环境 表位特异性B细胞分选和RT-PCR方法分析HIV感染者记忆B细胞亚群 个体，这导致了CD4bs的有效和bNAb，VRCO1，它中和了90% 传播的主要病毒。我们采用了这种特定于表位的记忆B细胞分选策略来扩展我们的 对非人类接种环境免疫原期间和接种后环境B细胞应答情况的分析 灵长类(NHP)。因此，本项目3的目标概述如下。在目标1中，我们将 开发包膜特异性，包括CD4bs特异性记忆B细胞分离和单抗 NHP动物Env免疫原的克隆及bNAb新型特异性探针的设计 与世隔绝。在目标2中，我们将用最先进的技术来表征env特异性的单抗。 结合和表征分析。在目标3中，我们将研究角色、组成和演变 环境特异性IgM记忆B细胞室的通路，记忆B细胞的一个亚群，最近 已被证明在长期的B细胞反应中起着重要作用。

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.