HIV Neutralizing Antibodies: Isolation, characterization, and interaction with viral variants

针对HIV-1的中和抗体(NAB)可能是疫苗诱导的有效免疫反应的主要组成部分。交叉反应NAB通常出现在HIV-1感染期间，尽管只有一小部分受感染的患者产生具有高广度和效力的NAB。相比之下，迄今为止测试的HIV-1包膜糖蛋白(Env)疫苗免疫原未能诱导交叉反应的中和抗体。因此，研究广谱中和抗体(BNAbs)在感染者体内的发展可能会为疫苗设计提供重要的经验教训。此外，从选定的供体和接种疫苗的动物中分离出bNAb极大地帮助了我们了解HIV-1环境病毒的结构和对中和抗体的易感性，这些抗体有可能预防或治疗HIV-1感染。 几年来，我们的实验室在分离和鉴定来自艾滋病毒感染者捐赠者的广泛中和抗体方面一直处于领先地位。我们率先开发了分离表位特异性B细胞的试剂，以及高通量筛选未选择的B细胞的方法。经其中一种方法鉴定后，通过单细胞聚合酶链式反应、亚克隆和在哺乳动物细胞中的表达，从B细胞中回收免疫球蛋白。对产生的抗体进行病毒结合和中和分析，并分析其广度、效力、表位和识别模式。我们还使用下一代深度测序来寻找抗体的克隆亲属，并了解它们在B细胞发育中的起源。对于后一项研究，我们拥有艾滋病毒感染时间的纵向样本的捐赠者特别有价值。此外，我们将这些技术应用于用候选疫苗免疫的动物的研究。 在过去的一年里，我们的工作包括：从三名艾滋病毒感染患者中分离出单抗，并开发出广泛的中和血清；针对一名这样的患者，评估了这些抗体针对患者自身病毒变异株的活性；对接种了候选艾滋病毒疫苗的恒河猴的抗体进行了分离和下一代测序分析；流式细胞仪分析了单抗与艾滋病毒被膜蛋白的结合，以支持与VRC和耶鲁大学的合作者进行的生物物理研究；以及分析了从接受抗体VRC01治疗的患者体内分离的艾滋病毒被膜蛋白。

Neutralizing antibodies (NAb) against HIV-1 are likely to be a major component of an effective vaccine-induced immune response. Cross-reactive NAbs commonly arise during HIV-1 infection, though only a small subset of infected patients produce NAbs with high breadth and potency. In contrast, the HIV-1 envelope glycoprotein (Env) vaccine immunogens tested to date have failed to elicit cross-reactive neutralizing antibodies. Thus, studying the development of broadly neutralizing antibodies (bNAbs) in infected individuals may provide important lessons for vaccine design. In addition, the isolation of bNAbs from selected donors and vvaccinated animals has greatly aided our understanding of HIV-1 Env structure and vulnerability to neutralizing antibodies and such antibodies have potential for prevention or treatment of HIV-1 infection. For several years our lab has been a leader in the field of isolating and characterizing broadly neutralizing antibodies from HIV-infected donors. We have pioneered the development of reagents for isolating epitope-specific B cells, as well as a method for high-throughput screening of unselected B cells. After identification by one of these methods, we recover IgG from the B cells by single-cell PCR, subcloning, and expression in mammalian cells. The resulting antibodies are assayed for virus binding and neutralization, and their breadth, potency, epitopes, and modes of recognition analyzed. We also use next-generation deep sequencing to find clonal relatives of the antibodies and to understand their origins in B cell development. For the latter studies, donors for whom we have longitudinal samples from the time of HIV infection are particularly valuable. In addition, we apply these techniques to the study of animals that were immunized with candidate vaccines. In the past year, our work has included: isolation of monoclonal antibodies from three HIV-infected patients that developed broadly neutralizing serum; for one such patient, assessment of the activity of such antibodies against the patients' own viral variants; isolation and next-generation sequencing analysis of antibodies from rhesus macaques vaccinated with candidate HIV vaccines; flow cytometric analysis of binding of monoclonal antibodies to HIV envelope proteins, in support of biophysical studies with collaborators at the VRC and Yale University; and analysis of HIV envelope proteins isolated from patients treated with the antibody VRC01.