Quantifying bNAb neutralization of the HIV latent reservoir

项目摘要 针对HIV env的广谱中和抗体(BNAb)代表了一类新的抗病毒药物，具有 有可能中和病毒入侵，同时激活非中和免疫效应器功能。 然而，它们临床应用的一个主要障碍是识别出病毒抑制的HIV+患者 最适合给定的bNAb或bNAb组合。事实上，使用bNAb的早期治疗研究并没有 在病毒生长试验序列(体外)和环境抗性之间发现一致的一致性 体内接受治疗的患者中出现的突变。我们的实验室已经开发出稳健和定量的流量 基于细胞学的中和无细胞感染和细胞间感染的检测方法。我们观察到 使用细胞相关和无细胞HIV-1中和的抗体效力和效力的降低 化验。这些细胞相关表型的检测可能对基于治愈的 BNAbs对感染细胞的功能反应性可能对体内疗效重要的方法。至 克服了基于PCR-First的方法通常识别非功能环境的局限性，我们建议 单细胞病毒突起中和试验方法的建立 BNAb的存在和缺失。这一scVONA将同时量化病毒潜伏储存库序列和 测定其对bNAb的敏感性。为了实现这一目标，scVONAs将利用并增强高度敏感的 单细胞报告细胞系，用于检测感染和鉴定细胞，以检测细胞间传播的中和性。 最终的读数是单细胞流式细胞术测量，使捕获抗药性env基因通过 对不敏感克隆和敏感克隆进行排序和排序。将采用长读环境测序 生物信息学分析，以确定与抗药性相关的连锁序列。BNAb的滴定也将 提供估计的IC50和序列可能允许测量敏感克隆的比例。我们相信 ScVONA将提供快速2周中和灵敏度读数，并同时提供 用于验证性分析的功能完整的env基因的测量，显著减少了时间、成本和 与从HIV+患者样本中识别bNAb耐药序列相关的工作。

Project Summary Broadly neutralizing antibodies (bNAb) against HIV Env represent a novel class of antivirals that have the potential to neutralize viral entry, while simultaneously activating non-neutralizing immune effector functions. However, a major obstacle for their clinical use is identifying the virologically suppressed HIV+ patients that are best suited for a given bNAb or bNAb combination. Indeed, early-stage therapeutic studies with bNAb have not found uniform concordance between viral outgrowth assay sequences (in vitro) and the Env resistance mutations that arise in treated patients in vivo. Our laboratory has developed robust and quantitative flow cytometry-based assays for neutralization of both cell-free and cell-to-cell infection. We have observed decreases in the potency and efficacy of Abs using cell-associated versus cell-free HIV-1 neutralization assays. The detection of these cell-associated phenotypes may be particularly important for cure-based approaches where functional reactivity of bNAbs against infected cells may be important for in vivo efficacy. To overcome the limitations of PCR-first based approaches that often identify non-functional Env, we propose the development of a single cell Viral Outgrowth Neutralization Assay (scVONA) that will be performed in the presence and absence of bNAb. This scVONA will simultaneously quantify viral latent reservoir sequences and determine their sensitivity to bNAb. To accomplish this, scVONAs will leverage and enhance a highly sensitive single cell reporter cell line to detect infection and identify cells to test for neutralization of cell-to-cell spread. The ultimate readout are single cell flow cytometry measures that enable capture of resistant env genes by sorting and sequencing insensitive versus sensitive clones. Long read Env sequencing will be employed for bioinformatic analyses to determine linked sequences that correlate with resistance. Titration of bNAb will also provide estimated IC50 and sequences may allow a measure of the fraction of sensitive clones. We believe that scVONA will provide a rapid <2 week readout of neutralization sensitivity and simultaneously provide a measure of functionally intact env genes for confirmatory analyses, significantly reducing the time, cost and labor associated with identifying bNAb resistant sequences from HIV+ patient samples.