Quantifying bNAb neutralization of the HIV latent reservoir

定量 HIV 潜伏库的 bNAb 中和作用

基本信息

批准号：
10676564
负责人：
BENJAMIN K CHEN
金额：
$ 46.33万
依托单位：
ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-04-01 至 2026-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10676564
关键词：
Antibody Therapy Antiviral Agents Bioinformatics Biological Assay CD4 Lymphocyte Count CD4 Positive T Lymphocytes Cell Line Cell model Cells Clinical Cloning Coculture Techniques DNA Detection Development Flow Cytometry HIV HIV-1 Immune Immune response In Vitro Individual Infection International Laboratories Length Link Maps Measures Mediating Neutralization Tests Patients Phase Phenotype Property Protocols documentation RNA RNA Splicing Recording of previous events Reporter Resistance Resolution Sampling Sensitivity and Specificity Sequence Analysis Site Sorting T-Cell Activation Testing Therapeutic Studies Time Titrations Transcript Transcriptional Activation Viral Viral Load result Viral reservoir Virus Virus Latency analysis pipeline barrier to testing cohort combinatorial cost env Genes genetic analysis genetic resistance high throughput screening immunoregulation in vivo latent HIV reservoir neutralizing antibody novel resistance mutation sex

项目摘要

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.

项目概要针对 HIV 包膜的广泛中和抗体 (bNAb) 是一类新型抗病毒药物，具有具有中和病毒进入的潜力，同时激活非中和免疫效应器功能。然而，其临床应用的一个主要障碍是识别病毒学受到抑制的 HIV+ 患者，这些患者是最适合给定的 bNAb 或 bNAb 组合。事实上，bNAb 的早期治疗研究尚未发现病毒生长测定序列（体外）和 Env 抗性之间存在一致的一致性接受治疗的患者体内出现的突变。我们的实验室开发了强大的定量流程基于细胞计数的测定，用于中和无细胞和细胞间感染。我们观察到使用细胞相关 HIV-1 中和与使用无细胞 HIV-1 中和相比，Ab 的效力和功效降低化验。这些细胞相关表型的检测对于基于治愈的治疗可能特别重要 bNAb 针对感染细胞的功能反应对于体内功效可能很重要。到克服基于 PCR 的方法的局限性（通常识别非功能性 Env），我们提出开发单细胞病毒生长中和测定（scVONA），该测定将在 bNAb 的存在和不存在。该 scVONA 将同时量化病毒潜伏库序列和确定他们对 bNAb 的敏感性。为了实现这一目标，scVONA 将利用并增强高度敏感的单细胞报告细胞系，用于检测感染并识别细胞以测试细胞间传播的中和作用。最终读数是单细胞流式细胞术测量，能够通过以下方式捕获耐药 env 基因对不敏感克隆与敏感克隆进行分选和测序。长读包络测序将用于生物信息分析以确定与耐药性相关的连锁序列。 bNAb 的滴定也将提供估计的 IC50 和序列可以测量敏感克隆的比例。我们相信 scVONA 将提供不到 2 周的快速中和灵敏度读数，并同时提供测量功能完整的 env 基因以进行验证性分析，显着减少时间、成本和与从 HIV+ 患者样本中识别 bNAb 耐药序列相关的劳动。