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.

项目摘要针对HIV ENV的广泛中和抗体（BNAB）代表了一种新型的抗病毒药，潜在的中和病毒进入，同时激活非中和免疫效应子功能。但是，其临床用途的主要障碍是确定病毒学抑制的HIV+患者最适合给定的BNAB或BNAB组合。实际上，使用BNAB的早期治疗研究尚未发现病毒生长测定序列（体外）与ENV电阻之间的一致性一致性在体内治疗的患者中出现的突变。我们的实验室发展了强大而定量的流动基于细胞仪的测定，用于中和无细胞和细胞对细胞感染。我们已经观察到使用与细胞相关的无细胞HIV-1中和的ABS的效力和功效降低测定。这些与细胞相关的表型的检测对于基于治疗的表型尤为重要 BNAB对感染细胞的功能反应性的方法对于体内功效可能很重要。到克服经常识别非功能性env的基于PCR-优先的方法的局限性，我们提出将在将在 BNAB的存在和不存在。此SCVONA将同时量化病毒潜在储层序列和确定它们对BNAB的敏感性。为此，Scvonas将利用和增强高度敏感的单细胞报告细胞系以检测感染并鉴定细胞以测试细胞间扩散的中和。最终的读数是单细胞流式细胞仪测量措施，可以通过分类和测序不敏感与敏感克隆。长期阅读env测序将用于生物信息学分析以确定与电阻相关的链接序列。 BNAB的滴定也将提供估计的IC50和序列可以允许衡量敏感克隆的比例。我们相信 SCVONA将提供中和敏感性的快速<2周读数，并同时提供衡量功能完整的ENV基因的量度用于确认性分析，大大降低了时间，成本和成本和与鉴定来自HIV+患者样品的BNAB抗性序列相关的劳动。