A novel PCR-based method for quantification of antibody-dependent clearance of HIV-1 reservoirs

一种基于 PCR 的新方法，用于量化 HIV-1 储存库的抗体依赖性清除

基本信息

批准号：
10012578
负责人：
LIANG SHAN
金额：
$ 23.63万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-08-10 至 2022-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10012578
关键词：
Affect Agonist Anti-Retroviral Agents Antibodies Antibody Therapy Biological Assay Bromodomain CD4 Positive T Lymphocytes Categories Cell Culture Techniques Cells Combined Modality Therapy Cytolysis DNA Data Deletion Mutation Evaluation Frequencies GAG Gene Genetic Transcription HIV HIV-1 Histone Deacetylase Inhibitor Immune response Infection Ionomycin Length Measurement Measures Mediating Messenger RNA Methods Monitor Mutation Open Reading Frames Patients Phagocytosis Pharmaceutical Preparations Pharmacology Plasma Production Proviruses RNA RNA Splicing Residual state Resistance Rest Testing Toll-like receptors Transcript Viral Viral Genome Viral Proteins Viral reservoir Virus Virus Activation Virus Latency antibody-dependent cell cytotoxicity antiretroviral therapy base bryostatin cost design env Gene Products experience genomic RNA inhibitor/antagonist latent HIV reservoir memory CD4 T lymphocyte neutralizing antibody novel novel strategies protein expression purge response treatment strategy viral RNA viral genomics viral rebound virtual

项目摘要

Abstract Combination antiretroviral therapy (cART) does not cure HIV-1 infection. HIV-1 mainly persists in a small pool of latently infected, resting memory CD4+ T cells. None of antiretroviral drugs can target latent HIV-1. Current approaches to purging the viral reservoirs involve pharmacologic reactivation of HIV-1 transcription by agents that reverse viral latency. Induction of viral-specific host immune responses is required to eliminate infected cells in which HIV-1 gene transcription has been induced by latency reversal agents (LRAs). Antibodies targeting HIV-1 envelope protein (Env) can mediate killing of HIV-1-infected cells through antibody effector functions such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP). Recent progress in broadly reactive neutralizing antibodies (bNAb) brought new hope to purge the HIV-1 latent reservoirs. Antibody dependent cytolysis or phagocytosis of HIV-1-infected cells relies on the induction of HIV-1 env expression. Standard quantitative PCR-based measurement of cell-associated HIV-1 RNA utilize primers and probe that target HIV-1 gag, which is part of the unspliced full-length viral genomic RNA. Vast majority of the integrated HIV-1 proviruses in patients under cART contain lethal mutations and/or deletions but still carry entire or part of the gag ORF and can transcribe truncated viral RNA. There are two major issues when using standard gag qPCR to evaluate bNAb-LRA efficacy: 1) it detects viral genomes that can be transcribed despite carrying lethal mutations or deletions which are irrelevant to the HIV-1 reservoirs; 2) it only detects unspliced viral genomic RNA which is always detectable at low levels even without LRA treatment, and is not well correlated with production of spliced viral mRNAs. To date, there is no quantitative assay to determine the induction of HIV-1 Env mRNA by LRAs. It is not possible to properly evaluate LRA and bNAb combination therapy for HIV-1 cure without confirming and quantifying HIV-1 Env production. We have developed a novel qPCR-based approach to quantitatively measure the singly spliced HIV-1 vpu/env mRNA. We propose to study whether this novel approach can faithfully monitor induction of HIV-1 Env at the transcriptional level by LRAs in patient CD4+ T cells and assess reduction of vpu/env transcripts by bNAb-LRA therapy. We also plan to test whether this assay can be used to measure the frequency of latent HIV-1 compared to the standard quantitative viral outgrowth assay. This new assay will provide guidance to the optimization of LRA and bNAb combination treatment strategies for HIV cure.

抽象的联合抗逆转录病毒疗法 (cART) 不能治愈 HIV-1 感染。 HIV-1主要持续存在于一小群潜伏感染的静息记忆 CD4+ T 细胞。任何抗逆转录病毒药物都不能针对潜在的 HIV-1。目前清除病毒库的方法涉及药理学通过逆转病毒潜伏期的药物重新激活 HIV-1 转录。病毒特异性的诱导需要宿主免疫反应来消除受感染的细胞，其中 HIV-1 基因转录是由潜伏期逆转剂（LRA）诱导的。针对 HIV-1 包膜蛋白的抗体 (Env) 可以通过抗体效应功能介导杀死 HIV-1 感染细胞，例如抗体依赖性细胞毒性（ADCC）和抗体依赖性细胞吞噬作用（ADCP）。广泛反应性中和抗体（bNAb）的最新进展给清除 HIV-1 潜伏库。 HIV-1 感染者的抗体依赖性细胞溶解或吞噬作用细胞依赖于 HIV-1 env 表达的诱导。基于标准定量 PCR 细胞相关 HIV-1 RNA 的测量利用针对 HIV-1 gag 的引物和探针，是未剪接的全长病毒基因组RNA的一部分。绝大多数整合的 HIV-1 cART 患者体内的原病毒含有致命突变和/或缺失，但仍携带完整或 gag ORF的一部分，可以转录截短的病毒RNA。当使用标准 gag qPCR 评估 bNAb-LRA 功效：1) 它检测到的病毒基因组可以尽管携带与 HIV-1 无关的致命突变或缺失，但仍被转录水库； 2) 它仅检测未剪接的病毒基因组 RNA，且始终可检测到低水平即使没有 LRA 处理，也与剪接病毒 mRNA 的产生没有很好的相关性。到迄今为止，还没有定量测定来确定 LRA 对 HIV-1 Env mRNA 的诱导。这是不可能正确评估 LRA 和 bNAb 联合疗法治疗 HIV-1 的效果，除非确认和量化 HIV-1 Env 的产生。我们开发了一种新型的基于 qPCR 的定量测量单剪接 HIV-1 vpu/env mRNA 的方法。我们建议学习这种新方法是否可以忠实地监测转录过程中 HIV-1 Env 的诱导患者 CD4+ T 细胞中 LRA 的水平，并评估 bNAb-LRA 对 vpu/env 转录物的减少治疗。我们还计划测试该测定是否可用于测量潜在的频率 HIV-1 与标准定量病毒生长测定法的比较。这种新的检测将提供指导优化 LRA 和 bNAb 联合治疗策略以治愈 HIV。