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.

抽象的抗逆转录病毒疗法（CART）无法治愈HIV-1感染。 HIV-1主要存在一小部分被潜在感染的静止记忆CD4+ T细胞。没有抗逆转录病毒药物可以靶标潜在HIV-1。清除病毒库的当前方法涉及药理逆转病毒潜伏期的药物对HIV-1转录的重新激活。诱导病毒特异性需要宿主免疫反应以消除受感染的细胞，其中HIV-1基因转录已由潜伏期逆转剂（LRAS）诱导。靶向HIV-1包膜蛋白的抗体（env）可以通过抗体效应子功能（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）的最新进展带来了新的希望清除HIV-1潜在水库。 HIV-1感染的抗体依赖性胞体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 与细胞相关的HIV-1 RNA的测量利用引物和探测器，以靶向HIV-1 GAG，这是未涂抹的全长病毒基因组RNA的一部分。绝大多数综合HIV-1 货车下的患者的病毒病毒中有致命的突变和/或缺失，但仍携带全部或一部分GAG ORF，可以转录截短的病毒RNA。有两个主要问题使用标准GAG QPCR评估BNAB-LRA功效：1）它检测到可以是的病毒基因组尽管携带致命突变或缺失，但与HIV-1无关水库； 2）它仅检测未填充的病毒基因组RNA，始终在低水平下可检测到即使没有LRA治疗，并且与剪接病毒mRNA的产生也不相关。到日期，没有定量测定来确定LRA诱导HIV-1 Env mRNA。这是无法正确评估LRA和BNAB联合疗法以治愈HIV-1 确认和量化HIV-1 Env的生产。我们已经开发了一种基于QPCR的新颖定量测量单剪接的HIV-1 VPU/ENV mRNA的方法。我们建议学习这种新颖的方法是否可以忠实地监测转录的HIV-1 env诱导 LRA在患者CD4+ T细胞中的水平，并评估BNAB-LRA的VPU/ENV转录本的降低治疗。我们还计划测试该测定是否可以用于测量潜在的频率 HIV-1与标准定量病毒生长测定法相比。这个新测定将提供针对HIV治疗的LRA和BNAB组合治疗策略优化的指导。