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

摘要 联合抗逆转录病毒疗法（cART）不能治愈HIV-1感染。HIV-1主要存在于 一小群潜伏感染的静息记忆CD 4 + T细胞。没有一种抗逆转录病毒药物 针对潜伏HIV-1。目前清除病毒库的方法包括药理学 通过逆转病毒潜伏期的试剂重新激活HIV-1转录。病毒特异性诱导 宿主的免疫反应需要消除感染细胞，其中HIV-1基因转录 潜伏期逆转剂（LRA）。靶向HIV-1包膜蛋白的抗体 (Env)可以通过抗体效应子功能介导HIV-1感染细胞的杀伤， 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）的最新进展为免疫治疗带来了新的希望。 清除HIV-1潜在的储存库。HIV-1感染者的抗体依赖性细胞溶解或吞噬 细胞依赖于HIV-1 env表达的诱导。基于标准定量PCR 利用靶向HIV-1gag引物和探针测量细胞相关HIV-1 RNA， 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 接受cART治疗的患者中的前病毒含有致死性突变和/或缺失，但仍携带完整或 gag ORF的一部分，并且可以转录截短的病毒RNA。有两个主要问题， 使用标准gag qPCR来评估bNAb-LRA功效：1）其检测可被 尽管携带与HIV-1无关的致命突变或缺失， 2）它只检测未剪接的病毒基因组RNA，其总是在低水平下可检测到 即使没有LRA治疗，也是如此，并且与剪接的病毒mRNA的产生没有很好的相关性。到 迄今为止，还没有定量测定LRA对HIV-1 Env mRNA的诱导作用。是 不可能正确评估LRA和bNAb联合治疗对HIV-1治愈的作用， 确认和定量HIV-1 Env的产生。我们开发了一种基于qPCR的新型 方法定量测量单剪接的HIV-1 vpu/env mRNA。我们建议研究 这种新的方法是否可以忠实地监测HIV-1 Env在转录水平的诱导， 通过LRA在患者CD 4 + T细胞中的水平，并评估通过bNAb-LRA的vpu/env转录物的减少 疗法我们还计划测试这种检测方法是否可以用来测量潜伏的频率。 HIV-1与标准定量病毒生长试验的比较。这种新的检测方法将提供 优化LRA和bNAb联合治疗策略以治愈艾滋病毒的指导。

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.