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主要持续在 一小群潜伏感染的、静止的记忆CD4T细胞。没有一种抗逆转录病毒药物可以 瞄准潜伏的HIV-1病毒。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药物重新激活HIV-1转录。诱导病毒特异性 宿主免疫反应是消除HIV-1基因转录的感染细胞所必需的 是由潜伏期反转剂(LRA)诱导的。针对HIV-1包膜蛋白的抗体 (Env)可以通过抗体效应器功能介导对艾滋病毒感染细胞的杀伤，如 抗体依赖性细胞毒性(ADCC)与抗体依赖性细胞吞噬 (ADCP)。广谱中和抗体(BNAb)的最新进展给人们带来了新的希望 清除HIV-1潜伏蓄水池。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。标准定量聚合酶链式反应 利用针对HIV-1Gag引物和探针测量与细胞相关的HIV-1RNA， 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病毒 CART患者中的前病毒包含致命性突变和/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。当出现以下两个主要问题时 使用标准的Gag qPCR来评估bNAb-LRA的有效性：1)它检测到可以 转录，尽管携带与HIV-1无关的致命性突变或缺失 2)它只检测到低水平始终可检测到的未剪接的病毒基因组RNA 即使没有LRA治疗，而且与剪接病毒mRNAs的产生没有很好的相关性。至 到目前为止，还没有定量的方法来确定LRAs对HIV-1Env mRNA的诱导作用。它是 如果没有LRA和bNAb联合疗法治愈HIV-1，就不可能正确评估 确认和量化HIV-1 env的生产。我们开发了一种新型的基于qPCR的 定量检测HIV-1VPU/env基因单剪接体的方法我们建议研究 这一新方法是否能在转录水平上忠实地监测HIV-1 env的诱导 LRAs在患者CD4T细胞中的水平及bNAb-LRA对VPU/env转录本的减少 心理治疗。我们还计划测试这种测试是否可以用来测量潜伏期的频率 将HIV-1与标准的病毒生长定量分析进行比较。这种新的化验方法将提供 指导优化LRA和bNAb联合治疗策略以治愈HIV。

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.