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

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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.