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Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA.

ABSTRACT: Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using peripheral blood mononuclear cells (PBMCs) from 5 viremic patients and 20 patients receiving effective ART. Total and resting CD4(+)T cells were isolated from a subset of patients and tested for comparison with PBMCs. The estimated standard deviations including interassay variability and intra-assay variability of the assays were modest, i.e., 0.15 and 0.10 log10copies/10(6)PBMCs, respectively, for CA HIV-1 DNA and 0.40 and 0.19 log10copies/10(6)PBMCs for CA HIV-1 RNA. Testing of longitudinally obtained PBMC samples showed little variation for either viremic patients (median fold differences of 0.80 and 0.88 for CA HIV-1 DNA and RNA, respectively) or virologically suppressed patients (median fold differences of 1.14 and 0.97, respectively). CA HIV-1 DNA and RNA levels were strongly correlated (r= 0.77 to 1;P= 0.0001 to 0.037) for assays performed using PBMCs from different sources (phlebotomy versus leukapheresis) or using total or resting CD4(+)T cells purified by either bead selection or flow cytometric sorting. Their sensitivity, reproducibility, and broad applicability to small numbers of mononuclear cells make these assays useful for observational and interventional studies that examine longitudinal changes in the numbers of HIV-1-infected cells and their levels of transcription.

SUBMITTER: Hong F

PROVIDER: S-EPMC4809955 | biostudies-literature | 2016 Apr

REPOSITORIES: biostudies-literature

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Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA.

Hong Feiyu F Aga Evgenia E Cillo Anthony R AR Yates Aarika L AL Besson Guillaume G Fyne Elizabeth E Koontz Dianna L DL Jennings Cheryl C Zheng Lu L Mellors John W JW

Journal of clinical microbiology 20160113 4

Although a number of PCR-based quantitative assays for measuring HIV-1 persistence during suppressive antiretroviral therapy (ART) have been reported, a simple, sensitive, reproducible method is needed for application to large clinical trials. We developed novel quantitative PCR assays for cell-associated (CA) HIV-1 DNA and RNA, targeting a highly conserved region in HIV-1pol, with sensitivities of 3 to 5 copies/1 million cells. We evaluated the performance characteristics of the assays using pe ...[more]

PMID: 26763968

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Project description:The HIV proviral reservoir is the major barrier to cure. The predominantly replication-defective proviral landscape makes the measurement of virus that is likely to cause rebound upon antiretroviral therapy (ART)-cessation challenging. To address this issue, novel assays to measure intact HIV proviruses have been developed. The intact proviral DNA assay (IPDA) is a high-throughput assay that uses two probes to exclude the majority of defective proviruses and determine the frequency of intact proviruses, albeit without sequence confirmation. Quadruplex PCR with four probes (Q4PCR) is a lower-throughput assay that uses limiting dilution long-distance PCR amplification followed by quantitative PCR (qPCR) and near-full-length genome sequencing (nFGS) to estimate the frequency of sequence-confirmed intact proviruses and provide insight into their clonal composition. To explore the advantages and limitations of these assays, we compared IPDA and Q4PCR measurements from 39 ART-suppressed people living with HIV. We found that IPDA and Q4PCR measurements correlated with one another, but frequencies of intact proviral DNA differed by approximately 19-fold. This difference may be in part due to inefficiencies in long-distance PCR amplification of proviruses in Q4PCR, leading to underestimates of intact proviral frequencies. In addition, nFGS analysis within Q4PCR explained that some of this difference is explained by proviruses that are classified as intact by IPDA but carry defects elsewhere in the genome. Taken together, this head-to-head comparison of novel intact proviral DNA assays provides important context for their interpretation in studies to deplete the HIV reservoir and shows that together the assays bracket true reservoir size.IMPORTANCE The intact proviral DNA assay (IPDA) and quadruplex PCR (Q4PCR) represent major advances in accurately quantifying and characterizing the replication-competent HIV reservoir. This study compares the two novel approaches for measuring intact HIV proviral DNA in samples from 39 antiretroviral therapy (ART)-suppressed people living with HIV, thereby informing ongoing efforts to deplete the HIV reservoir in cure-related trials.

Dataset Information

Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA.

Publications

Novel Assays for Measurement of Total Cell-Associated HIV-1 DNA and RNA.

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