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.

FIG 1

Levels of proviral HIV-1 DNA and unspliced cellular HIV-1 RNA in PBMCs and HIV-1 RNA in plasma. Long horizontal lines, median values; short horizontal lines, first and third quartile values. CA HIV-1 RNA and plasma HIV-1 RNA levels less than the lower limit of detection are represented as one-half of the corresponding limit of detection. CAD, cell-associated HIV-1 DNA (copies/10⁶ PBMCs); CAR, cell-associated HIV-1 RNA (copies/10⁶ PBMCs); SCA, plasma HIV-1 RNA as determined by single-copy assay (copies/ml).

FIG 2

Short-term variations in levels of proviral HIV-1 DNA and unspliced RNA in PBMCs and HIV-1 RNA in plasma collected at two time points. (A) Short-term variations in CA HIV-1 DNA (CAD) levels in cryopreserved PBMCs. (B) Short-term variations in CA HIV-1 RNA (CAR) levels in cryopreserved PBMCs. (C) Short-term variations in HIV-1 RNA levels in plasma (SCA). Solid lines, viremic samples; dashed lines, virologically suppressed samples. cps or cp, copies.

FIG 3

Correlations between HIV-1 DNA and HIV-1 RNA results according to cell purification method, i.e., beads alone versus beads plus sorting. (A) Correlation of CA HIV-1 DNA levels in tCD4 cells isolated by negative selection alone (bead) versus tCD4 cells isolated by negative selection followed by FACS. (B) Correlation of CA HIV-1 RNA levels in tCD4 cells isolated by beads versus tCD4 cells isolated by FACS. (C) Correlation of CA HIV-1 DNA levels in rCD4 cells isolated by beads versus rCD4 cells isolated by FACS. (D) Correlation of CA HIV-1 RNA levels in rCD4 cells isolated by beads versus rCD4 cells isolated by FACS. Long dashed lines, CA HIV-1 DNA; short dashed lines, CA HIV-1 RNA. cp, copies.

FIG 4

Correlations between HIV-1 DNA and HIV-1 RNA results according to collection method, i.e., leukapheresis versus phlebotomy. (A) Correlation of CA HIV-1 DNA levels in tCD4 cells from leukapheresis (LKP) versus tCD4 cells from large-volume phlebotomy (blood). (B) Correlation of CA HIV-1 RNA levels in tCD4 cells from leukapheresis versus tCD4 cells from blood. (C) Correlation of CA HIV-1 DNA levels in rCD4 cells from leukapheresis versus rCD4 cells from blood. (D) Correlation of CA HIV-1 RNA levels in rCD4 cells from leukapheresis versus rCD4 cells from blood. Long dashed lines, CA HIV-1 DNA; short dashed lines, CA HIV-1 RNA. cp, copies.