Development of a sensitive, quantitative assay with broad subtype specificity for detection of total HIV-1 nucleic acids in plasma and PBMC

C N Kibirige,Y Liu,D Wooding,M Manak,H Hack,D King,N Imami,N Fernandez,B Abel,Steve Kaye,L Jagodzinski,J Dalel,J Gilmour

doi:10.1038/s41598-021-03016-1

Abstract

An LTR-based quantitative PCR (qPCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and PBMC. TaqMan qPCR primers and probes were designed against the NCBI/LANL HIV-1 compendium database by analyzing sequences used in assays for sensitive cross-clade detection of HIV-1 as reported in the literature and elucidating regions of improved cross-subtype specificity. Inosine and mixed nucleotide bases were included at polymorphic sites. Real-time RT-qPCR and qPCR were performed on plasma viral RNA and cellular lysates. A step-up amplification approach to allow binding of primers across polymorphic regions showed improved sensitivity compared to universal cycling. Unlike a lead competing laboratory-developed assay, all major HIV-1 subtypes, and a wide range of recombinants from a 127-member diversity panel were detected and accurately quantified in spiked plasmas. Semi-nested PCR increased detection sensitivity even further. The assay was able to detect down to 88 copies/mL of HIV-1 in plasma with 95% efficiency or the equivalent of a single infected cell. The PCR assay will be valuable in studies that monitor very low viral levels including residual or break through HIV-1 in patients receiving antiretroviral therapy, in HIV-1 cure, and in other research studies.

Highlights

An long terminal repeat (LTR)-based quantitative polymerase chain reaction (PCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and peripheral blood mononuclear cells (PBMCs)
The use of highly sensitive laboratory developed assays (LDAs), such as the Single-Copy Assay (SCA) which detects down to 1 copy of HIV-1 RNA per ml of plasma, has shown that discontinuation of antiretroviral treatment by HIV-infected patients with plasma RNA levels above 10–15 copies/ml results in viremia rebound earlier and more often than patients who have lower or undetectable RNA5,6. These results indicate that candidates for viral control or cure studies may have better success if viral loads are driven below 15 copies/ml before treatment cessation
We describe a sensitive polymerase chain reaction (PCR)-based assay which can be used in various formats to detect residual HIV-1 nucleic acids in the plasma and PBMCs of HIV-1 infected individuals undergoing combination anti-retroviral therapy (cART)

Summary

Introduction

An LTR-based quantitative PCR (qPCR) assay was modified and optimized for the quantification of total HIV-1 nucleic acids in plasma and PBMC. The use of highly sensitive laboratory developed assays (LDAs), such as the Single-Copy Assay (SCA) which detects down to 1 copy of HIV-1 RNA per ml of plasma, has shown that discontinuation of antiretroviral treatment by HIV-infected patients with plasma RNA levels above 10–15 copies/ml results in viremia rebound earlier and more often than patients who have lower or undetectable RNA5,6 These results indicate that candidates for viral control or cure studies may have better success if viral loads are driven below 15 copies/ml before treatment cessation. HIV-1 DNA integrates into the human chromosome and persists in an inactive state even in the presence of ongoing antiretroviral therapy This means that reduction of plasma viral load to low levels, even below 10–15 copies/ml as indicated by the highly efficient SCA assay, does not lead to viral eradication. The quantitative virus outgrowth assay (QVOA) is considered the gold-standard assay for this type of assessment, but is complicated, expensive and time intensive, and despite recent improvements, is not practical for routine viral m onitoring[10]

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