Measuring Proviral HIV-1 DNA: Hurdles and Improvements to an Assay Monitoring Integration Events Utilising Human <i>Alu</i> Repeat Sequences
Eva Malatinkova,
Jordan Thomas,
Ward De Spiegelaere,
Sofie Rutsaert,
Anna Maria Geretti,
Georgios Pollakis,
William A. Paxton,
Linos Vandekerckhove,
Alessandra Ruggiero
Affiliations
Eva Malatinkova
HIV Cure Research Center, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium
Jordan Thomas
Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK
Ward De Spiegelaere
Laboratory of Veterinary Morphology, Faculty of Veterinary Sciences, Ghent University, B-9820 Ghent, Belgium
Sofie Rutsaert
HIV Cure Research Center, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium
Anna Maria Geretti
Fondazione PTV and Faculty of Medicine, University of Rome Tor Vergata, 00133 Rome, Italy
Georgios Pollakis
Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK
William A. Paxton
Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK
Linos Vandekerckhove
HIV Cure Research Center, Department of Internal Medicine, Faculty of Medicine and Health Sciences, Ghent University, B-9000 Ghent, Belgium
Alessandra Ruggiero
Department of Clinical Infection, Microbiology and Immunology, Institute of Infection, Veterinary and Ecological Sciences, University of Liverpool, Liverpool L69 7BE, UK
Integrated HIV-1 DNA persists despite antiretroviral therapy and can fuel viral rebound following treatment interruption. Hence, methods to specifically measure the integrated HIV-1 DNA portion only are important to monitor the reservoir in eradication trials. Here, we provide an up-to-date overview of the literature on the different approaches used to measure integrated HIV-1 DNA. Further, we propose an implemented standard-curve free assay to quantify integrated HIV-1 DNA, so-called Alu-5LTR PCR, which utilises novel primer combinations. We tested the Alu-5LTR PCR in 20 individuals on suppressive ART for a median of nine years; the results were compared to those produced with the standard-free Alu-gag assay. The numbers of median integrated HIV-1 DNA copies were 5 (range: 1–12) and 14 (5–26) with the Alu-gag and Alu-5LTR, respectively. The ratios between Alu-gag vs Alu-5LTR results were distributed within the cohort as follows: most patients (12/20, 60%) provided ratios between 2–5, with 3/20 (15%) and 5/20 (25%) being below or above this range, respectively. Alu-5LTR assay sensitivity was also determined using an “integrated standard”; the data confirmed the increased sensitivity of the assay, i.e., equal to 0.25 proviruses in 10,000 genomes. This work represents an improvement in the field of measuring proviral HIV-1 DNA that could be employed in future HIV-1 persistence and eradication studies.
