Sequence Evaluation and Comparative Analysis of Novel Assays for Intact Proviral HIV-1 DNA
Open Access
- 24 February 2021
- journal article
- research article
- Published by American Society for Microbiology in Journal of Virology
- Vol. 95 (6)
- https://doi.org/10.1128/jvi.01986-20
Abstract
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.Keywords
Funding Information
- HHS | National Institutes of Health (UM1AI100663, R01AI129795)
- HHS | National Institutes of Health (1P30AI124414-01A1)
- HHS | National Institutes of Health (UM1 AI126620)
- HHS | National Institutes of Health (UL1TR001866)
- HHS | National Institutes of Health (1U01AI129825)
- HHS | National Institutes of Health (UM1AI126619)
- HHS | National Institutes of Health (R01AI134363)
- HHS | National Institutes of Health (F30AI145588)
- Bill and Melinda Gates Foundation (OPP1092074)
This publication has 31 references indexed in Scilit:
- Proliferation of latently infected CD4+ T cells carrying replication-competent HIV-1: Potential role in latent reservoir dynamicsThe Journal of Experimental Medicine, 2017
- Paired quantitative and qualitative assessment of the replication-competent HIV-1 reservoir and comparison with integrated proviral DNAProceedings of the National Academy of Sciences of the United States of America, 2016
- Defective proviruses rapidly accumulate during acute HIV-1 infectionNature Medicine, 2016
- Precise Quantitation of the Latent HIV-1 Reservoir: Implications for Eradication StrategiesThe Journal of Infectious Diseases, 2015
- Replication-Competent Noninduced Proviruses in the Latent Reservoir Increase Barrier to HIV-1 CureCell, 2013
- Comparative Analysis of Measures of Viral Reservoirs in HIV-1 Eradication StudiesPLoS Pathogens, 2013
- Immediate antiviral therapy appears to restrict resting CD4 + cell HIV-1 infection without accelerating the decay of latent infectionProceedings of the National Academy of Sciences of the United States of America, 2012
- The Effect of Primer-Template Mismatches on the Detection and Quantification of Nucleic Acids Using the 5′ Nuclease AssayThe Journal of Molecular Diagnostics, 2010
- A universal real-time PCR assay for the quantification of group-M HIV-1 proviral loadNature Protocols, 2008
- Effects of primer-template mismatches on the polymerase chain reaction: Human immunodeficiency virus type 1 model studiesNucleic Acids Research, 1990