https://orcid.org/0000-0001-5714-0002
(.)
TO THE EDITOR—Human immunodeficiency virus type 1 (HIV) infection has become a manageable chronic condition as the safety and efficacy of antiretroviral therapy (ART) continue to improve. Long-acting therapies are emerging that may remove the stigma of daily dosing, but the many other burdens of chronic HIV infection still drive HIV cure research to seek an ultimate solution.
Central challenges to researchers seeking to understand persistent HIV infection and develop curative interventions are the demanding laboratory assays required to quantitate the stable but inducible viral reservoir in people with HIV infection (PWH) on ART. The long-lived reservoir of viral genomes that are competent to produce infectious virions resides principally in resting central memory CD4+ T cells, although during the process of T-cell differentiation and proliferation, proviral clones may expand to the effector memory pool as well [1–3]. However, the quantification of CD4+ T cells carrying intact proviruses that can drive viral rebound is obscured by a large population CD4+ cells carrying defective HIV deoxyribonucleic acid (DNA) genomes [4, 5]. Although the total population of the few replication-competent and many defective HIV genomes can be measured accurately and simply by DNA polymerase chain reaction (PCR) assays, direct measurement of the true rebound-competent reservoir requires the use of quantitative viral outgrowth assays ([QVOAs] [6, 7]), a definitive and useful metric that is too costly and demanding to be widely applied, particularly in broad clinical trial settings.
Bruner et al [8] have described a clever droplet digital PCR assay of HIV DNA designed to take advantage of probes that target 2 amplicons from relatively conserved regions of the HIV genome, and thereby identify genomes likely to lack mutations or deletions: the Intact Proviral DNA Assay [IPDA]. One IPDA amplicon interrogates the packaging signal region Ψ, and the other, an area in env 5’ deletions, often results in the loss of the Ψ amplicon, whereas 3’ deletions or hypermutations often result in the loss of the env amplicon. Detection of both amplicons (Figure 1, quadrant 2) indicate a viral genome likely to be intact and replication-competent. Bruner et al [8] found, in a limited cohort, that levels of intact proviral DNA measured with the IPDA correlate with levels of replication-competent HIV-1 assessed by the virus outgrowth assay but are, on average, 50- to 100-fold higher.
![Detection of both Intact Proviral DNA Assay amplicons indicates a proviral genome likely to be intact (Q2). From Figure 2 [8].](https://oup.silverchair-cdn.com/oup/backfile/Content_public/Journal/jid/223/2/10.1093_infdis_jiaa533/1/m_jiaa533_fig1.jpeg?Expires=1749904051&Signature=kU9-IPW5xcU1kfZgMb69P4fylP7k8uCih6Js47HPvYc3f-zTp7xu-VlzkN5hJO2Dw7FF7nZlgLSpImPcpSDwx027G~-b57J-1Pdy8F5X5vBwJv~TGrwmMjw0IWQPi7zOMyKWM~gCX-1j4vG5VeQaCSbEGmfuDRk3gEduC289cPpVU-~wQGOGP~~u0TDeh5zPJy-lUnh~mm8ZVrM4EnJge-lBr53swUJ6NXkCV33xKZpoF8Id5KVXayI7hN4vPbc0N7lHdTu2GT4dRBjnINLc4SJd9K63YfJbWYt0CoubjyFk2cOw~XWRz6a8HknQEGcVH7NW0B4A7VBBTJFp72JzhQ__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Detection of both Intact Proviral DNA Assay amplicons indicates a proviral genome likely to be intact (Q2). From Figure 2 [8].
In this issue of the Journal of Infectious Diseases, Gandhi et al ([9], pg X) expanded the assessment of the performance of the IPDA. They sought to determine whether the intact proviral DNA reservoir as measured by IPDA declines over time on ART and, if so, how this decline is correlated with other measures of HIV-1 persistence: HIV-1 DNA, cell-associated HIV-1 ribonucleic acid (RNA), and residual plasma viremia. Because it has been hypothesized that the persistence of HIV infection despite ART is driven by HIV-related persistent inflammation and T-cell activation that may persist despite ART, changes in these parameters over time were also assessed in longitudinal specimens from a cohort of people on ART with long-term suppression of viremia.
Gandhi et al obtained samples from 50 PWH who had enrolled in AIDS Clinical Trials Group Study A5321 and remained in follow-up with plasma HIV-1 RNA levels <50 copies/mL by commercial assays starting at week 48 of ART and at all subsequent time points. None reported ART interruptions. Of the 50 participants, IPDA failed to amplify viral DNA in 6 (12%), presumably due to viral sequence diversity. The IPDA is currently validated to amplify Clade B sequences, but even within Clade B cohorts assay failure is sometimes seen. Ongoing studies in diverse cohorts are expected to clarify the frequency of assay failure: one study of 400 patients finds a failure rate of 6.3% [9].
Samples from participants were assayed at 3 timepoints following durable ART: at a median of 7.1, 10.8, and 12.6 years after ART initiation. Ultimately, 40 of the remaining 44 participants were fully assessed, because samples from 4 were unavailable due to various technical and protocol-driven reasons. The study population was reflective of the current US epidemic, with 50% black and Hispanic participants, a mean age of 41 years, 27% women, a median gain of 429/µL CD4+ cells on ART to a median count of 748/µL, and a representative distribution of modern ART regimens.
The key finding in these 40 participants was a significant decline in intact provirus as measured by IPDA (Q2, Figure 1) with continued administration of ART. The rate of IPDA decline (median half-life 7.1 years; 95% confidence interval [CI], 3.9–18 years) was somewhat slower than the half-life described for replication-competent HIV, as measured by QVOA by Crooks et al [6] (3.6 years) and Siliciano et al [7] (3.6–3.7 years), but within the range observed by Crooks et al [6] (2.3–8.1 years) where reduced slopes of decline tended to be seen in participants studied after longer times on ART, as were the participants in the Gandhi et al study [9].
In contrast, defective provirus levels (Q1 and Q4, Figure 1) did not substantially decline. The median half-life of total HIV-1 DNA was 41.6 years (95% CI, 13.6–75 years). The proportion of all proviruses that were intact diminished over time on ART, from approximately 10% at the first on-ART time point to approximately 5% at the last. Intact provirus levels on ART correlated with total HIV-1 DNA and residual plasma viremia, but there was also no evidence of association between intact proviral levels and inflammation or immune activation, as has also been recently reported in a study that used QVOA and the IPDA to measure the latent reservoir in a cohort of men and women [11].
The progressive loss of cells containing intact, replication-competent proviruses during suppressive ART is, of course, expected. However, a head-to-head comparison of IPDA to QVOA over time would be informative, and such studies are underway. The assay may be challenged by sequence diversity in some cohorts or populations; in this study, only 12% of the participants could not be assessed. In addition, new primer/probe sets must be developed to assay populations infected with non-Clade B viruses. Primer/probe sets have been developed to allow the use of IPDA in simian immunodeficiency virus infection, simian HIV, and HIV-2 infection [12].
If efforts towards HIV eradication begin to find success, the relative ease of use of IPDA, and its requirement for a relatively low number of peripheral blood cells, may allow its broad use in HIV cure research and, ultimately, in clinical trials. Several other studies have been recently published to reinforce this view [10, 13–15].
Notes
Financial support. This work was funded by National Institutes of Health Grant UM1-AI126619 (to D.M.M.).
Potential conflicts of interest. D.M.M. reports personal fees from Merck and ViiV Healthcare and holds common stock in Gilead. All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
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