Size, Composition, and Evolution of HIV DNA Populations during Early Antiretroviral Therapy and Intensification with Maraviroc

Abstract

Residual viremia is common during antiretroviral therapy (ART) and could be caused by ongoing low-level virus replication or by release of viral particles from infected cells. ART intensification should impact ongoing viral propagation but not virion release. Eighteen acutely infected men were enrolled in a randomized controlled trial and monitored for a median of 107 weeks. Participants started ART with ( = 9) or without ( = 9) intensification with maraviroc (MVC) within 90 days of infection. Levels of HIV DNA and cell-free RNA were quantified by droplet digital PCR. Deep sequencing of C2-V3 ,, and (454 Roche) was performed on longitudinally collected plasma and peripheral blood mononuclear cell (PBMC) samples while on ART. Sequence data were analyzed for evidence of evolution by (i) molecular diversity analysis, (ii) nonparametric test for panmixia, and (iii) tip date randomization within a Bayesian framework. There was a longitudinal decay of HIV DNA after initiation of ART with no difference between MVC intensification groups (-0.08 ± 0.01 versus -0.09 ± 0.01 log copies/week in MVC versus MVC groups; = 0.62). All participants had low-level residual viremia (median, 2.8 RNA copies/ml). Across participants, medians of 56 (interquartile range [IQR], 36 to 74), 29 (IQR, 25 to 35), and 40 (IQR, 31 to 54) haplotypes were generated for, , and regions, respectively. There was no clear evidence of viral evolution during ART and no difference in viral diversity or population structure from individuals with or without MVC intensification. Further efforts focusing on elucidating the mechanism(s) of viral persistence in various compartments using recent sequencing technologies are still needed, and potential low-level viral replication should always be considered in cure strategies. Residual viremia is common among HIV-infected people on ART. It remains controversial if this viremia is a consequence of propagating infection. We hypothesized that molecular evolution would be detectable during viral propagation and that therapy intensified with the entry inhibitor maraviroc would demonstrate less evolution. We performed a randomized double-blinded treatment trial with 18 acutely infected men (standard ART versus standard ART plus maraviroc). From longitudinally collected blood plasma and cells, levels of HIV DNA and cell-free HIV RNA were quantified by droplet digital PCR, and HIV DNA (, , and coding regions) was deep sequenced (454 Roche). Investigating people who started ART during the earliest stages of their HIV infection, when viral diversity is low, provides an opportunity to detect evidence of viral evolution. Despite using a battery of analytical techniques, no clear and consistent evidence of viral propagation for over 90 weeks of observation could be discerned.

Clones of infected cells arise early in HIV-infected individuals

“…Second, eliminating the reservoir is not a problem of blocking viral replication, but is rather a problem of eliminating all the infected cells that carry infectious proviruses, at least some of which have clonally expanded. It is likely that the forces which drove the clonal expansion in the first place will still be present following the administration of a curative therapy, such as "kick and kill" (44). Even if a strategy is found that activates the expression of all of the infectious proviruses in the cells of a particular clone, leading to the elimination of that clone, it is not clear that such a strategy would be able to successfully activate all of the infectious proviruses that are in all of the different clones of expanded cells.…”

“…Every cell that carries a fully intact provirus has the potential to release infectious virus that could rekindle the infection. Although it has been possible to eliminate large masses of tumor tissue with immunotherapy, the tumor cells that respond to immunotherapy express antigens that make it possible to target them (44). If, as appears to be the case, most of the HIV-infected cells that carry intact proviruses do not express viral RNA or proteins, eliminating the HIV-infected cells that carry fully intact proviruses will require a strategy that activates the expression of the proviruses in all, or almost all, of the cells that carry latent infectious proviruses or an alternative strategy that prevents any expressed virus from rekindling a systemic infection.…”

HIV Diversity and Genetic Compartmentalization in Blood and Testes during Suppressive Antiretroviral Therapy

“…We employed one genetic distance-based test (56) and one tree-based test (57), both of which were implemented in the HyPhy (v2.22) program (58). For the distance-based test, we employed the Hudson, Boos, and Kaplan nonparametric test for population structure (56), a test that has been validated for within-host HIV data sets (59), including from the reservoir, where it is sometimes referred to as the "nonparametric test for panmixia" (59)(60)(61). This test compares the mean pairwise distances between sequences from different subpopulations versus the same subpopulation (compartmentalization is supported if the mean pairwise distances of sequences from the same subpopulation are smaller than those from different subpopulations) and computes the K ST statistic, whose values range from 0 (denoting no compartmentalization) to 1 (denoting complete compartmentalization).…”

Differences in HIV Markers between Infected Individuals Treated with Different ART Regimens: Implications for the Persistence of Viral Reservoirs

“…The possibility of ongoing HIV replication in sanctuaries, where drug penetration is suboptimal, and its potential contribution to the residual viremia roused a considerable interest in drug intensification: adding one or more antiretroviral drugs to boost the ART regimen [91][92][93][94][95][96][97][98][99][100]. Most intensification studies added an INSTI (raltegravir or dolutegravir) to the ART regimen.…”

“…Therefore, its effect on 2-LTR circles and RV was explained by possible HIV latency reversal and induction of residual replication by the drug [112][113][114]. However, other groups failed to show any effect of maraviroc on 2-LTR circles or RV [98,115]. Interestingly, the addition of maraviroc to a raltegravir-based ART regimen in individuals starting ART resulted in a faster decline of plasma viremia, as well as lower levels of total HIV DNA and 2-LTR circles in the intensified arm [116], suggesting that suppression of HIV replication by INSTI-based regimens may also be incomplete.…”