The aim of this study was to investigate whether ongoing viral replication contributes to HIV persistence under ART. To the best of our knowledge, this is the first study where ART intensification was achieved not by adding a new antiretroviral drug to the existing regimen but by increasing the dosage of a drug that was already part of the regimen pre-intensification. We evaluated the impact of doubling the dosage of DTG on HIV cellular and tissue reservoirs, immune activation, exhaustion, and inflammation in ART-suppressed PWH.
As was to be expected, plasma and tissue DTG concentrations significantly increased between day 0 and day 84 in the intensified group but not in the control group. Accordingly, meaningful decreases in total HIV DNA, intact HIV DNA, US HIV RNA, and US RNA/total DNA ratio in PBMCs were observed during the study period in the intensified group but not in the control group.This strongly suggests that the pre-intensification ART regimen was not entirely suppressive. The transient decrease in total DNA at day 28 may have been due to an initial decrease in newly infected cells upon ART intensification; however, at the subsequent time points, this affect was likely masked by proliferation (clonal expansion) of infected cells, mostly containing defective proviruses. In line with this, the number of intact proviruses decreased, but that of total proviruses did not change, between days 0 and 84.
In addition, intensification modestly yet significantly reduced percentages of CD4+ T cells expressing TIGIT, a T-cell exhaustion marker, and percentages of CD8+ T cells co-expressing CD38 and HLA-DR, T-cell activation markers. Interestingly, CD4+ cells expressing TIGIT have been shown to be enriched for HIV persistence in ART-treated PWH. Arguably,the reduction of HIV reservoir upon intensification has caused a corresponding reduction in chronic immune activation and exhaustion in this cohort. indeed, changes in US HIV RNA levels and US RNA/total DNA ratio during the study positively correlated with changes in levels of markers of immune activation, exhaustion, and inflammation, suggesting a link between changes in HIV reservoir activity and levels of immune activation and inflammation. These results are in line with previous studies reporting decreases in immune activation markers upon ART intensification.
In contrast, in the control group, we measured significant longitudinal increases in total HIV DNA, percentages of CD4+ T cells expressing PD-1, another T-cell exhaustion marker, and in the plasma levels of IL-6, an inflammatory cytokine, and of CRP, another marker of inflammation. Residual HIV replication, among other factors, could have contributed to these effects in the control group. However, in the control group, we also observed a reduction in the percentages of CD8+ T cells co-expressing a T-cell activation marker CD38 and a strong reduction in the plasma levels of sCD14. One possible explanation for these effects can be increased adherence of the participants to their ART regimens during the study.
Two previous studies have measured a transient increase in HIV episomal DNA (2-LTR circles) upon ART intensification with raltegravir, an INSTI. As 2-LTR circles are a by-product of HIV integration and are expected to accumulate if integration is blocked, this was interpreted as strong evidence of new infections pre-intensification.However, another study did not observe any change in 2-LTR circles upon ART intensification with DTG.
as a part of a standard regimen even in PWH without resistance. However, this has to be approached with caution as intensification has caused a transient decrease in the CD4/CD8 ratio in our study.
The main strength of our study is the measurement of a large number of virological and immunological parameters that allowed us to observe a significant impact of intensification on both virus and host. Our study is the first to observe significant reductions in as many as four HIV markers following ART intensification. Moreover, we measured plasma and tissue concentrations of DTG, allowing us to directly demonstrate their increase upon intensification. Our results are in line with some earlier studies that reported a decrease in cell-associated HIV RNA or a transient increase in 2-LTR circles upon intensification (buzón et al., 2010; Yukl et al., 2010; Hatano et al., 2013). However, they are in contrast with other studies, in which treatment intensification did not affect HIV markers (Dinoso et al., 2009; McMahon et al., 2010; Gandhi et al., 2010). As this is the first ART intensification study to increase the dosage of an existing antiretroviral drug rather of adding a new drug, the results of this study and those of previous intensification studies cannot be directly compared.
Some limitations can be highlighted. First, we included twenty PWH (10 participants per arm). Although this small sample size does somewhat limit the robustness of the conclusions, we would like to emphasize that in this cohort, we performed a very detailed analysis, longitudinally measuring 30 biomarkers at five time points during ART. Secondly, despite the randomization, the groups were still not perfectly balanced as levels of total HIV DNA in PBMCs were by chance higher at baseline in the intensified group. However, this is unlikely to have affected the conclusions of the study because (1) this study has a within-subject (repeated-measures) design, with the longitudinal change of a parameter within the same participant during the study being the main outcome, and (2) this difference most probably reflects a higher number of infected cells, and not a higher level of residual replication, at baseline in the intensified group. No significant differences between the groups were found at baseline in the levels of the other four virological markers we measured (US RNA in PBMCs,US RNA/total DNA ratio,intact DNA in PBMCs,and total DNA in tissue),as well as between the antiretroviral drug concentrations in plasma or in tissue. The lack of blinding and placebo control,the predominantly male study population,and the absence of post-intervention follow-up represent additional limitations of our study. Moreover, these findings should be considered exploratory and hypothesis-generating, warranting confirmation in larger, placebo-controlled, and blinded trials. Still,we believe that the convergence of the effect of intensification on multiple reservoir markers in the same direction indicates a possibly meaningful biological signal that merits further investigation.
doubling the DTG dosage in PWH who had been suppressed on DTG-containing ART for a number of years resulted in a reduction in the levels of four HIV reservoir markers in peripheral blood, and also in markers of T-cell activation and exhaustion. If confirmed in larger clinical trials, these results could have an impact on the clinical management of PWH. Moreover, if ongoing viral replication does indeed replenish HIV reservoirs over time, then improving ART regimens should be a necessary part of the curative strategies.
