Abstract
BackgroundPersistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication. Methylation of the proviral 5′ long terminal repeat (LTR) promoter region has been proposed as a possible mechanism contributing to HIV latency; however, conflicting observations exist regarding its relevance. We assessed 5′-LTR methylation profiles in total CD4+ T cells from blood of 12 participants on short-term ART (30 months) followed up for 2 years, and a cross-sectional group of participants with long-term ART (6–15 years), using next generation sequencing. We then looked for associations between specific 5′-LTR methylation patterns and baseline and follow-up clinical characteristics.Results5′-LTR methylation was observed in all participants and behaved dynamically. The number of 5′-LTR variants found per sample ranged from 1 to 13, with median sequencing depth of 16270× (IQR 4107×-46760×). An overall significant 5′-LTR methylation increase was observed at month 42 compared to month 30 (median CpG Methylation Index: 74.7% vs. 0%, p = 0.025). This methylation increase was evident in a subset of participants (methylation increase group), while the rest maintained fairly high and constant methylation (constant methylation group). Persons in the methylation increase group were younger, had higher CD4+ T cell gain, larger CD8% decrease, and larger CD4/CD8 ratio change after 48 months on ART (all p < 0.001). Using principal component analysis, the constant methylation and methylation increase groups showed low evidence of separation along time (factor 2: p = 0.04). Variance was largely explained (21%) by age, CD4+/CD8+ T cell change, and CD4+ T cell subpopulation proportions. Persons with long-term ART showed overall high methylation (median CpG Methylation Index: 78%; IQR 71–87%). No differences were observed in residual plasma viral load or proviral load comparing individuals on short-term (both at 30 or 42 months) and long-term ART.ConclusionsOur study shows evidence that HIV 5′-LTR methylation in total CD4+ T cells is dynamic along time and that it can follow different temporal patterns that are associated with a combination of baseline and follow-up clinical characteristics. These observations may account for differences observed between previous contrasting studies.
Highlights
Persistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication
Our study shows for the first time that HIV 5′-long terminal repeat (LTR) methylation is dynamic along time, with different methylation variation patterns possibly associated with baseline clinical characteristics of persons starting ART
We show strong evidence that HIV 5′-LTR methylation can vary significantly along time in proviruses from circulating CD4+ T cells and that methylation is observed from early stages of ART initiation
Summary
Persistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication. Modern combination antiretroviral treatment (ART) reduces human immunodeficiency virus (HIV) plasma viral load (pVL) to undetectable levels (< 40 HIV RNA copies/mL), achieving a 4–5 log reduction during the first year of treatment [1]. "Kick and kill" HIV cure strategies aim to achieve complete activation of latently-infected cells so that they can be eliminated by diverse immunologic/pharmacologic strategies. To this day, these strategies have shown limited success [6]
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