Medical Virology

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Browsing Medical Virology by Subject "AIDS-associated retrovirus"

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    Characterization of the persisting HIV-1 reservoir in early treated children on long-term suppressive ARV regimens
    (Stellenbosch : Stellenbosch University, 2019-12) Katusiime, Mary Grace Kato; Van Zyl, Gert Uves; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Pathology: Medical Virology.
    Introduction: The major barrier to curing HIV-1 infection is a latent reservoir of long-lived, replication-competent proviruses that persists despite suppressive antiretroviral therapy (ART). Initiating ART during acute infection limits the development of phylogenetically diverse reservoirs. Novel approaches for reservoir elimination are emerging, providing hope for a cure. Perinatally infected, early-treated children, are likely good candidates for cure interventions as they have low immune activation states and a low proportion of central memory T-cells. We studied the post-CHER cohort of perinatally infected children, who initiated ART during acute infection and are in long-term follow-up. To inform cure interventions, we aimed to: (i) quantify latently infected cells and describe longitudinal genetic diversity, (ii) describe mechanisms that enable long-term reservoir persistence, (iii) describe the extent to which early therapy shapes the proviral landscape. Methods: In Aim I, we used a sensitive quantitative PCR assay for HIV-1 cell associated DNA (iCAD), followed by cell-associated DNA-single genome sequencing (CAD-SGS) of 1200bp in HIV-1 gag-pol to investigate genetic evolution in the reservoir during long-term ART. We performed 3 tests for evolution: (i) average pairwise distance (APD) for intra-patient viral population diversity, (ii) panmixia for probability of shifts in viral population structure, (iii) maximum likelihood (ML) root-to-tip distances to detect emergence of new viral populations. In Aim II, we performed integration site analysis (ISA) on samples from close to therapy initiation (baseline) and after 6-9 years on ART to investigate clonal expansion as a mechanism for reservoir persistence despite early, suppressive therapy. In Aim III, near full length- proviral amplification and sequencing (NFL-PAS) was performed to determine the proportion of genetically intact vs defective proviruses after 6-9 years on ART. Findings: We found low iCAD levels (median iCAD:22.45cp/106) in 16 children who initiated ART within the first 18 months of life. No significant changes in intra-patient proviral diversity, shifts in viral population structure or emergence of new viral populations were detected in children who were fully suppressed on ART, suggesting that ART prevents ongoing replication that replenishes the reservoir. ISA detected expanded clones in baseline samples of 6 children treated as early as 2 months of age, suggesting that infected cells begin clonally expanding before ART. Furthermore, there was a significant increase in the proportion of expanded clones after several years. A total of 738 NFL amplicons were generated from 9 children. Of these, 72.9% had large internal deletions, 23.7% were hypermutated, 1.4% had small internal deletions, and 1% had deletions in the gag-leader region. Intact proviruses were detected at a frequency of 1%. This study showed that early therapy and long-term suppression in children leads to limited reservoir size and genetic diversity, factors that are favourable for cure interventions. The reservoir appears to be maintained by clonal expansion that begins before therapy is initiated. Although a large proportion of proviral DNA in long-term suppressed children is defective, genetically intact variants persist and likely form part of expanded clones. This suggests the need for novel approaches that target HIV reservoirs by reducing proliferation of cells that harbour replication-competent proviruses.