Characterization of the persisting HIV-1 reservoir in early treated children on long-term suppressive ARV regimens

Katusiime, Mary Grace Kato (2019-12)

Thesis (PhD)--Stellenbosch University, 2019.

Thesis

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.

Inleiding: Die groot hindernis in die soektog na ’n geneesmiddel vir MIV-1-infeksie is ’n latente reservoir langlewende, replikasievaardige provirusse wat ondanks doeltreffende antiretrovirale terapie (ART) oorleef. Die aanvang van ART gedurende akute infeksie beperk die saaiing en ontwikkeling van filogeneties diverse reservoirs. Namate die vakgebied verder ontwikkel, kom nuwe benaderings aan die lig om dié reservoir uit te wis, wat hoop bied vir ’n geneesmiddel. Kinders wat perinataal geïnfekteer is en vroeg reeds met behandeling begin, is waarskynlik goeie kandidate vir genesingsintervensies omdat hulle laer immuunaktiveringstoestande en minder sentralegeheue-T-selle as volwassenes het. Die CHER-kohort (“Children with HIV Early Antiretroviral Therapy”) is ’n groep perinataal geïnfekteerde kinders van wie sommige gedurende akute infeksie reeds met ART begin het en die afgelope dekade deur middel van opvolgbesoeke gemoniteer is. Om die soektog na ’n geneesmiddel te ondersteun, was die doel van hierdie studie (i) om latent geïnfekteerde selle te kwantifiseer en provirale genetiese diversiteit oor tyd te beskryf, (ii) om meganismes te beskryf wat die reservoir in staat stel om op lang termyn te oorleef, en (iii) om te beskryf watter invloed vroeë terapie op die provirale landskap by ’n substel van die CHER-kohort het. Metodes: Vir doelwit I is ’n sensitiewe kwantitatiewe PCR-toets vir MIV-1-selverwante DNS (CAD) by 16 kinders gebruik, gevolg deur selverwante DNS-enkelgenoomreeksvorming (CAD-SGS) van ’n 1 200 bp-fragment wat oor MIV-1-gag-pol strek, om die genetiese evolusie in die reservoir ná ses tot nege jaar op behandeling te ondersoek. Daarna is drie sensitiewe evolusietoetse uitgevoer: (i) gemiddelde paarsgewyse afstand (APD) om virale populasiediversiteit binne pasiënte te bepaal, (ii) panmiksie (lukrake paring) om die waarskynlikheid van verskuiwings in virale populasiestruktuur vas te stel, en (iii) maksimumaanneemlikheids- (ML-)afstande van wortel tot punt om die ontwikkeling van nuwe virale populasies op te spoor. Vir doelwit II is integrasiesetelontleding (ISA) op monsters van 12 kinders uitgevoer na aan terapieaanvang (basislyn) en weer ná ses tot nege jaar op ART. Die doel hiermee was om klonale uitbreiding te ondersoek as ’n meganisme vir reservoir-oorlewing ondanks vroeë, langdurige onderdrukkingsterapie. Vir doelwit III is byna-vollengte- provirale versterking en reeksvorming (NFL-PAS) op nege kinders uitgevoer om die MIV-1- provirale landskap te beskryf en die hoeveelheid geneties ongeskonde teenoor disfunksionele provirusse ná ses tot nege jaar op ART te bepaal. Bevindinge: CAD-ladings was laag (mediaan: 22,45 cp/106) vir die 16 kinders wat binne die eerste 18 maande ná geboorte met ART begin het, wat daarop dui dat vroeë behandeling die frekwensie van latent-geïnfekteerde selle beperk. Geen beduidende veranderinge in provirale diversiteit binne pasiënte, verskuiwings in virale populasiestruktuur óf ontwikkeling van nuwe virale populasies is opgemerk by kinders wie se toestand ten volle onderdruk is op ART nie. Dít gee te kenne dat ART voortdurende replikasiesiklusse, wat die reservoir kan aanvul, voorkom. ISA het op uitgebreide klone afgekom in voor-ART-monsters van ses kinders wat op so jonk as twee maande met terapie begin het, wat daarop dui dat geïnfekteerde selle klonaal begin uitbrei reeds voordat ART kort ná geboorte by kinders aangevoor word. Daarbenewens het die hoeveelheid uitgebreide klone ná ’n aantal jaar beduidend toegeneem. Van die 738 NFL-amplikone wat van nege kinders verkry is, het 72,9% groot interne delesies gehad, 23,7% hipermutasie, 1,4% klein interne delesies, en 1% delesies in die verpakkingsein/groot splitsskenkersetel. Seldsame ongeskonde provirusse is teen ’n frekwensie van 1% opgespoor, wat soortgelyk is aan volwassenes wat gedurende akute infeksie behandel word.

Please refer to this item in SUNScholar by using the following persistent URL: http://hdl.handle.net/10019.1/106914
This item appears in the following collections: