Intact HIV Proviruses Persist in Children Seven to Nine Years after Initiation of Antiretroviral Therapy in the First Year of Life

Abstract

In adults starting antiretroviral therapy (ART) during acute infection, 2% of proviruses that persist on ART are genetically intact by sequence analysis. In contrast, a recent report in children treated early failed to detect sequence-intact proviruses. In another cohort of children treated early, we sought to detect and characterize proviral sequences after 6 to 9 years on suppressive ART. Peripheral blood mononuclear cells (PBMC) from perinatally infected children from the Children with HIV Early antiRetroviral (CHER) study were analyzed. Nearly full-length proviral amplification and sequencing (NFL-PAS) were performed at one time point after 6 to 9 years on ART. Amplicons with large internal deletions were excluded (<9 kb). All amplicons of ≥9 kb were sequenced and analyzed through a bioinformatic pipeline to detect indels, frameshifts, or hypermutations that would render them defective. In eight children who started ART at a median age of 5.4 months (range, 2.0 to 11.1 months), 733 single NFL-PAS amplicons were generated. Of these, 534 (72.9%) had large internal deletions, 174 (23.7%) had hypermutations, 15 (1.4%) had small internal deletions, 3 (1.0%) had deletions in the packaging signal/major splice donor site, and 7 (1.0%) were sequence intact. These 7 intact sequences were from three children who initiated ART after 2.3 months of age, one of whom had two identical intact sequences, suggestive of a cell clone harboring a replication-competent provirus. No intact proviruses were detected in four children who initiated ART before 2.3 months of age. Rare, intact proviruses can be detected in children who initiate ART after 2.3 months of age and are probably, as in adults, maintained by clonal expansion of cells infected before ART initiation. IMPORTANCE There are limited data about the proviral landscape in children exhibiting long-term suppression after early treatment, particularly in Sub-Saharan Africa where HIV-1 subtype C predominates. Investigating the sequence-intact reservoir could provide insight on the mechanisms by which intact proviruses persist and inform ongoing cure efforts. Through nearly full-length proviral amplification and sequencing (NFL-PAS), we generated 733 NFL-PAS amplicons from eight children. We showed that rare, genetically intact proviruses could be detected in children who initiated ART after 2.3 months of age. The frequency of intact proviruses was lower (P < 0.05) than that reported for HIV subtype B-infected adults treated during early HIV infection. We show that cells harboring genetically intact HIV proviruses are rare in children exhibiting long-term suppression after early treatment and may require the processing of a large number of cells to assess reservoir size. This points to the need for efficient methods to accurately quantify latent reservoirs, particularly in pediatric studies where sample availability is limited.

Early Emergence and Long-Term Persistence of HIV-Infected T Cell Clones in Children

“…These results, in conjunction with previous studies showing that ongoing HIV-1 replication does not occur in children when viremia is fully suppressed on ART (3, 57, 58) and the fact that intact proviruses persist for years both in adults treated early (16) and in children treated early (20), supports the conclusion that the HIV-1 reservoir is maintained in vertically-infected children through the proliferation of cells infected prior to ART initiation, as it is in adults (6,32,35,59).…”

“…However, the available data are limited by the rarity of infected cells and the very small subset of HIV-infected cells that harbor intact, replication-competent proviruses in children (20). Although further studies are required to increase our understanding of the clonal expansion of intact proviruses as a mechanism by which the reservoir persists in both children and adults, it is possible that defective proviruses can undergo complementation upon ART interruption and contribute to viral rebound (60).…”

“…These defective proviruses do not directly contribute to the HIV-1 reservoir that persists on ART but complicate its measurement and may contribute to persistent immune activation. The fraction of infected cells that contains replication-competent (intact/infectious) proviruses has been estimated to be between 1 and 5% in individuals on longterm ART (16,17,20). Although the fraction of intact proviruses is small relative to the total number of infected cells, there are sufficient replication-competent proviruses, or defective proviruses that can be readily complemented, to fuel rapid viral rebound if ART is interrupted (21,22).…”

“…Although the fraction of intact proviruses is small relative to the total number of infected cells, there are sufficient replication-competent proviruses, or defective proviruses that can be readily complemented, to fuel rapid viral rebound if ART is interrupted (21,22). In both adults and children, when ART is initiated soon after infection, the number of infected cells is reduced, sometimes to levels below the detection limit of current assays (20,23,24) and rebound viremia can be significantly delayed (25)(26)(27)(28).…”

Residual Proviral Reservoirs: A High Risk for HIV Persistence and Driving Forces for Viral Rebound after Analytical Treatment Interruption

“…Early ART, even initiated as early as one day of infection in adult subjects or within 30 min after birth in infants, usually fails to achieve a sustained state of ART-free virologic remission, leading to HIV rebound after months or years of treatment interruption [ 50 , 104 , 105 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 ]. Although the Berlin, London, and Düsseldorf adult patients seemingly appear to have a “sterilizing HIV cure” [ 108 , 114 , 115 , 116 , 117 ], the treatment of these three cases, which involved toxic chemotherapy due to hematological malignancies followed by hematopoietic stem cell transplantation, is not feasible in others because of its complexity and risk.…”