Date Published: February 8, 2017
Publisher: Public Library of Science
Author(s): Kaitlin Rainwater-Lovett, Carrie Ziemniak, Douglas Watson, Katherine Luzuriaga, George Siberry, Ann Petru, YaHui Chen, Priyanka Uprety, Margaret McManus, Ya-Chi Ho, Susanna L. Lamers, Deborah Persaud, Cristian Apetrei.
The latent reservoir is a major barrier to HIV eradication. Reservoir size is emerging as an important biomarker to assess the likelihood of HIV remission in the absence of antiretroviral therapy (ART) and may be reduced by earlier initiation of ART that restricts HIV spread into CD4+ T cells. Reservoir size is traditionally measured with a quantitative viral outgrowth assay (QVOA) that induces replication-competent HIV production through in vitro stimulation of resting CD4+ T cells. However, the recent identification of replication-intact, non-induced proviral genomes (NIPG) suggests the QVOA significantly underestimates (by 62-fold) latent reservoir size in chronically-infected adults. Whether formation and persistence of Intact, NIPG is thwarted by early ART initiation and long-term virologic suppression in perinatal infection is unclear. Here, we show that the latent reservoir in 11 early treated, long-term suppressed perinatally infected children and adolescents was not inducible by QVOA and dominated by defective, NIPG. Single genome analysis of 164 NIPG from 232 million cultured resting CD4+ T cells revealed no replication-intact, near-full length sequences. Forty-three (26%) NIPG contained APOBEC3G-mediated hypermutation, 115 (70%) NIPG contained large internal deletions, one NIPG contained nonsense mutations and indels, and 5 (3%) NIPG were assigned as “Not Evaluable” due to multiple failed sequencing attempts that precluded further classification. The lack of replication competent inducible provirus and intact NIPG in this cohort indicate early, long-term ART of perinatal infection leads to marked diminution of replication-competent HIV-1 reservoirs, creating a favorable state towards interventions aimed at virologic remission.
Infection with HIV results in the rapid formation of a latent reservoir in resting memory CD4+ T cells (rCD4s) that cannot be eradicated by combination antiretroviral therapy (ART) and is capable of rekindling viremia after ART discontinuation [1–5]. The size of the rCD4 latent reservoir has been associated with the risk and timing of virologic rebound following ART cessation [6–9]. Traditionally, the presence of inducible latent provirus is detected by a quantitative viral outgrowth assay (QVOA) in which a single-round of CD4+ T cell activation is used to reverse HIV latency and induce infectious virus production from rCD4s to infect susceptible target cells, allowing quantitative estimates of latent reservoir size . However, replication-competent, non-induced proviral genomes (NIPG) were recently detected in culture- negative wells of the standard QVOA indicating that this measure underestimates the size of the replication-competent latent reservoir by up to 62-fold in chronically-infected adults .
We performed the first assessment of induced and non-induced replication-competent latent proviral genomes during early, long-term treatment to understand the distribution of replication-competent and non-induced, intact proviral genomes retained in perinatal HIV infection. Contrary to our hypothesis, HIV-infected rCD4s during early, long-term ART were dominated by replication-defective, non-intact NIPG. This profile is distinct from 12% of intact NIPG identified in chronically-infected adults, with virologic suppression [11,21], highlighting differences in HIV persistence between early and late ART initiation, but closer to the 2% recently identified with early ART in adults. A number of mechanisms governing genomic variation contribute to the generation of defective genomes during the course of HIV infection. By virtue of being a diploid virus, genome switching by reverse transcriptase results in large deletions that prove lethal [27–31]. In addition to a lack of proofreading activity that increases the rates of point mutation in HIV genomes, several innate immune factors contribute to mutagenesis, such as the cytidine deaminases APCOBEC3F and APOBEC3G that induces hypermutation . Our findings confirm that early ART initiation in the context of an infant immune system with long-term virologic suppression did not lead to preservation of intact HIV genomes but persistence of an overwhelming majority of replication-defective genomes.