The replication-competent HIV-1 latent reservoir is primarily established near the time of therapy initiation

Abstract

Although antiretroviral therapy (ART) is highly effective at suppressing HIV-1 replication, the virus persists as a latent reservoir in resting CD4⁺ T cells during therapy. This reservoir forms even when ART is initiated early after infection, but the dynamics of its formation are largely unknown. The viral reservoirs of individuals who initiate ART during chronic infection are generally larger and genetically more diverse than those of individuals who initiate therapy during acute infection, consistent with the hypothesis that the reservoir is formed continuously throughout untreated infection. To determine when viruses enter the latent reservoir, we compared sequences of replication-competent viruses from resting peripheral CD4⁺ T cells from nine HIV-positive women on therapy to viral sequences circulating in blood collected longitudinally before therapy. We found that, on average, 71% of the unique viruses induced from the post-therapy latent reservoir were most genetically similar to viruses replicating just before ART initiation. This proportion is far greater than would be expected if the reservoir formed continuously and was always long lived. We conclude that ART alters the host environment in a way that allows the formation or stabilization of most of the long-lived latent HIV-1 reservoir, which points to new strategies targeted at limiting the formation of the reservoir around the time of therapy initiation.

Fig 1.. Viral Load and Suppression History of Nine Participants from the CAPRISA 002 Acute Infection Cohort.

The graph shows the viral loads of HIV-1 RNA in the blood pre- and post-therapy, with the time of therapy initiation designated as T=0. Each participant is designated by a different color and the time of blood collection for assessing the reservoir is designated by verticle arrows.

Fig. 2.. The Majority of Outgrowth Viruses (OGVs) Are Most Closely Related to Viruses Replicating Near the Time of ART Initiation.

Phylogenetic analyses of one genomic region for each of two participants are shown. Pre-ART sequences are colored red (year after transmission) to blue (year before ART) and outgrowth viruses are shown in pink. The clustering of outgrowth sequences (pink) with sequences from the year before ART (blue) indicates that these participants each had a majority of OGVs with timing assignments for entry into the reservoir in the year preceding ART. (a) CAP257 had 44 unique OGVs of which 89% were most closely related to viruses replicating within one year pre-ART, and 7% to viruses replicating 142 weeks pre-ART. (b) CAP288 had 7 unique OGVs, all (100%) of which were most closely related to viruses replicating within one year pre-ART.

Fig. 3.. A Subset of Participants Had Outgrowth Viruses (OGVs) Most Closely Related to Viruses Replicating Throughout Untreated Infection.

Phylogenetic analyses of one genomic region for each of two participants are shown. Selected participants each had OGVs with timing assignments from a wide range of times pre-ART, as illustrated by the clustering of outgrowth sequences (pink) with sequences from different pre-ART time points (red to blue). (a) CAP217 had 17 OGVs, two of which were clones. Of the genetically unique OGVs, 44% were most closely related to viruses replicating within one year pre-ART, and 31% to viruses replicating more than 2 years before ART. (b) CAP302 had 6 unique OGVs of which one was from the year before ART and one was from the year after transmission.

Fig. 4.. Timing of Reservoir Entry for OGVs as Estimated by Combining Three Methods.

Three methods (patristic distance, clade support and phylogenetic placement) were used to generate estimates of OGV entry into the reservoir by comparing multiple regions of the OGV genome to that of viral RNA isolated from the plasma pre-ART. (a, b) Estimates are for two participants with OGVs that typically entered the reservoir near the time of ART initiation (CAP257 and CAP288) and (c, d) two with OGVs from throughout untreated infection (CAP217 and CAP302). Each OGV is represented as a vertical line with up to 15 estimates along that line. For each OGV, estimates of reservoir entry were summarized as a weighted median, shown as a horizonal line and represented in a colored bar along the x axis. Boxes indicate OGVs with highly variable estimates (SD > 1 yr). Additional phylogenetic analyses of OGVs with variable estimates found that one (designated with an ‘x’) was in a time-specific lineage that did not correspond to the weighted median. All other OGVs were found in time-specific lineages that corresponded to the weighted median.

Fig. 5.. Skewing of Reservoir Virus to Virus Replicating Near the Time of ART Initiation May be Explained by a Change in the Half-Life of Latently Infected Cells in the Presence of ART.

(a) All outgrowth virus (OGV) sequences are shown in an approximate maximum-likelihood tree. Branch tips are colored according to the estimated time when each OGV entered the reservoir. With OGVs from the year after transmission in red and OGVs from the year before ART in blue. For each participant, the OGV timing distribution is illustrated in a pie chart with the percentage of OGVs from the year pre-ART listed. Overall, 71% of OGVs were produced by cells infected near the time of ART initiation. (b) Two models were considered to explain this pattern. One model assumes that latently infected cells have a long half-life of 176 weeks (44 months) both prior to and during therapy, and predicts that the reservoir will contain variants from throughout untreated infection. The other model assumes that latently infected cells have a short half-life in untreated infection (here chosen as 2 weeks) that then stabilizes to a long half-life during therapy. For the majority of participants (four shown here), the observed data most closely resembles the predicted pattern if latently infected cells have a short half-life prior to ART initiation and then decay slowly after ART initiation.