Persistence of wild-type virus and lack of temporal structure in the latent reservoir for human immunodeficiency virus type 1 in pediatric patients with extensive antiretroviral exposure

Abstract

Although highly active antiretroviral therapy (HAART) for human immunodeficiency virus type 1 (HIV-1) infection can reduce levels of HIV-1 RNA in plasma to below the limit of detection, replication-competent forms of the virus persist in all infected individuals. One form of persistence involves a stable reservoir of latent but potentially infectious virus that resides in resting memory CD4(+) T cells. The mechanisms involved in maintaining this latent reservoir are incompletely understood. In the present study, we examined the dynamic characteristics of this reservoir in a cohort of children who developed drug-resistant HIV-1 as a result of extensive exposure to inadequately suppressive one- or two-drug regimens prior to the advent of HAART. We have previously shown that drug-resistant viruses selected by nonsuppressive pre-HAART regimens can enter and persist in this reservoir. We have extended these findings here by demonstrating that archival wild-type HIV-1 persists in this reservoir despite the fact that in these patients drug-resistant mutants have been favored by the selective conditions for many years. Phylogenetic analysis of replication-competent viruses persisting in resting CD4(+) T cells revealed a striking lack of temporal structure in the sense that isolates obtained at later time points did not show greater sequence divergence than isolates from earlier time points. The persistence of drug-sensitive virus and the lack of temporal structure in the latent reservoir provide genetic evidence for the idea that HIV-1 can persist in a latent form free of selective pressure from antiretroviral drugs in long-lived resting memory CD4(+) T cells. Although there may be other mechanisms for viral persistence, this stable pool of latently infected cells is of significant concern because of its potential to serve as a lasting source of replication-competent viruses, including the infecting wild-type form and all drug-resistant variants that have arisen subsequently.

FIG. 1.

Treatment, immunologic and virologic profiles of children in the study. Absolute CD4 counts (blue diamonds) and HIV-1 RNA levels (red squares) in plasma are shown as a function of time, with zero time defined by the initiation of HAART. Arrows (↓) indicate the time of birth. Open symbols for plasma HIV-1 RNA measurements indicate that the level was below the limit of detection; the plotted value is the limit of detection of the assay used (400, 200, or 50 copies/ml). Colored bars indicate denote treatment with the indicated drugs. For full names of antiretroviral drugs, see Table 1, footnote d.

FIG. 2.

Phylogenetic analysis of HIV-1 pol sequences obtained from resting CD4⁺ T cells of children receiving HAART. Sequences from study subjects are represented by colored boxes with the sampling time indicated in terms of the number of months since initiation of HAART. Lack of temporal structure is indicated by the fact that genetic distances (horizontal scale) do not correlate with sampling time (indicated by a colored bar). In the classic pattern of progressive evolutionary change, viruses obtained at later time points should have diverged further from the most recent common ancestor. Filled boxes indicate wild-type viruses with no antiretroviral resistance mutations. Open boxes indicate viruses with one or more antiretroviral resistance mutations. The numbers at the internal nodes indicate the percentage of 1,000 bootstrap replicates that reproduced the clade. Only values of >70% are indicated; these clades were supported by all three models of phylogenetic inference applied (neighbor-joining, maximum parsimony, and maximum likelihood). The maximum-likelihood tree is shown. The reference sequence HXB2 (GenBank accession no. K03455) was used as an outgroup.