A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children

Abstract

HIV-1 persists in a latent state in resting CD4(+) T lymphocytes of infected adults despite prolonged highly active antiretroviral therapy (HAART). To determine whether a latent reservoir for HIV-1 exists in infected children, we performed a quantitative viral culture assay on highly purified resting CD4(+) T cells from 21 children with perinatally acquired infection. Replication-competent HIV-1 was recovered from all 18 children from whom sufficient cells were obtained. The frequency of latently infected resting CD4(+) T cells directly correlated with plasma virus levels, suggesting that in children with ongoing viral replication, most latently infected cells are in the labile preintegration state of latency. However, in each of 7 children who had suppression of viral replication to undetectable levels for 1-3 years on HAART, latent replication-competent HIV-1 persisted with little decay, owing to a stable reservoir of infected cells in the postintegration stage of latency. Drug-resistance mutations generated by previous nonsuppressive regimens persisted in this compartment despite more than 1 year of fully suppressive HAART, rendering untenable the idea of recycling drugs that were part of failed regimens. Thus the latent reservoir for HIV-1 in resting CD4(+) T cells will be a major obstacle to HIV-1 eradication in children.

Figure 1

Representative flow cytometric analysis of CD4 and HLA-DR antigen expression on monocyte-depleted PBMCs before (a) and after (b) sequential magnetic bead depletion and cell sorting.

Figure 2

Correlation between the frequency of latently infected resting CD4 T cells (IUPM) and the plasma HIV-1 RNA (copies/mL). Closed symbols represent children who had detectable levels of plasma HIV-1 RNA at study entry. Open symbols represent children who had undetectable (< 400 copies/mL) levels of plasma HIV-1 RNA at the time of study entry. Values are arbitrarily plotted at the upper bound of the plasma virus assay at 400 copies/mL.

Figure 3

Decay in the frequency of latently infected resting CD4⁺ T lymphocytes (in IUPM) versus time on HAART in months. (a) Decay of latently infected T cells in 6 children followed longitudinally before and after the initiation of HAART. (b) Decay of latently infected T cells in 3 children studied after the initiation of HAART. Closed symbols represent the time points at which replication-competent HIV-1 was recovered from the resting CD4⁺ T lymphocytes. Open symbols represent time points at which viral cultures on resting CD4⁺ T lymphocytes were negative. An upper bound on the frequency of latently infected resting CD4⁺T lymphocytes was determined as described.

Figure 4

HIV-1 RT and protease sequences from viral isolates cultured from the resting CD4⁺ T lymphocytes. Dashes represent amino acids that are unchanged from the reference sequence HXB2R. Blanks represent codons not determined. Numbers in the top row indicate RT codons associated with drug resistance and protease codons associated with drug resistance, common polymorphisms, or polymorphism and accessory substitutions to protease inhibitors. Mutations associated with resistance to antiretroviral drugs are shown in blocks of the appropriate color. Positions in protease at which polymorphisms occur are shown in black type. Known polymorphisms that are also seen as accessory substitutions contributing to resistance to ritonavir (RTV) or nelfinavir (NFV) are shown in colored type.