Expanded cellular clones carrying replication-competent HIV-1 persist, wax, and wane

Abstract

The latent reservoir for HIV-1 in resting CD4⁺ T cells is a major barrier to cure. Several lines of evidence suggest that the latent reservoir is maintained through cellular proliferation. Analysis of this proliferative process is complicated by the fact that most infected cells carry defective proviruses. Additional complications are that stimuli that drive T cell proliferation can also induce virus production from latently infected cells and productively infected cells have a short in vivo half-life. In this ex vivo study, we show that latently infected cells containing replication-competent HIV-1 can proliferate in response to T cell receptor agonists or cytokines that are known to induce homeostatic proliferation and that this can occur without virus production. Some cells that have proliferated in response to these stimuli can survive for 7 d while retaining the ability to produce virus. This finding supports the hypothesis that both antigen-driven and cytokine-induced proliferation may contribute to the stability of the latent reservoir. Sequencing of replication-competent proviruses isolated from patients at different time points confirmed the presence of expanded clones and demonstrated that while some clones harboring replication-competent virus persist longitudinally on a scale of years, others wax and wane. A similar pattern is observed in longitudinal sampling of residual viremia in patients. The observed patterns are not consistent with a continuous, cell-autonomous, proliferative process related to the HIV-1 integration site. The fact that the latent reservoir can be maintained, in part, by cellular proliferation without viral reactivation poses challenges to cure.

Keywords: HIV persistence; clonal expansion; latent reservoir.

Fig. 1.

Proliferation of infected cells in response to TCR stimulation and cytokines. (A) Experimental setup with assay time course. Resting CD4⁺ T cells were isolated from participants on ART; stained with CFSE; and then cultured for 7 d with media alone, cultured with anti-CD3/CD28, or treated with IL-7. On day 7, cells that had proliferated in response to anti-CD3/CD28 or IL-7 were isolated based on CFSE dilution. Half of the sorted cells were then plated in a limiting dilution QVOA with PHA and irradiated allogeneic PBMCs. The other half of the cells were plated at the same dilutions without PHA or irradiated allogeneic PBMCs. After 24 h, PHA was removed and MOLT-4/CCR5⁺ cells were added to all culture wells to expand virus released from infected cells. On day 20, a p24 ELISA was performed to quantify viral outgrowth. Cells treated with media alone were cultured without sorting. (B) Activation marker expression and proliferation induced by anti-CD3/CD28 and IL-7 stimulations. Resting CD4⁺ T cells were stained with CFSE before stimulation. CFSE dilution and activation marker expression were quantitated by flow cytometry 7 d after stimulation. (C) TCR sequence analysis. TCRs of cells that proliferated with anti-CD3/CD28 stimulation or IL-7 stimulation were sequenced, and the percentage of each TCR was then calculated in each sample. (D) Resting CD4⁺ T cell expression of IL-7 receptor (CD127). Freshly isolated resting CD4⁺ T cells were stained with CD127 to quantify IL-7 receptor expression level before any treatment. (E) Induction of virus production by anti-CD3/CD28 and IL-7. Resting CD4⁺ T cells from individuals on ART (n = 10) were left untreated or simulated with anti-CD3/CD28 or IL-7 for 7 d. HIV-1 RNA levels in culture supernatants were measured by qRT-PCR. **P < 0.01. L.O.D., limit of detection; NS, P > 0.05. (F) Frequency of latently infected cells that proliferated in response to anti-CD3/CD28 or IL-7 as measured by QVOA. Sorted cells were analyzed by QVOA with or without an activating stimulus (PHA and allogeneic PBMCs). The frequency of cells producing replication-competent virus was determined by limiting dilution statistics 14 d later.

Fig. 2.

Expanded clones carrying replication-competent HIV-1 emerge and wane over time. (A) Phylogenetic trees of env sequences of independent isolates of replication-competent virus from eight subjects on ART (S01–S08) are shown. Sequencing was performed on genomic viral RNA in supernatants of p24⁺ wells. Different colors correspond to viruses recovered from different time points as indicated under the time line. Groups of identical sequences are indicated by symbols present on the same vertical “rake.” Sequences for the first time point were included in a previous study (18). Sequences that were previously shown to be identical by full-genome sequencing are grouped in boxes (18). The time scale indicates time in years from study entry. All patients were on suppressive ART for >6 mo before study entry. Black squares indicate the reference sequence HXB2. (B) Dynamics of expanded clones containing replication-competent HIV-1. Each pie figure shows how all of the replication-competent viruses (n) collected at a specific time point (shown on the x axis) are divided into clonal populations, with distinct colors representing different clones. Clones marked by M were identified at multiple time points. Starred lines indicate samples that are significantly different according to a test for difference in clone proportions when the null model is a random partition of the aggregated samples (Materials and Methods) (*P < 0.05; **P < 0.01; ***P < 0.001. NS, P > 0.05).

Fig. 2.

Expanded clones carrying replication-competent HIV-1 emerge and wane over time. (A) Phylogenetic trees of env sequences of independent isolates of replication-competent virus from eight subjects on ART (S01–S08) are shown. Sequencing was performed on genomic viral RNA in supernatants of p24⁺ wells. Different colors correspond to viruses recovered from different time points as indicated under the time line. Groups of identical sequences are indicated by symbols present on the same vertical “rake.” Sequences for the first time point were included in a previous study (18). Sequences that were previously shown to be identical by full-genome sequencing are grouped in boxes (18). The time scale indicates time in years from study entry. All patients were on suppressive ART for >6 mo before study entry. Black squares indicate the reference sequence HXB2. (B) Dynamics of expanded clones containing replication-competent HIV-1. Each pie figure shows how all of the replication-competent viruses (n) collected at a specific time point (shown on the x axis) are divided into clonal populations, with distinct colors representing different clones. Clones marked by M were identified at multiple time points. Starred lines indicate samples that are significantly different according to a test for difference in clone proportions when the null model is a random partition of the aggregated samples (Materials and Methods) (*P < 0.05; **P < 0.01; ***P < 0.001. NS, P > 0.05).

Fig. 3.

Neighbor-joining phylogenetic tree constructed with env sequences from the plasma and resting CD4⁺ T cells from subject S16. Samples were taken at the indicated times after study entry while the plasma HIV-1 RNA level was <50 copies per milliliter. Samples were processed for analysis of HIV-1 RNA in plasma (triangles) and proviral DNA in resting CD4⁺ T cells (circles).

Fig. 4.

Plasma virus clones wax and wane over a time scale of years. Neighbor-joining phylogenetic trees constructed with env sequences from the plasma and resting CD4⁺ T cells from subjects S14, S13, and S15. Samples were taken at the indicated times after study entry while the plasma HIV-1 RNA level was <50 copies per milliliter. Samples were processed for analysis of HIV-1 RNA in plasma (triangles) and proviral DNA in resting CD4⁺ T cells (circles).

Fig. 5.

Composite graph illustrating the appearance and longevity of clonal populations of free plasma virus and provirus derived from resting CD4⁺ T cells. Colors correspond to time of sampling and are derived directly from the corresponding phylogenies.