Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells

Abstract

The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4+ T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4+ T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4+ T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1-7), followed by rest (day 7-21), and then repeat stimulation (day 21-28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4+ T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.

Fig 1. Experimental system schematic.

PBMC or purified total CD4⁺ T-cells were cultured with 300 nM efavirenz (EFV), 300 nM raltegravir (RAL), and 20 IU/mL of recombinant human IL-2 (rhIL-2). Cells were sequentially stimulated with PMA (50 ng/mL) and ionomycin (500 ng/mL). The stimulation duration was seven days with an inter-stimulation period of fourteen days. Cells were washed twice weekly. Aliquots of cells from blood and from ex vivo cultures on days -1, 7, 21, and 28 were saved for HIV DNA qPCR and/or single-genome sequencing (SGS). Aliquots of supernatant were removed once weekly for virion-associated HIV RNA qRT-PCR and SGS.

Fig 2. Activation marker expression during cell culture.

The frequency of CD69⁺ and CD25⁺ cells is measured relative to the parent population of live, CD3⁺CD4⁺ lymphocytes.

Fig 3. Cell number changes during cell culture.

Cell numbers at the conclusion of each week are normalized relative to the total number of cells that were seeded in the flask at the beginning of each week.

Fig 4. Levels of HIV DNA during cell culture.

The frequency of HIV-infected cells was quantified using qPCR.

Fig 5. Levels of HIV virion production during cell culture.

HIV virion production was measured on cell-free supernatants using the Roche COBAS AmpliPrep/TaqMan HIV-1 test. The cumulative levels of virus produced spontaneously after 24 hours in separate experiments were < 20 copies/mL.

Fig 6. Neighbor-joining distance tree of virion-associated HIV RNA and proviral-associated HIV DNA sequences illustrates proviral population dynamics after CD4⁺ T-cell activation (Donor 1).

The tree was constructed using the neighbor-joining p-distance method. All sequences were rooted to a consensus sequence of HIV subtype B. Hypermutant sequences are boxed. The Viral Outgrowth Assay (VOA) was performed using day 7 and day 28 cells from Donor 1 (Experiment 2) total CD4⁺ T-cells. The day 7 cells were seeded into 6 wells at 1x10⁶ cells/well and the day 28 cells were seeded into 6 wells at 3x10⁵ cells/well.