Improving HIV Outgrowth by Optimizing Cell-Culture Conditions and Supplementing With all-trans Retinoic Acid

Abstract

The persistence of replication-competent HIV reservoirs in people living with HIV (PLWH) receiving antiretroviral therapy (ART) is a barrier to cure. Therefore, their accurate quantification is essential for evaluating the efficacy of new therapeutic interventions and orienting the decision to interrupt ART. Quantitative viral outgrowth assays (QVOAs) represent the "gold standard" for measuring the size of replication-competent HIV reservoirs. However, they require large numbers of cells and are technically challenging. This justifies the need for the development of novel simplified methods adapted for small biological samples. Herein, we sought to simplify the viral outgrowth procedure (VOP) by (i) using memory CD4 + T-cells, documented to be enriched in HIV reservoirs (ii) optimizing cell-culture conditions, and (iii) supplementing with all-trans retinoic acid (ATRA), a positive regulator of HIV replication. Memory CD4 + T-cells were sorted from the peripheral blood of ART-treated (HIV+ART; n = 14) and untreated (HIV+; n = 5) PLWH. The VOP was first performed with one original replicate of 1 × 10 6 cells/well in 48-well plates. Cells were stimulated via CD3/CD28 for 3 days, washed to remove residual CD3/CD28 Abs, split every 3 days for optimal cell density, and cultured in the presence or the absence of ATRA for 12 days. Soluble and intracellular HIV-p24 levels were quantified by ELISA and flow cytometry, respectively. Optimal cellculture density achieved by splitting improved HIV outgrowth detection. ATRA promoted superior/accelerated detection of replication-competent HIV in all HIV+ART individuals tested, including those with low/undetectable viral outgrowth in the absence of ATRA. Finally, this VOP was used to design a simplified ATRA-based QVOA by including 4 and 6 original replicates of 1 × 10 6 cells/well in 48-well plates and 2 × 10 5 cells/well in 96-well plates, respectively. Consistently, the number of infectious units per million cells (IUPM) was significantly increased in the presence of ATRA. In conclusion, we demonstrate that memory CD4 + T-cell splitting for optimal density in culture and ATRA supplementation significantly improved the efficacy of HIV outgrowth in a simplified ATRA-based QVOA performed in the absence of feeder/target cells or indicator cell lines.

Retinoic Acid Improves the Recovery of Replication-Competent Virus from Latent SIV Infected Cells

“…Our observation that RA is capable of reactivating replication-competent latent SIV is similar to the findings of Li et al, who showed that acitretin, an FDA-approved homologue of RA also leads to increased transcription of activated HIV-1 provirus [47]. Likewise, Zhang et al showed that supplementation of RA during QVOA improves the reactivation of the latent reservoir in sorted human memory CD4 + T cells [21].…”

“…Recently, Zhang et al, 2020, showed that QVOA estimates could be improved by refining cell culture conditions after T cell receptor (TCR) stimulation of sorted memory CD4 + T cells and later supplementation with retinoic acid (RA) for improved viral outgrowth [ 21 ]. However, it remains to be determined whether the supplementation with RA could be modified to improve QVOA estimates in latently-infected cells obtained in vivo from non-human primate (NHP) models such as rhesus macaques (RMs).…”

LILAC pilot study: Effects of metformin on mTOR activation and HIV reservoir persistence during antiretroviral therapy

“…Nested real-time PCR quantifications [43]), showed a minor but statistically significant increase in HIV-DNA integration between week 12 and Week 24; this may be explained by the recirculation of the T cells from tissues, consistent with the metformin-mediated decrease in the frequency of colon-infiltrating CD4 + T-cells and in the plasma levels of CCL20, a chemokine involved in CCR6 + T cell trafficking [74]. In contrast, changes in the frequency of cells carrying inducible MS HIV-RNA (measured by TILDA [66]), and replication-competent HIV reservoirs (measured by QVOA [75]) did not reach statistical significance between Baseline, Week 12, and Week 24, consistent with the low expression of phosphorylated mTOR in blood cells. These results are in line with the well-described stability of HIV reservoirs under ART and the difficulty to perturb HIV latency upon short time interventions [2À4].…”

Th17 cell master transcription factor RORC2 regulates HIV-1 gene expression and viral outgrowth

“…A simplified VOA was performed, as we previously described (50). Briefly, memory CD4+ T-cells were cultured at 1×10 6 cells/well in 1 ml of media (RPMI, 10% FBS, 1% Penicillin/Streptomycin) in a 48-well plate (Costar) coated with CD3 Abs (1 μg/ml; BD Biosciences, Clone UCHT1) and in the presence of soluble CD28 Abs (1 μg/ml; BD Biosciences, Clone CD28.2).…”