‘Shock and kill’ cure strategies rely on the efficient induction of HIV transcription in latently infected cells by latency reversing agents (LRAs). Since latently infected cells are highly heterogeneous in vivo, investigating the activity of latency reversal agents at the single-cell level in clinical samples is crucial.
We developed a novel single-cell multiplexed RNA flow cytometric fluorescent in situ hybridization (RNAflow-FISH) assay for co-detection of viral RNA (vRNA: gagRNA, nefRNA and HIV exons) and p24 protein. We applied this approach to PBMCs from 11 ART-suppressed people living with HIV. We examined HIV transcription and translation induced by PMA/Ionomycin, HDAC inhibitors (panobinostat, vorinostat; HDACi), and PKC agonists (bryostatin, PEP005; PKCa).
We detected a median of 115 vRNA+ cells/10^6 CD4+ T cells upon PMA/Ionomycin stimulation, a frequency only 6.8-fold lower than integrated HIV DNA (median 783). Only a small fraction of these cells produced the HIV protein p24 (median 3.9%), which all co-expressed gag and nef RNA. Reactivated cells were dominated by transcriptionally heterogeneous vRNA+p24- cells: gag+nef- (median 47%) > gag-nef+ (median 30%) > gag+nef+ (median 12%). PEP005 and panobinostat resulted in robust latency reversal (median of 54 and 55 vRNA+ cells/10^6 CD4+ T cells, respectively). While PEP005 recapitulated the profile obtained with PMA/ionomycin, panobinostat induced an homogenous gag+nef-p24- population (median of 88% of vRNA+) with few nef RNA. These profiles were consistent within LRA classes. Combining PEP005 and panobinostat boosted reactivation at a level surpassing PMA/ionomycin (median 240 vRNA+ cells/10^6 CD4+ T cells) with a profile similar to panobinostat, without p24 expression. We performed single-cell nested near full-length PCR on index sorted PMA/ionomycin -stimulated viral reservoirs to relate viral integrity to virus transcription. Highly deleted proviruses were enriched in gag-nef+p24- and gag+nef-p24- populations. Both gag+nef+p24- and p24+ populations harbored potentially intact proviruses.
We identified distinct single-cell patterns of viral reactivation upon stimulation by latency reversal agents in clinical samples. Dissociated expression of structural and regulatory genes was frequent, and differences between LRA classes were observed. Regardless of the agent tested, only a minority of the cells in which HIV transcription was efficiently induced produced detectable levels of the HIV protein p24.