Caroline Pereira Bittencourt Passaes, Timothée Bruel, Jérémie Decalf, et.al.
Journal of Virology
The existence of HIV reservoirs in infected individuals under cART represents a major obstacle towards cure. Viral reservoirs are assessed by quantification of HIV nucleic acids, which does not discriminate between infectious and defective viruses, or by viral outgrowth assays, which requires large number of cells and long-term cultures. Here, we used an ultrasensitive p24 digital assay, which we report to be 1000 fold more sensitive than classical ELISA in the quantification of HIV-1 Gag p24 production in HIV-infected individuals samples. Results from ultrasensitive p24 were compared to conventional viral RNA RT-qPCR based assays, and outgrowth assays readout by flow cytometry. Using serial dilutions and flow-based single cell sorting, we show that viral proteins produced by a single infected cell can be detected by ultrasensitive p24. This unique sensitivity allowed the early (as soon as day 1 in 49% of cases) and more efficient detection and quantification of p24 in PHA-stimulated CD4+ T cells from individuals under effective cART. When testing seven different classes of latency reversal agents (LRA) in resting CD4+ T cells from HIV-infected individuals, ultrasensitive p24 revealed differences in the extent of HIV reactivation. Of note, HIV RNA production was infrequently accompanied by p24 protein production (19%). Among the drugs tested, prostratin showed a superior capacity in inducing viral protein production. In summary, the ultrasensitive p24 assay allows the detection and quantification of p24 produced by single infected-CD4+ T cells and provides a unique tool to assess early reactivation of infectious virus from reservoirs in HIV-infected individuals.