Single-cell characterization and quantification of translation-competent viral reservoirs in treated and untreated HIV infection

The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins 47 and 41. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/10(6) cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin 41. Remarkably, 41 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir. Author summary HIV persists in a small pool of infected CD4+ T cells during ART. A better characterization of these cells is a pre-requisite to the development of HIV eradication strategies. We developed a novel assay, named HIV-Flow, to simultaneously quantify and characterize reservoir cells in individuals receiving ART. With this assay, we found that a median of only 5 cells/million have the ability to produce the HIV protein Gag in individuals on suppressive ART. These frequencies correlated with other assays aimed at measuring HIV reservoirs. Importantly, we show that the HIV reservoir is phenotypically diverse, with numerous cell subsets contributing to the pool of persistently infected cells. Nonetheless, we identified several markers preferentially expressed at the surface or these rare reservoir cells, including immune checkpoint molecules and homing receptors. By combining these markers, we identified discrete cellular subsets highly enriched in HIV-infected cells. This novel assay will facilitate the identification of markers expressed by cellular HIV reservoirs.