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Latency reversal agents (LRAs) have proven to induce HIV-1 transcription in vivo but are ineffective at decreasing the size of the latent reservoir in antiretroviral treated patients. The capacity of the LRAs to perturb the viral reservoir present in distinct subpopulations of cells is currently unknown. Here, using a new RNA FISH/flow ex vivo viral reactivation assay, we performed a comprehensive assessment of the viral reactivation capacity of different families of LRAs, and their combinations, in different CD4+ T cell subsets. We observed that a median of 16.28% of the whole HIV-reservoir induced HIV-1 transcripts after viral reactivation, but only 10.10% of these HIV-1 RNA+ cells produced the viral protein p24. Moreover, none of the LRAs were powerful enough to reactivate HIV-1 transcription in all CD4+ T cell subpopulations. For instance, the combination of Romidepsin and Ingenol was identified as the best combination of drugs at increasing the proportion of HIV-1 RNA+ cells, in most, but not all, CD4+ T cell subsets. Importantly, memory stem cells were identified as highly resistant to HIV-1 reactivation, and only the combination of Panobinostat and Bryostatin-1 significantly increased the number of cells transcribing HIV within this subset. Overall, our results validate the use of the RNA FISH/flow technique to assess the potency of LRAs among different CD4+ T cell subsets, manifest the intrinsic differences between cells that encompass the latent HIV reservoir, and highlight the difficulty to significantly impact the latent infection with the currently available drugs. Thus, our results have important implications for the rational design of therapies aimed at reversing HIV latency from diverse cellular reservoirs., Author summary HIV infection is an incurable disease. Despite antiretroviral therapy, a pool of cells with HIV in a latent state persists and precludes fully eradication of the viral infection. The cells that contain this latent viral reservoir are very diverse, and therefore different therapeutic strategies would be necessary to target and eliminate all infected cells. Latency Reversal Agents (LRAs) are compounds able to awake the latent virus from its dormant state with the purpose of making infected cells visible to the immune system. But the ability of the LRAs to target different cell types containing HIV is currently unknown. Here, using a novel methodology that interrogates individual cells, we found that current LRAs do not impact equally all infected cells. In fact, certain types of memory lymphocytes, recognized to harbor latent HIV for decades, are not fully impacted by most of the LRAs tested. Our study highlights the difficulty to cure HIV with the currently available LRAs. Different therapeutic approaches aimed at reversing HIV latency from diverse cellular reservoirs are needed to reduce HIV persistence.

The identification of exclusive markers to target HIV-reservoir cells will represent a significant advance in the search for therapies to cure HIV. Here, we identify the B lymphocyte antigen CD20 as a marker for HIV-infected cells in vitro and in vivo. The CD20 molecule is dimly expressed in a subpopulation of CD4-positive (CD4+) T lymphocytes from blood, with high levels of cell activation and heterogeneous memory phenotypes. In lymph node samples from infected patients, CD20 is present in productively HIV-infected cells, and ex vivo viral infection selectively upregulates the expression of CD20 during early infection. In samples from patients on antiretroviral therapy (ART) this subpopulation is significantly enriched in HIV transcripts, and the anti-CD20 monoclonal antibody Rituximab induces cell killing, which reduces the pool of HIV-expressing cells when combined with latency reversal agents. We provide a tool for targeting this active HIV-reservoir after viral reactivation in patients while on ART., Here, the authors identify B lymphocyte antigen CD20 as a marker for HIV-infected T cells and provide evidence for the potential use of anti-CD20 antibodies in combination with latency reversing agents for depletion of viral reactivated CD4 T cells in patients on antiretroviral therapy.

Cells that actively transcribe HIV-1 have been defined as the “active viral reservoir” in HIV-infected individuals. However, important technical limitations have precluded the characterization of this specific viral reservoir during both treated and untreated HIV-1 infections. Here, we used a novel single-cell RNA fluorescence in situ hybridization-flow cytometry (FISH-flow) assay that requires only 15 million unfractionated peripheral blood mononuclear cells (PBMCs) to characterize the specific cell subpopulations that transcribe HIV RNA in different subsets of CD4+ T cells. In samples from treated and untreated HIV-infected patients, effector memory CD4+ T cells were the main cell population supporting HIV RNA transcription. The number of cells expressing HIV correlated with the plasma viral load, intracellular HIV RNA, and proviral DNA quantified by conventional methods and inversely correlated with the CD4+ T cell count and the CD4/CD8 ratio. We also found that after ex vivo infection of unstimulated PBMCs, HIV-infected T cells upregulated the expression of CD32. In addition, this new methodology detected increased numbers of primary cells expressing viral transcripts and proteins after ex vivo viral reactivation with latency reversal agents. This RNA FISH-flow technique allows the identification of the specific cell subpopulations that support viral transcription in HIV-1-infected individuals and has the potential to provide important information on the mechanisms of viral pathogenesis, HIV persistence, and viral reactivation., IMPORTANCE Persons infected with HIV-1 contain several cellular viral reservoirs that preclude the complete eradication of the viral infection. Using a novel methodology, we identified effector memory CD4+ T cells, immune cells preferentially located in inflamed tissues with potent activity against pathogens, as the main cells encompassing the transcriptionally active HIV-1 reservoir in patients on antiretroviral therapy. Importantly, the identification of such cells provides us with an important target for new therapies designed to target the hidden virus and thus to eliminate the virus from the human body. In addition, because of its ability to identify cells forming part of the viral reservoir, the assay used in this study represents an important new tool in the field of HIV pathogenesis and viral persistence.