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Ex vivo activation of CD4+ T-cells from donors on suppressive ART can lead to sustained production of infectious HIV-1 from a subset of infected cells

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      Abstract

      The fate of HIV-infected cells after reversal of proviral latency is not well characterized. Simonetti, et al. recently showed that CD4 + T-cells containing intact proviruses can clonally expand in vivo and produce low-level infectious viremia. We hypothesized that reversal of HIV latency by activation of CD4 + T-cells can lead to the expansion of a subset of virus-producing cells rather than their elimination. We established an ex vivo cell culture system involving stimulation of CD4 + T-cells from donors on suppressive antiretroviral therapy (ART) with PMA/ionomycin (day 1–7), followed by rest (day 7–21), and then repeat stimulation (day 21–28), always in the presence of high concentrations of raltegravir and efavirenz to effectively block new cycles of viral replication. HIV DNA and virion RNA in the supernatant were quantified by qPCR. Single genome sequencing (SGS) of p6-PR-RT was performed to genetically characterize proviruses and virion-associated genomic RNA. The replication-competence of the virions produced was determined by the viral outgrowth assay (VOA) and SGS of co-culture supernatants from multiple time points. Experiments were performed with purified CD4 + T-cells from five consecutively recruited donors who had been on suppressive ART for > 2 years. In all experiments, HIV RNA levels in supernatant increased following initial stimulation, decreased or remained stable during the rest period, and increased again with repeat stimulation. HIV DNA levels did not show a consistent pattern of change. SGS of proviruses revealed diverse outcomes of infected cell populations, ranging from their apparent elimination to persistence and expansion. Importantly, a subset of infected cells expanded and produced infectious virus continuously after stimulation. These findings underscore the complexity of eliminating reservoirs of HIV-infected cells and highlight the need for new strategies to kill HIV-infected cells before they can proliferate.

      Author summary

      Stable latent reservoirs of HIV persist despite suppressive antiretroviral therapy (ART) and cause rebound viremia following interruption of ART. New HIV cure strategies aim to deplete the latent reservoir by reversing HIV latency and promoting the death of cells containing inducible proviruses. Multiple latency reversing agents have been discovered, with the most effective compounds also causing T-cell activation. We investigated the ex vivo effects of cell activation on HIV-infected cells from individuals on stable suppressive ART. Latency reversal through robust cell activation led to diverse outcomes of infected cells. Although many infected cells appeared to be eliminated following T-cell activation, a subset of HIV-infected cells persisted and could expand, including those that can produce infectious virus. These findings highlight the need for new therapies that kill HIV-infected cells before they can proliferate.

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      Most cited references 41

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      Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy.

      The hypothesis that quiescent CD4+ T lymphocytes carrying proviral DNA provide a reservoir for human immunodeficiency virus-type 1 (HIV-1) in patients on highly active antiretroviral therapy (HAART) was examined. In a study of 22 patients successfully treated with HAART for up to 30 months, replication-competent virus was routinely recovered from resting CD4+ T lymphocytes. The frequency of resting CD4+ T cells harboring latent HIV-1 was low, 0.2 to 16.4 per 10(6) cells, and, in cross-sectional analysis, did not decrease with increasing time on therapy. The recovered viruses generally did not show mutations associated with resistance to the relevant antiretroviral drugs. This reservoir of nonevolving latent virus in resting CD4+ T cells should be considered in deciding whether to terminate treatment in patients who respond to HAART.
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        Molecular and cellular insights into T cell exhaustion.

        In chronic infections and cancer, T cells are exposed to persistent antigen and/or inflammatory signals. This scenario is often associated with the deterioration of T cell function: a state called 'exhaustion'. Exhausted T cells lose robust effector functions, express multiple inhibitory receptors and are defined by an altered transcriptional programme. T cell exhaustion is often associated with inefficient control of persisting infections and tumours, but revitalization of exhausted T cells can reinvigorate immunity. Here, we review recent advances that provide a clearer molecular understanding of T cell exhaustion and reveal new therapeutic targets for persisting infections and cancer.
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          HIV reservoir size and persistence are driven by T cell survival and homeostatic proliferation.

          HIV persists in a reservoir of latently infected CD4(+) T cells in individuals treated with highly active antiretroviral therapy (HAART). Here we identify central memory (T(CM)) and transitional memory (T(TM)) CD4(+) T cells as the major cellular reservoirs for HIV and find that viral persistence is ensured by two different mechanisms. HIV primarily persists in T(CM) cells in subjects showing reconstitution of the CD4(+) compartment upon HAART. This reservoir is maintained through T cell survival and low-level antigen-driven proliferation and is slowly depleted with time. In contrast, proviral DNA is preferentially detected in T(TM) cells from aviremic individuals with low CD4(+) counts and higher amounts of interleukin-7-mediated homeostatic proliferation, a mechanism that ensures the persistence of these cells. Our results suggest that viral eradication might be achieved through the combined use of strategic interventions targeting viral replication and, as in cancer, drugs that interfere with the self renewal and persistence of proliferating memory T cells.
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            Author and article information

            Affiliations
            [1 ]Division of Infectious Diseases, Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, United States of America
            [2 ]Howard Hughes Medical Research Fellows Program, Howard Hughes Medical Institute, Bethesda, Maryland, United States of America
            [3 ]Advanced Biomedical Computing Center, Frederick National Laboratory for Cancer Research operated by Leidos Biomedical Research, Inc., Frederick, Maryland, United States of America
            [4 ]HIV Dynamics and Replication Program, National Cancer Institute, Frederick, Maryland, United States of America
            John Hopkins University, UNITED STATES
            Author notes

            I have read the journal's policy and the authors of this manuscript have the following competing interests: JWM is a consultant for Gilead Sciences and a shareholder of Cocrystal Pharma, Inc. WS and BL are employees of Leidos Biomedical Research, Inc.

            • Conceptualization: JWM MFK JKB EKH.

            • Data curation: JKB WS.

            • Formal analysis: JKB JWM WS BL.

            • Funding acquisition: JWM.

            • Investigation: JKB JWM.

            • Methodology: JKB JWM MFK EKH EF MDS DK WS BL FFH.

            • Project administration: JKB JWM.

            • Resources: JKB JWM WS.

            • Software: JKB WS BL.

            • Supervision: JKB JWM MFK.

            • Validation: JKB JWM.

            • Visualization: JKB JWM MFK EKH.

            • Writing – original draft: JKB.

            • Writing – review & editing: JKB JWM MFK EKH EF.

            Contributors
            Role: Editor
            Journal
            PLoS Pathog
            PLoS Pathog
            plos
            plospath
            PLoS Pathogens
            Public Library of Science (San Francisco, CA USA )
            1553-7366
            1553-7374
            22 February 2017
            February 2017
            : 13
            : 2
            28225830
            5338860
            10.1371/journal.ppat.1006230
            PPATHOGENS-D-16-02012
            (Editor)

            This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

            Counts
            Figures: 6, Tables: 2, Pages: 19
            Product
            Funding
            Funded by: funder-id http://dx.doi.org/10.13039/100000011, Howard Hughes Medical Institute;
            Award ID: Medical Research Fellows Program
            Award Recipient : John Khoa Bui
            Funded by: Leidos Biomedical Research
            Award ID: 12XS547
            Award Recipient :
            Funded by: funder-id http://dx.doi.org/10.13039/100000054, National Cancer Institute;
            Award ID: HHSN26120080001E
            Award Recipient :
            Funded by: funder-id http://dx.doi.org/10.13039/100000865, Bill and Melinda Gates Foundation;
            Award ID: OPP1115715
            Award Recipient :
            This work was supported by Leidos Biomedical Research (Grant Number: 12XS547 / National Cancer Institute HHSN26120080001E, https://www.leidos.com/about/companies/leidos-biomedical-research, http://www.cancer.gov/) and the Bill & Melinda Gates Foundation (Grant Number: OPP1115715, http://www.gatesfoundation.org/). JKB is a recipient of funding through the Howard Hughes Medical Institute (HHMI) Medical Research Fellows Program ( http://www.hhmi.org/programs/medical-research-fellows-program). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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            2017-03-06
            Relevant data are within the paper and the Supporting Information files. The sequences reported in this paper have been deposited in the GenBank database (accession numbers: KX829224-829753, KX830756-830801).

            Infectious disease & Microbiology

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