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      A Subset of Latency-Reversing Agents Expose HIV-Infected Resting CD4 + T-Cells to Recognition by Cytotoxic T-Lymphocytes

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          There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.

          Abstract

          Resting CD4 + T-cells harboring inducible HIV proviruses are a critical reservoir in antiretroviral therapy (ART)-treated subjects. These cells express little to no viral protein, and thus neither die by viral cytopathic effects, nor are efficiently cleared by immune effectors. Elimination of this reservoir is theoretically possible by combining latency-reversing agents (LRAs) with immune effectors, such as CD8 + T-cells. However, the relative efficacy of different LRAs in sensitizing latently-infected cells for recognition by HIV-specific CD8 + T-cells has not been determined. To address this, we developed an assay that utilizes HIV-specific CD8 + T-cell clones as biosensors for HIV antigen expression. By testing multiple CD8 + T-cell clones against a primary cell model of HIV latency, we identified several single agents that primed latently-infected cells for CD8 + T-cell recognition, including IL-2, IL-15, two IL-15 superagonists (IL-15SA and ALT-803), prostratin, and the TLR-2 ligand Pam 3CSK 4. In contrast, we did not observe CD8 + T-cell recognition of target cells following treatment with histone deacetylase inhibitors or with hexamethylene bisacetamide (HMBA). In further experiments we demonstrate that a clinically achievable concentration of the IL-15 superagonist ‘ALT-803’, an agent presently in clinical trials for solid and hematological tumors, primes the natural ex vivo reservoir for CD8 + T-cell recognition. Thus, our results establish a novel experimental approach for comparative evaluation of LRAs, and highlight ALT-803 as an LRA with the potential to synergize with CD8 + T-cells in HIV eradication strategies.

          Author Summary

          Although modern therapies have greatly improved the lives of HIV-positive people with access to care, a cure remains elusive. This leaves these individuals burdened by a lifelong commitment to medication, and fails to fully restore health. Curing infection would likely require therapies that combine the ability to force the virus out the ‘latent state’ in which it hides, with immune responses able to kill unmasked infected cells, the so called “shock and kill” strategy. A critical aspect of this strategy is identifying drugs that are effective at shocking virus out of latency, known as latency reversing agents. In this study, we took the novel approach of using CD8 + T-cells, immune cells responsible for killing infected cells, as biosensors able to detect the unmasking of latently-infected cells. Using this method, we screened a panel of potential latency reversing agents. We found that while a subset of these agents exposed infected cells to the immune system, others did not. Our results establish a new method for screening potential latency reversing agents, and support the prioritization of the agents that were shown to be effective for combination with CD8 + T-cells in shock and kill strategies aimed at curing HIV infection.

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          Most cited references 49

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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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            Stimulation of HIV-1-specific cytolytic T lymphocytes facilitates elimination of latent viral reservoir after virus reactivation.

            Highly active antiretroviral therapy (HAART) suppresses HIV-1 replication but cannot eliminate the virus because HIV-1 establishes latent infection. Interruption of HAART leads to a rapid rebound of viremia, so life-long treatment is required. Efforts to purge the latent reservoir have focused on reactivating latent proviruses without inducing global T cell activation. However, the killing of the infected cells after virus reactivation, which is essential for elimination of the reservoir, has not been assessed. Here we show that after reversal of latency in an in vitro model, infected resting CD4(+) T cells survived despite viral cytopathic effects, even in the presence of autologous cytolytic T lymphocytes (CTLs) from most patients on HAART. Antigen-specific stimulation of patient CTLs led to efficient killing of infected cells. These results demonstrate that stimulating HIV-1-specific CTLs prior to reactivating latent HIV-1 may be essential for successful eradication efforts and should be considered in future clinical trials. Copyright © 2012 Elsevier Inc. All rights reserved.
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              Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy.

              Although highly active antiretroviral therapy (HAART) in the form of triple combinations of drugs including protease inhibitors can reduce the plasma viral load of some HIV-1-infected individuals to undetectable levels, it is unclear what the effects of these regimens are on latently infected CD4+ T cells and what role these cells play in the persistence of HIV-1 infection in individuals receiving such treatment. The present study demonstrates that highly purified CD4+ T cells from 13 of 13 patients receiving HAART with an average treatment time of 10 months and with undetectable (<500 copies HIV RNA/ml) plasma viremia by a commonly used bDNA assay carried integrated proviral DNA and were capable of producing infectious virus upon cellular activation in vitro. Phenotypic analysis of HIV-1 produced by activation of latently infected CD4+ T cells revealed the presence in some patients of syncytium-inducing virus. In addition, the presence of unintegrated HIV-1 DNA in infected resting CD4+ T cells from patients receiving HAART, even those with undetectable plasma viremia, suggests persistent active virus replication in vivo.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, CA USA )
                1553-7366
                1553-7374
                15 April 2016
                April 2016
                : 12
                : 4
                Affiliations
                [1 ]The Ragon Institute of Massachusetts General Hospital, Massachusetts Institute of Technology, and Harvard University, Cambridge, Massachusetts, United States of America
                [2 ]Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, United States of America
                [3 ]Department of Microbiology Immunology and Tropical Medicine, The George Washington University, Washington, D.C., United States of America
                [4 ]Gilead Sciences, Foster City, California, United States of America
                [5 ]Altor BioScience Corporation, Miramar, Florida, United States of America
                [6 ]Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, United States of America
                [7 ]The Maple Leaf Medical Clinic, Toronto, Ontario, Canada
                [8 ]Department of Medicine, University of Toronto, Toronto, Ontario, Canada
                [9 ]Li Ka Shing Medical Institute, St. Michael’s Hospital, Toronto, Ontario, Canad
                [10 ]Howard Hughes Medical Institute, Chevy Chase, Maryland, United States of America
                [11 ]Department of Biological Engineering, MIT, Cambridge, Massachusetts, United States of America
                University of Wisconsin, UNITED STATES
                Author notes

                I have read the journal's policy and the authors of the manuscript have the following competing interests: DDS, AI, ATs, JPM, and HY are employed by, and own stock and/or stock options in Gilead Sciences, Inc. This does not alter our adherence to all PLoS Pathogens policies on sharing data and materials. HCW and EKJ are employees and stockholders of Altor BioScience Corporation. This does not alter our adherence to all PLOS Pathogens policies on sharing data and materials. The authors declare that they have no other competing financial interests.

                Conceived and designed the experiments: RBJ SM RO KR AST DK MJB DDS AI JPM ATs HY MAO DJI BDW ML RG. Performed the experiments: RBJ SM RO KR AST DK MJB DDS AI JPM ATs ATr HY SL SHH JBW SK. Analyzed the data: RBJ SM RO KR AST DK MJB DDS AI JPM ATs HY SHH JBW SL RG. Contributed reagents/materials/analysis tools: HCW EKJ EB CK. Wrote the paper: RBJ. Supervised the project: BDW DJI MAO ML RG.

                ‡ DJI and BDW are co-senior authors.

                Article
                PPATHOGENS-D-15-02228
                10.1371/journal.ppat.1005545
                4833318
                27082643
                © 2016 Jones et al

                This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

                Page count
                Figures: 7, Tables: 0, Pages: 25
                Product
                Funding
                This work was supported in part by the NIH (AI111860) and the Ragon Institute of MGH, MIT, and Harvard. We also gratefully acknowledge financial support from the GenerationCure initiative through the American Foundation for AIDS Research (amfAR). During the time that this study was performed, RBJ was a Banting Fellow of the Canadian Institutes for Health Research and a Junior Investigator of the Ontario HIV Treatment Network. BDW and DJI are Investigators of the Howard Hughes Medical Institute. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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