Viral Sanctuaries during Highly Active Antiretroviral Therapy in a Nonhuman Primate Model for AIDS

ABSTRACT Highly active antiretroviral therapy (HAART) enables long-term suppression of plasma HIV-1 loads in infected persons, but low-level virus persists and rebounds following cessation of therapy. During HAART, this virus resides in latently infected cells, such as resting CD4+ T cells, and in other cell types that may support residual virus replication. Therapeutic eradication will require elimination of virus from all reservoirs. We report here a comprehensive analysis of these reservoirs in fluids, cells, and tissues in a rhesus macaque model that mimics HAART in HIV-infected humans. This nonhuman primate model uses RT-SHIV, a chimera of simian immunodeficiency virus containing the HIV-1 reverse transcriptase (RT). Methods were developed for extraction, preamplification, and real-time PCR analyses of viral DNA (vDNA) and viral RNA (vRNA) in tissues from RT-SHIV-infected macaques. These methods were used to identify viral reservoirs in RT-SHIV-infected macaques treated with a potent HAART regimen consisting of efavirenz, emtricitabine, and tenofovir. Plasma virus loads at necropsy ranged from 11 to 28 copies of vRNA per ml. Viral RNA and DNA were detected during HAART, in tissues from numerous anatomical locations. Additional analysis provided evidence for full-length viral RNA in tissues of animals with virus suppressed by HAART. The highest levels of vDNA and vRNA in HAART-treated macaques were in lymphoid tissues, particularly the spleen, lymph nodes, and gastrointestinal tract tissues. This study is the first comprehensive analysis of the tissue and organ distribution of a primate AIDS virus during HAART. These data demonstrate widespread persistence of residual virus in tissues during HAART.

[1]  A S Perelson,et al.  Dynamics of HIV-1 and CD4+ lymphocytes in vivo. , 1997, AIDS.

[2]  M. Gardner.  Chapter 10 – Simian Retroviruses , 2004 .

[3]  N. Pedersen,et al.  Real-time TaqMan PCR as a specific and more sensitive alternative to the branched-chain DNA assay for quantitation of simian immunodeficiency virus RNA. , 2001, AIDS research and human retroviruses.

[4]  D. Richman,et al.  Reduction of HIV-1 in blood and lymph nodes following potent antiretroviral therapy and the virologic correlates of treatment failure. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Qingsheng Li,et al.  Peak SIV replication in resting memory CD4+ T cells depletes gut lamina propria CD4+ T cells , 2005, Nature.

[6]  M. Murphey-Corb,et al.  Distribution of SIV in lymph nodes of serially sacrificed rhesus monkeys. , 1995, AIDS research and human retroviruses.

[7]  M. Groudine,et al.  Cellular latency in human immunodeficiency virus-infected individuals with high CD4 levels can be detected by the presence of promoter-proximal transcripts. , 1994, Proceedings of the National Academy of Sciences of the United States of America.

[8]  S J Gange,et al.  Treatment intensification does not reduce residual HIV-1 viremia in patients on highly active antiretroviral therapy , 2009, Proceedings of the National Academy of Sciences.

[9]  D. Ho,et al.  Use of Real-Time PCR and Molecular Beacons To Detect Virus Replication in Human Immunodeficiency Virus Type 1-Infected Individuals on Prolonged Effective Antiretroviral Therapy , 1999, Journal of Virology.

[10]  C. Miller,et al.  Immunophenotypic characterization of simian immunodeficiency virus-infected dendritic cells in cervix, vagina, and draining lymph nodes of rhesus monkeys. , 1998, Laboratory investigation; a journal of technical methods and pathology.

[11]  B. Gazzard,et al.  Tenofovir Disoproxil Fumarate, Emtricitabine, and Efavirenz Versus Fixed-Dose Zidovudine/Lamivudine and Efavirenz in Antiretroviral-Naive Patients: Virologic, Immunologic, and Morphologic Changes-A 96-Week Analysis , 2006, Journal of acquired immune deficiency syndromes.

[12]  J. Verhoef,et al.  Binding of human immunodeficiency virus type-1 to follicular dendritic cells in vitro is complement dependent. , 1993, Journal of immunology.

[13]  Alan S. Perelson,et al.  Decay characteristics of HIV-1-infected compartments during combination therapy , 1997, Nature.

[14]  R. Hogg,et al.  Virologic and Immunologic Response, Clinical Progression, and Highly Active Antiretroviral Therapy Adherence , 2002, Journal of acquired immune deficiency syndromes.

[15]  D. Metcalfe,et al.  Human tissue mast cells are an inducible reservoir of persistent HIV infection. , 2007, Blood.

[16]  A. Trkola,et al.  HIV rebounds from latently infected cells, rather than from continuing low-level replication , 2008, Proceedings of the National Academy of Sciences.

[17]  J. Mellors,et al.  Low-level viremia persists for at least 7 years in patients on suppressive antiretroviral therapy , 2008, Proceedings of the National Academy of Sciences.

[18]  R. Singh,et al.  The Clinical Benefits of Tenofovir for Simian Immunodeficiency Virus–Infected Macaques Are Larger Than Predicted by its Effects on Standard Viral and Immunologic Parameters , 2004, Journal of acquired immune deficiency syndromes.

[19]  N. Pedersen,et al.  Efavirenz Therapy in Rhesus Macaques Infected with a Chimera of Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1 , 2004, Antimicrobial Agents and Chemotherapy.

[20]  A. Lackner,et al.  Getting to the Guts of HIV Pathogenesis , 2004, The Journal of experimental medicine.

[21]  R. Siliciano,et al.  A stable latent reservoir for HIV-1 in resting CD4(+) T lymphocytes in infected children. , 2000, The Journal of clinical investigation.

[22]  R. Doms,et al.  Derivation and biological characterization of a molecular clone of SHIV(KU-2) that causes AIDS, neurological disease, and renal disease in rhesus macaques. , 1999, Virology.

[23]  J. Palefsky,et al.  Highly tissue substructure-specific effects of human papilloma virus in mucosa of HIV-infected patients revealed by laser-dissection microscopy-assisted gene expression profiling. , 2004, The American journal of pathology.

[24]  Peter Reiss,et al.  Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. , 2008, JAMA.

[25]  J. Lisziewicz,et al.  Latent infection of CD4+ T cells provides a mechanism for lifelong persistence of HIV-1, even in patients on effective combination therapy , 1999, Nature Medicine.

[26]  J. Lifson,et al.  Suppression of Viremia and Evolution of Human Immunodeficiency Virus Type 1 Drug Resistance in a Macaque Model for Antiretroviral Therapy , 2007, Journal of Virology.

[27]  D. Richman,et al.  Heterogeneous clearance rates of long-lived lymphocytes infected with HIV: Intrinsic stability predicts lifelong persistence , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[28]  Amalio Telenti,et al.  Antiretroviral Treatment of Adult HIV Infection2010 Recommendations of the International AIDS Society–USA Panel , 2010 .

[29]  H. McClure,et al.  Sensitive and robust one-tube real-time reverse transcriptase-polymerase chain reaction to quantify SIV RNA load: comparison of one- versus two-enzyme systems. , 2000, AIDS research and human retroviruses.

[30]  R. Desrosiers,et al.  A nucleotide substitution in the tRNA(Lys) primer binding site dramatically increases replication of recombinant simian immunodeficiency virus containing a human immunodeficiency virus type 1 reverse transcriptase. , 2002, Journal of virology.

[31]  P. Johnson,et al.  Simian immunodeficiency virus infection of macaques: end-stage disease is characterized by widespread distribution of proviral DNA in tissues. , 1991, The Journal of infectious diseases.

[32]  R. Schinazi,et al.  Antiretroviral Therapy in Macrophages: Implication for HIV Eradication , 2009, Antiviral chemistry & chemotherapy.

[33]  S. Hughes,et al.  In Vitro Characterization of a Simian Immunodeficiency Virus-Human Immunodeficiency Virus (HIV) Chimera Expressing HIV Type 1 Reverse Transcriptase To Study Antiviral Resistance in Pigtail Macaques , 2004, Journal of Virology.

[34]  P. Crino,et al.  Simian immunodeficiency virus encephalitis: analysis of envelope sequences from individual brain multinucleated giant cells and tissue samples. , 2002, Virology.

[35]  Alan S. Perelson,et al.  Quantitative Image Analysis of HIV-1 Infection in Lymphoid Tissue , 1996, Science.

[36]  Douglas D. Richman,et al.  HIV chemotherapy , 2001, Nature.

[37]  K. Mansfield,et al.  Identifying the Target Cell in Primary Simian Immunodeficiency Virus (SIV) Infection: Highly Activated Memory CD4+ T Cells Are Rapidly Eliminated in Early SIV Infection In Vivo , 2000, Journal of Virology.

[38]  John W. Mellors,et al.  New Real-Time Reverse Transcriptase-Initiated PCR Assay with Single-Copy Sensitivity for Human Immunodeficiency Virus Type 1 RNA in Plasma , 2003, Journal of Clinical Microbiology.

[39]  R. Siliciano,et al.  A Simian Immunodeficiency Virus-Infected Macaque Model To Study Viral Reservoirs That Persist during Highly Active Antiretroviral Therapy , 2009, Journal of Virology.

[40]  R. Johnson,et al.  Characterization of gut-associated lymphoid tissue (GALT) of normal rhesus macaques. , 1997, Clinical immunology and immunopathology.

[41]  W. Haseltine,et al.  Animal model for the therapy of acquired immunodeficiency syndrome with reverse transcriptase inhibitors. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[42]  A. Lackner,et al.  Perivascular Macrophages Are the Primary Cell Type Productively Infected by Simian Immunodeficiency Virus in the Brains of Macaques , 2001, The Journal of experimental medicine.

[43]  R. Desrosiers,et al.  A Nucleotide Substitution in the tRNALys Primer Binding Site Dramatically Increases Replication of Recombinant Simian Immunodeficiency Virus Containing a Human Immunodeficiency Virus Type 1 Reverse Transcriptase , 2002, Journal of Virology.

[44]  R. Desrosiers,et al.  The Productive Infection of Alveolar Macrophages by Simian Immunodeficiency Virus , 1989, Journal of medical primatology.

[45]  Alan S. Perelson,et al.  The decay of the latent reservoir of replication-competent HIV-1 is inversely correlated with the extent of residual viral replication during prolonged anti-retroviral therapy , 2000, Nature Medicine.

[46]  Ashley T. Haase,et al.  Massive covert infection of helper T lymphocytes and macrophages by HIV during the incubation period of AIDS , 1993, Nature.

[47]  D. Nickle,et al.  Persistence of HIV in gut-associated lymphoid tissue despite long-term antiretroviral therapy. , 2008, The Journal of infectious diseases.

[48]  A. Lackner,et al.  Distribution of SIV infection in the gastrointestinal tract of rhesus macaques at early and terminal stages of AIDS. , 1993, Journal of medical primatology.

[49]  R. Siliciano,et al.  Long-term follow-up studies confirm the stability of the latent reservoir for HIV-1 in resting CD4+ T cells , 2003, Nature Medicine.

[50]  Claus O. Wilke,et al.  Residual Human Immunodeficiency Virus Type 1 Viremia in Some Patients on Antiretroviral Therapy Is Dominated by a Small Number of Invariant Clones Rarely Found in Circulating CD4+ T Cells , 2006, Journal of Virology.

[51]  J. Clements,et al.  From mice to macaques--animal models of HIV nervous system disease. , 2006, Current HIV research.

[52]  R. Siliciano,et al.  The challenge of viral reservoirs in HIV-1 infection. , 2002, Annual review of medicine.

[53]  E. De Clercq,et al.  SIV/HIV-1 hybrid virus expressing the reverse transcriptase gene of HIV-1 remains sensitive to HIV-1-specific reverse transcriptase inhibitors after passage in rhesus macaques. , 1997, Journal of acquired immune deficiency syndromes and human retrovirology : official publication of the International Retrovirology Association.

[54]  A. Lackner,et al.  Simian immunodeficiency virus infection of macaque bone marrow macrophages correlates with disease progression in vivo. , 1991, The American journal of pathology.

[55]  A. Fauci,et al.  The AIDS epidemic--considerations for the 21st century. , 1999, The New England journal of medicine.

[56]  J. Lifson,et al.  Plasma SIV RNA viral load determination by real-time quantification of product generation in reverse transcriptase-polymerase chain reaction. , 1998, AIDS research and human retroviruses.

[57]  M A Nowak,et al.  Presence of an inducible HIV-1 latent reservoir during highly active antiretroviral therapy. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[58]  N. Pedersen,et al.  Suppression of Virus Load by Highly Active Antiretroviral Therapy in Rhesus Macaques Infected with a Recombinant Simian Immunodeficiency Virus Containing Reverse Transcriptase from Human Immunodeficiency Virus Type 1 , 2005, Journal of Virology.

[59]  T. Chun,et al.  Latent reservoirs of HIV: obstacles to the eradication of virus. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[60]  M A Fischl,et al.  A controlled trial of two nucleoside analogues plus indinavir in persons with human immunodeficiency virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS Clinical Trials Group 320 Study Team. , 1997, The New England journal of medicine.

[61]  R Brookmeyer,et al.  Identification of a reservoir for HIV-1 in patients on highly active antiretroviral therapy. , 1997, Science.

[62]  D. Ho,et al.  Genetic characterization of rebounding HIV-1 after cessation of highly active antiretroviral therapy. , 2000, The Journal of clinical investigation.

[63]  E A Emini,et al.  Treatment with indinavir, zidovudine, and lamivudine in adults with human immunodeficiency virus infection and prior antiretroviral therapy. , 1997, The New England journal of medicine.

[64]  R. Siliciano,et al.  Resting CD4+ T Lymphocytes but Not Thymocytes Provide a Latent Viral Reservoir in a Simian Immunodeficiency Virus-Macaca nemestrina Model of Human Immunodeficiency Virus Type 1-Infected Patients on Highly Active Antiretroviral Therapy , 2003, Journal of Virology.

[65]  J. Lifson,et al.  Simian immunodeficiency virus infection of monkeys as a model system for the study of AIDS pathogenesis, treatment, and prevention. , 2000, Advances in pharmacology.

[66]  A. Lackner,et al.  Early events in tissues during infection with pathogenic (SIVmac239) and nonpathogenic (SIVmac1A11) molecular clones of simian immunodeficiency virus. , 1994, The American journal of pathology.

[67]  J. Hoxie,et al.  Biological characterization of a simian immunodeficiency virus-like retrovirus (HTLV-IV): evidence for CD4-associated molecules required for infection , 1988, Journal of virology.

[68]  Yang Wang,et al.  Evidence that Low-Level Viremias during Effective Highly Active Antiretroviral Therapy Result from Two Processes: Expression of Archival Virus and Replication of Virus , 2005, Journal of Virology.

[69]  E. De Clercq,et al.  Sensitivity/resistance profile of a simian immunodeficiency virus containing the reverse transcriptase gene of human immunodeficiency virus type 1 (HIV-1) toward the HIV-1-specific non-nucleoside reverse transcriptase inhibitors. , 1995, Biochemical and biophysical research communications.

[70]  T. Reinhart,et al.  Restricted SIV replication in rhesus macaque lung tissues during the acute phase of infection. , 2002, The American journal of pathology.

[71]  Libin Rong,et al.  Modeling HIV persistence, the latent reservoir, and viral blips. , 2009, Journal of theoretical biology.

[72]  Mario Clerici,et al.  Productive Infection Maintains a Dynamic Steady State of Residual Viremia in Human Immunodeficiency Virus Type 1-Infected Persons Treated with Suppressive Antiretroviral Therapy for Five Years , 2003, Journal of Virology.

[73]  T. O'Leary,et al.  Immunohistochemical localization of human and simian immunodeficiency viral antigens in fixed tissue sections. , 1987, The American journal of pathology.