RESUMO
UNLABELLED: Human immunodeficiency virus (HIV) persistence in latently infected resting memory CD4+ T-cells is the major barrier to HIV cure. Cellular histone deacetylases (HDACs) are important in maintaining HIV latency and histone deacetylase inhibitors (HDACi) may reverse latency by activating HIV transcription from latently infected CD4+ T-cells. We performed a single arm, open label, proof-of-concept study in which vorinostat, a pan-HDACi, was administered 400 mg orally once daily for 14 days to 20 HIV-infected individuals on suppressive antiretroviral therapy (ART). The primary endpoint was change in cell associated unspliced (CA-US) HIV RNA in total CD4+ T-cells from blood at day 14. The study is registered at ClinicalTrials.gov (NCT01365065). Vorinostat was safe and well tolerated and there were no dose modifications or study drug discontinuations. CA-US HIV RNA in blood increased significantly in 18/20 patients (90%) with a median fold change from baseline to peak value of 7.4 (IQR 3.4, 9.1). CA-US RNA was significantly elevated 8 hours post drug and remained elevated 70 days after last dose. Significant early changes in expression of genes associated with chromatin remodeling and activation of HIV transcription correlated with the magnitude of increased CA-US HIV RNA. There were no statistically significant changes in plasma HIV RNA, concentration of HIV DNA, integrated DNA, inducible virus in CD4+ T-cells or markers of T-cell activation. Vorinostat induced a significant and sustained increase in HIV transcription from latency in the majority of HIV-infected patients. However, additional interventions will be needed to efficiently induce virus production and ultimately eliminate latently infected cells. TRIAL REGISTRATION: ClinicalTrials.gov NCT01365065.
Assuntos
Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Ácidos Hidroxâmicos/uso terapêutico , Ativação Viral/efeitos dos fármacos , Adulto , Linfócitos T CD4-Positivos/imunologia , Feminino , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/imunologia , HIV-1/fisiologia , Humanos , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , RNA Viral/genética , Transcrição Gênica/efeitos dos fármacos , Latência Viral/efeitos dos fármacos , VorinostatRESUMO
Eradication of HIV-1 with highly active antiretroviral therapy (HAART) is not possible due to the persistence of long-lived, latently infected resting memory CD4(+) T cells. We now show that HIV-1 latency can be established in resting CD4(+) T cells infected with HIV-1 after exposure to ligands for CCR7 (CCL19), CXCR3 (CXCL9 and CXCL10), and CCR6 (CCL20) but not in unactivated CD4(+) T cells. The mechanism did not involve cell activation or significant changes in gene expression, but was associated with rapid dephosphorylation of cofilin and changes in filamentous actin. Incubation with chemokine before infection led to efficient HIV-1 nuclear localization and integration and this was inhibited by the actin stabilizer jasplakinolide. We propose a unique pathway for establishment of latency by direct HIV-1 infection of resting CD4(+) T cells during normal chemokine-directed recirculation of CD4(+) T cells between blood and tissue.
Assuntos
Linfócitos T CD4-Positivos/virologia , Quimiocinas/imunologia , Infecções por HIV/imunologia , HIV-1/fisiologia , Integração Viral/imunologia , Latência Viral/imunologia , Actinas/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Núcleo Celular/imunologia , Quimiocinas/farmacologia , Citoesqueleto/metabolismo , Humanos , Receptores de Quimiocinas/imunologia , Integração Viral/efeitos dos fármacos , Internalização do Vírus , Replicação ViralRESUMO
Combination antiretroviral therapy (cART) has led to a very substantial reduction in morbidity and mortality in HIV-infected patients; however, cART alone is unable to cure HIV and therapy is lifelong. Therefore, a new strategy to cure HIV is urgently needed. There is now a concerted effort from scientists, clinicians and funding agencies to identify ways to achieve either a functional cure (long-term control of HIV in the absence of cART) or a sterilizing cure (elimination of all HIV-infected cells). Multiple strategies aiming at achieving a cure for HIV are currently being investigated, including both pharmacotherapy and gene therapy. In this review, we will review the rationale as well as in vitro and clinical trial data that support the role of histone deacetylase inhibitors as one approach to cure HIV.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Inibidores de Histona Desacetilases/uso terapêutico , Fármacos Anti-HIV/efeitos adversos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Ensaios Clínicos como Assunto , Infecções por HIV/imunologia , Infecções por HIV/virologia , Inibidores de Histona Desacetilases/efeitos adversos , Humanos , Ácidos Hidroxâmicos/efeitos adversos , Ácidos Hidroxâmicos/uso terapêutico , Latência Viral/efeitos dos fármacos , VorinostatRESUMO
Combination antiretroviral therapy (cART) has led to a reduction in morbidity and mortality in HIV-infected patients but therapy is lifelong and there is no cure for HIV. The major barriers to cure include HIV latency, which has been identified in different T-cell subsets, as well as persistence of HIV in anatomical reservoirs. We review recent developments in our understanding of the major reservoirs of HIV in patients on cART as well as how latency is established and maintained in T cells. Finally, we review the scientific rationale of and clinical experience with pharmacotherapeutic strategies aimed at eliminating latently infected cells.
Assuntos
Antirretrovirais/uso terapêutico , Reservatórios de Doenças/virologia , Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral , Animais , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Humanos , Modelos Animais , Primatas , Latência Viral/imunologia , Latência Viral/fisiologiaRESUMO
BACKGROUND: We recently described that HIV latent infection can be established in vitro following incubation of resting CD4+ T-cells with chemokines that bind to CCR7. The main aim of this study was to fully define the post-integration blocks to virus replication in this model of CCL19-induced HIV latency. RESULTS: High levels of integrated HIV DNA but low production of reverse transcriptase (RT) was found in CCL19-treated CD4+ T-cells infected with either wild type (WT) NL4.3 or single round envelope deleted NL4.3 pseudotyped virus (NL4.3- Δenv). Supernatants from CCL19-treated cells infected with either WT NL4.3 or NL4.3- Δenv did not induce luciferase expression in TZM-bl cells, and there was no expression of intracellular p24. Following infection of CCL19-treated CD4+ T-cells with NL4.3 with enhanced green fluorescent protein (EGFP) inserted into the nef open reading frame (NL4.3- Δnef-EGFP), there was no EGFP expression detected. These data are consistent with non-productive latent infection of CCL19-treated infected CD4+ T-cells. Treatment of cells with phytohemagluttinin (PHA)/IL-2 or CCL19, prior to infection with WT NL4.3, resulted in a mean fold change in unspliced (US) RNA at day 4 compared to day 0 of 21.2 and 1.1 respectively (p = 0.01; n = 5), and the mean expression of multiply spliced (MS) RNA was 56,000, and 5,000 copies/million cells respectively (p = 0.01; n = 5). In CCL19-treated infected CD4+ T-cells, MS-RNA was detected in the nucleus and not in the cytoplasm; in contrast to PHA/IL-2 activated infected cells where MS RNA was detected in both. Virus could be recovered from CCL19-treated infected CD4+ T-cells following mitogen stimulation (with PHA and phorbyl myristate acetate (PMA)) as well as TNFα, IL-7, prostratin and vorinostat. CONCLUSIONS: In this model of CCL19-induced HIV latency, we demonstrate HIV integration without spontaneous production of infectious virus, detection of MS RNA in the nucleus only, and the induction of virus production with multiple activating stimuli. These data are consistent with ex vivo findings from latently infected CD4+ T-cells from patients on combination antiretroviral therapy, and therefore provide further support of this model as an excellent in vitro model of HIV latency.
Assuntos
Linfócitos T CD4-Positivos/virologia , Quimiocina CCL19/farmacologia , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Infecções por HIV/virologia , HIV-1/fisiologia , Latência Viral/efeitos dos fármacos , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linhagem Celular , Células Cultivadas , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , HIV-1/genética , Humanos , Modelos Biológicos , Integração Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Liver-related mortality is increased in the setting of HIV-hepatitis B virus (HBV) coinfection. However, interactions between HIV and HBV to explain this observation have not been described. We hypothesized that HIV infection of hepatocytes directly affects the life cycle of HBV. We infected human hepatic cell lines expressing HBV (Hep3B and AD38 cells) or not expressing HBV (Huh7, HepG2, and AD43 cells) with laboratory strains of HIV (NL4-3 and AD8), as well as a vesicular stomatitis virus (VSV)-pseudotyped HIV expressing enhanced green fluorescent protein (EGFP). Following HIV infection with NL4-3 or AD8 in hepatic cell lines, we observed a significant increase in HIV reverse transcriptase activity which was infectious. Despite no detection of surface CD4, CCR5, and CXCR4 by flow cytometry, AD8 infection of AD38 cells was inhibited by maraviroc and NL4-3 was inhibited by AMD3100, demonstrating that HIV enters AD38 hepatic cell lines via CCR5 or CXCR4. High-level infection of AD38 cells (50%) was achieved using VSV-pseudotyped HIV. Coinfection of the AD38 cell line with HIV did not alter the HBV DNA amount or species as determined by Southern blotting or nucleic acid signal amplification. However, coinfection with HIV was associated with a significant increase in intracellular HBsAg when measured by Western blotting, quantitative HBsAg, and fluorescence microscopy. We conclude that HIV infection of HBV-infected hepatic cell lines significantly increased intracellular HBsAg but not HBV DNA synthesis and that increased intrahepatic HBsAg secondary to direct infection by HIV may contribute to accelerated liver disease in HIV-HBV-coinfected individuals.
Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/fisiologia , Hepatite B/virologia , Hepatócitos/virologia , Linhagem Celular , HIV-1/genética , Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , HumanosRESUMO
Following treatment of hepatitis B virus (HBV) monoinfection, HBV-specific T-cell responses increase significantly; however, little is known about the recovery of HBV-specific T-cell responses following HBV-active highly active antiretroviral therapy (HAART) in HIV-HBV coinfected patients. HIV-HBV coinfected patients who were treatment naïve and initiating HBV-active HAART were recruited as part of a prospective cohort study in Thailand and followed for 48 weeks (n = 24). Production of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in both HBV- and HIV-specific CD8(+) T cells was quantified using intracellular cytokine staining on whole blood. Following HBV-active HAART, the median (interquartile range) log decline from week 0 to week 48 for HBV DNA was 5.8 log (range, 3.4 to 6.7) IU/ml, and for HIV RNA it was 3.1 (range, 2.9 to 3.5) log copies/ml (P < 0.001 for both). The frequency of HIV Gag-specific CD8(+) T-cell responses significantly decreased (IFN-gamma, P < 0.001; TNF-alpha, P = 0.05). In contrast, there was no significant change in the frequency (IFN-gamma, P = 0.21; TNF-alpha, P = 0.61; and IFN-gamma and TNF-alpha, P = 0.11) or magnitude (IFN-gamma, P = 0.13; TNF-alpha, P = 0.13; and IFN-gamma and TNF-alpha, P = 0.13) of HBV-specific CD8(+) T-cell responses over 48 weeks of HBV-active HAART. Of the 14 individuals who were HBV e antigen (HBeAg) positive, 5/14 (36%) lost HBeAg during the 48 weeks of follow-up. HBV-specific CD8(+) T cells were detected in 4/5 (80%) of patients prior to HBeAg loss. Results from this study show no sustained change in the HBV-specific CD8(+) T-cell response following HBV-active HAART. These findings may have implications for the duration of treatment of HBV in HIV-HBV coinfected patients, particularly in HBeAg-positive disease.
Assuntos
Terapia Antirretroviral de Alta Atividade , Linfócitos T CD8-Positivos/virologia , Infecções por HIV , HIV-1/imunologia , Vírus da Hepatite B/imunologia , Hepatite B , Adulto , Linfócitos T CD8-Positivos/imunologia , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , Infecções por HIV/virologia , Hepatite B/tratamento farmacológico , Hepatite B/imunologia , Hepatite B/virologia , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , TailândiaRESUMO
BACKGROUND: Naive T cell recovery is critical for successful immune reconstitution after antiretroviral therapy (ART), but the relative contribution of CD31(+) and CD31â» naive T cells to immune reconstitution and viral persistence is unknown. METHODS: In a cross-sectional (n = 94) and longitudinal (n = 10) study of human immunodeficiency virus (HIV)-infected patients before and after ART, we examined the ratio of CD31(+) to CD31â» naive CD4(+) T cells. In the longitudinal cohort we then quantified the concentration of HIV-1 DNA in each cell subset and performed single-genome amplification of virus from memory and naive T cells. RESULTS: Patients receiving ART had a higher proportion of CD31(+) CD4(+) T cells than HIV-1-infected individuals naive to ART and uninfected control subjects (P < .001 and .007, respectively). After 24 months of ART, the proportion of CD31(+) naive CD4(+) T cells did not change, the concentration of HIV-1 DNA in memory CD4(+) T cells significantly decreased over time (P < .001), and there was no change in the concentration of HIV-1 DNA in CD31(+) or CD31â» naive CD4(+) T cells (P = .751 and .251, respectively). Single-genome amplification showed no evidence of virus compartmentalization in memory and naive T cell subsets before or after ART. CONCLUSIONS: After ART, both CD31(+) and CD31â» naive CD4(+) T cells expand, and both subsets represent a stable, persistent reservoir of HIV-1.
Assuntos
Antirretrovirais/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Infecções por HIV/sangue , HIV-1/efeitos dos fármacos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Adulto , Contagem de Linfócito CD4 , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Estudos Transversais , Feminino , Citometria de Fluxo , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Estudos Longitudinais , Masculino , Reação em Cadeia da Polimerase , Análise de Regressão , Singapura , VitóriaRESUMO
UNLABELLED: Hepatitis B virus (HBV)-specific T cells play a key role in clearance of the virus and in the pathogenesis of liver disease. Peripheral blood (n = 25) and liver biopsies (n = 19) were collected from individuals with chronic untreated HBV infection. Whole blood, cultured peripheral blood mononuclear cells (PBMCs), and cultured liver-infiltrating lymphocytes (LILs) were each stimulated with an overlapping peptide library to the whole HBV genome. The expression of T helper 1 (Th1) cytokines [interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin 2 (IL-2)] and interleukin 10 (IL-10) was analyzed by intracellular cytokine staining and flow cytometry. In ex vivo whole blood, more lymphocytes produced Th1 cytokines than IL-10. When comparing cultured LILs with cultured PBMCs, we found a significantly higher magnitude of CD8(+) T cells from the liver producing IL-10 (P = 0.044), primarily in hepatitis B e antigen positive (HBeAg(+)) individuals. A positive correlation resulted between the magnitude of HBV-specific TNF-alpha(+) CD4(+) T cells in the liver and the degree of liver inflammation and fibrosis (P = 0.002 and P = 0.006, respectively). CONCLUSION: The differences in cytokine production from HBV-specific T cells in blood and liver may explain the capacity for HBV to persist in the absence of significant hepatic destruction and highlights the balance between cytokine-mediated viral control and liver damage.
Assuntos
Citocinas/metabolismo , Vírus da Hepatite B/imunologia , Hepatite B Crônica/imunologia , Fígado/imunologia , Linfócitos T/citologia , Adulto , Idoso , DNA Circular/metabolismo , Feminino , Fibrose , Antígenos E da Hepatite B/imunologia , Hepatite B Crônica/patologia , Humanos , Interleucina-10/metabolismo , Fígado/patologia , Masculino , Pessoa de Meia-Idade , Fenótipo , Coloração e Rotulagem , Linfócitos T/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
PURPOSE OF REVIEW: This article provides an overview of anticancer therapies in various stages of clinical development as potential interventions to target HIV persistence. RECENT FINDINGS: Epigenetic drugs developed for cancer have been investigated in vitro, ex vivo and in clinical trials as interventions aimed at reversing HIV latency and depleting the amount of virus that persists on antiretroviral therapy. Treatment with histone deacetylase inhibitors induced HIV expression in patients on antiretroviral therapy but did not reduce the frequency of infected cells. Other interventions that may accelerate the decay of latently infected cells, in the presence or absence of latency-reversing therapy, are now being explored. These include apoptosis-promoting agents, nonhistone deacetylase inhibitor compounds to reverse HIV latency and immunotherapy interventions to enhance antiviral immunity such as immune checkpoint inhibitors and Toll-like receptor agonists. SUMMARY: A curative strategy in HIV will likely need to both reduce the amount of virus that persists on antiretroviral therapy and improve anti-HIV immune surveillance. Although we continue to explore advances in the field of oncology including cancer immunotherapy, there are major differences in the risk-benefit assessment between HIV-infected individuals and patients with malignancies. Drug development specifically targeting HIV persistence will be the key to developing effective interventions with an appropriate safety profile.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Infecções por HIV/complicações , Imunoterapia/métodos , Neoplasias/terapia , Fármacos Anti-HIV/isolamento & purificação , Pesquisa Biomédica/tendências , Infecções por HIV/tratamento farmacológico , HumanosRESUMO
OBJECTIVE: To determine the long-term effects of vorinostat on safety and virological parameters in HIV-infected individuals on suppressive antiretroviral therapy (ART). DESIGN: Prospective longitudinal observational extended follow-up of 20 HIV-infected individuals on ART previously enrolled in a clinical trial of daily vorinostat 400âmg for 14 days. Extended follow-up included visits at 6, 12, 18 and 24 months postenrolment in the initial clinical trial. METHODS: Cell-associated unspliced HIV RNA, total HIV DNA and plasma HIV RNA were quantified by PCR, and CD4 and CD8 T cells quantified by flow cytometry. Changes over time in each parameter were assessed using the Wilcoxon matched pair signed-rank test and generalized estimating equations for trend modelling. RESULTS: We recorded a total of 31 adverse events (26 grade 1 and five grade 2) in all study participants (nâ=â20). There were no significant changes in the number of CD4 or CD8 T cells or plasma HIV RNA over time. In 12 participants for whom baseline samples were available, there were no significant changes in total HIV DNA, cell-associated unspliced HIV RNA, plasma RNA or CD4 and CD8 T cells at 6, 12, 18 or 24 months. CONCLUSION: Extended follow-up for 24 months did not reveal any long-term toxicity or changes in markers of HIV persistence or transcription in participants on ART who had received 14 days of vorinostat.
Assuntos
Antirretrovirais/uso terapêutico , Antineoplásicos/uso terapêutico , Interações Medicamentosas , Infecções por HIV/tratamento farmacológico , Ácidos Hidroxâmicos/uso terapêutico , Neoplasias/tratamento farmacológico , Carga Viral , Antirretrovirais/efeitos adversos , Contagem de Linfócito CD4 , Relação CD4-CD8 , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/epidemiologia , Infecções por HIV/complicações , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/efeitos adversos , Estudos Longitudinais , Estudos Prospectivos , Resultado do Tratamento , VorinostatRESUMO
Developing robust in vitro models of HIV latency is needed to better understand how latency is established, maintained and reversed. In this study, we examined the effects of donor variability, HIV titre and co-receptor usage on establishing HIV latency in vitro using two models of HIV latency. Using the CCL19 model of HIV latency, we found that in up to 50% of donors, CCL19 enhanced latent infection of resting CD4+ T-cells by CXCR4-tropic HIV in the presence of low dose IL-2. Increasing the infectious titre of CXCR4-tropic HIV increased both productive and latent infection of resting CD4+ T-cells. In a different model where myeloid dendritic cells (mDC) were co-cultured with resting CD4+ T-cells, we observed a higher frequency of latently infected cells in vitro than CCL19-treated or unstimulated CD4+ T-cells in the presence of low dose IL-2. In the DC-T-cell model, latency was established with both CCR5- and CXCR4-tropic virus but higher titres of CCR5-tropic virus was required in most donors. The establishment of latency in vitro through direct infection of resting CD4+ T-cells is significantly enhanced by CCL19 and mDC, but the efficiency is dependent on virus titre, co-receptor usage and there is significant donor variability.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Latência Viral/imunologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Quimiocina CCL19/imunologia , Quimiocina CCL19/farmacologia , Técnicas de Cocultura , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/virologia , Infecções por HIV/sangue , Infecções por HIV/virologia , HIV-1/fisiologia , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Interleucina-2/imunologia , Interleucina-2/farmacologia , Modelos Imunológicos , Receptores CCR5/imunologia , Receptores CXCR4/imunologia , Latência Viral/efeitos dos fármacosRESUMO
We describe here a severe flare of hepatitis caused by lamivudine-resistant hepatitis B virus(HBV) in an HIV/HBV co-infected individual.Lamivudine-resistant HBV was detected 6 months before the development of severe hepatitis. Sequencing of the HBV genome isolated from the patients' serum did not identify compensatory mutations in the HBV polymerase that may have restored viral replication. However, a strong HBV-specific CD8 T-cell response was identified and may have resulted in the severe hepatitis.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Vírus da Hepatite B/genética , Hepatite B Crônica/imunologia , Adulto , Terapia Antirretroviral de Alta Atividade , Sequência de Bases , DNA Viral/análise , Farmacorresistência Viral , Vírus da Hepatite B/imunologia , Hepatite B Crônica/complicações , Hepatite B Crônica/genética , Humanos , Lamivudina/uso terapêutico , Masculino , Mutação , Inibidores da Transcriptase Reversa/uso terapêutico , Replicação Viral/genética , Replicação Viral/imunologiaRESUMO
High throughput studies of hepatitis B virus (HBV) replication are limited by the absence of convenient and reliable in vitro systems that can be utilised to examine mutations emerging as a consequence of selective pressures such as drug therapy, immune response and genotypic evolution. We have developed an efficient and reproducible method of transfecting a human hepatoma cell line (Huh7) with a replication competent clone of HBV, which utilises an internal reporter of transfection efficiency. Wild type (WT) and a common lamivudine (LMV) resistant mutant virus characterised by a methionine to isoleucine mutation in the highly conserved YMDD motif of the polymerase gene were used to highlight the utility of this in vitro transfection procedure in studying common HBV mutations. The presence of an internal GFP reporter increased the efficiency of generating highly reproducible results and facilitated multiple forms of analysis from a single transfection accurately.
Assuntos
Genes Reporter , Vírus da Hepatite B/fisiologia , Transfecção , Replicação Viral , Antivirais/farmacologia , Northern Blotting , Linhagem Celular Tumoral , DNA Viral/análise , Farmacorresistência Viral , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/genética , Humanos , Lamivudina/farmacologia , Mutação , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Viral/análise , Reprodutibilidade dos TestesRESUMO
Histone deacetylase inhibitors (HDACi) can induce human immunodeficiency virus (HIV) transcription from the HIV long terminal repeat (LTR). However, ex vivo and in vivo responses to HDACi are variable and the activity of HDACi in cells other than T-cells have not been well characterised. Here, we developed a novel assay to determine the activity of HDACi on patient-derived HIV LTRs in different cell types. HIV LTRs from integrated virus were amplified using triple-nested Alu-PCR from total memory CD4+ T-cells (CD45RO+) isolated from HIV-infected patients prior to and following suppressive antiretroviral therapy. NL4-3 or patient-derived HIV LTRs were cloned into the chromatin forming episomal vector pCEP4, and the effect of HDACi investigated in the astrocyte and epithelial cell lines SVG and HeLa, respectively. There were no significant differences in the sequence of the HIV LTRs isolated from CD4+ T-cells prior to and after 18 months of combination antiretroviral therapy (cART). We found that in both cell lines, the HDACi panobinostat, trichostatin A, vorinostat and entinostat activated patient-derived HIV LTRs to similar levels seen with NL4-3 and all patient derived isolates had similar sensitivity to maximum HDACi stimulation. We observed a marked difference in the maximum fold induction of luciferase by HDACi in HeLa and SVG, suggesting that the effect of HDACi may be influenced by the cellular environment. Finally, we observed significant synergy in activation of the LTR with vorinostat and the viral protein Tat. Together, our results suggest that the LTR sequence of integrated virus is not a major determinant of a functional response to an HDACi.
Assuntos
Infecções por HIV/sangue , Repetição Terminal Longa de HIV/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Linfócitos T/virologia , Adulto , Idoso , Fármacos Anti-HIV/uso terapêutico , Benzamidas/farmacologia , Linhagem Celular , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Células HeLa , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Estudos Observacionais como Assunto , Panobinostat , Filogenia , Piridinas/farmacologia , Linfócitos T/efeitos dos fármacos , Vorinostat , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologiaRESUMO
OBJECTIVE: We aimed to compare the potential for inducing HIV production and the effect on T-cell activation of potent HDAC inhibitors undergoing clinical investigation. DESIGN: In vitro study RESULTS: The various HDAC inhibitors displayed significant potency differences in stimulating HIV-1 expression from the latently infected cell lines with panobinostat>givinostat ≈belinostat>vorinostat>valproic acid. Panobinostat was significantly more potent than all other HDAC inhibitors and induced virus production even in the very low concentration range 8-31 nM. The proportion of primary T-cells expressing the early activation marker CD69 increased moderately in all HDAC inhibitor-treated cells compared with untreated cells. Finally, proof was obtained that panobinostat, givinostat and belinostat induce virus production in latently infected primary cells at therapeutic concentrations with panobinostat being the most potent stimulator. METHODS: The latently infected cell lines ACH2 and U1 were treated with the HDAC inhibitors panobinostat, givinostat, belinostat, vorinostat and valproic acid. Viral induction was estimated by p24 production. Peripheral blood mononuclear cells from uninfected donors were treated with the HDAC inhibitors and the expression of activation markers on T-cell phenotypes was measured using flow cytometry. Finally, the ability of givinostat, belinostat and panobinostat to reactivate latent HIV-1 expression in primary T-cells was investigated employing a CCL19-induced latent primary CD4+ T cell infection model. CONCLUSION: At therapeutic concentrations panobinostat stimulate HIV-1 expression in latently infected cells with greater potency than other HDAC inhibitors undergoing clinical investigation. These findings warrant further investigation and panobinostat is now being advanced into clinical testing against latent HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , HIV-1/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Ativação Linfocitária , Linfócitos T/imunologia , Latência Viral/efeitos dos fármacos , Antígenos CD/análise , Antígenos de Diferenciação de Linfócitos T/análise , Células Cultivadas , Proteína do Núcleo p24 do HIV/análise , HIV-1/crescimento & desenvolvimento , HIV-1/imunologia , Humanos , Ácidos Hidroxâmicos/farmacologia , Indóis/farmacologia , Lectinas Tipo C/análise , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/virologia , PanobinostatRESUMO
OBJECTIVES: To compare the potency, toxicity and mechanism of action of multiple histone deacetylase inhibitors (HDACi) in activating HIV production from latency. DESIGN: In-vitro analysis of HDACi in a primary T-cell model of HIV latency and latently infected cell lines. METHODS: Latently infected chemokine ligand 19 (CCL19)-treated CD4⺠T cells and the latently infected cell lines ACH2 and J-Lat were treated with a panel of HDACi, including entinostat, vorinostat, panonbinostat and MCT3. Viral production and cell viability were compared. Expression of cellular HDACs was measured by western blot and PCR. Association of HDACs with the HIV long-terminal repeat (LTR) using latently infected CCL19-treated primary CD4⺠T cells in the presence and absence of specific HDACi was determined by chromatin immunoprecipitation (ChIP). RESULTS: We demonstrated considerable variation in the potency and toxicity of HDACi in latently infected primary CD4⺠T cells and cell lines. All HDACi tested activated HIV production in latently infected primary T cells with greatest potency demonstrated with entinostat and vorinostat and greatest toxicity with panobinostat. Following the addition of HDACi in vitro, there were no changes in markers of T-cell activation or expression of the HIV coreceptors chemokine (C-X-C motif) receptor 4 (CXCR4) or chemokine (C-C motif) receptor type 5 (CCR5). ChIP analysis of latently infected CCL19-treated primary CD4⺠T cells showed binding by HDAC1, HDAC2 and HDAC3 to the LTR with removal of HDAC1 and HDAC2 following treatment with the HDACi vorinostat and HDAC1 only following treatment with entinostat. CONCLUSION: The HDACi entinostat, selective for inhibition of class I HDACs, induced virus expression in latently infected primary CD4⺠T cells making this compound an attractive novel option for future clinical trials.
Assuntos
Benzamidas/farmacologia , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/virologia , HIV/fisiologia , Inibidores de Histona Desacetilases/farmacologia , Piridinas/farmacologia , Latência Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Western Blotting , Células Cultivadas , Imunoprecipitação da Cromatina , Humanos , Reação em Cadeia da PolimeraseAssuntos
Anticorpos Monoclonais/uso terapêutico , Infecções por HIV/tratamento farmacológico , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Terapia Combinada , Infecções por HIV/complicações , Infecções por HIV/imunologia , Humanos , Ipilimumab , Masculino , Melanoma/secundário , Pessoa de Meia-Idade , Prognóstico , Neoplasias Cutâneas/patologia , Resultado do TratamentoRESUMO
We investigated the ability of several novel class I histone deacetylase inhibitors to activate HIV-1 transcription in latently infected cell lines. Oxamflatin, metacept-1 and metacept-3 induced high levels of HIV-1 transcription in latently infected T cell and monocytic cells lines, were potent inhibitors of histone deacetylase activity and caused preferential cell death in transcriptionally active cells. Although these compounds had potent in-vitro activity, their cytotoxicity may limit their use in patients.
Assuntos
HIV-1/efeitos dos fármacos , Ácidos Hidroxâmicos/farmacologia , Sulfonamidas/farmacologia , Linfócitos T/virologia , Ativação Transcricional/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos/métodos , HIV-1/genética , HIV-1/fisiologia , Inibidores de Histona Desacetilases , Histona Desacetilases/metabolismo , Humanos , Latência Viral/efeitos dos fármacosRESUMO
BACKGROUND: Latently infected resting memory CD4 T cells are thought to be the major reservoir that contributes to rebound viremia after cessation of antiretrovirals (ARVs). Commencing ARVs during primary HIV-1 infection (PHI) may limit the size of the latent pool and lead to improved control of viral replication during structured treatment interruption (STI). METHODS: Individuals with PHI (n = 59) were randomized to receive ARVs with or without hydroxyurea. After STI, a good response was defined as maintenance of HIV-1 RNA <5000 copies/mL for 24 weeks off therapy. In a detailed prospective virologic substudy (n = 19), integrated HIV-1 DNA, total HIV-1 DNA, and cell-associated HIV-1 unspliced (US) RNA were quantified using a real-time polymerase chain reaction assay. RESULTS: The plasma HIV-1 RNA 12 weeks after the initiation of ARVs was significantly lower in good responders (n = 7) compared with poor responders (n = 12) (P = 0.005). There were no significant differences between good and poor responders in integrated HIV-1 DNA, HIV-1 DNA, and HIV-1 US RNA. Integrated HIV-1 DNA before initiation of ARVs was strongly correlated with plasma HIV-1 RNA at week 12 (P = 0.006, r = 0.81). CONCLUSION: HIV-1 RNA measured 12 weeks after initiation of ARV was the only virologic variable associated with viral rebound after treatment interruption in PHI.