MicroRNA expression profiling in HCV-infected human hepatoma cells identifies potential anti-viral targets induced by interferon-α.

OBJECTIVE: Increasing evidence suggests that miRNAs have a profound impact on host defense to Hepatitis C virus (HCV) infection and clinical outcome of standard HCV therapy. In this study, we investigated modulation of miRNA expression in Huh7.5 hepatoma cells by HCV infection and in vitro interferon-αtreatment. METHODS: MiRNA expression profiling was determined using Human miRNA TaqMan® Arrays followed by rigorous pairwise statistical analysis. MiRNA inhibitors assessed the functional effects of miRNAs on HCV replication. Computational analysis predicted anti-correlated mRNA targets and their involvement in host cellular pathways. Quantitative RTPCR confirmed the expression of predicted miRNA-mRNA correlated pairs in HCV-infected Huh7.5 cells with and without interferon-α. RESULTS: Seven miRNAs (miR-30b, miR-30c, miR-130a, miR-192, miR-301, miR-324-5p, and miR-565) were down-regulated in HCV-infected Huh7.5 cells (p<0.05) and subsequently up-regulated following interferon-α treatment (p<0.01). The miR-30(a-d) cluster and miR-130a/301 and their putative mRNA targets were predicted to be associated with cellular pathways that involve Hepatitis C virus entry, propagation and host response to viral infection. CONCLUSIONS: HCV differentially modulates miRNAs to facilitate entry and early establishment of infection in vitro. Interferon-α appears to neutralize the effect of HCV replication on miRNA regulation thus providing a potential mechanism of action in eradicating HCV from hepatocytes.

Augmentation of hepatitis B virus-specific cellular immunity with programmed death receptor-1/programmed death receptor-L1 blockade in hepatitis B virus and HIV/hepatitis B virus coinfected patients treated with adefovir.

The immunological parameters leading to viral persistence in chronic hepatitis B (CHB) are not clearly established. We analyzed HBV-specific immunoregulatory mechanisms in HIV-infected and HIV-uninfected HBeAg(+) CHB patients to determine (1) the roles of immunoregulatory pathways, (2) the effect of anti-HBV therapy on immunoregulatory pathways, and (3) the role of immunomodulatory therapy to overcome the effect of T regulatory cells (Tregs, CD4(+)CD25(+)FoxP3(+)) in HBV-infected individuals. A prospective, double blind, randomized, placebo-controlled trial treated HBV (HIV(+/-))-infected patients with adefovir 10 mg daily or placebo for 48 weeks. HBV viral load (VL), immunophenotying, and functional studies were performed at multiple time points. Suppression of HBV VL with adefovir leads to decreased peripheral expansion of Tregs. While declining, Tregs significantly inhibit cytokine-secreting HBV-specific CD8(+) T cell responses over 48 weeks of anti-HBV adefovir therapy (p<0.05). A large proportion of these Tregs express programmed death receptor-1 (PD-1), blockade of which in vitro leads to improved cytokine-secreting HBV-specific CD8(+) T cell responses, particularly in HIV/HBV-coinfected patients (p<0.05). Peripheral expansion of Treg levels correlated with HBV viral load and decreased HBV-specific CD8(+) T cells. PD-1 blockade increased survival of HBV-specific CD8(+) T cells, removing the inhibitory effect of PD-1(+) peripheral Tregs. Hence therapies involving PD-1 blockade in combination with directly acting antivirals should be investigated to reduce the need for life-long directly acting antiviral therapy.

Human immunodeficiency virus enhances hepatitis C virus replication by differential regulation of IFN and TGF family genes.

HIV co-infection significantly impacts the natural history of hepatitis C virus (HCV) by increasing plasma HCV viral load, accelerating liver disease progression, and reducing rates of HCV clearance. Cytokines play an important role in regulating hepatic inflammation and fibrogenesis during chronic HCV infection, yet the impact of HIV on cytokine expression is unknown. In this study, an HCV continuous infection cell culture system was modified to permit co-infection with HIV to test the hypothesis that virus-induced disregulation of immune-response genes, particularly interferons and TGF-ß, may create a permissive environment for the initial establishment of HIV/HCV co-infection in the host. CCR5-expressing Huh-7.5 hepatoma cells were transduced with human CD4 antigen to allow HIV infection in vitro. Co-infection of CD4⁺ Huh-7.5 cells with HIV and HCV or co-culture of HIV-infected CD4⁺ Huh-7.5 cells and HCV-infected Huh-7.5 cells increased the level of HCV RNA compared to HCV mono-infection. Quantitative gene expression analysis revealed HIV-induced up regulation of most tested IFN family genes when compared to HCV or co-infection. HCV infection induced up regulation of many TGF family genes that were subsequently down-regulated in the presence of HIV or HIV/HCV. Interestingly, co-infection resulted in down regulation of several IFN genes and significant up regulation of TGF-ß genes leading to an overall enhancement of HCV replication. These data suggest that HIV infection may influence HCV replication in vitro by increasing levels of HCV RNA, possibly through the differential regulation of endogenous IFN and TGF family genes.

A randomized, controlled, trial of short cycle intermittent compared to continuous antiretroviral therapy for the treatment of HIV infection in Uganda.

BACKGROUND: Short cycle treatment interruption could reduce toxicity and drug costs and contribute to further expansion of antiretroviral therapy (ART) programs. METHODS: A 72 week, non-inferiority trial enrolled one hundred forty six HIV positive persons receiving ART (CD4+ cell count > or =125 cells/mm(3) and HIV RNA plasma levels <50 copies/ml) in one of three arms: continuous, 7 days on/7 days off and 5 days on/2 days off treatment. Primary endpoint was ART treatment failure determined by plasma HIV RNA level, CD4+ cell count decrease, death attributed to study participation, or opportunistic infection. RESULTS: Following enrollment of 32 participants, the 7 days on/7 days off arm was closed because of a failure rate of 31%. Six of 52 (11.5%) participants in the 5 days on/2 days off arm failed. Five had virologic failure and one participant had immunologic failure. Eleven of 51 (21.6%) participants in the continuous treatment arm failed. Nine had virologic failure with 1 death (lactic acidosis) and 1 clinical failure (extra-pulmonary TB). The upper 97.5% confidence boundary for the difference between the percent of non-failures in the 5 days on/2 days off arm (88.5% non-failure) compared to continuous treatment (78.4% non failure) was 4.8% which is well within the preset non-inferiority margin of 15%. No significant difference was found in time to failure in the 2 study arms (p = 0.39). CONCLUSIONS: Short cycle 5 days on/2 days off intermittent ART was at least as effective as continuous therapy. TRIAL REGISTRATION: ClinicalTrials.gov NCT00339456.

Altered regulation of extrinsic apoptosis pathway in HCV-infected HCC cells enhances susceptibility to mapatumumab-induced apoptosis.

BACKGROUND: Hepatitis C virus (HCV)-infected patients, including those co-infected with human immunodeficiency virus (HIV), are at increased risk of developing hepatocellular carcinoma (HCC). We evaluated the ability of agonistic human monoclonal antibodies to tumor necrosis factor-related apoptosis inducing ligand (TRAIL) receptors, mapatumumab and lexatumumab, respectively, to induce TRAIL-receptor mediated apoptosis (TRMA) in HCC (HCV-infected and -uninfected) cells and in peripheral blood cells (HIV-infected and -uninfected). METHODS: Susceptibility to antibody-mediated TRMA was measured by caspase 3/7 activity and by confocal microscopy. Surface expression of receptors on HCV-uninfected and -infected Huh7.5 cells was measured by flow cytometry and confocal microscopy. Inhibitor of Apoptosis Protein (IAP) RNA levels were quantified by RT-PCR. DNA Microarray was performed using RNA isolated from Huh7.5 cells (HCV-infected and uninfected) using Affymetrix U133A chips. RESULTS: Mapatumumab preferentially induces TRMA of HCV-infected Huh7.5 cells by binding to TRAIL-R1. Higher basal expression of TRAIL-R2 compared to that of TRAIL-R1 on HCV-uninfected Huh7.5 cells were observed. Lexatumumab induces TRMA of both HCV-infected and -uninfected cells by binding to TRAIL-R2. IFN-alpha has minimal effect on mapatumumab- and lexatumumab-induced TRMA. HCV infection of Huh7.5 cells up-regulates TRAIL-R1 expression and X-linked Inhibitor of apoptosis protein and survivin gene expression. Neither antibody had a pro-apoptotic effect on PBMCs from patients with HIV infection ex vivo. CONCLUSION: Both mapatumumab and lexatumumab are excellent candidates for therapy of HCC. HCV infection of Huh7.5 cells selectively up-regulates TRAIL-R1 receptor, associated with increased susceptibility to mapatumumab-mediated TRMA. HCV infection up-regulated IAP genes, offering promise for future combination therapy using TRAIL agonists and IAP inhibitors.

Human immunodeficiency virus and hepatitis C infections induce distinct immunologic imprints in peripheral mononuclear cells.

UNLABELLED: Coinfection with hepatitis C virus (HCV) is present in one-third of all human immunodeficiency virus (HIV)-infected individuals in the United States and is associated with rapid progression of liver fibrosis and poor response to pegylated interferon (IFN) and ribavirin. In this study we examined gene expression profiles in peripheral blood mononuclear cells (PBMCs) from different groups of individuals who are monoinfected or coinfected with HIV and HCV. Data showed that HIV and HCV viremia up-regulate genes associated with immune activation and immunoregulatory pathways. HCV viremia is also associated with abnormalities in all peripheral immune cells, suggesting a global effect of HCV on the immune system. Interferon-alpha-induced genes were expressed at a higher level in PBMCs from HIV-infected individuals. HCV and HIV infections leave distinct profiles or gene expression of immune activation in PBMCs. HIV viremia induces an immune activated state; by comparison, HCV infection induces immunoregulatory and proinflammatory pathways that may contribute to progression of liver fibrosis. CONCLUSION: An aberrant type-I IFN response seen exclusively in HIV-infected individuals could be responsible for the poor therapeutic response experienced by HIV/HCV coinfected individuals receiving interferon-alpha-based current standard of care.

Evaluation of the pathogenesis of decreasing CD4(+) T cell counts in human immunodeficiency virus type 1-infected patients receiving successfully suppressive antiretroviral therapy.

Most human immunodeficiency virus (HIV)-infected individuals experience increases in peripheral CD4(+) T cell counts with suppressive antiretroviral therapy (ART) that achieves plasma HIV RNA levels that are less than the limit of detection. However, some individuals experience decreasing CD4(+) T cell counts despite suppression of plasma viremia. We evaluated 4 patients with a history of CD4(+) T cell decline despite successfully suppressive ART, from a median of 719 cells/mm(3) (range, 360-1141 cells/mm(3)) to 227 cells/mm(3) (range, 174-311 cells/mm(3)) over a period of 18-24 months; 3 of the patients were receiving tenofovir and didanosine, which may have contributed to this decrease. There was no evidence of HIV replication, nor of antiretroviral drug resistance in the blood or lymphoid tissue, or increased proliferation or decreased thymic production of naive CD4(+) T cells. All 4 patients had significant fibrosis of the T cell zone of lymphoid tissue, which appeared to be an important factor in the failure to reconstitute T cells.

Virological outcome after structured interruption of antiretroviral therapy for human immunodeficiency virus infection is associated with the functional profile of virus-specific CD8+ T cells.

A clear understanding of the antiviral effects of CD8(+) T cells in the context of chronic human immunodeficiency virus (HIV) infection is critical for the development of prophylactic vaccines and therapeutics designed to support T-cell-mediated immunity. However, defining the potential correlates of effective CD8(+) T-cell immunity has proven difficult; notably, comprehensive analyses have demonstrated that the size and shape of the CD8(+) T-cell response are not necessarily indicative of efficacy determined by measures of plasma viral load. Here, we conducted a detailed quantitative and qualitative analysis of CD8(+) T-cell responses to autologous virus in a cohort of six HIV-infected individuals with a history of structured interruption of antiretroviral therapy (ART) (SIT). The magnitude and breadth of the HIV-specific response did not, by themselves, explain the changes observed in plasma virus levels after the cessation of ART. Furthermore, mutational escape from targeted epitopes could not account for the differential virological outcomes in this cohort. However, the functionality of HIV-specific CD8(+) T-cell populations upon antigen encounter, determined by the simultaneous and independent measurement of five CD8(+) T-cell functions (degranulation and gamma interferon, macrophage inflammatory protein 1beta, tumor necrosis factor alpha, and interleukin-2 levels) reflected the emergent level of plasma virus, with multiple functions being elicited in those individuals with lower levels of viremia after SIT. These data show that the quality of the HIV-specific CD8(+) T-cell response, rather than the quantity, is associated with the dynamics of viral replication in the absence of ART and suggest that the effects of SIT can be assessed by measuring the functional profile of HIV-specific CD8(+) T cells.

HIV-1 gp120 induces NFAT nuclear translocation in resting CD4+ T-cells.

The replication of human immunodeficiency virus (HIV) in CD4+ T-cells is strongly dependent upon the state of activation of infected cells. Infection of sub-optimally activated cells is believed to play a critical role in both the transmission of virus and the persistence of CD4+ T-cell reservoirs. There is accumulating evidence that HIV can modulate signal-transduction pathways in a manner that may facilitate replication in such cells. We previously demonstrated that HIV gp120 induces virus replication in resting CD4+ T cells isolated from HIV-infected individuals. Here, we show that in resting CD4+ T-cells, gp120 activates NFATs and induces their translocation into the nucleus. The HIV LTR encodes NFAT recognition sites, and NFATs may play a critical role in promoting viral replication in sub-optimally activated cells. These observations provide insight into a potential mechanism by which HIV is able to establish infection in resting cells, which may have implications for both transmission of HIV and the persistence of viral reservoirs.

HIV-infected individuals receiving effective antiviral therapy for extended periods of time continually replenish their viral reservoir.

The persistence of latently infected, resting CD4+ T cells is considered to be a major obstacle in preventing the eradication of HIV-1 even in patients who have received effective antiviral therapy for an average duration of 5 years. Although previous studies have suggested that the latent HIV reservoir in the resting CD4+ T cell compartment is virologically quiescent in the absence of activating stimuli, evidence has been mounting to suggest that low levels of ongoing viral replication persist and in turn, prolong the overall half-life of HIV in patients receiving antiviral therapy. Here, we demonstrate the persistence of replication-competent virus in CD4+ T cells in a cohort of patients who had received uninterrupted antiviral therapy for up to 9.1 years that rendered them consistently aviremic throughout that time. Surprisingly, substantially higher levels of HIV proviral DNA were found in activated CD4+ T cells when compared with resting CD4+ T cells in the majority of patients we studied. Phylogenetic analyses revealed evidence for cross infection between the resting and activated CD4+ T cell compartments, suggesting that ongoing reactivation of latently infected, resting CD4+ T cells and spread of virus by activated CD4+ T cells may occur in these patients. Such events may allow continual replenishment of the CD4+ T cell reservoir and resetting of the half-life of the latently infected, resting CD4+ T cells despite prolonged periods of aviremia.

Identification of NKG2A and NKp80 as specific natural killer cell markers in rhesus and pigtailed monkeys.

Investigations of natural killer (NK) cells in simian models of disease have been hampered by a lack of appropriate phenotypic markers and by an inadequate understanding of the regulation of NK cell activities. In the present study, a panel of monoclonal antibodies (mAbs) specific for various human NK receptors was screened for cross-reactivity with NK cells from rhesus macaques and pigtailed macaques. Flow cytometric analyses using anti-human NKG2A and anti-human NKp80 mAbs individually, and particularly in combination with anti-CD16 mAb, allowed for the identification of the entire NK cell population in both species. NK cells in monkeys were generally identified by negative selection of peripheral blood mononuclear cells (PBMCs) for the absence of T-cell, B-cell, and monocyte markers. mAb-mediated ligation of NKp80 induced NK cell cytotoxicity, while in the case of NKG2A it displayed a clear capability to inhibit the lysis of target cells by NK cells from macaques, as well as from humans. This new phenotypic and functional characterization of NKG2A and NKp80 in rhesus and pigtailed macaque NK cells provides a new approach in the analysis of their innate immune system.

Targeted lysis of HIV-infected cells by natural killer cells armed and triggered by a recombinant immunoglobulin fusion protein: implications for immunotherapy.

Natural killer (NK) cells play an important role in both innate and adaptive antiviral immune responses. The adaptive response typically requires that virus-specific antibodies decorate infected cells which then direct NK cell lysis through a CD16 mediated process termed antibody-dependent cellular cytotoxicity (ADCC). In this report, we employ a highly polymerized chimeric IgG1/IgA immunoglobulin (Ig) fusion protein that, by virtue of its capacity to extensively crosslink CD16, activates NK cells while directing the lysis of infected target cells. We employ HIV as a model system, and demonstrate that freshly isolated NK cells preloaded with an HIV gp120-specific chimeric IgG1/IgA fusion protein efficiently lyse HIV-infected target cells at picomolar concentrations. NK cells pre-armed in this manner retain the capacity to kill targets over an extended period of time. This strategy may have application to other disease states including various viral infections and cancers.

CD25(+)CD4(+) regulatory T cells from the peripheral blood of asymptomatic HIV-infected individuals regulate CD4(+) and CD8(+) HIV-specific T cell immune responses in vitro and are associated with favorable clinical markers of disease status.

Human immunodeficiency virus (HIV) disease is associated with loss of CD4(+) T cells, chronic immune activation, and progressive immune dysfunction. HIV-specific responses, particularly those of CD4(+) T cells, become impaired early after infection, before the loss of responses directed against other antigens; the basis for this diminution has not been elucidated fully. The potential role of CD25(+)CD4(+) regulatory T cells (T reg cells), previously shown to inhibit immune responses directed against numerous pathogens, as suppressors of HIV-specific T cell responses was investigated. In the majority of healthy HIV-infected individuals, CD25(+)CD4(+) T cells significantly suppressed cellular proliferation and cytokine production by CD4(+) and CD8(+) T cells in response to HIV antigens/peptides in vitro; these effects were cell contact dependent and IL-10 and TGF-beta independent. Individuals with strong HIV-specific CD25(+) T reg cell function in vitro had significantly lower levels of plasma viremia and higher CD4(+): CD8(+) T cell ratios than did those individuals in whom this activity could not be detected. These in vitro data suggest that CD25(+)CD4(+) T reg cells may contribute to the diminution of HIV-specific T cell immune responses in vivo in the early stages of HIV disease.

A proof-of-concept study of short-cycle intermittent antiretroviral therapy with a once-daily regimen of didanosine, lamivudine, and efavirenz for the treatment of chronic HIV infection.

BACKGROUND: We previously demonstrated that short-cycle structured intermittent therapy (SIT; 7 days without therapy followed by 7 days with antiretroviral therapy [ART]) with a ritonavir-boosted, indinavir-based, twice-daily regimen maintained suppression of plasma HIV viremia while reducing serum levels of lipids. Adherence to such a regimen may be problematic for certain patients. METHODS: Eight patients with a history of receiving combination ART that maintained suppression of plasma HIV RNA to <50 copies/mL received a once-daily SIT regimen of didanosine, lamivudine, and efavirenz. RESULTS: For 7 patients, suppression of plasma HIV RNA to <50 copies/mL was maintained for 60-84 weeks. Four patients with adequate samples had no evidence for an increase in plasma viremia for up to 72 weeks, by use of an assay with a limit of detection of <1 copy/mL. The lack of rebound viremia may be the result of the persistence of efavirenz in plasma on day 7 of the no-therapy period, as was detected in 7 of 7 patients. There was no significant change in CD4(+) T cell counts or serum hepatic transaminase or lipid levels. CONCLUSION: A once-daily short-cycle SIT regimen maintained suppression of plasma HIV RNA while preserving CD4(+) T cell counts. Such a regimen may have importance in resource-limited settings where the monetary cost of continuous ART is prohibitive.

Natural killer cells in HIV-1 infection: dichotomous effects of viremia on inhibitory and activating receptors and their functional correlates.

Natural killer (NK) cells play a central role in host defense against various pathogens. Functional defects of NK cells in HIV-1 infection as a direct effect of abnormal expression or function of inhibitory NK receptors (iNKRs), activating natural cytotoxicity receptors (NCRs), and NKG2D have not yet been described. This study demonstrates an expansion of the functionally defective CD56-/CD16+ population of NK cells in viremic versus aviremic patients. We also demonstrate that in HIV-infected viremic patients, expression of iNKRs was well conserved and that in most cases, there was a trend toward increased expression on NK cells as compared with healthy donors. It was also demonstrated that the major activating NK receptors, with the exception of NKG2D, were significantly down-regulated. In contrast, the expression of iNKRs and activating receptors in HIV-infected individuals whose viremia was suppressed to below detectable levels by highly active antiretroviral therapy for 2 years or longer was comparable to that of healthy donors. Functional tests confirmed that the abnormal expression of the activating receptors and of iNKRs was associated with a markedly impaired NK cytolytic function. This phenomenon is not attributed to a direct HIV-1 infection of NK cells; thus, this study may provide insight into the mechanisms of impaired host defenses in HIV-1 viremic patients.

Long-cycle structured intermittent versus continuous highly active antiretroviral therapy for the treatment of chronic infection with human immunodeficiency virus: effects on drug toxicity and on immunologic and virologic parameters.

We evaluated the effect of long-cycle structured intermittent therapy (SIT; 4 weeks without highly active antiretroviral therapy [HAART] followed by 8 weeks with HAART) versus continuous HAART. The study was prematurely terminated to new enrollment because of the emergence of genetic mutations associated with resistance to antiretroviral drugs in 5 patients. After 48 weeks, there was no significant difference between groups in lipid, hepatic transaminase, and C-reactive protein levels in 41 patients. Although there were no differences in CD4(+) or CD8(+) T cell counts or the percentage of cells that were CD4(+)CD25(+), CD8(+)CD25(+), or CD4(+)DR(+), patients who received SIT had a significantly higher percentage of CD8(+)CD38(+) and CD8(+)DR(+) cells. There was no clear autoimmunization effect by immunologic or virologic parameters. There was no benefit to long-cycle SIT versus continuous HAART with regard to certain toxicity, immunologic, or virologic parameters.

Genetic characterization of rebounding human immunodeficiency virus type 1 in plasma during multiple interruptions of highly active antiretroviral therapy.

Various strategies of interrupting highly active antiretroviral therapy (HAART) are being investigated for the treatment of human immunodeficiency virus (HIV) infection. Interruptions of greater than 2 weeks frequently result in rebound of plasma HIV RNA. In order to discern changes in the viral population that might occur during cycles of treatment interruption, we evaluated the homology of HIV-1 envelope gene sequences over time in 12 patients who received four to seven cycles of 4 weeks off HAART followed by 8 weeks on HAART by using the heteroduplex tracking assay and novel statistical tools. HIV populations in 9 of 12 patients diverged from those found in the first cycle in at least one subsequent cycle. The substantial genetic changes noted in HIV env did not correlate with increased or decreased log changes in levels of plasma HIV RNA (P > 0.5). Thus, genetic changes in HIV env itself did not contribute in a systematic way to changes in levels of plasma viremia from cycle to cycle of treatment interruption. In addition, the data suggest that there may be multiple compartments contributing to the rebound of plasma viremia and to viral diversity from cycle to cycle of intermittent therapy.

HIV envelope induces a cascade of cell signals in non-proliferating target cells that favor virus replication.

Certain HIV-encoded proteins modify host-cell gene expression in a manner that facilitates viral replication. These activities may contribute to low-level viral replication in nonproliferating cells. Through the use of oligonucleotide microarrays and high-throughput Western blotting we demonstrate that one of these proteins, gp120, induces the expression of cytokines, chemokines, kinases, and transcription factors associated with antigen-specific T cell activation in the absence of cellular proliferation. Examination of transcriptional changes induced by gp120 in freshly isolated peripheral blood mononuclear cells and monocyte-derived-macrophages reveals a broad and complex transcriptional program conducive to productive infection with HIV. Observations include the induction of nuclear factor of activated T cells, components of the RNA polymerase II complex including TFII D, proteins localized to the plasma membrane, including several syntaxins, and members of the Rho protein family, including Cdc 42. These observations provide evidence that envelope-mediated signaling contributes to the productive infection of HIV in suboptimally activated T cells.