Achieving sustained virologic response after interferon-free hepatitis C virus treatment correlates with hepatic interferon gene expression changes independent of cirrhosis.

Chronic hepatitis C virus (HCV) infection can now be treated with oral directly acting antiviral agents, either with or without ribavirin (RBV). Virologic relapse after treatment can occur, and in some studies was more common in cirrhotic subjects. We previously observed changes in hepatic immunity during interferon (IFN)-free therapy that correlated with favourable outcome in subjects with early liver disease. Here, we compared changes in endogenous IFN pathways during IFN-free, RBV-free therapy between cirrhotic and noncirrhotic subjects. mRNA and microRNA (miRNA) expression analyses were performed on paired pre- and post-treatment liver biopsies from genotype-1 HCV subjects treated with sofosbuvir/ledipasvir (SOF/LDV) for 12 weeks (n = 4, 3 cirrhotics) or SOF/LDV combined with GS-9669 or GS-9451 for 6 weeks (n = 6, 0 cirrhotics). Nine of ten subjects achieved a sustained virologic response (SVR), while one noncirrhotic subject relapsed. Hepatic IFN-stimulated gene expression decreased with treatment in the liver of all subjects, with no observable impact of cirrhosis. Hepatic gene expression of type III IFNs (IFNL1, IFNL3, IFNL4-ΔG) similarly decreased with treatment, while IFNA2 expression, undetectable in all subjects pretreatment, was detected post-treatment in three subjects who achieved a SVR. Only the subject who relapsed had detectable IFNL4-ΔG expression in post-treatment liver. Other IFNs had no change in gene expression (IFNG, IFNB1, IFNA5) or could not be detected. Although expression of multiple hepatic miRNAs changed with treatment, many miRNAs previously implicated in HCV replication and IFN signalling had unchanged expression. In conclusion, favourable treatment outcome during IFN-free HCV therapy is associated with changes in the host IFN response regardless of cirrhosis.

Hepatic compartmentalization of exhausted and regulatory cells in HIV/HCV-coinfected patients.

Accelerated intrahepatic hepatitis C virus (HCV) pathogenesis is likely the result of dysregulation within both the innate and adaptive immune compartments, but the exact contribution of peripheral blood and liver lymphocyte subsets remains unclear. Prolonged activation and expansion of immunoregulatory cells have been thought to play a role. We determined immune cell subset frequency in contemporaneous liver and peripheral blood samples from chronic HCV-infected and HIV/HCV-coinfected individuals. Peripheral blood mononuclear cells (PBMC) and biopsy-derived liver-infiltrating lymphocytes from 26 HIV/HCV-coinfected, 10 chronic HCV-infected and 10 HIV-infected individuals were assessed for various subsets of T and B lymphocytes, dendritic cell, natural killer (NK) cell and NK T-cell frequency by flow cytometry. CD8(+) T cells expressing the exhaustion marker PD-1 were increased in HCV-infected individuals compared with uninfected individuals (P = 0.02), and HIV coinfection enhanced this effect (P = 0.005). In the liver, regulatory CD4(+) CD25(+) Foxp3(+) T cells, as well as CD4(+) CD25(+) PD1(+) T cells, were more frequent in HIV/HCV-coinfected than in HCV-monoinfected samples (P < 0.001). HCV was associated with increased regulatory T cells, PD-1(+) T cells and decreased memory B cells, regardless of HIV infection (P ≤ 0.005 for all). Low CD8(+) expression was observed only in PD-1(+) CD8(+) T cells from HCV-infected individuals and healthy controls (P = 0.002) and was associated with enhanced expansion of exhausted CD8(+) T cells when exposed in vitro to PHA or CMV peptides. In conclusion, in HIV/HCV coinfection, ongoing HCV replication is associated with increased regulatory and exhausted T cells in the periphery and liver that may impact control of HCV. Simultaneous characterization of liver and peripheral blood highlights the disproportionate intrahepatic compartmentalization of immunoregulatory T cells, which may contribute to establishment of chronicity and hepatic fibrogenesis in HIV coinfection.

Baseline CD4+ T-cell counts predict HBV viral kinetics to adefovir treatment in lamivudine-resistant HBV-infected patients with or without HIV infection.

BACKGROUND: Coinfection with HIV and hepatitis B virus (HBV) substantially alters the course of HBV. Directly acting anti-HBV agents suppress HBV viral levels; however, the kinetics of HBV decline in mono- and coinfected persons have not been evaluated. We investigated the role of baseline CD4+ T-cell counts as a predictor of HBV response to adefovir (ADV) therapy in chronic HBV with and without HIV coinfection. METHODS: We conducted a double-blind, randomized, placebo-controlled study of HIV-infected (n = 12) and uninfected (n = 5) chronic HBV patients treated with ADV. Five HIV uninfected patients received ADV; the HIV+ patients received ADV or placebo for a total of 48 weeks. At the end of 48 weeks, all patients received open-label ADV for an additional 48 weeks. HBV, HIV viral loads, CD4+ T-cell counts, and safety labs were performed on days 0, 1, 3, 5, 7, 10, 14, and 28 and then every 4 weeks. RESULTS: Lower HBV slopes were observed among coinfected compared to monoinfected patients (P = .027 at 4 weeks, P = .019 at 24 weeks, and P = .045 at 48 weeks). Using a mixed model analysis, we found a significant difference between the slopes of the 2 groups at 48 weeks (P = .045). Baseline CD4+ T-cell count was the only independent predictor of HBV decline in all patients. CONCLUSION: HIV coinfection is associated with slower HBV response to ADV. Baseline CD4+ T-cell count and not IL28B genotype is an independent predictor of HBV decline in all patients, emphasizing the role of immune status on clearance of HBV.

Effect of HIV on liver fibrosis among HCV-infected African Americans.

Degree of liver fibrosis largely determines treatment urgency for hepatitis C virus (HCV). This retrospective study examined fibrosis stages and predictive factors in African Americans with HCV monoinfection and human immunodeficiency virus (HIV)/HCV coinfection. Nearly 50% of patients had early-stage fibrosis in the study, despite the long duration of infection in many patients. HIV was associated with the early fibrosis group. These results indicate that a large proportion of patients with HCV infection, including those with HIV, could possibly await more-effective and better-tolerated treatment.

Distinct and overlapping genomic profiles and antiviral effects of Interferon-λ and -α on HCV-infected and noninfected hepatoma cells.

Recently, several SNPs in the region of the IL28B (IFN-λ) gene have been associated with spontaneous clearance of hepatitis C virus (HCV) and enhanced cure rates for IFN-alfa-based therapies, suggesting a potential correlation between IFN-λ and the ability to clear HCV. To understand the mechanism of IFN-λ's as compared to IFN-α's antiviral activity, we performed a comprehensive analysis of their anti-HCV effects, whole genome transcriptome profiling with validation, and signalling of IFN-α and IFN-λ using J6/JFH-1 and Huh7.5 cells in vitro. IFN-λ and IFN-α exhibited comparable anti-HCV activity and gene expression profiles in Huh7.5 cells. While the majority of genes induced by IFN-α and IFN-λ were similar, IFN-λ exhibits profound, but delayed kinetics of IFN-stimulated genes (ISG) induction, while IFN-α induced more rapid induction of ISGs. Furthermore, the increased induction of ISG expression by IFN-λ correlated with up-regulation of IFN-λ receptor (IL-28RA) expression and more prolonged activation of the Jak-STAT signalling pathway. The findings from our comparative analysis of IFN-α and IFN-λ in HCV-infected and noninfected cells support the clinical use of IFN-λ as a potential alternative to IFN-α in the treatment of chronic hepatitis C.

Therapeutic response to peg-IFN-alpha-2b and ribavirin in HIV/HCV co-infected African-American and Caucasian patients as a function of HCV viral kinetics and interferon pharmacodynamics.

METHOD: In this study we sought to characterize the relationship between several pharmacokinetic and pharmacodynamic parameters and virologic responses among HIV/hepatitis C virus genotype-1 co-infected patients receiving pegylated interferon-alpha-2b (peg-IFN2b) and ribavirin. We also tried to establish the underlying mechanisms that lead to poor sustained virologic responder rates observed with African-Americans against Caucasians and compared their results with those observed in a cohort of hepatitis C virus mono-infected patients. RESULTS: Among our studied population, a viral decline of more than 1.0 log at day 3 combined with viral load of less than 5.0 log IU/ml at day 28 predicted sustained virologic responders with negative predictive value 100% and positive predictive value 100%. African-Americans had significantly (P < 0.01) slower hepatitis C virus viral kinetics as compared to Caucasians. However, peg-IFN2b concentrations and pharmacokinetic parameters, peg-IFN2b(max) and peg-IFN2b half-life, were similar in both groups and did not predict sustained virologic responders. Nevertheless, the pharmacodynamic parameter EC(50), estimated from nonlinear fitting of the viral kinetics together with peg-IFN2b concentration data, showed that HIV/ hepatitis C virus co-infected African-Americans have lower sensitivity to interferon-alpha thus giving rise to slower viral decline. The combined pharmacokinetic/pharmacodynamic parameter IFN(max)/EC(90) was an excellent predictor of sustained virologic responders, thus showing the importance of maintaining peg-IFN2b levels above EC(90) to achieve successful treatment. CONCLUSION: Further studies are needed to evaluate whether these pharmacodynamic predictions are a result of differential host response to peg-IFN2b or other viral factors conferring relative resistance to peg-IFN2b.

Differential antiviral effect of PEG-interferon-alpha-2b on HIV and HCV in the treatment of HIV/HCV co-infected patients.

OBJECTIVE: The major antiviral effect of interferon (IFN)-alpha on hepatitis C virus (HCV) is blocking of virion production from infected cells. We now investigate the previously unknown mechanism of action of IFN-alpha against HIV. METHODS: HIV kinetics in parallel to HCV kinetics and IFN pharmacokinetics during pegylated-IFN-alpha-2b (1.5 microg/Kg q.w., PEG-IFN) and ribavirin (1-1.2 g daily) treatment in nine HIV patients co-infected with HCV genotype 1 were analyzed. In vivo modeling predictions of suppression of HIV replication by PEG-IFN in CD8-depleted peripheral blood mononuclear cells were verified by in vitro experiments. RESULTS: HCV and HIV show different viral decline patterns after administration of PEG-IFN. Unlike the bi-phasic decline shown by HCV, HIV shows a slow continuous decline during the first week, with no rebound when PEG-IFN levels decline. Fitting of HIV kinetics with known half-lives of free virus and infected cells indicates that the major effect of IFN on HIV is to block de novo infection rather than to block virion production. The magnitude of the antiviral effect is similar (mean 1.1 log10 decline at 7 days) to those of direct anti-HIV drugs, but shows an inverse correlation with baseline viremia. In vitro studies show that preincubation with IFN renders a suppression of HIV replication superior to that of treatment postinfection, thus corroborating the mathematical analysis in vivo. CONCLUSION: The complimentary antiviral properties of IFN-alpha and antiretroviral therapy suggest a role for pharmacokinetically improved formulations of IFN as part of salvage therapy for HIV-infected individuals.

Cytochrome P450 2B6 (CYP2B6) G516T influences nevirapine plasma concentrations in HIV-infected patients in Uganda.

OBJECTIVES: Polymorphisms in the cytochrome P450 (CYP) 2B6 gene have been shown to influence nevirapine plasma concentrations in HIV-infected European Caucasians. Although nevirapine is used extensively in Africa, the influence of CYP2B6 genotype on nevirapine exposure has not been assessed in this population. We aimed to determine the influence of CYP2B6 genotype at position 516 on nevirapine trough concentrations in HIV-infected patients in Kampala, Uganda. Additional polymorphisms in the CYP and multidrug resistance protein-1 (MDR-1) genes were also assessed for their impact on nevirapine concentrations. METHODS: The following genotypes were determined in all subjects using polymerase chain reaction-restriction fragment length polymorphism: CYP2B6 G516T, MDR-1 C3435T and G2677T, CYP3A4(*)1B and CYP3A5(*)3. Nevirapine plasma concentrations were determined using high-performance liquid chromatography in 23 HIV-infected patients who were generally healthy and had been taking nevirapine 200 mg twice daily for at least 14 days. Analysis of variance with post hoc testing was used to compare nevirapine concentrations among CYP2B6 genotype groups. RESULTS: The median nevirapine trough concentration in individuals homozygous for the variant allele (TT) was 7607 ng/mL vs 4181 and 5559 ng/mL for GG and GT individuals, respectively (GG vs TT median ratio=1.82; P=0.011). The mean ratio for TT vs GG individuals (95% confidence interval) was 1.51 (1.18, 1.84). No associations were observed between the other polymorphisms studied and nevirapine concentrations. CONCLUSIONS: CYP2B6 G516T significantly influenced nevirapine trough concentrations in HIV-infected patients in Uganda. Additional studies in larger patient populations are necessary to further define the potential clinical impact of these preliminary findings.

Gene expression profiles in hepatitis C virus (HCV) and HIV coinfection: class prediction analyses before treatment predict the outcome of anti-HCV therapy among HIV-coinfected persons.

Therapy for hepatitis C virus (HCV) infection in human immunodeficiency virus (HIV)-infected patients results in modest cure rates. Gene expression patterns in peripheral blood mononuclear cells from 29 patients coinfected with HIV and HCV were used to predict virological response to therapy for HCV infection. Prediction analysis using pretherapy samples identified 79 genes that correctly classified all 10 patients who did not respond to therapy, 8 of 10 patients with a response at the end of treatment, and 7 of 9 patients with sustained virological response (86% overall). Analysis of 17 posttreatment samples identified 105 genes that correctly classified all 9 patients with response at the end of treatment and 7 of 8 patients with sustained virological response (94% overall). Failure of anti-HCV therapy was associated with elevated expression of interferon-stimulated genes. Gene expression patterns may provide a tool to predict anti-HCV therapeutic response.

New insights into transmission, diagnosis, and drug treatment of Pneumocystis carinii pneumonia.

Pneumocystis carinii has been recognized as a human pathogen for nearly 50 years. We present a case of P carinii infection that typifies clinical presentation in the era of the acquired immunodeficiency syndrome epidemic. The high incidence of P carinii pneumonia in persons infected with human immunodeficiency virus (HIV) has served to focus laboratory and clinical research efforts on better understanding the biology of the organism and on improving diagnosis, treatment, and prevention of this disease. Although inability to culture P carinii has hampered research efforts, molecular and immunologic approaches have led to the recognition that the organism represents a family of fungi with a very restricted host range and have allowed characterization of clinically relevant antigens and enzymes. Molecular epidemiologic studies have identified more than 50 strains of human-derived P carinii and have suggested that recently acquired infection, as opposed to reactivation of latent infection, may account for many cases of clinical disease. Diagnosis has been improved by the development of organism-specific monoclonal antibodies and, more recently, by polymerase chain reaction using multicopy gene targets, together with induced sputum or oral wash samples. Chemotherapeutic prophylaxis is very effective in preventing P carinii pneumonia; the combination of trimethoprim-sulfamethoxazole remains the first-line agent for both therapy and prophylaxis. Prophylaxis needs to be administered only during periods of high risk; in HIV-infected patients responding to effective antiretroviral therapies, prophylaxis no longer needs to be lifelong. Molecular studies have identified mutations in the target of sulfa drugs that appear to represent emerging resistance in P carinii. Resistance to atovaquone, a second-line agent, may also be developing.

The use of oral washes to diagnose Pneumocystis carinii pneumonia: a blinded prospective study using a polymerase chain reaction-based detection system.

Pneumocystis carinii pneumonia (PCP) can be diagnosed by direct microscopic examination of induced sputum or by bronchoalveolar lavage (BAL). However, many institutions have little diagnostic success with induced sputum, and BAL is invasive and expensive. This prospective, blinded study assessed oral washes as a more convenient specimen than either sputum or BAL fluid and used a dissociation-enhanced lanthanide fluoroimmunoassay time-resolved fluorescent hybridization polymerase chain reaction (PCR) detection system that is feasible for clinical laboratories. The study assessed 175 oral washes, each paired with either an induced sputum that was positive for Pneumocystis or a BAL sample. The PCR test based on the Pneumocystis major surface glycoprotein primers had a sensitivity of 91% and a specificity of 94%, compared with a test based on mitochondrial large subunit rRNA primers, which had a sensitivity of 75% and a specificity of 96%. These results suggest that oral washes can provide a useful sample for diagnosis of PCP when a sensitive PCR detection system is used.

Identification of dynamically distinct subpopulations of T lymphocytes that are differentially affected by HIV.

We examined the effects of human immunodeficiency virus infection on the turnover of CD4 and CD8 T lymphocytes in 17 HIV-infected patients by 30 min in vivo pulse labeling with bromodeoxyuridine (BrdU). The percentage of labeled CD4 and CD8 T lymphocytes was initially higher in lymph nodes than in blood. Labeled cells equilibrated between the two compartments within 24 h. Based on mathematical modeling of the dynamics of BrdU-labeled cells in the blood, we identified rapidly and slowly proliferating subpopulations of CD4 and CD8 T lymphocytes. The percentage, but not the decay rate, of labeled CD4 or CD8 cells in the rapidly proliferating pool correlated significantly with plasma HIV RNA levels for both CD4 (r = 0.77, P < 0.001) and CD8 (r = 0.81, P < 0.001) T cells. In six patients there was a geometric mean decrease of greater than 2 logs in HIV levels within 2 to 6 mo after the initiation of highly active antiretroviral therapy; this was associated with a significant decrease in the percentage (but not the decay rate) of labeled cells in the rapidly proliferating pool for both CD4 (P = 0.03) and CD8 (P < 0.001) T lymphocytes. Neither plasma viral levels nor therapy had an effect on the decay rate constants or the percentage of labeled cells in the slowly proliferating pool. Monocyte production was inversely related to viral load (r = -0.56, P = 0.003) and increased with therapy (P = 0.01). These findings demonstrate that HIV does not impair CD4 T cell production but does increase CD4 and CD8 lymphocyte proliferation and death by inducing entry into a rapidly proliferating subpopulation of cells.

Death due to bioterrorism-related inhalational anthrax: report of 2 patients.

On October 9, 2001, a letter containing anthrax spores was mailed from New Jersey to Washington, DC. The letter was processed at a major postal facility in Washington, DC, and opened in the Senate's Hart Office Building on October 15. Between October 19 and October 26, there were 5 cases of inhalational anthrax among postal workers who were employed at that major facility or who handled bulk mail originating from that facility. The cases of 2 postal workers who died of inhalational anthrax are reported here. Both patients had nonspecific prodromal illnesses. One patient developed predominantly gastrointestinal symptoms, including nausea, vomiting, and abdominal pain. The other patient had a "flulike" illness associated with myalgias and malaise. Both patients ultimately developed dyspnea, retrosternal chest pressure, and respiratory failure requiring mechanical ventilation. Leukocytosis and hemoconcentration were noted in both cases prior to death. Both patients had evidence of mediastinitis and extensive pulmonary infiltrates late in their course of illness. The durations of illness were 7 days and 5 days from onset of symptoms to death; both patients died within 24 hours of hospitalization. Without a clinician's high index of suspicion, the diagnosis of inhalational anthrax is difficult during nonspecific prodromal illness. Clinicians have an urgent need for prompt communication of vital epidemiologic information that could focus their diagnostic evaluation. Rapid diagnostic assays to distinguish more common infectious processes from agents of bioterrorism also could improve management strategies.

Interleukin-2 induced immune effects in human immunodeficiency virus-infected patients receiving intermittent interleukin-2 immunotherapy.

To characterize the immunological effects of intermittent IL-2 therapy, which leads to selective increases in CD4+ T lymphocytes in HIV-infected patients, 11 patients underwent extensive immunological evaluation. While IL-2 induced changes in both CD4+ and CD8+ cell number acutely, only CD4+ cells showed sustained increases following discontinuation of IL-2. Transient increases in expression of the activation markers CD38 and HLA-DR were seen on both CD4+ and CD8+ cells, but CD25 (a chain of the IL-2 receptor) increased exclusively on CD4+ cells. This increase in CD25 expression was sustained for months following discontinuation of IL-2, and was seen in naive as well as memory cells. IL-2 induced cell proliferation, but tachyphylaxis to these proliferative effects developed after 1 week despite continued IL-2 administration. It thus appears that sustained CD25 expression selectively on CD4+ cells is a critical component of the immunological response to IL-2, and that intermittent administration of IL-2 is necessary to overcome the tachyphylaxis to IL-2-induced proliferation.

Immunotherapy of HIV-infected patients with intermittent interleukin-2: effects of cycle frequency and cycle duration on degree of CD4(+) T-lymphocyte expansion.

The ability of IL-2 to induce expansion of the CD4(+) T lymphocyte pool has made it the most studied cytokine in the treatment of HIV infection. The majority of trials have used an empirical regimen of 5-day IL-2 cycles given every 8 weeks--a regimen based upon early pharmacodynamic studies and patient preference. To better define optimal duration and frequency of cycles, a randomized trial was conducted in which patients who received this "standard" regimen were compared to patients who received cycles of variable duration (based on individual patterns of cell cycle progression) and to patients who received cycles of variable frequency (based on individual CD4(+) T lymphocyte responses to previous cycles). Twenty-two patients with HIV-1 infection and CD4(+) T lymphocyte counts > 200 cells/mm(3) were randomized to one of three treatment groups for 32 weeks of study. Eight participants received four 5-day IL-2 cycles (controls) every 8 weeks; 7 participants received four cycles of longer duration (mean 7.7-days); and 7 participants received an increased frequency of 5-day cycles (every 4.1 weeks on average). All three groups experienced significant increases in mean CD4(+) T lymphocytes. However, there were no statistically significant differences in CD4(+) T lymphocyte increases between the group that received longer cycles (median increase 239 cells/mm(3), P = 0.78) or between the group that received more frequent cycles (median increase 511 cells/mm(3), P = 0.54) and the control group (median 284 cells/mm(3)). HIV-1 viral loads decreased during the study period in all three groups. Our inability to demonstrate a significant advantage of increased frequency or duration of IL-2 administration provides corroborating experimental evidence for the use of an IL-2 regimen consisting of 5-day cycles administered no more frequently than every 8 weeks in future clinical trials aimed at expanding the CD4(+) T lymphocyte pool.

Rapid activation of lymph nodes and mononuclear cell HIV expression upon interrupting highly active antiretroviral therapy in patients after prolonged viral suppression.

OBJECTIVE: To compare the architecture and HIV-1 RNA and Gag p24 protein expression in lymph nodes (LN) excised from individuals during chronic highly active antiretroviral therapy (HAART) with LN removed from the same patient after plasma virus rebound following the interruption of HAART. MATERIALS AND METHODS: Six HIV-1-infected patients on HAART, with CD4 cell counts greater than 350 cells/microl, and plasma HIV-1 RNA less than 50 copies/ml, underwent inguinal LN excision upon discontinuation of HAART, and again after rebound of plasma virus. Lymph nodes were evaluated by immunohistochemical staining for Gag p24 antigen and Ki67, in-situ hybridization for HIV-1 RNA and H3-histone, and transmission electron microscopy (TEM). RESULTS: LN at baseline were quiescent to mildly hyperplastic and generally contained more primary than secondary follicles. Only one LN had detectable follicular dendritic cell (FDC)-associated p24 antigen, none had HIV RNA. Few mononuclear cells (MNC) expressed RNA or p24 antigen. Plasma virus at the second biopsy ranged from 329 to 3.2 x 10(6) copies/ml. CD4 cell count decline ranged from 5 to 51% during drug hiatus, and was greatest in patients with highest viral rebound. Four of six of the second LN were more hyperplastic than the initial LN, two showed paracortical hyperplasia. MNC expression of HIV RNA in the second LN paralleled the level of plasma viremia. Increased Ki67 and H3-histone signal occurred in the second LN. CONCLUSION: Quiescent LN from individuals on HAART rapidly become hyperplastic and activated within 1-2 months after treatment interruption. As in acute HIV infection, virus expression by LN MNC parallels the rebound in plasma viremia and fall in CD4 cell count.