Influence of HIV-related immunodeficiency on the histopathology of chronic hepatitis C

OBJECTIVE: There is substantial evidence demonstrating the aggravating effect of human immunodeficiency virus (HIV) infection on the progression of chronic hepatitis C virus (HCV) infection. There is however, little data on the affect of certain factors which could affect liver pathology findings in patients with concomitant HIV infection such as the duration of HIV infection or T-cell subpopulation counts. We examined pathology findings in patients with concomitant HIV and HCV infections to determine the impact of immunodepression. PATIENTS AND METHODS: We reviewed liver pathology data collected in patients with concomitant HIV and HCV infections grouping patients according to severity of the liver pathology: group 1 = cirrhosis or active hepatitis; group 2 = minimally active hepatitis or histologically normal liver. Transparietal liver biopsies were obtained for the work-up of viral hepatitis or because of long-term unexplained fever or suspected lymphoma. Epidemiological and biological data were obtained from medical files. The duration of the liver disease was estimated from the date of exposure to risk of immunodepression as determined by the peripheral CD4+ and CD8+ counts. All pathology specimens were read by two pathologists who established the Knodell score for each patient. RESULTS: Fifty patients were included: 23 were classed in group 1 and 28 in group 2. The Knodell score was significantly different between the two groups, 11 +/- 4 and 4 +/- 3 respectively (p < 0.0001). Disease duration was similar for the two groups: mean 8 years. Mean CD4+ count was significantly higher in group 1: 312/mm3 versus 110/mm3 for group 2 (p = 0.0057); as was the mean CD8+ count (758/mm3 versus 360/mm3, p = 0.0013). For the entire study population, there was a significantly negative correlation (p < 0.05) between the Knodell score and the CD4+ count (r = 0.31) and for the CD8+ count (r = 0.41). CONCLUSION: HCV-related liver pathology in patients co-infected with HIV depends on the level of immunodepression. CD8+ counts are better correlated with pathology findings than with CD4+ counts.     

Relationship between interferon therapy and variability in nonstructural gene 5b of hepatitis C virus

The hepatitis C virus (HCV) quasispecies nature in the hypervariable region (HVR) has been reported and found to relate to the effectiveness of interferon (IFN) treatment. However, the quasispecies nature in the nonstructural (NS) 5b region remains to be addressed. To examine this characterization and relationship with IFN therapy, we sequenced six independent HCV clones from each of eight patients. The eight patients were classified as responders or nonresponders to IFN. In the four responders, we found one to three isolates in each of the six clones. In the nonresponders, the six clones consisted of four, five, six, and six isolates, respectively. Compared the (NS) 5b genes of the isolates obtained from the patients with that of the reported hepatitis C virus HC-C2 or HC-J6 isolate the ratio of nonsynonymous to total substitutions ranged from 17.61% to 30.95% in the responders and from 33.11% to 76.47% in the nonresponders. We also compared posttreatment with pretreatment sequences. The average number of varying amino acids ranged from 5.5 to 9.0 in isolates remaining after IFN treatment and from 4.3 to 5.5 in the isolates that disappeared with IFN treatment. Two changed amino acids (glycine to arginine and valine to isoleucine) (compared with the pretreatment clones) were found in the posttreatment clones of one of the responders and one amino acid change (valine to alanine) was found in another responder. These results suggest that the NS5b quasispecies correlates with IFN treatment effectiveness. These results also implied that the heterogeneity in different hierarchical strata has a common impact on IFN treatment, making infected patients resistant to IFN. Our study also provides evidence that HCV elimination and mutation may occur simultaneously during IFN therapy.     

Influence of hepatitis C virus genotypes and HIV infection on histological severity of chronic hepatitis C. The Hepatitis/HIV Spanish Study Group

OBJECTIVES: The factors influencing the histological severity of chronic hepatitis C (CHC) have not been well established. We therefore investigated the effect of hepatitis C virus (HCV) genotypes and human immunodeficiency virus (HIV) infection on histological liver damage in a cohort of intravenous drug users with CHC. METHODS: We analyzed the histological activity score and the HCV genotypes in 59 HCV-RNA-positive patients with biopsy-proven CHC. Forty-eight (81%) of them had concomitant HIV infection with a CD4+ cell count above 200 x 10(6) cells/L and an absence of AIDS-defining conditions. Multivariate analysis was performed to determine the features associated with the histological severity. RESULTS: Minimal/mild hepatitis was found in 16 patients (27%), moderate chronic hepatitis in 29 (49%), and severe chronic hepatitis in 14 (24%). Patients with HCV subtype 1b had a higher histological score than others (8.7 +/- 3.3 vs. 6.5 +/- 3.2, p = 0.012), either as single or mixed infections. In multivariate analysis, HIV-infected individuals had a higher score of piecemeal necrosis (OR = 21.7, p = 0.002) and a higher stage of fibrosis (OR = 17.9, p = 0.004) than patients without HIV infection. HIV infection and HCV genotype 1b were found to be independent factors of histological severity. CONCLUSIONS: Liver damage in patients with CHC seems to be directly influenced by HCV subtypes. Infection by HCV subtype 1b is closely associated with more severe forms of liver pathology. Furthermore, the presence of HIV infection is an independent factor associated with more aggressive histological damage. In these patients, higher degrees of piecemeal necrosis and fibrosis are commonly seen.     

Replication of hepatitis C virus in cultured non-neoplastic human hepatocytes

We established a replication system for hepatitis C virus (HCV) using the PH5CH non-neoplastic human hepatocyte line that had been immortalized with simian virus 40 large T antigen. In cells inoculated with sera derived from two HCV-positive blood donors, positive-stranded HCV RNA was detected up to 30 days postinoculation (p.i.). Semi-quantitative analysis of HCV RNA revealed that HCV multiplied during the period of culture. Sequence analysis of the HCV hypervariable region 1 (HVR1) in both cases indicated that HVR1 populations from the cells at 8 days p.i. were apparently different from those of the original inocula. HVR1 populations in infected cells became homogeneous or just a few species were selected over time. These results suggest that HCV is replicating in the human hepatocyte PH5CH cells. This culture system will be useful for detailed studies of the biological effects of HCV in human hepatocytes.     

Variation of hepatitis C virus hypervariable region 1 in immunocompromised patients

The viral variability of 5 hepatitis C virus (HCV)-infected immunocompromised patients was analyzed and compared with that in isolates from immunocompetent subjects. The patients were followed longitudinally with regard to changes in hypervariable region 1 (HVR1) of HCV using a direct DNA sequencing approach. For the immunocompromised patients, viral nucleotide sequence variability was markedly lower than in immunocompetent HCV-positive patients. For 1 agammaglobulinemic patient and 1 AIDS patient, no variation in the major amino acid sequence of HCV HVR1 could be observed, while another agammaglobulinemic patient exhibited transient variations and amino acid substitutions despite the lack of functioning humoral immune response. The study supports the general hypothesis of humoral immune selection as the main force of sequence variation in the HVR1 region but suggests that other selection mechanisms may contribute to modulation of the composition of the viral population.     

Gradual loss of IgG antibodies against GB virus C/hepatitis G virus in a patient with AIDS

GB virus C/hepatitis G virus (GBV-C/HGV) is a positive-sense, single-stranded RNA virus belonging to the family Flaviviridae and is distantly related to hepatitis C virus (HCV). GBV-C/HGV can be transmitted by the parenteral and the sexual route. Among individuals infected with human immunodeficiency virus type 1 (HIV-1) by the sexual route, we and others have demonstrated a high prevalence of GBV-C/HGV infection. Recently, Woolley and colleagues reported that AIDS patients co-infected with GBV-C/HGV had a significantly lower mean CD4 cell count than AIDS patients without GBV-C/HGV infection, suggesting that GBV-C/HGV antibody may be lost with progression to AIDS. To our knowledge no data are available on the loss of antibody against GBV-C/HGV in AIDS patients. We now report on an HIV-infected patient who exhibited gradual loss of IgG antibodies against GBV-C/HGV, as well as HCV, with progression of HIV disease.     

Slower evolution of human immunodeficiency virus type 1 quasispecies during progression to AIDS

The evolution of human immunodeficiency virus type 1 (HIV-1) quasispecies at the envelope gene was studied from the time of infection in 11 men who experienced different rates of CD4+ cell count decline and 6 men with unknown dates of infection by using DNA heteroduplex mobility assays. Quasispecies were genetically homogeneous near the time of seroconversion. Subsequently, slower proviral genetic diversification and higher plasma viremia correlated with rapid CD4+ cell count decline. Except for the fastest progressors to AIDS, highly diverse quasispecies developed in all subjects within 3 to 4 years. High quasispecies diversity was then maintained for years until again becoming more homogeneous in a subset of late-stage AIDS patients. Individuals who maintained high CD4+ cell counts showed continuous genetic turnover of their complex proviral quasispecies, while more closely related sets of variants were found in longitudinal samples of severely immunocompromised patients. The limited number of variants that grew out in short-term PBMC cocultures were rare in the uncultured proviral quasispecies of healthy, long-term infected individuals but more common in vivo in patients with low CD4+ cell counts. The slower evolution of HIV-1 observed during rapid progression to AIDS and in advanced patients may reflect ineffective host-mediated selection pressures on replicating quasispecies.     

Responsiveness to interferon alpha treatment in patients with chronic hepatitis C coinfected with hepatitis G virus

BACKGROUND/AIMS: Patients with chronic hepatitis C are often coinfected with the new identified Flaviviridae-like agent, termed hepatitis G virus (HGV). The aim of the study was to investigate the responsiveness of hepatitis G virus to interferon alpha and to evaluate whether a hepatitis G virus coinfection negatively influences the outcome of treatment in chronic hepatitis C. METHODS: One hundred and fifteen patients with histologically proven chronic hepatitis C were treated with interferon alpha and investigated for the presence of hepatitis G virus coinfection by nested polymerase chain reaction with primers from the helicase region of hepatitis G virus. All patients received at least 3 MU (range 3-6) interferon alpha thrice weekly for at least 6 months (mean 8, range 6-12). Polymerase chain reaction products of seven pre- and post-treatment hepatitis G virus positive patients were directly sequenced for identification of sequence variability during the follow-up. RESULTS: Eighteen (16%) patients were coinfected with hepatitis G virus. Although nine (50%) of these patients became HGV RNA negative during interferon alpha therapy, only three patients (17%) remained HGV RNA negative at the end of follow-up (mean 24 months). The rate of sustained response of chronic hepatitis C was not significantly different between patients with hepatitis C virus infection and HCV/HGV coinfection (19% vs 28%). Severity of liver disease as determined by alanine aminotransferase levels, histology and hepatitis C virus viremia was not significantly different in patients with hepatitis C virus or HCV/HGV coinfection. Sequence analysis of the helicase region revealed that our isolates all belonged to the hepatitis G virus and not to the GBV-C like genotype. No amino acid exchanges during the observation period of up to 48 months were observed, indicating that this region is highly conserved. CONCLUSIONS: The responsiveness of hepatitis G virus to interferon alpha in chronic HCV/HGV coinfected patients is similar to that observed in chronic hepatitis C. Hepatitis G virus coinfection seems not to interfere with the efficacy of interferon alpha treatment in patients with chronic hepatitis C.     

Effect of human immunodeficiency virus on hepatitis C virus infection among injecting drug users

To assess the effect of human immunodeficiency virus (HIV) immunosuppression on ongoing hepatitis C virus (HCV) infection, CD4 lymphocyte counts and serum concentrations of HCV RNA, HIV RNA, and alanine aminotransferase (ALT) were evaluated among members of a cohort of injecting drug users (IDUs). With 100 participants randomly selected at various stages of HIV-related immunosuppression, serum HCV RNA concentrations increased with age (P = .007) and were higher in HIV-positive IDUs with 201-500 (P = .026) and 51-200 (P = .004) CD4 cells/mL than in HIV-negative participants. Among 27 HCV-infected IDUs who acquired HIV infection, serum HCV RNA concentrations varied between semiannual visits by a mean of 0.45 logs, increasing by 0.60 logs after HIV seroconversion (P < .0001), by 0.12 logs each subsequent year (P = .006), and by 0.36 logs per log increase in CD4 cells (P = .01). Serum ALT levels were similar between HIV-positive (40.1 IU/mL) and HIV-negative (45.4 IU/mL) patients (P > .10). While HIV infection and possibly HIV progression are associated with increased HCV RNA levels, other factors appear to affect biochemical and virologic markers of HCV infection in some dually infected persons.     

Quantitation of hepatitis G and C viruses in the liver: evidence that hepatitis G virus is not hepatotropic

Hepatitis G virus (HGV) is prevalent in patients with chronic liver disease and has been previously detected in liver specimens. However, it is unknown whether the virus is replicating in the liver or is simply a contaminant from serum. We sought to determine whether HGV was hepatotropic and to determine whether coinfection with HGV and hepatitis C virus (HCV) influenced the level of either virus. Virus was quantitated using branched DNA (bDNA) assay for both HGV and HCV in the liver explants and pretransplant serum samples from 30 transplant recipients: Group I, HGV/HCV coinfection (n = 10); group II, HCV infection alone, (n = 8); group III, HGV alone (n = 12). In patients with coinfection HCV (RNA) titers in liver were consistently higher than those for HGV RNA (median 1.13 x 10(8) and 360,000 Eq/g respectively, P < .01). The ratio of liver/serum viral RNA was significantly higher for HCV than for HGV (median 129 and 0.3 respectively, P < .01). Levels of HCV RNA were similar in patients with HCV infection alone versus those with HGV/HCV coinfection (median; liver = 1.15 x 10(7) vs. 1.13 x 10(8) Eq/g, serum = 500,000 vs. 200,000 Eq/mL) and levels of HGV RNA in liver and serum were similar in patients with HGV infection alone compared to those with HGV/HCV coinfection (median; liver = 1.2 x 10(6) vs. 4.0 x 10(5) Eq/g, serum = 4.5 x 106 vs. 2.6 x 10(6) Eq/mL). Levels of either virus appeared unaffected by the presence of an additional virus. The high ratio of HCV RNA levels in liver compared to serum is consistent with its known hepatotropism, but this pattern was not observed for HGV. The median liver/serum ratio of HGV RNA was less than unity, a finding consistent with serum contamination of liver tissue. Thus we conclude that the liver is not the main site of HGV replication.     

T cell recognition of hypervariable region-1 from hepatitis C virus envelope protein with multiple class II MHC molecules in mice and humans: preferential help for induction of antibodies to the hypervariable region

Hypervariable region-1 (HVR1) from the hepatitis C virus (HCV) envelope protein is thought to be a target for neutralizing Abs. To explore HVR1 recognition by helper T cells, and their role in Ab responses, we attempted to generate helper T cells specific for HVR1 in mice of three MHC types, and with PBMC from HCV-infected HLA-diverse humans. In both species, HVR1 was presented by >1 class II MHC molecule to CD4+ helper T cells and showed surprising interisolate cross-reactivity. The epitope for two DR4+ patients was mapped to a more conserved C-terminal sequence containing a DR4 binding motif, possibly accounting for cross-reactivity. Strikingly, Abs to patients' own HVR1 sequences were found only in patients with T cell responses to HVR1, even though all had Abs to envelope protein, suggesting that induction of Abs to HVR1 depends on helper T cells specific for a sequence proximal to the Ab epitope. Thus, helper T cells specific for HVR1 may be functionally important in inducing neutralizing Abs to HCV. These results may be the first example of "T-B reciprocity," in which proximity of a helper T cell epitope determines Ab epitope specificity, in a human disease setting.     

Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.     

Immunosuppression may lead to progression of hepatitis C virus-associated liver disease in hemophiliacs coinfected with HIV

OBJECTIVE: The aim of this study was to examine the interaction between HIV and hepatitis C virus (HCV) in hemophiliacs coinfected with the viruses and to investigate the possible relationship between immunosuppression and liver failure. METHODS: To identify risk factors for impending liver failure in hemophiliacs coinfected with HIV and HCV, we analyzed clinical and laboratory parameters, including CD4 count, aminotransferases (ALT, AST), cholinesterase, alkaline phosphatase, bilirubin, and gamma-glutamyltransferase, during 3 yr of follow-up (1990-1993) in four groups of patients: hemophiliacs with progressive immunodeficiency who were coinfected with HCV and HIV (group A, n = 49); hemophiliacs with stable immune function who were seropositive for HIV and HCV (group B, n = 95); hemophiliacs who were infected with HCV but not HIV (group C, n = 72); and homosexuals with progressive immunodeficiency who were infected with HIV but not HCV (group D, n = 24). RESULTS: Univariate analysis of data for group A showed a significant rise in gamma-glutamyltransferase and alkaline phosphatase (p < 0.01) that was not seen in groups B, C, and D. In a multivariate Cox regression analysis, age (odds ratio, 1.054 per yr; 95% confidence interval, 1.014-1.096 per yr), decline in CD4 count (odds ratio, 1.063 per cell/microl; 95% confidence interval, 1.037-1.091 per cell/microl), and alkaline phosphatase level (odds ratio, 1.012 per U/L; 95% confidence interval, 1.002-1.021 per U/L) emerged as independent determinants of death. CONCLUSIONS: Our data suggest that progressive immune dysfunction in hemophiliacs coinfected with HIV and HCV may influence progression of liver failure. In these patients cholestasis is an additional prognostic marker for survival that may reflect both exhausted immunity and impaired liver function.     

Role of bronchoalveolar lavage in predicting survival of patients with human immunodeficiency virus infection

In this multicenter study, we investigated the prognostic factors that influence the risk of death in patients with human immunodeficiency virus (HIV) infection. Clinical and laboratory indices obtained from 161 HIV-seropositive patients who underwent a detailed morphologic and immunophenotypic evaluation of bronchoalveolar lavage (BAL) and peripheral blood cell populations were retrospectively analyzed. In 155 patients, death occurred within the 48-mo follow-up (mean follow-up: 14.8 mo; range: 1 to 48 mo). In the univariate analysis, the patient's age (> 30 yr), HIV disease status, HIV transmission category, number of opportunistic pathogens isolated from the BAL, percentage of BAL neutrophils, and low number of BAL CD4 T cells were predictive of increased mortality. In contrast, the presence of an alveolitis or an increase in the numbers of alveolar macrophages and CD3 T cells was associated with a decreased mortality. In the multivariate analysis, significant independent predictors were age, risk factor for HIV, and presence of an alveolitis. Furthermore, patients with a low number of BAL CD4 T cells had a particularly poor prognosis while the CD4 T-cell count in the peripheral blood (< 50 cells/mm3 in the majority of our patients) had a negligible effect on predicting survival. Our findings suggest the clinical utility of BAL analysis in patients infected with HIV.     

The prognostic relevance of the nonstructural 5A gene interferon sensitivity determining region is different in infections with genotype 1b and 3a isolates of hepatitis C virus

Hepatitis C virus (HCV) RNA serum concentration, quasispecies complexity, and sequence and phylogenetic analysis of the nonstructural 5A gene (NS5A) interferon sensitivity determining region (ISDR) were determined in pretreatment serum samples from 47 patients with chronic hepatitis C (36 infected by HCV genotype 1b and 11 by 3a). Among HCV genotype 1b-infected patients, virus load was lower (P = .003) and the number of NS5A-ISDR amino acid changes was higher (P = .001) in long-term responders than in non-long-term responders, but there were no differences in quasispecies complexity. Multivariate analysis showed a close association between response to interferon and NS5A-ISDR phenotype. Phylogenetic analysis showed that isolates from non-long-term responders clustered apart from the majority of isolates from long-term responders. There was no association between virologic features and therapeutic response in HCV genotype 3a-infected patients. In conclusion, low virus load and mutant NS5A-ISDR phenotype are closely associated with long-term response to interferon in HCV genotype 1b- but probably not in 3a-infected patients.     

Differential sensitivity of hepatitis C virus quasispecies to interferon-alpha therapy

The quasispecies nature of hepatitis C virus was investigated in a patient with chronic hepatitis C virus infection who underwent interferon-alpha therapy. The hepatitis C virus E2/NS1 region was amplified and cloned, and multiple clones were sequenced before and after interferon-alpha therapy. The hepatitis C virus quasispecies can be grouped into three groups by phylogenetic tree analysis. Quasispecies from all the groups were present before interferon-alpha therapy. However, only group 3 remained after interferon-alpha therapy. In addition, only group 3 hepatitis C virus quasispecies were present during the early biochemical relapse. These data indicate that various groups of hepatitis C virus quasispecies may have different sensitivity to interferon-alpha.