Daniel G. Sullivan

Prospective Characterization of Full-Length Hepatitis C Virus NS5A Quasispecies during Induction and Combination Antiviral Therapy

ABSTRACT The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein has been controversially implicated in the inherent resistance of HCV to interferon (IFN) antiviral therapy in clinical studies. In this study, the relationship between NS5A mutations and selection pressures before and during antiviral therapy and virologic response to therapy were investigated. Full-length NS5A clones were sequenced from 20 HCV genotype 1-infected patients in a prospective, randomized clinical trial of IFN induction (daily) therapy and IFN plus ribavirin combination therapy. Pretreatment NS5A nucleotide and amino acid phylogenies did not correlate with clinical IFN responses and domains involved in NS5A functions in vitro were all well conserved before and during treatment. A consensus IFN sensitivity-determining region (ISDR237–276) sequence associated with IFN resistance was not found, although the presence of Ala245 within the ISDR was associated with nonresponse to treatment in genotype 1a-infected patients (P < 0.01). There were more mutations in the 26 amino acids downstream of the ISDR required for PKR binding in pretreatment isolates from responders versus nonresponders in both HCV-1a- and HCV-1b-infected patients (P < 0.05). In HCV-1a patients, more amino acid changes were observed in isolates from IFN-sensitive patients (P < 0.001), and the mutations appeared to be concentrated in two variable regions in the C terminus of NS5A, that corresponded to the previously described V3 region and a new variable region, 310 to 330. Selection of pretreatment minor V3 quasispecies was observed within the first 2 to 6 weeks of therapy in responders but not nonresponders, whereas the ISDR and PKR binding domains did not change in either patient response group. These data suggest that host-mediated selective pressures act primarily on the C terminus of NS5A and that NS5A can perturb or evade the IFN-induced antiviral response using sequences outside of the putative ISDR. Mechanistic studies are needed to address the role of the C terminus of NS5A in HCV replication and antiviral resistance.

Epidemiology and risk factors for hepatitis C in Alaska Natives

Large cohorts of persons infected with hepatitis C virus (HCV) that include patients with multiple risk exposures and behaviors have been rarely reported. We herein describe a population‐based cohort of 759 Alaska Natives (AN) with HCV who were recruited into a long‐term follow‐up study. History of injection drug use (IDU) was reported by 60.1% and blood transfusion by 14.0%. The most common genotype was 1a (42.0%), followed by 1b (20.3%), 2b (14.7%), 3a (14.3%), and 2a (7.8%). By multivariable analysis, risk exposures (blood transfusion vs. other; P < 0.01; odds ratio [OR], 2.87; 95% confidence interval [CI], 1.51–5.45) and year of infection (P < 0.01; OR, 3.47; 95% CI, 1.34–8.96) were significantly associated with HCV RNA‐positivity. Having an RNA concentration ≥2 million copies/mL was associated with male gender (OR, 1.94) and genotype (P < 0.01 overall; 1a vs. 3a: OR, 1.92; 2b vs. 3a: OR, 3.17) by multivariable analysis. In conclusion, the two principal risk exposures for AN infected with HCV (IDU and blood transfusion) are the same as the overall U.S. population. Persons with a history of blood transfusion were more likely to be HCV RNA positive than those without such history. Higher RNA levels found in males may explain the more severe disease previously reported in this group. (HEPATOLOGY 2004;39:325–332.)

Hepatitis C Virus-Specific Immune Responses and Quasi-Species Variability at Baseline Are Associated with Nonresponse to Antiviral Therapy during Advanced Hepatitis C

Pretreatment hepatitis C virus (HCV)-specific lymphoproliferative (LP) responses, neutralizing antibody (NA) responses, intrahepatic cytotoxic T lymphocyte (CTL) responses, and HCV quasi-species (QS) diversity and complexity were examined in patients with advanced hepatic fibrosis (Ishak fibrosis score of > or = 3) and prior nonresponse to interferon (IFN)- alpha therapy who were enrolled in the initial phase of the Hepatitis C Antiviral Long-Term Treatment against Cirrhosis Trial. Positive baseline HCV E1- and/or E2-specific NA responses (P = .01) and higher baseline HCV QS diversity (P = .01) were more commonly found in patients who did not become sustained virologic responders (SVRs) at week 72 (W72) than they were in those who did. No patients with positive results for both the LP and NA assays achieved a sustained virologic response. Multiple logistic regression analysis revealed that, when the presence of cirrhosis, prior ribavirin therapy, genotype 1 infection, log serum HCV RNA level, and receipt of >80% of the prescribed medication were controlled for, a sustained virologic response (W72) was negatively correlated with positive baseline LP assay results (P = .02) and with 1 or more positive assays (LP, NA, or CTL) (P = .02). No differences were noted in baseline intrahepatic CTL activity between SVRs and non-SVRs. Thus, in patients with advanced hepatic fibrosis due to HCV infection, pretreatment HCV-specific immune responses and increased QS variability appear to hinder viral clearance by pegylated IFN- alpha 2a and ribavirin combination therapy.

Daily interferon therapy for hepatitis C virus infection in liver transplant recipients.

Background. Hepatitis C virus infection persists after liver transplantation and causes recurrent liver injury in the majority of patients. Standard dose interferon therapy has been largely unsuccessful for hepatitis C in transplant recipients. Methods. Twelve patients, at least 7 months posttransplant, with detectable hepatitis C virus RNA in serum and features of hepatitis C on liver biopsy were randomized to interferon-&agr;2a, 3 mU daily for 12 months (n=8) or no treatment (n=4). The tolerability of daily interferon dosing in liver transplant recipients was evaluated and effects on hepatitis C virus RNA level, quasispecies evolution, and liver histology were studied. Results. Treated patients had an improvement in histological activity index at the end of therapy relative to controls (median reduction of 2 versus median increase of 1.5) (P =0.04). Four treated patients had a virological response (all bDNA negative, one qualitative polymerase chain reaction negative) compared with none of the untreated patients. Only two of six treated patients tested had evidence of quasispecies diversification on therapy. Seven of eight patients in the treatment group required dose reduction for fatigue and/or depression. They tolerated 1.5 mU of interferon-&agr;2a daily. Two treated patients developed graft dysfunction, one of who had histological evidence of rejection and subsequent graft loss. Conclusions. Low daily doses of interferon were tolerated by liver transplant recipients and provided histological benefit without associated quasispecies diversification in most cases. These findings provide a rationale to study low dose daily or pegylated interferon maintenance therapy for the management of hepatitis C posttransplant.

High Rate of Spontaneous Negativity for Hepatitis C Virus RNA after Establishment of Chronic Infection in Alaska Natives

BACKGROUND Hepatitis C virus (HCV) leads to chronic infection in 70%-85% of exposed patients. Spontaneous clearance of the virus after chronic infection is believed to occur rarely. METHODS Alaska Natives who tested positive for HCV antibodies were enrolled in a prospective study that began in 1994 and were followed up on a regular basis. Individuals who tested positive for HCV RNA on 3 separate dates, each of which were at least 1 year apart, were included. Being negative for the virus was defined as having at least 1 negative HCV RNA test result after chronic viremia had been established. RESULTS Of the 815 patients enrolled in the cohort, 139 met entry criteria and were observed for a mean period of 7.0 years. Eleven (8%) of the persons had at least 1 test in which HCV RNA was undetectable; 7 were classified as having either possible or probable clearance of the virus, corresponding to an annualized clearance rate of 0.74% per person-year (95% CI, 0.30%-1.53%). Of 9 patients who underwent subsequent HCV RNA testing, 5 (56%) had negative test results. A low HCV RNA level was significantly associated with spontaneous nondetectability of HCV RNA. CONCLUSION Spontaneous HCV RNA negativity during chronic HCV infection is a surprisingly frequent event and is associated with low HCV RNA titers. Knowledge of immunologic determinants of clearance may open up avenues of novel therapy.

Social Structural and Behavioral Underpinnings of Hyperendemic Hepatitis C Virus Transmission in Drug Injectors

BACKGROUND Hepatitis C virus (HCV) is hyperendemic in drug injectors, yet social structural and behavioral factors underlying transmission are not well established. METHODS We conducted a case-control study of HCV seroconversion in drug injectors, focusing on transmission within networks. Incident case subjects (n=17) and seronegative control subjects (n=42) reported injection and sex partners and referred as many as 5 for interviewing and blood testing. We performed nucleotide sequencing of HCV isolates from infected individuals. RESULTS Seventy-eight percent of recent injection partnerships involved behavior that could transmit HCV. Case subjects and control subjects were similar demographically and behaviorally. Case subjects, however, had more HCV-infected partners and consequently engaged in injection risk behavior with more infected partners. The injection network was mostly connected, dense, and cyclic, but the sexual network was highly fragmented. Although participants generally injected with partners of similar age, most HCV-uninfected participants recently had injected with infected partners. In at least 1 of 4 pairs of genetically linked infections, transmission appeared to be due to sharing of injection equipment other than syringes. Except for transmission pairs, network distance between incident case subjects and genetic distance between their HCV variants were uncorrelated. CONCLUSIONS Without dramatic reductions in injection risk behaviors, shattering of cohesive injection networks, and/or broad coverage of an effective vaccine, HCV will likely remain hyperendemic in drug injectors.

Hepatitis C Virus Dynamics during Natural Infection Are Associated with Long-Term Histological Outcome of Chronic Hepatitis C Disease

BACKGROUND The long-term dynamics of hepatitis C virus (HCV) infection and their association with hepatitis C disease are unknown. METHODS Fifty-two treatment-naive subjects with chronic HCV genotype 1 infection were selected from the Alaska Natives and American Indians cohort. Viral RNA levels were measured in 223 specimens (mean, 4.3 specimens/subject) over 457 patient-years. Viral quasispecies diversity was analyzed in 187 specimens (mean, 3.6 specimens/subject) over 365 patient-years. RESULTS Thirty-three subjects had minimal hepatic fibrosis, and 19 developed bridging fibrosis or cirrhosis. There was no significant difference in host variables, including alcohol consumption, between disease groups. Subjects with mild disease had higher serum RNA levels after 2 decades of infection (P=.013), greater fluctuations in RNA levels over time (P=.04), higher intraspecimen quasispecies diversity (P=.001), and higher rates of quasispecies diversification (P=.004) than did subjects with severe disease. On multivariate analysis, the odds of having severe disease were 15.3 (95% confidence interval, 2.3-99.6) times higher among persons with low quasispecies diversification rates compared with the odds among persons with high diversification rates. CONCLUSIONS Histological progression of hepatitis C is tightly associated with homogenization of HCV quasispecies, perhaps reflecting immune failure and/or selective outgrowth of aggressive viral variants.

Associations among clinical, immunological, and viral quasispecies measurements in advanced chronic hepatitis C

The relationships among host immune and viral factors and the severity of liver disease due to hepatitis C virus (HCV) are poorly understood. Previous studies have focused on individual components of the immune response to HCV, often in relatively small numbers of patients. We measured HCV‐specific lymphoproliferation (LP), intrahepatic cytotoxic T lymphocyte (CTL), and neutralizing antibody (NA) responses and HCV quasispecies (QS) diversity and complexity in a large cohort of subjects with advanced liver fibrosis (Ishak stages 3‐6) on entry into the HALT‐C (Hepatitis C Antiviral Long‐term Treatment against Cirrhosis) trial. We correlated LP, CTL, NA, and QS results with clinical characteristics, including serum alanine aminotransferase (ALT), HCV RNA level, HCV genotype, and hepatic histopathology. LP, CTL, and NA responses were detected in 37%, 22%, and 22% of subjects tested, respectively. The only association that was statistically significant was higher mean serum ALT values in patients with detectable HCV‐specific CTL responses (P = .03). In conclusion, immune responses to HCV and viral diversity showed little relationship to clinical or histological features at a single time point in this selected population of patients with advanced chronic hepatitis C for whom prior interferon treatment had failed. (HEPATOLOGY 2005;41:617–625.)

Adverse outcomes in Alaska natives who recovered from or have chronic hepatitis C infection.

BACKGROUND & AIMS The factors associated with adverse outcome from hepatitis C virus (HCV) infection are incompletely understood. To determine the incidence and risk factors associated with the development of end-stage liver disease (ESLD) and liver-related death (LRD), we conducted a retrospective/prospective population-based study in a cohort of Alaska Native persons chronically infected with HCV from 1994 to 2005. METHODS We followed 960 persons prospectively for an average of 7.2 years and retrospectively for 12.1 years with data from medical records and serum samples. We compared data from subjects that were chronically infected with those who recovered from HCV infection, stratified by alcohol use. Survival models were used to examine factors associated with ESLD and LRD in chronically infected patients. RESULTS During prospective follow-up, 80 (8.8%) and 47 (5.2%) patients developed ESLD and LRD, respectively. In examining incidence per 100 person-years, no difference was found among heavy alcohol users in the incidence of LRD (2.28 versus 3.50; P = .34) or ESLD (3.21 versus 5.69; P = .13) in persons with chronic HCV compared with those recovered from HCV infection. In subjects that consumed <50 g alcohol/d, the incidences of LRD were 0.77 and 0.09 (P = .01) and of ESLD were 1.58 versus 0.36 (P = .002), respectively, in subjects with chronic infection versus those that recovered. Multivariate analysis showed that older age, heavy alcohol use, and HCV genotype 3 were associated with ESLD. CONCLUSIONS A history of heavy alcohol use is associated with the highest incidence of LRD and ESLD, regardless of whether patients are chronically infected or recover from HCV infection.

HIV Infection and Antiretroviral Therapy: Effect on Hepatitis C Virus Quasispecies Variability

BACKGROUND Hepatitis C virus (HCV) quasispecies variability has been associated with liver disease progression. The effects of human immunodeficiency virus (HIV) coinfection and highly active antiretroviral therapy (HAART) on HCV quasispecies variability have not been firmly established. METHODS We determined HCV quasispecies complexity and diversity in 69 subjects, 28 of whom were HIV infected, using clonal frequency analysis via heteroduplex mobility analysis of the second envelope gene hypervariable region. Nucleotide sequencing was performed for a small subset of subjects. RESULTS HIV-positive, HAART-naive subjects had significantly lower HCV quasispecies complexity and diversity than did both HIV-negative and HIV-positive HAART-treated subjects. In multivariate analysis, HIV infection predicted decreased complexity (P < .0001) and diversity (P = .001) of HCV quasispecies, whereas HAART predicted increased complexity (P = .013) and diversity (P = .026). For 4 of 6 patients, sequence analysis yielded data supporting the model that positive host pressure drives HCV quasispecies heterogeneity, although data favoring the hypothesis of selective outgrowth of the most fit variants were also observed. CONCLUSION HIV coinfection is associated with decreased HCV quasispecies variability, which appears to be reversed by effective HAART. Although HIV- and HAART-related effects on host immune pressure are likely to play a role in the observed differences in HCV genetic heterogeneity, other mechanisms may be operative.