Within-host quantitation of anellovirus genome complexity from clinical samples.

Anellovirus (AV) is a ubiquitous and diverse virus in the human population. An individual can be infected with multiple AV genera and species that form a heterogeneous repertoire, called the anellome. Due to its exceptional genetic diversity, efficient evaluation of anellome complexity remains a methodological challenge. In the current study, AV genome was first enriched from patient serum samples through two-phase rolling circle amplification. Following Illumina sequencing, anellome was analyzed with an advanced bioinformatics pipeline, including read extraction at three similarity levels, de novo assembly, species assignment, and determination of relative abundance among AV variants. The method was validated in the mock sample and then applied to 21 hepatitis C virus (HCV) patients with and without hepatocellular carcinoma (HCC). Overall, there was a large variance regarding AV richness, ranging from 2 to 51 AV species. In contrast to HCV patients without HCC, HCC incidence was associated with reduced richness (12.6 ± 14.4 vs. 35.4 ± 13.6, p = 0.001) and Shannon entropy (0.4 ± 0.34 vs. 0.61 ± 0.12, p = 0.095) at the AV species level. Interestingly, AV genus beta and gamma expanded in the anellome in 7 of 10 HCC patients. These observations shed light on the potential association between anellome and HCC incidence in patients with chronic HCV infection. The method presented here represents a valuable tool to investigate the role of anellome in human health and disease.

A Real-World Observational Cohort of Patients with Hepatocellular Carcinoma: Design and Rationale for TARGET-HCC.

This study describes the design of the TARGET-hepatocellular carcinoma (HCC) cohort and descriptive characteristics of the patient population at diagnosis among those who were enrolled in the cohort across academic and community clinical centers. TARGET-HCC is a 5-year, longitudinal, observational cohort of patients with HCC receiving care in usual clinical practice. Redacted clinical information, obtained from medical records, captures the natural history and management of the disease, including the safety and efficacy of treatment interventions used in usual clinical practice. Patients can complete patient-reported outcome measures and provide biological specimens for future translational studies. The TARGET-HCC study includes adults with histologic, cytologic, or radiologic diagnosis of HCC from academic and community centers in both the United States and Europe. A total of 1,841 participants were enrolled between January 9, 2017, and July 23, 2019, at 67 sites in the United States and Europe. To date, the most common liver disease etiology in the cohort continues to be hepatitis C, although nearly half had a nonviral etiology, including alcohol-related liver disease or nonalcoholic steatohepatitis. Most included patients were diagnosed at an early stage (Barcelona Clinic Liver Cancer Stage [BCLC] 0/A), but only approximately one third underwent curative treatment. Systemic therapy has been used in 7.3% of enrolled patients, including 45.7% of those with BCLC stage C tumors. Conclusion: Overall, the TARGET-HCC cohort allows for the assessment of patient characteristics and investigation of new treatment paradigms and sequencing with existing agents as well as novel regimens for HCC.

Low Incidence of Adverse Outcomes in Adults With Chronic Hepatitis B Virus Infection in the Era of Antiviral Therapy.

BACKGROUND AND AIMS: Outcomes of persons with chronic hepatitis B virus (HBV) infection in the era of antiviral therapy (AVT) are not well characterized. We determined the incidence and factors associated with clinical outcomes in a multiethnic, North American cohort of adults with chronic HBV infection, who were not on AVT at enrollment. APPROACH AND RESULTS: Adults with chronic HBV infection, not receiving AVT, and without a history of decompensation, HCC, or liver transplantation (LT), were prospectively followed. Participants with known human immunodeficiency virus (HIV), hepatitis C virus, or hepatitis D virus (HDV) coinfection were excluded. During follow-up, treatment could be initiated per standard of care. Clinical outcomes included: incident cirrhosis, decompensation, HCC, OLT, and HBV-related death. Among 1,418 participants analyzed, 51.5% were women, median age was 41.1 years, 75% were Asian, 10% White, 13% Black, 24% HBeAg(+), and 1.5% cirrhosis at baseline. During the study, 274 started treatment, 83 had an alanine aminotransferase flare, 118 of 330 initially HBeAg(+) became HBeAg(-), and 90 of 1,329 became HBsAg(-). After 6,641 person-years follow-up, 8 participants (4 of 21 with baseline cirrhosis) had 12 clinical outcomes (2 decompensation, 5 HCC, 2 OLT, and 3 HBV-related deaths) and 19 of 1,397 had incident cirrhosis. Twenty-one of 26 participants had first outcome before treatment, none had become HBsAg(-), whereas 5/9 HBeAg(+) had become HBeAg(-) at time of first outcome. Cumulative percentage of clinical outcomes was 16% at year 4 in participants with baseline cirrhosis and 2% (including incident cirrhosis) at year 7 in those without. CONCLUSIONS: Incidence of adverse outcomes was low in this closely monitored, large cohort of North American adults with predominantly inactive, chronic HBV without cirrhosis. Our data highlight the benefits of HBsAg loss and the importance of early diagnosis and treatment to prevent cirrhosis and other complications of chronic HBV infection.

Best practices for detection, assessment and management of suspected immune-mediated liver injury caused by immune checkpoint inhibitors during drug development.

Immune checkpoint inhibitors (ICIs) have shown significant efficacy in patients with various malignancies, however, they are associated with a wide range of immune-related toxicities affecting many organs, including the liver. Immune-mediated liver injury caused by checkpoint inhibitors (ILICI) is a distinctive form of drug induced liver injury (DILI), that differs from most DILI types in presumed underlying mechanism, incidence, and response to therapeutic interventions. Despite increased awareness of ILICI and other immune-related adverse effects of ICIs reflected by recent guidelines for their management in post marketing clinical practice, there is lack of uniform best practices to address ILICI risk during drug development. As efforts to develop safer and more effective ICIs for additional indications grow, and as combination therapies including ICIs are increasingly investigated, there is a growing need for consistent practices for ILICI in drug development. This publication summarizes current best practices to optimize the monitoring, diagnosis, assessment, and management of suspected ILICI in clinical trials using ICI as a single agent and in combination with other ICIs or other oncological agents. It is one of several publications developed by the IQ DILI Initiative in collaboration with DILI experts from academia and regulatory agencies. Recommended best practices are outlined pertaining to hepatic inclusion and exclusion criteria, monitoring of liver tests, ILICI detection, approach to a suspected ILICI signal, causality assessment, hepatic discontinuation rules and additional medical treatment.

Treatment Status of Hepatocellular Carcinoma Does Not Influence Rates of Sustained Virologic Response: An HCV-TARGET Analysis.

Recent studies have suggested a negative impact of hepatocellular carcinoma (HCC) on sustained virologic response (SVR) to hepatitis C virus (HCV) direct acting antivirals (DAAs). We compared the effectiveness of DAAs in patients with cirrhosis, with and without HCC, and in those with HCC partially treated or untreated (PT/UT-HCC) versus completely treated (CT-HCC). HCC status was based on imaging 6 months before or 2 months after start of DAA therapy. Absence and presence of enhancing lesions after HCC treatment defined CT-HCC and PT/UT-HCC, respectively. Using minimally adjusted logistic regression, the association between the presence of HCC and SVR rates was estimated. Among the 1,457 patients with cirrhosis from HCV-TARGET with complete virologic data (per-protocol population) who did not undergo liver transplantation during treatment and followup, 1,300 were without HCC, 91 with CT-HCC, and 66 with PT/UT-HCC. Most patients were genotype 1 (81%) and treatment-experienced (56%), 41% had history of prior decompensation, and the median pretreatment Model for End-Stage Liver Disease was 9 (range 6-39). The SVR rates were 91% for patients without HCC, 84% for CT-HCC, and 80% for PT/UT-HCC. The presence of HCC (versus not having HCC) was associated with significantly lower odds of achieving SVR (odds ratio [OR] = 0.51, 95% confidence interval [CI]: 0.33-0.81; P = 0.003). However, among those with HCC, HCC treatment status (PT/UT-HCC versus CT-HCC) did not show association with SVR (OR = 0.79, 95% CI: 0.35-1.79, P = 0.569). Conclusions: The presence of HCC reduces the likelihood of SVR by 50%, but with no evident difference in those with completely treated HCC versus partially treated/untreated HCC.

Current and Future Systemic Therapies for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is a common cancer with unmet needs and limited effective therapeutic options. The management strategy for diagnosed HCC is based on Barcelona Clinic Liver Cancer (BCLC) staging. Advanced HCC is treated with systemic therapy comprising oral tyrosine kinase inhibitors (TKIs) and intravenous immune checkpoint inhibitors, provided that liver function is reasonable. Five new agents have been approved by the US Food and Drug Administration (FDA) in the past 2 years for the treatment of HCC: lenvatinib in the first-line setting, and regorafenib, nivolumab, pembrolizumab, and cabozantinib as second-line therapies. The FDA is considering a label expansion of ramucirumab to include its use in HCC. These therapies have all been shown to extend overall patient survival and appear to have a reasonable safety profile. Multiple ongoing trials are studying immune checkpoint inhibition alone or in combination with TKIs. The results of these trials will help determine the optimal choice, timing, and sequence of agents. This article reviews the role of currently approved systemic therapies for HCC and highlights potential future combination therapeutic strategies. The article also brings forward the concept of a developing shift to the left for therapy, as mapped out in the BCLC staging and treatment algorithm, marking earlier use of systemic therapy prior to advanced progression of the disease.

Age, race and viral genotype are associated with the prevalence of hepatitis B e antigen in children and adults with chronic hepatitis B.

Hepatitis B e antigen (HBeAg) is an important serological marker of hepatitis B virus (HBV) infection and is associated with higher levels of viraemia, increased risk of infectivity to others and increased risk of hepatocellular carcinoma. We analysed HBeAg status in a large cohort of adults and children enrolled in Cohort Studies of the Hepatitis B Research Network, long-term natural history studies of chronic HBV infection. A cross-sectional analysis examined factors associated with HBeAg positivity, including demographic and virologic data, across the age spectrum. Among 2241 enrolled participants who met criteria for this analysis, 825 (37%) were seropositive for HBeAg. The prevalence of HBeAg was lower in those with older age, ranging from 85% among those up to 10 years of age to only 12% among those older than 50 years. In addition to age, both race and HBV genotype were independently associated with HBeAg positivity. There was a significant interaction between age and race; the prevalence of HBeAg was significantly higher among Asians > 10-30 years old vs Whites or Blacks who were >10 to 30 years old and those infected with HBV genotype C. Conversely, the presence of the basal core promoter and precore variants was associated with significantly lower prevalence of HBeAg, even when adjusted for age, race and genotype. These data will provide a better understanding of factors associated with seropositivity for HBeAg and may lead to better strategies for preventing HBV infection and broader indications for antiviral therapy.

Association Between Severe Serum Alanine Aminotransferase Flares and Hepatitis B e Antigen Seroconversion and HBV DNA Decrease in Untreated Patients With Chronic HBV Infection.

BACKGROUND & AIMS: The incidence and outcomes of alanine aminotransferase (ALT) flares during the natural history of chronic HBV infection has not been determined in a large, racially heterogeneous group of patients in North America. METHODS: We collected data from the Hepatitis B Research Network-an observational cohort study of untreated adults with chronic HBV infection enrolled at 21 sites in the United States and Canada. Clinical and laboratory data were collected from 1587 participants (49.9% male, 73.7% Asian, 35.2% genotype B infection, mean age of 42.6 years) at enrollment, at weeks 12 and 24, and every 24 weeks thereafter for a planned 5 years of follow up (from January 2011 through May 2016). Participants were excluded if they had a history of hepatic decompensation, hepatocellular carcinoma, solid organ or bone marrow transplantation, chronic immune suppression, or antiviral therapy within 6 months before enrollment. Levels of ALT were measured in serum samples and flares were defined as at least 10 times the upper limit of normal (300 U/L in males and 200 U/L in females). RESULTS: ALT flares occurred in 102 participants (6%), with 31 flares (30%) occurring at baseline. The 4-year cumulative incidence of ALT flares was 5.7%. The median peak level of ALT was 450 U/L (25th-75th percentile, 330 U/L to 747 U/L) with a maximum of 2578 U/L. In multivariable analysis, factors associated with the occurrence of an ALT flares were: male sex (odds ratio [OR], 3.02; P=.0007), higher baseline HBV DNA values (OR per log10, 1.41; P<.0001), at risk alcohol use (OR, 2.27 vs none or moderate; P=.02), and higher FIB-4 values (OR, 1.85 per log2; P<.0001). Older age was associated with lower odds of an ALT flare (OR, 0.63 per 10 years; P=.004). Rate of decrease in level of HBV DNA by 1 log10 or more (59 vs 23 per 100 person-years for HB e antigen (HBeAg)-positive vs HBeAg-negative patients; P=.003) and HBeAg loss (47 vs 15 per 100 person-years; P=.002) were higher in patients with an ALT flare than in patients without, but the rate of HBsAg loss was similar (4 vs 2 per 100 person-years; P=.26). No hepatic decompensation, liver transplants, or deaths were observed in participants with ALT flares. CONCLUSION: In a large racially heterogeneous cohort of adults with chronic HBV infection, the cumulative incidence of severe ALT flares was low and associated with greater decreases in HBV DNA and loss of HBeAg, but not with loss of HBsAg.

Template-dependent multiple displacement amplification for profiling human circulating RNA.

Multiple displacement amplification (MDA) is widely used in whole-genome/transcriptome amplification. However, template-independent amplification (TIA) in MDA is a commonly observed phenomenon, particularly when using high concentrations of random hexamer primers and extended incubation times. Here, we demonstrate that the use of random pentamer primers with 5´ ends blocked by a C18 spacer results in MDA solely in a template-dependent manner, a technique we have named tdMDA. Together with an optimized procedure for the removal of residual genomic DNA during RNA extraction, tdMDA was used to profile circulating RNA from 0.2 mL of patient sera. In comparison to regular MDA, tdMDA demonstrated a lack of quantifiable DNA amplification in the negative control, a remarkable reduction of unmapped reads from Illumina sequencing (7 ± 10.9% versus 58.6 ± 39%, P = 0.006), and increased mapping rates of the serum transcriptome (26.9 ± 7.9% versus 5.8 ± 8.2%, P = 3.8 × 10-4). Transcriptome profiles could be used to separate patients with chronic hepatitis C virus (HCV) infection from those with HCV-associated hepatocellular carcinoma (HCC). We conclude that tdMDA should facilitate RNA-based liquid biopsy, as well as other genome studies with biological specimens having ultralow amounts of genetic material.

Differential Expression of MicroRNAs in Hepatitis C Virus-Mediated Liver Disease Between African Americans and Caucasians: Implications for Racial Health Disparities.

African Americans (AAs) have higher hepatocellular carcinoma (HCC) mortality rates than Caucasian Americans (CAs). Chronic hepatitis C virus (HCV) infection leads to cirrhosis and HCC. HCV infection is highly prevalent in the AA population compared to other racial groups. AAs are also less likely to naturally clear HCV, potentially contributing to higher prevalence of HCV. However, the explanation for this disparity is currently unknown. Circulating microRNAs (miRNAs) in the blood are emerging as biomarkers for pathological conditions. Expression analysis of miRNAs in major racial groups would be important for optimizing personalized treatment strategies. Here we assessed the differential expression of circulatory miRNAs from HCV-infected AA and CA patients. We identified increased expression of miR-146a, miR-150, and miR-155 in HCV-infected AA patient sera compared to that of CA. Further analysis demonstrated that these miRNAs were significantly elevated in AA patients diagnosed with HCV-mediated HCC. Higher expression of miR-150 was also noted in cirrhosis and HCC in AA patients, which may serve as a predictor of liver disease progression in this population. The differential expression of miRNAs suggests that these miRNAs and their target genes could be useful to gain further mechanistic insight of racial disparity associated with HCV-mediated pathogenesis.

Distinct CD55 Isoform Synthesis and Inhibition of Complement-Dependent Cytolysis by Hepatitis C Virus.

CD55/DAF, one of the regulators of complement activation, is known to limit excess complement activation on the host cell surface by accelerating the decay of C3 convertase. We reported previously that hepatitis C virus (HCV) infection or virus core protein expression upregulates CD55 expression. CD55 associates with HCV particles, potentially protecting HCV from lysis in circulation. An increase in CD55 on the surface of HCV-infected cells may inhibit complement-mediated cell killing. In this study, we show that Abs against cancer cell surface proteins induce complement-dependent cytolysis or Ab-dependent cell-mediated cytotoxicity of immortalized human hepatocytes in the presence of CD55-blocking Ab. CD55 has a secreted isoform (sCD55) that is generated by alternative splicing. We observed that sCD55 is induced in HCV-infected or HCV replicon-harboring cells, as well as in liver biopsy samples from chronically HCV-infected patients. Conditioned medium from HCV-infected hepatoma cells (Huh7.5 cells) or immortalized human hepatocytes inhibited C3 convertase activity and complement-dependent cytolysis of sheep blood erythrocytes. Chronically HCV-infected patient sera inhibited C3 convertase activity, further implicating HCV-specific impairment of complement function in infected humans. CD55-blocking Ab inhibited erythrocyte lysis by conditioned medium, suggesting that CD55/sCD55 impairs convertase activity. Together, our data show that HCV infection induces sCD55 expression in HCV-infected cell culture-conditioned medium and inhibits C3 convertase activity. This may have implications for modulating complement-mediated immune function in the microenvironment and on HCV-harboring cells.

Serum miR-30e and miR-223 as Novel Noninvasive Biomarkers for Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is one of the most aggressive cancers and is the third leading cause of all cancer-related death. Limited noninvasive biomarkers are available for HCC detection. Early detection is the key in improving the survival of HCC patients. In this study, we tested the hypothesis that serum miRNAs can be used as a potential biomarker for HCC. Quantitative RT-PCR for miRNA analysis was performed using 70 serum samples. Receiver operating characteristic analysis was performed to measure the prognostic power of the miRNAs. The miRNA expression level was also measured from liver biopsy samples. Our study revealed that two miRNAs, miR-30e and miR-223, were expressed at significantly lower levels (P < 0.003) in the sera of HCC patients compared with healthy volunteers. Furthermore, expression of these miRNAs was compared between sera from chronic liver disease and sera from HCC patients. miR-30e and miR-223 expression was significantly lower in HCC sera compared with sera from chronic liver disease patients. Both miRNA expression levels were lower in HCC liver biopsy specimens compared with normal liver RNA. Taken together, our results suggested that serum miR-30e and miR-223 are useful biomarkers of HCC, irrespective of etiology, and deserve further study for their diagnostic value.

Promotion of Cancer Stem-Like Cell Properties in Hepatitis C Virus-Infected Hepatocytes.

UNLABELLED: We have previously reported that hepatitis C virus (HCV) infection of primary human hepatocytes (PHH) induces the epithelial mesenchymal transition (EMT) state and extends hepatocyte life span (S. K. Bose, K. Meyer, A. M. Di Bisceglie, R. B. Ray, and R. Ray, J Virol 86:13621-13628, 2012, http://dx.doi.org/10.1128/JVI.02016-12). These hepatocytes displayed sphere formation on ultralow binding plates and survived for more than 12 weeks. The sphere-forming hepatocytes expressed a number of cancer stem-like cell (CSC) markers, including high levels of the stem cell factor receptor c-Kit. The c-Kit receptor is regarded as one of the CSC markers in hepatocellular carcinoma (HCC). Analysis of c-Kit mRNA displayed a significant increase in the liver biopsy specimens of chronically HCV-infected patients. We also found c-Kit is highly expressed in transformed human hepatocytes (THH) infected in vitro with cell culture-grown HCV genotype 2a. Further studies suggested that HCV core protein significantly upregulates c-Kit expression at the transcriptional level. HCV infection of THH led to a significant increase in the number of spheres displayed on ultralow binding plates and in enhanced EMT and CSC markers and tumor growth in immunodeficient mice. The use of imatinib or dasatinib as a c-Kit inhibitor reduced the level of sphere-forming cells in culture. The sphere-forming cells were sensitive to treatment with sorafenib, a multikinase inhibitor, that is used for HCC treatment. Further, stattic, an inhibitor of the Stat3 molecule, induced sphere-forming cell death. A combination of sorafenib and stattic had a significantly stronger effect, leading to cell death. These results suggested that HCV infection potentiates CSC generation, and selected drugs can be targeted to efficiently inhibit cell growth. IMPORTANCE: HCV infection may develop into HCC as an end-stage liver disease. We focused on understanding the mechanism for the risk of HCC from chronic HCV infection and identified targets for treatment. HCV-infected primary and transformed human hepatocytes (PHH or THH) generated CSC. HCV-induced spheres were highly sensitive to cell death from sorafenib and stattic treatment. Thus, our study is highly significant for HCV-associated HCC, with the potential for developing a target-specific strategy for improved therapies.

Hepatitis C virus-mediated enhancement of microRNA miR-373 impairs the JAK/STAT signaling pathway.

UNLABELLED: Hepatitis C virus (HCV) is a serious global health problem and establishes chronic infection in a significant number of infected humans worldwide. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients, and the mechanism is not fully understood. MicroRNAs (miRNAs) have been implicated in the control of many biological processes, including IFN signaling. To gain more insights into the role of cellular miRNAs in possible countermeasures of HCV for suppression of the host antiviral response, a miRNA array was performed by using primary human hepatocytes infected with in vitro cell culture-grown HCV. A group of miRNAs were modulated in HCV-infected primary human hepatocytes. We focused on miR-373, as this miRNA was significantly upregulated in HCV-infected primary human hepatocytes. Here, we analyzed the function of miR-373 in the context of HCV infection. HCV infection upregulates miR-373 expression in hepatocytes and HCV-infected liver biopsy specimens. Furthermore, we discovered that miR-373 directly targets Janus kinase 1 (JAK1) and IFN-regulating factor 9 (IRF9), important factors in the IFN signaling pathway. The upregulation of miR-373 by HCV also inhibited STAT1 phosphorylation, which is involved in ISG factor 3 (ISGF3) complex formation and ISG expression. The knockdown of miR-373 in hepatocytes enhanced JAK1 and IRF9 expression and reduced HCV RNA replication. Taken together, our results demonstrated that miR-373 is upregulated during HCV infection and negatively regulated the type I IFN signaling pathway by suppressing JAK1 and IRF9. Our results offer a potential therapeutic approach for antiviral intervention. IMPORTANCE: Chronic HCV infection is one of the major causes of end-stage liver disease worldwide. Although the recent introduction of direct-acting antiviral (DAA) therapy is extremely encouraging, some infected individuals do not respond to this therapy. Furthermore, these drugs target HCV nonstructural proteins, and with selective pressure, the virus may develop a resistant strain. Therefore, understanding the impairment of IFN signals will help in designing additional therapeutic modalities. In this study, we provide evidence of HCV-mediated upregulation of miR-373 and show that miR-373 impairs IFN signaling by targeting JAK1/IRF9 molecules. The knockdown of miR-373 inhibited HCV replication by upregulating interferon-stimulating gene expression. Together, these results provided new mechanistic insights into the role of miR-373 in HCV infection and suggest a new potential target against HCV infection.

Recent US Food and Drug Administration warnings on hepatitis B reactivation with immune-suppressing and anticancer drugs: just the tip of the iceberg?

UNLABELLED: Reactivation of hepatitis B in the context of immunosuppressive therapy may be severe and potentially fatal. The US Food and Drug Administration has recently drawn attention to the potentially fatal risk of hepatitis B reactivation in patients receiving the anti-CD20 agents ofatumumab or rituximab. This action focuses attention on the broader issue of hepatitis B virus reactivation, which may occur with a wide variety of immunosuppressive therapies in benign or malignant disease. This article summarizes the data behind this issue. These data support the recommendation that all patients undergoing chemotherapy, immunosuppressive therapy, hematopoietic stem cell transplantation, or solid organ transplantation be screened for active or prior hepatitis B viral infection by testing for hepatitis B surface antigen and the antibody to hepatitis B core antigen in serum. Those who are found to be hepatitis B surface antigen-positive should start appropriate antiviral therapy to prevent reactivation. Additionally, even those who have recovered from hepatitis B will benefit from antiviral therapy in certain circumstances because of the risks associated with a form of hepatitis B virus reactivation referred to as "reverse seroconversion." There remain many uncertain areas that warrant further study, and further advances will benefit from close interactions between various medical specialties, regulatory agencies, and researchers. CONCLUSIONS: There is good evidence to support routine screening of all patients for hepatitis B prior to undergoing chemotherapy or immunosuppressive treatment; use of prompt antiviral treatment appears to diminish the risk of severe or fatal reactivation of hepatitis B.

Characteristics of adults in the hepatitis B research network in North America reflect their country of origin and hepatitis B virus genotype.

BACKGROUND & AIMS: Chronic hepatitis B virus (HBV) infection is an important cause of cirrhosis and hepatocellular carcinoma worldwide; populations that migrate to the United States and Canada might be affected disproportionately. The Hepatitis B Research Network (HBRN) is a cooperative network of investigators from the United States and Canada, created to facilitate clinical, therapeutic, and translational research in adults and children with hepatitis B. We describe the structure of the network and baseline characteristics of adults with hepatitis B enrolled in the network. METHODS: The HBRN collected data on the clinical characteristics of 1625 adults with chronic HBV infection who are not receiving antiviral therapy from 21 clinical centers in North America. RESULTS: Half of the subjects in the HBRN are men, and the median age is 42 years; 72% are Asian, 15% are black, and 11% are white; with 82% born outside of North America. The most common HBV genotype was B (39%); 74% of subjects were negative for the hepatitis B e antigen. The median serum level of HBV DNA when the study began was 3.6 log10 IU/mL; 68% of male subjects and 67% of female subjects had alanine aminotransferase levels higher than the normal range. CONCLUSIONS: The HBRN cohort is used to address important clinical and therapeutic questions for North Americans infected with chronic HBV and to guide health policies on HBV prevention and management in North America.

Inhibition of c3 convertase activity by hepatitis C virus as an additional lesion in the regulation of complement components.

We have previously reported that in vitro HCV infection of cells of hepatocyte origin attenuates complement system at multiple steps, and attenuation also occurs in chronically HCV infected liver, irrespective of the disease stage. However, none of these regulations alone completely impaired complement pathways. Modulation of the upstream proteins involved in proteolytic processing of the complement cascade prior to convertase formation is critical in promoting the function of the complement system in response to infection. Here, we examined the regulation of C2 complement expression in hepatoma cells infected in vitro with cell culture grown virus, and validated our observations using randomly selected chronically HCV infected patient liver biopsy specimens. C2 mRNA expression was significantly inhibited, and classical C3 convertase (C4b2a) decreased. In separate experiments for C3 convertase function, C3b deposition onto bacterial membrane was reduced using HCV infected patient sera as compared to uninfected control, suggesting impaired C3 convertase. Further, iC3b level, a proteolytically inactive form of C3b, was lower in HCV infected patient sera, reflecting impairment of both C3 convertase and Factor I activity. The expression level of Factor I was significantly reduced in HCV infected liver biopsy specimens, while Factor H level remained unchanged or enhanced. Together, these results suggested that inhibition of C3 convertase activity is an additional cumulative effect for attenuation of complement system adopted by HCV for weakening innate immune response.

Hepatitis C virus infection upregulates CD55 expression on the hepatocyte surface and promotes association with virus particles.

CD55 limits excessive complement activation on the host cell surface by accelerating the decay of C3 convertases. In this study, we observed that hepatitis C virus (HCV) infection of hepatocytes or HCV core protein expression in transfected hepatocytes upregulated CD55 expression at the mRNA and protein levels. Further analysis suggested that the HCV core protein or full-length (FL) genome enhanced CD55 promoter activity in a luciferase-based assay, which was further augmented in the presence of interleukin-6. Mutation of the CREB or SP-1 binding site on the CD55 promoter impaired HCV core protein-mediated upregulation of CD55. HCV-infected or core protein-transfected Huh7.5 cells displayed greater viability in the presence of CD81 and CD55 antibodies and complement. Biochemical analysis revealed that CD55 was associated with cell culture-grown HCV after purification by sucrose density gradient ultracentrifugation. Consistent with this, a polyclonal antibody to CD55 captured cell culture-grown HCV. Blocking antibodies against CD55 or virus envelope glycoproteins in the presence of normal human serum as a source of complement inhibited HCV infection. The inhibition was enhanced in the presence of both the antibodies and serum complement. Collectively, these results suggest that HCV induces and associates with a negative regulator of the complement pathway, a likely mechanism for immune evasion.

Hepatitis C virus suppresses C9 complement synthesis and impairs membrane attack complex function.

Hepatitis C virus (HCV) proteins inhibit complement component expression, which may attenuate immunity against infection. In this study, we examined whether HCV regulates the membrane attack complex (MAC) via complement component C9. MAC is composed of C5b to C9 (C5b-9) and mediates cell lysis of invaded pathogens. Liver biopsy specimens from chronically HCV-infected patients exhibited a lower level of C9 mRNA expression than liver biopsy specimens from unrelated disease or healthy control human liver RNA. Hepatocytes infected with cell culture-grown HCV or expressing HCV core protein also displayed significant repression of C9 mRNA and protein levels. Promoter analysis suggested that the T cell factor-4 (TCF-4E) transcription factor is responsible for HCV core-mediated C9 promoter regulation. Sera from chronically HCV-infected patients displayed a lower level of C5b-9 and a reduced antimicrobial effect on model organisms compared to unrelated patient sera or sera from healthy volunteers. Together, these results for C9 regulation by HCV core protein coupled with functional impairment of the membrane attack complex underscore HCV-mediated attenuation of immune mechanisms.