Bernadette Guerra

Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees

ABSTRACT Hepatitis C virus (HCV) infections represent a global health problem and are a major contributor to end-stage liver disease including cirrhosis and hepatocellular carcinoma. An improved understanding of the parameters involved in disease progression is needed to develop better therapies and diagnostic markers of disease manifestation. To better understand the dynamics of host gene expression resulting from persistent virus infection, DNA microarray analyses were conducted on livers from 10 chimpanzees persistently infected with HCV. A total of 162 genes were differentially regulated in chronically infected animals compared to uninfected controls. Many genes exhibited a remarkable consistency in changes in expression in the 10 chronically infected animals. A second method of analysis identified 971 genes altered in expression during chronic infection at a 99% confidence level. As with acute-resolving HCV infections, many interferon (IFN)-stimulated genes (ISGs) were transcriptionally elevated, suggesting an ongoing response to IFN and/or double-stranded RNA which is amplified in downstream ISG expression. Thus, persistent infection with HCV results in a complex and partially predictable pattern of gene expression, although the underlying mechanisms regulating the different pathways are not well defined. A single genotype 3-infected animal was available for analysis, and this animal exhibited reduced levels of ISG expression compared to levels of expression with genotype 1 infections and increased expression of a number of genes potentially involved in steatosis. Gene expression data in concert with other observations from HCV infections permit speculation on the regulation of specific aspects of HCV infection.

Antiviral Effect and Virus-Host Interactions in Response to Alpha Interferon, Gamma Interferon, Poly(I)-Poly(C), Tumor Necrosis Factor Alpha, and Ribavirin in Hepatitis C Virus Subgenomic Replicons

ABSTRACT The recently developed hepatitis C virus (HCV) subgenomic replicon system was utilized to evaluate the efficacy of several known antiviral agents. Cell lines that persistently maintained a genotype 1b replicon were selected. The replicon resident in each cell line had acquired adaptive mutations in the NS5A region that increased colony-forming efficiency, and some replicons had acquired NS3 mutations that alone did not enhance colony-forming efficiency but were synergistic with NS5A mutations. A replicon constructed from the infectious clone of the HCV-1 strain (genotype 1a) was not capable of inducing colony formation even after the introduction of adaptive mutations identified in the genotype 1b replicon. Alpha interferon (IFN-α), IFN-γ, and ribavirin exhibited antiviral activity, while double-stranded RNA (dsRNA) and tumor necrosis factor alpha did not. Analysis of transcript levels for a series of genes stimulated by IFN (ISGs) or dsRNA following treatment with IFN-α, IFN-γ, and dsRNA revealed that both IFNs increased ISG transcript levels, but that some aspect of the dsRNA response pathway was defective in Huh7 cells and replicon cell lines in comparison to primary chimpanzee and tamarin hepatocytes. The colony-forming efficiency of the replicon was reduced or eliminated following replication in the presence of ribavirin, implicating the induction of error-prone replication. The potential role of error-prone replication in the synergy observed between IFN-α and ribavirin in attaining sustained viral clearance is discussed. These studies reveal characteristics of Huh7 cells that may contribute to their unique capacity to support HCV RNA synthesis and demonstrate the utility of the replicon system for mechanistic studies on antiviral agents.

GS-9620, an oral agonist of toll-like receptor-7, induces prolonged suppression of hepatitis B virus in chronically infected chimpanzees

BACKGROUND & AIMS Direct-acting antiviral agents suppress hepatitis B virus (HBV) load, but they require life-long use. Stimulation of the innate immune system could increase its ability to control the virus and have long-lasting effects after a finite regimen. We investigated the effects of immune activation with GS-9620--a potent and selective orally active small molecule agonist of Toll-like receptor 7--in chimpanzees with chronic HBV infection. METHODS GS-9620 was administered to chimpanzees every other day (3 times each week) for 4 weeks at 1 mg/kg and, after a 1-week rest, for 4 weeks at 2 mg/kg. We measured viral load in plasma and liver samples, the pharmacokinetics of GS-9620, and the following pharmacodynamics parameters: interferon-stimulated gene expression, cytokine and chemokine levels, lymphocyte and natural killer cell activation, and viral antigen expression. Clinical pathology parameters were monitored to determine the safety and tolerability of GS-9620. RESULTS Short-term oral administration of GS-9620 provided long-term suppression of serum and liver HBV DNA. The mean maximum reduction of viral DNA was 2.2 logs, which occurred within 1 week of the end of GS-9620 administration; reductions of >1 log persisted for months. Serum levels of HBV surface antigen and HBV e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of interferon-α and other cytokines and chemokines, and activated interferon-stimulated genes, natural killer cells, and lymphocyte subsets. CONCLUSIONS The small molecule GS-9620 activates Toll-like receptor 7 signaling in immune cells of chimpanzees to induce clearance of HBV-infected cells. This reagent might be developed for treatment of patients with chronic HBV infection.

Cross-Genotype Immunity to Hepatitis C Virus

ABSTRACT Recent studies in humans and chimpanzees suggest that immunity can be induced to diminish the incidence of chronic hepatitis C virus (HCV) infection. However, the immunity that promotes viral recovery is poorly understood, and whether the breadth of this adaptive immunity is sufficient to overcome the substantial intergenotype antigenic diversity represents a final obstacle to demonstrating the feasibility of vaccine development. Here we demonstrate that recovery from a genotype 1 HCV infection protects chimpanzees against infection with representatives of other genotypes that exhibit up to 30% divergence at the amino acid level, including challenges with genotype 4, a mixture of genotypes 2 and 3, and a complex inoculum containing genotypes 1, 2, 3, and 4. In each instance, the level and duration of viremia were markedly reduced in comparison to the primary infection in the same animal. The data indicate that epitopes conserved between genotypes must play an essential role in immunity. The inocula used in the rechallenge studies induced typical primary infection profiles in naïve chimpanzees. Rechallenge infections were associated with rapid increases in the intrahepatic transcripts of interferon-stimulated genes, even in animals exhibiting apparent sterilizing immunity. Protective immunity was often associated with an early increase in gamma interferon transcripts in the liver and increases in intrahepatic transcripts of Mig, a T-cell chemokine that is a gamma interferon response gene. These studies are the first to show that cross-genotype immunity can be induced to HCV, demonstrating the feasibility of developing a vaccine protective against all HCV strains.

Ribavirin Induces Error-Prone Replication of GB Virus B in Primary Tamarin Hepatocytes

ABSTRACT GB virus B (GBV-B) is the closest relative of hepatitis C virus (HCV) and is an attractive surrogate model for HCV antiviral studies. GBV-B induces an acute, resolving hepatitis in tamarins. Utilizing primary cultures of tamarin hepatocytes, we have previously developed a tissue culture system that exhibits high levels of GBV-B replication. In this report, we have extended the utility of this system for testing antiviral compounds. Treatment with human interferon provided only a marginal antiviral effect, while poly(I-C) yielded >3 and 4 log units of reduction of cell-associated and secreted viral RNA, respectively. Interestingly, treatment of GBV-B-infected hepatocytes with ribavirin resulted in an approximately 4-log decrease in viral RNA levels. Guanosine blocked the antiviral effect of ribavirin, suggesting that inhibition of IMP dehydrogenase (IMPDH) and reduction of intracellular GTP levels were essential for the antiviral effect. However, mycophenolic acid, another IMPDH inhibitor, had no antiviral effect. Virions harvested from ribavirin-treated cultures exhibited a dramatically reduced specific infectivity. These data suggest that incorporation of ribavirin triphosphate induces error-prone replication with concomitant reduction in infectivity and that reduction of GTP pools may be required for incorporation of ribavirin triphosphate. In contrast to the in vitro studies, no significant reduction in viremia was observed in vivo following treatment of tamarins with ribavirin during acute infection with GBV-B. These findings are consistent with the observation that ribavirin monotherapy for HCV infection decreases liver disease without a significant reduction in viremia. Our data suggest that nucleoside analogues that induce error-prone replication could be an attractive approach for the treatment of HCV infection if administered at sufficient levels to result in efficient incorporation by the viral polymerase.

Genomic response to interferon-α in chimpanzees: Implications of rapid downregulation for hepatitis C kinetics

The mechanism of the interferon‐alpha (IFN‐α)‐induced antiviral response during hepatitis C virus (HCV) therapy is not completely understood. In this study, we examined the transcriptional response to IFN‐α in uninfected chimpanzees after single doses of chimpanzee, human, or human‐pegylated IFN‐α. Liver and peripheral blood mononuclear cell (PBMC) samples were used for total genome microarray analysis. Most induced genes achieved maximal response within 4 hours, began to decline by 8 hours, and were at baseline levels by 24 hours post‐inoculation, a time when high levels of circulating pegylated IFN‐α were still present. The rapid downregulation of the IFN‐α response may be involved in the transition between the observed phase I and phase II viral kinetics during IFN‐α therapy in HCV‐infected patients. The response to all three forms of IFN‐α was similar; thus, the reasons for previous failures in antiviral treatment of chimpanzees with human IFN‐α were not due to species specificity of IFN‐α. The response to IFN‐α was partially tissue‐specific. A total of 1,778 genes were altered in expression by twofold or more by IFN‐α, with 538 and 950 being unique to the liver or PBMC, respectively. Analysis of the IFN‐α and IFN‐γ responses in primary chimpanzee and human hepatocytes were compared as well. IFN‐α and IFN‐γ induced partially overlapping sets of genes in hepatocytes. In conclusion, the response to IFN‐α is largely tissue‐specific, and the response is rapidly downregulated in vivo, which may have a significant influence on the kinetics of antiviral response. (HEPATOLOGY 2006;43:961–972.)

Mapping of the Hepatitis B Virus Pre-S1 Domain Involved in Receptor Recognition

ABSTRACT Hepatitis B virus (HBV) and woolly monkey hepatitis B virus (WMHBV) are primate hepadnaviruses that display restricted tissue and host tropisms. Hepatitis D virus (HDV) particles pseudotyped with HBV and WMHBV envelopes (HBV-HDV and WM-HDV) preferentially infect human and spider monkey hepatocytes, respectively, thereby confirming host range bias in vitro. The analysis of chimeric HBV and WMHBV large (L) envelope proteins suggests that the pre-S1 domain may comprise two regions that affect infectivity: one within the amino-terminal 40 amino acids of pre-S1 and one downstream of this region. In the present study, we further characterized the role of the amino terminus of pre-S1 in infectivity by examining the ability of synthetic peptides to competitively block HDV infection of primary human and spider monkey hepatocytes. A synthetic peptide representing the first 45 residues of the pre-S1 domain of the HBV L protein blocked infectivity of HBV-HDV and WM-HDV, with a requirement for myristylation of the amino terminal residue. Competition studies with truncated peptides suggested that pre-S1 residues 5 to 20 represent the minimal domain for inhibition of HDV infection and, thus, presumably represent the residues involved in virus-host receptor interaction. Recombinant pre-S1 proteins expressed in insect cells blocked infection with HBV-HDV and WM-HDV at a concentration of 1 nanomolar. The ability of short pre-S1 peptides to efficiently inhibit HDV infection suggests that they represent suitable ligands for identification of the HBV receptor and that a pre-S1 mimetic may represent a rational therapy for the treatment of HBV infection.

Acute hepatitis A virus infection is associated with a limited type I interferon response and persistence of intrahepatic viral RNA

Hepatitis A virus (HAV) is an hepatotropic human picornavirus that is associated only with acute infection. Its pathogenesis is not well understood because there are few studies in animal models using modern methodologies. We characterized HAV infections in three chimpanzees, quantifying viral RNA by quantitative RT-PCR and examining critical aspects of the innate immune response including intrahepatic IFN-stimulated gene expression. We compared these infection profiles with similar studies of chimpanzees infected with hepatitis C virus (HCV), an hepatotropic flavivirus that frequently causes persistent infection. Surprisingly, HAV-infected animals exhibited very limited induction of type I IFN-stimulated genes in the liver compared with chimpanzees with acute resolving HCV infection, despite similar levels of viremia and 100-fold greater quantities of viral RNA in the liver. Minimal IFN-stimulated gene 15 and IFIT1 responses peaked 1–2 wk after HAV challenge and then subsided despite continuing high hepatic viral RNA. An acute inflammatory response at 3–4 wk correlated with the appearance of virus-specific antibodies and apoptosis and proliferation of hepatocytes. Despite this, HAV RNA persisted in the liver for months, remaining present long after clearance from serum and feces and revealing dramatic differences in the kinetics of clearance in the three compartments. Viral RNA was detected in the liver for significantly longer (35 to >48 wk) than HCV RNA in animals with acute resolving HCV infection (10–20 wk). Collectively, these findings indicate that HAV is far stealthier than HCV early in the course of acute resolving infection. HAV infections represent a distinctly different paradigm in virus–host interactions within the liver.

Development of a Primary Tamarin Hepatocyte Culture System for GB Virus-B: a Surrogate Model for Hepatitis C Virus

ABSTRACT GB virus-B (GBV-B) causes an acute hepatitis in tamarins characterized by increased alanine transaminase levels that quickly return to normal as the virus is cleared. Phylogenetically, GBV-B is the closest relative to hepatitis C virus (HCV), and thus GBV-B infection of tamarins represents a powerful surrogate model system for the study of HCV. In this study, the course of infection of GBV-B in tamarins was followed using a real-time 5′ exonuclease (TaqMan) reverse transcription-PCR assay to determine the level of GBV-B in the serum. Peak viremia levels exceeded 109 genome equivalents/ml, followed by viral clearance within 14 to 16 weeks. Rechallenge of animals that had cleared infection resulted in viremia that was limited to 1 week, suggestive of a strong protective immune response. A robust tissue culture system for GBV-B was developed using primary cultures of tamarin hepatocytes. Hepatocytes obtained from a GBV-B-infected animal maintained high levels of cell-associated viral RNA and virion secretion for 42 days of culture. In vitro infection of normal hepatocytes resulted in rapid amplification of cell-associated viral RNA and secretion of up to 107 genome equivalents/ml of culture supernatant. In addition, infection could be monitored by immunofluorescence staining for GBV-B nonstructural NS3 protein. This model system overcomes many of the current obstacles to HCV research, including low levels of viral replication, lack of a small primate animal model, and lack of a reproducible tissue culture system.

Lack of response to exogenous interferon‐α in the liver of chimpanzees chronically infected with hepatitis C virus

The mechanism of the interferon‐alpha (IFNα)–induced antiviral response is not completely understood. We recently examined the transcriptional response to IFNα in uninfected chimpanzees. The transcriptional response to IFNα in the liver and peripheral blood mononuclear cells (PBMCs) was rapidly induced but was also rapidly down‐regulated, with most interferon‐alpha–stimulated genes (ISGs) returning to the baseline within 24 hours. We have extended these observations to include chimpanzees chronically infected with hepatitis C virus (HCV). Remarkably, using total genome microarray analysis, we observed almost no induction of ISG transcripts in the livers of chronically infected animals following IFNα dosing, whereas the response in PBMCs was similar to that in uninfected animals. In agreement with this finding, no decrease in the viral load occurred with up to 12 weeks of pegylated IFNα therapy. The block in the response to exogenous IFNα appeared to be HCV‐specific because the response in a hepatitis B virus–infected animal was similar to that of uninfected animals. The lack of a response to exogenous IFNα may be due to an already maximally induced ISG response because chronically HCV‐infected chimpanzees already have a highly up‐regulated hepatic ISG response. Alternatively, negative regulation may block the response to exogenous IFNα, yet it does not prevent the continued response to endogenous ISG stimuli. The IFNα response in chronically HCV‐infected chimpanzees may be mechanistically similar to the null response in the human population. Conclusion: In chimpanzees infected with HCV, the highly elevated hepatic ISG expression may prevent the further induction of ISGs and antiviral efficacy following an IFNα treatment. (HEPATOLOGY 2007.)