Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure and hepatocellular carcinoma. Evidence exists that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with ARC-520, a RNA interference (RNAi)-based therapeutic targeting HBV transcripts, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients that were HBeAg negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several independent lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome, but also from transcripts arising from HBV DNA integrated into the host genome. The latter was the dominant source in HBeAg negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the siRNAs in ARC-520, explaining the reduced response in HBeAg negative chimpanzees and by extension in HBeAg negative patients. Our results uncover a heretofore under-recognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immune surveillance and could alter trial design and endpoint expectations of new therapies for chronic HBV.

Background & Aims Direct-acting anti-viral agents suppress hepatitis B virus (HBV) load but must be given lifelong. 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 (TLR)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 (IFN)-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 greater than 1 log persisted for months. Serum levels of HB surface antigen and HB e antigen, and numbers of HBV antigen-positive hepatocytes, were reduced as hepatocyte apoptosis increased. GS-9620 administration induced production of IFN-α and other cytokines and chemokines, and activated ISGs, natural killer cells, and lymphocyte subsets. Conclusions The small molecule GS-9620 activates TLR-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.

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.innate immunity | viral persistence | immune evasion H epatitis A virus (HAV) is a small, hepatotropic positivestrand RNA virus. Although it is classified among the Picornaviridae, it differs in several important respects from most other human picornaviral pathogens in having a very slow and nonlytic replication cycle (reviewed in ref. 1). Several primatederived cell lines are permissive for infection by HAV, and in the absence of extensive adaptation of the virus to cell culture, these infections are typically noncytopathic. Despite this, HAV causes only acute disease in humans and has never been documented to establish long-term persistent infection within the liver (1-3). However, relatively little is known about the pathogenesis of HAV infections and how the virus is cleared from the liver as there has been little impetus for research on hepatitis A since the development of effective inactivated vaccines almost 2 decades ago.The absence of long-term persistent HAV infection is well documented by disappearance of the virus from isolated human populations over time (4, 5) and differs dramatically from the outcome of hepatitis C virus (HCV) infections, which persist for life in the majority of persons (6). HCV is the cause of considerable human morbidity and mortality. Like HAV, it...

Hepatitis B virus (HBV) infections are a major worldwide health problem with chronic infections leading to cirrhosis and liver cancer. Viruses related to human HBV have been isolated from birds and rodents, but despite efforts to find hepadnaviruses that infect species intermediate in evolution between rodents and humans, none have been described. We recently isolated a hepadnavirus from a woolly monkey (Lagothrix lagotricha) that was suffering from fulminant hepatitis. Phylogenetic analysis of the nucleotide sequences of the core and surface genes indicated that the virus was distinct from the human HBV family, and because it is basal (ancestral) to the human monophyletic group, it probably represents a progenitor of the human viruses. This virus was designated woolly monkey hepatitis B virus (WMHBV). Analysis of woolly monkey colonies at five zoos indicated that WMHBV infections occurred in most of the animals at the Louisville zoo but not at four other zoos in the United States.The host range of WMHBV was examined by inoculation of one chimpanzee and two black-handed spider monkeys (Ateles geoffroyi), the closest nonendangered relative of the woolly monkey. The data suggest that spider monkeys are susceptible to infection with WMHBV and that minimal replication was observed in a chimpanzee. Thus, we have isolated a hepadnavirus with a host intermediate between humans and rodents and establishes a new animal model for evaluation of antiviral therapies for treating HBV chronic infections.

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-␣), IFN-␥, 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-␣, IFN-␥, 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-␣ 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.Chronic hepatitis C virus (HCV) infections are one of the leading causes of liver disease worldwide (2). The prevalence of HCV infections is 1 to 2%, although certain geographical regions, age groups, and ethnic groups have much higher rates of infection (3). Although symptoms may be mild for decades, 20% of persistently infected individuals may eventually develop serious liver disease including cirrhosis and liver cancer (2). HCV infection is the leading cause for liver transplantation in the United States (12). Although the initial use of interferon (IFN) for treatment of chronic infections yielded marginal results, the current therapeutic regimen of pegylated alpha 2b IFN (IFN-␣2b) and ribavirin provides substantially improved rates of sustained viral clearance of 42 and 82% for genotype 1 and genotype 2 and 3, respectively (45). Treatment of acute infections with standard IFN therapy without ribavirin is highly efficacious and approaches 100% sustained viral clearance (33). Nonetheless, a great need exists for improved antiviral agents, since many patients still do not benefit from IFN therapy, and IFN therapy is associated with undesirable side effects. The lack of a suitable tissue culture system has previously hampered the development of antiviral agents, but the re...

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.

To further explore the controversial potential for extrahepatic replication of hepatitis C virus (HCV), the highly strand-specific rTth method of reverse transcriptase PCR was used to examine sera, liver, peripheral blood mononuclear cells, and other extrahepatic tissues from HCV-infected chimpanzees and humans. Positive-strand HCV RNA was present in the liver at approximately 10-fold-higher levels than negative-strand HCV RNA. No negative-strand RNA was detected in peripheral blood mononuclear cells or other extrahepatic tissues despite the presence of abundant positive-strand RNA. These data demonstrate that within the limits of sensitivity of this highly strand-specific reverse transcriptase PCR method, no extrahepatic replication of HCV was detected.

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.