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.

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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 C virus (HCV) infections were evaluated in chimpanzees that had previously cleared HCV and were rechallenged. Animals that had previously cleared HCV infection rapidly cleared homologous and heterologous virus upon rechallenge, indicative of a strong protective immunity. In one animal, sterilizing immunity was observed with regard to viremia, although viral RNA was transiently detected in the liver. Accelerated viral clearance following rechallenge with HCV was observed in animals that had not been exposed to HCV for over 16 Hepatitis C virus (HCV) infections represent a serious health problem. The majority of HCV infections develop into chronic infections that may progress to cirrhosis and hepatocellular carcinoma. 1 HCV is classified in the Hepacivirus genus of the Flaviviridae family. 2 The HCV genome is approximately 9.6 kb and consists of single-stranded RNA of positive polarity. The viral RNA has a single large open reading frame that encodes for a polyprotein of approximately 3,000 amino acids. 3 The structural proteins are located at the amino terminal end of the polyprotein and include the capsid protein and 2 envelope glycoproteins, E1 and E2. The nonstructural proteins are preceded by a p7 domain of unknown function and include NS2-NS5. The NS2 domain forms an autoprotease with the amino-terminal portion of NS3. The amino terminus of NS3 encodes a serine protease and the carboxy terminus encodes a helicase, which plays a role in viral RNA replication. NS4A is a cofactor for the serine protease. The viral RNAdependent RNA polymerase is encoded by NS5B. 4 The functions of NS4B and NS5A are unknown.The chimpanzee is the only animal model for studying HCV infection. Humans and chimpanzees with persistent HCV infections mount an antibody response to most HCV proteins. 5 HCV-specific antibody does not appear to protect humans and chimpanzees from infection and is actually associated with active viremia rather than viral clearance. The kinetics of antibody production to HCV proteins and the pattern of antibodies to individual proteins do not appear to predict disease outcome (clearance versus persistence).The humoral immune response to the nonstructural HCV proteins appears to be similar in humans and chimpanzees. 5 In contrast, antibody responses to HCV structural proteins are observed less frequently in chimpanzees than in humans for reasons not understood. [5][6][7][8][9][10][11] Studies in chimpanzees have revealed that antibody neutralization of HCV is not easily attained. 12,13 Recently, Cooper et al. observed that strong antibody responses to HCV proteins were not necessary for viral clearance in HCV-inoculated chimpanzees. 14 Several investigators have also observed that circulating HCV-specific antibodies do not prevent reinfection of chimpanzees with HCV. [15][16][17][18] Therefore, T cells may play a more critical role than antibodies in the resolution of HCV infection.HCV antigen-specific CD8 ϩ T cells have been observed in the peripheral blood and liver of humans and chimpanzees durin...

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...