The liver-expressed microRNA-122 (miR-122) is essential for hepatitis C virus (HCV) RNA accumulation in cultured liver cells, but its potential as a target for antiviral intervention has not been assessed. Here, we show that treatment of chronically infected chimpanzees with a locked nucleic acid (LNA)-modified oligonucleotide (SPC3649) complementary to miR-122 leads to long-lasting suppression of HCV viremia with no evidence for viral resistance or side effects in the treated animals. Furthermore, transcriptome and histological analyses of liver biopsies demonstrated derepression of target mRNAs with miR-122 seed sites, down-regulation of interferon-regulated genes (IRGs) and improvement of HCV-induced liver pathology. The prolonged virological response to SPC3649 treatment without HCV rebound holds promise of a new antiviral therapy with a high barrier to resistance.

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane1, attributes that profoundly affect stability, transmission, and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically-transmitted hepatitis2–4. HAV infects in a stealth-like manner and replicates efficiently in the liver5. Virus-specific antibodies appear only after 3–4 weeks of infection, and typically herald its resolution3,4. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure6, when virus replication is well established in the liver5. Here, we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses (“eHAV”) resemble exosomes7, small vesicles that are increasingly recognized to play important roles in intercellular communications. They are fully infectious, sensitive to chloroform extraction, and circulate in the blood of infected humans. Their biogenesis is dependent upon host proteins associated with endosomal-sorting complexes required for transport (ESCRT)8, VPS4B and ALIX. While the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and likely promotes virus spread within the liver, anti-capsid antibodies restrict replication following infection with eHAV, suggesting a possible explanation for post-exposure prophylaxis. Membrane hijacking by HAV blurs the classic distinction between “enveloped” and “nonenveloped” viruses, and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.

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.

Hepatitis C virus (HCV) is a major cause of chronic liver disease, frequently progressing to cirrhosis and increased risk of hepatocellular carcinoma. Current therapies are inadequate and progress in the field has been hampered by the lack of efficient HCV culture systems. By using a recently described HCV genotype 2a infectious clone that replicates and produces infectious virus in cell culture (HCVcc), we report here that HCVcc strain FL-J6͞JFH can establish long-term infections in chimpanzees and in mice containing human liver grafts. Importantly, virus recovered from these animals was highly infectious in cell culture, demonstrating efficient ex vivo culture of HCV. The improved infectivity of animal-derived HCV correlated with virions of a lower average buoyant density than HCVcc, suggesting that physical association with low-density factors influences viral infectivity. These results greatly extend the utility of the HCVcc genetic system to allow the complete in vitro and in vivo dissection of the HCV life cycle.animal model ͉ pathogenesis ͉ reverse genetics ͉ viral hepatitis A major limitation in hepatitis C virus (HCV) research has been the lack of virus culture systems. After identification of the viral genome in 1989 (1), early efforts focused on understanding the structure and function of individual viral gene products. HCV is an enveloped, positive-strand RNA virus classified in the family Flaviviridae (2). The 9.6-kb ssRNA genome encodes three structural (virion-associated) and seven nonstructural (intracellular) genes within a single ORF.The first functional cDNA clones of HCV were constructed in 1997, allowing chimpanzees to be infected after intrahepatic transfection with recombinant viral RNA (3, 4). Unfortunately, these infectious genomes failed to replicate in cell culture. By engineering HCV replicons to express a drug-selectable gene, it became possible to select for HCV RNA replication in cell culture (5). However, efficient replication required cell cultureadaptive mutations in the viral RNA (6). Moreover, only the intracellular aspects of HCV replication were modeled by these systems. For unknown reasons, cell culture-adaptive mutations can inhibit virion production in culture (T. Pietschmann and R. Bartenschlager, personal communication) and attenuate RNA infectivity in vivo (7).Recent progress in the field has come from the identification of JFH-1, a genotype 2a subgenomic replicon that does not require adaptive mutations for efficient RNA replication in culture (8). Based on this sequence, we constructed a chimeric JFH-1 genome containing the core to nonstructural protein 2 (NS2) region of HCV strain J6. This genome, FL-J6͞JFH, replicated and produced high levels of infectious virus in cell culture (HCVcc) (9), allowing us to study new aspects of the viral life cycle in tissue culture (9, 10). Similarly, full-length JFH-1 clones produced HCVcc, albeit with delayed kinetics of virus release (11, 12). HCVcc strain JFH-1 was able to transiently infect a chimpanzee, although replication le...

Hepatitis C virus (HCV) poses a worldwide health problem in that the majority of individuals exposed to HCV become chronically infected and are predisposed for developing significant liver disease. DNA microarray technology provides an opportunity to survey transcription modulation in the context of an infectious disease and is a particularly attractive approach in characterizing HCV-host interactions, since the mechanisms underlying viral persistence and disease progression are not understood and are difficult to study. Here, we describe the changes in liver gene expression during the course of an acute-resolving HCV infection in a chimpanzee. Clearance of viremia in this animal occurred between weeks 6 and 8, while clearance of residual infected hepatocytes did not occur until 14 weeks postinfection. The most notable changes in gene expression occurred in numerous interferon response genes (including all three classical interferon antiviral pathways) that increased dramatically, some as early as day 2 postinfection. The data suggest a biphasic mechanism of viral clearance dependent on both the innate and adaptive immune responses and provide insight into the response of the liver to a hepatotropic viral infection.

No abstract

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.

The intrinsic variability of hepatitis C virus (HCV) envelope proteins E1 and E2 complicates the identification of protective antibodies. In an attempt to identify antibodies to E2 proteins from divergent HCV isolates, we produced HCV E2 recombinant proteins from individuals infected with HCV genotypes 1a, 1b, 2a, and 2b. These proteins were then used to characterize 10 human monoclonal antibodies (HMAbs) produced from peripheral B cells isolated from an individual infected with HCV genotype 1b. Nine of the antibodies recognize conformational epitopes within HCV E2. Six HMAbs identify epitopes shared among HCV genotypes 1a, 1b, 2a, and 2b. Six, including five broadly reactive HMAbs, could inhibit binding of HCV E2 of genotypes 1a, 1b, 2a, and 2b to human CD81 when E2 and the antibody were simultaneously exposed to CD81. Surprisingly, all of the antibodies that inhibited the binding of E2 to CD81 retained the ability to recognize preformed CD81-E2 complexes generated with some of the same recombinant E2 proteins. Two antibodies that did not recognize preformed complexes of HCV 1a E2 and CD81 also inhibited binding of HCV 1a virions to CD81. Thus, HCV-infected individuals can produce antibodies that recognize conserved conformational epitopes and inhibit the binding of HCV to CD81. The inhibition is mediated via antibody binding to epitopes outside of the CD81 binding site in E2, possibly by preventing conformational changes in E2 that are required for CD81 binding.Hepatitis C virus (HCV), a member of the family Flaviviridae, expresses its proteins from a 9.5-kb positive-sense RNA genome (18). The virus is highly variable, with more than nine distinct genotypes (1, 18). Most patients progress from acute to chronic disease in spite of a robust immune response. Nonetheless, evidence for a humoral immune response providing at least partial protection in clinical and animal model studies is accumulating (6, 9-11, 29, 37) and suggests that neutralizing antibodies have a role in the containment of HCV infection. For a protective immune response, the important viral gene products are the envelope proteins, designated E1 and E2. Both sequence analyses of different isolates and sequential studies of virus isolates in infected patients suggest that the HCV E2 protein is under immune selection leading to selection of variants in the amino-terminal domain of HCV E2, designated hypervariable region 1 (HVR-1) (1, 9, 16-18, 20, 37, 39, 40). Antibodies to HVR-1 appear to mediate virus neutralization in cell culture and chimpanzee protection studies (10, 37). Unfortunately, antibodies to HVR-1 tend to be isolate specific and over time drive the selection of new viral variants that the existing immune response does not recognize (9,20,37,40). Although there has been progress at inducing a broader immune response to HVR-1-related sequences (31), the high mutability of HVR-1 sequences in vivo may allow for the selection of immune escape mutants even against antibodies that recognize the majority of HVR-1 isolates.Studies using HCV ...