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...

Chemically synthesized combinatorial libraries of unmodified or modified nucleic acids have not previously been used in methods to rapidly select oligonucleotides binding to target biomolecules such as proteins. Phosphorothioate oligonucleotides (S-ODNs) or phosphorodithioate oligonucleotides (S2-ODNs) with sulfurs replacing one or both of the non-bridging phosphate oxygens bind to proteins more tightly than unmodified oligonucleotides and have the potential to be used as diagnostic reagents and therapeutics. We have applied a split synthesis methodology to create one-bead one-S-ODN and one-bead one-S2-ODN libraries. Binding and selection of specific beads to the transcription factor NF-kappaB p50/p50 protein were demonstrated. Sequencing both the nucleic acid bases and the positions of any 3'-O-thioate/dithioate linkages was carried out by using a novel PCR-based identification tag of the selected beads. This approach allows us to rapidly and conveniently identify S-ODNs or S2-ODNs that bind to proteins.

The chimpanzee (Pan troglodytes) is the only experimental animal susceptible to infection with hepatitis C virus (HCV). The chimpanzee model of HCV infection was instrumental in the initial studies on non-A, non-B hepatitis, including observations on the clinical course of infection, determination of the physical properties of the virus, and eventual cloning of the HCV nucleic acid. This review focuses on more recent aspects of the use of the chimpanzee in HCV research. The chimpanzee model has been critical for the analysis of early events in HCV infection because it represents a population for which samples are available from the time of exposure and all exposed animals are examined. For this reason, the chimpanzee represents a truly nonselected population. In contrast, human cohorts are often selected for disease status or antibody reactivity and typically include individuals that have been infected for decades. The chimpanzee model is essential to an improved understanding of the factors involved in viral clearance, analysis of the immune response to infection, and the development of vaccines. The development of infectious cDNA clones of HCV was dependent on the use of chimpanzees, and they will continue to be needed in the use of reverse genetics to evaluate critical sequences for viral replication. In addition, chimpanzees have been used in conjunction with DNA microarray technology to probe the entire spectrum of changes in liver gene expression during the course of HCV infection. The chimpanzee will continue to provide a critical aspect to the understanding of HCV disease and the development of therapeutic modalities.

The clinical course of hepatitis C virus (HCV) infections in a chimpanzee cohort was examined to better characterize the outcome of this valuable animal model. Results of a cross-sectional study revealed that a low percentage (39%) of HCV-inoculated chimpanzees were viremic based on reverse transcription (RT-PCR) analysis. A correlation was observed between viremia and the presence of anti-HCV antibodies. The pattern of antibodies was dissimilar among viremic chimpanzees and chimpanzees that cleared the virus. Viremic chimpanzees had a higher prevalence of antibody reactivity to NS3, NS4, and NS5. Since an unexpectedly low percentage of chimpanzees were persistently infected with HCV, a longitudinal analysis of the virological profile of a small panel of HCV-infected chimpanzees was performed to determine the kinetics of viral clearance and loss of antibody. This study also revealed that a low percentage (33%) of HCV-inoculated chimpanzees were persistently viremic. Analysis of serial bleeds from six HCV-infected animals revealed four different clinical profiles. Viral clearance with either gradual or rapid loss of anti-HCV antibody was observed in four animals within 5 months postinoculation. A chronic-carrier profile characterized by persistent HCV RNA and anti-HCV antibody was observed in two animals. One of these chimpanzees was RT-PCR positive, antibody negative for 5 years and thus represented a silent carrier. If extrapolated to the human population, these data would imply that a significant percentage of unrecognized HCV infections may occur and that silent carriers may represent potentially infectious blood donors.

Previously, we reported the in vitro combinatorial selection of phosphorothioate aptamers or "thioaptamers" targeting the transcription factor NF-IL6. Using the same approach and purified recombinant human NF-kappa B proteins RelA(p65) and p50, duplex thioaptamers have been selected that demonstrate high-affinity, competitive binding with the duplex 22-mer binding site, Ig kappa B. Binding energetics of RelA(p65) and p50 homodimers were studied using a quantitative electrophoretic mobility shift assay or EMSA. As a reference system for competitive aptamer binding, the duplex 22-mer phosphoryl binding site known as Ig kappa was determined to bind each p65 and p50 homodimer with a 1:1 stoichiometry and with affinities, determined by global analysis, K(d) = 4.8 +/- 0.2 nM for p65 and K(d) = 0.8 +/- 0.2 nM for p50. A global analysis tool for competitive NF-kappa B/Ig kappa binding was developed and utilized to measure the affinity of thioaptamers selected by both p65 and p50. The competition results indicate that the thioaptamers bind and compete for the same NF-kappa B site as the known promoter element Ig kappa B (K(d) = 78.9 +/- 1.9 nM for a p65-selected aptamer and 19.6 +/- 1.3 nM for a p50-selected thioaptamer). Qualitative gel shift binding experiments with p50 also demonstrate that the nature of enhanced affinity and specificity can be attributed to the presence of sulfur. Collectively, these results demonstrate the feasibility of the thioaptamer in vitro combinatorial selection technology as a method for producing specific, high-affinity ligands to proteins.

An immunofluorescence assay was developed to identify proteins specifically binding to oligonucleoside phosphorodithioate (ODN) aptamers from a bead-bound ODN library. Accordingly, NF-kappaB p50 protein was incubated with either bead-bound NF-kappaB consensus sequence or a bead-bound ODN combinatorial library and adsorption was then assessed using a specific primary antibody and a secondary antibody conjugated with Alexa 488 fluorescent dye. This assay avoids any problems related to fluorescently labeling target proteins. The method is straightforward and readily applicable to other transcription factors and proteins, and the feasibility of its application for high-throughput screening of large aptamer bead-based libraries by flow cytometry is demonstrated.

The putative envelope 2 (E2) gene of hepatitis C virus (HCV) contains a highly variable region referred to as hypervariable region 1 (HVR1). We hypothesized that this genetic variability is driven by immune selection pressure, rather than representing the accumulation of random mutations in a region with relatively little functional constraint. To test this hypothesis, we examined the E2 sequence of a human inoculum that was passaged through eight chimpanzees, which appear to have a replicative rate (opportunity for chance mutation) similar to that of humans. Acute-phase plasma samples from a human (the inoculum) and six of eight serially infected chimpanzees were studied. For each, 33 cloned cDNAs were examined by a combined heteroduplex-single-stranded conformational polymorphism assay to assess quasispecies complexity and optimize selection of clones with unique gel shift patterns (clonotypes) for sequencing. The sequence diversity of HCV was significantly lower in the chimpanzees than in the humans, and during eight serial passages there was no change in the sequence of the majority clonotype from each animal examined. Similarly, the rates of protein sequence altering (nonsynonymous) substitution were lower in the chimpanzees than in the humans. These findings demonstrate that nonsynonymous mutations indicate selection pressure rather than being an incidental result of HCV replication.An estimated 170 million people worldwide are infected with hepatitis C virus (HCV) (4). More than 80% of infections result in persistent viremia, which may be associated with chronic hepatitis, cirrhosis, liver failure, and hepatocellular cancer (3,41,44). HCV-associated disease is responsible for more than 10,000 deaths each year in the United States alone, and this mortality is expected to rise (8). Because of limitations of in vitro replication systems, studies of HCV pathogenesis have been limited to observation of natural infection of humans and experimental infection of chimpanzees.The chimpanzee is the best experimental model of HCV infection. HCV does not grow efficiently in tissue culture, and the only other candidate model is a tree shrew that is too small to permit adequate sampling with current technology (52). In chimpanzees, HCV replication has been demonstrated within days of experimental infection, and the potential for reinfection has been demonstrated (14,15,37,51). Serum HCV RNA levels and the extent of hepatic HCV involvement are similar in chimpanzees and humans, suggesting that replication rates are similar (1). The chimpanzee has also been used to test the infectivity of HCV clones and vaccine candidates. However, Bassett et al. recently demonstrated that the natural history of HCV infection in a cohort of experimentally infected chimpanzees differed from what is typically found in humans (5, 6). These chimpanzees had a higher rate of clearance of viremia, lower rate of antibody production to envelope proteins, and lower rate of envelope amino acid change. Similarly, others have reported little change in...

Elevated iron levels have been associated with raised serum alanine transaminase (ALT) levels in hepatitis C virus (HCV)-infected humans. However, it is not clear if HCV infection causes increased iron accumulation by the liver or if the severity of HCV infection is actually worsenedby higher iron levels in the host. To better understand the relationship between iron and persistent HCV infections, we examined the effect of excess dietary iron on disease severity in HCV-infected chimpanzees. Iron was supplemented in the diets of four HCV-infected and two uninfected chimpanzees for 29 weeks to achieve iron loading. Iron loading was confirmed by increases in serum iron levels, percentages of transferrin saturation, ferritin levels, elevations in hepatic iron concentration (HIC), and by histological examination. The majority of HCV-infected chimpanzees had higher iron levels before iron feeding than the uninfected animals. Hepatitis C virus (HCV) infections are a significant health problem worldwide. HCV infections are particularly problematic as a result of the high rate of chronicity. Persistent infection has been estimated to occur in approximately 85% of HCV-infected individuals. 1-3 Individuals with persistent HCV infection may develop cirrhosis and hepatocellular carcinoma, usually after several decades. 1,3 The transmission of HCV is primarily associated with percutaneous routes 4 and is commonly associated with injection drug use. However, the route of transmission for many HCV infections is unclear, because risk factors are not always identified. Although recipients of blood transfusions were at risk for acquiring HCV in the past, obligatory anti-HCV screening of blood donors has significantly decreased this risk. Other routes of infection may include sexual and perinatal transmission. However, the significance of these routes is unclear, and the efficiency is very low compared with hepatitis B virus or human immunodeficiency virus.The pathogenesis of hepatitis C has been difficult to study because of the lack of small animal models and conventional tissue culture systems. These obstacles have hindered the development of vaccines and antiviral treatments. Current antiviral treatments are associated with unpleasant side effects and are ineffective in the majority of HCV-infected patients. 1 Treatments for chronic HCV infection include interferon alfa (IFN-␣) alone or in combination with ribavirin. The majority (80%-85%) of patients fail to normalize serum alanine transaminase (ALT) and lose HCV RNA in the serum at 6 months' posttreatment when treated with IFN-␣ alone. The combination of IFN-␣ and ribavirin is more promising, because 40% to 50% of individuals experience a sustained response. 1 Iron reduction therapy is under investigation as a potential approach to lessening the severity of HCV infections. Elevated serum iron, transferrin saturation, and ferritin levels are often observed in individuals with chronic HCV infection, and although few have severe hepatic iron overload, a high percentage of indivi...