NIH Public Access Author Manuscript Hepatology. Author manuscript; available in PMC 2008 May 19. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Hepatology. 2007 October ; 46(4): 999±1008. Lack of Response to Exogenous Interferon-α in the Liver of HCV Chronically Infected Chimpanzees Robert E. Lanford1,2, Bernadette Guerra1, Catherine B. Bigger3, Helen Lee1, Deborah Chavez1, and Kathleen M. Brasky2 1 Department of Virology and Immunology, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227 2 Southwest National Primate Research Center, 7620 NW Loop 410, San Antonio, TX 78227 3 Children's Research Institute, Columbus, OH 45205 Abstract 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 (PBMC) was rapidly induced but was also rapidly down-regulated, with most IFNα stimulated genes (ISGs) returning to baseline within 24 hr. We have extended these observations to include chimpanzees chronically infected with hepatitis C virus (HCV). Remarkably, using total genome microarray analysis, almost no induction of ISG transcripts was observed in the liver of chronically infected animals following IFNα dosing, while the response in PBMC was similar to that in uninfected animals. Consistent with this finding, no decrease in viral load occurred in up to 12 weeks of pegylated (peg)-IFNα therapy. The block in response to exogenous IFNα appeared to be HCV specific, since the response in an HBV infected animal was similar to that of uninfected animals. The lack of response to exogenous IFNα may be due to an already maximally induced ISG response, since HCV chronically infected chimpanzees already have a highly up-regulated hepatic ISG response. Alternatively, negative regulation may block the response to exogenous IFNα, yet does not prevent the continued response to endogenous ISG stimuli. The IFNα response in HCV chronically 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 further induction of ISGs and antiviral efficacy following IFNα treatment. Keywords microarray; hepatitis; innate immunity; virus; antiviral Approximately 170 million people worldwide are chronically infected with HCV which frequently progresses to serious liver disease, including cirrhosis and hepatocellular carcinoma 1,2. Combination therapy with pegIFNα and ribavirin results in sustained viral clearance for approximately 40–50% and 80–90% of patients infected with genotypes 1 and 2/3, respectively 3–5. Little is understood regarding the factors leading to successful or failed viral clearance during IFNα therapy, but studies on gene expression are beginning to illustrate differences in these populations. Gene expression studies in chimpanzees with acute-resolving and chronic HCV infections have revealed elevated ISG expression in the liver, indicative of a response to Address reprint requests to: Robert E. Lanford, Department of Virology and Immunology, Southwest Foundation for Biomedical Research, 7620 NW Loop 410, San Antonio, TX 78227. Phone: (210) 258-9445 FAX: (210) 670-3329 Email: [email protected]. full names of genes indicated by gene symbols can be found at www.genecards.org. Lanford et al. Page 2 dsRNA and/or IFNα 6–8, and similar observations have been made in chronically infected humans 9,10. More recently, an inverse correlation has been observed between pretreatment 11 NIH-PA Author Manuscript NIH-PA Author Manuscripthepatic NIH-PA Author Manuscript levels for some ISG transcripts and virologic response to therapy , while a positive correlation has been observed between the magnitude of the ISG response in IFNα treated PBMC and virologic response to therapy 12. The early kinetics of viral RNA loss from the circulation during IFNα therapy suggests the presence of two phases. Phase I occurs during the first 24–48 hr and is presumably due to the decrease in secretion of new virions, while phase II kinetics vary between individuals, are predictive of the outcome of therapy, and are thought to be a measurement of loss of infected cells 13–15. Our recent data on gene expression in uninfected chimpanzees dosed with IFNα suggests that the rapid down-regulation of the IFNα-induced ISG response may be partially responsible for the rapid change in kinetics from phase I and II 16. Peak expression for most genes occurred by 4 hr after IFNα dosing and was declining or at baseline by 8hr. The transcriptional response to IFNα in vivo was largely tissue specific with significant differences in the response in liver and PBMC. Previous studies with IFNα in chimpanzees have failed to demonstrate a reduction in viral load using either traditional IFNα therapy with ribavirin, or adenovirus-based gene therapy to induce high-level expression of IFNα in the liver 17,18 (Lanford, unpublished data). Our recent studies comparing human and chimpanzee IFNα in uninfected animals indicated that IFNα from both species was equally effective with regard to ISG induction in the liver and PBMC. Thus, the reason for the lack of antiviral effect of IFNα in the chimpanzee remains unresolved. Here, we have examined human peg-IFNα in three HCV chronically infected chimpanzees and have performed total genome DNA microarray analysis to characterize changes in liver and PBMC gene expression. No decline in viral load was observed with up to 12 weeks of therapy. The induction of ISGs in PBMC from infected animals was similar to that observed in uninfected animals, while the liver of HCV infected animals was resistant to exogenous IFNα. The implications of these findings to HCV infected humans and possible mechanisms of resistance to exogenous IFNα are discussed. MATERIALS AND METHODS Chimpanzees Chimpanzees were housed at the Southwest National Primate Research Center at the Southwest Foundation for Biomedical Research. The animals were cared for in accordance with the Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Institutional Animal Care and Use Committee. Biopsies were obtained in the morning on fasting animals to avoid postprandial and diurnal changes in liver gene expression. The chimpanzees used in this study had been chronically infected for 16–28 years, had been monitored on a regular basis in recent years and had stable levels of viremia. The three HCV infected animals were 4x0081(Genotype 1b; infected in 1983 with NANB); 4 × 0119(Genotype 3a infected in 1981 with NANB contaminated factor VIII); and 4 × 0341(Genotype 1a infected in 1991 with strain HCV1). The HBV infected animal was 4 × 0139 (infected in 1979). Microarray and RT-PCR Analyses Total RNA prepared from liver and PBMC was used to perform microarray analyses 6,7 and to monitor viral and cellular transcripts by quantitative reverse transcription-PCR (RT-PCR; TaqMan) 19, as described in Supplemental Methods. Microarray analysis was performed on 71 microarrays which included a previous data set from 36 arrays from uninfected animals 16. A more comprehensive data set is available at Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 3 http://www.sfbr.org/pages/virology/lanford including Excel versions of the Supplemental Tables that can be searched, sorted and downloaded. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript IFNα dosing Peg-IFNα2a (Roche; Nutely, NJ) was partially a gift of Dr. David Thomas (Johns Hopkins University) and partially purchased from a pharmacy. Chimpanzees were inoculated subcutaneously with 5 million IU of IFNα, and blood and liver samples were obtained at 2 and 4 weeks prior to dosing, at 4, 8 and 24 hr and 7 days post-inoculation. The genotype 1 animals were continued on weekly administration of peg-IFNα for 8 or 12 weeks as indicated. RESULTS Lack of antiviral response in chimpanzees to peg-IFNα In the current study, we examined the relationship of the antiviral response to peg-IFNα in HCV infected chimpanzees and the induction of gene expression in the liver and PBMC. Three chimpanzees chronically infected with HCV were dosed with peg-IFNα weekly for 1–12 weeks, and viral load was determined by quantitative RT-PCR (TaqMan). The genotype 1a chimpanzee (4 × 0341) was dosed for 12 weeks, while the genotype 1b chimpanzee (4 × 0081) was stopped at 8 weeks due to unrelated health issues, and the genotype 3a chimpanzee (4 × 0119) was given a single dose of IFNα to examine ISG induction in the liver and was only followed for 14 days. No significant decline in viral RNA was observed in the serum (Fig. 1) or liver (data not shown). Lack of IP-10 transcriptional response to IFNα in the liver of HCV infected chimpanzees To evaluate the possible reasons for failed antiviral response to IFNα in chimpanzees, the induction of IP-10 (CXCL10) transcription was examined in the liver and PBMC by quantitative RT-PCR. We have previously demonstrated that this chemokine is highly elevated during acute 7 and chronic 6 HCV infection and following IFNα dosing in uninfected chimpanzees 16, primary hepatocytes 16 and Huh7 cells 19. At 4 hr post-inoculation with peg- IFNα in an uninfected chimpanzee (4 × 0363), IP-10 transcripts were elevated by 394- and 667-fold in PBMC and liver, respectively. Similarly, in the PBMC of an HCV infected animal (4 × 0081), IP-10 transcripts were increased by 107-fold, but surprisingly no induction was observed in the liver of this animal (Fig. 2). As previously observed in uninfected chimpanzees 16, the transcriptional response to IFNα was rapidly down-regulated. IP-10 transcripts were reduced to near baseline in the liver and PBMC of uninfected animals and in PBMC of HCV infected animals by 8–24 hr post-inoculation.