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. The response in the other HCV infected animals was similar to 4 × 0081, and examination of several other ISGs by quantitative RT-PCR revealed patterns of expression similar to IP-10. To determine if the lack of ISG response in the liver was unique to HCV or common to other chronic viral infections, a chimpanzee chronically infected with HBV (4x0139) was dosed with peg-IFNα using the identical protocol. IP-10 transcripts were elevated by 107- and 24-fold at 4 hr post-inoculation in the PBMC and liver, respectively (Fig. 2). We also examined the serum levels of IP-10 protein by ELISA. In uninfected animals, the levels are highly induced and mimic the induction and down regulation of the transcript observed in liver and PBMC. In HCV chronic animals, there was no significant increase in serum IP10 levels despite the transient increase in transcripts in PBMC. This suggests that due to its size, the liver may represent the primary source of IP-10 in the serum when induced by HCV infection or IFNα treatment. In the HBV infected animal, the serum levels of IP-10 did not increase as much as observed in the uninfected animals. This correlates with the lower induction of IP-10 transcripts in both liver and PBMC in comparison to the uninfected animals and is likely due to animal to animal variation, although HBV infection may influence IP-10 induction.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 4

Lack of ISG response to exogenous IFNα in the liver of HCV infected chimpanzees To evaluate the extent of non-responsiveness to IFNα in the liver of HCV infected chimpanzees,

NIH-PA Author Manuscript NIH-PA Author Manuscripttotal NIH-PA Author Manuscript genome microarray analysis was performed on the liver and PBMC at two time points prior to dosing and at 4, 8 and 24 hr post-dosing. A near total lack of ISG response to IFNα was observed in the liver of all three HCV infected animals, while the PBMC fraction of infected and uninfected animals exhibited very similar ISG induction profiles. Table 1 illustrates the lack of ISG response in the liver by selecting 28 genes that were highly induced in the liver of uninfected animals for comparison with HCV infected liver. The genes were selected from 500 hepatic ISGs in uninfected animals based on the magnitude of fold induction in the liver and PBMC and/or name recognition in various aspects of the IFN antiviral pathway 16 (Supplement Table 1). The values shown are the average fold change at 4 hr post-dosing in relation to pretreatment baselines for three uninfected (two independent experiments each) and three HCV infected animals. Only five of the ISGs that were highly induced in uninfected livers were induced in the liver of HCV infected chimpanzees, and for these five ISGs, the fold induction was lower than that observed in uninfected animals. All but five of the ISGs in Table 1 were induced in the liver of the HBV infected chimpanzee, and the magnitude of induction was similar to that observed in uninfected animals. Microarray analysis of liver and PBMC from one HCV infected animal (4 × 0081) over 8 weeks of peg-IFNα therapy revealed that the ISGs in PBMC remained down-regulated after the initial burst of induction and that ISG transcripts in the liver were not induced even after repeated exposure to IFNα (Fig. 3).

In contrast to selecting the genes most highly induced in uninfected liver for comparison to infected liver, when all genes significantly up-regulated in the liver of HCV infected animals in response to IFNα were selected, only 24 ISGs (from 500 in uninfected liver) met the criteria for inclusion (FC ≥ 2.0, p ≤ 0.02) (Table 2). Of particular interest was the induction in HCV infected liver of both SOCS1 and SOCS3 which are inhibitors of the JAK-STAT pathways. SOCS1 was not detected in the uninfected liver, but SOCS3 was similarly induced in the liver of all animals. Despite the remarkable differences in hepatic response to IFNα, the response in PBMC was similar in uninfected, HCV infected and HBV infected animals. All 25 of the ISGs shown in Table 1 that were induced in PBMC of uninfected animals were also induced in the PBMC of HCV infected animals. Thus, the lack of hepatic response to exogenous IFNα appeared to involve nearly all ISGs in HCV infected animals, but was not observed in PBMC of HCV infected animals or HBV infected liver and PBMC.

Baseline ISG expression in HCV chronically infected animals Although IFNα did not induce an increase in ISG expression in the liver of HCV infected animals, the ISG response was already highly induced due to HCV infection. We have previously characterized the persistent ISG induction in HCV chronically infected animals by microarray analysis 6. These results were confirmed during the current study due to the analysis of the baseline infected and uninfected liver samples prior to dosing. Examination of the IP-10 RT-PCR data for all of the animals in this study, using copies of transcript rather than fold change from baseline, revealed that IP-10 transcripts were elevated at baseline in the liver of HCV infected animals in comparison to the uninfected animals, and that the hepatic IP-10 baseline values for HCV chronic animals were similar to the IFNα induced level in uninfected and HBV infected animals (Fig. 4, black bars). The PBMC IP-10 baseline levels were similar for infected and uninfected animals and were highly induced by IFNα in both infected and uninfected animals (Fig. 4, gray bars).

To better illustrate the elevated baseline levels of ISGs in the liver of HCV chronically infected chimpanzees, the microarray data were examined as signal intensity rather than fold change from baseline. A set of 822 genes were identified as significantly induced by IFNα using ANOVA (signal log ratio ≥ 1 = fold change of 2; and p ≤ 0.02) and PAM clustering (Supplement

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 5

Table 2). This analysis revealed that total ISG response followed the same trend as IP-10. In Fig. 5, a heat map illustrates the results from this analysis with one dimensional hierarchical

NIH-PA Author Manuscript NIH-PA Author Manuscriptclustering. NIH-PA Author Manuscript In uninfected animals, most genes are induced at 4 and 8 hr post-IFNα (shift from green to red/black) and are down regulated by 24hr (shift back to green). In contrast, most genes were already up-regulated in the chronically infected animals and were either not further induced by IFNα or were minimally induced. The trend for elevated baseline values for ISG transcripts in the liver of HCV infected animals is further illustrated when the selected set of the most highly induced ISGs from Table 1 were subjected to the same type of analysis (Fig. 6). Although only one HBV infected animal was available for this study, comparison of the baseline values for ISG transcripts in the liver of uninfected animals and the HBV infected animal failed to detect an significant increase in ISG expression due to HBV infection (data not shown) which is consistent with previous studies on HBV infection in chimpanzees 20.

The relationship of the total gene profiles from each sample type can best be visualized using a hierarchical cluster analysis on the entire gene set (Fig. 7). As expected, liver and PBMC samples cluster separately, independent of HCV infection and IFNα dosing, due to their divergent transcriptomes. The uninfected (shaded area) and chronic liver samples cluster separately from each other due to baseline ISG induction in the chronic liver, while this is not true for PBMC samples, where the chronic and uninfected pretreatment samples cluster together. For the uninfected liver (shaded area), the 0 and 24 hr samples cluster separately from 4 and 8 hr post-IFNα dosing, due to the induced ISGs at 4 and 8 hr and the down-regulation to baseline levels by 24 hr. For HCV infected liver, little difference is observed in the pre- and post-treatment samples; the 4 and 8 hr post-IFNα samples do not cluster together.

Discussion In this study, we have demonstrated that the liver of HCV infected chimpanzees is non- responsive to exogenous IFNα. Previous studies with human IFNα in HCV chronically infected chimpanzees failed to demonstrate an antiviral response 18 (unpublished data, REL). These studies included conventional IFNα with ribavirin for 28 days and the use of adenovirus-based gene therapy 17,18. The lack of antiviral response was assumed to be due to the use of human IFNα. However, our recent studies comparing chimpanzee and human IFNα in uninfected chimpanzees demonstrated that the two forms of IFNα were essentially equivalent with regard to induction of ISGs in the liver and PBMC 16. Thus, additional studies using peg-IFNα were warranted in the hopes of developing a chimpanzee model for examination of combined therapies with newly developed antivirals. Although peg-IFNα treatment of HCV infected chimpanzees provided no antiviral efficacy, important aspects of the reason for lack of antiviral response were revealed.

The most notable change in hepatic gene expression during acute and chronic HCV infection in chimpanzees is the up-regulation of ISGs 6–8, suggestive of an ongoing type I IFN or dsRNA response. Similar findings have been observed in human liver during HCV chronic infection 9–11,21. During acute infection in chimpanzees, the level of ISG transcript induction in the liver clearly parallels the increase and decrease of viral RNA in the serum and liver 7,8,22. However, in the analysis of ten HCV chronically infected chimpanzees with viral loads differing over 1000-fold (104 to 107 ge/ml) no correlation was observed in the hepatic ISG transcript levels and the viral load suggesting that this response maybe maximally induced even at relatively low levels of viral replication 6. The stimulus for the ISG induction during HCV infection has not been unambiguously defined and may be multifactorial. An increase in type 1 IFN transcripts was not detectable by microarray; however, if a small fraction of cells are producing IFN, the dilution of the transcript by total liver RNA may render detection of an increase in IFN transcripts unlikely. Viral RNA can interact with a number of cellular proteins to trigger the innate response, most notably TLR3, RIGI and MDA5. The elevated hepatic ISG

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 6

levels during HCV chronic infection occur despite the capacity of the NS3/4a protease to block activation of IRF3 and secretion of type I IFN by the RIGI, MDA5 and TLR3 pathways 23– 25

NIH-PA Author Manuscript NIH-PA Author Manuscript. Newly NIH-PA Author Manuscript infected hepatocytes cells may produce type I IFN until sufficient viral protein is available to block these pathways. In addition, the response of dendritic cells and macrophages to dsRNA and the resulting production of type I IFN presumably would not be subject to NS3 inhibition.

Recently, a correlation was observed between sustained virologic response to IFNα and reduced pretreatment levels for some hepatic ISG transcripts 11. Of the eight genes showing more than 90% correlation with treatment outcome, five were ISGs. Lower pretreatment serum levels of IP-10 have also been found to correlate with treatment outcome 26–29. The levels of IP-10 decrease further during therapy 27,28 which suggests that the liver and PBMC are no longer responding to the exogenous IFNα, which is consistent with our observations that the transcriptional response to IFNα is down-regulated after the initial burst of transcription despite the continued presence of high levels of circulating IFNα (Fig. 1) 16. The decrease in IP-10 levels during IFNα therapy in responders also implies that the original stimulus for IP-10 induction, presumably a dsRNA response, is declining.

At this time, no data are available in humans on the hepatic ISG transcript levels after IFNα dosing, due to the difficultly in obtaining multiple biopsies in humans. Our data on the rapid up-regulation and subsequent down-regulation of the ISG response in chimpanzees following IFNα dosing 16 suggests that studies in humans will be further complicated by the difficulty in obtaining biopsies during the narrow time frame of maximum ISG induction. Currently, the post-dosing ISG response in humans has only been examined in PBMC. An increased ISG response was noted in responders using PMBC exposed in vitro to IFNα 12 and in PBMC taken during treatment 30; however, unlike the liver, no relationship between ISG baseline values and response to therapy was observed. Some differences in the response of the liver and PBMC might be expected, since HCV primarily replicates in the liver. Influences of viral replication on the IFNα response may be confined to the liver, while the response in PBMC may reflect individual genetic differences that precede HCV infection.

The mechanism for the lack of hepatic response to IFNα in HCV infected chimpanzees is not known, but it is possible that during chronic HCV infection the hepatic ISG response is already maximally induced. In a study of ten chronically infected chimpanzees with over 1000-fold difference in viral load, the level of hepatic ISG response did not correlate with viral load and was similar in all animals 6, suggesting a possible saturation of the response to the virus. The persistence of the virus in the presence of an elevated hepatic ISG response suggests that the endogenous ISG response may have limited antiviral activity. However, an alternative view would be that the endogenous ISG response is functioning appropriately in limiting replication and spread of the virus. Determination of the percentage of infected hepatocytes has been problematic and the results varied. Strand specific in situ hybridization has detected negative strand RNA in up to 40% of cells 31, although the exact percentage of positive cells varied between biopsies and different fields of the same biopsy. We have used the level of viral RNA in the liver as an indirect measurement of the level of infection. Based on our assumptions, on average less than 10% of cells were infected during acute and chronic infections, while the range of hepatic viral RNA in different animals suggest that 0.1 to 30% of hepatocytes may be infected 6,16. Thus, our estimations are not inconsistent with in situ hybridization data and would allow for up to 100% infection of hepatocytes in individuals with very high viremia. A low percentage of infected hepatocytes is also consistent with calculations on the total daily production of virus in humans. Assuming that the liver contains 5 × 1011 hepatocytes and produces 1012 HCV particles per day 13, only 2 particles per cell need be produced per day. However, at best, the endogenous ISG response only suppresses replication, and thus there may be qualitative and quantitative differences between the ISG response to HCV infection

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 7

(endogenous response) and that induced by exogenous IFNα in sustained responders. Presumably, humans that respond to therapy have a lower endogenous hepatic ISG response

NIH-PA Author Manuscript NIH-PA Author Manuscriptand NIH-PA Author Manuscript an increased response to exogenous IFNα. The recent finding that nonresponders have higher baseline levels for some hepatic ISGs 11 suggests a possible correlation between nonresponders in the human population and chimpanzees, both of which have elevated hepatic baseline ISG levels.

A number of factors are involved in negative regulation of the IFNα response and our studies in uninfected chimpanzees demonstrated that negative regulation is rapidly invoked, with the ISG response being down-regulated within 8–24 hr of peg-IFNα dosing 16. The transcriptional inhibitors involved in down-regulation of the IFN response include: IRF2, IRF4 (ICSAT), IRF8 (ICSBP), and STAT1β. IRF2 was up-regulated in uninfected chimpanzee liver, PBMC and primary hepatocytes 16 following treatment with IFNα. STAT1β was up-regulated in uninfected chimpanzee liver following IFNα dosing and during acute and chronic HCV infection 6,7. In addition, the proteins involved in signal transduction are subject to post- translational regulation by ubiquitination (suppressor of cytokine signaling; SOCS), sumoylation (protein inhibitor of IFN alpha signaling; PIAS), ISGylation and tyrosine- dephosphorylation. Following IFNα treatment, SOCS3, was up-regulated in both infected and uninfected liver, but SOCS1 was significantly up-regulated only in infected liver.

A precise understanding of the mechanism for the lack of response to exogenous IFNα may require detailed studies in the chimpanzee model, and analysis of the IFNα hepatic response in null responders in the human population will be required to determine if the chimpanzee actually mimics this response in humans.

Supplementary Material Refer to Web version on PubMed Central for supplementary material.

Acknowledgements The authors would like Dr. David Thomas, Johns Hopkins University, for the kind gift of peg-IFNα.

Supported by grants from the National Institutes of Health (R21 DK066755, U19 AI40035 and a pilot grant from P51 RR13986).

Abbreviations IFNα Interferon-alpha ISG interferon stimulated gene peg pegylated HCV hepatitis C virus

References 1. Alter HJ, Seeff LB. Recovery, persistence, and sequelae in hepatitis C virus infection: a perspective on long-term outcome. Semin Liver Dis 2005;20:17–35. [PubMed: 10895429] 2. Thomas D, Seeff LB. Natural history of hepatitis C. Clin Liver Dis 2005;9:383–398. [PubMed: 16023972]

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 8

3. Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, et al. Peginterferon alfa-2b plus ribavirin compared with interferon alfa-2b plus ribavirin for initial treatmentof chronic hepatitis C: a randomised trial. The Lancet 2001;358:958–965. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 4. Fried MW, Shiffman ML, Reddy KR, Smith C, Marinos G, Goncales FL, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002;347(13):975–982. [PubMed: 12324553] 5. Feld J, Hoofnagle JH. Mechanism of action of interferon and ribavirin in treatment of hepatitis C. Nature 2005;436:967–972. [PubMed: 16107837] 6. Bigger CB, Guerra B, Brasky KM, Hubbard G, Beard MR, Luxon BA, et al. Intrahepatic gene expression during chronic hepatitis C virus infection in chimpanzees. J Virol 2004;78:13779–13792. [PubMed: 15564486] 7. Bigger CB, Brasky KM, Lanford RE. DNA microarray analysis of chimpanzee liver during acute resolving hepatitis C virus infection. J Virol 2001;75(15):7059–7066. [PubMed: 11435586] 8. Su AI, Pezacki JP, Wodicka L, Brideau AD, Supekova L, Thimme R, et al. Genomic analysis of the host reponse to hepatitis C virus infection. Proc Natl Acad Sci USA 2002;99:15669–15674. [PubMed: 12441396] 9. Helbig KJ, Lau DTY, Semendric L, Harley HAJ, Beard MR. Analysis of ISG expression in chronic hepatitis C identifies viperin as a potential antiviral effector. Hepatology 2005;42(3):702–710. [PubMed: 16108059] 10. Smith MW, Yue ZN, Korth MJ, Do HA, Boix L, Fausto N, et al. Hepatitis C virus and liver disease: global transcriptional profiling and identification of potential markers. Hepatology 2003;38:1458– 1467. [PubMed: 14647057] 11. Chen LM, Borozan I, Feld J, Sun J, Tannis LL, Coltescu C, et al. Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection. Gastroenterology 2005;128(5):1437–1444. [PubMed: 15887125] 12. He XS, Ji X, Hale MB, Cheung R, Ahmed A, Guo Y, et al. Global transcriptional response to interferon is a determinant of HCV treatment outcome and is modified by race. Hepatology 2006;44:352–359. [PubMed: 16871572] 13. Neumann AU, Lam NP, Dahari H, Gretch DR, Wiley TE, Layden TJ, et al. Hepatitis C viral dynamics in vivo and the antiviral efficacy of interferon-α therapy. Science 1998;282(5386):103–107. [PubMed: 9756471] 14. Perelson AS, Herrmann E, Micol F, Zeuzem S. New kinetic models for the hepatitis C virus. Hepatology 2005;42:749–754. [PubMed: 16175615] 15. Lam NP. Dose-dependent acute clearance of hepatitis C genotype 1 virus with interferon alfa. Hepatology 1997;26(1):226–231. [PubMed: 9214474] 16. Lanford RE, Guerra B, Lee H, Chavez D, Brasky K, Bigger CB. Genomic response to interferon- alpha in chimpanzees: Implications of rapid downregulation for hepatitis C kinetics. Hepatology 2006;43:961–972. [PubMed: 16628626] 17. Demers GW, Sugarman BJ, Beltran JC, Westreich LN, Iqbal Ahmed CM, Lau JY, et al. Interferon- a2b secretion by adenovirus-mediated gene delivery in rat, rabbit, and chimpanzee results in similar pharmacokinetic profiles. Toxicol Appl Pharmacol 2002;180:36–42. [PubMed: 11922775] 18. Lanford RE, Guerra B, Demers GW, Chang S, Sugarman BJ, Maneval DC, et al. Gene therapy for chronic hepatitis C based on adenoviral-mediated transfer of interferon-2b gene study based on the chimpanzee model. Hepatology Abtracts to AASLD Meeting. 2000 19. Lanford RE, Guerra B, Lee H, Averett DR, Pfeiffer B, Chavez D, et al. Antiviral effect and virus- host interactions in response to alpha interferon, gamma interferon, poly(I)-poly(C), tumor necrosis factor alpha, and ribavirin in hepatitis C virus subgenomic replicons. J Virol 2003;77(2):1092–1104. [PubMed: 12502825] 20. Wieland S, Thimme R, Purcell RH, Chisari FV. Genomic analysis of the host response to hepatitis B virus infection. Proceedings of the National Academy of Sciences 2004;101(17):6669–6674. 21. MacQuillan GCMCRW, Jeffrey GP, Allan JE. Upregulation of endogenous intrahepatic interferon stimulated genes during chronic hepatitis C virus infection. J Med Virol 2003;70:219–227. [PubMed: 12696108]

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 9

22. Thimme R, Bukh J, Spangenberg HC, Wieland S, Pemberton J, Steiger C, et al. Viral and immunological determinants of hepatitis C virus clearance, persistence, and disease. Proc Nat Acad Sci Usa 2002;99(24):15661–15668. [PubMed: 12441397] NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript 23. Foy E, Li K, Sumpter R, Loo YM, Johnson CL, Wang CF, et al. Control of antiviral defenses through hepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc Natl Acad Sci USA 2005;102(8):2986–2991. [PubMed: 15710892] 24. Foy E, Li K, Wang CF, Sumpter R, Ikeda M, Lemon SM, et al. Regulation of interferon regulatory factor-3 by the hepatitis C virus serine protease. Science 2003;300(5622):1145–1148. [PubMed: 12702807] 25. Li K, Foy E, Ferreon JC, Nakamura M, Ferreon ACM, Ikeda M, et al. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci USA 2005;102(8):2992–2997. [PubMed: 15710891] 26. Lagging M, Romero A, Westin J, Norkrans G, Dhillon A, Pawlotsky J, et al. IP-10 Predicts Viral Response and Therapeutic Outcome in Difficult-to-Treat Patients With HCV Genotype 1 Infection. Hepatology 2006;44:1617–1625. [PubMed: 17133471] 27. Diago M, Castellano G, Garcia-Samaniego J, Perez C, Fernandez I, Romero M, et al. Association of pretreatment serum interferon gamma inducible protein 10 levels with sustained virological response to peginterferon plus ribavirin therapy in genotype 1 infected patients with chronic hepatitis C. Gut 2006;55(3):374–379. [PubMed: 16150856] 28. Butera D, Marukian S, Iwamaye AE, Hembrador E, Chambers TJ, Di Bisceglie AM, et al. Plasma chemokine levels correlate with the outcome of antiviral therapy in patients with hepatitis C. Blood 2005;106(4):1175–1182. [PubMed: 15860662] 29. Romero AI, Lagging M, Westin J, Dhillon AP, Dustin LB, Pawlotsky JM, et al. Interferon (IFN)- [gamma]-Inducible Protein-10: Association with Histological Results, Viral Kinetics, and Outcome during Treatment with Pegylated IFN-[alpha]2a and Ribavirin for Chronic Hepatitis C Virus Infection. J Infect Dis 2006;194(7):895–903. [PubMed: 16960776] 30. Luo SY, Cassidy W, Jeffers L, Reddy KR, Bruno C, Howell CD. Interferon-stimulated gene expression in black and white hepatitis C patients during peginterferon alfa-2a combination therapy. Clinical Gastroenterology and Hepatology 2005;3(5):499–506. [PubMed: 15880320] 31. Pal S, Shuhart MC, Thomassen L, Emerson SS, Su T, Feuerborn N, et al. Intrahepatic Hepatitis C Virus Replication Correlates with Chronic Hepatitis C Disease Severity In Vivo. The Journal of Virology 2006;80(5):2280–2290.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 10 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 1. Lack of antiviral response to peg-IFNα in HCV infected chimpanzees. Three chimpanzees were dosed weekly with peg-IFNα2a and monitored for reductions in serum viral RNA levels by quantitative RT-PCR. The genotype 1a animal was dosed for 12 weeks, while the genotype 1b animal was stopped at 8 weeks of therapy due to unrelated health issues. The genotype 3 animal was given a single dose of peg-IFNα in order to examine ISG response and was monitored for 14 days.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 11 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 2. Lack of transcriptional induction of IP-10 in the liver of HCV chronic chimpanzees. Total cellular RNA was purified from liver and PBMCs at 0, 4, 8 and 24 hr post-inoculation of IFNα. The levels of IP-10 (CXCL10) transcripts were determined by quantitative, real-time RT-PCR and were expressed as the fold change in comparison to time 0, pre-treatment. The response of uninfected chimpanzees was contrasted to HCV and HBV chronically infected chimpanzees. Serum was analyzed by ELISA for IP-10 protein levels (R&D Systems; Minneapolis, MN) and expressed as fold increase from pretreatment levels. The data set from uninfected chimpanzees has been previously published 16.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 12 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 3. Weekly IFNα treatment fails to induce hepatic IP-10 transcripts. HCV chronically infected chimpanzee 4 × 0081 (genotype 1b) was treated with 5 million IU peg-IFN weekly for 8 weeks. Microarray analysis was performed on liver and PBMC at two time points prior to IFNα treatment (-3 and -2 weeks) and at 4, 8 and 24 hr after the first IFNα inoculation and at 1, 2, 4 and 8 weeks of treatment. The values represent the fold increase in IP-10 transcripts from the array data in comparison to the average of both pretreatment samples. No increase in hepatic IP-10 transcripts occurred even with repeated IFNα dosing and ISGs in the PBMC remained down regulated after the initial burst of transcription.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 13 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 4. Comparison of baseline and induced IP-10 levels in infected and uninfected chimpanzees. Baseline (−) and 4 hr post-IFNα (+) samples from liver (black bars) and PBMC (grey bars) were examined for IP-10 transcript levels by quantitative RT-PCR. Values from three uninfected, three HCV infected and one HBV infected chimpanzee are shown. Values are expressed as copies of transcript per microgram of total cell RNA, rather than as fold change (Fig. 1) to illustrate differences in hepatic baseline values of chronic and uninfected animals. The baseline values for hepatic IP-10 transcripts in HCV infected animals was equivalent to the IFNα-induced levels in uninfected animals.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 14 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 5. Heatmap of IFNα induced liver gene expression. A data set of 822 genes were obtained by ANOVA (SLR ≥ 1, p ≤ 0.02) and PAM clustering (Supplement Table 2). The heatmap compares the signal intensity between the pretreatment baseline liver samples and the 4, 8 and 24 hr post-IFNα dosing samples from three uninfected (each with two independent dosing studies) and three HCV infected chimpanzees. The average value for all samples for a given gene are assigned the color black. Red and green intensity indicate positive and negative changes from the average values. One dimensional hierarchical clustering analysis with gene symbols is shown to the left. In uninfected animals, this gene set is induced at 4 and 8 hr post-

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 15

IFNα dosing (green to red), while in infected animals the genes are induced at baseline and no or minimal increase occurs post-IFNα dosing. NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 16 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig. 6. Heatmap of selected ISGs. The heatmap compares the signal intensity between the pretreatment baseline liver samples and the 4, 8 and 24 hr post-IFNα dosing samples from three uninfected (each with two independent dosing studies) and three HCV infected chimpanzees. The average value for all samples for a given gene are assigned the color black. Red and green intensity indicate positive and negative changes from the average values. One dimensional hierarchical clustering analysis with gene symbols is shown to the left. The genes for this analysis are the same as shown in Table 1 and were selected from 500 of the most highly up-regulated ISGs at 4 hr in uninfected animals (Supplement Table 1).

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 17 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

Fig 7. Hierarchical clustering of the total gene sets. One dimensional hierarchical clustering was performed (Gene Sifter) on the total gene sets (rather than individual genes as shown in Fig. 5 and 6) to determine the interrelationship of the different samples. Samples included all genes either positively or negatively regulated by ≥ 2.0 fold and p ≤ 0.02 (Affymetrix, DMT 2.0) at 4, 8 and 24 hr post-IFNα dosing for liver and PBMC derived from three uninfected (two independent experiments each) and three HCV infected animals. Note that liver and PBMC samples cluster independently, as do uninfected (shaded area) and chronic liver, but that uninfected (shaded area) and chronic PBMC clustering is dependent on IFNα dosing.

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 18 - - - 6 7 8 5 7 8 5 5 48 69 13 24 22 29 10 24 10 16 22 13 13 18 14 14 10 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Liver HBV Infected - - - 6 5 4 6 5 7 6 5 8 3 6 5 4 5 5 4 6 4 2 4 8 10 37 11 32 PBMC α HCV Infected ------3 6 7 10 Liver Average Fold Change at 4 hr Post-pegIFN 0.02. ≤ Table 1 - - 8 7 4 9 8 5 8 9 6 7 5 3 8 18 10 12 14 19 31 19 14 12 16 14 36 10 2.0 and p PBMC ≥ for three uninfected (each with two independent dosing studies), HCV infected and a single HBV chimpanzee. The α Uninfected 9 8 8 7 7 6 6 4 47 34 30 29 28 26 24 24 20 20 19 18 16 15 14 14 13 13 12 11 Liver Alias IFI54, G10P2 I-TAC RIG-G, CIG-49 C1orf29 IP-10 cig5, viperin IFI56 MCP-2 CIS3, Cish3 RIG-I MDA5 OIASI, IFI-4 MMTRA1B CEM15, MDS019 TRAIL, Apo-2L PTX1 indicates that the gene did not meet our criteria for inclusion; average fold change Gene Symbol IFIT2 CXCL11 HERC6 OAS3 ISG20 IFIT3 IFI44L CXCL10 RSAD2 ISG15 MX1 IFIT1 CCL8 IRF7 SOCS3 DDX58 IFIH1 INDO OAS1 STAT1 OASL ISG20 GBP1 PLSCR1 APOBEC3G LGP2 TNFSF10 CRP * The values are the average fold-change between 0 and 4 hr post-IFN ISGs were selected from the 500 most highly induced genes at 4 hr in uninfected animals. Lack of ISG Induction in HCV Infected Liver

Hepatology. Author manuscript; available in PMC 2008 May 19. Lanford et al. Page 19 ------3 8 2 6 4 3 3 3 4 2 4 4 4 2 48 13 18 10 NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript Liver HBV Infected ------6 4 3 4 4 4 5 4 2 8 10 11 11 PBMC HCV Infected . Of the 500 genes up-regulated in uninfected liver, only 24 were upregulated α 3 6 7 3 2 5 3 5 3 2 3 3 3 3 3 2 3 2 3 3 2 2 3 10 Liver 0.02. ≤ Table 2 2.0 and p ≥ ------4 9 5 4 2 7 5 3 4 2 6 18 14 15 PBMC Uninfected - - - 6 5 5 5 4 4 4 3 3 3 3 3 3 3 3 2 2 47 16 14 11 Liver α Alias IFI54, G10P2 MCP-2 CIS3, Cish3 C7orf6 IL1RA, IL1F3 ADRP MDR2/3 LRG IFI53 TNFSF20 PROL2 CPT1 MT1 onzin, C15 CD64 (-) indicates that the gene did not meet our criteria for inclusion; average fold change Gene Symbol IFIT2 CCL8 SOCS3 SAMD9L IL1RN ADFP ABCB4 SECTM1 CRP LRG1 PBEF1 ABLIM3 WARS MX2 TNFSF13B APOL6 PNRC1 CPT1A STAT3 SLC25A30 MT1F SOCS1 PLAC8 FCGR1A * In contrast to Table 1, this table contains all ISGs that were up-regulated in the liver of HCV infected chimpanzees by peg-IFN in HCV chronically infected liver. The values are the average fold-change between 0 and 4 Average Fold Change at 4hr Post-pegIFN

Hepatology. Author manuscript; available in PMC 2008 May 19.