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Transcriptomic analysis of the woodchuck model of chronic hepatitis B: Liver gene expression in uninfected, resolved and chronically infected woodchucks

ABSTRACT: The Eastern woodchuck (Marmota monax) is naturally infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to the human hepatitis B virus (HBV). The woodchuck is used as an animal model for studying chronic hepatitis B (CHB) and HBV-associated hepatocellular carcinoma (HCC) in humans, but the lack of sequence information has hitherto precluded functional genomics analysis. To address this major limitation of the model, we report here the sequencing, assembly and annotation of the woodchuck transcriptome, together with the generation of custom woodchuck microarrays. Using this new platform, we characterized the transcriptional response to persistent WHV infection and WHV-induced HCC. This revealed that chronic WHV infection, like HBV, is associated with (i) a limited intrahepatic type I interferon response, (ii) intrahepatic induction of markers associated with T cell exhaustion, (iii) elevated levels of suppressor of cytokine signaling 3 (SOCS3) in the liver, and (iv) intrahepatic accumulation of neutrophils. Underscoring the translational value of the woodchuck model, this study also determined that WHV-induced HCC shares molecular characteristics with a subtype of human HCC with poor prognosis. Conclusion: Our data establish the translational value of the woodchuck model and provide new insights into immune pathways which may play a role either in the persistence of HBV infection or the sequelae of CHB.

ORGANISM(S): Marmota monax

PROVIDER: GSE36533 | GEO | 2012/04/11

SECONDARY ACCESSION(S): PRJNA155579

REPOSITORIES: GEO

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Project description:Background & Aims: New therapies for chronic hepatitis B (CHB) are urgently needed since current treatments rarely lead to cure. We evaluated whether the oral small molecule toll-like receptor (TLR7) agonist GS-9620 could induce durable anti-viral efficacy in woodchucks chronically infected with woodchuck hepatitis virus (WHV), a hepadnavirus closely related to human hepatitis B virus (HBV). Methods: After evaluating the pharmacokinetics, pharmacodynamics and tolerability of oral GS-9620 in uninfected woodchucks, adult woodchucks chronically infected with WHV (n=7 per group) were dosed with GS-9620 or placebo for 4 or 8 weeks with different treatment schedules. Results: GS?9620 treatment induced rapid, marked and sustained reduction in serum viral DNA (mean maximal 6.2 log10 reduction), and hepatic WHV DNA replicative intermediates, WHV cccDNA and WHV RNA, as well as loss of detectable serum WHV surface antigen (WHsAg). GS-9620 treatment also induced a sustained antibody response against WHsAg in a subset of animals. Strikingly, treatment reduced the incidence of hepatocellular carcinoma (HCC) from 71% in the placebo group to 8% in GS-9620-treated woodchucks with sustained viral load reduction. GS-9620 treatment was associated with reversible increases in serum liver enzymes and thrombocytopenia, and induced intrahepatic CD8+ T cell, NK cell, B cell and interferon (IFN) response transcriptional signatures. Conclusions: The data demonstrate that short duration, finite treatment with the oral TLR7 agonist GS-9620 can induce a sustained antiviral response in the woodchuck model of CHB, and support investigation of this compound as a therapeutic approach to attain a functional cure in CHB patients. Liver biopsies were taken at various time points pre-treatment, during treatment and post-treatment from placebo-treated group and GS9620-treated group (5mg/kg QW). Total RNAs were extracted and analyzed by RNA-Seq. The number of animals analyzed at each time-point for each group was as follows: group 1 (placebo) day -14, n=7; day 43, n=7; day 71, n=7; day 120, n=7; day 250, n=7; group 5 (5 mg/kg QW) day -14, n=7; day 23, n=6; day 57, n=5; day 78, n=4; day 250, n=5.

Project description:The woodchuck model of hepatitis B virus (HBV) infection displays many characteristics of human infection and has particular value for characterizing the host immune responses during the development of chronic infection. Using the newly developed custom woodchuck microarray platform, we compared the intrahepatic transcriptional responses of neonatal woodchucks with self-limiting and progressing persistent infection with woodchuck hepatitis virus (WHV). This revealed that WHV does not induce intrahepatic gene expression during the early acute stage of infection (8 weeks), suggesting it is a M-bM-^@M-^\stealthM-bM-^@M-^] virus. At the mid-acute phase of infection (14 weeks), resolution was associated with induction of a prominent cytotoxic T cell signature, with perforin and other markers of immune-mediated cytotolytic response being strongly expressed. Strikingly, this was accompanied by high level expression of PD-1 and various other inhibitory T cell receptors, which likely act to minimize liver damage by cytotoxic T cells during viral clearance. Conversely, self-limiting infection was not associated with a strong interferon-M-NM-1/M-NM-2 (IFN-M-NM-1/M-NM-2) transcriptional response, while the IFN-M-NM-3 signaling response (as measured by expression of CXCL9) in the mid-acute phase was comparable to that in chronically infected adult animals. Nevertheless, viperin and other antiviral genes were differentially expressed during resolving infection, suggesting that a subset of interferon-stimulated genes (ISGs) may play a role in the control of WHV replication. Conclusion: This study identifies new immune pathways associated with the clearance of hepadnavirus infection and reveals novel targets with potential for the treatment of chronic hepatitis B. Neonatal woodchucks of both genders were infected at 3 days of age with the same WHV7P1 inoculum containing 5 x 106 WID50 of WHV strain WHV7-11. Custom microarrays were generated from sequences obtained in transcriptome sequencing of woodchuck liver and PBMCs, and were used to examine liver gene expression in animals which eventually become chronically infected with WHV (8 weeks, n=5; 14 weeks, n=9), animals that eventually resolve WHV infection (8 weeks, n=10;14 weeks, n=7) and uninfected animals (8 weeks, n=5;14 weeks,n=5).

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Transcriptomic analysis of the woodchuck model of chronic hepatitis B: Liver gene expression in uninfected, resolved and chronically infected woodchucks

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