Achieving sustained virologic response after interferon-free hepatitis C virus treatment correlates with hepatic interferon gene expression changes independent of cirrhosis

Abstract

Chronic hepatitis C virus (HCV) infection can now be treated with oral directly acting antiviral agents, either with or without ribavirin (RBV). Virologic relapse after treatment can occur, and in some studies was more common in cirrhotic subjects. We previously observed changes in hepatic immunity during interferon (IFN)-free therapy that correlated with favourable outcome in subjects with early liver disease. Here, we compared changes in endogenous IFN pathways during IFN-free, RBV-free therapy between cirrhotic and noncirrhotic subjects. mRNA and microRNA (miRNA) expression analyses were performed on paired pre- and post-treatment liver biopsies from genotype-1 HCV subjects treated with sofosbuvir/ledipasvir (SOF/LDV) for 12 weeks (n = 4, 3 cirrhotics) or SOF/LDV combined with GS-9669 or GS-9451 for 6 weeks (n = 6, 0 cirrhotics). Nine of ten subjects achieved a sustained virologic response (SVR), while one noncirrhotic subject relapsed. Hepatic IFN-stimulated gene expression decreased with treatment in the liver of all subjects, with no observable impact of cirrhosis. Hepatic gene expression of type III IFNs (IFNL1, IFNL3, IFNL4-ΔG) similarly decreased with treatment, while IFNA2 expression, undetectable in all subjects pretreatment, was detected post-treatment in three subjects who achieved a SVR. Only the subject who relapsed had detectable IFNL4-ΔG expression in post-treatment liver. Other IFNs had no change in gene expression (IFNG, IFNB1, IFNA5) or could not be detected. Although expression of multiple hepatic miRNAs changed with treatment, many miRNAs previously implicated in HCV replication and IFN signalling had unchanged expression. In conclusion, favourable treatment outcome during IFN-free HCV therapy is associated with changes in the host IFN response regardless of cirrhosis.

Keywords: cirrhosis; endogenous interferons; hepatitis C virus; interferon-free therapy; treatment relapse.

Figure 1

Endogenous interferon signalling declines with treatment. Paired liver biopsies from nine subjects (eight sustained virologic response, one relapse) were analysed by microarray gene expression analysis. The top 1% of genes identified by a rank‐order approach were analysed using Ingenuity Pathway Analysis. (a) The top 4 canonical pathways as ranked by P‐value. All had predicted reduced activity. (b) The top 10 significant upregulated and downregulated genes (IFN‐stimulated genes are shown with bold/italicized font) based on fold change. (c) The top significant 10 upregulated and downregulated ‘predicted activation state’ based on z‐score.

Figure 2

IFN‐stimulated gene (ISG) expression is downregulated irrespective of cirrhosis. Select ISGs were chosen to compare relative gene expression in noncirrhotic (shown in black, n = 5) vs cirrhotic subjects (shown in green, n = 3) achieving sustained virologic response. Data from the subject who relapsed (noncirrhotic) are shown in red (n = 1). All shown ISGs had significantly decreased expression by the Wilcoxon paired test (P < 0.05) over the course of treatment with the exception of SOCS3 (NS = not significant), with no detectable difference based on fibrosis stage. Data from pre‐ and post‐treatment liver biopsies are indicated, with individual genes separated by dashed lines.

Figure 3

Hepatic interferon gene expression changes with treatment. Select interferon genes were amplified from liver mRNA before and after treatment. (a) Subjects with detectable IFNA2 increased during treatment while IFNL1 and IFNL4‐ΔG were less frequently detected after treatment. Data are shown as number of subjects with detectable transcript, as defined in the methods, as in many cases transcripts could not be quantified, precluding quantitative statistics. IFNL4 results are shown for six subjects carrying at least one ΔG allele and from six subjects carrying at least one TT allele (Table 1), with allele specific gene expression displayed. SVR subjects are shown in black/blue and the subject who relapsed in red/green. (b) IFNs with quantifiable pre‐ and post‐treatment expression. IFNL3 decreases over the course of treatment while IFNA1/4/6/8/16/17 and IFNL2 were not detectable. Noncirrhotic SVR subjects are shown in black, cirrhotic SVR subjects are shown in green, and the noncirrhotic relapser is shown in red. Statistical analysis for panel B is by Wilcoxon paired test considering all subjects.

Figure 4

Changes in miR‐122 expression over the course of treatment. Shown are RNA‐Seq data derived from analysis of paired liver biopsy RNA using the Illumina TruSeq platform. N = 10 subjects, with three cirrhotic subjects shown in red and one subject who relapsed shown in green. Pre‐ and post‐treatment means with standard error are shown. Statistical analysis is by paired t‐test with a multiple‐test correction, as described in the methods.