HEPATOLOGY, VOL. 68, NO. 3, 2018 VIRAL HEPATITIS Impact of Interferon Lambda 4 Genotype on Interferon-Stimulated Gene Expression During Direct-Acting Antiviral Therapy for Hepatitis C 1* 1* 1-3 2 4 1 Narayan Ramamurthy , Emanuele Marchi, M. Azim Ansari, Vincent Pedergnana, Angela Mclean, Emma Hudson, 2 2 1 1 STOP HCV consortium, Rory Bowden, Chris C.A. Spencer, Eleanor Barnes, and Paul Klenerman New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16-week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon- stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non-CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 68:859-871). epatitis C virus (HCV) infects approxi- Interferon (IFN) and ribavirin combination ther- mately 180 million people globally. An esti- apy was, for many years, the mainstay of treatment for (4) mated 20% of patients are able to control patients with chronic hepatitis C (CHC). However, Hthe infection spontaneously after exposure—that regimes using combined pegylated interferon (Peg-IFN) is, through a combination of innate and adaptive and ribavirin therapy result in only modest sustained (1,2) responses. In the remainder, chronic hepatitis virological response (SVR) rates and are associated with leads to HCV-related cirrhosis in around 15%-30% significant adverse effects. Recently, many new directly of patients and 1%-4% of patients with cirrhosis acting antiviral (DAA) therapies have been developed, (3) develop hepatocellular carcinoma. The develop- which provide significantly higher cure rates in different ment of treatments for HCV infection has therefore patient groups. Such drugs may be used in combination (5) been an important priority in recent years and huge with IFN (e.g., the protease inhibitor, telapravir) or, advances have been made. most recently, in IFN-free regimens, using combinations Abbreviations: DAA, direct-acting antiviral; GSEA, gene set enrichment analysis; HCV, hepatitis C virus; IFN, interferon; IFNL4, interferon lambda 4; ISG, interferon-stimulated gene; NS, nonstructural protein; OR, odds ratio; Peg-IFN, pegylated interferon; RNAseq, RNA sequencing; SNP, single-nucleotide polymorphism; SVR, sustained virological response. Received November 1, 2017; accepted March 8, 2018. Additional Supporting Information may be found at onlinelibrary.wiley.com/10.1002/hep.29877/suppinfo. This work was funded by a grant from the Medical Research Council (MRC) (MR/K01532X/1; to the STOP-HCV Consortium). The work was supported by the Wellcome Trust Fund WT109965MA, NIH (U19AI082630), and NIHR Senior Fellowship to P.K. and the NIHR Biomedical Research Centre, Oxford. *These authors contributed equally to the work. © 2018 The Authors. HEPATOLOGY published by Wiley Periodicals, Inc. on behalf of American Association for the Study of Liver Diseases. This is an open access article under the terms of the Creative Commons Attribution License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. View this article online at wileyonlinelibrary.com. DOI 10.1002/hep.29877 Potential conflict of interest: Nothing to report. 859 RAMAMURTHY, MARCHI, ET AL. HEPATOLOGY, September 2018 of inhibitors of nonstructural protein (NS) 3, NS5A, a greater than 2-fold increase in the likelihood of (6) (12) and NS5B, with or without ribavirin. achieving SVR compared to the TT genotype. The HCV is genetically highly diverse and has evolved CC genotype was also associated with changes in the (12,16-19) into seven major genotypes and multiple subtypes. dynamics of viral control in genotypes 1 and 3. Response rates to both the IFN-based and, more The mechanism by which the IFNL4 locus affects recently, the DAA based regimes are genotype depen- hepatitis C outcomes has not been fully elucidated. dent. In particular, HCV genotype 3 (one of the most Although the CC genotype is associated with bet- common in the UK) has been associated with lower ter immune control and response to therapy, it has response rates to several DAA regimens, compared to also been associated with higher viral load than the (7-9) (13-16,18) genotype 1 infection. Defining optimal treatment non-CC genotypes. This elevated viral load for such patient groups is an issue that was addressed in is associated with a reduced host innate immune (10) the recent BOSON trial, which tested the efficacy response during chronic infection. Hepatic interfer- of the NS5B (polymerase) inhibitor, sofosbuvir, on 594 on-stimulated genes (ISGs) are also up-regulated in patients, mainly genotype 3, with a small subgroup of CHC patients resistant to antiviral therapy and higher (20) genotype 2 infections, and including patients with and in patients with the non-CC with IFNL4 genotype. without cirrhosis and with or without previous treat- These associations may be attributed to modulation of ment. Patients were divided into three groups, given 16 the IFNL4 gene itself or of linked genes. SNPs in the or 24 weeks of sofosbuvir with ribavirin or 12 weeks of IFNL4 region may influence expression, transcription sofosbuvir with Peg-IFN and ribavirin. A key result of factor binding, or mRNA stability, but overall appear (21) the study was that patients with cirrhosis who received to reduce IFNL3 gene expression. The C allele at 16 weeks of sofosbuvir and ribavirin had the lowest SNP rs12979860 tags a dinucleotide insertion poly- (22) rates of SVR—at 51%, compared to >80% attaining morphism (rs368234815-TT) that prevents IFNL4 (23) cure in other treatment groups. This prompted us to expression. Using human genotyping arrays com- ask: In such a setting (and in the absence of exoge- bined with whole-genome viral sequencing, we recently neous interferon), what is the role of the host immune reported viral “footprints” of host IFNL4 genotypes response and host genetics in defining outcome? (CC vs. non-CC at rs12979860). We identified the Host genetics play an important role in both the association of 11 sites in the HCV polyprotein with spontaneous control of HCV and response to therapy. IFNL4 genotypes, including one (serine vs. nonserine In particular, a single-nucleotide polymorphism (SNP; at position 2414 in NS5A) that was also associated (24) rs12979860) in the interferon lambda 4 (IFNL4) with viral load in vivo and viral fitness in vitro. (11-13) gene has been associated with higher probability of Based on these data, we hypothesized that host spontaneous clearance of the virus and of SVR follow- IFNL4 genotype might particularly affect viral outcome (12,14,15) ing Peg-IFN and ribavirin combination therapy. in patients with cirrhosis treated with the shortest proto- Those carrying the CC genotype at rs12979860 had col (16 weeks). We aimed to test whether transcriptional ARTICLE INFORMATION: 1 From the Peter Medawar Building for Pathogen Research and Translational Gastroeneterology Unit, Nuffield Department of 2 Medicine, University of Oxford, Oxford, United Kingdom; Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, 3 4 United Kingdom; Oxford Martin School, University of Oxford, Oxford, United Kingdom; Department of Zoology, University of Oxford, Oxford, United Kingdom. ADDRESS CORRESPONDENCE AND REPRINT REQUESTS TO: Paul Klenerman, M.R.C.P., FRCPath Narayan Ramamurthy, Ph.D. Peter Medawar Building for Pathogen Research and Translational Peter Medawar Building for Pathogen Research Gastroeneterology Unit, Nuffield Department of Medicine Nuffield Department of Medicine University of Oxford University of Oxford South Parks Road South Parks Road Oxford, OX1 3SY, United Kingdom Oxford, OX1 3SY, United Kingdom E-mail: [email protected] E-mail: [email protected] Tel: +44 1865 281885 Tel: +44 1865 281885 or 860 HEPATOLOGY, Vol. 68, No. 3, 2018 RAMAMURTHY, MARCHI, ET AL. changes in the host response were detectable in blood BIOINFORMATIC ANALYSIS and linked to outcome. We therefore analyzed the host response to HCV by microarray analysis of gene expres- Illumina bead chip output files were processed and analyzed using the R package (http://www.r-project. sion in whole blood over time in a subset of patients from (25) the BOSON study. We make a report of a time-depen- org), wherein samples were normalized and gene dent molecular signature detectable in blood associated expression levels calculated to determine statistical sig- with IFNL4 genotypes, which, in itself, is associated with nificance. Differential gene expression between different SVR in patients with cirrhosis. We provide a model that groups was assessed by generating relevant contrasts cor- explains the paradoxical observations related to HCV responding to the possible comparisons. Statistical testing and its interaction with the IFNL4 region. was performed using the Linear Models for Microarray Analysis (LIMMA) package (http://bioconductor.org/ (26) packages/release/bioc/html/limma.html). Raw P val- Materials and Methods ues were corrected for multiple testing using the false discovery rate controlling procedure of Benjamini and (27) STUDY DESIGN, PATIENTS, AND Hochberg. Following this correction, adjusted P val- ETHICS ues <0.01 were considered significant. Pathway over-rep- resentation analysis and plotting was conducted using This study was part of the study described ear- (28) (10,22) Reactome PA. We also used the CAMERA pack- lier.