Dysregulation of innate immunity in hepatitis C virus genotype 1 IL28B-unfavorable genotype patients: impaired viral kinetics and therapeutic response

Abstract

Recent studies have shown that a single-nucleotide polymorphism upstream of the interleukin-28B (IL28B) gene plays a major role in predicting therapeutic response in hepatitis C virus (HCV)-infected patients treated with pegylated interferon (PEG-IFN)/ribavirin. We sought to investigate the mechanism of the IL28B polymorphism, specifically as it relates to early HCV viral kinetics, IFN pharmacokinetics, IFN pharmacodynamics, and gene expression profiles. Two prospective cohorts (human immunodeficiency virus [HIV]/HCV-coinfected and HCV-monoinfected) completing treatment with IFN/ribavirin were enrolled. Patients were genotyped at the polymorphic site rs12979860. In the HIV/HCV cohort, frequent serum sampling was completed for HCV RNA and IFN levels. DNA microarray of peripheral blood mononuclear cells and individual expression of IFN-stimulated genes (ISGs) were quantified on IFN therapy. The IL28B-favorable (CC) genotype was associated with improved therapeutic response compared with unfavorable (CT or TT) genotypes. Patients with a favorable genotype had greater first- and second-phase viral kinetics (P = 0.004 and P = 0.036, respectively), IFN maximum antiviral efficiency (P = 0.007) and infected cell death loss (P = 0.009) compared with unfavorable genotypes. Functional annotation analysis of DNA microarray data was consistent with depressed innate immune function, particularly of natural killer cells, from patients with unfavorable genotypes (P <0.004). Induction of innate immunity genes was also lower in unfavorable genotypes. ISG expression at baseline and induction with IFN was independent of IL28B genotype.

Conclusion: Carriers of the IL28B-favorable genotype were more likely to have superior innate immune response to IFN therapy compared with unfavorable genotypes, suggesting that the unfavorable genotype has aberrant baseline induction of innate immune response pathways resulting in impaired virologic response. IL28B genotype is associated with more rapid viral kinetics and improved treatment response outcomes independent of ISG expression.

Conflict of interest statement

Conflict of Interest Statement

Eva Herrmann serves as a research consultant to Gilead Sciences, Roche Pharma and Novartis. John McHutchison and Alex Thompson are co-investigators on the IL28B rs12979860 patent. None of the other authors have any conflicts of interest to report.

Copyright © 2012 American Association for the Study of Liver Diseases.

Figures

Figure 1. Modeling

A: Pharmacokinetics AUC of fitted PK function for the first week and the first 4 weeks are not significantly different by IL28B genotype. B: Pharmacodynamics Maximum efficiency (which determines first phase viral decay) is higher for CC favorable genotype compared to CT/TT unfavorable genotype (P=0.007). Mean efficiency is also higher for the CC favorable genotype (P=0.061), although this did not reach statistical significance. C: Viral Kinetics First phase (24 hours) and second phase (after day 2) viral decay is greater for the CC favorable genotype. Phases of viral decay are a function of maximum efficiency and delta (infected cell loss), respectively.

Figure 1. Modeling

A: Pharmacokinetics AUC of fitted PK function for the first week and the first 4 weeks are not significantly different by IL28B genotype. B: Pharmacodynamics Maximum efficiency (which determines first phase viral decay) is higher for CC favorable genotype compared to CT/TT unfavorable genotype (P=0.007). Mean efficiency is also higher for the CC favorable genotype (P=0.061), although this did not reach statistical significance. C: Viral Kinetics First phase (24 hours) and second phase (after day 2) viral decay is greater for the CC favorable genotype. Phases of viral decay are a function of maximum efficiency and delta (infected cell loss), respectively.

Figure 1. Modeling

A: Pharmacokinetics AUC of fitted PK function for the first week and the first 4 weeks are not significantly different by IL28B genotype. B: Pharmacodynamics Maximum efficiency (which determines first phase viral decay) is higher for CC favorable genotype compared to CT/TT unfavorable genotype (P=0.007). Mean efficiency is also higher for the CC favorable genotype (P=0.061), although this did not reach statistical significance. C: Viral Kinetics First phase (24 hours) and second phase (after day 2) viral decay is greater for the CC favorable genotype. Phases of viral decay are a function of maximum efficiency and delta (infected cell loss), respectively.

Figure 2. DNA Microarray Gene Expression Profiles

A: HCV /HIV Coinfection (N=20) PRE=baseline, prior to IFN therapy; POST=trough after the fourth dose of IFN therapy; G=favorable genotype (CC); HET=unfavorable genotype (CT); A=unfavorable genotype (TT); NR=non-responder to IFN therapy; SVR=responder to IFN therapy. Cluster 1 genes were up regulated in both NR and unfavorable genotypes at PRE and POST time points. These genes belong to regulation of both adaptive and innate immune responses (MHC class I receptor activity, p=4.7 × 10−8; genes involved in antigen processing and presentation p=1.2 × 10−7 and NK cell mediated cytotoxicity p=1.9 × 10−7 Cluster 2 genes were up regulated in favorable, but not in the unfavorable genotypes at PRE and POST time points with the major functional category being genes involved in host-virus interaction (p=3.4 × 10−4), and epigenetic modification of DNA (3.5 × 10−3). Cluster 3 genes were up regulated in all groups compared to baseline expression in the SVR group and represent post-translational modification and protein metabolism genes (p= 7.1 × 10−9) suggesting an increased metabolic state in these patients compared to SVR. Cluster 4 included genes upregulated in NR or unfavorable genotypes, at both PRE and POST time points. This cluster reveals upregulation of genes that are directly involved in tissue injury and inflammatory response (p=3.1 × 10−4), which may reflect sustained tissue damage without HCV clearance. Cluster 5 genes were expressed at higher levels among those who were NR or favorable genotype. This cluster included protein transport, endosomal and lysosomal functions (p=6.1 × 10−4) and is of unclear significance. B: HCV monoinfection (N=26) PRE=baseline, prior to IFN therapy; POST=12 hours after first dose of IFN therapy; G=favorable genotype (CC); HET=unfavorable genotype (CT); A=unfavorable genotype (TT); NR=non-responder to IFN therapy; SVR=responder to IFN therapy. Cluster 2 included the genes that were up regulated in both PRE and POST treatment samples of the favorable genotype. The functional category of genes in this cluster included many coding for the ras pathway genes (p=1 × 10−8). Cluster 3 were genes up regulated only in unfavorable genotypes or NRs at PRE but not POST time points and included phosphoproteins involved in cellular signaling and activation (p= 1.5 × 10−8). Cluster 4 genes were up regulated only in unfavorable genotypes or NRs at PRE and POST time points. These genes clearly induced cellular signaling pathways and RNA editing (p=3.1 × 10−7 and p1 × 10−3 respectively). Cluster 5 genes were upregulated at PRE and POST time points for all IL28B genotypes but were different from NRs. These genes are involved in cellular signal transduction and histone modulation (p=4.2 × 10−5) with unclear significance. Cluster 6 genes were upregulated in subjects who were NR or unfavorable genotypes at PRE and POST time points. Genes encoding structural and microtubule formation were clearly overrepresented in this cluster (p=6.7 × 10−5). Cluster 7 included genes that were expressed at higher levels in favorable genotype rather than SVR or unfavorable genotype. Interestingly, these genes included those involved in protection from cell death and apoptosis and negative regulation of apoptotic pathway (p=6.9 × 10−4).

Figure 2. DNA Microarray Gene Expression Profiles

A: HCV /HIV Coinfection (N=20) PRE=baseline, prior to IFN therapy; POST=trough after the fourth dose of IFN therapy; G=favorable genotype (CC); HET=unfavorable genotype (CT); A=unfavorable genotype (TT); NR=non-responder to IFN therapy; SVR=responder to IFN therapy. Cluster 1 genes were up regulated in both NR and unfavorable genotypes at PRE and POST time points. These genes belong to regulation of both adaptive and innate immune responses (MHC class I receptor activity, p=4.7 × 10−8; genes involved in antigen processing and presentation p=1.2 × 10−7 and NK cell mediated cytotoxicity p=1.9 × 10−7 Cluster 2 genes were up regulated in favorable, but not in the unfavorable genotypes at PRE and POST time points with the major functional category being genes involved in host-virus interaction (p=3.4 × 10−4), and epigenetic modification of DNA (3.5 × 10−3). Cluster 3 genes were up regulated in all groups compared to baseline expression in the SVR group and represent post-translational modification and protein metabolism genes (p= 7.1 × 10−9) suggesting an increased metabolic state in these patients compared to SVR. Cluster 4 included genes upregulated in NR or unfavorable genotypes, at both PRE and POST time points. This cluster reveals upregulation of genes that are directly involved in tissue injury and inflammatory response (p=3.1 × 10−4), which may reflect sustained tissue damage without HCV clearance. Cluster 5 genes were expressed at higher levels among those who were NR or favorable genotype. This cluster included protein transport, endosomal and lysosomal functions (p=6.1 × 10−4) and is of unclear significance. B: HCV monoinfection (N=26) PRE=baseline, prior to IFN therapy; POST=12 hours after first dose of IFN therapy; G=favorable genotype (CC); HET=unfavorable genotype (CT); A=unfavorable genotype (TT); NR=non-responder to IFN therapy; SVR=responder to IFN therapy. Cluster 2 included the genes that were up regulated in both PRE and POST treatment samples of the favorable genotype. The functional category of genes in this cluster included many coding for the ras pathway genes (p=1 × 10−8). Cluster 3 were genes up regulated only in unfavorable genotypes or NRs at PRE but not POST time points and included phosphoproteins involved in cellular signaling and activation (p= 1.5 × 10−8). Cluster 4 genes were up regulated only in unfavorable genotypes or NRs at PRE and POST time points. These genes clearly induced cellular signaling pathways and RNA editing (p=3.1 × 10−7 and p1 × 10−3 respectively). Cluster 5 genes were upregulated at PRE and POST time points for all IL28B genotypes but were different from NRs. These genes are involved in cellular signal transduction and histone modulation (p=4.2 × 10−5) with unclear significance. Cluster 6 genes were upregulated in subjects who were NR or unfavorable genotypes at PRE and POST time points. Genes encoding structural and microtubule formation were clearly overrepresented in this cluster (p=6.7 × 10−5). Cluster 7 included genes that were expressed at higher levels in favorable genotype rather than SVR or unfavorable genotype. Interestingly, these genes included those involved in protection from cell death and apoptosis and negative regulation of apoptotic pathway (p=6.9 × 10−4).

Figure 3. Individual Interferon Stimulated Gene Expression for Treatment Response Stratified by IL28B Genotype

VR=Virologic Responders (SVR+relapsers); NR=Non-Virologic Responders (NR+viral breakthrough) A: Mean baseline ISG expression in Favorable CC Genotype B: Mean baseline ISG expression in Unfavorable CT/TT Genotype C: Mean delta ISG expression in Favorable CC Genotype D: Mean delta ISG expression in Unfavorable CT/TT Genotype

Figure 4. Total Interferon Stimulated Gene Expression for Treatment Response Stratified by IL28B Genotype

VR=Virologic Responders (SVR+relapsers); NR=Non-Virologic Responders (NR+viral breakthrough) A: Mean baseline ISG expression B: Mean delta ISG expression

Figure 5. Total Interferon Stimulated Gene Expression by Race and IL28B Genotype

A: Baseline ISG expression by Race and IL28B Genotype B: Delta ISG expression by Race and IL28B Genotype