IL28B and the Control of Hepatitis C Virus Infection

Gastroenterology
Volume 139, Issue 6 , Pages 1865-1876, December 2010

Ashwin Balagopal, David L. Thomas, Chloe L. Thio

Received 6 August 2010; accepted 8 October 2010. published online 14 October 2010.

John P. Lynch and David C. Metz, Section Editors

Treatment-induced control and spontaneous clearance of hepatitis C virus (HCV) infection are affected by various host factors. Polymorphisms in the region of the gene IL28B are associated with HCV clearance, implicating the gene product, interferon (IFN)-λ3, in the immune response to HCV. Although it is not clear how the IL28B haplotype affects HCV clearance, IFN-λ3 up-regulates interferon-stimulated genes, similar to IFN-α and IFN-β but via a different receptor. There is also evidence that IFN-λ3 affects the adaptive immune response. The IL28B genotype can be considered, along with other factors, in predicting patient responses to therapy with pegylated IFN-α and ribavirin. We review the genetic studies that uncovered the association between IL28B and HCV clearance, the biology of IFN-λ3, the clinical implications of the genetic association, and areas of future research.

Keywords: IL28B, Hepatitis C Virus, Interferon Lambda, Interferon Sensitivity, HCV Treatment

Abbreviations used in this paper: GWAS, genome-wide association studies, HIV, human immunodeficiency virus, IFN, interferon, IL-10Rβ, interleukin-10 receptor beta chain, IL-28Rα, interleukin-28 receptor alpha chain, ISG, interferon-stimulated gene, PEG, pegylated, RBV, ribavirin, SNP, single nucleotide polymorphism, STAT, signal transducers and activators of transcription, SVR, sustained virologic response

Spontaneous clearance of hepatitis C virus (HCV) occurs in ∼30% of patients with acute infections; the remaining patients develop chronic infections and have predispositions to cirrhosis and hepatocellular carcinoma.1, 2, 3 Among chronically infected patients, HCV can be cleared by interferon (IFN)-α–based treatment in some cases. Researchers have searched for factors responsible for natural and treatment-induced HCV clearance for more than 10 years. In the past year, genetic studies have identified several single nucleotide polymorphisms (SNPs) in and near IL28B (which encodes IFN-λ3) that are associated with clearance. We review the role of IL28B in HCV infection, clinical implications, and directions for future research.

Overview of the IFN-λ Family

IFN-λ3 belongs to the IFN-λ family, along with IFN-λ1 and IFN-λ2, which are encoded by IL29 and IL28A, respectively. IFN-λs are categorized as type 3 IFNs and are potent, endogenous antiviral cytokines. Although they are structurally most homologous to members of the IL10 family, IFN-λs are more functionally similar to type 1 IFNs4; they signal via Jak/signal transducers and activators of transcription (STAT) intracellular pathways and up-regulate transcription of IFN-stimulated genes (ISGs) that are required to control viral infection. IFN-λ–like sequences have been found in most mammalian species, but many are pseudogenes.5 Phylogenetic studies of type 1 and type 3 IFNs and the interleukin-10 family of cytokines indicate the existence of a common, ancestral IFN gene that shared the multi-exon genomic structure of the IFN-λ family members; it was putatively identified in ancient fish, evolved in a series of gene duplication and retrotransposon events, and gave rise to the IFNs observed in mammals.5 In humans, the IFN-λ genes cluster on chromosome 19 (Figure 1).5

IFN-λs inhibit HCV replication in vitro,6, 7, 8 and trials of IFN-λ1 in patients with chronic HCV infections have shown promising results9; 86% of treatment naive-patients who received combined pegylated (PEG) IFN-λ and ribavirin (RBV) for 4 weeks had a >2 log10 IU/mL decrease in HCV RNA. Therefore, associations made between IL28B variants and HCV clearance in large-scale genetic studies provide an exciting mechanistic link between innate immunity and viral clearance.

IL28B and Control of HCV Infection With Therapy

Many of the first studies that linked IL28B and HCV clearance came from studies of large cohorts of patients with chronic HCV infection who were treated with PEG-IFN-α and RBV. These cohorts were investigated in genome-wide association studies (GWAS), which allow an unbiased sampling of variations in genes across the entire genome without a hypothesis. The first GWAS was performed using an Illumina Human610Quad BeadChip (San Diego, CA) in the IDEAL study, in which patients infected with HCV genotype 1 were randomly assigned to groups that were treated with PEG-IFN-α2a or PEG-IFN-α2b; the study included only subjects who were treated with a minimum number of total doses.10 Although more than 500,000 SNPs were considered, the investigators found that the strongest predictor of sustained virologic response (SVR) was a SNP (rs12979860) located on the long arm of chromosome 19, within the IFN-λ gene cluster (Figure 2). IL28B is upstream and in the reverse orientation of IL28A; rs12979860 is upstream of both of these genes, closer to IL28B (Figure 1). At this position, the C allele is the most frequently observed in the white population (but not in the black population) and is associated with SVR; people with the CC genotype have SVR rates more than 2-fold higher than those with the minor T allele (Table 1). In addition to this SNP, the 6 SNPs most strongly associated with SVR were also found at the IFN-λ gene cluster, although the effects of these were no longer observed after adjusting for the presence of rs12979860. After adjusting for the association of rs12979860, the next SNP most strongly associated with SVR found in the GWAS was rs8099917, a noncoding SNP found ∼7.5 kilobases upstream of the IL28B start codon. These findings were validated in a study that genotyped the rs12979860 SNP in patients from the Duke Hepatology Clinical Research Database and Respository, a registry of patients with HCV infections who were followed up longitudinally.11 The subjects had HCV genotype 1 (80.5%) or genotype 2 or 3 (19.5%) infections and had received complete courses of PEG-IFN-α and RBV therapy. White subjects, who made up most of the study group, with the CC genotype were >5 times more likely to achieve an SVR than subjects with the CT or TT genotypes (P = 9.0 × 10−6). Differences in treatment outcomes among black subjects were not significantly associated with the IL28B genotype, although the investigators acknowledged the limited power of the study given the smaller numbers of black subjects included (n = 106). The association of IL28B genotype with SVR was independent of treatment history. In contrast to treatment response, relapse was not associated with IL28B genotype.

Three other groups used similar approaches to study the genetic basis for SVR and found a group of SNPs near IL28B that were in strong linkage disequilibrium, indicating that they were inherited as a block rather than independently. Depending on the technology used and the racial composition of the study population, the specific findings of each study varied (detailed in the following text; Figure 1 and Table 1). Collectively, the results showed that there is a genomic region comprising IL28B and its potential regulatory sequences that is strongly associated with IFN response (Figure 3). Suppiah et al studied Australians of European descent with HCV genotype 1 infections who received PEG-IFN-α and RBV.12 In the first phase, a GWAS was performed on all participants and results were compared between patients who achieved an SVR while receiving treatment and those who did not respond. Several SNPs were associated with clearance, but the strongest was rs8099917. Compared with the T allele, heterozygosity for the minor G allele was associated with a 1.64-fold increase in risk for not responding to therapy and homozygosity was associated with a 2.39-fold increase in risk.

Tanaka et al performed a GWAS using an Affymetrix SNP Array (Santa Clara, CA) of DNA from HCV genotype 1–infected Japanese patients who achieved an SVR to therapy with PEG-IFN-α2a or PEG-IFN-α2b and RBV, comparing data with that of nonresponders.13 The SNPs rs8099917 and rs12980275 segregated with treatment response (Figure 1). Rauch et al performed a GWAS of the Swiss Hepatitis C Cohort, a population of European white subjects infected with HCV genotypes 1 (48%), 2 (10%), 3 (29%), and 4 (9%) who received PEG-IFN-α and RBV.14 The most significant markers were clustered at the IFN-λ gene loci. The SNP rs8099917 was most highly associated with response to treatment, also reported by Suppiah et al and Tanaka et al (Figure 1). Only 73.9% of patients homozygous for the T allele achieved an SVR, but patients with the minor G allele were 5-fold less likely to respond to therapy (P = 3.11 × 10−8). It is important to note that these investigators used different GWAS platforms than Ge et al10 (Figure 1). The rs12979860 was associated with SVR in subsequent studies, but this association was not fully tested; Tanaka et al did not include rs12979860 in their GWAS platform, and Suppiah et al and Rauch et al used multiple platforms with only limited representation of this SNP. Rauch et al did report that where data were available for rs12979860 it was linked to rs8099917.

IL28B Mapping and HCV Control

It appears that an IL28B haplotype can be a strong determinant of a patient's response to treatment of HCV infection and can be represented by a single SNP (or a small number of SNPs) (Figure 3). Several groups performed more finely tuned genetic studies to clarify which SNP(s) had the greatest associations with treatment response. Ge et al sequenced the IL28B gene in a subset of patients to find genetic markers that were in high linkage disequilibrium with the SNP rs12979860, identified in their GWAS.10 Two additional markers were found: a nonsynonymous SNP (rs8103142) within the IL28B gene that encodes a lysine → arginine substitution at position 70 (K70R) and a G → C substitution (rs28416813) 37 base pairs upstream of the translation initiation site (Figure 1). These 3 SNPs were tightly linked, so it was a challenge to associate any one, individually, with treatment response. Suppiah et al genotyped 20 additional SNPs within the IL28B gene in an expanded cohort that included white subjects from Europe and Australia and compared frequencies between subjects who did and did not achieve an SVR.12 A G allele at rs12980275 had the strongest association with nonresponse (Figure 2). In this study, a 6-allele haplotype was identified that also included K70R (T > C on the coding strand); the haplotype with an arginine (R) was associated with nonresponse.

Similarly, Tanaka et al sequenced 16 SNPs in a validation cohort, based on HapMap data that characterized chromosome 19.13 Although they associated the haplotype, which included K70R, with response to therapy, it did not have a significantly greater association with SVR than any of the individual SNPs; thus, any single SNP is sufficient to mark the association with an SVR. In a logistic regression model, rs8099917 was most predictive of an SVR. Rauch et al sequenced the IL28B gene in subjects with the TT genotype at rs8099917 (associated with response) who achieved the expected SVR and those who did not achieve an SVR (the unexpected outcome).14 They also sequenced DNA from subjects with the risk GG genotype with the expected outcome of nonresponse to therapy and from those who achieved an SVR despite carrying this genotype. Twenty-one additional SNPs were identified that fell into 1 of 2 haplotype families. The first family was associated with HCV clearance, and the second was associated with persistence. As with Suppiah et al, an R at amino acid position 70 was found in the risk haplotype, along with another SNP (rs28416813) identified by Ge et al.10, 12, 14 These genetic mapping studies identified the specific alleles associated with response (or lack of response) to anti-HCV therapy (Figure 3).

IL28B and Spontaneous Clearance

The same IL28B haplotypes associated with treatment response are also associated with spontaneous clearance of HCV. Thomas et al genotyped the rs12979860 SNP in more than 1000 people from 6 cohorts with well-characterized spontaneous clearance of HCV or viral persistence and found that the CC genotype was strongly associated with HCV clearance (odds ratio, 0.33; P < 10−12).15 The investigators also showed that the clearance was mediated by linkage of this genotype with other identified markers, because their inclusion in a multivariate model did not reduce the association between the CC genotype and viral clearance. Ge et al had observed the clearance effect of the C allele at a higher frequency (73%) in a population with unknown HCV status compared with patients with chronic infections who went on to receive treatment (63%; P = 2.48 × 10−6), suggesting that this allele occurred more frequently among patients with spontaneous clearance of HCV.10 Further evidence that IL28B is involved in spontaneous clearance was provided by Rauch et al using a GWAS.14 They associated the T allele of rs8099917 with spontaneous clearance; no SNPs outside the IL28B/A gene loci were associated with clearance. In studies of a cohort of Spanish patients with acute HCV infection and a cohort of German patients with acute HCV infections (from the anti-D common source outbreak), researchers reported that the CC genotype at rs12979860 was observed more frequently in subjects with spontaneous resolution of HCV.16, 17 These results indicate the involvement of the same IL28B SNPs in both spontaneous and treatment-induced control of HCV infection (Figure 3).

IL28B and Racial Differences in HCV Control

Because SNPs in IL28B have varied distributions among ethnic groups (Table 2; http://www.hapmap.org/), it is intriguing to consider whether the differences at this locus account for the association between white ethnicity and increased spontaneous and treatment-induced clearances. Of the 2 alleles most strongly associated with HCV clearance (rs12979860 C and rs8099917 T), only the former is more common in people of European compared with African descent (Table 2 and Figure 4; hapmap.org); it might underlie the racial differences observed. Thomas et al genotyped the rs12979860 SNP in more than 2000 people from 51 ethnic populations worldwide and showed that East Asian populations have the highest frequencies of the alleles associated with clearance, sub-Saharan African populations have the lowest frequencies, and European populations have intermediate frequencies (Figure 4).15 In their study population, Ge et al also found the lowest frequencies of the allele associated with clearance among black subjects (allele frequency, ∼0.42), the highest frequencies in East Asian subjects (∼0.95), and intermediate frequencies in European-American (∼0.73) and Hispanic (∼0.7) subjects; these findings were validated in a subsequent study.10, 11 This distribution of alleles could account for the high rates of treatment-induced SVR observed in East Asian subjects.18, 19 When this SNP was studied in multivariate models of spontaneous and treatment-induced clearance, however, it accounted for only ∼50% to 60% of the ethnic differences observed in HCV control (D. L. Thomas, personal communication, August, 2010).10 It is notable that only 53% of black subjects with this genotype achieved an SVR compared with 82% of white subjects. Other genetic factors beyond IL28B genotype mediate spontaneous and treatment-associated clearance of HCV, although within a given race, IL28B genotype does predict outcome.

IL28B in HCV Genotype 2 and 3 Infections

Most of the initial studies of IL28B and HCV control were centered on people with HCV genotype 1 infections; studies of IL28B in patients infected with HCV of other genotypes have produced conflicting data and included small numbers. The largest study included 268 subjects infected with HCV genotype 2/3 who were randomly assigned to groups that were given PEG-IFN-α and RBV for a standard (24 weeks) or variable duration (12 weeks if they had a rapid virologic response or 24 weeks if they did not).20 Surprisingly, the genotype of IL28B was not associated with SVR in subjects who received standard duration therapy or who achieved a rapid virologic response and received variable therapy (12 weeks). There was a strong association between IL28B and treatment response only among subjects who did not achieve a rapid virologic response and received variable therapy (24 weeks). Intriguingly, in this subset, there appeared to be an effect of gene dose; IL28B heterozygotes had an intermediate rate of SVR, between that of patients with homozygosity for alleles that were and were not associated with clearance.

Several other studies have produced mixed results. Montes-Cano et al associated the IL28B haplotype with an SVR in a cohort of Spanish patients with non–genotype 1 infections.16 McCarthy et al found that the effect of the IL28B haplotype on SVR was similar between subjects with HCV genotypes 1 and 2/3.11 The inclusion of IL28B genotype in a multivariate model, however, reduced only slightly the influence of HCV genotype on SVR. In contrast, Rauch et al noted a trend in the effect of the IL28B haplotype in subjects infected with HCV genotypes 2/3, but this was not statistically significant,14 whereas Rallon et al did not associate the IL28B haplotype with treatment response in people infected with HCV genotype 3.21 The effect of the IL28B haplotype status on treatment response might therefore be attenuated for genotypes 2 and 3, but further research is required to clarify this relationship.

IL28B in Patients Coinfected With Human Immunodeficiency Virus and HCV

About one third of people infected with human immunodeficiency virus (HIV) are also infected with HCV. HIV complicates HCV infection by increasing rates of HCV persistence.2 Despite the immunologic changes associated with HIV infection, it does not affect the association between SNPs near the IFN-λ gene cluster and clearance of HCV. Thomas et al reported that stratification based on HIV status did not modulate the effect of the SNP rs12979860 on HCV clearance.15 Similarly, in a subset of patients in the Swiss Hepatitis C Cohort who were infected with HIV, Rauch et al found that the SNP rs8099917 was still associated with HCV clearance; effects of this SNP did not differ markedly between HCV-infected patients with or without HIV infection.14 Rallon et al found that among patients coinfected with HIV and HCV who completed PEG-IFN-α and RBV treatment, the CC genotype at locus rs12979860 was associated with an SVR and the association was strongest among patients infected with HCV genotypes 1 or 4.21 Given the low rates of spontaneous clearance and treatment-induced SVR among people coinfected with HIV and HCV, it is striking that IL28B is still associated with HCV control in this population.

IL28B and Other Infectious Diseases

Martin et al investigated whether IL28B SNPs are important in the control of other chronic viral infections where IFN-α is important in the immune response.22 A cohort of 226 individuals with persistent hepatitis B virus (HBV) infection was compared with 384 individuals who had recovered from HBV infection; recovery was not associated with the rs12979860 SNP. They also studied a cohort of 2548 individuals with, or who were at high risk for, HIV infection and found that the rs12979860 SNP was not associated with infection with HIV or disease progression. In GWAS with the Illumina HumanHap550 BeadChip, Kamatani et al studied patients with chronic HBV infection and found no association of chronic infection with IL28B genotype.23 Similarly, Fellay et al studied HIV-infected subjects using the Illumina BeadChip and found no association of IL28B with either HIV-1 viral RNA set point or with disease progression.24 Polymorphisms in IL28B have therefore not been associated with clearance of other viral infections.

The Effect of IL28B on HCV RNA Levels, Viral Kinetics, and IFN Responsiveness

Several groups have studied the correlation of IL28B genotype, baseline levels of HCV RNA, and treatment response. Ge et al found that although the C allele of rs12979860 was associated with SVR, it was also, paradoxically, associated with higher viral RNA levels compared with the T allele (CC patients had 6.35 log10 IU/mL, TC patients had 6.33 log10 IU/mL, and TT patients had 6.16 log10 IU/mL; P = 1.21 × 10−10).10 Higher levels of HCV RNA levels were also observed in patients who were off therapy who had the C allele.11 Although the differences are modest (<0.5 log10), the higher levels of HCV RNA among patients with the CC genotype might facilitate innate immune detection and control of the virus during treatment.

In addition to baseline level of HCV RNA, the kinetics of the treatment response appears to be influenced by IL28B genotype. Thompson et al compared viral kinetic data between subjects with CC, CT, and TT genotypes at the rs12979860 SNP 2 weeks after they began therapy with PEG-IFN-α and RBV.25 Irrespective of race, subjects with the CC genotype had the largest reductions in levels of HCV RNA (white subjects with CC genotype had the greatest decrease in HCV RNA). This translated to greater rapid virologic response and early virologic response rates among subjects with the CC genotype. Much of the effect of the IL28B genotype is evident in the first 48 hours after treatment, indicating that IL28B genotype somehow primes the host response to HCV, decreasing the threshold for virologic control with treatment. Alternatively, IL28B genotype could simply be a marker for greater baseline levels of the IFN response, consistent with the findings in patients with spontaneous clearance of HCV.

Intriguingly, Honda et al found that subjects with the TT genotype at the SNP rs8099917 (associated with SVR) had pretreatment hepatic expression levels of ISGs that were lower than those of subjects with the TG or GG genotypes.26 The fact that subjects with genotypes associated with SVR have reduced expression of ISGs might account for the higher levels of HCV RNA observed before treatment. Sensitivity to exogenous IFN is inversely associated with levels of ISGs; IL28B genotypes may affect expression levels of ISGs, accounting for the association between IL28B genotype and response to therapy.27, 28 Despite the association of IL28B with clearance, there are people who carry alleles that are not associated with clearance who clear the virus, as well as patients with alleles associated with clearance whose infection persists. Further studies should be performed with these patients to investigate IL28B genetics, ISG expression levels, and other genetic factors involved in the response to anti-HCV therapy.

IFN-λ Biology and Role in HCV Infection

IL28B can determine the outcome of HCV infection, but the mechanisms that mediate the association between the different SNPs and HCV control are unclear, especially in light of the lack of association of IL28B polymorphisms with HBV or HIV infection outcomes. Investigating the biology of IFN-λ in HCV and other viral infections could provide mechanistic insight.

The main cellular sources of IFN-λs are believed to be plasmacytoid dendritic cells, although macrophages and conventional dendritic cells probably also produce IFN-λs.29 It is not known which cells produce IFN-λs in the liver, but candidates include Kupffer cells, dendritic cells, and liver sinusoidal endothelial cells. Hepatocytes also have active innate immune responses and probably release IFN-λs on viral infection.6, 7 Although IFN-λs have many effects on a number of viruses, their site of action is constrained by expression of their cognate receptor. IFN-λs signal through a heterodimer that comprises the interleukin-28 receptor α chain (IL-28Rα) and the interleukin-10 receptor β chain (IL-10Rβ). In contrast to the distribution of IFN-α receptor and even IL-10Rβ, which are found on a wide variety of cell types, IL-28Rα is found primarily on epithelial cells. This has implications on which cells IFN-λs can act on; in one study, livers from mice were found to express only low levels of IL-28Rα.30

Downstream signaling after IFN-λ receptor ligation, however, is similar to type 1 IFN signaling and occurs via the covalently bound tyrosine kinases Tyk2 and Jak1 (Figure 5).29 These binding partners phosphorylate each other and also phosphorylate STAT1 and STAT2 proteins. A consequence of phosphorylation of STAT1 and STAT2 is the formation of the IFN-stimulated gene factor 3 complex along with activated IFN-regulatory factor 9, which leads to the up-regulation of canonical ISGs. ISG up-regulation causes many of the innate cellular defenses against viral infection. Overall, IFN-λ signaling is believed to be proinflammatory and unlike responses to IL-10, despite sharing the IL-10 receptor subunit.

In addition to phosphorylation of STAT1 and STAT2, STAT3, STAT4, and STAT5 can also be activated via the IL-10Rβ chain, which can have immunomodulatory effects.29 IFN-λ1 and IFN-λ2 inhibit IL-13 production by T cells following stimulation with concanavalin A, indicating that IFN-λs promote the T-helper cell 1 response.31, 32 IFN-λ3 increases the T-helper cell 1 response to an HIV DNA vaccine and simultaneously inhibits regulatory T-cell responses.33 More investigations into the effect of IFN-λs on adaptive immunity could reveal that HCV control by IFN-λ is the result of a multi-level response.

It is not clear how the SNPs in IL28B affect the IFN signaling pathways. The rs12979860 is 3 kilobases upstream of IL28B, whereas rs8099917 is nearly 8 kilobases upstream (Figure 1). Although it is possible that these SNPs modulate IL28B transcription, it is more likely that they are in linkage disequilibrium with one or more SNPs in the IL28B coding or promoter regions. Alternatively, the SNPs could modify transcription factor binding sites. IL29 has multiple IFN-regulatory factor and nuclear factor κB binding sites (eg, −214 to −172 and −98 to −89 upstream of the transcription initiation site), although those that have been reported are not polymorphic.34 The SNP that encodes K70R is important to study because it is tightly linked with SNPs associated with SVR and natural clearance. K70R is not predicted to affect binding to the receptor or signaling, but it could be involved in interactions with other signaling factors that affect viral control.4 The SNPs in IL28B might lead to expression of forms of IFN-λ3 that do not function or have weak function or even hyperfunctional variants that reduce the antiviral response by negative feedback. Research is required to determine how IL28B and its variants affect HCV persistence and response to therapy.

Because IFN-λs inhibit viral replication, it is logical to consider that expression of different amounts of endogenous IFN-λ3 could determine whether a patient controls the virus or remains infected. Two studies compared messenger RNA levels of IFN-λ in whole blood or peripheral blood mononuclear cells from subjects with the T allele at position rs8099917 (associated with clearance) with that of subjects with the G allele (associated with viral persistence).12, 13 It has been difficult to quantify IFN-λ2 and IFN-λ3 messenger RNA levels by polymerase chain reaction because of their sequence homology; levels of IFN-λ2 and IFN-λ3 measured by quantitative polymerase chain reaction are combined when reported. Each group found the highest expression of IFN-λ2 and IFN-λ3 in subjects with the TT genotype, compared with TG and GG genotypes, associating higher amounts of endogenous IFN-λs with HCV clearance. Using information from the SNPExpress database, Ge et al, in contrast, did not observe differences in IFN-λ3 expression among subjects not infected with HCV who were homozygous for an allele in linkage disequilibrium with the rs12979860 SNP (see supplemental data for Ge et al).10 Similarly, Honda et al found no association between hepatic expression of IFN-λ2 and IFN-λ3 and rs8099917 genotype.26 Studies of plasmacytoid dendritic cells and cells that produce IFN-λ in the liver should provide insight into the relationship between the IL28B genotype and expression.

Studies of animals with viral infections indicate that the organ-specific distribution of IL-28Rα determines response to IFN-λ. While some studies observed that IFN-λ protects mice against respiratory but not hepatic viruses,35, 36 others found protection against hepatic viral infection.37 One study made the surprising observation that IFN-λ did not induce expression of ISGs in livers of mice.30 Studies in mice should be interpreted cautiously, however; in human cells and tissues, IFN-λ expression was shown to affect hepatotropic viruses. Robek et al found that IFN-λ1 and IFN-λ2 inhibited HBV replication.6 Moreover, recombinant IFN-λ3 had a more potent antiviral effect than IFN-λ1 or IFN-λ2 against encephalomyocarditis virus in hepatocyte cell lines.38 There has not been an extensive, published study of whether primary human hepatic cells respond to IFN-λ, although in one report HCV-infected liver tissue had higher amounts of IL-28Rα than uninfected liver tissue.39 Interestingly, treatment of macrophages with IFN-λ1 inhibited HIV-1 infection, possibly through production of competitive ligands for HIV coreceptors; it is not clear how this finding might relate to HCV clearance in patients.40

Several in vitro studies support a direct role for IFN-λ for the control of HCV replication through the innate immune pathway. Robek et al showed that subgenomic and full-length HCV replicons were inhibited by recombinant IFN-λ1 and IFN-λ2, which up-regulated a representative ISG.6 In a cell culture system, Marcello et al7 showed that IFN-λ1 inhibited HCV replication with similar kinetics to that of IFN-α but that IFN-λ1–induced up-regulation of ISGs was stronger and lasted longer. Combinations of IFN-λ1 and IFN-α had the greatest inhibitory effect on HCV replication compared with individual agents.8 IFN-λ and IFN-α might therefore have synergistic effects in controlling HCV infection. Type 1 IFN potentiated IFN-λ release in an animal model of viral infection.41 It is possible that the putative SNPs alter the interaction of IFN-λ with IFN-α.

Clinical Implications

The IL28B genotype provides important, independent information about a patient's likelihood of achieving an SVR, and a commercial test became available in the United States in July 2010. Results from this test could be used in combination with algorithms based on HCV genotype and viral load to predict patients' responses to treatment; IL28B genotype could be a factor that patients and their physicians use to decide whether to initiate therapy or wait until direct antiviral agents become available. Unfortunately, IL28B genotype does not have a positive predictive value of 100% for SVR, so it cannot be used as the only predictor of response (Table 1); HCV treatment should not be withheld based solely on IL28B genotype. In addition, the positive predictive value is influenced by the prevalence of SVR, which is difficult to extrapolate from published studies in which patients with ambiguous outcomes were removed from the analyses and the proportions of patients that achieve SVRs varied. To put some numbers in perspective, the SVR rate for black subjects with the rs12979860 CC genotype (associated with clearance) was 53%10; this is similar to that of white subjects with genotype 1 HCV, irrespective of IL28B genotype. Among white subjects in the same study, the SVR rate for those with the rs12979860 CC genotype was ∼82%. Interestingly, when the likelihood of SVR approaches 80%, IL28B genotype can also affect the decision for liver disease staging before treatment. Just as patients with genotype 2 or 3 HCV infection can elect to undergo treatment without consideration of fibrosis stage, people who have IL28B genotypes associated with viral control might decide not to undergo biopsy evaluation. In either case, noninvasive staging is still recommended to determine whether patients should be screened for hepatocellular carcinoma.42

IL28B genotype can affect how long a clinician should monitor someone with an acute HCV infection before treatment. Those with a haplotype associated with HCV clearance might be monitored longer, because they are more likely to spontaneously clear the virus; those with haplotypes associated with persistence might be better off receiving therapy during the acute period and be monitored for a shorter period beforehand.43 The association between kinetics of HCV response to IFN treatment and IL28B genotype might be used to identify patients who require shorter durations of therapy; further studies are required to determine if this is the case. The association between sensitivity to IFN therapy and IL28B genotype could also affect how clinicians use direct antiviral agents. For example, IFN lead-in dosing might be the best option for patients with IL28B haplotypes that are not associated with HCV clearance; they are more likely to develop resistance to direct antiviral agents because their response to IFN-α therapy is slower.

As therapeutic agents, IFN-λs might have longer and more potent effects than type 1 IFNs, with fewer adverse events, because distribution of IFN-λ receptors is more restricted. Phase 1 trials of IFN-λ1 in treatment-naive patients and those with chronic HCV who experienced a relapse after therapy have shown significant reductions in HCV viremia after 4 weeks.9 It will be interesting to see if polymorphisms in IL28B predict a response to IFN-λs in these trials; later-stage trials are under way.

Future Directions

Basic science studies help us understand how specific genetic features relate to immunologic function and HCV clearance. It is important to determine exactly which SNP or specific genetic feature of IL28B affects clearance, and this could require sequencing of IL28A and IL29 in different ethnic groups of patients with natural and treatment-induced clearance to understand linkage in this region. Comparing the human IL28B sequence with that of chimpanzees and other primates might also provide important information about linkage. Although the same gene cluster and SNPs are associated with clearance versus persistence and an SVR versus no response to therapy, these gene variants might affect responses via different mechanisms. The structure of IFN-λ3 has been determined, but the active-site amino acids have only been inferred from alanine scans; the role of SNPs in the IL28B coding region might be more precisely defined by resolving the structure of IFN-λ3 bound to its heterodimeric receptor. Investigations into the genetics of HCV control are hampered by inadequate model systems of HCV infection; animals with phenotypes that more closely resemble patients with HCV infection would improve our understanding of the role of IFN-λ3 in HCV infection. Several HCV cell culture systems exist and studies are under way to examine the role of IL28B in HCV replication using site-directed mutagenesis to compare major and minor alleles for several SNPs. Similarly, knockout mice with humanized livers can be used to study the effects of IFN-λ3 on the immune response and HCV control. Because IFN-λs can have redundant effects, responses to IFN-λ might need to be fully suppressed by interfering with IL-28Rα signaling. Using combined approaches, ISG responses to HCV can be determined using cell culture systems, animal models, small interfering RNAs, and antibodies that inhibit IFN-λ signaling.

In situ studies of liver tissues from patients with chronic HCV infection are necessary to delineate which cells release or respond to IFN-λs, especially given findings from mouse studies that hepatic expression of IL28Rα is limited. Studies should be performed in human hepatic tissue to compare expression of IFN-λ with its receptor. IL28B genotype is assumed to predict the early stages of HCV control, but other immunologic factors, such as pretreatment levels of ISG, might also predict response. It will be important to determine whether IL28B SNPs also predict response to small molecule therapeutics, their utility in patients with acute HCV infections, and optimal treatment duration. There is evidence that IL28B is associated with an SVR in subjects with chronic HCV who were treated with the protease inhibitor telaprevir in addition to standard therapy.44 The paradox of the association of IL28B genotypes that promote HCV clearance and higher baseline levels of HCV RNA should be further evaluated; usually patients with poor response to treatment have high pretreatment levels of HCV RNA. IL28B genotype might not predict clearance of all HCV genotypes, and the interaction between host and viral genotypes should be further explored. Interest in IL28B genotype has extended to other chronic viral infections, and researchers are investigating whether genotypes associated with HCV clearance have other effects in the immune response to pathogens. The identification of IL28B heralds the era of genomic medicine in HCV and opens the door to understanding HCV clearance. The finding has spurred intense bidirectional investigation into the clinic and the bench with the hopes of enhanced therapies against HCV. The coming months and years promise rapid fulfillment of some of these goals and healthy excitement in the field.

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Conflicts of interest The authors disclose no conflicts.

Funding Supported by National Institutes of Health grants DA13324 and 1K08AI081544.

PII: S0016-5085(10)01462-9
doi:10.1053/j.gastro.2010.10.004
© 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

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