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.

References

1.Wilson LE, Torbenson M, Astemborski J, et al. Progression of liver fibrosis among injection drug users with chronic hepatitis C. Hepatology. 2006;43:788–795
2.Thomas DL, Astemborski J, Rai RM, et al. The natural history of hepatitis C virus infection: host, viral, and environmental factors. JAMA. 2000;284:450–456
3.Thomas DL, Seeff LB. Natural history of hepatitis C. Clin Liver Dis. 2005;9:383–398vi
4.Gad HH, Dellgren C, Hamming OJ, et al. Interferon-lambda is functionally an interferon but structurally related to the interleukin-10 family. J Biol Chem. 2009;284:20869–20875
5.Fox BA, Sheppard PO, O'Hara PJ. The role of genomic data in the discovery, annotation and evolutionary interpretation of the interferon-lambda family. PLoS One. 2009;4:e4933
6.Robek MD, Boyd BS, Chisari FV. Lambda interferon inhibits hepatitis B and C virus replication. J Virol. 2005;79:3851–3854
7.Marcello T, Grakoui A, Barba-Spaeth G, et al. Interferons alpha and lambda inhibit hepatitis C virus replication with distinct signal transduction and gene regulation kinetics. Gastroenterology. 2006;131:1887–1898
8.Pagliaccetti NE, Eduardo R, Kleinstein SH, et al. Interleukin-29 functions cooperatively with interferon to induce antiviral gene expression and inhibit hepatitis C virus replication. J Biol Chem. 2008;283:30079–30089
9.Muir AJ, Shiffman ML, Zaman A, et al. Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection. Hepatology. 2010;52:822–832
10.Ge D, Fellay J, Thompson AJ, et al. Genetic variation in IL28B predicts hepatitis C treatment-induced viral clearance. Nature. 2009;461:399–401
11.McCarthy JJ, Li JH, Thompson A, et al. Replicated association between an IL28B gene variant and a sustained response to pegylated interferon and ribavirin. Gastroenterology. 2010;138:2307–2314
12.Suppiah V, Moldovan M, Ahlenstiel G, et al. IL28B is associated with response to chronic hepatitis C interferon-alpha and ribavirin therapy. Nat Genet. 2009;41:1100–1104
13.Tanaka Y, Nishida N, Sugiyama M, et al. Genome-wide association of IL28B with response to pegylated interferon-alpha and ribavirin therapy for chronic hepatitis C. Nat Genet. 2009;41:1105–1109
14.Rauch A, Kutalik Z, Descombes P, et al. Genetic variation in IL28B is associated with chronic hepatitis C and treatment failure: a genome-wide association study. Gastroenterology. 2010;138:1338–13451345.e1-7
15.Thomas DL, Thio CL, Martin MP, et al. Genetic variation in IL28B and spontaneous clearance of hepatitis C virus. Nature. 2009;461:798–801
16.Montes-Cano MA, Garcia-Lozano JR, Abad-Molina C, et al. Interleukin-28B genetic variants and hepatitis virus infection by different viral genotypes. Hepatology. 2010;52:33–37
17.Tillmann HL, Thompson AJ, Patel K, et al. A polymorphism near IL28B is associated with spontaneous clearance of acute hepatitis C virus and jaundice. Gastroenterology. 2010 Jul 14;[epub ahead of print]
18.Yan KK, Guirgis M, Dinh T, et al. Treatment responses in Asians and Caucasians with chronic hepatitis C infection. World J Gastroenterol. 2008;14:3416–3420
19.Liu CH, Liu CJ, Lin CL, et al. Pegylated interferon-alpha-2a plus ribavirin for treatment-naive Asian patients with hepatitis C virus genotype 1 infection: a multicenter, randomized controlled trial. Clin Infect Dis. 2008;47:1260–1269
20.Mangia A, Thompson AJ, Santoro R, et al. An IL28B polymorphism determines treatment response of hepatitis C virus genotype 2 or 3 patients who do not achieve a rapid virologic response. Gastroenterology. 2010;139:821–827
21.Rallon NI, Naggie S, Benito JM, et al. Association of a single nucleotide polymorphism near the interleukin-28B gene with response to hepatitis C therapy in HIV/hepatitis C virus-coinfected patients. AIDS. 2010;24:F23–F29
22.Martin MP, Qi Y, Goedert JJ, et al. IL28B polymorphism does not determine hepatitis B virus or HIV outcomes. J Infect Dis (in press).
23.Kamatani Y, Wattanapokayakit S, Ochi H, et al. A genome-wide association study identifies variants in the HLA-DP locus associated with chronic hepatitis B in Asians. Nat Genet. 2009;41:591–595
24.Fellay J, Shianna KV, Ge D, et al. A whole-genome association study of major determinants for host control of HIV-1. Science. 2007;317:944–947
25.Thompson AJ, Muir AJ, Sulkowski MSet a. Interleukin-28B polymorphism improves viral kinetics and is the strongest pretreatment predictor of sustained virologic response in genotype 1 hepatitis C virus. Gastroenterology. 2010;139:120–129
26.Honda M, Sakai A, Yamashita T, et al. Hepatic ISG expression is associated with genetic variation in interleukin 28B and the outcome of interferon therapy for chronic hepatitis C. Gastroenterology. 2010;139:499–509
27.Chen L, Borozan I, Feld J, et al. Hepatic gene expression discriminates responders and nonresponders in treatment of chronic hepatitis C viral infection. Gastroenterology. 2005;128:1437–1444
28.Sarasin-Filipowicz M, Oakeley EJ, Duong FH, et al. Interferon signaling and treatment outcome in chronic hepatitis C. Proc Natl Acad Sci U S A. 2008;105:7034–7039
29.Ank N, West H, Paludan SR. IFN-lambda: novel antiviral cytokines. J Interferon Cytokine Res. 2006;26:373–379
30.Sommereyns C, Paul S, Staeheli P, et al. IFN-lambda (IFN-lambda) is expressed in a tissue-dependent fashion and primarily acts on epithelial cells in vivo. PLoS Pathog. 2008;4:e1000017
31.Jordan WJ, Eskdale J, Srinivas S, et al. Human interferon lambda-1 (IFN-lambda1/IL-29) modulates the Th1/Th2 response. Genes Immun. 2007;8:254–261
32.Srinivas S, Dai J, Eskdale J, et al. Interferon-lambda1 (interleukin-29) preferentially down-regulates interleukin-13 over other T helper type 2 cytokine responses in vitro. Immunology. 2008;125:492–502
33.Morrow MP, Pankhong P, Laddy DJ, et al. Comparative ability of IL-12 and IL-28B to regulate Treg populations and enhance adaptive cellular immunity. Blood. 2009;113:5868–5877
34.Onoguchi K, Yoneyama M, Takemura A, et al. Viral infections activate types I and III interferon genes through a common mechanism. J Biol Chem. 2007;282:7576–7581
35.Mordstein M, Kochs G, Dumoutier L, et al. Interferon-lambda contributes to innate immunity of mice against influenza A virus but not against hepatotropic viruses. PLoS Pathog. 2008;4:e1000151
36.Mordstein M, Neugebauer E, Ditt V, et al. Lambda interferon renders epithelial cells of the respiratory and gastrointestinal tracts resistant to viral infections. J Virol. 2010;84:5670–5677
37.Ank N, West H, Bartholdy C, et al. Lambda interferon (IFN-lambda), a type III IFN, is induced by viruses and IFNs and displays potent antiviral activity against select virus infections in vivo. J Virol. 2006;80:4501–4509
38.Dellgren C, Gad HH, Hamming OJ, et al. Human interferon-lambda3 is a potent member of the type III interferon family. Genes Immun. 2009;10:125–131
39.Doyle SE, Schreckhise H, Khuu-Duong K, et al. Interleukin-29 uses a type 1 interferon-like program to promote antiviral responses in human hepatocytes. Hepatology. 2006;44:896–906
40.Hou W, Wang X, Ye L, et al. Lambda interferon inhibits human immunodeficiency virus type 1 infection of macrophages. J Virol. 2009;83:3834–3842
41.Ank N, Iversen MB, Bartholdy C, et al. An important role for type III interferon (IFN-lambda/IL-28) in TLR-induced antiviral activity. J Immunol. 2008;180:2474–2485
42.Ghany MG, Strader DB, Thomas DL, et al. Diagnosis, management, and treatment of hepatitis C. Hepatology. 2009;494:1335–1374
43.Grebely J, Petoumenos K, Hellard M, et al. Potential role for interleukin-28B genotype in treatment decision-making in recent hepatitis C virus infection. Hepatology. 2010;52:1216–1224
44.Akuta N, Suzuki F, Hirakawa M, et al. Amino acid substitution in hepatitis C virus core region and genetic variation near the interleukin 28B gene predict viral response to telaprevir with peginterferon and ribavirin. Hepatology. 2010;52:421–429
45.Thio CL, Thomas DL. Interleukin-28b: a key piece of the hepatitis C virus recovery puzzle. Gastroenterology. 2010;138:1240–1243

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.

Source

Phase 1b study of pegylated interferon lambda 1 with or without ribavirin in patients with chronic genotype 1 hepatitis C virus infection - publication pdf attached

publication pdf attached, figure 5 is very useful

Andrew J. Muir 1,2,*,, Mitchell L. Shiffman 3, Atif Zaman 4, Boris Yoffe 5, Andrew de la Torre 6, Steven Flamm 7, Stuart C. Gordon 8, Paul Marotta 9, John M. Vierling 10, Juan Carlos Lopez-Talavera 11, Kelly Byrnes-Blake 12, David Fontana 12, Jeremy Freeman 12, Todd Gray 12, Diana Hausman 12, Naomi N. Hunder 12, Eric Lawitz 13

Hepatology Article first published online: 14 MAY 2010

"The findings from this study offer evidence that PEG-IFN-λ, given QW for 4 weeks, exhibits potent antiviral effects against HCV with the potential for an improved tolerability profile with respect to that traditionally observed for PEG-IFN-α. These results are being tested in larger randomized controlled trials enrolling treatment-naive patients."

ABSTRACT

Interferon lambda 1 (IFN-λ1) is a type III IFN that produces intracellular responses similar to those of IFN-α but in fewer cell types because of differences in the receptor distribution pattern, and this could potentially result in an improved safety profile. This was an open-label three-part study of patients with chronic hepatitis C virus (HCV) genotype 1 infection. Part 1 evaluated single-agent pegylated interferon lambda (PEG-IFN-λ) at 1.5 or 3.0 µg/kg administered every 2 weeks or weekly for 4 weeks in patients who had relapsed after previous IFN-α-based treatment. Part 2 evaluated weekly doses of PEG-IFN-λ ranging from 0.5 to 2.25 µg/kg in combination with ribavirin (RBV) for 4 weeks in treatment-relapse patients. Part 3 evaluated weekly PEG-IFN-λ at 1.5 µg/kg in combination with RBV for 4 weeks in treatment-naive patients. Fifty-six patients were enrolled: 24 patients in part 1, 25 patients in part 2, and 7 patients in part 3. Antiviral activity was observed at all PEG-IFN-λ dose levels (from 0.5 to 3.0 µg/kg). Two of seven treatment-naive patients (29%) achieved rapid virological response. Treatment was well tolerated with minimal flu-like symptoms and no significant hematologic changes other than RBV-associated decreases in hemoglobin. The most common adverse events were fatigue (29%), nausea (12%), and myalgia (11%). Six patients experienced increases in aminotransferases that met protocol-defined criteria for dose-limiting toxicity (DLT) or temporarily holding therapy with PEG-IFN-λ. Most DLT occurred in patients with high PEG-IFN-λ exposure. Conclusion: Weekly PEG-IFN-λ with or without daily RBV for 4 weeks is well tolerated with minimal adverse events and hematologic effects and is associated with clear antiviral activity across a broad range of doses in patients with chronic HCV. (HEPATOLOGY 2010;)

The World Health Organization estimates that 180 million people worldwide (3% of the world population) are infected with hepatitis C virus (HCV), and 130 million of these are chronic HCV carriers.1 Chronic HCV infection is responsible for 50% to 76% of all liver cancer cases, two-thirds of all liver transplants in the developed world, and considerable morbidity and mortality. Identifying effective treatments for chronic HCV infection is therefore a global health priority.1

Consensus guidelines for the treatment of hepatitis C recommend a regimen of pegylated interferon alfa (PEG-IFN-α) and ribavirin (RBV).2 However, this treatment regimen results in sustained virological response (SVR) rates of only 40% in patients with genotype 1 HCV, and associated adverse events include flu-like symptoms, fatigue, depression, anxiety, and bone marrow suppression, which results in anemia, neutropenia, and thrombocytopenia.2-6 PEG-IFN-α is contraindicated in patients with major uncontrolled depressive illness and should be used with caution in patients with any psychiatric illness.2, 5-7 Other contraindications for the use of interferon alpha (IFN-α)-based regimens include hepatic decompensation, autoimmune disease, and severe concurrent medical disease such as chronic obstructive pulmonary disease, congestive heart failure, or significant coronary artery disease.2, 6, 7 In addition to the negative impact on quality of life, adverse events and laboratory abnormalities often lead to dose reductions or discontinuation of IFN-α therapy, which further compromises efficacy.2, 3, 8, 9

IFN-λ1, also known as interleukin-29 (IL-29), is a type III IFN with functional similarities to type I IFNs, which include IFN-α and IFN-ß. Like IFN-α and IFN-ß, IFN-λ1 is induced in response to viral infections such as hepatitis C and has demonstrated antiviral activity in vitro, including inhibition of HCV RNA replication in the replicon model.10 IFN-λ1 interacts with the structurally unique IFN-λ1 receptor complex to stimulate an intracellular response through phosphorylation of the Janus kinase/signal transducer and activator of transcription pathway (similar to the mechanism of action of IFN-α) and leads to the up-regulation of IFN-stimulated genes and an antiviral effect.11, 12 Unlike the widely distributed IFN-α receptor, expression of the IFN-λ1 receptor is more restricted. Although all cell types in the liver express the IFN-α receptor, the IFN-λ1 receptor is found only in hepatocytes. Similarly, although all peripheral blood leukocytes, including B, T, and natural killer cells, neutrophils, and monocytes, express the IFN-α receptor, messenger RNA of the IFN-λ1 receptor is not expressed in hematopoietic cells with the exception of B lymphocytes.10, 13 The limited distribution of the IFN-λ1 receptor suggests the potential for reduced adverse events with IFN-λ1-based therapy in comparison with IFN-α-based therapy along with preservation of the antiviral effect in HCV.

PEG-IFN-λ, a conjugate of a recombinant form of human IFN-λ1 and a 20-kDa linear polyethylene glycol chain, is currently under development for the treatment of chronic HCV infection. A phase 1a, placebo-controlled, dose escalation study of single subcutaneous doses of PEG-IFN-λ in healthy subjects was recently completed.14 PEG-IFN-λ was well tolerated at pharmacologically active doses without the toxicities typically associated with PEG-IFN-α. The estimated half-life was 50 to 80 hours, and the time to the maximum concentration was 8 to 24 hours. PEG-IFN-λ, starting at the 1.5 µg/kg dose, demonstrated dose-dependent biological activity with the induction of increases in serum ß2-microglobulin (ß2M). Here we describe the results of a three-part study assessing the safety and antiviral activity of PEG-IFN-λ with or without RBV over 4 weeks in patients infected with genotype 1 chronic HCV.

Patients and Methods

Study Design.

This was a three-part dose and schedule escalation study of PEG-IFN-λ administered subcutaneously as a single agent or in combination with RBV in patients chronically infected with HCV genotype 1 who had relapsed after IFN-α-based treatment (parts 1 and 2) or who were naive to treatment (part 3). Part 1 of the study evaluated escalating doses of PEG-IFN-λ monotherapy administered either every 2 weeks (Q2W) or weekly (QW) for a total of 4 weeks. Parts 2 and 3 of this study evaluated a range of doses of PEG-IFN-λ administered QW in combination with RBV twice daily for 4 weeks. All patients were followed for at least 4 weeks after the completion of treatment.

Figure 1 summarizes the treatment schema for the three parts of the study. PEG-IFN-λ doses ranging from 0.5 to 3.0 µg/kg were evaluated. PEG-IFN-λ was supplied at a concentration of 10 mg/mL, and a two-step dilution was required to achieve the final dose for subcutaneous administration. No dose modifications were allowed. In parts 2 and 3, RBV (Copegus, Roche Laboratories, Inc., Nutley, NJ) was administered orally twice daily to achieve a total dose of 1000 mg for patients < 75 kg or 1200 mg for patients ≥ 75 kg. The primary endpoints of the study were safety and tolerability. Secondary endpoints included HCV RNA reduction and pharmacokinetic analysis of PEG-IFN-λ serum concentrations.

Each cohort consisted of at least six evaluable patients. To be considered evaluable, a patient had to have completed all study visits through day 29 (Q2W cohorts) or day 36 (QW cohorts) unless the reason for not doing so was PEG-IFN-λ-related toxicity. A dose level or schedule was considered not tolerated if two or more patients experienced dose-limiting toxicity (DLT) or if two or more patients were unable to receive all planned doses because of treatment-related toxicity. Data from each cohort were reviewed by a safety monitoring committee.

Patients.

Parts 1 and 2 enrolled patients with chronic HCV infection who had relapsed after at least 12 weeks of prior treatment for HCV with either PEG-IFN-α or another IFN-α in combination with RBV. Part 3 enrolled treatment-naive patients. Patients had HCV genotype 1 infection and serum HCV RNA levels ≥ 100,000 IU/mL at enrollment. Inclusion criteria included alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels ≤ 2.5 times the upper limit of normal with no evidence of decompensated liver disease or hepatocellular carcinoma and documented liver biopsy within 2 years of study enrollment with an Ishak score ≤ 4.15 Patients were excluded if they had significant cardiac disease or a medical condition requiring immunosuppressive therapy. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and was consistent with good clinical practice guidelines and requirements of the regulatory authorities at each institution. All patients provided written, informed consent.

Safety Assessments.

All patients who received at least one dose of PEG-IFN-λ were included in the safety analyses. The evaluation of safety included adverse event monitoring, physical examination, clinical laboratories (hematology and serum chemistry), electrocardiogram, and echocardiogram. The National Cancer Institute's Common Terminology Criteria for Adverse Events (version 3.0) was used to evaluate adverse event severity and laboratory toxicities.

DLT included any clinical adverse event ≥ grade 3 in severity that was at least possibly related to PEG-IFN-λ treatment, with the exceptions of transient (<72-hour) grade 3 fatigue, fever, or rigor. In addition, a >5-fold increase in ALT or AST levels from the baseline (≥grade 2) or a >2-fold increase in ALT or AST levels from the baseline (≥grade 2) with a grade 2 elevation in bilirubin was considered DLT. If the ALT or AST level increased to more than 3 times the baseline (≥grade 2) or more than 7 times the upper limit of normal, but the bilirubin level or international normalized ratio did not increase to grade 2, PEG-IFN-λ was to be held until the ALT or AST level returned to less than 2 times the baseline value.

Pharmacokinetics.

Pharmacokinetic samples were collected at selected time points throughout the study. Samples were analyzed with a fully validated method developed at ZymoGenetics with mesoscale discovery electrochemiluminescent technology to quantify the PEG-IFN-λ serum concentration. The lower limit of quantification of the assay was 0.125 ng/mL in human serum. The PEG-IFN-λ serum concentration versus time profile for each patient was evaluated by noncompartmental methods with the software WinNonlin 5.2.1 (Pharsight Corp., Cary, NC). The maximum serum concentration (Cmax) and the area under the curve (AUC0-t) were estimated for each patient.

Immunogenicity.

Serum samples for evaluating antibody responses directed against PEG-IFN-λ were collected and analyzed with a fully validated method developed at ZymoGenetics with mesoscale discovery electrochemiluminescent technology to detect antibodies to PEG-IFN-λ1. Analysis was performed by a tiered approach designed to confirm reactivity, quantify the antibody titer, and demonstrate specificity. Samples that were confirmed to be reactive and demonstrated specificity to the drug product were assayed for neutralizing activity in a cell-based assay. The lower limit of assay detection was 0.1 µg/mL. The cell-based neutralizing bioassay was qualitative in nature.

Pharmacodynamics.

Samples were collected for pharmacodynamic studies at selected time points during the study. ß2M levels were assessed with a competitive binding assay (R&D Systems, Minneapolis, MN). The lower limit of assay quantification was 0.4 µg/mL.

Efficacy Assessments.

HCV RNA levels were measured with the COBAS TaqMan HCV test (version 2.0, Roche Molecular Diagnostics, Pleasanton, CA) with a lower limit of detection of 25 IU/mL. A central laboratory with expertise in the serial measurement of HCV RNA levels was used (Covance, Indianapolis, IN).

Data Analysis Methods.

No formal hypothesis was proposed; however, a cohort size of six patients would allow for a greater than 80% probability of detecting an event with a true population incidence of 25% and allow an evaluation of antiviral activity. The presented data represent an intent-to-treat population. All statistical analyses were performed with SAS (version 9.1.3 or higher, SAS Institute, Inc., Cary, NC) or other commercially available validated software.

RESULTS

Patient Demographics and Baseline Characteristics.

Fifty-six patients were enrolled from 10 sites in the United States: 24 with treatment-relapse disease in part 1, 25 with treatment-relapse disease in part 2, and 7 with treatment-naive disease in part 3 (Fig. 1). All but one patient completed the study through day 59. One patient in the cohort receiving 1.5 µg/kg PEG-IFN-λ QW plus RBV discontinued dosing after receiving PEG-IFN-λ through day 8 because of an adverse drug reaction to meperidine that was deemed unrelated to PEG-IFN-λ. Data from this patient were excluded from the efficacy assessment.

Patient demographics are shown in Table 1. All treatment-relapse patients had received at least one previous course of therapy with a PEG-IFN-α plus RBV except for two patients, one of whom had previously received treatment with albinterferon alfa-2b plus RBV and one of whom had previously received interferon alfacon-1.

Safety and Tolerability.

PEG-IFN-λ was well tolerated by most patients. Four of 56 patients (7%) had one or more doses withheld because of treatment-related toxicity. Most adverse events were mild or moderate in severity. Table 2 summarizes adverse events occurring in at least three patients in all cohorts combined. The most common adverse events were fatigue (29%), nausea (12%), myalgia (11%), and headache (9%). Among treatment-relapse patients, fatigue, nausea, myalgia, headache, diarrhea, irritability, pruritis, and insomnia were more commonly observed in patients treated with PEG-IFN-λ in combination with RBV versus those receiving single-agent therapy. PEG-IFN-λ plus RBV therapy was very well tolerated by treatment-naive patients, with fatigue, headache, and chills each reported by one of seven patients (14%) and with no patients reporting nausea, myalgia, diarrhea, irritability, pruritis, anorexia, influenza-like illness, or insomnia. The incidence of adverse events did not appear to be dose-related; adverse events were reported in 33.3%, 50%, 50%, and 50% of patients in part 1 cohorts receiving 1.5 µg/kg PEG-IFN-λ Q2W, 3.0 µg/kg PEG-IFN-λ Q2W, 1.5 µg/kg PEG-IFN-λ QW, and 3.0 µg/kg PEG-IFN-λ QW, respectively. Similarly, in part 2 (PEG-IFN-λ plus RBV), adverse events were reported in 83.3%, 83.3%, 85.7%, and 83.3% of patients receiving 0.5 µg/kg PEG-IFN-λ Q2W, 0.75 µg/kg PEG-IFN-λ Q2W, 1.5 µg/kg PEG-IFN-λ QW, and 2.25 µg/kg PEG-IFN-λ QW, respectively. Adverse events were reported in 42.9% of treatment-naive patients receiving 1.5 µg/kg PEG-IFN-λ QW plus RBV.

Two patients experienced other clinically important events considered related to PEG-IFN-λ. One patient treated with 3.0 µg/kg PEG-IFN-λ QW as a single agent experienced grade 3 idiopathic thrombocytopenic purpura, which occurred 2 weeks after the final dose; this event was considered DLT. The patient responded rapidly to therapy with oral prednisone. Another patient, treated with 1.5 µg/kg PEG-IFN-λ QW plus RBV in part 2, experienced elevated ALT, AST, and bilirubin levels, which met the protocol-defined criteria for withdrawing therapy. However, this patient received an additional dose of PEG-IFN-λ in violation of the protocol and subsequently experienced further elevation of ALT, AST, and bilirubin levels, which resulted in suspected medication-associated hepatotoxicity, pruritis, and elevated lipase and amylase levels (without associated clinical symptoms of pancreatitis).

Clinical Laboratory Evaluations.

Five patients experienced aminotransferase elevations that met the protocol-defined criteria for DLT, and an additional patient met the criteria for temporarily holding of a dose of PEG-IFN-λ (Table 3). Of these patients, four were in cohorts receiving the highest PEG-IFN-λ dose evaluated [3.0 µg/kg QW (n = 3) or Q2W (n = 1)], and two were in cohorts receiving 1.5 µg/kg PEG-IFN-λ QW with RBV (n = 1) or without RBV (n = 1). In three patients, the ALT and AST elevations were accompanied by a bilirubin elevation. All elevations in ALT, AST, and bilirubin were reversible, generally at or before the end of the study visit (day 59).

Grade 3 or 4 elevations in lipase and/or amylase not associated with abdominal pain or nausea were observed in four patients during the study; all were resolved after the withholding or discontinuation of PEG-IFN-λ. One patient had the day 15 dose held and was subsequently successfully treated on day 22, with no loss of antiviral effect observed that was related to the interruption. No other clinically important changes were noted in serum chemistry values.

Mean absolute neutrophil counts and platelet counts over time in cohorts receiving PEG-IFN-λ plus RBV are displayed in Fig. 2(A1,A2), respectively. There were no clinically significant changes in absolute neutrophil counts. Three patients with baseline absolute neutrophil counts in the low normal range of 2.04 to 2.30 x 109/L had decreases to 1.62 to 1.80 x 109/L; these were all isolated values that required no change in the studied therapy. One patient (described previously) experienced a grade 3 decrease in platelets during the posttreatment follow-up period, and this was concurrent with a diagnosis of idiopathic thrombocytopenic purpura; platelet decreases were not observed in other patients. Figure 2(B1,B2) displays the mean hemoglobin values in the cohorts that received PEG-IFN-λ as a single agent and PEG-IFN-λ plus RBV, respectively. No significant changes were observed in patients treated with single-agent PEG-IFN-λ, whereas decreases in hemoglobin consistent with the known effects of RBV were observed in patients treated with combination therapy.

Antiviral Activity.

Virological responses are summarized in Table 4. Antiviral activity was observed at all PEG-IFN-λ dose levels. Among the 43 patients dosed QW and evaluable for efficacy, all but 4 achieved a >1-log10 decline in HCV RNA during the study. The first day of treatment yielded a rapid decline in HCV RNA that continued in a biphasic manner from days 4 through 29; this was similar to the pattern observed with IFN-α.16 Higher PEG-IFN-λ doses were associated with greater declines in HCV RNA when PEG-IFN-λ was used both as a single agent and in combination with RBV (Fig. 3). At PEG-IFN-λ doses ≥ 1.5 µg/kg QW with or without RBV, 23 of 24 treatment-relapse patients (96%) achieved a >2-log10 decline in HCV RNA levels, and 4 of 24 (17%) achieved undetectable HCV RNA. Among treatment-naive patients, six of seven patients (86%) achieved a >2-log10 decline in HCV RNA levels, and they included two (29%) who achieved undetectable HCV RNA. Among patients dosed QW with PEG-IFN-λ with or without RBV, a >2-log10 decline in HCV was achieved by 21 of 28 white patients (75%), 8 of 8 African American patients (100%), and 6 of 7 Hispanic patients (86%).

Four treatment-relapse patients who received PEG-IFN-λ QW did not achieve a >1-log10 reduction in HCV RNA. Three of these patients were in the lowest PEG-IFN-λ dose cohorts; two were treated with 0.5 µg/kg PEG-IFN-λ QW plus RBV, and one was treated with 0.75 µg/kg PEG-IFN-λ QW plus RBV. The remaining patient without a detectable antiviral response was treated with 1.5 µg/kg PEG-IFN-λ QW plus RBV; this patient had preexisting neutralizing antibodies against PEG-IFN-λ and very low or undetectable serum levels of the study drug throughout the study period.

Pharmacokinetics.

The first-dose Cmax and AUC0-t values are summarized in Table 5. Two patients were excluded from the pharmacokinetic analyses; one patient who received PEG-IFN-λ (1.5 µg/kg) was excluded because of premature discontinuation of the study, and one patient who received 0.5 µg/kg PEG-IFN-λ was excluded because of Cmax and AUC0-t values grossly inconsistent with the nominal dose administered.

Overall, the exposure of PEG-IFN-λ appeared to be dose-dependent and ranged from a mean AUC0-t value of 11.6 h*ng/mL at the 0.5 µg/kg dose level to 148 h*ng/mL at the 3.0 mg/kg dose level. Normalization of AUC0-t by dose (µg/kg) appeared to remove the effect of dose on exposure, and this suggested that exposure may increase linearly with dose. A modest accumulation from week 1 to week 4 was observed with average patient Cmax and AUC0-t accumulation index values of 1.12 and 1.34, respectively. No obvious effect of RBV on the exposure to PEG-IFN-λ was detected. To explore the effect of body weight on exposure, AUC0-t/dose (µg) estimates were compared to the body weight. No trend was detected, and linear regression analysis indicated no apparent effect of weight on exposure.

Although there was no continuous relationship between exposure and change in ALT, very high exposure was observed in patients who demonstrated significant ALT elevations. Among patients dosed QW, six had a PEG-IFN-λ AUC0-t value > 225 h ng/mL; four of these patients (67%) experienced DLT. Conversely, there appeared to be a continuous relationship between PEG-IFN-λ exposure and decreases in HCV RNA, with robust antiviral activity observed at exposures both above and below 225 h*ng/mL (Fig. 4).

Pharmacodynamics.

A rapid rise in ß2M levels was observed after the first dose of PEG-IFN-λ; levels peaked on day 4 and decreased toward the baseline values by day 8. Increases in ß2M were observed at all dose levels, and this confirmed pharmacological activity. A potential relationship between ß2M induction and decreases in HCV RNA was observed. In general, patients who had the greatest HCV RNA decline on day 8 had the strongest induction of ß2M. In addition, minimal or no induction of ß2M was observed in patients with only minimal changes in HCV RNA on day 8, and this suggested that the lack of a virological response in these patients was likely related to an absence of pharmacological activity.

Immunogenicity.

Low-titer antibodies specific to PEG-IFN-λ developed in 3 of 56 patients (5.4%) during the study (all in the treatment-relapse group). Neutralizing activity was observed on day 59 in one of these patients (1.8%); this patient achieved a ≥2-log decrease from the baseline in HCV RNA on days 22 and 29.

Two additional treatment-relapse patients had detectable antibodies to PEG-IFN-λ prior to treatment and throughout the study period; neither had a titer increase during the study. One of these patients had neutralizing antibodies and had very low or unmeasurable PEG-IFN-λ serum levels and no antiviral response throughout the study period. The second patient had preexisting but nonneutralizing antibodies to PEG-IFN-λ and had measurable serum levels of PEG-IFN-λ as well as an antiviral response (a 3.81-log10 decrease on day 29).

DISCUSSION

This study was the first designed to assess the safety, tolerability, and efficacy of PEG-IFN-λ in patients chronically infected with genotype 1 HCV. The results indicate that a broad range of PEG-IFN-λ doses exhibit antiviral activity with limited toxicity when it is administered QW, and this supports the hypothesis that PEG-IFN-λ may be a useful agent in future HCV treatment regimens

SVR, the most definitive measure of antiviral activity, was not evaluated in this study because patients received only 4 weeks of therapy with PEG-IFN-λ with or without RBV. However, relationships between early viral kinetics and long-term response are well established and add clinical relevance to the 4-week antiviral results observed in the current study. Predictive markers of response, including rapid virological response (defined as undetectable HCV RNA at week 4) and early virological response (defined as a >2-log10 decrease in HCV RNA by week 12), have been demonstrated to predict response in treatment-naive patients treated with PEG-IFN-α plus RBV and in treatment-relapse patients as well.17-21 A recent report demonstrated that a ≥2-log10 decrease in HCV RNA by week 4 had a positive predictive value of 52% and a negative predictive value of 94% for predicting SVR in treatment-relapse patients receiving PEG-IFN-α in combination with RBV.19

Parts 1 and 2 of the study included patients who had previously responded to therapy with PEG-IFN-α, and this allowed a close examination of the safety profile in a group of patients who were expected to demonstrate antiviral activity (proof of concept) within a short treatment period. As anticipated, antiviral activity was observed in the majority of treatment-relapse patients, especially in those who received a dose of PEG-IFN-λ ≥ 1.5 µg/kg QW with or without RBV, with 23 of 24 patients (96%) achieving at least a >2-log10 decrease in HCV RNA. Although information on the viral kinetics of the patients' previous responses to IFN-α-based therapies was not collected as part of the study, this magnitude of HCV RNA decrease in the first 4 weeks of therapy indicates clear antiviral activity potentially comparable to that of PEG-IFN-α.

Unambiguous antiviral activity was also observed in the cohort of treatment-naive patients who were all treated with 1.5 µg/kg PEG-IFN-λ QW plus RBV. Six of the seven treatment-naive patients (86%) achieved a >2-log10 decrease in HCV RNA, and two of seven (29%) achieved undetectable HCV RNA; this was an encouraging result despite the small sample size. Historically published rates of >2-log10 decreases in HCV RNA or undetectable HCV RNA after 4 weeks of therapy with PEG-IFN-α plus RBV in treatment-naive patients range from 42% to 67% and from 7.4% to 11.7%, respectively.3, 16, 22

Recent evidence indicates that the viral response to PEG-IFN-α plus RBV may have a genetic basis.23, 24 Single nucleotide polymorphisms upstream from the IL-28B gene on chromosome 19 have been found to be associated with SVR to PEG-IFN-α plus RBV treatment as well as natural clearance of HCV infection.23-27 These links to IL-28B were initially found through genome-wide association studies, so the mechanism of action was not determined. The connection to PEG-IFN-λ is of interest because IL-28B is also a type III IFN known as IFN-λ3, which shares approximately 70% sequence identity with IFN-λ1 and shares the same receptor with IFN-λ1. The genes for IFN-λ1 (IL-29), IFN-λ2 (IL-28A), and IFN-λ3 (IL-28B) are all located in this same region of chromosome 19. It is possible that the IFN-λ family has unique antiviral effects important to the control of HCV infection, and studies to assess the impact of the different polymorphisms on the antiviral response to PEG-IFN-λ plus RBV are being conducted.

Overall, PEG-IFN-λ, given QW for 4 weeks, was safe and was well tolerated by most patients. Minimal constitutional symptoms or hematologic effects were observed with PEG-IFN-λ as a single agent. As expected, the addition of RBV increased the incidence of fatigue and other constitutional symptoms in treatment-relapse patients. Previously untreated patients experienced a low incidence of adverse events, despite combination therapy with RBV, and this suggested a possible experience effect from previous treatment in the treatment-relapse patients. The majority of significant aminotransferase elevations occurred in patients with high PEG-IFN-λ exposure values, which were well above the mean for the individual cohorts. Dose reductions were not allowed in the current study. However, because the aminotransferase elevations were reversible and may be related to high exposure, it may be possible in future studies to manage these effects with dose reductions.

Pharmacokinetic data suggest that the relationship between dose and exposure may be linear and that body weight is not an important determinant of exposure. Future studies will explore fixed microgram doses of PEG-IFN-λ; this is expected to be less complicated for self-administration than weight-based dosing. In addition, the use of a ready-to-use formulation in future studies may also reduce the interpatient variability in PEG-IFN-λ exposure estimates.

Only one patient developed a neutralizing antibody to PEG-IFN-λ during the study. Because the antibody was not observed until day 59, 4 weeks after the last dose of PEG-IFN-λ, no conclusions can be drawn regarding a potential effect on the PEG-IFN-λ serum concentration or antiviral activity. The incidence of neutralizing antibody formation with PEG-IFN-α is approximately 2% to 3%, although direct comparisons between different products may not be valid because the observed incidence of antibody positivity is highly dependent on the sensitivity of the assay.5, 6

These safety and tolerability results observed with the 4-week PEG-IFN-λ treatment compare favorably with the results of published studies of 48-week PEG-IFN-α therapy, in which up to 67% of patients have experienced fatigue, 62% have experienced headache, 56% have experienced myalgia, 48% have experienced rigors, 46% have experienced fever, 43% have experienced nausea, and 40% have experienced insomnia.3, 28 Although direct comparisons with the safety of PEG-IFN-α therapy are not possible because of the 4-week treatment duration in the current study, the incidence of constitutional side effects observed with PEG-IFN-λ therapy appears to be lower. The myelosuppression associated with IFN-α treatment is related to its binding to leukocytes.29 Although adverse hematologic events may appear after 4 weeks of therapy, the absence of IFN-λ binding to leukocytes may explain why such adverse effects have not yet been observed with PEG-IFN-λ therapy.

Key limitations in the current study include the lack of a direct comparison between PEG-IFN-λ and PEG-IFN-α, and this restricts any conclusions regarding the relative effects of one agent with respect to the other. Because the current study encompassed only 4 weeks of dosing, the long-term effects of PEG-IFN-λ could not be determined. The open-label design of the study may have influenced adverse event reporting. Lastly, the study included only patients with genotype 1 HCV infection, and it is not known if these results will translate to patients with other genotypes. Future studies will explore these questions.

The findings from this study offer evidence that PEG-IFN-λ, given QW for 4 weeks, exhibits potent antiviral effects against HCV with the potential for an improved tolerability profile with respect to that traditionally observed for PEG-IFN-α. These results are being tested in larger randomized controlled trials enrolling treatment-naive patients.

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