Hepatitis C Virus and Interferon Type

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REVIEW 10.1111/1469-0691.12797

Hepatitis C virus and interferon type III (interferon-k3/interleukin-28B and interferon-k4): genetic basis of susceptibility to infection and response to antiviral treatment

E. Riva1, C. Scagnolari2, O. Turriziani2 and G. Antonelli2 1) Department of Integrated Research, Virology Section, University Campus Bio-Medico of Rome and 2) Department of Molecular Medicine, Virology Section, Sapienza University of Rome, Rome, Italy

Abstract

There has been a signiﬁcant increase in our understanding of the host genetic determinants of susceptibility to viral infections in recent years. Recently, two single-nucleotide polymorphisms (SNPs), rs12979860 T/C and rs8099917 T/G, upstream of the interleukin (IL)-28B/interferon (IFN)-k3 gene have been clearly associated with spontaneous and treatment-induced viral clearance in hepatitis C virus (HCV) infection. Because of their power in predicting the response to IFN/ribavirin therapy, the above SNPs have been used as a diagnostic tool, even though their relevance in the management of HCV infection will be blunt in the era of IFN-free regimens. The recent discovery of a new genetic variant, ss469415590 TT/DG, upstream of the IL-28B gene, which generates the novel IFN-k4 protein, has opened up a new and alternative scenario to understand the functional architecture of type III IFN genomic regions and to improve our knowledge of the pathogenetic mechanism of HCV infection. A role of ss469415590 in predicting responsiveness to antiviral therapy has also been observed in HCV-infected patients receiving direct antiviral agents. The underlying biological mechanism that links the above IL-28B polymorphisms (in both IFN-k3 and IFN-k4)to spontaneous and treatment-induced clearance of HCV infection remains to be discovered. Despite this, shedding some light on this issue, which is the main aim of this review, may provide new insights into the general topic of ‘host genetics and viral infections’.

Keywords: HCV infection, host genetics, interferon-k3, interferon-k4, single-nucleotide polymorphisms Article published online: 1 October 2014 Clin Microbiol Infect 2014; 20: 1237–1245

Corresponding author: E. Riva, Campus Bio-Medico University, via A del Portillo 200, 00128 Rome, Italy E-mail: [email protected]

Introduction variants involved in treatment response in patients suffering from chronic viral infections. In recent years, there has been a significant improvement in Nevertheless, it should be mentioned that, due to the our understanding of the host genetic and genomic determi- rapid moving of the issue, it is quite difficult to obtain a full nants of susceptibility to viral infections, disease progression, picture of the field at present. This is why this review will and response to antiviral therapy. Indeed, common variants in focus only on the specific and limited issue of the some host loci that clearly influence viral disease progression association of common single-nucleotide polymorphisms have been documented at all levels, i.e. viral receptors, (SNPs) in the interferon (IFN)-k gene with disease outcome Toll-like receptors, DNA sensors, restriction factors, induc- and treatment response in chronic hepatitis C (CHC). We tion and production of cytokines, and/or the immune function believe that this is one of the best characterized examples of specific cellular effectors. A long list of host genes of the interplay between host genetics and chronic viral potentially affecting the natural course of viral diseases and infection that strongly suggests the consideration of individ- the effectiveness of antiviral therapy can therefore be ualization of antiviral therapy to effectively control viral provided. Table 1 shows the main consolidated host genetic infections.

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TABLE 1. Main host genomic determinants and therapy outcome

Acronym Infection Effect References

Interleukin-28B IL-28B HCV Spontaneous clearance and treatment response (SVR) 15–18 rs12979860 (T/C) and rs8099917 (T/G) Interferon-k4 IFN-k4 HCV Spontaneous clearance and treatment response (SVR) 13,64–66 ss469415590 or rs368234815 IFN-k4-TT/DG Inosine triphosphate tyrophosphatase ITPA HCV Ribavirin reduced anaemia induced by ITP deﬁciency 70 rs6051702 (A/C) rs1127354 (C/C, A/C, A/A), s7270101 (A/A, A/C, C/C) Vitamin D receptor VDR HCV Treatment response (SVR) 71 rs2228570 (T/C) Human leukocyte antigen C HLA-C HCV Treatment response 72 HLA-C (C1/C2) Killer immunoglobulin-like receptors KIR HCV Treatment response (SVR) 72 KIR2DL2 or 2DL3 KIR2DL3 in combination with HLAC2C2 Human leukocyte antigen A HLA HCV Treatment response (SVR) 73 HLA-DQB1*0301 and HLA-A*0201 in synergism with rs12979860 CC Low-density lipoprotein cholesterol LDLR HCV Treatment response (SVR) 74 receptor LDRL levels Human cytochrome P450 2B6 CYP2B6 HIV Efavirenz treatment decision and discontinuation 75 CYP2B6 516 (T/T) genotype is associated with higher efavirenz plasma exposure and increased CNS toxicity Human leukocyte antigen B HLAB*5701 HIV Abacavir treatment decision and discontinuation 76 Development of immune-mediated hypersensitivity reaction to abacavir Human leukocyte antigen A HLA-DRB*0101 HIV Nevirapine treatment decision and discontinuation 75 Nevirapine-associated hypersensitivity

CNS, central nervous system; HCV, hepatitis C virus; ITP, inosine triphosphate; SVR, sustained virological response.

IFN and Hepatitis C Virus (HCV) Infection ance of the virus from the blood) to be obtained in approximately 50% of the patients infected with HCV genotype 1 (GT1 HCV), the most widely distributed and Infection by HCV, which was discovered only in 1989 with IFN-a-resistant genotype of HCV. Actually, several new drugs molecular techniques [1], represents a significant global health (with both host and virus targets), which are expected to cure issue, with an estimated prevalence of 130–200 million >90% of HCV infections, have reached or are going to reach infected individuals worldwide. So far, on the basis of their the market, probably making IFN-free regimens available in the genome sequences, six major genotypes (designated 1–6) and near future. several subtypes (named in alphabetical order from ‘a’) of HCV Since the beginning of IFN therapy, it was evident that a have been characterized. It is well known that the genotypes of proportion of patients did not respond to the therapy or HCV differ mainly in their response to antiviral therapy. relapsed after an initial response, and a significant variability in Acute HCV infections are usually asymptomatic and, even in response, even early after the first injection of IFN [2], could western countries, very rarely diagnosed. In some of the be demonstrated. This issue was addressed very intensely, and infected subjects, the infection is spontaneously eradicated, but soon many viral and host factors affecting the treatment the virus may persist in a high percentage (70–80%) of the response were identified (e.g. viral genotype, viral load level, infected individuals, leading to CHC. These subjects are at risk and wideness of viral quasi-species variability at the beginning (15–25%) of developing liver cirrhosis and hepatocarcinoma of therapy; acute vs. chronic infection; gender and age; level of after 10–40 years of infection, owing to the continuing fibrosis; body mass index and weight; insulin resistance; and necrosis and inflammation in the liver. presence of comorbidities). In the meantime, it was being Until recently, the standard antiviral therapy was based on recognized that the above factors could only partially explain IFN-a, a virus-induced cytokine that is rapidly produced in the the IFN response variability, and that, on the basis of the liver of HCV-infected subjects during the first 4–10 weeks of relevance of ethnicity to the outcome of the disease, the infection, and that is able to induce the expression of a number response to IFN may also have a genetic basis. Thus, of specific genes, collectively called IFN-stimulated genes (ISGs), researchers started to believe that there could be other coding for proteins with direct or indirect antiviral properties. important factors, mainly host genetic factors, that played a This therapy has undergone significant evolution and improve- pivotal role in the natural course of the disease, and that could ment over the years [from 1989 to 2012, when pegylated IFN also profoundly affect the response to antiviral therapy based (PEG) and the nucleoside analog ribavirin (RBV) were used], on a biological modifier, such as IFN. allowing a complete virological response (i.e. the disappear-

ª2014 The Authors Clinical Microbiology and Infection ª2014 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, 1237–1245 CMI Riva et al. Interferon Type III and HCV infection 1239

A number of studies were tentatively published on the issue gene resides on the short arm of chromosome 19 (19q13.13) [3–11], until genome-wide association studies (GWASs), which and encodes IFN-k3, which, together with IFN-k1 (IL-29), allowed study of the association between mapped SNPs and IFN-k2 (IL-28A) [12], and IFN-k4 [13], constitutes the IFN-k the presence of common conditions in large patient cohorts, family. Owing to their molecular structure, type III IFNs provided evidence that host genetic factors may profoundly belong to the IL-10 superfamily, but functionally they are affect the natural history of many diseases, including CHC and closely related to type I IFNs, which play a major role in its treatment. Specifically, these studies demonstrated a strong antiviral immunity [14]. Like type I IFNs, IFN-ks can be association between the presence of variants in the IFN-k3 triggered by viral infections and, through interaction with (also known as interleukin (IL)-28B) genetic region and the specific receptors distinct from those of type I IFNs, may PEG/RBV treatment response in CHC. induce antiviral, immunomodulatory and antitumour activity This article reviews the history and the impact of the and the expression of ISGs, although to a different extent to breathtaking discoveries that led to the characterization of this type I IFNs and with different kinetics. issue and to the discovery of a completely new and unexpected way to consider the response to IFN therapy. Genetics of IFN-k3/IL-28B: association with treatment of chronic hepatitis and the natural course of HCV infection IFN-k family As already stated, in 2009 and 2010, four independent GWASs Briefly, IFNs are currently categorized into three different on treatment response to PEG/RBV in CHC patients were families: type I IFNs (IFN-a and IFN-b, with least 13 published [15–18]. In each of these GWASs, only SNPs around subtypes), type II IFN (IFN-c), and type III IFN (IFN-k1–4) the IL-28B gene reached genome-wide significance for treat- (Table 2). The last of these types has only recently been ment outcome and were significantly associated with sponta- characterized. IFN-ls 1–3 overlap with the previously neous and treatment-induced viral clearance in GT1 identified IL-28 and IL-29, and their genes all mapped HCV-infected patients, called the sustained virological together on chromosome 19 (Fig. 1). Specifically, the IL-28B response (SVR).

TABLE 2. Main characteristics of human interferons (IFNs)

IFN-a IFN-b IFN-e IFN-j IFN-x IFN-c IFN-k

Type of IFN I I I I I II III Chromosomal localization 9 9 9 9 9 12 19 Gene structure Intronless Intronless Intronless Intron Intronless Introns Introns No. of subtypes 13 1 1 1 1 1 4a Receptor IFN-AR1/R2 IFN-AR1/R2 IFN-AR1/R2 IFN-AR1/R2 IFN-AR1/R2 IFN-GR1/R2 IL-28R1/IL-10R2

aIFN-k1 (interleukin (IL)-29), IFN-k2 (IL-28A), IFN-k3 (IL-28B), IFN-k4 (dinucleotide genetic variant ss469415590 DG; ref. 13 in the text).

Chromosome 19

p13.3 p13.2 p13.1 p12 q12 q13.2 q13.3

IFN-λ3 (IL-28B) IFN-λ4 IFN-λ2 (IL-28A) IFN-λ1 (IL-29A)

rs 12979860 SNP ss 469415590 ΔG rs 8099917 SNP ss 469415590 TT Frameshift

k k FIG. 1. Interferon (IFN)- 3 and IFN- 4 T T/G G T/C C TT/ΔG T variants on chromosome 19. IL, G C Good response TT interleukin; SNP, single-nucleotide Good response CC Poor response GG polymorphism. Poor response TT

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Although many different SNPs were initially tagged in or treatment response in GT1 HCV patients than the rs8099917 near the IL-28B region (i.e. rs12980275, rs8103142, rs8105790, TT genotype, whereas the unfavourable rs8099917 GG rs1188222, rs8099917, rs12979860, etc.) only two of them, genotype seems to be more important in predicting failure namely rs12979860 T/C and rs8099917 T/G, were subse- of treatment response. quently confirmed to be relevant in treatment outcome Data supporting a role for IL-28B genetic variation in (Fig. 1). Because all tagged SNPs were in strong linkage spontaneous viral clearance followed rapidly after, or were disequilibrium, it was concluded that the association initially obtained concomitantly with, the discovery of its role in the observed for the other SNPs was mainly driven by the two treatment response. Indeed, between 2009 and 2010, major SNPs, rs12979860 and rs8099917 [16], although, at that rs12979860 was shown to be strongly associated with time, it was not possible to conclude which was the causal spontaneous HCV clearance in patients with acute hepatitis C variant. Consistently, rs12979860 C (good-response allele) vs. [23], with the favourable rs12979860 CC genotype being T (poor-response allele) and rs8099917 T (good-response mainly linked to jaundice and spontaneous HCV resolution allele) vs. G (poor-response allele) showed the strongest [24]. These data were extended to rs8099917 by a second association among those so far characterized. group, who suggested that the non-favourable rs8099917 GG Both rs12979860 and rs8099917 SNPs are in linkage genotype is a common genetic variant negatively associated disequilibrium, and their relative allele and genotype frequen- with the spontaneous clearance of HCV infection [18]. cies vary among races. In particular, the favourable CC In addition to their striking role in spontaneous and genotype was more common in Caucasians than in African– treatment-induced clearance, IL-28B genetic variations seem Americans, and it was estimated that frequency of this to be linked to various phenotypes in HCV infection. genotype was responsible for approximately half of the Good-response alleles were, indeed, associated with the recognized discrepancy in the treatment response rates activity of CHC in terms of more severe necroinflammation between the two populations. Random sampling of healthy in liver biopsies, and with high alanine aminotransferase, populations then identified the rs12979860 CC genotype as aspartate aminotransferase [25], apolipoprotein B100, and the most common in Asian patients, suggesting that this c-glutamyl transpeptidase [26]. Moreover, a significant associ- genetic variant may also contribute to the high response rates ation of the rs12979860 CC genotype with higher low-density that have been reported in Asians [19]. Also, the allele lipoprotein levels and other serum lipid parameters was frequencies of rs8099917 differ worldwide, making its power observed in HCV-infected patients [27]. in predicting an SVR weak in African–Americans [15]. In most Conflicting data have been published on the association studies, the rs12979860 CC genotype was associated with a between IL-28 polymorphisms and the risk of HCV-related more than two-fold higher chance of achieving an SVR than the cirrhosis and fibrosis; however, it is generally accepted that TT genotype. These findings were comparable in patients of IL-28 SNPs may influence the frequency of steatosis [28]. The European–American, African–American and Hispanic ancestry, effect of genetic IL-28B variations on spontaneous and although SVR rates in African–American patients were gener- treatment-induced clearance of HCV infection, as well as on ally lower than those in European–American patients. A more the natural course of infection, points to an important role of recent study on selected populations confirmed that the IFN-ks in the pathogenesis and management of CHC, but the rs12979860 CC genotype is the strongest pretreatment underlying mechanism and its effectors are largely unknown. predictor of response [20–22], and that it is independent of In this regard, it has been intriguingly suggested that other other pretreatment clinical predictors, including HCV geno- host factors, related or not related to IL-28, may have an type. Some of these studies also demonstrated that the additive effect with IL-28B SNPs in predicting spontaneous rs12979860 genotype is strongly associated with very early virus clearance and/or treatment response in both acute and on-treatment viral kinetics [21], which, in turn, leads to a more chronic HCV infection. Among these are: pretreatment plasma rapid virological response (RVR). Thus, the association of levels of IFN-c-inducible protein 10 [29,30]; serum levels of IL-28B SNPs with RVR and SVR appears to be related to a vitamin D [31]; HLA-C antigen (HLAC2C2) or immunoglob- strong impact on very early kinetics during treatment, which ulin-like receptors (KIR2DL3 and KIR2DS1) [32]; and expres- may reflect the antiviral effectiveness of IFN-a. Some GWASs sion levels of specific HLA class II genes (DQB1*0301) [33]. [16–18] and recently published studies [22] have indepen- Other studies suggest that SNP rs10903035 at IL-28 recep- dently identified the rs8099917 GG genotype as the strongest tor-a is associated with the early treatment failure in human marker of treatment failure, independently of other clinical immunodeficiency virus–HCV-coinfected patients, and it has variables. All together, these data support that the idea that been proposed that the combination of IL-28B and IL-28 the rs12979860 CC genotype is more efficient in predicting receptor-a polymorphisms might be a valuable tool for

predicting early treatment failure before starting HCV treat- protein production or structure, these polymorphisms may ment [34]. exert a functional effect through regulation of gene expression, Altogether, the above evidence supports the existence of a mRNA splicing of receptors, and/or binding/activation of other very complex interplay between host genetic and biochemical, cytokines. inflammatory and immune factors, among which IFN-k finally Because IFN-ks seems to affect viral replication and control leads to the regulation of the HCV infection response. the infection [43], it was reasonable to consider the possibility that the expression level of endogenous IFN-k3 could be Genetics of IFN-k3/IL-28B in infection with HCV genotypes influenced by genetic variants. Recent data suggest that IL-28B other than genotype 1 SNPs may affect IFN-k expression via altering DNA methyl- Most of the initial studies on IL-28B and HCV were focused GT1 ation and transcription factor-binding sites [44]. Nevertheless, HCV infections, because this HCV genotype was the most data on the expression levels in patients with different IL-28B resistant to IFN-based therapy. Several studies have clearly genotypes remain conflicting, and only a few studies have shown that the associations between IL-28B SNPs and RVR/SVR shown higher IFN-k3 mRNA levels in the liver and/or blood of in HCV genotype 4 patients were comparable to those obtained patients with good-response alleles [16,17,45]. for GT1 HCV subjects [18,35]. In contrast, data on IL-28B in In contrast, and perhaps more importantly, a number of patients infected with HCV genotype 2 (GT2 HCV) and HCV studies have clearly demonstrated that the IL-28B poor-response genotype 3 (GT3 HCV) have produced conflicting data and are minor alleles (both rs120979860 and rs8099917) are significantly few in number. All studies in GT2 HCV/GT3 HCV patients associated with high levels of ISG expression[42,46,47]. The yielded mixed results, although most of them failed to show a initially unexpected high expression levels of pretreatment ISGs significant association between IL-28B and an SVR [36–39]. The in non-responders have been repeatedly reported [3,48–51], scenario is different if one considers GT3 HCV separately or and the confirmed association between IL-28B genetic variations uses an adequate sample size. In fact, it has been recently and ISG expression, other than confirming that, paradoxically, reported from the largest studies that the rs12979860 CC pre-activation of the IFN system and/or high expression levels of genotype is an independent predictive factor for an SVR also in ISGs may represent one of the strongest predictors of GT3 HCV patients, whereas no association with an SVR in GT2 non-response to IFN therapy, indicates that the presence of HCV infection was observed [40,41]. These data clearly suggest minor alleles of IL-28B may result in a phenotype with a limited the relevance of sample size in determining the correlation action of IFN-mediated and IFN-inducible defence. between IL-28B genotype and RVR/SVR, and suggest a rationale In light of the above considerations, it could be speculated for testing IL-28B genotyping also in patients with GT3 HCV. that the poor-response allele might be associated with high expression levels of IFN-signalling inhibitors, leading to a Effects of genetics of IFN-k3/IL-28B on the mechanism of IFN persistent, but ineffective, high level of activation of ISG action expression triggered by the intracellular HCV genome. By Although the associations between IL-28B SNPs and the contrast, the good-response allele might be associated with outcome of treated and untreated CHC patients have been fully functional IFN-related endogenous effectors that are able repeatedly confirmed, the mechanism by which these changes to clear HCV and are still able to respond to IFN when it is influence the treatment response and the spontaneous clear- administered exogenously [26,52]. However, considering also ance of HCV is still unknown, and no resulting phenotype has that, in uninfected subjects, no association between ISG been associated with specific SNPs. Below, some consider- expression and IL-28 genotype has been demonstrated [53], ations are presented that make clear that the phenomenon is such an explanation might not provide any insights into the very complex and that further studies are warranted to draw direct link between IL-28B genetic variants and IFN-k3. Thus, definite conclusions on the issue. the underlying biological mechanism that links IL-28B poly- First, it is important to note that nearly all IL-28B genetic morphisms to spontaneous and treatment-induced clearance variations associated with spontaneous and treatment-induced of HCV infection remains to be discovered. clearance are not located within coding regions; they are located only in close proximity to the IL-28B coding region. Genetics of IFN-k3/IL-28B in the era of direct antiviral agents With fine-mapping strategies, only a non-synonymous coding (DAAs) variation (rs8103142) in exon 2 of IL-28B was identified, but As stated, the standard of care for patients with chronic thus far no functional differences between the resulting hepatitis C has consisted, until recently, of a combination of variants (Lys70Arg) have beeen recorded [42]. Thus, it has PEG and RBV administered for 24–48 weeks, depending on been proposed that, even without a direct impact on IFN the HCV genotype. The SVR rate for this standard of care has

ª2014 The Authors Clinical Microbiology and Infection ª2014 European Society of Clinical Microbiology and Infectious Diseases, CMI, 20, 1237–1245 1242 Clinical Microbiology and Infection, Volume 20 Number 12, December 2014 CMI

been only approximately 50% in patients infected with GT1 Genetics of IFN-k4: association with treatment of chronic HCV, the most prevalent genotype in Europe and North hepatitis and the natural course of HCV infection America. The therapy of CHC was revolutionized by the Recently, a dinucleotide frameshift variant in ss469415590 TT/ approval in 2011 of two direct antiviral agents (DAAs): the DG (also designated rs368234815 TT/DG; IFN-k4-TT/DG) NS3/4A serine protease inhibitors boceprevir and telaprevir. upstream of and in the same orientation as IFN-k3 was described The use of these DAAs in combination therapy with PEG/ on chromosome 19q13.13; this generates a novel IFN-k pro- RBV opened a new era in the field of CHC therapy, mainly in tein, designated IFN-k4, which shares only 29% homology at the GT1 HCV-infected patients. Triple therapy in previously amino acid level with the other three IFN-ks [13,64]. IFN-k4 can untreated patients raised overall SVR rates from 40–45% to be produced by individuals who carry the minor DG allele of the 68–79% [54,55]. All studies on boceprevir/telaprevir showed ss469415590 variant (IFN-k4-DG), whereas the presence of the an attenuated impact of IL-28B variants on the SVR, even major TT allele leads to a frameshift in exon 1 and disrupts the though patients with a favourable rs12979869 CC genotype IFN-k4 open reading frame, favouring HCV clearance and are more likely to be eligible for shorter treatment duration. improving the outcome after IFN–RBV combination treatment. In treatment-na€ıve patients or those subsequently treated The rs12979860 and rs8099917 IL-28B SNPs are located 367 bp with a combination of the ‘second wave’ NS3 protease downstream (intron 1) and 4 kb upstream of ss469415590, inhibitor simeprevir and PEG/RBV, IL-28B genotype has shown respectively. It has also been observed that the IFN-k4-creating a significant impact on the outcome of treatment [56–58]. ss469415590 DG allele is perfectly correlated with the unfa- Furthermore, the NEUTRINO study (NS5B nucleoside inhib- vourable rs12979860 T allele in Asians and is well correlated in itor sofosbuvir plus PEG/RBV) [59] identified the rs12979860 Europeans, whereas in Africans this correlation is only moderate SNP as an independent pretreatment predictor of an SVR. [13]. The correlation between ss469415590 and rs8099917 was More recently, data from INFORM-1 (the nucleoside poly- high in Asians and moderate in Europeans, but very low in merase inhibitor mericitabin plus the protease inhibitor Africans [13]. The ss469415590 SNP seems to be a better danoprevir) [60] and SOUND-C2 (the protease inhibitor marker than the rs12979860 SNP in predicting PEG/RBV faldaprevir plus the non-nucleoside polymerase inhibitor treatment response in CHC and possibly spontaneous HCV delobuvir) [61] seemed to support a significant impact of clearance in African–Americans, whereas these variants seem to IL-28B genotype also in IFN-free regimens, owing to the effects be similarly informative in European–Americans [13]. This of such polymorphisms on the kinetics of viral decay. polymorphism also appears to be a better marker than It is commonly believed that the effect of the genetics of rs12979860 in identifying human immunodeficiency virus– IL-28B will be less effective as more potent agents and HCV-co-infected patients who do not respond to antiviral combinations (with response rates up to almost 100%) become therapy [65]. Furthermore, IFN-k4 polymorphism has been available. In fact, data from phase III studies with new DAA shown to be independently associated with RVR in all viral combinations, especially those involving sofosbuvir in combina- genotypes (GT1 HCV to GT4 HCV4) and with an SVR in GT1 tion with NS5A inhibitors (daclatasvir or ledipasvir), have shown HCV/GT4 HCV, but not in GT2 HCV/GT3 HCV [66]. Interest- that it is possible to achieve high SVR rates independently of ingly a role of ss469415590 (TT or DG) in predicting response to IL-28B genotype [62,63]. Thus, the impact of IL-28B polymor- antiviral therapy has also been observed in HCV-infected phism in some all-oral IFN-free regimens seems to be moderate patients receiving DAAs in regimens without IFN. In particular, even more data are needed to evaluate the actual influence of in patients treated with sofosbuvir and RBV, IFN-k4-DG is IL-28B genotype on treatment success in such regiments, associated with slower early viral decay, owing to slower loss of especially for highly active regimens combining two or three free virus and decreased drug efficacy, suggesting a functional different DAA classes. It is, however, important to note that, relevance of IFN-k4 in IFN-free DAA therapies [67]. despite the moderate effect in some regimens, pretreatment Although the predictive power of this additional SNP was determination of IL-28B genotype might still have some use, rapidly confirmed by several studies [13,68], it is difficult to especially considering that IFN-free treatment will not afford- explain conceptually why the production of this novel IFN-k able in many parts of the world in the near future. Indeed, it is to protein, which shows anti-HCV activity [64] and triggers an be expected that, in developing countries and even in most antiviral response by inducing STAT1 and STAT2 phosphor- countries of eastern Europe, IFN and RBV will constitute the ylation and upregulating the expression of ISGs [13,64], may backbone of antiviral treatment for CHC in the coming years. have a deleterious effect on antiviral treatment. In this regard, The determination of IL-28B genotype might maintain its value in new findings have been recently published that make more individualizing therapy to optimize cost-effectiveness and avoid evident the complexity of the phenomenon: (i) ss469415590 side effects. TT/DG polymorphisms promote the methylation of adjacent

cytosine residues, as already suggested for IL-28B SNPs [44], appear anywhere in the genome, within, near or far away from thereby influencing gene expression [68]; and (ii) ss469415590 any coding sequence. Therefore we believe that the greatest genotypes seem to be not related to IFN-k2/3 or IFN-k1 gene challenge lies in combining GWAS findings with additional expression [69]. Further studies are needed to provide new molecular data to functionally characterize the associations. The information so that we can start to understand the function of advances in various -omics techniques have made possible to this new IFN-k. investigate the effects of risk variants at intermediate molecular levels, such as gene expression, methylation, protein abundance, and metabolite levels. As disease pathogenesis involves a Closing Remarks complex interplay between host and virus (and this perfectly applies to the issue described here, namely HCV infection), no The recent identification of the genetic polymorphisms in the single molecular analysis is expected to fully unravel the disease IL-28B gene region (in both the IFN-k3 gene and the IFN-k4 mechanism. Multiple molecular levels can interact and also show gene) as predictors of CHC treatment outcome is certainly an plasticity in different physiological conditions and disease stages. exciting finding. It provides new insights into virus–host There is therefore a great need for new integrative approaches interaction, and appears to have immediate clinical utility, that can combine data from different molecular levels and can not only as a diagnostic tool, but also as potential aid for both help to determine the causal path from genotype to phenotype. patients and physicians in considering IFN-based therapy. However, the treatment paradigm, especially in GT1 HCV Transparency Declaration infection, is rapidly evolving, owing to the introduction of DAAs in new highly active regimens. The impact of IFN-k3 and IFN-k4 polymorphisms and their interplay in this setting appear The authors declare no conflict of interest.. to be complex and need further investigation. Nevertheless, it is evident that these findings could References represent an intriguing example of host genomic contribution to disease progression and outcome, and provide the basis for 1. Choo QL, Kuo G, Weiner AJ, Overby LR, Bradley DW, Houghton M. individualization of antiviral therapy. Isolation of a cDNA clone derived from a blood-borne non-A, non-B Although, in the recent past, the assay for analysing the viral hepatitis genome. Science 1989; 244: 359–362. so-called IL-28 polymorphism has been utilized and has 2. Antonelli G, Riva E, Maggi F, Vatteroni ML, Simeoni E. 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