Interleukin-28B (IL-28B) genotype has a strong impact on both spontaneous hepatitis C virus (HCV) clearance and HCV clearance induced by treatment [1–10]. Studies focusing on single nucleotide polymorphisms (SNPs) near the IL-28B gene have shown that the C allele of the SNP rs12979860 is an important predictor of treatment response both in HCV-monoinfected [1,4,10] and coinfected patients [7–9]. Interestingly, the effect of variations in IL-28B genotype is mainly seen in carriers of HCV genotype 1 or 4, while the impact on genotype 3 carriers, if any, is minimal [5,7,8].
In several reports [4,5,7,8,10], infection with HCV genotype 3 in those with chronic hepatitis C (CHC) has been shown to be significantly more prevalent among patients with the rs12979860 CC genotype than among those with non-CC genotypes. Theoretically, there are two possible explanations for this finding. On the one hand, the rs12979860 CC genotype might exert more protection against the acquisition of infection with HCV genotype 1 or 4 than against the acquisition of infection with HCV genotype 3. On the other hand, once HCV infection has been acquired, the rs12979860 CC genotype may protect against evolution to CHC during the acute phase of infection to a greater extent in those carrying HCV genotype 1 or 4 than in patients infected with HCV genotype 3. Comparative data on the distribution of HCV genotypes according to IL-28B genotype in acute hepatitis C (AHC) vs. CHC may help us to clarify which of these two hypotheses is correct. In addition, this information may lead to a better knowledge of the determinants of the immune response to HCV. However, there are still no data available on the HCV genotype distribution in patients with AHC. To elucidate the influence of the IL-28B genotype on acquisition and chronification of infection with different HCV genotypes, we compared the HCV genotype distributions within subpopulations with different IL-28B genotypes in two cohorts of patients, one with AHC and the other with CHC.
Patients and methods
Patients with AHC
This group was part of a cohort of 85 HIV-infected patients with AHC, defined according to the criteria stated below, who were recruited in several German hospitals from May 2002 to September 2006 [9,11]. Eighty of these patients (94%) had an available HCV genotype determination and were included in the analysis. Eight (10%) patients experienced spontaneous clearance and 54 (67.5%) started therapy with pegylated interferon plus ribavirin. Frozen peripheral blood mononuclear cells (PBMCs) from all these patients were available.
Patients with CHC
This subpopulation consisted of 476 HCV treatment-naïve patients, who had been consecutively enrolled in one German and two Spanish cohorts of HIV/HCV-coinfected patients with CHC from October 2001 to June 2008. One hundred and fifty-four individuals had been recruited in the infectious diseases units of two university hospitals in southern Spain and 160 individuals in a reference HIV clinic located in Madrid. The remaining 162 patients belonged to a cohort followed in the Department of Internal Medicine I at the University of Bonn, Germany. Further details of these cohorts have been reported elsewhere [7–9,12]. A whole-blood or PBMC sample was collected from each patient and cryopreserved for genetic determinations.
A patient was defined as harbouring AHC if at least two of the following criteria were met within 4 months prior to diagnosis: known or suspected exposure to HCV, documented anti-HCV antibody seroconversion, or serum alanine aminotransferase (ALT) >350 IU/L with normal levels during the year before infection. Spontaneous clearance in AHC was considered to have occurred if HCV RNA became negative without treatment. Patients in whom HCV RNA remained detectable 12 weeks after diagnosis and who started therapy thereafter were considered not to have cleared HCV spontaneously. CHC was defined as a persistent elevation of serum transaminases for >6 months, along with positive serum antibodies against HCV and detectable plasma HCV RNA.
Determination of HCV infection markers
Serum HCV antibodies were determined using an enzyme immunoassay (EIA) test (ADVIA Centaur XP; Siemens Healthcare Diagnostics S.L., Tarrytown, NY, USA). Plasma HCV-RNA load was measured using a polymerase chain reaction (PCR) assay according to the available technique at the respective hospital and the moment of treatment (Cobas Amplicor HCV Monitor; Roche Diagnostic Systems Inc., Branchburg, NJ, USA: detection limit 600 IU/mL; Cobas AmpliPrep-Cobas TaqMan; Roche Diagnostic Systems Inc., Meylan, France: detection limit 50 IU/mL; Cobas TaqMan; Roche Diagnostic Systems Inc., Pleasanton, CA, USA: detection limit 10 IU/mL). HCV genotyping was performed using a real-time PCR hybridization assay (Versant HCV Genotype2.0 LIPA; Siemens Healthcare Diagnostics S.L.).
Determination of the IL-28B genotype
DNA was extracted from whole blood or PBMCs using the automated MagNA Pure DNA extraction method (Roche Diagnostics Corporation, Indianapolis, IN, USA). In patients with CHC from the Spanish cohorts, isolated DNA was genotyped for the rs12979860 SNP using a custom TaqMan genotyping assay (Applied Biosystems, Foster City, CA, USA), according to the manufacturer's instructions, and a Stratagene MX3005 thermocycler with mxpro software (Stratagene, La Jolla, CA, USA). In subjects with CHC from the German cohort, as well as those with AHC, IL-28B genotyping was performed using the LightSNiP Typing Assay (TIB MOLBIOL, Berlin, Germany) after amplification of isolated DNA using a LightCycler Instrument (Roche Diagnostics, Mannheim, Germany).
Hardy–Weinberg equilibrium was determined using haploview software (http://www.broadinstitute.org/haploview/haploview). In the descriptive analysis, qualitative variables are expressed as a percentage and quantitative variables as a median [first–third quartiles (Q1–Q3)]. The significance of differences between the study subpopulations in terms of demographic and clinical characteristics was evaluated using the χ2 test for categorical variables and the Mann–Whitney U-test for continuous variables. The association between HCV genotype and IL-28B genotype, as well as their impact on spontaneous clearance, was analysed. Also, the relationship between the IL-28B genotype and the following parameters was assessed: age, sex, HCV viral load, undetectable HIV viral load, CD4 cell count and plasma ALT level. The statistical analysis was carried out using the spss statistical software package release 15.0 (SPSS Inc., Chicago, IL, USA).
The study was designed and performed according to the Helsinki Declaration and was approved by the Ethics Committee of the Hospital Universitario de Valme.
Characteristics of the study population
In the group with CHC, one patient (0.2%) was Afro-American and the remaining 475 (99.8%) were Caucasians, mainly of Spanish (62.4%) and German (36.3%) origin. Among the patients with AHC, all were Caucasians of German ancestry. Three hundred twenty-five subjects with CHC (68.3%) had acquired HCV infection through drug injection, 35 patients (7.4%) were infected through sexual transmission, three (0.6%) were infected through blood transfusion and 113 (23.9%) were infected by unknown routes. Among subjects with AHC, all 80 patients with information available were infected through sexual contact. The median (Q1–Q3) time from diagnosis to starting therapy in this group was 6 (2–11) weeks. Forty-two (77.8%) of 54 patients with AHC who received therapy started it before week 12. Further characteristics of both populations are listed in Table 1.
Table 1. Patient characteristics
|Age (years)*||39.1 (36.1–43.7)||43 (39–47)||<0.001|
|Male gender [n (%)]||79 (98.8)||388 (81.5)||<0.001|
|rs12979860 genotype [n (%)]|
| CC||38 (47.5)||218 (45.8)||0.954|
| TT||7 (8.8)||45 (9.5)|| |
| CT||35 (43.8)||213 (44.7)|| |
|HCV genotype [n (%)]|
| 1||53 (66.3)||274 (57.6)||<0.001|
| 2||7 (8.8)||10 (2.1)|| |
| 3||10 (12.5)||147 (30.9)|| |
| 4||10 (12.5)||45 (9.5)|| |
|HCV RNA (log10 IU/mL)*||5.9 (5.34–6.38)||6.44 (5.64–6.84)||0.007|
|Undetectable HIV RNA [n (%)]#||40 (52)||250 (71)||<0.001|
|CD4 cell count (cells/μL)*||440 (346–610)||460 (333–647)||0.645|
|ALT (U/L)*||448 (248–891)||67 (45–105)||<0.001|
Relationship between IL-28B and HCV genotype
The IL-28B genotypes were in Hardy–Weinberg equilibrium (P=0.791 for AHC and 0.821 for CHC). The prevalence of the rs12979860 CC genotype was 47.5% among patients with AHC and 45.8% in those with CHC (P=0.778) (Table 1). In the group of individuals with AHC, 31 subjects with genotype CC (81.6%) were infected with HCV genotype 1 or 4 and 7 (18.4%) with genotype 2 or 3. Among CT/TT patients with AHC, 32 (76.2%) were infected with genotype 1 or 4 and 10 (23.8%) with genotype 2 or 3, respectively (P=0.948). In the group of patients with CHC, 119 (54.6%) of those with rs12979860 genotype CC were carriers of HCV genotype 1 or 4, while 99 (45.4%) were infected with HCV genotype 2 or 3. Of those harbouring rs12979860 genotype CT/TT, 200 (77.5%) bore HCV genotype 1 or 4 and 58 (22.5%) genotype 2 or 3 (P<0.001). A more detailed genotype distribution is shown in Table 2.
Table 2. Hepatitis C virus (HCV) genotype distribution in relation to interleukin-28B (IL-28B) genotype in patients with acute and chronic hepatitis C
|1||26 (68.4)||27 (64.3)||0.948||104 (47.7)||170 (65.9)||<0.001|
|2||3 (7.9)||4 (9.5)|| ||7 (3.2)||3 (1.2)|| |
|3||4 (10.5)||6 (14.3)|| ||92 (42.2)||55 (21.3)|| |
|4||5 (13.2)||5 (11.9)|| ||15 (6.9)||30 (11.6)|| |
In the subpopulation of patients with CHC enrolled in the German cohort, the distribution of HCV genotypes was also significantly different from that found in patients with AHC. Specifically, 41 (53.9%) German patients with CHC and rs12979860 CC harboured HCV 1 or 4 vs. 65 (75.6%) of those bearing CT/TT (P=0.034).
Relationship between IL-28B genotype and other factors
There were no significant differences in HCV plasma load among patients with different IL-28B genotypes in the overall population. Thus, the median (Q1–Q3) HCV-RNA level was 6.36 (5.68–6.88) log10 IU/mL in carriers of rs12979860 CC and 6.27 (5.59–6.79) log10 IU/mL in those harbouring CT or TT (P=0.458). However, HCV-RNA load was significantly higher in patients with AHC and the CC genotype than in those with AHC and rs12979860 CT/TT (Table 3). ALT levels in the entire population were higher in patients with the CC genotype [83 (58–165) in CC carriers vs. 74 (45–126) in CT/TT carriers; P=0.022]. The relationships between the IL-28B genotype and several parameters in the AHC and CHC groups are listed in Table 3.
Table 3. Relationship between rs12979860 genotypes and other factors
|Male gender [n (%)]||37 (97.4)||42 (100)||0.29||171 (78.4)||217 (84.1)||0.112|
|HCV RNA (log10 IU/mL)*||6.35 (5.61–6.53)||5.81 (5.25–6.36)||0.033||6.46 (5.75–6.92)||6.41 (5.61–6.83)||0.981|
|Undetectable HIV RNA [n (%)]†||19 (51.4)||21 (52.5)||0.92||113 (71.1)||137 (71)||0.986|
|CD4 cell count (cells/mL)*||395 (301–519)||486 (374–693)||0.029||456 (320–612)||464 (342–698)||0.174|
|ALT (U/L)*||483 (291–1007)||423 (194–626)||0.24||72 (50.8–112)||63 (43–99.3)||0.016|
Spontaneous clearance of HCV, as defined in this study, was documented in eight (10.1%) of the 79 patients in whom this information was available. There was no relationship between spontaneous clearance of the virus and HCV genotype. Thus, the numbers of patients who cleared HCV were as follows: six (11.3%) with genotype 1, one (12.5%) with genotype 2, one (10%) with genotype 3 and none with genotype 4 (P=0.746). The association between IL-28B genotype and spontaneous clearance did not reach statistical significance. Five (13.5%) of the patients with rs12979860 genotype CC and three (7.1%) of the patients with genotype CT or TT (P=0.349) showed spontaneous HCV clearance. The associations between IL-28B genotype and other factors are displayed in Table 3.
In this study, we found that the distribution of HCV genotypes in patients with CHC depended on the IL-28B genotype. Conversely, the proportions of HCV genotypes were similar, whatever the IL-28B genotype, in patients with AHC. The prevalence of HCV genotype 3 in CHC patients who were rs12979860 CC carriers was higher than that in subjects with genotypes other than CC. This finding provides indirect evidence suggesting that the favourable impact of IL-28B CC on spontaneous clearance of HCV is stronger in patients infected with genotype 1 or 4 than in those bearing genotype 3, similar to findings obtained for treatment-induced clearance [5,7,8].
In recent studies focusing on the impact of variations in the IL-28B gene on HCV treatment, it has been observed that the HCV genotype distribution is different for CC and non-CC genotypes in CHC patients [5,7,8,10]. However, no potential underlying mechanism for this finding has been reported to date. Our data confirm that the prevalence of genotype 3 is over twofold higher in genotype CC carriers among patients with CHC. Furthermore, this is the first study that has analysed the HCV genotype distribution in patients with AHC, according to IL-28B genotype. The finding that there was no difference in the HCV genotype distribution in AHC patients with different IL-28B genotypes supports the hypothesis that the susceptibility to infection with specific HCV genotypes is similar for patients with different IL-28B genotypes. However, the marked shift of the HCV genotype distribution in CHC suggests that the genotype CC provides greater protection against chronification of genotype 1/4 infection than against chronification of HCV genotype 3 infection.
Unfortunately, the population of patients with AHC included in this study was not large enough to allow direct testing of the hypothesis that the impact of the IL-28B genotype on spontaneous clearance is greater in patients with HCV genotype 1 or 4 than in those with genotype 3. Indeed, of the patients with AHC included in the study, only eight fulfilled the criteria for spontaneous clearance. This was probably mainly attributable to the fact that the rate of spontaneous clearance of HCV during AHC in HIV-coinfected patients is estimated to be below 20%, which is even lower than in HCV monoinfection [13,14]. In addition, a relatively high number of patients in the cohort with AHC started therapy against HCV earlier than 12 weeks after diagnosis, perhaps precluding the identification of some patients who would have cleared HCV spontaneously. Because of a lack of statistical power, even the impact of the IL-28B CC genotype on spontaneous clearance of all HCV genotypes, considered as a whole, which has previously been well documented [5,6,15], did not reach statistical significance in this analysis. However, studies with a sufficient sample size to demonstrate a significant impact of IL-28B genotype on the likelihood of self-clearance of specific HCV genotypes are very unlikely to be carried out, given the very limited number of patients with AHC who are identified, and the fact that a significant proportion of these patients receive early antiviral therapy.
Another potential limitation of this study is the different origins of the populations. While the AHC group mainly consisted of Central European individuals, who were infected by sexual transmission, the majority of patients in the CHC group were Southern European injecting drug users (IDUs). In both groups, the HCV genotype distribution was in accordance with the results of the EuroSIDA cohort study , which reported a slightly lower prevalence of genotypes 1 and 2 relative to genotype 3 in the Southern European CHC population, as compared with Central Europe. In addition, the ethnicity of patients in the two cohorts, a factor strongly associated with the prevalence of different IL-28B genotypes [1,4], might have differed. However, most patients were Caucasian in this study, and accordingly the prevalence of the rs12979860 CC genotype was very similar in patients with AHC and CHC (47.5%vs. 45.2%). Furthermore, similar differences in HCV genotype distribution in relation to the IL-28B genotype were found within the group of German patients with CHC. Therefore, it is unlikely that demographic differences had an impact on the study results.
Relationships between rs12979860 genotype CC and a higher baseline HCV viral load [1,4] and between genotype CC and higher transaminase levels  have previously been found in HCV-monoinfected patients. The IL-28B genotype CC is associated with lower expression of interferon-stimulated genes . The presence of the IL-28B CC genotype may therefore lead to elevated HCV replication and higher levels of necrosis and inflammation, in response to higher activity of HCV. However, data on the impact of these SNPs on viral replication are contradictory [6,8,10]. Recently, Lindh et al. proposed that the higher viral load in CHC patients with the CC genotype may be attributable to a significantly higher clearance rate in CC carriers with a low viral load, causing a higher proportion of those with the CC genotype and a higher viral load in the CHC population . In our study, the plasma HCV viral load was higher in patients with the CC genotype and AHC, while in those with CHC there was no significant difference in this parameter according to IL-28B genotype. This may be attributable to the fact that HIV/HCV-coinfected patients show higher levels of viraemia than HCV-monoinfected subjects with CHC . In this setting, a subtle effect of IL-28B genotype on HCV viral load may not be detected. Finally, significantly higher ALT levels were observed in patients with IL-28B CC, supporting the above theory.
Most homosexual male patients with AHC carried HIV before becoming infected with HCV, whereas IDU patients with CHC are presumed to be infected with HCV before, or at the same time as, HIV. Because of this, the immunodeficiency in patients with AHC could have been more profound. Whether this immunodeficiency has an impact on the different distributions of HCV genotypes according to IL-28B genotype in patients with AHC and CHC is uncertain. However, the association between IL-28B and viral genotypes has also been reported in several studies carried out in HCV-monoinfected patients with CHC [4,5,7,8,10]. Therefore, it is likely that our findings are applicable to patients without immunodeficiency.
In patients with AHC, the mechanism whereby the impact of the IL-28B genotype on the likelihood of evolution to CHC depends on HCV genotype remains unclear. The IL-28B genotype is a marker of the innate immune response to HCV . The variability of HCV is extremely high, and genomic sequences of different HCV genotypes vary by as much as 35% . Accordingly, the relevance of specific aspects of the immune response to such different viral variants could vary. Thus, we hypothesize that the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway, through which IL-28B may exert its effect , would be less important for HCV genotype 3 clearance than for clearance of genotype 1 or 4.
The findings presented in this study have clinical implications. In some developed countries, AHC in HIV-infected individuals is a growing problem [11,22,23]. It is unclear if antiviral therapy in HIV-infected patients with AHC should be started immediately or deferred until 12 weeks after diagnosis, given the chance of spontaneous clearance . These findings may help in the identification of patients for whom treatment could be deferred, as the likelihood of spontaneous clearance of HCV is higher, such as genotype CC carriers who are infected with HCV genotype 1 or 4. In the same way, new treatment strategies based on the manipulation of the JAK/STAT pathway by new compounds and/or the interferon λ itself, should be focused on carriers of HCV genotype 1 or 4, as little improvement in the success rate of currently available drugs using such strategies is expected in patients with genotype 3.
In summary, the IL-28B genotype CC seems to prevent HCV infection evolving to CHC mainly in patients bearing HCV genotype 1 or 4. This finding may help us to better understand the immune response to HCV and to design new therapeutic strategies against this infection.
This study was supported in part by grants from the Spanish Health Ministry (ISCIII-RETIC RD06/006), the European NEAT project, the Instituto de Salud Carlos III (grant for health research projects reference PI10/01664), the Fundación Progreso y Salud, Consejería de Salud (grants for health research projects, references 0133/08 and PI-0247-2010), the Fondo de Investigaciones Sanitarias (reference PI10/01664) and the Fundación para la Investigación y la Prevención del Sida en España (FIPSE). JAP is the recipient of a research extension grant from the Fundación Progreso y Salud of the Consejería de Salud de la Junta de Andalucía (Reference AI-0021). VS is the recipient of an intensification grant from the Instituto de Salud Carlos III. JN is the recipient of a research grant from the H.W. & J. Hector-Stiftung (Project M42). KN is the recipient of a ‘Sara Borrell’ postdoctoral perfection grant from the Instituto de Salud Carlos III (SCO/523/2008).
Conflicts of interest: The authors have no conflicts of interest to declare.