2015 APMIS. Published by John Wiley & Sons Ltd.

DOI 10.1111/apm.12414

APMIS

Interleukin- 28B: a prognostic marker in interferon

based therapy of chronic HCV patients of the Pakistan
with variable treatment response
SALEHA RESHAM, SOBIA MANZOOR, MUHAMMAD IMRAN, MUHAMMAD SAALIM,
SIDRAH NASEEM and SIKANDAR AZAM
Atta-Ur-Rahman School of Applied Bio-Sciences, Department of Healthcare Biotechnology, National
University of Sciences and Technology (NUST), Islamabad, Pakistan

Resham S, Manzoor S, Imran M, Saalim M, Naseem S, Azam S. Interleukin- 28B: A prognostic marker in
interferon based therapy of chronic HCV patients of the Pakistan with variable treatment response. APMIS 2015.
Unfortunately Pakistan carries one of the worlds highest burdens of chronic hepatitis along with mortality due to liver
failure and hepatocellular carcinoma. Scientists after extensive research have come up with this outcome that host genetics
play a vital role in dictating the type of treatment response produced by the patients. In 2009, a genome wide association
study (GWAS) revealed that genetic variants in close proximity to the IL28B (IFNL3) gene predicted greater likelihood of
achieving sustained virological response (SVR) following treatment with pegylated IFN-alpha (peg INF-a) and ribavirin.
IL28B (rs12979860 and rs8099917) single nucleotide polymorphisms (SNPs) have been recently found among the Pakistani
population associated with response to chronic HCV infection INF-a + ribavirin therapy. Therefore, this study was aimed
to investigate the IL-28B protein levels in the HCV infected patients. The ndings showed that the serum IL28B protein
level was higher in HCV infected patients as compared to healthy controls (7.743
1.519 pg/mL versus 1.600
0.06054
[mean
SEM], p < 0.05). When the chronic hepatitis C (CHC) patients were further categorized into SVR and NR (nonresponders) on the basis of treatment outcomes, the mean IL28B protein level was higher in NRs (15.54
3.609) than
SVRs (4.259
0.3405). Thus, there was a signicant correlation between IL28B protein level in varied treatment response
(p < 0.05). However, the ndings can lead us to propose that IL28B could be used as a prognostic marker. It can help the
clinicians to take better pre-informed decisions whether to take combinational therapy of peg IFN
ribavirin or not. This
will in turn prove benecial for the patient by saving patients health, treatment cost and undesirable treatment side eects.
Key words: Sustained virological response; non-responders; IL28B; chronic HCV.
Sobia Manzoor, Atta-Ur-Rahman School of Applied Bio-Sciences, Department of Healthcare Biotechnology, National
University of Sciences and Technology (NUST), Islamabad 44000, Pakistan. e-mails: lcianunique@yahoo.com;
dr.sobiamanzoor@asab.nust.edu.pk

Hepatitis C virus (HCV) is a small enveloped RNA

virus belonging to the family Flaviviridae. HCV
genome is 9.6 kb in length and encodes a single
poly-protein which is post-translationally cleaved
into 10 polypeptides including multiple structural
(C, El, and E2, P7) and non-structural proteins
(NS: NS2 to NS5) (1).
Approximately 3.3% of the worlds population is
infected with HCV. According to the recent epidemiological data, 4.9% (9 million) of the Pakistani
population is chronically infected with HCV (1, 2).
Pakistan is ranked as the country with second

Received 28 January 2015. Accepted 1 June 2015

highest rate of HCV infection with subgenotype 3a

as the most predominant in the world (24).
In 2007, IL-28 and IL-29 were introduced as the
members of a new cytokine family that shares the
same JAK-STAT signaling pathway with type I
interferon (IFN), driving the expression of set of
common genes. Interferon lambda (IFNL) exhibits
several common features with type I IFNs: antiviral
activity, anti-proliferative activity and in vivo antitumor activity (5).
Under current scenario patients varied treatment
response is among one of the major serious concerns regarding HCV management and control.
HCV is the major health concern in Pakistan
because about 75% of patients do not receive the
1

RESHAM et al.

standard anti HCV therapy (Interferon + Ribavirin) and of the 25% that do receive treatment, the
sustained virological response rate (SVR) is 60
70% (6). Among HCV infected individuals, only
15% have the natural tendency to overcome acute
viral infection, whereas 85% of individuals are
unable to overcome the infection (6). Patients
treatment response to HCV therapy is multifactorial and inuenced by both host and viral factors.
Adding to the dilemma, more than 50% of people
who are at highest risk of HCV transmission are
currently unaware of their disease status, thereby
aggravating viral infection and limiting therapeutic
opportunities (7).
In 2009, a GWAS (genome wise association
study) revealed that genetic variants in close proximity to the IL28B (IFNL3) gene predicts the
greater likelihood of achieving SVR following treatment with pegylated IFN-alpha (peg INF-a) and
ribavirin among HCV genotype 1 infected patients.
Genetic variation in IL28B is associated with treatment outcomes of CHC (chronic hepatitis C)
patients (8). The genetic polymorphism of IL28B is
also found in the study on Western population and
in Chinese population which signicantly associated
with varied treatment response to the HCV therapy
(9). The strongest association, among a predominantly caucasian population, was noted for the
rs12979860 SNP where the CCrs12979860, had 2fold increased likelihood of achieving SVR as compared to the TT variant (10, 11). However, in a
recently published study from Pakistan, a signicant association of HCV genotypes and genetic
variants of IFNL (IL28B) have been shown with
IFN a + Rib treatment of HCV infection (4).
An interesting nding reported the discovery of a
novel IFNL4 gene generated by frame shift change
within the promoter region of IL28B gene. Upstream
of IFNL3 (IL28B) on chromosome 19q13.13, a
dinucleotide variant ss469415590 (TT or DG), has
been established as potent genetic marker strongly
associated with HCV clearance, is in the high linkage
disequilibrium with rs12979860.ss469415590 [DG] is
a three frame shift variant that creates a novel gene,
designated IFNL4, encoding the interferon-k4 protein (IFNL4), which is moderately similar to IFNL3.
Thus, disruption of the IFNL4 gene is benecial for
humans in the context of HCV infection, although
the reason for this remains unclear (12).
Various viral and host factors have been identied
as signicant determinants of the outcome of IFNbased treatments. Viral genotype and baseline viral
load are well-known predictors of response to therapy. Other viral factors include amino acid substitutions at positions 70 and 91 in the HCV core region
(13) and in the IFN sensitivity-determining region in

NS5A (14) in patients infected with HCV genotype

1. So far the strongest predictor for SVR related to
HCV treatment is viral genotype. Depending upon
viral genotype, the treatment response varies signicantly. The response rate of the patients infected
with HCV genotype 2,3 and genotype 1 are 80%
(for both 2 & 3 genotype) and 4050%, respectively
(15, 16). Expression levels of chemokines and cytokines (for e.g. as reported in 2011 for IL-8) (17) proposed to have role in HCV antiviral resistance, virus
persistence and pathogenesis (18).
Several host factors related to failure of treatment-induced viral clearance include older age,
insulin resistance, advanced brosis and hepatic
steatosis (19, 20). Ethnicity is also a factor in treatment outcome. The proportion of African American patients achieving SVR on treatment with
PEG-IFN/RBV is lower than Caucasian patients,
(2123) indicating that host genetic factors can be
an important determinant of treatment outcome. It
has been reported that high-level expression of intrahepatic ISGs aected poor response to PEGIFN/RBV therapy (24). Thus, both viral and host
factors are associated with disease progression and
treatment-associated complications.
This study was designed to investigate the IL28B
at translational level (protein). The rst objective of
this study was to investigate the expression levels of
IL28B (IFN k 3) among HCV patients. Patients
infected with HCV genotype 3a (HCVG3a) received
the treatment for 24 weeks and HCVG1 and 4
received the treatment for 48 weeks. The second
objective was to establish if there exists a signicant
correlation between IL28B protein levels and treatment response (SVRs, NRs) in chronic HCV
infected patients.
MATERIALS AND METHODS
Samples collection
Initially, 150 chronic HCV patients at Gambatt Hospital
Sindh, Pakistan were enrolled for this study. However,
nally we were left with 68 patients who completed the
proposed duration of combinational therapy of pegylated
IFN-alpha (peg INF-a) and ribavirin and possessed any
nal fate of therapy. Of 68 patients, two samples were
missed out due to lack of serum for IL-28B quantication
hence reducing the sample size from 68 to 66. The ethical
committee of Atta-Ur-Rahman School of Applied Biosciences (ASAB), National University of Sciences and
Technology (NUST) approved the study proposal and
the protocols used. Appropriate clinical examination
for each participant was conducted while individuals
with severe health complications were excluded from the
study. Patients demographics and consent forms were
documented. Serum was extracted and stored at 20 C
for further analysis.

2015 APMIS. Published by John Wiley & Sons Ltd

IL28B AS A MARKER TO DETERMINE THE OUTCOME OF HCV THERAPY.

Patient selection (inclusion/exclusion criteria)

The inclusion criteria were patients infected with HCV
(initially conrmed by the detection of HCV antibody in
patients serum). Moreover, all the patients had HCV
RNA level higher than 1000 IU/mL as quantied by realtime reverse transcriptase Polymerase chain reaction
(qRT-PCR). Only those patients were included in the
study that had completed the therapy and were followed
up to 6 months after the cessation of combinational therapy (IFN + Ribavirin). Patients with liver damage caused
by infection other than HCV, patients co-infected with
other diseases such as HBV, HIV, patients with decompensated liver, patients with hepatocellular carcinoma and
patients who discontinued the planned course of treatment, were all excluded from this study.

Patient monitoring
All patients that received antiviral treatment were monitored for HCV viral load and alanine aminotransferase
(ALT) levels during treatment and post-treatment
(24 weeks). Treatment ecacy was assessed with normalization of ALT and undetectable HCV RNA in serum
measured at 4, 8, 12 and 24 weeks.

Control
Blood samples from 20 healthy volunteers were obtained
upon their consent. All the control blood samples were
examined for absence of HCV. After that, blood samples
were processed for IL28B protein quantication and compared with HCV infected patients.

with 40 lL Master Mix (Gene Proof Hepatitis C Virus

PCR kit). HCV RNA was quantied in 24 weeks posttreatment sera using real-time PCR Bio Rad MyiQTM 2
two color Real-time PCR detection system with an internal RNA standard derived from the 50 un-translated
regions (50 -UTR). Accuracy of measurement lies within
the range of 103 IU/lL - 101 IU/lL. Sensitivity (Limit of
Detection) is 36.173 IU/mL.

Statistical analysis
Using GRAPH PAD PRISM 5.01 summary statistics were calculated. The results were presented as the Mean
SEM.
Statistical correlation and variation were observed. Correlation and variations were found statistically signicant at
p < 0.05 and in some cases p < 0.01.

RESULTS
Demographic characteristics - patients and control

The demographic characteristics of the infected

patients enrolled for the study are shown in Table 1.
A total of 66 HCV infected patients underwent the
treatment and were available till the completion of
present study. There mean age ranged from 19 to
65 years data shown in Table 1. HCV infected sera
samples were utilized for analysis of the study
parameters. Moreover, serum samples from 20
healthy volunteers were processed as controls for
IL28B level. Table 2 is illustrating the complete
demographic data for 20 control individuals.

Estimation of expression level of IL28B

The CUSABIO ELISA (Enzyme-Linked Immunosorbant
Assay) Kit (China) was used to estimate the expression of
IL-28B in both control and HCV positive patients according to procedure given in the kit protocol. The assay
employs the quantitative sandwich enzyme immunoassay
technique. The monoclonal antibody specic for IL28B
has been pre-coated onto a 96-well plate. Standards
(100 pg/mL, 50 pg/mL, 25 pg/mL, 12.5 pg/mL, 6.25 pg/
mL, 3.12 pg/mL, and 1.56 pg/mL) and samples were
added to the corresponding wells. The wells were washed
and avidin horseradish peroxidase conjugate mixed with
biotinylated anti-human IL28B antibody was added that
produced an antigen-antibody sandwich. The wells were
washed and TMB (tetramethylbenzidine) substrate solution was added, which produced a blue color in direct
proportion to the amount of IL28B present in the initial
sample. The stop solution changes the color from blue to
yellow and absorbance were noted at 450 nanometer (nm)
within 5 min along with the reference lter of 570 nm.
The mean absorbance for each set of duplicate standards,
controls and samples were calculated.

24 weeks pre and post-treatment viral load

quantification

All the HCV infected patients were categorized

into sustained virological response (SVRs) and
non-responders (NRs). SVRs were dened as those
patients who were negative for HCV RNA using
real-time PCR 6 months after the end of therapy.
The NRs were those patients who possessed
detectable HCV RNA using real-time PCR after
the end of therapy. The pre-treatment viral load
of SVR and NR CHC patients were (Mean

SEM = 5.0 9 105
8.2 9 104) and (Mean

SEM = 5.7 9 105
9.9 9 104), respectively. While
the post-treatment viral loads for SVRs and
NRs were (Mean
SEM = 1 9 102
4.0 and
4.2 9 104
2.2 9 104), respectively. Table 3
shows the detailed summary.
Expression level of IL-28B (IFN-k3)

HCV RNA extraction and viral quantification

HCV RNA was extracted from 100 mL serum sample
using RTA Viral RNA isolation kit following the provided protocol. The extracted RNA (10 lL) was mixed
2015 APMIS. Published by John Wiley & Sons Ltd

The protein level of IL28B (IFN-k3) in the sera of

HCV infected patients ranged from 1.786 pg/mL to
86.93 (pg/mL) (Mean
SEM = 7.743
1.519). In
controls it ranged from 0.930 pg/mL to 2.338 pg/

RESHAM et al.

Table 1. Demographic characteristics, viral genotype, mean viral load and IL28B level (IFN-k3) of HCV infected patients
Gender
Age (years)
Number of samples
Genotype
Viral load (IU/lL)
IL28B (pg/mL)
Mean
Mean
SEM
Mean
SEM
Females
35
26
3a (13)
9.1 9 103
7.0 9 103
8.7
1.93
1a (3)
3b (4)
1b (1)
4 (1)
Coinfection (4)
Untypeable (2)
5.1 9 103
4.3 9 103
Males
31
40
3a (21)
7.527
2.151
1a (5)
3b (7)
Untypeable (3)
Coinfection (2)
Total
66
6.5 9 103
3.7 9 103
7.743
1.519

Table 2. Controls demographic prole with IL28B expression levels

Gender
Age
Health status
Number of samples
Female
Male
Total

2022
1730
1730

Normal
Normal

5
15
20

IL28B Level (pg/mL)

Mean
SEM
1.625
0.09738
1.637
0.07018
1.600
0.0635

Table 3. Mean viral load (pre-treatment & post-treatment) and IL28B (IFN-k3) levels in SVRs and NRs along with
response rates
Type of response Total number of patients Pre-treatment viral load Post-treatment (24 weeks) IL28B levels (pg/mL)
Mean
SEM
viral load (IU/lL)
(n) (response rate %)
(IU/lL)
Mean
SEM
Mean
SEM
SVR
n = 56 (79%)
5.0 9 105
8.2 9 104
1 9 102
4.0
4.529
0.3405
NR
n = 10 (15%)
5.7 9 105
9.9 9 104
4.2 9 104
2.2 9 104
15.54
3.609
Controls
n = 20
NA
NA
1.600
0.0605

mL (1.600
0.0605). Figure 1(A) shows the IFNk3 (IL28-B) concentration among HCV infected
patients (66 patients) and 20 healthy controls
whereas Fig. 1(B) shows its concentration in dierent response categories.
Correlation of IL28B (IFN-k3) levels and response to
IFN therapy

Figure 2 shows the mean IL28B (IFN-k3) levels for

SVRs, NRs, and Controls compared with standard
known concentration of IL28B. The two patient
response categories were analyzed for varied IL28B
expression levels through statistical analysis. ANOVA among IL-28B levels of SVRs, NRs, and Controls gave signicant p value (p < 0.05) p = 0.0001.
There has been found signicant association
between IL28B levels and response to antiviral therapy i.e. increase in IL28B levels are associated with
decrease in response to therapy (response rate).No
association was observed between IL28B level and
4

gender (p > 0.05). It was observed that NRs had

signicantly higher IL-28B levels. There was nonsignicant variation between response and viral
load (p 0.05). It was conrmed that non-responsive patients had signicantly higher baseline IL28B
level than responsive patients (p < 0.05), and the
responders (SVRs) had signicantly lower baseline
IL28B level than non-responders (NRs) (p < 0.05).
DISCUSSION
Since the discovery of HCV in 1989, it has been
implicated in causing acute to chronic hepatitis
infections that ultimately leads to the development
of end stage liver disease i.e. hepatocellular carcinoma (HCC). Genetic variation in both the HCV
genome and its human host aect the natural history of the infection and the degree of response to
treatment (9, 16). Because HCV infection has a
high propensity to persist in the host therefore,
2015 APMIS. Published by John Wiley & Sons Ltd

IL28B AS A MARKER TO DETERMINE THE OUTCOME OF HCV THERAPY.

Fig. 1. (A) IL 28B (IFN-lambda) concentration in HCV infected and controls. (B) IL28B (IFN-k3) is shown separately
for dierent response categories. Mean
SEM = 15.54
3.609 (NRs), (SVRs) 4.529
0.3405 and control
group = 1.600
0.0605.

Fig. 2. Expression level of IL28B in SVRs, NRs and controls compared to the standard concentration provided in the kit.
The serum samples of all the 66 HCV infected patients and 20 controls were evaluated for IL28Bs concentration. Expression levels were monitored through using IL28B specic ELISA kit according to the given protocol.

acutely infected patients fails to eradicate the virus

in approximately 80% of cases and subsequently
develop chronic infection. The condition leads to
extra hepatic and hepatic disorders, mainly chronic
liver inammation, cirrhosis and liver cancer.
It is well-known that Pakistan carries one of the
worlds highest burdens of chronic hepatitis and
mortality due to liver failure and hepatocellular carcinomas (25). The two major SNPs of the IL28B
genes, rs12979860 and rs8099917, were considered as
the strongest predictor of natural clearance and
treatment response. An important nding that both
2015 APMIS. Published by John Wiley & Sons Ltd

SNPs are in linkage disequilibrium and exhibits signicant ethnic diversity (26). These small genetic
variations are also reported to inuence IL-28B
mRNA expression and has possible role in the regulation of intra-hepatic expression of interferon stimulated genes (ISGs) (19, 20).
It is not necessary that all of the patients who
receive treatment for infection get treated with it.
Now-a-days, IFN-based treatment (approved by
FDA) is currently available but the virologists
and physicians together are still unable to tackle
and manage such a life threatening HCV infec-

RESHAM et al.

tion somehow. There are a commendable number

of factors that intervene and make the treatment
complicated. The factors are classied as viral
factors; genotypes 2 and 3, viral load <2 million
IU, rapid virological response etc. and host factors; age <40, gender: female, ethnic race, immune
response; genetic variations in IL28B gene etc.
studied yet to be most important in varying treatment responses (18).So this leads to a conclusion
that HCV is dicult to treat, with only IFN and
ribavirin. The therapeutic eects of IFN-a are
mediated by transcriptional induction and activation of hundreds of interferon stimulated genes
(ISGs). HCV has been reported to neutralize the
antiviral response by binding and inhibiting the
antiviral action of protein kinase R (PKR), a key
IFN stimulated gene (ISG) in IFN antiviral cascade (27). The IL28B polymorphism has been
associated with predicting treatment-induced viral
clearance (10). It has also been implicated as
strong pretreatment predictors of early viral clearance and SVR in patients infected with HCV
genotype 1(10, 28).
Collectively, the ndings of this study demonstrate a signicant correlation between IL28B protein expression and response to antiviral therapy. A
signicantly increased level of IL28B (IFN-k3) was
observed in the sera of the HCV patients as compared to the normal healthy volunteers. The CHC
patients with high level of IL28B compared to controls showed poor response to antiviral therapy;
they were NRs while the CHC patients with lower
expression of IL28B than controls exhibited better
response to the therapy were classied as SVRs.
ANOVA between IL-28B levels of SVRs, NRs
and Controls gave signicant p value (p < 0.05)
p = 0.0001. It was observed that NRs had signicantly higher IL-28B levels. The IL28B expression
was high with the value of 7.743
1.519 pg/mL
(Mean
SEM)
for
HCV
infected
and
1.600
0.0635 pg/mL (Mean
SEM) compared
to healthy controls. Findings of the study are in
agreement with the previously published study (29).
Relating to our previously published study (4)
HCV genotypes and IL28B rs12979860 SNP found
among the Pakistani HCV patients as predictive
markers for the eciency of (IFN + ribavirin)
combinational therapy. The results of this study were
found consistent with the study published recently,
which reports that IL28B contributes to viral resistance and is known to be up regulated as a result of
interferons and RNA virus infection (30).
However, in conclusion to our current study,
we propose to add IL28B protein expression levels in addition to IL28B genotyping (SNPs) (i.e.
both the genotyping and protein levels in the

blood of the infected patients) as suggestive biological (prognostic) markers before initiation of
IFN-based therapy. We recommend testing of
IL28B (either at transcriptional level or translational level) among chronic HCV patients helping
the clinicians to take better, pre-informed decisions regarding their more diverse treatment
options. This would help save patients health,
avoiding unusual treatment side eects and heavy
treatment cost.
CONCLUSION
Our ndings lead us to conclude that IL28Bs
expression levels are highly variable among treated
HCV infected patients. IL28B expression levels
were found to be higher among NR. Host genetics
may be useful for the prediction of drug response
or treatment response beforehand. So it can be
inferred from the results of this study that this varied expression of IL-28B could have a predictive
value for the outcome of IFN a plus ribavirin therapy. HCV patients having high IL-28B levels will
show no response to interferon therapy compared
to those having low levels of IL-28B. Therefore, IL28B levels can be used as predictive/prognostic
marker for IFNa
ribavirin treatment outcome
for the patients infected with HCV genotype 3a.
The polymorphism has already been reported
among Chinese, Western and African American
population. But the polymorphism has not yet been
reported for Asian ancestry.

The study was supported by National University of Sciences and Technology.

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