Role of Interferon-gamma and Immune Response Biomarkers in Predicting IFN-alpha Responsiveness and Treatment Outcome in Patients with Hepatitis C Virus
Amira F. Barakat,
Raghda E. Farag,
Azza Abdul Baky,
Lamiaa F. Arafa
The aim of this study was to examine interferon-gamma (IFN-γ), anti-C1q
antibodies, myeloperoxidase antineutrophil cytoplasmic antibodies (MPO-ANCA)
and anticardiolipin antibodies in the serum of biopsy-proven chronic hepatitis
C patients before and after interferon (IFN)-alpha and ribavirin therapy to
address whether or not viral clearance is related to these biomarkers and to
explore a possible association between the pattern of these immune response
parameters with the virological and biochemical status of Hepatitis C Virus
(HCV). The serum levels of IFN-γ, anti-C1q, myeloperoxidase ANCA and anticardiolipin
antibodies were assayed on 64 patients with chronic hepatitis C virus infection
before and 48 weeks after treatment with pegylated IFN-α plus ribavirin
and compared with sera from 20 normal control subjects. Serum levels of ICN-γ,
anti-C1q, myeloperoxidase ANCA and anticardiolipin antibodies were significantly
higher in HCV patients in comparison to healthy controls (p<0.0001). IFN-γ
levels were significantly increased after 48 weeks of antiviral treatment when
compared to pretreatment serum levels and it was significantly more elevated
in responder HCV patients than non responders. While as, both anti-C1q antibodies
and myeloperoxidase ANCA levels were significantly decreased after 48 weeks
antiviral treatment in the HCV patients. Moreover, IFN-γ levels (but not
other studied biomarkers) in HCV patients correlated significantly with high
alanine aminotransferase (ALT) levels as well as with high viral load (r = 0.675,
p≤0.05, r = 0.912, p≤0.001, respectively). In conclusion, IFN-gamma might
be useful in predicting the clinical outcome of the combination therapy of pegylated-IFN
alpha and ribavirin, as well as responsiveness to IFN-alpha-based therapy may
be improved by using easily assessed immune response biomarkers such as interferon
gamma, anti-C1q antibodies. Furthermore, early treatment in HCV patients with
multiple serological abnormalities will prevent further autoimmune response
which eventually could prevent marked extrahepatic complications.
to cite this article:
Amira F. Barakat, Asmaa Hegazy, Raghda E. Farag, Azza Abdul Baky, Lamiaa F. Arafa and Amal Farouk, 2012. Role of Interferon-gamma and Immune Response Biomarkers in Predicting IFN-alpha Responsiveness and Treatment Outcome in Patients with Hepatitis C Virus. International Journal of Virology, 8: 288-298.
Received: April 14, 2012;
Accepted: May 24, 2012;
Published: October 01, 2012
Infection with hepatitis C virus (HCV) may be associated with a wide spectrum
of immunological abnormalities. Hepatitis C virus tends to induce nonspecific
autoimmune reactions, as demonstrated by the high prevalence of various autoantibodies.
In addition, interferon-gamma (IFN-γ), as one of the T helper related cytokine,
has elicited great interest in chronic viral infections because it is abundantly
produced and has direct antiviral activity. The pathogenesis of viral chronic
liver disease that leads to liver damage is suggested to be immune-mediated
(Feng et al., 2009). The immunopathogenesis of
chronic Hepatitis B (HBV) or HCV infection occur through the participation of
CD8 T cells, CD4 T cells and Natural Killer (NK) cells as well as cytokines
(Kondo et al., 2004). A previous study indicates
that HBV and HCV could induce both early and late immune responses (Feng
et al., 2009). The immunological and genetic aspects of patient defenses
which determine the outcome of virus/host interaction will lead to persistent
viraemia as have been suggested by previous studies (Khakoo
et al., 2004; Cooper et al., 1999;
Thimme et al., 2001). Moreover, innate immune
mechanisms are involved also in the development of chronic persistent replication
of intracellular pathogens (Lodoen and Lanier, 2005;
Ahmad and Alvarez, 2004).
Infection with hepatitis C virus may be associated with a wide variety of immunological
abnormalities that could be demonstrated by high prevalence of various autoantibodies
(Kisiel and Kryczka, 2007) as well as the presence of
circulating immune complexes (Jablonska et al., 2008).
Most extrahepatic manifestations of the hepatitis C virus infection occur through
the influence of virus on the host's immune system. From the commonly encountered
serological immune response to HCV infection is the development of cryoglobulinemia,
rheumatoid factor, anticardiolipin, antinuclear antibodies (Mouelhi
et al., 2008).
Antiphospholipid antibodies (aPL) (lupus anticoagulant and anticardiolipin
antibodies) are a heterogeneous family of immunoglobulins that react with complexes
of phospholipids and plasma proteins (Kisiel and Kryczka,
2007). Previous reports have mentioned the increased prevalence of aPL antibodies
in several bacterial, parasitic and viral infections (Sene
et al., 2009). Most of the previous studies confirms that anticardiolipin
antibodies are frequently found in patients with chronic HCV infection but mostly
of no clinical importance (Kisiel and Kryczka, 2007;
Sene et al., 2008, 2009).
Some studies however, have found an increased incidence of thrombotic disorders
in patients with chronic hepatitis C virus who manifest aPL positivity (Rafai
et al., 2006; Cojocaru et al., 2005).
Thus, the clinical significance of antiphospholipid antibodies in patients with
chronic hepatitis C virus and some other viral infections is controversial (Habibagahi
et al., 2007). Moreover, their amelioration under antiviral therapy
and correlation with the virological load remains to be determined (Sene
et al., 2009).
Autoantibodies against a variety of self-antigens can be detected in the sera
of patients with HCV infection. C1q is the first component of the classical
pathway of complement activation and its main function is to clear immune complexes
from tissues and self-antigens generated during apoptosis (Walport,
2001). Autoantibodies against C1q have been described in many immune-complex
diseases including hypocomplementemic urticarial vasculitis and Systemic Lupus
Erythematosus (SLE) (Tsirogianni et al., 2009;
Wisnieski et al., 1995). Few previous studies
had focused on the presence of anti-C1q antibodies in hepatitis C virus infection
and aimed at evaluation of the prevalence of anti-C1q antibodies in HCV infection
(Saadoun et al., 2006; Lienesch
et al., 2006). However, assessing the role of antiviral treatment
of HCV patients on anti-C1q and its correlation with the viral load and liver
status was not yet studied.
Regarding the role of Anti-neutrophil Cytoplasmic Antibodies (ANCA) in HCV
patients, a previous study by DeRiva and colleagues suggests that the finding
of ANCA by ELISA is common not only in autoimmune Chronic Liver Disease (CLD)
but, also in viral-related CLD (Valentina et al.,
2009). Furthermore, the positivity for ANCA might have a prognostic value
in patients with viral-related as well as autoimmune-related cirrhosis (Valentina
et al., 2009). Recently, a high prevalence of ANCA has been reported
in patients with HCV (Yasuda et al., 2011). But
again, assessing the role of antiviral treatment of HCV patients on ANCA needs
Natural Killer (NK) cells are innate immune cells known for their immediate
effectors functions against virus-infected cells and tumor cells. These effectors
functions include the destruction of target cells via the production of cytokines,
such as tumor necrosis factor alpha (TNF-α) and interferon gamma (IFN-γ)
(Vivier et al., 2011). IFN-gamma, was particularly
studied because it is abundantly produced and has direct antiviral activity
(Billiau and Matthys, 2009). It has been recently shown
that patients with chronic hepatitis C virus (HCV) infection display a polarized
NK cell phenotype with increased cytotoxicity and IFN-γ production (Ahlenstiel
et al., 2010; Oliviero et al., 2009;
Stegmann et al., 2010). Furthermore, other studies
stated that the kinetics of the in vivo responsiveness of NK cells, with
IFN-γ production, to the IFN-α therapy in human are not yet known
and it will be important for the therapeutic use of IFN-α in chronic HCV
infection (Edlich et al., 2012). It is well documented
that imbalance of T helper 1 (Th1) and T helper 2 (Th2) may exert a critical
influence on the inflammatory environment of the host as well as the final outcome
of infection (Sobue et al., 2001; Bertoletti
et al., 1997). Previous reports have shown that IFN-γ may directly
inhibit virus replication and mediate liver injury (Shin
et al., 2005; Penna et al., 1997;
Byrnes et al., 2007; Jo and
Combination therapy of pegylated interferon-α (Peg-IFN-α) plus ribavirin
is standard treatment for patients with chronic hepatitis C nowadays. This combination
has led to a Sustained Virological Response rate (SVR) of 50 to 80% depending
on genotype. This percentage still unsatisfactory if we consider the side effects
of the treatment, overall costs and the prolonged duration of therapy. So far,
different strategies have been developed to predict SVR in HCV infected patients
on antiviral therapy such as genotype, fibrosis stage, viral load and genetic
polymorphism related to race, insulin resistance and viral kinetics (Moraes
Coelho and Villela-Nogueira, 2010). Studying further predictive factors
that are readily accessible from the peripheral blood and easily to be assessed
in the laboratory might help the decision about starting or discontinuing therapy
in chronic HCV infected patients.
Although, serological auto-immune manifestations in HCV infected patients were
explained by the lymphotropism of HCV, interferon-based treatment of HCV infection
where accused by some studies to precipitate or exacerbate the associated auto-immune
disease (Mouelhi et al., 2008). Thus, in patients
with serological auto-immune disorders associated with HCV infection careful
interpretation of clinical and biological features is necessary. Furthermore,
using more specific antibodies can be helpful in differentiating whether these
serologic autoimmune manifestations are induced by HCV infection or related
to the use of antiviral treatment in such patients (Mouelhi
et al., 2008).
The aim of the study was to investigate the features of immune response in
patients with HCV infection before and after IFN-α and ribavirin therapy,
through assessing one of the T helper-related cytokines (interferon-γ),
plus a variety of autoantibodies which are prevalent in HCV patients including;
autoantibodies against C1q, myeloperoxidase ANCA and anticardiolipin antibodies.
Moreover, we aimed to address whether or not viral clearance is related to the
studied immune mediators and to explore the possible association between these
immune biomarkers and both virological and biochemical status of HCV patients.
MATERIALS AND METHODS
This study was conducted on 64 chronically viraemic HCV genotype 4 patients
(M/F: 50/14; aged 36.71±14.12 years) from Internal Medicine and Tropical
departments, Mansoura University Hospital, in the period between October, 2009
to November 2011. In addition to twenty age and sex matched healthy subjects
served as controls.
Clinical and immunologic status of HCV patients were prospectively studied
before and then 48 weeks after antiviral treatment with combination of pegylated
IFN-γ and ribavirin.
Patients were selected according to Egyptian International Program Guideline
Selection Criteria with the following inclusion criteria: Age 18-60 years, HCV
RNA positive in serum compensated liver disease (total serum bilirubin <1.5
mg dl-1; INR 1.5, serum albumin ≥3.5, platelet count (not less
than) ≥90,000 mm with no evidence of hepatic decompensation (hepatic encephalopathy,
esophageal varices or ascites) and acceptable hematological and biochemical
indices (Hemoglobin ≥13 g dl-1 for men and 12 g dl-1
for women; neutrophil count >1500 mm-3 and serum creatinine <1.5
mg dl-1, with Body Mass Index ≤30, liver biopsy findings compatible
with chronic viral hepatitis in the preceding 12 months. The exclusion criteria
were: Age less than 18 or more than 60 years; previous treatment with interferon
or ribavirin or neutropenia (<than 1500 neutrophils mm-3), thrombocytopenia
(<than 90. 000 platelets mm-3), anemia, serum creatinine more
than 1.5 times above the upper limit of normal; history of alcohol or hemolytic
disease; decompensated cirrhosis; autoimmune hepatitis; hepatitis B infection;
HIV infection; current intravenous drug use; severe depressive illness; severe
comorbid disease; organ transplant; pregnancy; unwilling to practice contraception
and hepatocellular carcinoma. Informed consents were obtained from subjects
included in the study.
Chronic HCV patients with determined genotype 4 were selected for the study
while as other genotypes were excluded from the study.
Patients were given pegylated interferon alpha-2a in a fixed dose of 180 μg
weekly by subcutaneous injection. All of them received ribavirin in an adjusted
dose according to body weight; patients <75 kg were given 1000 mg and those
>75 kg were given 1200 mg. The safety was assessed by clinical evaluation
and laboratory tests at week 1, 2, 4 and monthly thereafter during treatment.
Stepwise reductions in the interferon and ribavirin dosages were allowed according
to international guidelines to manage adverse events or laboratory abnormalities
that had reached predetermined thresholds of severity. Patients were given growth
factors (erythropoietin hormone and macrophage colony stimulating factor when
possible) to avoid dose reduction in IFN and/or ribavirin.
Complete physical examination and laboratory investigations including liver
function tests (ALT, alanine aminotransferase; AST, aspartate aminotransferase;
TBIL, total bilirubin; ALB, albumin; PT, prothrombin time) and complete blood
count were done for all participants.
Serologic evidence of chronic HCV infection was determined by the detection
of antibodies to HCV (anti-HCV) using fourth generation enzyme immunoassay (HCV
antibody by ELISA using Cobas/Core supplied by Roch Germany). While active viral
replication was defined by the detection of HCV RNA using a quantitative assay
based on real-time PCR using Tag PCR master kit, Qiagen, Hilden, Germany. Viral
load was assessed by HCV-RNA at baseline and 24 weeks after treatment. Subjects
were considered to have a Sustained Virological Response (SVR) if they had negative
HCV RNA 24 weeks after completing the 48 weeks of treatment. Subjects were considered
relapsers if they had positive HCV RNA at week 72 after negative HCV RNA at
48 weeks of treatment. Subjects who failed to attain a negative HCV RNA at week
24 from the start of treatment or a decline of HCV RNA of >2 log10 IU ml-1
at week 12 of treatment were considered Non Responders (NR) and all were enrolled
in our study.
Immunological study was done for all participants and includes: Interferon-gamma
(IFN-γ) cytokine, serum anti-C1q antibodies, anticardiolipin IgG antibodies
(ACL) and myeloperoxidase anti-neutrophil cytoplasmic antibodies MPO-ANCA.
Serum IFN-γ was assayed by a sandwich enzyme linked immunosorbent assay
technology supplied by Boster Biological Technology Co. Results were expressed
as pg ml-1.
Anti-C1q was assayed by immunometric enzyme immunoassay supplied by Orgentec
Diagnostika GmbH (Germany) (Walport et al., 1998).
Results were expressed as unit ml-1 (U ml-1) and positive
anti-C1q was considered if the serum level was more than 10 U ml-1,
as recommended by the manufacturer as the cutoff value.
Anticardiolipin IgG antibodies, was assayed by immunometric enzyme immunoassay,
using Demeditec Diagnostics GmbH (Germany). Results were expressed as GPL unit
ml-1. Normal serum levels were considered normal if 10 GPL U ml-1.
Serum myeloperoxidase anti-neutrophil cytoplasmic antibodies (MPO-ANCA) was
assayed by immunometric enzyme immunoassay (IgG against myeloperoxidase using
Orgentec Diagnostika GmbH (Germany). Results were expressed as U ml-1.
Normal serum levels were considered normal if <5.0 U ml-1.
Statistical analysis: The statistical analysis of data was done using
SPSS (SPSS, Inc. Chicago, IL), program statistical package for Social Science
(version 16). To test the normality of data distribution K-S (Kolmogorov-Smirnov)
test was done only significant data revealed to be nonparametric. The description
of the data was done in form of mean±standard deviation (mean±SD)
for quantitative data. Nonparametric data were expresses as median and range.
For quantitative data student t-test was used to compare between two groups.
Mann-Whitney test and Kruskal-Wallis were used for non parametric data. To test
the association between variables Pearson correlation co-efficient test was
used. The p-value is considered significant if ≤0.05 at confidence interval
95% (Munro, 2002).
In the present study serum levels of IFN-γ, anti-C1q, myeloperoxidase
ANCA and anticardiolipin antibodies were significantly higher in HCV patients
at baseline (before treatment) in comparison to healthy controls (p<0.0001).
Data are shown in Table 1.
IFN-γ levels were significantly increased after 48 weeks antiviral treatment
when compared to pretreatment serum levels and it was significantly more elevated
in responder (SVR) HCV patients than Non Responders (NR). While as, both anti-C1q
antibodies and myeloperoxidase ANCA levels were significantly decreased after
48 weeks antiviral treatment in the HCV patients Table 2.
Regarding anticardiolipin antibodies (ACL), there was reduction in the serum
ACL levels after antiviral treatment but it was not statistically significant
In this study IFN-γ levels (but not other studied immune biomarkers) in
HCV patients correlated significantly with high ALT levels as well as with high
viral load (r = -0.675, p<0.05, r = -0.912, p<0.001, respectively).
All the pretreatment serum levels of the studied immunological parameters were
significantly correlated with their post treatment serum levels (Table
|| Comparison between serum levels of immunologic parameters
in HCV patients at baseline (before treatment) versus controls
|| Comparison of serum levels of immunologic parameters in HCV
patients before (at baseline) and after treatment
|NS: Non significant
|| Correlations between the studied immunologic parameters in
|IFN-γ: Interferon-γ, ACL-Abs: Anticardiolipin antibodies,
MPO: Myeloperoxidase, treatment, after: after treatment, ns: Non significant,
*p<0.05, **p<0.001, ***p<0.0001, r: Correlation coefficient, Abs:
Interestingly, anticardiolipin antibodies were the main immune response parameter
that significantly correlated with other studied parameters. Firstly, ACL serum
levels after antiviral treatment was significantly negatively correlated with
MPO -ANCA serum levels before and after treatment (r = -0.234, p<0.05, r
= -0.258, p<0.04, respectively). Secondly, ACL serum levels before antiviral
treatment was positively correlated with IFN-γ levels after treatment (r
= 0.303, p<0.01). Data are shown in Table 3.
In spite of recent progress for HCV treatment, there remains significant room
for improvement. To date, a variety of viral factors and host factors that correlate
with SVR in the combination therapy have been noted. In order to establish the
better treatment, the detail mechanism of HCV elimination should be elucidated
(Bertoletti et al., 1997).
In the present study it was found that interferon-γ levels were significantly
higher in HCV patients when compared to healthy controls which was in agreement
with recent studies by Zhang et al. (2011) and
Fathy et al. (2011). On the other hand, a previous
study by Li and colleagues stated that there were no significant differences
between IFN-γ in both HCV patients and controls (Li et
al., 2010). Interestingly, we found that after a 48 weeks treatment
by combination interferon alpha and ribavirin the serum IFN-γ was significantly
increased in comparison to its serum levels at baseline (before start of treatment)
and in responders to treatment more than non responders. Moreover, there was
a significant correlation between serum IFN-γ and HCV viral load as assessed
by PCR as well as with liver condition as assessed by high ALT levels. These
results were in accordance with that mentioned by Zhang
et al. (2011) but not in agreement with Fathy
et al. (2011) who stated that there are significant reduction in
IFN-gamma serum levels after 3 months of IFN alpha and ribavirin therapy and
it also stated that IFN-gamma was not correlated with high viral load. These
discrepancies in results could be attributed to the differences in numbers;
which was relatively small in Fathy et al. (2011)
(26 patients), as well as to the time of assessment of IFN-gamma serum levels
which was earlier (12 weeks) than the usual proper duration to have the expected
antiviral response. Thus, from the present study we can suggest that IFN-γ
can be regarded as an attempt by the immune system to inhibit viral replication
and to eradicate the infection as well as a predictive outcome factor for the
responsiveness of HCV patients to IFN alpha therapy, thus reducing treatment
costs and decrease side effects of long duration of therapy.
The interaction between the core protein of HCV and the C1q receptor has been
shown to suppress the T cell immune response which may have implications in
HCV persistence (Kittlesen et al., 2000). C1q
protein and C1q binding activity are enriched substantially in the cryoprecipitates
of HCV infected patients (Sansonno et al., 2003).
The wide expression of C1q receptor on the surface of blood cells and endothelial
cells favors their specific binding to immune complexes containing HCV core
protein. Efficient engagement of the C1q protein by cryoglobulins may represent
an important pathogenic mechanism in the cryoglobulins-related pathway (Feng
et al., 2002). In the present study we found a significantly higher
serum level of these autoantibodies in the studied HCV patients when compared
to controls. Moreover, there was no significant correlation between anti-C1q
antibodies and HCV viral load or ALT levels in the studies HCV patients. These
results were in agreement with previous studies (Saadoun
et al., 2006; Lienesch et al., 2006).
Concerning the effect of interferon alpha plus ribavirin treatment over the
serum levels of anti-C1q antibodies it was not yet probably evaluated in the
previous studies. Thus, in the present study the effect of interferon alpha
therapy on anti-C1q antibodies was assessed after 48 weeks treatment and interestingly
it was found that serum levels were significantly decreased after interferon
alpha and ribavirin combination therapy. But there were no significant differences
in anti-C1q serum levels between responders or non responders HCV patients after
48 weeks treatment. These observations suggest the significant role of antiviral
treatment in HCV patients in eliminating immune response autoantibodies such
as those against C1q.
In the present study although, none of the participants had a previous history
of thrombosis, higher serum levels of anticardiolipin antibodies was detected
in HCV patients when compared to healthy controls. This was in agreement with
previous studies (Sene et al., 2009; Habibagahi
et al., 2007). Regarding the effect of antiviral treatment of HCV
(combination of IFN-α and ribavirin) on ACL serum levels, there were no
statistically significant changes between pre and post treatment ACL antibodies.
There were a controversial data about anticardiolipin antibodies (ACL Abs) serum
levels of HCV patients in the previous studies, some found decrease in the ACL
Abs after period of interferon therapy (Rajan and Liebman,
2001), others described increased in the positivity of ACL Abs after 6 month
course of interferon alpha treatment (Leroy et al.,
1998). These controversies in results could be explained by the different
patient population and probably genotypes of HCV studied.
Regarding MPO-ANCA serum levels in the present study, although none of the
studied chronic HCV patients had clinical signs of vasculitis, MPO-ANCA was
significantly higher in HCV patients when compared to healthy controls. This
result was in accordance with that of Valentina et al.
(2009) who stated that ANCA is common not only in autoimmune Chronic Liver
Disease (CLD) but also in viral-related CLD. It was also in agreement with a
recent study by Bonaci-Nikolic and colleagues who found increased serum levels
of MPO-ANCA in chronic HCV patients and that in patients with positive ANCA,
HCV infection should be excluded (Bonaci-Nikolic et al.,
2010). Furthermore, in the present study we found that MPO-ANCA was significantly
reduced after 48 weeks treatment with interferon alpha plus ribavirin. It is
found that a very small number of patients with HCV and positive ANCA, stated
that with alpha interferon treatment for such patients the positivity of ANCA
had decreased but still positive after end of treatment and that associated
vasculitic symptoms and signs had been improved by using corticosteroids or
immunosuppressive therapy but no sufficient data was available on alpha interferon
therapy in such patients (Bonaci-Nikolic et al.,
2010). Thus, early diagnosis and therapy for patients with HCV infection
and ANCA positivity considered a big challenge in preventing enhancement of
the autoimmune response and induction of further inflammation and extrahepatic
In conclusion, the present study confirms that presence of anticardiolipin
antibodies has no pathologic significance in patients with HCV and that it is
not affected by interferon alpha treatment in such patients. Moreover, we suggest
that in patients with positive ANCA HCV infection should be excluded. Moreover,
it was found that IFN-gamma might be useful in predicting the clinical outcome
of the combination therapy of peg-IFN alpha and ribavirin. Thus the present
study opens the interesting possibility that responsiveness to IFN-alpha-based
therapy may be improved by using easily assessed immune response biomarkers
such as interferon gamma, anti-C1q antibodies which could help in predicting
responsiveness to IFN alpha therapy. Furthermore, early treatment in HCV patients
with multiple serological abnormalities will prevent further autoimmune response
which eventually could prevent marked extrahepatic complications.
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