Hepatitis C virus (HCV) is a main cause of both acute and chronic hepatitis
which is a disease with a significant global impact. Based on WHO report, about
170 million individual (consisting 3% of world population) are the victims of
HCV infection (Murata et al., 2003; Frank
et al., 2010). However the prevalence of the HCV are different in
various regions which is higher among Asians and Africans, on the other hand,
humeral immune response, which plays the key role in protective immunity of
various viral infections and it has not been observed that this immune response
could have any actual role in viral clearance in patients with acute HCV infection
or chronic hepatitis C treatment (Murata et al.,
2003), to find a proper vaccine which could stimulate the Cellular responses
specially cytokine secretions, could help to prevent or decrease the incidence
of the infection among these populations.
One of the viral features is its acute reaction by the infected cell which
included the activation of previous antiviral defense mechanism, commitment
to apoptosis and cytokine secretion which leaded to decrease the level of viral
replication and to control the viral spread (Mogensen and
Paludan, 2010). Helper T cells (Th) are the main regulators that help to
destruct the antigen and to reinforce the antibody (Najafizadeh
et al., 2007). Th1 cytokines such as IL-2, IF-Gamma and TNF-alpha
are essential for antiviral activities of immune system. The highest interleukin
activity begins at the site of their production however, when entering the circulation
they are biologically active, so the IL s could be measured from the target
tissue and the Peripheral blood (Lapinski, 2001; Najafizadeh
et al., 2007).
The HCV core protein is an extremely basic protein that constructs the viral
nucleocapsid by binding to the RNA. The biochemical properties of HCV-core protein
have not been well described. It is consisted of 191 aa and three distinctive
domains (Boulant et al., 2005; Torbati
et al., 2012). HCV core protein was considered as immunogenic counterpart
of the HCV vaccine. Serum antibodies against HCV core protein epitopes have
been detected in HCV patients (Torbati et al., 2012).
According to the conservative feature of the core gene in different HCV genotypes
and the low mutation incidence in this genomic region , make this protein an
ideal candidate for HCV vaccine (Torbati et al.,
The capability to monitor hepatitis C virus (HCV)-specific T cell immunity
and cytokine secretion in immunized mice could provide new information about
the privation of HCV infection. Elispot assay was used as well as the ELISA
technique to evaluate a cross-section of HCV-core immunized mice based on our
previous study (Torbati et al., 2012). To this
purpose The amount of IL-6, TNF-α and IFN-γ in immunized mice with
the recombinant protein were measured, these cytokines are responsible to prevent
the viral infection.
MATERIALS AND METHODS
Recombinant protein: The confirmed recombinant protein from our previous
study (Torbati et al., 2012) was used in order
to evaluate the specified cytokines secretion.
Immunization of balb/c mice: As there is a great similarity between the balb/c mice and human immunity system mechanisms, identical (gender, age, weight, etc.,) mice were used in this study. 6-8 weeks old Female BALB/c mice (Razi Institute of Iran) were housed in approved animal care facilities during the experimental period. According to the Guiding Principles in the Care and Use of Laboratory Animals, the immunization performed as follow:
Control group: A group of 6 female balb/c mice was immunized with a total volume of 150 μL Phosphate-Buffered Salin-1X (PBS), divided in 3 footpad injections (50 μL per injection), with 2 weeks intervals.
Subject group: This group consisted of 10 balb/c mice, 50 ng of recombinant protein in complete Freunds adjuvant (Sigma, Germany) were injected s.c in to the footpad on the first day, a booster with the same amount of protein in incomplete Freunds adjuvant (Sigma, Germany) were injected 2 weeks later and on day 28, the mice were immunized with 50 ng of the protein in PBS. On day 42, before splenocytes isolation, the serum samples were collected by retro-orbital puncture.
Ex vivo direct enzyme-linked immunospot (ELISPOT) Assay: In order
to assess the amount of T lymphocytes responsible for cytokine secretion, the
splenocytes were collected using the available protocol in laboratory (BDB
2011) Fig. 1. ELISpot assay employs a capture antibody
specific for Mouse, pre-coated onto a PVDF-backed microplate. Stimulated cells
are directly pipetted into the wells and the immobilized antibody binds to the
nearest vicinity of the cells secreting IL-6 or IFN-γ.
IFN-γ Elispot test: Elispot assay for detecting IFN-γ secreting
cells specific for HCV core epitopes has been formerly described by Duenas-Carrera
et al. Acosta-Rivero et al. (2009),
PVDF-bottomed well plate (Millipore, Cat No. MAIPS45510) coated with 100 μL
murine IFN-γ-specific antibody (Mouse IF gamma Elispot Ready-SET-Go, bioscience,
CatNo.88-7384) the test carried out using the kit instructions. At the end,
the plates were dried in dark room, the spots were counted using dissection
microscope (Zisse, Germany).Specific spots were red round with a dark center
and fairly fuzzy edges.
IL-6 Elispot test: The collected splenocytes, were then platted in to 96-well PVDF-backed coated with polyclonal antibody specific for mouse/rat IL-6 (R and D System, Cat No. EL406, USA), the procedure performed according to the kit instructions (Mouse/Rat IL-6 ELISpot Kit, Cat No. EL406, USA), after the final wash, the developed microplate analyzed, using dissection microscope. Specific spots are blue round with a dark center and slightly fuzzy edges.
TNF-α Enzyme-linked Immunosorbent assay: The sera of immunized mice were collected before scarification. Pre-immune sera were considered as negative controls. The test was performed based on the kit instruction (Mouse TNF-α Immunoassay Elisa kit, R and D System, USA) and the optical density was measured at 640 nm using the microplate reader (Sunrise Tecan, Austria).
|| The collected splenocytes of immunized mice
MTT test: In order to assess the viability of the cultured splenocytes as a complementary test, cells (5x103 cells well-1) were plated onto 96-well plates and cultured for 3 days (every 24 h the cells were transferred to a new culture), then 100 μL of MTT (Sigma, USA) (5 mg mL-1 MTT ,90 μL RPMI) added to each well and were incubated at 37°C for 4 h, following centrifuged at 1000 g for 5 min. The supernatant emptied and 200 μL of Dimethyl sulfoxide (DMSO) (Sigma, Germany) was added to each well. The stained cells were eluted with methanol (Merck, Germany) and the optical density was measured at 590 nm using the microplate reader (Sunrise Tecan, Austria).
Dot blot: In order to evaluate the humoral immune response against the recombinant HCV-core protein, the protein in different concentrations (1:100 and 1:500) were dotted on a nitrocellulose paper, the primary (Immunized sera) and secondary antibodies (conjugatedhuman anti-IgG peroxidase) were added, subsequently the presence of antibodies against recombinant HCV-core protein were detected by adding substrate (30% H2O2 and DAB) (Merck, Germany) diluted in 1 M Tris (Merck, Germany) and TBS.
Statistical analysis: Data were analyzed using SPSS version 16. The one-way ANOVA was used to compare the means. The values are significant at p<0.05.
According to the result of the Elispot tests there is a significant difference between the IFN-γ level in case and control group (18.53±3.87, p = 0.001) as well as IL-6 level (22.9±1.26, p = 0.000). Moreover, similar to pervious cytokines it has been observed that the TNF-α had dramatically higher level in recombinant protein treated group compared to the control group (53.96±4.54, p = 0.000) (Fig. 2).
In this study, the MTT assay reproducibility was considerable. The cells proliferation and viability were defined by color change. The absorbance was measured at 590 nm and it was higher in test group in comparison to control group with p<0.05.
The result of dot blotting approved the humoral immune system response of immunized mice against HCV core protein. The dot formation was shown in two different concentrations (1:100 and 1:500) in test group while there was no dot formed by the control group (Fig. 3).
|| IFN-γ, IL-6 and TNF-α in test and control groups
||Dot Blot result, the sera of the control group and the immunized
HCV causes silent infection and it has been reported that its existence would
possibly be destructive for host cells; moreover, the core protein has been
mostly detected in chronically infected HCV patients (Torbati
et al., 2012). It has also been reported that Hepaciviruses core
protein is unique among the Flaviviridae (Boulant et al.,
2005). On the other hand, it has observed that the Cytokines and chemokines
play key roles in responses against viral infections and viral immunopathology
related to specific cellular receptors, a vast number of viral glycoproteins
could stimulate the cytokines and chemokines secretion directly. Moreover, viral
RNA and some viral proteins interfere with cellular signal transduction and
transcription factor activities, Such as NF-κB that plays an important
role such as expression of cytokines and chemokines is considered (Mogensen
and Paludan, 2010). As a result, a protein which could stimulate the cytokine
secretion would have a critical role in viral clearance and prevention. Thus
the recombinant HCV-Core protein which was reported in our previous study was
used and the cytokine secretion in immunized mice was analyzed in order to assess
the capability of the recombinant HCV-core protein as an effective vaccine candidate
(Torbati et al., 2012).
Interleukin 6 (IL-6) is a multifunctional cytokine that has critical roles
in acute phase reactions, hematopoiesis, host defense and bone remodeling (Hirano
et al., 1986), it has been reported by Feldmann
et al. (2006) that elevated secretion of IL-6 through stimulation
of TLR2 by HCV core protein can affect the pathogenesis of hepatitis C-associated
B-cell non-Hodgkin's lymphoma . In another research conducted by Sekiguchi
et al. (2012) they have stated that the amount of inflammatory cytokines
(tumor necrosis factor) TNF-α and IL-6 is related to the onset of chronic
hepatitis in mice.
Mouse TNF-α is a pleiotrophic cytokine that plays a crucial role in the
hepatitis C inflammatory processes and apoptosis, TNF-α induces cell death
that can be enhanced by nuclear factor kappa B (NF-κB) activation (Park
et al., 2012). Mouse cells known to express TNF-α include neurons,
CD8+T cells, mast cells, macrophages, monocytes and dendritic cells (Lees
et al., 1999). Park et al (2012).
have been reported that HCV infection increases TNF-α-induced cell death
by suppressing NF-κB activation via the action of core, NS4B and NS5B proteins
which could lead to immune-mediated liver injury in HCV infection (Park
et al., 2012).
IFN-γ is the only member of the type II class of interferons and it is
a dimerized soluble cytokine (Thiel et al., 2000).
IFN-γ is critical for innate and adaptive immunity against viral and intracellular
bacterial infections as well as tumor control, IFN-γ is largely produced
by Natural Killer T (NKT) cells and Natural Killer (NK) and by CD4 Th1 and CD8
cytotoxic T lymphocyte (CTL) effector T cells (Schoenborn
and Wilson 2007). Kang et al. (2003) has
examined the HCV-specific interferon-γ responses to HCV core and NS3A proteins
using enzyme-linked immunosorbent spot and it has been observed that the level
of IFN-γ is increased in the immunized group as well as patients with Hepatitis
It has been concluded that the amounts of IL-6, TNF-α and IFN-γ are increased in patients with acute HCV infection, thus a vaccine which could stimulate the secretion of these cytokines in advance may have a preventive role. Based on the results of this study, a significant difference between the amounts of cytokine secretion in immunized mice in response to recombinant protein of HCV-core protein and the control group was observed.
We would like to thank the staff of Shahid Beheshti University of medical sciences and Tehran university of medical sciences.