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1 Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3
2 Department of Oncology, Research Unit, Saskatchewan Cancer Agency, 20 Campus Drive, Saskatoon, SK, Canada S7N 0W0
Correspondence
Sylvia van Drunen Littel-van den Hurk
sylvia.vandenhurk{at}usask.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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production and stronger lymphocyte proliferation. Upon challenge with a recombinant vaccinia virus expressing NS3, all mice immunized with NS3-pulsed DCs showed a significant reduction in vaccinia virus titres when compared with mock-immunized mice. However, the NS3-pulsed DCs matured with CpG ODN induced higher levels of protection compared with the untreated NS3-pulsed DCs. These data are the first to show that NS3-pulsed DCs induce specific immune responses and provide protection from viral challenge, and also demonstrate that CpG ODNs, which have a proven safety profile, would be useful in the development of DC vaccines. Published online ahead of print on 12 October 2005 as DOI 10.1099/vir.0.81423-0.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Treatment with interferon-
(IFN-) and ribavirin is the only effective therapy against HCV infection, but the overall response rate is only 40–50 % (Fried et al., 2002; Manns et al., 2001). Currently, there is no licensed vaccine against HCV; therefore, developing strategies for vaccination as well as for treatment of HCV infection is of great importance.
Dendritic cells (DCs) are the most potent type of antigen presenting cells (APCs) and are responsible for the initiation and maintenance of immune responses. Situated in peripheral tissues and in lymphoid organs, DCs are uniquely suited to detect and capture pathogens. They express members of the recently identified Toll-like receptor (TLR) family, which bind common chemical moieties associated with microbial organisms. TLR ligands include bacterial lipopolysaccharide, lipopeptides, hypomethylated CpG DNA motifs, dsRNA and flagellin (Akira et al., 2001; Iwasaki & Medzhitov, 2004). TLR signalling triggers a maturation programme in DCs that leads to the upregulation of major histocompatibility complex (MHC) and co-stimulatory molecules and the expression of pro-inflammatory cytokines. As a result, DCs acquire the unique ability to prime naive T cells (Kaisho & Akira, 2003; Pulendran, 2004). Because of their pivotal functions, DCs have begun to be appreciated as a mandatory target in the creation of new adjuvants. Most importantly, DCs can be used as exogenous adjuvants by loading them with the antigen of interest ex vivo and injecting them back into animals or humans to manipulate the immune response (Ardavin et al., 2004; Berger & Schultz, 2003; Ludewig, 2003; Nieda et al., 2003; Walsh et al., 2003).
The therapeutic potential of DC-based vaccines has been demonstrated for numerous murine tumour models and some human clinical trials (Banchereau et al., 2001; Cerundolo et al., 2004). Recently, DCs have been examined for their capacity to serve as adjuvants and vaccine carriers mediating protection against bacterial, viral, parasitic or fungal pathogens (Moll & Berberich, 2001a, b). For example, DC-based vaccination improved immunity to malaria (Pouniotis et al., 2004), human immunodeficiency virus (Brown et al., 2003), Candida albicans (d'Ostiani et al., 2000) and Leishmania major (Berberich et al., 2003; Ramirez-Pineda et al., 2004).
An effective vaccine against HCV infection should be capable of inducing strong, cross-reactive, helper T-cell (Th) and cytotoxic T-lymphocyte (CTL) responses (Esser et al., 2003; Neumann-Haefelin et al., 2005). A major inherent problem in the design of an effective vaccine against HCV infection is the heterogeneity of its genome (Pawlotsky, 2003; Simmonds, 1999). Thus, the antigens included in HCV vaccines should not only be immunogenic but also conserved between HCV genotypes. HCV NS3 protein has serine proteins and helicase activity and is one of the most conserved protease of HCV (Grakoui et al., 1993). It contains an immunodominant CD4+ T-helper epitope and several CTL epitopes, which have been associated with control of HCV in patients with self-limiting infection (Diepolder et al., 1997; Jiao et al., 2003; Takaki et al., 2000). These characteristics make NS3 an appropriate vaccine candidate for HCV.
In this study, we tested the hypothesis that adoptive transfer of DCs transduced ex vivo with HCV NS3 protein can initiate potent HCV-specific protective immune responses in vivo. DCs were generated from murine bone marrow, pulsed with NS3 protein and stimulated with a CpG oligodeoxynucleotide (ODN). Mice vaccinated with these NS3-pulsed, CpG ODN stimulated DCs developed stronger cellular immune responses and were better protected from a challenge with vaccinia virus expressing NS3 protein than animals immunized with unstimulated NS3-pulsed DCs or DCs pulsed with a control protein.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Briefly, the NS3 gene (HCV 1b strain) consisting of aa 1027–1657 of the polyprotein was cloned into the expression vector pRSETA (Invitrogen). The recombinant clone was transformed into Escherichia coli BL21 (DE3) and the NS3 protein plus a six histidine amino-terminal linker from the vector sequence was expressed as inclusion bodies. Recombinant NS3 protein (rNS3) was purified by nickel-chelate affinity resin under denaturing conditions according to the recommendations of the supplier (Invitrogen). The NS3-containing fraction was renatured by reduction of the urea concentration by dialysis (Jin & Peterson, 1995). The purity of the NS3 protein was analysed by SDS-PAGE and determined to be about 90 %. The endotoxin level, determined using the QCL-1000 Chromogenic Limulus amoebocyte lysate test (BioWhittaker), was 51 ng NS3 protein mg–1. ODN 1826 (5'-CCATGACGTTCCTGACGTT-3') (Qiagen), which is a strong immune cell mitogen known to stimulate mouse splenocytes in vitro (Davis et al., 1998; Rankin et al., 2001), was used in this study. The CpG ODN was phosphorothioate modified to increase resistance to nuclease degradation (Samani et al., 2001).
DC generation from bone marrow.
Murine DCs were generated following the protocol described by Lutz et al. (1999). Briefly, bone marrow cells prepared from the femora and tibiae of normal BALB/c mice (H-2d) were depleted of red blood cells with ammonium chloride (17 mM Tris, 144 mM NH4Cl, pH 7·2) and cultured in RPMI 1640 medium (Gibco-BRL) supplemented with 10 % fetal bovine serum (JRH Biosciences), 1 mM sodium pyruvate, 1 mM non-essential amino acids, 100 IU penicillin ml–1, 100 µg streptomycin ml–1, 5x10–5 M 2-mercaptoethanol and 10 mM HEPES (complete RPMI) containing 20 ng GM-CSF (PeproTech) ml–1 at 37 °C and 5 % CO2. On day 3, the non-adherent granulocytes and T and B cells were gently removed and fresh medium was added. On days 5 and 7, 50 % of the medium was replaced with fresh culture medium containing 20 ng GM-CSF ml–1. On day 9, non-adherent cells were harvested for protein pulsing.
DC pulsing.
DCs harvested on day 9 were washed twice in RPMI 1640. One hundred microlitres of the liposomal transfection reagent N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl-sulfate (DOTAP; Roche) and 20 µg rNS3 or control protein, human serum albumin (HSA; Sigma), were mixed with 500 µl RPMI 1640 at room temperature in polystyrene tubes for 20 min (Liu et al., 2001; Nonn et al., 2003; Santin et al., 1999). DCs (2x107) in 2 ml RPMI 1640 were added to the DOTAP/protein mixtures. The DCs were incubated for 3 h at 37 °C, washed three times, resuspended in complete RPMI 1640 containing 20 ng GM-CSF ml–1 and then cultured for 18 h in the absence or presence of 25 µg ODN 1826 ml–1. These pulsed DCs were used for in vitro phenotypic analysis, immunohistochemical analysis, mixed lymphocyte reaction (MLR) assay, cytokine measurements and in vivo immunization.
Phenotypic analysis of DCs.
After the DCs were subjected to different treatments they were collected on day 10 and incubated with FITC-labelled monoclonal antibodies (anti-mouse CD11c, I-Ad, CD86, CD54 and CD40; BD PharMingen) for 30 min at 4 °C in PBS (0·137 M NaCl, 0·003 M KCl, 0·008 M Na2HPO4, 0·001 M NaH2PO4, pH 7·3). After three washes, the cells were resuspended in PBS. Analysis was performed on a FACSCalibur flow cytometer (BD Biosciences).
Immunohistochemistry.
The NS3- and HSA-pulsed DCs were plated in four-well LAB-TEK chamber slides (Nalge Nunc International). After 6 h, the DCs were fixed for 20 min in 4 % paraformaldehyde in PBS. Subsequently, they were permeabilized for 10 min in 0·5 % Triton X-100 in PBS. The cells were blocked for 20 min with PBS containing 1 % goat serum, and incubated with NS3-specific polyclonal rabbit serum (1 : 200) for 1·5 h. Biotinylated goat anti-rabbit immunoglobulin G (IgG) (1 : 5000) (Zymed Laboratories) was added and then the cells were incubated again for 1·5 h. Finally, the cells were incubated for 45 min with ABC Reagent (Vector Laboratories) and incubated with peroxidase substrate solution (DAB substrate kit SK-4100; Vector Laboratories) until the desired stain intensity developed. The slides were rinsed in dH2O, counterstained with 0·1 % toluidine blue, and again rinsed in dH2O. All incubations were performed at room temperature and the slides were washed in PBS three times between incubations. The slides were observed and the images were captured with a Zeiss Axiovert 200M microscope (Carl Zeiss).
MLR assay.
Splenocytes from C57BL/6 (H-2b) and BALB/c (H-2d) mice (Charles River Laboratories) were passed over nylon wool fibre columns (Polyscience) and T cells were separated and used as responder cells at 2x105 cells per well in U-bottom 96-well plates. The DCs from BALB/c mice were collected on day 10, irradiated at 50 Gy and added to the responder cells in varying cell numbers as stimulator cells. Cells were cultured for 5 days in complete RPMI 1640 at 37 °C and 5 % CO2. The cell cultures were pulsed with 0·4 µCi (14·8 kBq) [methyl-3H]thymidine (Amersham Pharmacia Biotech) per well during the last 18 h. The cells were harvested and radioactivity was determined by scintillation counting. Data are expressed as mean c.p.m. of triplicate wells.
Cytokine measurements.
The culture supernatants of the treated DCs were collected on day 10 and analysed with respect to interleukin 12 (IL-12) and IL-10 production with a sandwich ELISA using corresponding specific capture and detection antibodies. Cytokine levels were calculated using standard curves constructed by recombinant murine cytokines (BD PharMingen).
Immunization of mice.
Eight groups of 12 eight-week-old female BALB/c mice (Charles River Laboratories) were immunized twice with a 2 week interval, subcutaneously in the base of the tail with 5x106 DCs or 1 µg rNS3 formulated with 25 µg alum (2 % Alhydrogel; Superfos Biosector) or PBS in a 100 µl volume. The DCs were transduced either with rNS3 or HSA and then left either untreated or incubated with ODN 1826. Ten days after the last immunization, half of the mice of each group were sacrificed to isolate splenocytes for lymphocyte proliferation, enzyme-linked immunospot (ELISPOT) and CTL assays. The other half of the mice were used for viral challenge. The experiments were carried out according to the guidelines provided by the Canadian Council for Animal Care.
ELISPOT assay.
A cytokine-specific ELISPOT assay was performed as described previously (Ioannou et al., 2002; Lewis et al., 1999). Briefly, 96-well MultiScreen-HA filtration plates (Millipore) were coated overnight at 4 °C with 0·1 µg murine IFN- or IL-4-specific monoclonal antibodies (BD PharMingen) per well. Splenocytes were isolated from the mice as described previously (Baca-Estrada et al., 1996), resuspended in AIM-V medium (Gibco-BRL) and added to the coated plates at 106 cells per well in the absence or presence of rNS3 at a final concentration of 1 µg ml–1. After a 20 h incubation at 37 °C and 5 % CO2, the plates were washed extensively and incubated with biotinylated anti-murine IFN- or IL-4 monoclonal antibodies (BD PharMingen) at 2 µg ml–1. This was followed by incubation with streptavidin–alkaline phosphatase (Gibco-BRL) at a 1 : 1000 dilution. The spots were visualized with a substrate consisting of nitro blue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Sigma). The number of cytokine-secreting cells was expressed as the difference between the number of spots per 106 cells in rNS3-stimulated wells and the number of spots per 106 cells in non-stimulated wells.
Proliferation assay.
The splenocytes isolated from the immunized mice were dispensed at 3·5x106 cells ml–1 in AIM-V medium and cultured in 96-well tissue culture plates at 3·5x105 cells per well in the absence or presence of 1 µg rNS3 ml–1. After 72 h in culture, the cells were pulsed with 0·4 µCi (14·8 kBq) [methyl-3H]thymidine (Amersham Pharmacia Biotech) per well. The cells were harvested 18 h later and radioactivity was determined by scintillation counting (TopCount NXT Microplate Scintillation & Luminescence Counters; Packard Instrument Company). The 
c.p.m. was determined by subtracting background activity of cells incubated with medium only without antigen.
CTL assay.
To prepare effector cells, splenocytes were isolated from each group of mice. Syngeneic splenocyte stimulators were prepared by infection for 1 h at 37 °C with a recombinant vaccinia virus VP1461, which encodes NS3/NS4/NS5 from HCV-1b strain BK (kindly provided by Sanofi Pasteur MSD, Toronto, Canada), at an m.o.i. of 10. After infection, the stimulators were suspended at a concentration of 106 cells ml–1 and irradiated with 30 Gy. The splenocytes from each group were cultured with the stimulators at 37 °C and 5 % CO2 for 5 days in AIM-V medium. Mouse IL-2 (Roche) was added to a final concentration of 5 U ml–1 after 2 days. To generate target cells, P815 cells (H-2d) (ATCC) were stably transformed with NS3. NS3-transformed and control P815 cells were labelled for 1 h with 100 µCi (3·7 MBq) Na251CrO4 per 106 cells. Cells were washed four times and used as targets at 5x104 cells ml–1. One hundred microlitres of labelled target cells was added to each well of a U-bottom 96-well plate and 100 µl of effector cells were added to the target cells in triplicate wells at various effector-to-target (E : T) ratios. Plates were incubated for 4 h at 37 °C and 5 % CO2. The supernatant from each well was counted in a 1470 Wizard gamma counter (Perkin Elmer). The percentage specific cytotoxicity was calculated as [(experimental 51Cr release–spontaneous release)/(total 51Cr release–spontaneous release)]x100.
Recombinant vaccinia virus challenge and plaque assay.
Ten days after the last immunization, mice were challenged intraperitoneally with 5x106 p.f.u. of recombinant vaccinia virus encoding NS3/NS4/NS5, designated VP1461. Five days after challenge, mice were scarified and the ovaries were harvested, homogenized and sonicated. Recombinant vaccinia virus titres were determined by plating 10-fold dilutions of the homogenized ovaries onto BSC-1 cells. The BSC-1 cells were stained with 0·075 % (w/v) crystal violet (Murata et al., 2003; Pancholi et al., 2003) to identify the vaccinia virus-infected cells.
Statistical analysis.
All data were analysed with the aid of a software program (GraphPad Prism 3.0). Differences between the means of experimental groups were analysed using an independent two-tailed t-test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), whereas no signal was detected in the HSA-pulsed DCs as expected (Fig. 1b).
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). No IL-10 production was detected (data not shown) by any DCs. The production of high levels of IL-12, but no IL-10 by DCs stimulated with CpG ODN suggests the development of a Th1-biased response, which is likely to clear HCV most efficiently. The results also showed that DCs matured with CpG ODN induced a stronger allogeneic T-cell response compared with untreated DCs, indicating a more efficient antigen presenting capacity of the CpG ODN-treated DCs (Fig. 3b). There were no differences between rNS3-pulsed DCs and HSA-pulsed DCs with respect to cytokine production and MLR (Fig. 3).
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and low numbers of IL-4-secreting cells, indicating that a Th1-biased immune response was induced. Furthermore, the animals immunized with CpG ODN-matured, rNS3-pulsed DCs produced significantly (P<0·05) higher numbers of IFN--secreting cells than the animals vaccinated with untreated rNS3-pulsed DCs. In contrast, the group of mice immunized with rNS3 formulated with alum developed relatively high numbers of IL-4-secreting cells and low numbers of IFN--secreting cells, indicating that a Th2-biased immune response was induced (Fig. 5).
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, both groups of mice immunized with rNS3-pulsed DCs developed a CTL response. However, the CTL response obtained by immunization with rNS3-pulsed DCs stimulated with CpG ODN was significantly stronger than that observed after immunization with unstimulated rNS3-pulsed DCs (P<0·05). In contrast, as expected, the mice immunized with HSA-pulsed DCs or rNS3 protein formulated with alum did not develop a NS3-specific CTL response. These results were confirmed in a second trial and demonstrate the induction of a NS3-specific CTL response in mice immunized with rNS3-pulsed DCs. In addition, the fact that P815 is an Ia-negative cell line (Mozdzanowska et al., 2000) indicates that these cytotoxic T-cell responses are mediated by NS3-specific CD8+ T cells.
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) to assess the efficacy of adoptive transfer of rNS3-pulsed DCs in the protection of mice against virus infection. Ten days after the last immunization, all mice were challenged with 5x106 p.f.u. of recombinant vaccinia virus expressing NS3/NS4/NS5. Five days after challenge, the mice were sacrificed and the presence of recombinant vaccinia virus in the ovaries was determined. As shown in Fig. 7, the mean vaccinia virus titres decreased at least five orders of magnitude in mice vaccinated with rNS3-pulsed DCs stimulated with CpG ODN (P<0·001) and decreased four orders of magnitude in mice vaccinated with unstimulated rNS3-pulsed DCs (P<0·01) compared with the mice immunized with PBS. The two groups of animals immunized with HSA-pulsed DCs also shed less virus than the PBS group (P<0·05), but the reduction in virus titre was at most 1 log. This was most likely due to the development of natural killer cell activity (van den Broeke et al., 2003). No reduction in virus titre was seen in the group immunized with NS3 and alum.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Larsson et al., 2004; Sarobe et al., 2002, 2003). If we assume that this results in impaired antigen presentation, which in turn fails to result in an expansion of the Th cell population, then the result will be a limited expansion of the humoral and cell-mediated immune response. Indeed, characteristics of HCV infection include low HCV-specific antibody (Chen et al., 1999) and a low frequency of HCV-specific CTLs (Rehermann & Chisari, 2000), both of which are consistent with a failure of CD4+ Th cells to induce proliferation of the B- and CTL-effector cells. IFN treatment of HCV carriers indicates that the frequency of HCV core-specific Th cell precursors is significantly higher in sustained responders than in transient or non-responders (Lasarte et al., 1998), suggesting that the expansion of Th cell precursors correlates with and is critical for HCV elimination. There is therefore a high probability that DCs from HCV carriers that are loaded and matured ex vivo with HCV proteins followed by autologous transfusion will be able to prime naive T-cells and/or stimulate existing HCV-specific cellular immunity. The aim then is to change the equilibrium between the virus and the immune response in patients that will result in viral clearance. In addition, it has been suggested that the number of impaired DCs is small (Sarobe et al., 2002), so autologous transfusion of a large number of HCV antigen-loaded matured DCs may overcome the impairment.
The therapeutic potential of DC-based immune interventions has been reported for a variety of murine tumour models and more recently in human clinical trials. However, only two studies have explored the in vivo efficacy of DC-mediated vaccination against HCV. One study showed that core-specific CTL are effectively primed in mice by injecting DCs treated in vitro with an anthrax toxin fusion protein composed of a lethal factor (LF) and the HCV-core 133–142 epitope (Moriya et al., 2001). However, epitope-based DC immunotherapies induce limited CTL responses and are only applicable in patients with the appropriate HLA haplotye. In the other study, DCs transfected with recombinant adenovirus expressing HCV core (Adex1SR3ST) more efficiently prime core-specific CTLs than Adex1SR3ST-transfected macrophages or DCs treated with an anthrax toxin fusion protein mentioned above. Upon challenge with recombinant HCV-core-expressing vaccinia virus, vaccinia virus titres were significantly reduced in mice immunized with Adex1SR3ST-transfected DCs (Matsui et al., 2002). Although the strategy of vaccination with DCs transfected with recombinant virus containing HCV genes might result in efficient transfection and high levels of transgene expression in DCs, it may also negatively impact DC functions. In addition, immunodominant viral products could suppress an immune response against the transgene in an unpredictable manner. Since a variety of practical and theoretical concerns may limit the utility of these methods in patients, DCs pulsed with HCV proteins as an alternative strategy would be more efficient, safer and more feasible for human immunotherapy.
DCs are capable of internalizing macromolecules by macropinocytosis and receptor-mediated endocytosis (Sallusto et al., 1995). Thereby, exogenous proteins are usually processed and presented via the MHC class II pathway. However, DCs are able to channel antigenic peptides efficiently towards MHC class I, a process termed ‘cross priming’ (Rodriguez et al., 1999). In this study, we used the cationic liposomal transfection reagent DOTAP to deliver HCV NS3 protein to DCs. The rNS3-pulsed DCs induced strong cellular immune responses and protection against recombinant vaccinia virus containing the HCV NS3 gene. Several studies reported that if proteins were precomplexed with the DOTAP, cytoplasmic uptake of proteins was increased, and processing and presentation of incorporated protein by the MHC class I pathway was further enhanced (Nonn et al., 2003; Santin et al., 1999).
Recently, CpG ODNs have attracted a great deal of attention as a novel and safe adjuvant. Indeed, CpG DNA induces stronger immune responses with less toxicity than other adjuvants (Lonsdorf et al., 2003; Oumouna et al., 2005; Weeratna et al., 2000). CpG ODNs bind to TLR9 and preferentially induce Th1-biased immune responses with the production of cytokines such as IL-12 and IFN- (Harandi et al., 2003; Rao et al., 2004). In this study, we used ODN 1826, a mouse-specific CpG ODN, to mature protein-pulsed DCs. The results show that the CpG ODN caused upregulation of MHC class II, CD86 and CD40 and triggered cytokine release such as IL-12. CpG ODN 1826, in particular, dramatically increased the expression of CD40 on DCs. CD40 has emerged as a key signalling molecule for the function of DCs in the immune system. CD40 is expressed by DCs and is upregulated when DCs migrate from the periphery to draining lymph nodes in response to microbial challenge. CD40 functions in the adaptive immune response as a trigger for expression of co-stimulatory molecules, and for potent T-cell activation (O'Sullivan & Thomas, 2003). DC stimulation via CD40 also has the capacity to induce high levels of the cytokine IL-12, which can activate natural killer cells to enhance proliferation of CD8+ T cells, and polarize CD4+ T cells toward a Th1 type (O'Sullivan & Thomas, 2003), which plays a major role in clearing HCV. In this study, DCs matured with CpG ODN produced a greater allogeneic T-cell response compared with DCs not treated with CpG ODN, indicating that CpG ODN-mediated DC maturation/activation is associated with a more efficient functional transition to professional APCs. Notably, there were no differences between rNS3-pulsed DCs and HSA-pulsed DCs with respect to phenotype, cytokine production and MLR implying that transduction with rNS3 protein does not impair DC functions.
Our study also demonstrates that compared with the unstimulated rNS3-pulsed DCs, the rNS3-pulsed DCs matured with CpG ODN 1826 induced more robust cellular immune responses including enhanced cytotoxicity, higher IFN- production and stronger lymphocyte proliferation in mice. Upon challenge with a recombinant vaccinia virus expressing HCV NS3, the two groups of mice vaccinated with rNS3-pulsed DCs showed a remarkable reduction in vaccinia virus titre compared with mock-immunized controls, and the mice immunized with the rNS3-pulsed DCs matured with CpG ODN showed stronger immune protection than those not matured with CpG ODN.
These data have important clinical implications and are the first to show that NS3-pulsed DCs can induce specific immune responses and provide antiviral protection. CpG ODNs as non-toxic, safe agent would be useful in the development of DC vaccines. Thus, this study provides a novel vaccination strategy against hepatitis C that ultimately should be transferable to humans.
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ACKNOWLEDGEMENTS |
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Received 9 August 2005;
accepted 27 September 2005.
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