Dendritic cell‐based immunity and vaccination against hepatitis C virus infection

Hepatitis C virus (HCV) has chronically infected an estimated 170 million people worldwide. There are many impediments to the development of an effective vaccine for HCV infection. Dendritic cells (DC) remain the most important antigen‐presenting cells for host immune responses, and are capable of either inducing productive immunity or maintaining the state of tolerance to self and non‐self antigens. Researchers have recently explored the mechanisms by which DC function is regulated during HCV infection, leading to impaired antiviral T‐cell responses and so to persistent viral infection. Recently, DC‐based vaccines against HCV have been developed. This review summarizes the current understanding of DC function during HCV infection and explores the prospects of DC‐based HCV vaccine. In particular, it describes the biology of DC, the phenotype of DC in HCV‐infected patients, the effect of HCV on DC development and function, the studies on new DC‐based vaccines against HCV infection, and strategies to improve the efficacy of DC‐based vaccines.


Introduction
Hepatitis C virus (HCV) is a blood-borne pathogen and has led to chronic infection in an estimated 170 million people worldwide. It is a major cause of chronic liver diseases with a substantial morbidity and mortality. 1 People infected with HCV serve as a reservoir for transmission to others and are at risk for developing chronic liver diseases, such as liver cirrhosis and primary hepatocellular carcinoma. It has been estimated that HCV accounts for 27% of cirrhosis and 25% of hepatocellular carcinoma worldwide. 2 Therapy for chronically HCV-infected patients has involved a combination of a pegylated interferon-a and ribavirin (pegIFN/RBV). 3 The choice of this regimen was based upon the results of three pivotal, randomized, clinical trials that demonstrated the superiority of this combination treatment over standard IFN-a and RBV. [4][5][6] However, this therapy is expensive, non-specific, toxic, and only effective in about 50% of genotype-1 HCV patients. 7 Specific targeted antiviral therapies for HCV using directly acting antiviral agents or inhibitors are at different phases of development and clinical trials. 8 These inhibitors target HCV receptors, HCV-IRES, NS3/ 4A, NS5A and NS5B. 9 Two protease inhibitors (boceprevir and teleprevir) have recently been approved and are increasingly used in combination with pegIFN/RBV for type-1 HCV mono-infection.
An effective HCV vaccine would reduce the number of new infections and thereby reduce the burden on healthcare systems. However, there are many impediments to the development of an effective HCV vaccine including the existence of multiple HCV genotypes, limited availability of animal models and the complex nature of the immunological response to HCV. 10 Clearance of HCV infection appears to require strong and broadly crossreactive CD4 + , CD8 + T-cell resonsese [11][12][13] and neutralizing antibody responses. 14 With the variability of HCV, a combination approach including vaccination and antiviral therapy or immune modulation might be necessary for management of HCV infection. 15 Several HCV vaccines have been developed. Although most of them are still at the preclinical stages, some have advanced into phase I or phase II clinical trials to determine the safety and efficacy of the candidate vaccines. The approaches or classifications of HCV vaccine development include: (i) recombinant proteins such as HCV core protein and non-structural proteins emulsified with MF59, 16 HCV gpE1/E2 emulsified with MF59, 17 GI-5005: HCV NS3 and core proteins, 18 HCV core protein/ISCOMATRIX; 19 (ii) synthetic peptides such as IC41 20 and a peptide (core) emulsified with ISA51; 21 (iii) DNA-based vaccine such as CIGB-230 22 and others; 23-26 (iv) virus-based vaccine such as modified vaccinia Ankara virus-based HCV vaccine: TG4040, 27,28 recombinant adenoviral HCV vaccines, [29][30][31] lentiviral vector-based HCV vaccine. 32 These approaches have limited effectiveness for a number of reasons including: the delivery of a limited number of protective viral epitopes, the inclusion of incorrectly folded recombinant proteins, the limited humoral and cell-mediated responses that are associated with DNA vaccines, and the use of adjuvants with relatively poor potency.
Recently, dendritic cell (DC) -based vaccines against HCV has been developed. [31][32][33][34][35][36][37] Dendritic cells (DC) are the most effective antigen-presenting cells and are professionalized to capture and process antigens, converting proteins to peptides that are presented on MHC molecules and recognized by T cells. 38,39 The medical implications of DC that control a spectrum of innate and adaptive responses have been reviewed. 40 The present review summarizes the current understanding of DC functions in HCV infection and explores the prospects of DC-based HCV vaccine development. In particular, it describes the biology of DC, the phenotype of DC in HCV-infected patients, the effect of HCV on DC, the studies on new DC-based vaccines against HCV, and strategies to improve the efficacy of DC-based vaccines.

DC function and generation in culture
Dendritic cells are the most efficient inducers of all immune responses, and are capable of either inducing productive immunity or maintaining a state of tolerance to self and non-self antigens. Two major DC subsets have been characterized to date in humans, based on their development from myeloid or lymphoid precursors of bone marrow pluripotent cells. 41 Myeloid dendritic cells (MDC) are CD1a, CD11c, CD13, CD14, CD33 + , whereas lymphoid descendants, also called plasmacytoid dendritic cells (PDC) express CD123 and BDCA-2 on their surface. Both MDC and PDC are derived from bone marrow and can be found in peripheral blood in an immature stage. Immature dendritic cells (iDC) express low levels of MHC class I and II and co-stimulatory molecules on their surface and are proficient in endocytosis and antigen processing. Maturation of DC occurs after detecting microbial or host-derived danger signals, or upon contact with pro-inflammatory cytokines, such as tumour necrosis factor-a (TNF-a), interleukin-1 (IL-1), or after engagement of the CD40/CD40 ligand (CD40L) system. The DC play a key role in regulating immunity, serving as the sentinels that capture antigens in the periphery, process these antigens into peptides, and present these peptides to lymphocytes within lymph nodes. The maturation process includes a series of transformations that lead to a reduction of antigen-capturing capacity, an increase in MHC and co-stimulatory molecule expression and, most importantly, the development of an exceptional efficiency in presenting antigens to T cells, activating natural killer cells, and producing interferons, so linking the innate and adaptive immune responses. 42 Although both MDC and PDC are potent in antigen uptake, processing and presentation, they have fairly distinct cytokine profiles: MDC produce large amounts of IL-12 and IL-10 and make small amounts of IFNs, while PDC are specialized type-I IFN-producing machines and express much lower levels of other cytokines ( Table 1).
As the frequencies of DC in the peripheral circulation are low, alternative approaches to DC generation for research purposes were sought. 43 The classic strategy for the ex vivo generation of monocyte-derived DC (MDDC) consists of a two-step culture protocol, in which monocytes are differentiated towards iDC, followed by the induction of mature DC. Monocytes may be isolated from blood by adherence or positive selection using immunomagnetic beads. 44 Differentiation of DC is induced by using granulocyte-macrophage colony-stimulating factor and IL-4, 45 but the doses of each reagent, the culture conditions (flask or closed plastic bag 46,47 ), the composition of the culture medium, the cocktail of reagents such as CD40L 48 and poly(I:C) 49 used to induce maturation, and the methods used to antigen-load DCs all vary substantially. 50 The total in vitro culture duration lasts 1 week but there is increasing evidence that maturation of MDDC can be generated even after short-term cell culture for 2-3 days 51-54 with several advantages: it simplifies the laborious and time-consuming process of DC manufacture and it reduces the actual risk of microbial contamination related to in vitro culture.

DC in HCV-infected patients
Many researchers have explored the hypothesis that the failure of HCV-infected individuals to mount an effective T-cell response, and so lead to the development of chronic HCV infection, is the result of a virus-mediated impairment of DC function. This impairment may include a reduced frequency of MDC and PDC, reduced IL-12 and IFN-a, and increased IL-10 production, accompanied by an impaired capacity to prime naive T cells. 37,55,56 In human studies, findings related to DC functions are controversial. Complex defects such as reduced number of DC, deficiency in co-stimulatory molecules, decreased Tcell stimulatory capacity, overproduction of the immunoregulatory cytokine IL-10/transforming growth factor-b and proliferation of regulatory T lymphocytes were detected in patients with chronic HCV infection, [57][58][59][60][61][62][63][64][65][66][67][68][69][70][71][72] while others failed to identify any DC abnormalities. [73][74][75][76][77] One analysis suggested that DC from HCV-infected subjects have a normal capacity to stimulate CD4 + T cells, and so the functional effectiveness of DCs derived from HCV-infected individuals provides a rationale for the DCbased immunotherapy of chronic HCV infection. 78 Another study demonstrated that DC retained the same allostimulatory capacity before and following the establishment of persistent HCV infection. The surface phenotype and the amount of IL-10 and IL-12p70 produced during DC maturation did not differ between HCV-infected individuals and healthy controls. Maturation of DC from HCV-infected individuals performed comparably in an allogeneic MLR compared with healthy individuals. Mature MDDC from HCV-infected individuals stimulated the expansion of peptide-specific naive CD8 + T cells. The MDDC from HCV-infected and healthy individuals were phenotypically indistinguishable and performed comparably in functional assays. 79 Such discrepancies most possibly derive from different patient cohorts who had taken ribavirin either at the time of study or in the past, or from cohorts with different amounts of liver inflammation/ fibrosis, assessment of non-human primate models of HCV infection, different experimental approaches, and distinct read-outs. 80 Various approaches have been used to clarify the discrepancies and possible underlying mechanisms, including generation of MDDC or the analysis of peripheral blood DCs in patients with chronic HCV, by studying the effectiveness of recombinant HCV proteins or cell-cultureadapted strains of HCV on DC in vitro. Some researchers also reported that HCV-infected cells trigger a robust IFN response in PDC by a mechanism that requires active viral replication, direct cell-cell contact, and Toll-like receptor 7 (TLR7) signalling, and showed that the activated PDC supernatant inhibits HCV infection in an IFN receptor-dependent manner. 81 As there is clearly controversy regarding MDC's ability to activate T cells, it is unclear whether on a per cell basis MDCs from HCVinfected individuals are able to prime naive T cells. Additionally, reduced numbers of peripheral blood MDC have been observed in HCV-infected individuals and may play a role in the defective response to vaccine. Canaday et al. 82 specifically focused on analysis of peptide-MHC complex formation and presentation, the culmination of uptake, degradation and trafficking of antigen. They found that this specific antigen-presenting cell function is preserved in the setting of chronic HCV infection.

DC in liver microenvironment
As the liver is the primary site for HCV replication, DC changes in peripheral blood may or may not reflect what is happening locally at the site of infection. Several studies demonstrated enrichment of DC in the liver compartment compared with peripheral blood. 80 Galle et al. 83 employed electron microscopy, immunohistochemistry and immunofluorescence to show that DC are indeed enriched in the livers of HCV-infected individuals. Wertheimer et al. 84 also showed a clear enrichment of DC in the intrahepatic compartment compared with the peripheral circulation. To investigate the contribution of intrahepatic PDC and MDC to local immune responses during HCV infection, Lai et al. 85,86 developed methods to isolate and characterize MDC and PDC from human liver. The MDC from HCV-infected liver demonstrated greater expression of MHC class II, CD86 and CD123, that were more efficient stimulators of allogeneic T cells and secreted less IL-10. In contrast, PDC were present at lower frequencies in HCV-infected liver and expressed higher levels of the regulatory receptor BDCA-2. In HCV-infected liver, the combination of enhanced MDC function and a reduced number of PDC might contribute to viral persistence in the face of persistent inflammation. Nattermann et al. 87 demonstrated that chronic HCV infection, associated with intrahepatic DC enrichment, migration of DC is markedly affected by interaction of HCV E2 with CD81. A twophoton confocal microscopic analysis revealed that DC and T lymphocytes were rapidly recruited within human liver slices undergoing an ex vivo HCV infection. 88 However, liver MDC enrichment is not unique to HCV infection, as similar trends were seen in non-HCV-infected liver disease, such as hepatitis B virus infection. 89

HCV on DC development and function
Several studies have suggested that DC can be infected with HCV, but the role of HCV in DC development and function is still elusive. 59,90,91 Virologically, HCV first attaches itself to the host cell surface by means of weak interactions with glycosylaminoglycans or the low-density lipoprotein receptor. Once bound and concentrated on the cell surface, virions are able to interact with entry receptors such as CD81 and SR-BI with high affinity. The virus-receptor complex then translocates to the tight junctions where claudin and occludin act as cofactors and induce receptor-mediated endocytosis. 92 Barth et al. 35 used HCV-like particles (HCV-LPs) to study the interaction of HCV with human DC. The iDC exhibited an envelope-specific and saturable binding of HCV-LPs, indicating receptor-mediated DC-HCV-LP interaction. They revealed that HCV-LPs were rapidly taken up by DC in a temperature-dependent manner, and C-type lectins such as mannose receptor or DC-SIGN (DC-specific intercellular adhesion molecule 3-grabbing non-integrin) were not sufficient for mediating HCV-LP binding. Lambotin et al. 93 suggested that HCV cell entry factors, which are crucial for viral uptake in hepatocytes, do not support the cell culture-produced HCV (HCVcc) uptake in DC subsets. HCVcc acquisition by DC subsets does not depend on the C-type lectin DC-SIGN, but is partially mediated by HCVcc E2 protein interaction at the cell surface.
To date, the mechanisms whereby HCV affects DC function remain largely elusive. 55 It is possible that HCV proteins play a role in suppressing protective immunity through interactions with host immune cells, such as DC. Indeed, the HCV core protein has been reported to impair the function of DC. The HCV core protein was able to selectively inhibit TLR4-induced IL-12 production after interacting with the gC1q receptor on the surface of MDDC by activating the phosphatidyl inositol 3-kinase pathway, leading to reduced T helper type 1 (Th1) cell development. 94,95 Dolganiuc et al. 96 demonstrate that HCV core and NS3 proteins, but not envelope 2 proteins (E2), activate monocytes and inhibit DC differentiation in the absence of the intact virus, and induced production of the anti-inflammatory cytokine IL-10 associated with elevated IL-10 and decreased IL-2 levels during T-cell proliferation. They also found that treatment-naive patients with chronic HCV infection had a reduced frequency of circulating PDC as the result of increased apoptosis and showed diminished IFN-a production after stimulation with TLR9 ligands. 97 The HCV core protein reduced TLR9-triggered IFN-a and increased TNF-a and IL-10 production in peripheral blood mononuclear cells (PBMCs) but not in isolated PDC, suggesting that HCV core induces PDC defects. The addition of rTNF-a and IL-10 induced apoptosis and inhibited IFN-a production in PDC. Neutralization of TNF-a or IL-10 prevented HCV core-induced inhibition of IFN-a production. Anti-TLR2-blocking antibody, but not anti-TLR4-blocking antibody, prevented the HCV core-induced inhibition of IFN-a production. These results suggest that HCV interferes with antiviral immunity through TLR2-mediated monocyte activation triggered by the HCV core protein to induce cytokines, which in turn lead to PDC apoptosis and inhibit IFN-a production. These mechanisms may contribute to viral escape by HCV from immune responses. Consistent with these studies, Liang et al. 98 treated freshly purified human MDC and PDC with HCV JFH1 strain (HCV genotype 2a). They found that HCV up-regulated MDC maturation marker (CD83, CD86 and CD40) expression and did not inhibit TLR3 ligand [poly(I:C)]-induced MDC maturation whereas HCV JFH1 inhibited the ability of poly(I:C)-treated MDC to activate naive CD4 + T cells. The HCV JFH1 also inhibited TLR7 ligand (R848) -induced PDC CD40 expression, and this was associated with an impaired ability to activate naive CD4 + T cells. Parallel experiments with recombinant HCV proteins indicated that HCV core protein may be responsible for a portion of the activity.
It has recently been shown that TLR7 may be implicated in anti-HCV immunity, HCV encodes G/U-rich ssRNA TLR7 ligands that induce immune activation of PBMCs and PDC. 99 Studies suggested that a TLR7-dependent impairment of co-stimulatory molecule expression caused by HCV persistence may affect DC activity in non-responder patients. 100 Exploitation of the MHC class I antigenprocessing pathway by HCV core 191 impairs the ability of DC to stimulate CD8 + T cells and may contribute to the persistence of HCV infection. 101 However, Landi et al.'s results 102 show that HCV core does not have an inhibitory effect on human DC maturation, and could be a target for the immune system. To evaluate the effects of core and NS3 proteins on DC, they transfected monocyte-derived iDC with in vitro transcribed HCV core or NS3 RNA and treated with maturation factors. Neither core nor NS3 had an inhibitory effect on DC maturation; however, transfection of iDC with in vitro transcribed core RNA appeared to result in changes compatible with maturation confirmed by a DC-specific membrane array. The effects of core on maturation of iDC were confirmed with a significant increase in surface expression of CD83 and HLA-DR, a reduction of phagocytosis, as well as an increase in proliferation and IFN-c secretion by T cells in a mixed lymphocyte reaction assay. 102 Similarly, in Li et al.'s studies, 103 the phenotype and function (determined by expression of various DC surface markers and co-stimulatory molecules, allo-T-cell stimulation and processing and presentation of a foreign antigen) of DC expressing HCV NS3 or core were similar to those of the uninfected or control vectorinfected DC, suggesting that the HCV NS3 or core proteinexpressing DC are phenotypically and functionally normal and stimulate T cells efficiently. Others reported that NS3dependent suppression of Toll/IL-1 domain-containing adapter-inducing IFN-b-coupled and IFN-b promoter stimulator-1-coupled pathogen recognition receptorinduced synthesis of pro-inflammatory cytokines (IL-12 and TNF-a) from DC by HCV is a distinctive feature of a subgroup of chronically infected patients. 104 Beyond HCV core protein and NS3, NS4 also suppressed T-cell responses as a result of the effect on monocytes or DC. The DCs produce high levels of type I IFN in response to double-stranded RNA generated upon viral replication. 105 However, HCV suppresses this response via the NS3-NS4A viral protein, which blocks IFN regulatory factor 3-mediated induction of type I IFN. 106 In Brady et al.'s study, 107 supernatants from NS4-stimulated monocytes inhibited LPS-induced maturation of DC and sup-pressed their capacity to stimulate proliferation and IFN-c production by allospecific T cells. Their data suggested that HCV subverts cellular immunity by inducing IL-10 and inhibiting IL-12 production by monocytes, which in turn inhibits the activation of DC that drive the differentiation of Th1 cells. Takaki et al. 108 also found that HCV non-structural proteins, particularly NS4, change the iDC phenotype and reduce antigen-specific T-cell stimulatory function with Th1 cytokine reductions. HCV NS5 was also shown to impair PDC function with several other in vivo studies indicating decreased numbers and impaired function of PDC in chronically HCV-infected patients. 109 Over-expression of HCV core, NS3, NS5A or NS5B proteins induced apoptosis in mature DC. 110 Likewise, individual HCV proteins, Core, NS3, NS4, NS5 as well as fused polyprotein (Core-NS3-NS4) were found to impair functions of both iDC and mDC by regulating the expression of co-stimulatory and antigen presentation molecules, strikingly reducing IL-12 secretion, inducing the expression of FasL to mediate apoptosis, interfering with allo-stimulatory capacity, inhibiting TLR signalling and inhibiting nuclear translocation of nuclear factor-jB in DC. 111 It is reported that increased PD-L1 expression and PD-L1/CD86 ratio on DC was associated with impaired DC function in HCV infection. 112 Further indications that HCV affects DC function came directly from studies using the cell culture-produced HCV (HCVcc). Culture with HCVcc demonstrated inhibition of maturation of MDDC induced by a cocktail of cytokines (IL-1b, TNF, IL-6, prostaglandin E 2 ) while enhancing the production of IL-10. In addition, DC exposed to HCVcc were impaired in their ability to stimulate antigen-specific T-cell responses. 71 Similar experiments performed by Shiina and Rehermann 113 proved that HCVcc inhibited TLR-9 mediated IFN-a production by PBMC and PDC. In contrast to its effect on PDC, HCVcc did not inhibit TLR3-mediated and TLR4-mediated maturation and IL-12, IL-6, IL-10, IFN-c and TNF-a production by MDCs and MDDCs. Likewise, HCVcc altered the capacity of neither MDCs nor MDDCs to induce CD4 T-cell proliferation. Gondois-Rey et al. 114 also proved that HCVcc and HCV-LPs but not HCV core or envelope glycoprotein E2 inhibit PDC-associated production of IFN-a stimulated via TLR9.

DC-based vaccine against HCV infection
Several approaches involving DC-based vaccines were developed as early-stage attempts to manage/cure HCV infection, some of them being developed at the experimental level while some advanced towards the translational level. 37 The DC-based HCV vaccine development is summarized in Table 2.
Moriya et al. 115 employed the anthrax toxin fusion protein containing the HCV-core epitope as a vehicle for antigen loading on DC, and reported that immunization with the fusion protein-treated DC induced HCV-corespecific cytotoxic lymphocytes (CTL) in mice. Later, they immunized mice with DC transduced with recombinant adenovirus expressing HCV-core protein effectively induced HCV-core-specific CTL. Hence, adenovirustransduced DC may be a promising candidate for a CTLbased vaccine against HCV infection. 116 Racanelli et al. 36 present a system to induce cellular immunity and to study the immunological implications of time-delayed DC apoptosis and antigen reprocessing in vivo. They generated a self-replicating cytopathic pestivirus RNA to enhance production and presentation of HCV antigens and to induce apoptosis in DC 24-48 hr after transfection. Replicon-transfected H-2b DC used to immunize HLA-A2 transgenic mice induced protection upon challenge with a vaccinia virus expressing HCV antigens. Induction of cell death enhanced the immunogenicity of DC-associated antigen. Transfer of cellular material from vaccine DC to endogenous antigen-presenting cells was visualized in lymph nodes and spleen, and cross-primed CD8 + T cells were characterized.
Dendritic cells pulsed with HCV-LPs stimulated HCV core-specific CD4 + T cells, indicating that uptake of HCV-LPs by DC leads to antigen processing and presentation on MHC class II molecules. The HCV-LP-derived antigens were efficiently cross-presented to HCV core-specific CD8 + T cells. These findings demonstrate that HCV-LPs represent a novel model system to study HCV-DC interaction allowing definition of the molecular mechanisms of HCV uptake, DC activation, and antigen presentation to T cells. Furthermore, HCV-LP may be a potent vaccine candidate for the induction of antiviral cellular immune responses in humans. 35 By using recombinant adenoviral vectors, 103 DC expressing HCV NS3 or core proteins expressed several inflammatory cytokine mRNAs, had a normal phenotype, and effectively stimulated allogeneic T cells, as well as T cells specific for another foreign antigen (tetanus toxoid). These findings are important for the rational design of cellular-vaccine approaches for the immunotherapy of chronic HCV. Zabaleta et al. 117 proved that immunization with DC transfected with an adenovirus encoding NS3 protein, from HCV (AdNS3), induced multi-epitopic CD4 Th1 and CD8 + T-cell responses in different mouse strains. Moreover, immunization with AdNS3-transfected DC did not induce anti-adenoviral antibodies, as compared with direct immunization with AdNS3, but elicited T-cell responses even in the presence of pre-existing antiadenoviral antibodies. Finally, responses induced by this protocol down-regulated the expression of HCV RNA in the liver. By using recombinant adeno-associated virus (rAAV) vectors, DC expressing core (49-180) can generate significant antigen-specific CTL. 118 The researchers believe that direct manipulation of professional antigen-presenting DC may provide new clinical treatments through the forced feeding of antigens into DC coupled with their stimulation and manipulation towards an effective Th1 response, and AAV-loading appears to naturally stimulate a Th1 response in vitro. By using lentiviral vectors, Jirmo et al. 32 demonstrated the high capability of lentiviral vectors to transfer whole sets of HCV structural or non-structural gene clusters in vitro into monocytes before their differentiation into DC. Notably, gene delivery of the HCV-NS cluster into monocytes resulted in its persistent expression in differentiated DC leading to potent stimulation of CD4 + and CD8 + allogeneic and autologous responses. Hence, lentiviral-mediated expression of the multi-antigenic HCV-NS cluster in monocytes subsequently differentiated into DC is a novel potential anti-HCV vaccine modality. Gehring et al. 119 generated immune responses against HCV by DC containing NS5 protein-coated microparticles. They revealed that it was essential to use microbeads as carriers to achieve efficient uptake of the immunogen by DCs because intravenous injection of soluble NS5 protein did not induce detectable T-cell responses as demonstrated in the tumour challenge experiments and Th1-type cytokine secretion. 120 Because DC are essential for T-cell activation and viral clearance in HCV-infected patients is associated with a vigorous T-cell response, vaccination with HCV antigenloaded DC may constitute an efficient and important antiviral therapy for HCV. Encke et al. 121 proposed a new type of HCV vaccine based on ex vivo stimulated and matured DC loaded with HCV-specific antigens. This vaccine circumvents the impaired DC maturation and the down-regulated DC function of HCV-infected patients in vivo by giving the necessary maturation stimuli and the HCV antigens in a different setting and location ex vivo. Strong humoral and cellular immune responses were detected after HCV core DC vaccination. Furthermore, DC vaccination shows partial protection in a therapeutic and prophylactic model of HCV infection. In conclusion, mice immunized with HCV core-pulsed DC generated a specific antiviral response in a mouse HCV challenge model.
The use of HCV-primed DC for vaccination in chronically infected patients as a prophylactic vaccine seems to Specific CD8+ T cell responses in HLA-A2 transgenic mice and six patients 125,126 CTL, cytotoxic T lymphocyte; DC, dendritic cell; HCV, hepatitis C virus; HCV-LP, HCV-like particle; MDC, myeloid DC; be a new promising modality for immunotherapy of HCV. Ito et al. 122 started with the premise that self-DC could be used to deliver HCV antigens in vivo; hence, they pulsed MDC with peptides from structural (core) and non-structural (NS3 and NS4) HCV proteins to successfully trigger the generation of CTLs. Explored by Kuzushita et al., 34 DC were substantially transduced with recombinant HCV core or NS5 protein by using a protein delivery based on a short amphipathic peptide carrier, Pep1. This DC vaccine induced HCV-specific T-cell priming (Th1 type) with high efficacy and duration and protection against tumour challenge. All evidence suggesting that a vaccine consisting of HCV protein transfected DCs should be useful as both prophylactic and therapeutic vaccination against HCV. Lasarte and colleagues reported that fusion of an antigen with the extra domain A from fibronectin (EDA) leads to antigen targeting TLR4-expressing DC, enhancing cross-presentation and immunogenicity. 123 To test if EDA-NS3 might behave as an immunogen capable of eliciting robust anti-HCV responses, they prepared a fusion protein and tested its capacity to activate DC maturation in vitro and its immunogenicity in vivo. Their results suggested that EDA-NS3 combined with these adjuvants may be considered for the development of a vaccine against HCV infection. 124 Gowans et al. took the DC-based approach one step forward and performed a phase I clinical trial of selfderived DC immunotherapy in HCV-infected individuals who had failed conventional therapy. The lipopeptides they employed contained a single CD4 + Th-cell epitope, an HLA-A2-restricted cytotoxic T-cell epitope and the lipid Pam2Cys. 125 Lipopeptides were able to induce specific CD8 + T-cell responses in HLA-A2 transgenic mice and consistently activated human MDDC from both healthy individuals and HCV-infected patients. Lipopeptide-pulsed human DC were also found to secrete the pro-inflammatory cytokine IL-12p70 and were able to activate antigen-specific IFN-c production by autologous CD8 + T cells obtained from a patient with hepatitis C. These results show that DC from HCV-infected patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8 + T-cell epitopes; the use of autologous lipopeptide-pulsed DC or direct lipopeptide vaccination may be successful approaches for the priming or boosting of anti-HCV CD8 + T-cell responses to aid in the clearance of the virus in chronically infected individuals. 126 They examined the potential of autologous MDDC, presenting HCV-specific HLA A2.1-restricted cytotoxic T-cell epitopes, to influence the course of infection in six patients who failed conventional therapy. In this phase 1 dose escalation study, no patient showed a severe adverse reaction although all experienced transient minor adverse effects. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines. They suggested that immunotherapy using autologous MDDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit. 33 Strategies to enhance the efforts of DC-based vaccine During the last decade, DC have been regarded as promising tools for the development of more effective therapeutic vaccines in cancer patients. For patients with late-stage disease, strategies that combine novel highly immunogenic DC-based vaccines and immunomodulatory antibodies may have a significant effect on enhancing therapeutic immunity by simultaneously enhancing the potency of beneficial immune arms and offsetting immunoregulatory pathways. These optimized therapeutic modalities include the following.

Adjuvant
Glucopyranosyl lipid A (GLA) is a new synthetic nontoxic analogue of lipopolysaccharide. Pantel et al. 127 studied DC directly from vaccinated mice. Within 4 hr, GLA caused DC to up-regulate CD86 and CD40 and produce cytokines including IL-12p70 in vivo. Importantly, DC removed from mice 4 hr after vaccination became immunogenic, capable of inducing T-cell immunity upon injection into naive mice. These data indicate that a synthetic and clinically feasible TLR4 agonist rapidly stimulates full maturation of DCs in vivo, allowing for adaptive immunity to develop many weeks to months later. Relative to several other TLR agonists, Longhi et al. 128 found polyinosinic : polycytidylic acid (poly I:C) to be the most effective adjuvant for Th1 CD4 + T-cell responses to a DC-targeted HIV gag protein vaccine in mice. Spranger et al. 129 described a new method for preparation of human DCs that secrete bioactive IL-12p70 using synthetic immunostimulatory compounds as TLR7/8 agonists R848 or CL075. Maturation mixtures included the TLR7/ 8 agonists, combined with the TLR3 agonist poly I:C, yielded 3 days mature DC that secreted high levels of IL-12p70, showed strong chemotaxis to CCR7 ligands, and had a positive co-stimulatory potential. They also had excellent capacity to activate natural killer cells, effectively polarized CD4 + and CD8 + T cells to secrete IFN-c and to induce T-cell-mediated cytotoxic function. Thereby, mature DCs prepared within 3 days using such maturation mixtures displayed optimal functions required for vaccine development.
Synthetic oligodeoxynucleotides (ODNs) containing unmethylated CpG motifs trigger cells that express TLR9 (including human PDCs and B cells) to mount an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. When used as vaccine adjuvants, CpG ODNs improve the function of professional antigen-presenting cells and boost the generation of humoral and cellular vaccine-specific immune responses. Preclinical studies indicate that CpG ODNs improve the activity of vaccines targeting infectious diseases and cancer. Clinical trials demonstrate that CpG ODNs have a good safety profile and increase the immunogenicity of co-administered vaccines. 130 Rizza et al. 131 predicted that IFN-a itself, as well as IFN-a-conditioned DC, can represent valuable components in the coming years of new and clinically effective protocols of therapeutic vaccination in patients with cancer and some chronic infectious diseases, whose immune suppression status can be restored by a selective use of these cytokines targeted to DCs and specific T-cell subsets under different experimental conditions.

Blockade of regulatory/suppressive pathways
In chronic HCV infection, virus-specific dysfunctional CD8 T cells often over-express various inhibitory receptors. Programmed cell death 1 (PD-1) was the first among these inhibitory receptors that were identified to be over-expressed in functionally impaired T cells. The roles of other inhibitory receptors such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) and T-cell immunoglobulin and mucin domain-containing molecule 3 (Tim-3) have also been demonstrated in T-cell dysfunctions that occur in patients with chronic HCV infection. Blocking these inhibitory receptors in vitro restores the functions of HCV-specific CD8 T cells and allows enhanced proliferation, cytolytic activity and cytokine production. Therefore, the blockade of the inhibitory receptors is considered as a novel strategy for the treatment of chronic HCV infection. 132 Recently, Zhang et al. 133 demonstrated that up-regulation of PD-1 and suppressor of cytokine signalling-1 (SOCS-1) correlates with IL-12 inhibition by HCV core protein and that blockade of PD-1 or SOCS-1 signalling may improve TLR-mediated signal transducer and activator of transcription 1 (STAT-1) activation and IL-12 production in monocytes/macrophages. Blocking PD-1 or silencing SOCS-1 gene expression also decreases Tim-3 expression and enhances IL-12 secretion and STAT-1 phosphorylation. 134 These findings suggest that Tim-3 plays a crucial role in negative regulation of innate immune responses, through cross-talk with PD-1 and SOCS-1 and limiting STAT-1 phosphorylation, and may be a novel target for immunotherapy to HCV infection.
The high levels of IL-10 present in chronic HCV infection have been suggested as responsible for the poor antiviral cellular immune responses found in these patients. To overcome the immunosuppressive effect of IL-10 on antigen-presenting cells such as DC, Diaz-Valdes et al. 135 developed peptide inhibitors of IL-10 to restore DC functions and concomitantly induce efficient antiviral immune responses. The results suggest that IL-10-inhibiting peptides may have important applications to enhance anti-HCV immune responses by restoring the immunostimulatory capabilities of DC.
Regulatory T cells (Treg cells) suppress autoreactive immune responses and limit the efficacy of vaccines, however, it remains a challenge to selectively eliminate or inhibit Treg cells. The zinc-finger A20, a negative regulator of the TLR and TNF receptor signalling pathways, was found to play a crucial part in controlling the maturation, cytokine production and immunostimulatory potency of DC. 136 A20-silenced DC showed spontaneous and enhanced expression of co-stimulatory molecules and pro-inflammatory cytokines and had different effects on T-cell subsets: they inhibited Treg cells and hyperactivated tumour-infiltrating cytotoxic T lymphocytes and T helper cells that produced IL-6 and TNF-a and were refractory to Treg-cell-mediated suppression. Mechanistic studies revealed that A20 regulated DC production of retinoic acid and pro-inflammatory cytokines, inhibiting the expression of gut-homing receptors on T and B cells. Their work provided a strategy for the development of an efficient vaccination. 137

Optimal of antigen loading
When compared with other cell types, DC are not easily transduced by adenoviruses, requiring high multiplicities of infection to obtain expression of antigen in most cells. Pereboev et al. 138 have reported that CFm40L, an adapter molecule combining the coxsackie-adenovirus receptor fused to the ecto-domain of CD40L by way of a trimerization motif, was able to efficiently target adenoviruses to DC. Moreover, direct immunization with adenoviral particles coated with this adapter molecule was able to induce stronger immune responses than uncoated adenoviral particles. In their studies, targeting of an adenovirus encoding HCV NS3 protein (AdNS3) to DC with CFm40L strongly enhanced NS3 presentation in vitro, activating IFN-c-producing T cells. Immunization of mice with these DC promoted strong CD4 and CD8 T-cell responses against HCV NS3. CFh40L, a similar adapter molecule containing human CD40L, enhanced transduction and maturation of human MDDC from patients with chronic HCV infection and healthy donors revealed similar maturation levels. DC transduced with AdNS3 and the adapter molecule CFm/h40L exhibit enhanced immunostimulatory functions, induced robust anti-HCV NS3 immunity in animals, and can induce antiviral immune responses in subjects with chronic HCV infection. This strategy may serve as therapeutic vaccination for patients with chronic hepatitis C. 31 To determine whether T-cell responses induced by the protein vaccines could be enhanced after boosting with a viral vector, non-human primates were boosted with a replication defective, recombinant New York vaccinia virus (NYVAC)-HIV Gag/Pol/Nef vector. Boosting with recombinant NYVAC strongly enhances IFN-c-producing T cells following priming with DEC-HIV Gag p24 or HIV Gag p24 plus Poly ICLC. The NYVAC boosting generates multifunctional CD4 + and CD8 + cytokine-producing T cells with a similar breadth to those elicited by protein priming. Hence, a robust, broad, durable and polyfunctional CD4 + and CD8 + T-cell response is generated by boosting a relatively low frequency of cross-primed CD8 + T cells induced by a protein vaccine with a single immunization with NYVAC-HIV Gag/Pol/Nef. 139 These studies can be extended to other diseases to verify whether heterologous prime-boost immunization with vectors and protein vaccines is a logical vaccine approach to optimize both humoral and cellular immunity.
Despite initially encouraging data from preclinical and clinical studies, the efficacy of human adenoviral vector serotype 5 (AdHu5) was hampered by a strong pre-existing anti-vector immunity among vaccinated macaques, in which transgene-specific T cells homed to different organs in the presence of anti-vector immunity. 140 Listeria monocytogenes is known to induce strong cellular immune responses. Listeria monocytogenes induces multiple effector mechanisms, including antigen presentation via MHC class I and II pathways as well as induction of innate immune responses. 141 As L. monocytogenes is a ubiquitous bacterium, anti-L. monocytogenes immune responses are likely to be present among the majority of individuals. Sciaranghella et al. 142 constructed a live-attenuated L. monocytogenes vector, which encodes SIVmac239 gag. The novel, live-attenuated L. monocytogenes vector may be an attractive platform for oral vaccine delivery.

Conclusions
Although HCV leads to impairment of both MDC and PDC according to many researchers, the mechanisms how HCV affects DC function remains elusive. 55 Further research is needed in regard to the mechanisms of HCVinduced DC impairment and the correlation between DC function and HCV persistence. Dendritic cell-based vaccination/therapeutic approaches are safe and promising in terms of their propensity to establish anti-HCV adaptive immune responses. However, possible side-effects of DCbased therapeutic vaccine should be carefully evaluated, especially those possibly inducing a strong T-cell-mediated immunity, because of the dual role of virus-specific cytotoxic T cells mediating both viral clearance and tissue damage. Nevertheless, the achievements in this field of studies brought us the hope of opening new routes to the prevention and treatment of HCV infection. Prospects of a DC-based vaccine against HCV infection include employment of adjuvants, the blockage of negative regulatory signal and enhancement of positive regulatory signals, so as to improve the vaccine immune response against HCV infection, reduce HCV viral load, and hinder progression of chronic liver disease.