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Immunology. Sep 2007; 122(1): 15–27.
PMCID: PMC2265985

Induction of potent cellular immune response in mice by hepatitis C virus NS3 protein with double-stranded RNA


Double-stranded RNA is produced during virus replication and, together with the viral antigen, is responsible for inducing host antivirus immunity. The hepatitis C virus (HCV) non-structural protein-3 (NS3) has been implicated in the immune evasion of HCV, and is one of the prime targets for inducing immunity against HCV infection. Mice were immunized with recombinant NS3 protein (rNS3) and poly (I:C) emulsified in Montanide ISA 720 (M720). Cytokine production was assayed by enzyme-linked immunospot assay, and CD4+ IFN-γ+ T helper (Th) cells or CD8+ IFN-γ+ cytotoxic T lymphocytes were detected by flow cytometry. Anti-NS3 titre and immunoglobulin G2a (IgG2a) and IgG1 levels were monitored by enzyme-linked immunosorbent assay. Administration of rNS3 formulated in poly (I:C) and M720 induced anti-NS3 titres with a predominantly IgG2a isotype comparable to those induced by rNS3 in CpG-ODN and M720. The cytokine profiles showed that this formulation induced a Th1-biased immune response with several-fold more interferon-γ (IFN-γ)-producing cells than interleukin-4-producing cells. In contrast, rNS3 in M720 induced a Th2-biased immune response. The frequency of IFN-γ-producing CD4+ and CD8+ cells induced by rNS3 in poly (I:C) and M720 was significantly higher than that induced by rNS3, rNS3 in M720, or rNS3 in poly (I:C), and was comparable to that induced by rNS3 in CpG-ODN with M720. The antigen-specific CD8+ T-cell immune response persisted for up to 7 months after immunization. In conclusion, poly (I:C) with rNS3 in M720 can elicit a strong and persistent Th1-biased immune response and a cytotoxic T-lymphocyte response through cross-priming in mice. This study highlighted a promising formulation for inducing an efficient cellular immune response against HCV that has potential for HCV vaccine development.

Keywords: ELISA/ELSIPOT, hepatitis C virus, intracellular cytokine staining, vaccine


Hepatitis C virus (HCV) infection is a serious health problem worldwide. It is estimated that as many as 170 million people, approximately 3% of the world's population, may be infected with HCV, and among those infected, 3·9 million reside in the USA.13 Up to 75–85% of acutely infected persons do not resolve their HCV infection and become persistent carriers despite a vigorous humoral immune response by the host.3

Neutralizing antibody directed to the E2 hypervariable region may be effective in eliminating HCV viraemia in some circumstances.47 However, HCV displays a continued evolution of neutralizing antibody-resistant mutants. The cellular immune response does seem to play a role in the virological outcome during acute infection based on the strong association of a sustained vigorous and multispecific antiviral CD4 and CD8 T-cell response with HCV clearance during acute infection. Thus, it is essential for HCV vaccine development to elicit cellular immunity specific for HCV.

Double-stranded RNA (dsRNA) is produced in virus-infected cells during viral replication8 and can activate Toll/interleukin-1 receptor domain-containing adapter inducing interferon-β (TRIF or TICAM-1)9,10 through its receptor, toll-like receptor 3 (TLR3). These interactions link to the nuclear factor-κB pathway via MyD88, interleukin-1 RI-associated protein kinases (IRAKs), and the tumour necrosis factor receptor-associated factor 6 (TRAF6), leading to the production of cytokines including tumour necrosis factor-α, interleukin-6 (IL-6) and IL-12p40.1113 In addition, dsRNA acting through the TLR pathway activates interferon regulatory factor 3 (IRF-3) leading to the production of type I interferons (IFN-α/IFN-β) that have potent antiviral effects. Recent research suggested that virus-infected cells bearing dsRNA or cells containing synthetic dsRNA can activate dendritic cells and elicit cytotoxic T lymphocyte (CTL) responses through cross-priming.14 Both natural and synthetic dsRNA can also induce type I interferons that play a critical role in antiviral host defence.15 Polyriboinosinic : polyribocytidylic acid [poly (I:C)] is the most potent known IFN inducer among synthetic dsRNA analogues and has been administered safely in previous clinical trials.16

HCV non-structural protein 3 (NS3), acting non-covalently with its cofactor, HCV NS4A, possesses a serine protease activity. Recent studies suggested that this serine protease can recognize TRIF as its substrate for hydrolysis. The proteolysis of TRIF by NS3/NS4A protease can disrupt the dsRNA-TLR3 pathway and facilitate the immune evasion of HCV.17,18 Therefore, immunity against NS3 will eliminate the cells that present NS3-specific epitopes in the context of major histocompatibility complex (MHC) class I and potentially diminish the ability of HCV to evade the immune system, which is helpful for viral clearance by the host. These mechanisms support a strategy that incorporates dsRNA into an NS3 vaccine to promote pathways through TLR3 to elicit HCV-specific cellular and humoral immunity.

In vitro studies have demonstrated that poly (I:C) can inhibit HCV replicons in human embryonic kidney 293 cells.19 Because dsRNA is readily degraded by ribonuclease in vivo, we incorporated poly (I:C) and HCV NS3 protein into Montanide ISA 720 (M720), an adjuvant composed of a non-mineral oil of vegetable origin with a surfactant from the mannide mono-oleate family,20 creating a water-in-oil emulsion to protect poly (I:C) from ribonuclease degradation. With this formulation, we immunized mice and showed a strong and persistent T helper type 1 (Th1)-biased immune response and CTL response, hence demonstrating the much more potent action as adjuvant of this protected form of poly (I:C).

Materials and methods

Expression and purification of recombinant HCV NS3 (rNS3) and NS5b (rNS5b) proteins

The construction of an HCV NS3 expression vector in an Escherichia coli system, and the expression and purification of recombinant HCV NS3 protein were performed as previously described.2123 A full-length HCV NS5b gene encoding amino acids 2421–3011 (nucleotides 7602–9374 plus the TGA stop codon, HCV strain H of genotype 1a) was inserted into the BamHI restriction enzyme site of plasmid pQE-11 (Qiagen Inc., Valencia, CA) and expressed in E. coli DH5αF'IQ cells. The expression and purification procedures were essentially the same as those described for HCV NS3 protein.23 The endotoxin levels in both purified proteins were <0·02 EU per μg protein.

Preparation of rNS3 with poly (I:C) or CpG-ODN emulsified in M720

M720 was obtained from Seppic, Paris, France. The poly (I:C) was purchased from Sigma (St. Louis, MO), and dissolved in diethylpyrocarbonate-treated water. Oligodeoxynucleotide (ODN) containing unmethylated cytosine preceding guanosine (CpG) motifs sequenced as: 5′-GACGTTGACGTTAGCGT-3′ was synthesized with full phosphorothioate backbone by Keystone Lab (Camarillo, CA).24 The rNS3 antigen diluted in diethylpyrocarbonate-treated 1 × phosphate-buffered saline and mixed with or without poly (I:C) or CpG-ODN was used in its aqueous phase and mixed with M720 in accordance with the manufacturer's instructions.20

Animals and immunization

Female BALB/c mice from The Jackson Laboratory (Bar Harbor, ME) were housed in approved facilities and handled following the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH publication 86-23). All animal study protocols were approved by the NIH Clinical Center Animal Care and Use Committee. Eight- to 10-week-old mice were immunized with formulations using an intramuscular injection to the quadriceps muscle of the two posterior legs under 2% isoflurane inhalation anaesthesia.

To determine a suitable dosage of poly (I:C) for immunization, groups of five mice each were immunized with 100 μg, 50 μg, 25 μg, or 10 μg poly (I:C) formulated with M720 and 10 μg rNS3. The blood samples were collected preimmunization and 5 weeks postimmunization from the orbital venous sinus. To make a rough assessment of the poly (I:C) stability in this formulation, the mixture of 10 μg rNS3 + 50 μg poly (I:C) + M720 was kept at 4° for 24 or 48 hr before injection. The anti-HCV NS3 immunoglobulin G (IgG) and its isotypes induced by 10 μg rNS3 + 50 μg poly (I:C) + M720 with different oil to water ratios (70 : 30 versus 50 : 50) were also tested.

For evaluation of early immune response elicitation, six groups of 10 mice each were immunized with 10 μg rNS3, 10 μg rNS3 + M720, 10 μg rNS3 + 50 μg poly (I:C) + M720, 10 μg rNS3 + 10 μg CpG-ODN + M720, 50 μg poly (I:C) + M720, and 10 μg rNS3 + 50 μg poly (I:C), respectively. Splenocytes for CTL and enzyme-linked immunospot (ELISPOT) assays were recovered from mice at 2 and 3 weeks after immunization. Another six groups were immunized with 10 μg rNS3 + M720, 10 μg rNS3 + 50 μg poly (I:C) + M720, 10 μg rNS3 + 10 μg CpG-ODN + M720, 10 μg rNS3 + 50 μg poly (I:C) (n = 10, respectively), 50 μg poly (I:C) + M720, and 10 μg CpG-ODN + M720 (n = 5 each), respectively. Splenocytes were obtained at 4 weeks after immunization.

To evaluate T-cell memory, three groups of 10 mice each were immunized once with 10 μg rNS3 + M720, 10 μg rNS3 + 50 μg poly (I:C) + M720, 10 μg rNS3 + 10 μg CpG-ODN + M720, respectively. CTL and ELISPOT assays were conducted at 4 and 7 months after immunization.

Assay of anti-HCV NS3 antibodies by ELISA

Anti-HCV NS3 IgG was assayed by enzyme-linked immunosorbent assay (ELISA) as previously described.23 The cut-off value was defined as the mean value of the optical density (OD) from preimmune serum samples plus two standard deviations. Anti-HCV NS3 IgG titre was determined by the highest dilution at which the OD value was greater than the cut-off value. The anti-HCV NS3 IgG isotype was determined by ELISA as previously described.23 IgG1 and IgG2a concentrations (ng/ml) were calibrated against a standard curve using purified mouse IgG1k or IgG2ak as standards, respectively.

Assay for cells secreting IL-4, IL-2, or IFN-γ by ELISPOT

The splenocytes were expanded for 40 hr by coculture with rNS3 or antigen-dissolving buffer as background in a 24-well culture plate (Corning Incorporated, Corning, NY) at 37°. In some experiments, rNS5b was used as an irrelevant antigen in the splenocyte expansion for ELISPOT assay to detect non-specific cytokine production. A sterile 96-well filtration plate (Millipore, Beverly, MA) was coated with rat anti-mouse IL-4, IL-2, or IFN-γ antibodies (BD Pharmingen, San Diego, CA). The expanded cell suspension containing 4 × 105 splenocytes was transferred to the antibody-coated filtration plate, and incubated at 37° overnight. After the cell suspension had been removed, biotin-labelled rat anti-mouse IL-4, IL-2 or IFN-γ antibodies (BD Pharmingen) were added for the detection of captured cytokines. Diluted streptavidin–horseradish peroxidase complex (SA-HRP) and peroxidase substrate Opti-4 CN (Bio-Rad Laboratories, Hercules, CA) were used for colour development. All the detections were performed in quadruplicate cultures. The developed plate was read on an AID EliSpot Reader System (Autoimmun Diagnostika, Strassberg, Germany). The spot-forming unit (SFU) value was expressed as mean of the quadruplicated cultures minus the mean value of its individual background.

Synthetic peptides pool selection for T-cell analysis by flow cytometry

A set of 441 overlapping peptides spanning the entire amino acid sequences of HCV strain H77 of genotype 1a was obtained from the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, NIH. All of these peptides were 18-mers except for four of them, which were 14- or 15-mers, and were overlapped by 11 amino acids each. Ninety-three overlapping peptides that cover the whole sequences of HCV NS3 were used in the current experiment (Table 1). The peptides were pooled into 20 individual pools containing 1 mg/ml per peptide (Table 2).

Table 1
Synthetic HCV NS3 overlapping peptide sequence
Table 2
HCV-NS3 peptide pool matrix

Ten mice were immunized once with 10 μg rNS3 + 50 μg poly (I:C) + M720 or 10 μg rNS3 + 10 μg CpG-ODN + M720, and were killed 1 month after immunization for HCV NS3 peptide pool mapping by ELISPOT assay. Another three naive mice were treated as controls. The splenocytes from each animal were stimulated by each peptide pool at 2 μg/ml per peptide for ELISPOT assay. Another six mice were similarly immunized for intracellular cytokine staining.

Assay for intracellular staining of IFN-γ+/CD8+ T cells (CTL) and IFN-γ+/CD4+ T cells (Th cell) by flow cytometry

Splenocytes, at a concentration of 2 × 106/ml and prepared on Ficoll–Paque, were cultured with anti-mouse CD28, anti-mouse CD49d antibodies, mouse IL-2 (all from BD Pharmingen), and the HCV NS3 peptide pool (2 μg/ml for each individual peptide) in a 48-well plate at 37° for 60 hr, followed by another 4-hr incubation after adding GolgiPlug (BD Pharmingen). Cells were treated with dimethylsulphoxide (DMSO) as negative control, or with DMSO plus ionomycin and phorbol 12-myristate 13-acetate as positive control. The cells were stained with phycoerythrin-labelled anti-mouse CD4 and allophycocyanin-conjugated anti-mouse CD8a antibodies as previously reported.24 The stained cells were fixed and permeabilized by Cytofix/Cytoperm solution and further stained by fluorescein isothiocyanate-conjugated anti-mouse IFN-γ antibody (BD Pharmingen). The stained cells were analysed by flow cytometry (FACSort, Becton Dickinson, CA) using CellQuest software. Data were analysed on WinMDI 2·8 software. From each sample, 500 000 events were acquired, and the percentage of IFN-γ+/CD8+ T cells or the percentage of IFN-γ+/CD4+ T cells was defined as the percentage detected in peptide-stimulated cells after subtraction of its negative control from the same animal.

Statistical analysis

Comparisons were analysed by Wilcoxon–Mann–Whitney rank sum test using Stata 7·0 software, and a difference was considered as statistically significant when a P-value was <0·05.


Poly (I:C) dosage determination and the stability of vaccine formulation

To optimize the dosage of poly (I:C), we incorporated poly (I:C) at dosages of 0, 10, 25, 50, and 100 μg, respectively, in a vaccine formulated with rNS3 and M720 for mouse immunization. Serum was collected 5 weeks after immunization to test the anti-HCV NS3 antibody titre and its isotype by ELISA. All the immunized mice developed an anti-HCV NS3 IgG antibody. The highest anti-HCV NS3 antibody titres were reached in mice vaccinated with 25 or 50 μg poly (I:C) or 10 μg CpG, and these responses were significantly greater than immunization without poly (I:C) (P = 0·01, 0·04, 0·03, respectively) (Table 3). The immune response elicited in vivo is a mixed system rather than a pure Th1 or Th2 response, wherein the IgG2a : IgG1 ratio represents the ratio of the Th1 response to the Th2 response.25,26 Anti-HCV NS3 IgG isotype production revealed that the mice immunized with rNS3 and M720 without poly (I:C) produced low levels of IgG2a and IgG1, and the ratio of IgG2a : IgG1 was 0·08 – indicating a Th2-dominated immune response. Most of the mice vaccinated with different amounts of poly (I:C) and all of those inoculated with CpG showed an IgG2a : IgG1 ratio >1, suggesting Th1 polarization. The greatest geometric mean value of IgG2a and IgG1 was achieved in mice immunized with vaccine incorporating 25 or 50 μg poly (I:C) or CpG, and their IgG2a production was significantly increased compared to that in mice receiving vaccine without poly (I:C) (Table 3). A poly (I:C) dosage of 50 μg was selected as optimal for subsequent experiments. A previous study has demonstrated that a CpG dose of 10 μg is suitable for mouse immunization.27

Table 3
Anti-HCV NS3 titre and IgG isotype production by different doses of poly (I:C) adjuvant

The M720-based vaccine emulsion is stable in storage at 4° for at least 1 year but the protein will undergo a gradual modification, the immunopotency will therefore be diminished by long-term storage.28 To test dsRNA stability in the M720-based rNS3 vaccine, a potency stability study was performed by immunization of mice with the formulation stored at 4° for 0, 24, or 48 hr, respectively. All the immunized mice displayed an IgG2a : IgG1 ratio >1, suggesting a Th1-biased immune response, and no significant difference was observed in anti-HCV NS3 titre, IgG2a or IgG1 production over time (data not shown).

Compared with the classical formulation ratio of 70 : 30 of M720 continuous phase to aqueous dispersed phase, a formulation with a 50 : 50 ratio was equally immunogenic (data not shown) but had increased viscosity, as reported previously.29

Selection of synthetic peptide pool in T-cell expansion for flow cytometry

Since both poly (I:C) and CpG adjuvants can elicit a Th1-polarized cellular immune response, these adjuvants were chosen for immunizing mice in a peptide pool selection study. ELISPOT results in 10 mice showed that pool 3 had the highest IFN-γ SFU among the total of 20 peptide pools (Fig. 1a). All three naive mice had less than 6 IFN-γ SFU upon stimulation by each of the 20 individual peptide pools (data not shown). Peptide pool 3 was also found in flow cytometry analysis to induce the highest percentage of CD4+ IFN-γ+ (Fig. 1c) and CD8+ IFN-γ+ cells (Fig. 1b) when compared with other peptide pools. The splenocytes from immunized mice stimulated by peptide pool 3 still had a much higher CD8+ IFN-γ+ cell percentage (Fig. 2b) than the splenocytes from naive mice (Fig. 2e). Although peptide pool 7 displayed a comparable percentage of CD4+ IFN-γ+, the CD8+ IFN-γ+ cell percentage produced by pool 7 was relatively lower than that produced by pool 3. Based on these results, peptide pool 3, with the biggest mean value of CD8+ IFN-γ+ cell percentage and IFN-γ SFU, was selected for subsequent T-cell stimulation and flow cytometry assays although there was no statistical significance among the several peptide pools with higher mean values.

Figure 1
Peptide pool selection for rNS3-specific T-cell stimulation. Mice were immunized with rNS3 + poly (I:C) + M720 or rNS3 + CpG-ODN + M720, and were killed 1 month after immunization. Splenocytes were expanded with 20 individual overlapping peptide pools ...
Figure 2
rNS3-specific CD8+ T cells percentage detected in splenocytes from immunized or naive mice expanded with peptide pool 3. (a) and (d) Splenocytes from mouse immunized with M720 + rNS3 + poly (I:C) (a) or from naive mouse (d) were expanded with DMSO as ...

Enhancement of antigen-specific CD4+ as well as CD8+ responses by poly (I:C)

As early as 2 weeks after vaccination, the splenocytes from mice immunized with rNS3 and poly (I:C) emulsified in M720 produced more IFN-γ SFU than those from mice immunized with rNS3 only (P < 0·01), or rNS3 emulsified in M720 (P < 0·01), or rNS3 and poly (I:C) without M720 (P < 0·01), and reached similar levels of IFN-γ SFU as those produced by the cells from mice immunized with rNS3 and CpG emulsified in M720 (Fig. 3a). An IFN-γ : IL-4 ratio >1 was also considered to be an indicator of Th1-biased immunity of the host and was correlated with the IgG2a : IgG1 ratio, whereas a ratio <1 represents Th2-polarization.30,31 The IFN-γ : IL-4 ratios recorded in the current study revealed that 95% of mice vaccinated with rNS3 and poly (I:C) (38/40), and all mice vaccinated with rNS3 and CpG had a ratio >1, demonstrating a Th1-biased immune response (Table 4). The ratio in mice receiving rNS3 only or rNS3 with M720 showed that 90% (27/30) had a ratio <1, suggesting Th2 polarization (Table 4). At 2 and 4 weeks after immunization, the number of IFN-γ SFU from mice vaccinated with rNS3 and poly (I:C) emulsified in M720 was significantly higher than that from mice immunized with rNS3 and poly (I:C) without M720, suggesting that M720 increases the immunogenicity of the rNS3 and poly (I:C) formula (Fig. 3a,c). The IL-2 SFU results, as another cytokine indicating a Th1-biased immune response, were similar to IFN-γ production. Mice immunized with rNS3 emulsified in M720 without poly (I:C) produced significantly more IL-4 SFU compared to mice vaccinated with other rNS3 formulations (P < 0·01), demonstrating that M720 contributes to the Th2-dominated immune response. Similar results were obtained from mice 3 or 4 weeks after immunization (Fig. 3b,c).

Figure 3
Cytokine production of splenocytes in response to rNS3 stimulation from mice immunized with different formulations of rNS3 and assayed by ELISPOT. Mice were immunized with rNS3 plus poly (I:C) or CpG emulsified in M720, or antigen only or adjuvant only, ...
Table 4
The IFN-γ : IL-4 ratio in ELISPOT assay from mice after 2, 3 or 4 weeks of immunization

Two weeks after immunization, a significantly higher percentage of CD8+ IFN-γ+ T cells was observed in mice immunized with rNS3 and poly (I:C) emulsified in M720 compared to mice immunized with rNS3 alone (P < 0·01), rNS3 emulsified in M720 (P < 0·01), or rNS3 and poly (I:C) without M720 (P < 0·01) and was comparable to that in mice immunized with rNS3 and CpG emulsified in M720 (Fig. 4a). Mice immunized with either poly (I:C) and M720, or CpG-ODN and M720 without antigen, had negligible CD8+ IFN-γ+ T cells specific for NS3 peptides (Fig. 4). The results from mice tested at 3 or 4 weeks postimmunization were similar except for a lower percentage of CD8+ IFN-γ+ cells in mice immunized with the poly (I:C) formulation when compared to the CpG formulation (Fig. 4b,c).

Figure 4
Potent cellular immune response specific to HCV NS3 at the early stage of vaccination with rNS3 in M720 and poly (I:C). (a) Two weeks after vaccination, the mice immunized with M720 + rNS3 + poly (I:C) had a significantly higher percentage of CD8+ IFN-γ ...

Sustained immune response in mice vaccinated with poly (I:C) adjuvant

Mice immunized with rNS3 and poly (I:C) emulsified in M720 were killed 4 or 7 months after vaccination to investigate the memory of the cellular immune response (Fig. 5). Four months after immunization, mice vaccinated with rNS3 and poly (I:C) emulsified in M720 maintained a significantly higher percentage of CD8+ IFN-γ+ T cells compared to mice immunized with rNS3 in M720 or rNS3 and CpG in M720 (P < 0·01 and P < 0·05, respectively). Similar differences were observed for CD4+ IFN-γ+ T cells (P < 0·01 and P < 0·05, respectively). The Th cell population from mice immunized with rNS3 and CpG in M720 was significantly greater than that from mice immunized with rNS3 in M720 without CpG (P < 0·05), though it was smaller than that from mice administered rNS3 in M720 with poly (I:C) (Fig. 5a). Seven months after immunization, mice vaccinated with poly (I:C) or CpG adjuvant had a significantly higher percentage of CD4+ IFN-γ+ T helper cells and CD8+ IFN-γ+ CTL compared with mice vaccinated with only rNS3 in M720 (P < 0·05). Overall, these results suggest that both poly (I:C) and CpG enhance the HCV-specific immune response but the poly (I:C) may elicit a stronger sustained response (Fig. 5).

Figure 5
Persistent cell-mediated immune response after vaccination with rNS3 in M720 and poly (I:C). (a) After 4 months of immunization, the mice vaccinated with M720 + rNS3 + poly (I:C) showed a significantly higher percentage of CD8+ IFN-γ+ and CD4 ...


Adaptive immunity after virus infection is mediated in part by dsRNA, which is produced during virus replication in infected cells and is capable of eliciting cellular immunity against the virus by cross-priming,13 activation of myeloid dendritic cells32 and induction of type I IFN production through either the TLR3 pathway33 or retinoic acid-inducible gene I (RIG-I) signalling by cytosolic DExD/H box RNA helicase.34,35 Type I IFN can further activate dendritic cells for cross-priming of CTL36 and can up-regulate TLR3 expression,37 forming an IFN amplification loop in a positive feedback pattern. Poly (I:C) is a synthetic analogue of dsRNA with potent ability to induce IFN production and has been used previously in patients.38 However, early studies demonstrated that application of poly (I:C) alone brought no conclusive evidence of its effectiveness in viral diseases or cancers in humans.38 HIV or influenza virus vaccines that incorporate poly (I:C) have shown promising Th1-dominated immunogenicity.32,39,40

Recent studies have shown that HCV NS3/NS4A, an enzyme complex possessing protease and RNA helicase activities, can cause proteolysis of TRIF, which links TLR3 to kinases and type I IFN production, inhibiting dsRNA-activated signal transduction through the TLR3 pathway.10,17,18,38,41 HCV NS3/NS4A can also disrupt the RIG-I pathway by proteolytic inactivation of components of a RIG-I signalling complex, ablating the downstream IRF-3 and nuclear factor-κB activation, diminishing type I IFN production and attenuating the host antiviral defence immunity.42 Both mechanisms eventually enable immune evasion of the virus. Theoretically, strong cell-mediated immunity against NS3, particularly a CD8+ CTL response, will eliminate cells that present NS3-specific epitopes in the context of MHC class I and diminishing ability of HCV to evade the host immune response. Development of an HCV vaccine targeted at NS3 has attracted considerable interests.2123,4347 In these previous reports, a vigorous Th1-biased cellular immune response against NS3 has been achieved in mice by enhancing vaccines with CpG, liposomes, or viral vectors. Nevertheless, these approaches are far from becoming a clinical application although they possess strong immunomodulatory properties.

Our results demonstrate that poly (I:C), as an adjuvant to an HCV NS3-based vaccine, can elicit a Th1-polarized specific cellular immune response and CTL responses against HCV NS3 in a murine model. However, in contrast to the studies that reported effective adaptive immunity from poly (I:C) adjuvant alone,39,48 our results show that the immune response was only slightly enhanced by the addition of poly (I:C), compared to mice administered antigen alone. This may be attributed to the degradation of poly (I:C) after injection because there is a ribonuclease capable of hydrolysis of dsRNA both in serum and tissue.49 To protect poly (I:C) from rapid degradation, the vaccine utilized in this study was emulsified with M720 to produce a water-in-oil formulation. In addition to its protection of poly (I:C) from degradation, M720 also has adjuvant effects by the mechanisms of slow antigen release, recruitment and stimulation of antigen-presenting cells, and diffusion of oil droplets to draining lymph nodes.29 Our results demonstrated that splenocytes from mice vaccinated with NS3 with M720 produced more IL-4 SFU and IFN-γ SFU than did cells from mice immunized with NS3 alone but the IFN-γ : IL-4 ratio did not change. This result demonstrated that M720 can significantly expand the immune response induced by protein antigen, but does not change the immune polarity induced by the priming antigen.

Unmethylated CpG and dsRNA are pathogen-associated molecular patterns of bacteria and viruses that elicit an immune response with a Th1 bias through TLR9 and TLR3 pathways, respectively. Our previous studies suggested that vaccination with rNS3 and CpG can induce a Th1-dominated immune response;23 however, dsRNA adjuvant may be an alternative protocol for HCV vaccine development, therefore CpG was used in the current study as a control adjuvant. This study showed that emulsification of HCV NS3 antigen with poly (I:C) or CpG-ODN adjuvant in M720 significantly augmented the anti-HCV NS3-specific Th1 and CTL responses. In the immune response elicitation studies, no significant difference in the percentage of CD8+ IFN-γ+ T cells was observed between vaccines with poly (I:C) or CpG adjuvant in M720 except in the assay 4 weeks after immunization when the percentage in the poly (I:C) adjuvant group was lower than in the group given CpG adjuvant. In the Th cell assay by ELISPOT, the IFN-γ SFU was similar between these two groups at all three time-points. In the persistent immune response assay, the CD8+ IFN-γ+ T-cell percentage and CD4+ IFN-γ+ T-cell percentage in poly (I:C) group mice were significantly higher than in the CpG group when measured 4 months after vaccination, suggesting that the cellular immune response induced by the vaccine with poly (I:C) adjuvant may persist longer than that induced by the vaccine with CpG adjuvant. However, 7 months after immunization, the differences in CD8+ IFN-γ+ T-cell percentage and CD4+ IFN-γ+ T-cell percentage between the poly (I:C) and CpG groups became non-significant. Whether the cellular immune response elicited by vaccine with CpG adjuvant is similar to that induced by vaccine with poly (I:C) adjuvant 7 months or more after immunization deserves further investigation. Most importantly, vaccines incorporating either poly (I:C) or CpG when combined with M720 were far superior to protein-M720 vaccination lacking these adjuvants.

Poly (I:C) has been used clinically for many years; the clinical drug Ampligen is available, which is composed of poly I : poly C12U, a specific form of mismatched dsRNA where uridylic acid (U) substitutions in the polycytidylic acid chain create periodic regions of non-hydrogen-bonding in the helical configuration.50 This mismatched dsRNA drug is well tolerated clinically and can be administered on a long-term basis.16,51 M720 is also currently undergoing clinical trials.52,53 Therefore, the rNS3 vaccine with poly (I:C) adjuvant emulsified in M720 possesses great potential for clinical trials if this potent immunomodulatory effect in mice can be demonstrated in the chimpanzee.

In summary, we investigated the adjuvant potential of poly (I:C), a synthetic dsRNA analogue, in a vaccine formulation with HCV NS3 emulsified in M720. The results showed that this vaccine formulation can elicit a strong and persistent antigen-specific Th1-biased immune response and CTL responses in mice. The potential of this immunization strategy for the control of HCV infection should be further explored in the chimpanzee model.


unmethylated cytosine preceding guanosine motif
cytotoxic T lymphocytes
dendritic cells
double-stranded RNA
enzyme-linked immunosorbent assay
enzyme-linked immunospot assay
hepatitis C virus
interferon regulatory factor 3
Montanide ISA 720
major histocompatibility complex
non-structural protein 3
optical density
poly (I:C)
polyriboinosinic:polyribocytidylic acid
retinoic acid-inducible gene I
recombinant HCV NS3 protein
recombinant HCV non-structural protein 5b
streptavidin-horseradish peroxidase complex
spot-forming units
T helper
toll-like receptor 3
Toll/IL-1 receptor domain-containing adapter inducing IFN-β


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