Vaccine and Infectious Disease Organization, University of Saskatchewan, 120 Veterinary Road, Saskatoon, SK, Canada S7N 5E3
Correspondence
Sylvia van Drunen Littel-van den Hurk
vandenhurk{at}sask.usask.ca
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ABSTRACT |
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INTRODUCTION |
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HCV is an enveloped, plus-stranded RNA virus of the family Flaviviridae. Its genome is 9·5 kb in length with one open reading frame encoding a polyprotein comprising structural (core and envelope glycoproteins E1 and E2) and non-structural (NS2, NS3, NS4a/b and NS5a/b) proteins (Grakoui et al., 1993; Hijikata et al., 1991
). Since HCV exists as numerous genotypes and within an infected host as numerous quasispecies, it is a notoriously difficult target for immunization (Inchauspe & Feinstone, 2003
). There is growing evidence that Th1 and cytotoxic T-lymphocyte (CTL) responses to HCV proteins play a major role in recovery (Thimme et al., 2001
; Cooper et al., 1999
). The NS3 protein, which has serine protease and helicase activity (Grakoui et al., 1993
) and is one of the most conserved proteins of HCV, seems to play a key role in virus clearance (Jiao et al., 2003
). NS3 contains an immunodominant CD4+ T helper epitope and several CTL epitopes. Since these epitopes have been associated with control of HCV in patients with self-limiting infection (Diepolder et al., 1997
; Kurokohchi et al., 1996
; Battegay et al., 1995
), NS3 represents a potential vaccine candidate expected to induce both CD4+ and CD8+ lymphocyte-mediated protective immune responses.
DNA vaccines preferentially induce Th1 immunity and CTL responses (Boyer et al., 1996; Raz et al., 1996
; Ulmer et al., 1993
) and elicit protective immunity to a variety of pathogens (Tedeschi et al., 1997
; Donnelly et al., 1995
; Fynan et al., 1993
). However, DNA immunization has also been demonstrated to generate weaker antibody and CTL responses than protein and live attenuated vaccines (Manickan et al., 1997
). Several approaches have been explored to enhance immune responses induced by DNA vaccines, including co-administration of cytokine-expressing plasmids (Kim et al., 2001
; Geissler et al., 1997
), delivery in a Salmonella vector (Wedemeyer et al., 2001
), targeting of DNA to dendritic cells (You et al., 2001
), incorporation of immunostimulatory DNA sequences (Klinman, 2003
; Pontarollo et al., 2002
) and heterologous prime and boost vaccination regimes (Pancholi et al., 2003
; Song et al., 2000
).
In general, recombinant protein vaccines stimulate primarily Th2 cells and thus elicit strong humoral responses, but weak cell-mediated immune responses. However, if proteins are formulated with appropriate adjuvants, it is also possible to induce Th1-type responses. Oligodeoxynucleotides containing unmethylated CpG dinucleotides (CpG ODNs) are novel adjuvants known to promote Th1-biased immune responses (Chu et al., 1997). Indeed, out of 19 different adjuvants tested, CpG ODN was most effective at eliciting a Th1-type immune response to a tumour antigen (Davis, 2000
). CpG ODNs are capable of augmenting antigen-specific humoral and cellular immune responses against peptides, viral and bacterial proteins and tumour antigens in a number of different species (Klinman, 2003
; Ioannou et al., 2002a
; Davis et al., 2000
). Furthermore, CpG ODNs cause minor adverse reactions in comparison to most immunostimulatory agents used to date (Ioannou et al., 2003
).
Unlike many other vaccine adjuvants, the saponin-derived adjuvant Quil A promotes a broad immune response, by simultaneously inducing strong antibody and T-cell responses including enhanced cytokine secretion and activation of CTL responses (Cox & Coulter, 1997; Barr & Mitchell, 1996
). One of our previous studies showed that Quil A promotes a balanced immune response to a truncated form of bovine herpesvirus-1 glycoprotein D (BHV-1 tgD), while causing minimal tissue damage. When Quil A was co-administered with CpG ODN, the immune response was further enhanced and shifted to a Th1-type response (Ioannou et al., 2002b
). Since the purity of Quil A-based adjuvants has significantly improved, virtually non-reactogenic vaccines can be made with these highly purified Quil A components (Rimmelzwaan & Osterhaus, 1995
). This makes it one of the few adjuvants safe enough to be licensed for human use.
There is increasing evidence that the cellular immune response is of major importance for the control of HCV infection. Therefore, our goal was to enhance cell-mediated immunity to HCV NS3 by testing different vaccine formulations and strategies in both in-bred laboratory and outbred large animal species. A comparison of various vaccination regimes demonstrated that priming with plasmid encoding NS3, followed by boosting with rNS3 formulated with CpG ODN and Quil A results in optimal NS3-specific immune responses.
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METHODS |
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Construction of NS3 expression vectors.
Plasmid pMASIA and plasmids enriched with different numbers of CpG motif GTCGTT, pBISIA24, pBISIA40, pBISIA88 and pBISIA160, were constructed as described previously (Pontarollo et al., 2002; Krieg et al., 1998
). To create plasmids encoding NS3, the NS3 gene was cut from pRSET-NS3 with BamHI and HindIII restriction enzymes and inserted into pMASIA, pBISIA24, pBISIA40, pBISIA88 and pBISIA160 digested with the same enzymes. The recombinant plasmids were transformed into E. coli DH5
, purified using an EndoFree Plasmid Giga kit (Qiagen), and stored at 20 °C. In vitro expression of NS3 protein in COS-7 cells transfected by these plasmids was confirmed by immunohistochemistry, as described previously by Bryan et al. (1988)
.
CpG ODN and Quil A.
The adjuvants used in this study were Quil A (Accurate Chemical and Scientific) and two synthetic ODNs containing unmethylated CpG dinucleotides (Qiagen). Although generally conserved, there is a certain degree of species specificity in the recognition of CpG motifs (Hartmann et al., 2000). ODN 1826 (5'-TCCATGACGTTCCTGACGTT-3'), which is a strong B cell mitogen known to stimulate mouse splenocytes in vitro (Rankin et al., 2001
; Davis et al., 1998
), was used for mice. ODN 2007 (5'-TCGTCGTTGTCGTTTTGTCGTT-3'), which stimulates porcine and human peripheral blood mononuclear cells (PBMCs) in vitro (Rankin et al., 2001
; Hartmann et al., 2000
), was used for pigs. These two CpG ODNs were phosphorothioate modified to increase resistance to nuclease degradation (Samani et al., 2001
).
Immunization of mice and piglets.
Eight-week-old female BALB/c (H-2d) mice were used in all mouse trials. In the first trial, groups of six mice were immunized three times intradermally (i.d.) with saline or 50 µg plasmid in the back as follows: (1) saline; (2) pMASIA-NS3; (3) pBISIA24-NS3; (4) pBISIA40-NS3; (5) pBISIA88-NS3; or (6) pBISIA160-NS3. In the second trial, four groups of five mice were immunized three times subcutaneously (s.c.) in the back with one of the following formulations: (1) saline; (2) 20 µg rNS3 with 10 µg ODN 1826 (rNS3+CpG); (3) 20 µg rNS3 with 10 µg Quil A (rNS3+Quil A); or (4) 20 µg rNS3 with 10 µg ODN 1826 and 10 µg Quil A (rNS3+CpG+Quil A). In the third trial, six groups of six mice were immunized in the back as follows: (1) three times with saline; (2) three times with 50 µg pBISIA24-NS3 (3) twice with 50 µg pBISIA24-NS3 followed by 5 µg rNS3 with 10 µg ODN 1826 and 10 µg Quil A; (4) three times with 5 µg rNS3, 10 µg ODN 1826 and 10 µg Quil A; (5) twice with 50 µg pBISIA24-NS3 followed by 20 µg rNS3 with 10 µg ODN 1826 and 10 µg Quil A; or (6) three times with 20 µg rNS3, 10 µg ODN 1826 and 10 µg Quil A. The protein formulations were injected s.c., whereas the plasmids were delivered i.d.
In the pig trial, 5-week-old cross-bred piglets were randomly allocated to four groups of six piglets each, and immunized with pBISIA24-NS3 and/or rNS3 with CpG ODN and Quil A. The vaccination regimes were the same as treatments 14 in the second mouse trial. However, CpG ODN 2007 instead of CpG ODN 1826 was used. Secondly, i.d. immunizations were performed in the ears of the piglets. Thirdly, the doses of plasmid (500 µg), protein (50 µg), CpG ODN (100 µg) and Quil A (100 µg) in the pig trial were 10-fold higher than the doses in the mouse trial.
In all animal trials, vaccinations were given on days 0, 28 and 49. All mice were bled at regular intervals for ELISAs and they were sacrificed on day 63 to isolate splenocytes for ELISPOT and CTL assays. The piglets were also bled at regular intervals for ELISA, ELISPOT and lymphocyte proliferation assays. All experiments were carried out according to the guidelines provided by the Canadian Council for Animal Care.
ELISA.
In order to determine antibody responses in mice and pigs, 96-well polystyrene microtitre plates (Immulon 2; Dynatech) were coated with 0·1 µg per well of purified rNS3 and incubated with serially diluted murine or porcine sera. Alkaline phosphatase (AP)-conjugated goat anti-mouse IgG or AP-conjugated goat anti-porcine IgG (Kirkegaard & Perry Laboratories) was used at a dilution of 1 : 5000 to detect bound murine and porcine antibodies, respectively. The reactions were visualized with p-nitrophenyl phosphate (PNPP) (Sigma). The NS3-specific antibody titres are expressed as the reciprocal of the highest dilution resulting in a reading of two standard deviations above the value of negative control sera.
Immunoglobulin isotyping ELISA.
To determine the NS3-specific IgG subtypes in mice and pigs, serially diluted murine and porcine sera were incubated in rNS3-coated 96-well plates. Murine antibodies were detected with biotinylated goat anti-murine IgG1 and IgG2a (Caltag Laboratories) at a dilution of 1 : 4000, followed by streptavidinAP (Gibco) at a dilution of 1 : 2000. Porcine antibodies were detected with tissue culture supernatants containing mouse anti-porcine IgG1 and IgG2 (Serotec) diluted 1 : 200 and 1 : 100, respectively, followed by AP-conjugated goat anti-mouse IgG (Kirkegaard & Perry Laboratories) diluted 1 : 5000. The reaction was visualized with PNPP (Sigma). The results are expressed as murine IgG1 : IgG2a ratio of titres and porcine IgG1 : IgG2 ratio of titres.
Cytokine ELISPOT.
A cytokine-specific enzyme-linked immunospot (ELISPOT) assay was performed as described previously (Ioannou et al., 2002a; Lewis et al., 1999
). Briefly, 96-well MultiScreen-HA filtration plates (Millipore) were coated overnight at 4 °C with 0·1 µg per well of murine IFN-
or IL-4 specific monoclonal antibodies (Pharmingen) or porcine IFN-
specific monoclonal antibodies (BioSource International). Splenocytes isolated from mice (Baca-Estrada et al., 1996
) or PBMCs isolated from piglets (Rankin et al., 2002
) in AIM-V medium were added to the coated plates at 106 cells per well in the absence and presence of rNS3 at a final concentration of 1 µg ml1 for mice and 0·1 µg ml1 for piglets. After 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 (Pharmingen) or biotinylated anti-porcine IFN-
monoclonal antibodies (BioSource International) at 2 µg ml1. This was followed by incubation with streptavidinAP (Gibco) at a 1 : 1000 dilution. The spots were visualized with a substrate consisting of 5-bromo-4-chloro-3-indolyl phosphate and nitro blue tetrazolium (Sigma). The number of cytokine-secreting cells is 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.
CTL assay.
To prepare effector cells, splenocytes were isolated from each group of mice and pooled. 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 Aventis), at an m.o.i. of 10. After infection, the stimulators at a concentration of 106 cells ml1 were irradiated with 3000 rads. The pooled 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 (Boehringer Mannheim) was added to a final concentration of 10 U ml1. To generate target cells, we stably transformed P815 cells (H-2d) (ATCC) with NS3. NS3-transformed and control P815 cells were labelled for 1 h with 100 µCi of Na251CrO4 per 106 cells. Cells were washed four times and used as targets at 5x104 cells ml1. 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 releasespontaneous release)/(total 51Cr releasespontaneous release)]x100.
Lymphocyte proliferation assay.
PBMCs from piglets were dispensed at 3·5x106 cells ml1 in AIM-V medium and cultured in 96-well tissue culture plates at 3·5x105 cells per well in the absence and presence of 0·1 µg rNS3 ml1. After 72 h in culture, the cells were pulsed with 0·4 µCi (14·8 kBq) of [methyl-3H]thymidine (Amersham) per well. The cells were harvested 18 h later and radioactivity was determined by scintillation counting. Proliferative responses were calculated as the means of triplicate wells and are expressed as a stimulation index (SI) where SI represents the counts per minute (c.p.m.) in the antigen-stimulated wells divided by the c.p.m. in wells with medium alone.
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 at the level of P<0·05.
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RESULTS |
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Immune responses of mice immunized with rNS3 formulated with CpG ODN and/or Quil A
Since we observed previously that BHV-1 tgD formulated with Quil A and CpG ODN induces a Th1-type response, BALB/c mice were immunized with rNS3 formulated with Quil A, ODN 1826, or both Quil A and ODN 1826. All mice developed high NS3-specific antibody titres and there was no significant difference between the three vaccinated groups (Fig. 3a). In contrast, there was a difference in isotype profiles. Although all three groups had similar IgG1 titres, the rNS3+CpG group developed a significantly lower IgG2a titre (P<0·05) when compared to the rNS3+CpG+Quil A and rNS3+Quil A groups. Although the rNS3+Quil A group tended to have a lower IgG2a titre than the rNS3+CpG+Quil A group, there was no significant difference (Fig. 3a
).
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Humoral immune responses of mice immunized with pBISIA24-NS3 and/or rNS3 formulated with CpG ODN and Quil A
To further enhance the cell-mediated immune response to NS3, a regime of priming with pBISIA24-NS3 followed by boosting with rNS3+CpG+Quil A was explored. A second plasmid immunization was given prior to immunization with protein, because previous reports have demonstrated that this greatly enhances the immune response (Song et al., 2000). After the first, second and third immunization, the groups immunized with 5 µg rNS3+CpG+Quil A or 20 µg rNS3+CpG+Quil A had significantly higher total IgG titres (P<0·01) than the other groups. Furthermore, the mice primed with pBISIA24-NS3 and boosted with rNS3+CpG+Quil A developed stronger antibody responses compared to the mice immunized with plasmid alone (P<0·05) (Fig. 4
a). The NS3-specific IgG1 : IgG2a ratios were determined to evaluate the type of responses induced. Immunization with pBISIA24-NS3 resulted in the lowest IgG1 : IgG2a ratio, whereas the IgG1 : IgG2a ratio was highest in mice immunized with 20 µg rNS3+CpG+Quil A. Furthermore, the mice primed with pBISIA24-NS3 and boosted with rNS3+CpG+Quil A developed a significantly (P<0·05) lower IgG1 : IgG2a ratio compared to the mice immunized with rNS3+CpG+Quil A. Noticeably, immunization with higher doses of rNS3 did not increase the total IgG titre, but tended to increase the IgG1 : IgG2a ratio in the protein groups and significantly (P<0·05) increased the IgG1 : IgG2a ratio in the groups primed with plasmid and boosted with protein (Fig. 4b
).
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Humoral immune responses of piglets immunized with pBISIA24-NS3 and/or rNS3 formulated with CpG ODN and Quil A
Since DNA vaccines tend to be less effective in large, outbred species, it was critical to confirm the efficacy of the NS3 DNA vaccine in another model species. Furthermore, the rNS3 vaccine formulation needed to be validated with a pan-activating CpG ODN. Because of the strong similarity with humans in size, physiology and immunology, we used pigs to validate these vaccination strategies. In contrast to the responses in mice, even after three immunizations with pBISIA24-NS3, only low NS3-specific antibody levels were detected in the sera of the piglets (Fig. 6a). However, antibody responses to NS3 increased dramatically (P<0·01) in piglets immunized with pBISIA24-NS3 followed by rNS3+CpG+Quil A. In the piglets immunized with rNS3+CpG+Quil A, there was a significant increase in antibody titre after the second immunization, but little change after the third immunization. There was no difference in antibody titres between the protein-vaccinated piglets and the piglets immunized with plasmid followed by protein.
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Cellular immune responses of piglets immunized with pBISIA24-NS3 and/or rNS3 formulated with CpG ODN and Quil A
To assess NS3-specific cell-mediated immune responses in the piglets, lymphocyte proliferation and IFN- ELISPOT assays were performed after in vitro stimulation of PBMCs with rNS3. All vaccinated groups showed significantly (P<0·05) stronger NS3-specific lymphocyte proliferation than the saline group on days 63 and 84 (Fig. 7
a). Furthermore, both the plasmid group and the plasmid primeprotein boost group had a significantly stronger (P<0·05) lymphocyte proliferative response than the protein group on day 84.
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DISCUSSION |
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A previous study has demonstrated that plasmid encoding NS3 induces strong CTL responses in BALB/c mice, whereas NS3 formulated with complete Freund's adjuvant (CFA) elicited antibody, but no CTL responses (Encke et al., 1998). In another report, NS3 in PBS or CFA was shown to induce primarily IgG1 in BALB/c mice, whereas plasmid encoding NS3 elicited predominantly IgG2a. Furthermore, T-cells from DNA-vaccinated mice tended to produce more IFN-
and IL-2 than those from rNS3-immunized mice (Lazdina et al., 2001
). In the current study, rNS3 was formulated for the first time with CpG ODN and/or Quil A. Generally, either CpG ODN or Quil A can enhance both cellular and humoral immune responses (Klinman, 2003
; Stittelaar et al., 2000
). However, we showed that only when rNS3 was formulated with both CpG ODN and Quil A was a balanced immune response induced with production of high numbers of IFN-
- and IL-4-secreting cells, while rNS3 combined with either CpG ODN or Quil A produced high levels of IL-4, but low IFN-
. The dramatic increase of IFN-
production in the rNS3+CpG+Quil A group may be related to either the dose or the properties of the protein. NS3 is a non-structural protein, which tends to be less immunogenic compared to structural proteins. Therefore, the dose of NS3 was higher than that of, for example, BHV-1 tgD, which was formulated with 10 µg of ODN 1826 at 0·2 µg per dose, and induced a Th1-type response (Ioannou et al., 2002b
). In support of this contention, a reduction of the rNS3 dose from 20 to 5 µg in the second mouse trial tended to shift the response to be more Th1-biased. Formulation of NS3 with CpG ODN and Quil A might be an excellent approach to induce strong and balanced immune responses in humans, since both CpG ODN and purified fractions of Quil A cause low side-effects (Ioannou et al., 2003
; Rimmelzwaan & Osterhaus, 1995
). Furthermore, this adjuvant combination will be useful in general for proteins that elicit low or Th2-biased immune responses.
As an approach to induce stronger cell-mediated immune responses to HCV NS3, we evaluated two additional vaccination regimes consisting of either vaccination with pBISIA24-NS3 or priming with pBISIA24-NS3 followed by boosting with rNS3+CpG+Quil A. Previous studies have demonstrated varying efficacy of heterologous primeboost strategies for NS3 compared to a single DNA vaccination regime. A DNA primecanarypox virus boost regime elicited stronger cellular immune responses to HCV structural and non-structural proteins when compared to vaccination with HCV DNA alone (Pancholi et al., 2003). In contrast, no difference was observed between the immune responses induced by a DNA-based NS3 vaccine, recombinant Semliki Forest virus (SFV) particles expressing NS3 (rSFV), or a DNA primerSFV boost regime (Brinster et al., 2002
). In our study, the DNA-based NS3 vaccine and the DNA primeprotein boost regime elicited very similar cell-mediated responses, both inducing a Th1-type response characterized by a predominance of IFN-
and high cytotoxicity in mice.
Interestingly, compared to previous reports (Ladzina et al., 2001; Pancholi et al., 2003
; Encke et al., 1998
), pBISIA24-NS3 induced relatively high NS3-specific antibody titres, which may be due to the selection of the intradermal route for immunization. Furthermore, the overall antibody responses induced by the DNA primeprotein boost regime were significantly stronger than the responses elicited by the DNA vaccine alone. Several studies suggest a role for virus neutralizing antibodies in resolution of HCV infection (Shimizu et al., 1996
; Farci et al., 1994
; Choo et al., 1994
), whereas two other studies rule out the role of such antibodies in recovery (Takaki et al., 2000
; Cooper et al., 1999
). This suggests that humoral immunity might have an impact on prevention of infection. Although NS3-specific antibody production is not expected to neutralize the virus, the DNA primeprotein boost regime might be the appropriate approach for induction of both humoral and cellular immunity to the surface proteins E1 and E2.
Because the natural host and the closely related primates are not readily available, only a few HCV vaccine studies have been carried out in large animal species. In addition to non-human primate and monkey trials, most of which have tested the envelope proteins of HCV (Bukh et al., 2001; Duenas-Carrera et al., 2004
), sheep have been used in a vaccine study on HCV core (Acosta-Rivero et al., 2002
). Furthermore, plasmids encoding other human products such as human growth hormone have been tested in both dogs and pigs (Anwer et al., 1999
). We chose pigs as our model because they are more closely related to humans and are used for xenografts. The NS3-specific cellular immune responses produced in piglets after vaccination with pBISIA24-NS3 followed by rNS3+CpG+Quil A were very strong and appeared to be at least as good as those in mice vaccinated with the same regime. The mean number of IFN-
-secreting cells in the ELISPOT assay was more than 200 per 106 cells and this number was maintained on day 35 after the last immunization, suggesting that, as in the mice, excellent and long-lasting immunity to NS3 was produced in this group. Interestingly, the humoral immune responses induced to NS3 in mice and pigs were quite different. As expected from previous large animal studies, the mice produced much higher serum IgG titres than the pigs after vaccination with pBISIA24-NS3. However, after vaccination with pBISIA24-NS3 followed by rNS3+CpG+Quil A, the NS3-specific IgG titre increased about fivefold in mice and 100-fold in pigs compared to immunization with plasmid alone. These data suggest that the piglets were very well primed by the DNA vaccine, resulting in significantly enhanced NS3-specific antibody responses following the protein boost. Thus, immune responses developed in the mouse and pig model may not entirely correlate, which supports the need for testing vaccine strategies in an outbred large animal. Based on the excellent immune responses induced in the pig model, it will be of interest to test the DNA primeprotein boost vaccination strategy in primates.
In conclusion, our study demonstrated that a vaccination regime of priming with pBISIA24-NS3 followed by boosting with rNS3 formulated with CpG ODN and Quil A elicited a strong cell-mediated, as well as humoral, immune response, both in mice and in piglets. This strategy might be of great value in general for vaccine development against HCV as well as many other pathogens.
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ACKNOWLEDGEMENTS |
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Received 18 November 2003;
accepted 20 January 2004.