Enhanced hepatitis C virus NS3 specific Th1 immune responses induced by co-delivery of protein antigen and CpG with cationic liposomes

Xuanmao Jiao, Richard Yan-Hui Wang, Qi Qiu, Harvey J. Alter and J. Wai-Kuo Shih

Department of Transfusion Medicine, Warren G. Magnuson Clinical Center, Building 10, Room 1C711, National Institutes of Health, Bethesda, MD 20892-1184, USA

Correspondence
J. Wai-Kuo Shih
jshih{at}mail.cc.nih.gov


   ABSTRACT
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Mice were immunized intramuscularly with free recombinant hepatitis C virus (HCV) NS3 (non-structural protein 3) protein, liposomes encapsulating rNS3 or rNS3 and CpG mixture, liposomes co-encapsulating rNS3 and CpG or liposomes co-encapsulating rNS3 and GpC. Liposomes co-encapsulating rNS3 and CpG induced a much higher titre of anti-HCV NS3 IgG and the dominant IgG subtype was IgG2a. Liposomes co-encapsulating rNS3 and GpC also induced high levels of anti-HCV NS3 IgG antibody, but the dominant IgG subtype was still IgG1, the same as in free HCV/NS3 immunized mice. Liposomes encapsulating rHCV NS3 and the mixture of rHCV NS3 and CpG did not increase the antibody response but switched the IgG subtype. A cytokine profile analysis revealed that the levels of Th1 cytokines in the mice immunized with liposomes co-encapsulating rHCV NS3 and CpG were significantly higher than in other mice while the levels of Th2 cytokine were significantly lower than in the mice immunized with naked rNS3. IL-12 in the mice immunized with liposome-NS3-CpG was significantly higher than in other mice. In conclusion, liposomes co-encapsulating HCV NS3 and CpG are a good candidate vaccine to induce strong Th1 immune responses against hepatitis C viruses.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Hepatitis C virus (HCV ) infects approximately 1 % of the world's population. Over 75 % of acutely infected individuals progress to a persistent infection that can result in cirrhosis, liver failure and hepatocellular carcinoma (Choo et al., 1989; Cohen, 1999). The development of a screening test in 1990 has virtually eliminated the spread of HCV through blood transfusion, but 36 000–150 000 new cases of HCV infection continue to occur in the USA every year through other transmission routes (Cohen, 1999; Stratton et al., 2000). Currently, the most effective treatment for chronic HCV infection is a combination therapy with interferon-{alpha} and ribavirin, but across all genotypes only 50 % of treated patients have sustained benefit from antiviral therapy (Poynard et al., 1998; McHutchison et al., 1998). Treatment efficiency might be considerably enhanced by direct stimulation of HCV-specific T-cell responses using a therapeutic vaccine.

Clinical observations of HCV-specific immune responses in patients with acute self-limited HCV infection or patients who have recovered from chronic HCV infection suggest that T-cell-mediated immune responses may determine the outcome of HCV infection (Rehermann & Chisari, 2000). During the first few weeks of acute hepatitis C infection, HCV-specific CD4+ T cells that proliferate after in vitro stimulation with recombinant HCV core, NS3 (non-structural protein 3) and NS4 (non-structural protein 4) antigens have been detected (Rehermann & Chisari, 2000). An NS3-specific CD4+ T cell immune response is much stronger and more frequently found in patients who resolve acute hepatitis than in patients who develop chronic infection and this response may be necessary for virus clearance (Diepolder et al., 1995). Similarly, IFN plus ribavirin treatment-induced control of hepatitis C viraemia is associated with the development of HCV-specific T-cell responses with enhanced IFN-{gamma} and low IL-10 production (Cramp et al., 2000). The effective immune response frequently displays a Th1 or Th0 cytokine profile while the activation of Th2 responses seems to be involved in the development of chronic hepatitis (Tsai et al., 1997). An immunodominant epitope recognized by NS3-specific Th cells has been described at aa 1251–1259 within HCV NS3 (Diepolder et al., 1997) and is completely conserved within HCV 1a, 1b, 1c, 2a and 2b genotypes (Rehermann & Chisari, 2000). These findings suggest that a vigorous HCV-specific CD4+ Th1 response, particularly against the non-structural proteins of the virus, may be associated with virus clearance and protection from disease progression. Accordingly, the development of a vaccine that increased Th1 immune responses could bring an important advance to overcoming established HCV infection. At least, this vaccine should include components that could induce Th1 immune responses against NS3.

It is well known that immunostimulatory DNA containing CpG motifs is sensed by immune cells as a sign of the presence of pathogens and evokes host defence mechanisms such as the activation of macrophages and dendritic cells to secrete IL-12 and the induction of Th1 cell differentiation (review, Krieg, 1999). The direct association of CpG with a protein antigen, either via biotin–avidin linkage or covalent linkage, can further enhance the immune response (Klinman et al., 1999; Shirota et al., 2000; Tighe et al., 2000). It is most likely that CpG and antigen are engulfed by the same APCs. A previous study in this laboratory found that the plasmids encapsulated in cationic liposomes induced much stronger IL-12 secretion in immunized mice than naked DNA, suggesting that the target of the cationic liposome–DNA complex was either macrophages or dendritic cells (Jiao et al., 2003). The ultimate goal of our laboratory's HCV vaccine programme is to develop a more effective recombinant protein vaccine that would stimulate cell-mediated as well as humoral immunity or to utilize such a lipid-encapsulated, CpG-enhanced recombinant protein as a booster vaccination following an optimized DNA prime (Jiao et al., 2003). In this study, we used liposomes as an alternative method to deliver CpG oligodeoxynucleotides (ODN) in close proximity to the antigen and found this to be invaluable for enhancing the Th1 type immune response.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Expression and purification of recombinant HCV NS3 protein.
The full-length HCV NS3 protein was produced in an E. coli expression system. Plasmid pQE-HCV/NS3 was constructed by inserting the HCV NS3 gene encoding aa 1027–1657 (nt 3420–5312, HCV strain H of genotype 1a) into the BamHI restriction enzyme site of pQE-11 plasmid (Qiagen). The recombinant plasmid DNA was introduced into E. coli DH5{alpha}F'IQ cells by transformation. The positive clone was identified by Western blot analysis using an anti-HCV-positive serum sample. To increase the expression level of HCV NS3 recombinant protein, pACYC-based plasmid containing extra copies of argU, ileY and leuW tRNA genes (Kane, 1995; Carstens, 2003), which recognize AGA and AGG codons for arginine, the AUA codon for isoleucine and the CUA codon for leucine, was introduced into the HCV NS3 expression clone by transformation. The pACYC-based plasmid was obtained from BL21-CodonPlus-RIL (Stratagene). One of the high-level expression clones, designated pQE-HCV/NS3-RIL-3, was selected for production of HCV NS3 protein.

The recombinant NS3 protein was purified by SDS-PAGE (10 %) and the residual endotoxin was removed by washing with 0·9 % NaCl, 10 mM sodium deoxycholic acid (Pestch & Anspach, 2000). The NS3 protein precipitates were dissolved in 10 mM Tris/HCl pH 8·0, 1 mM EDTA, 0·1 % SDS, 10 mM DTT. The yield of purified NS3 protein was 40 mg from 1 litre of pQE-HCVNS3-RIL-3 culture. The endotoxin activity in the purified NS3 protein was determined by a kinetic chromogenic Limulus amoebocyte lysate assay (BioWhittaker), and found to be less than 20 EU per mg of NS3 protein.

Preparation of liposome co-encapsulated recombinant HCV NS3 and CpG ODN.
Recombinant HCV NS3 protein and CpG were entrapped into cationic liposomes by a dehydration and rehydration procedure (Kirby & Gregoriadis, 1984). Cationic liposomes were composed of equimolar egg yolk phosphatidylcholine (EPC) (Avanti Polar Lipids) and a cationic lipid, dimethyldioctadecylammonium bromide (DDAB) (Avanti Polar Lipids). Briefly, 3·96 µmol of each lipid was dissolved in chloroform and dried under a gentle stream of nitrogen. The mixture of lipids was hydrated with 60 µl of injectable grade water and then sonicated in a bath-type sonicator until the suspension was translucent, creating a small unilamellar vesicle (SUV) suspension. The liposome preparation was determined to be endotoxin free by the Limulus amoebocyte lysate Pyrogent-5000 method (BioWhittaker). To prepare a dehydration–rehydration vesicle (DRV) containing recombinant HCV NS3 protein (liposome-rNS3), 60 µg of the recombinant HCV NS3 protein was added to the SUV suspension and the mixture was frozen on dry ice and dried in a freeze-drier after which 60 µl of 1x PBS was added to rehydrate the lipid–DNA mixture. The liposome-rNS3 combination was diluted with 1x PBS to a final volume of 600 µl. To make liposome-rNS3-CpG or liposome-rNS3-GpC, prior to freeze-drying, the SUV and recombinant HCV NS3 protein mixture was sonicated briefly to clarity and then 300 mg of CpG (5'-GACGTTGACGTTAGCGT-3', Keystone Labs) or GpC (5'-GAGCTTGAGCTTAGGCT-3', Keystone Labs) in 150 µl water was added to the mixture with vortexing. The sequence of CpG design was based on the results of Kusakabe et al., (2000). Liposome-rNS3-CpG and liposome-rNS3-GpC were diluted with 1x PBS to a final volume of 600 µl. All the final liposome preparations were DRV.

Immunization of mice.
Female BALB/c mice were housed in approved animal care facilities during the experimental period and were handled following the international guidelines required for experimentation with animals. All animal study protocols were approved by NIH Clinical Center Animal Care and Use Committee. Six- to 8-week-old mice were immunized under general anaesthesia by direct intramuscular injection into the tibialis anterior muscle of 10 µg recombinant HCV NS3 proteins with or without 50 µg CpG or GpC either in free form or encapsulated in cationic liposomes.

Assay of anti-HCV/NS3 antibodies.
Mice were bled from the tail vein and sera were collected at weeks 0, 1, 2, 3 and 4 after immunization. All sera were kept at –70 °C before assay. Anti-HCV/NS3 IgG was assayed by ELISA. The mice were bled from the tail and serum was prepared at 0, 2, 4, 6 and 8 weeks after primary DNA immunization. All sera were kept at –70 °C before assay. Anti-HCV/NS3 IgG was assayed by ELISA. MaxiSorp Nunc-Immuno plates were coated with recombinant HCV NS3 protein at 2 µg ml–1 in coating buffer (20 mM NaHCO3/Na2CO3 pH 9·6, 0·15 M NaCl) and overcoated with PBS containing 0·1 % BSA. The tested sera were added at 10 µl per well in 0·3 % NP-40 diluent (PBS pH 7·5, 2 % BSA, 10 % normal goat serum containing 0·3 % NP-40) at a final dilution of 1 : 50. Biotinylated goat anti-mouse IgG{gamma} (Kirkegaard & Perry Laboratories) and strepavidin–horseradish peroxidase (SA-HRP) (Kirkegaard & Perry Laboratories) were added sequentially. One hundred microlitres per well TMB micro-well peroxidase substrate was used to develop the colour and 100 µl per well peroxidase stop solution (Kirkegaard & Perry Laboratories) was added to stop the reaction. Absorbance was read at 450 nm. The IgG titre was determined by end-point dilution. The IgG subtype was determined by the ELISA assay described above except 1 : 1000 diluted biotinylated rat anti-mouse IgG1 or biotinylated rat anti-mouse IgG2a (both from BD Pharmingen) was used as second antibody. IgG1 and IgG2a concentration (ng ml–1) was calibrated against a standard curve using purified mouse IgG1{kappa} or IgG2a{kappa} (2·44–1250 ng, both from BD Pharmingen) as standards, respectively.

ELISPOT assay.
The number of IL-2-, IL-4-, IL-5-, IL-10-, IL-12- and IFN-{gamma}-secreting cells was determined by ELISPOT (Shevach, 1994; Jiao et al., 2003) from the spleen cells of mice immunized with different immunogens including free rNS3, the mixture of rNS3 and CpG ODN, cationic liposomes encapsulating rNS3, cationic liposomes co-encapsulating rNS3 and CpG ODN and liposomes co-encapsulating rNS3 and GpC while under the stimulation of rNS3 protein in vitro. Spleen cells from immunized mice were separated by Ficoll–Paque, the concentration adjusted to 4x106 cells ml–1, and then the cells were cultured with rNS3 protein at 3 µg ml–1 in a 24-well plate. Filtration plates (96-well) with 0·45 µm surfactant-free mixed cellulose ester membrane, type MAHA S45 (Millipore), were coated with purified anti-cytokine antibody [IL-2, IL-4, IL-5, IL-10, IL-12 or IFN-{gamma} (BD Pharmingen)] at a concentration of 10 µg ml–1 in 20 mM borate buffer (pH 8·4), incubated overnight at room temperature and then overcoated with 5 % BSA in PBS. At day 2 or day 4, 4x105 spleen cells in a 100 µl volume were added to each well and incubated with antigen for another 24 h to allow production and capture of the released cytokines. All the determinations were run in duplicate. After incubation, cells were removed by washing six times with PBS containing 0·05 % NP-40 and twice with distilled water. One microgram ml–1 of biotin-labelled anti-cytokine monoclonal antibody (BD Pharmingen) was added followed by 1 : 4000 diluted SA-HRP for antibody detection. Opti-4CN peroxidase substrate (Bio-Rad) was used to develop the spots. The spots were counted automatically using an ELISPOT reader (Carl Zeiss Vision). The frequency of antigen-specific cytokine secreted cells was defined as the frequency of the cytokine-secreting cells in the mice immunized with plasmid encoding antigen minus the frequency in mice immunized with control plasmid.

Statistical analysis.
Statistical significance was determined with the Mann–Whitney Rank-Sum test in the experiments to determine the titre of anti-HCV/NS3 IgG, with Fisher's exact test in analysis of the ratio based on individual mice and with Student's t-test in ELISPOT experiments.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Anti-HCV NS3 IgG responses to protein immunization in different formulations
The titres of anti-NS3 IgG from the mice 4 weeks after immunization with free recombinant HCV NS3 protein (rNS3), liposomes encapsulating rNS3 (liposome-rNS3), rNS3 and CpG ODN mixture (rNS3+CpG), liposomes co-encapsulating rNS3 and CpG ODN (liposome-rNS3-CpG) or liposomes co-encapsulating rNS3 and GpC ODN (liposome-rNS3-GpC) are shown in Table 1. All of the five mice immunized with rNS3, four of the five mice immunized with liposome-rNS3, and three of the five mice immunized with rNS3+CpG developed anti-NS3 IgG but the titres were relatively low. In contrast, all animals immunized with liposome-NS3-CpG produced strong antibody responses. The mean titre of NS3-specific IgG in the mice immunized with liposome-rNS3-CpG was 675 times higher than the mean titre of the mice immunized with naked rNS3. As a control, GpC ODN, a nucleotide with reversed base alignment, was used in place of CpG ODN. Anti-NS3 IgG developed in all five mice immunized with liposome-rNS3-GpC but the mean titre, although 147 times higher than rNS3 immunized mice, was only one-fifth of the mean titre in the mice immunized with liposome-rNS3-CpG.


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Table 1. Titres of anti-HCV NS3 IgG in mice immunized with various formulations of recombinant HCV NS3 proteins

The titre of anti-NS3 IgG in sera of mice immunized with free- or liposome-encapsulated pCI-HCV/NS3-4748 was determined by end-point dilution. Sera were collected 4 weeks after immunization. The cut-off for positive results was defined as the mean A450 adding 2 SD of control mice sera at 1 : 50 dilution.

 
The onset of anti-HCV/NS3 IgG in the mice immunized with different NS3 formulations varied (Fig. 1). In mice immunized with rNS3 or the mixture of rNS3 and CpG ODN, the onset of anti-HCV/NS3 IgG was at week two. NS3 in both of these formulations was free form. In liposome-rNS3-, liposome-rNS3-CpG- or liposome-rNS3-GpC-immunized mice, the onset of anti-HCV/NS3 IgG was delayed until week three.



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Fig. 1. Anti-NS3 IgG responses induced by immunization with different formulations of recombinant HCV NS3 protein. The mice were immunized by direct i.m. injections. All of the sera were tested at 1 : 50 dilution. (A) Anti-NS3 IgG response in mice immunized with the formulations containing free recombinant NS3. (B) Response induced by the formulations containing cationic liposomes. Each data point shown is a geometric mean of five mice. The figure demonstrates that the onset of anti-HCV/NS3 IgG in mice immunized with the formulation containing cationic liposomes occurred 1 week later than in mice immunized with the formulation without liposomes.

 
Anti-HCV/NS3 IgG subtype switch induced by protein immunization in different formulations
Anti-HCV/NS3 IgG1 and IgG2a responses were measured in all immunized mice (Table 2). Naked rNS3 protein induced low levels of both IgG1 and IgG2a. The ratio of IgG1 : IgG2a was 7·2, indicating that the predominant IgG isotope was IgG1, as previously reported (Jiao et al., 2004). This suggests that the immune responses induced by free rNS3 protein immunization favour the Th2 pathway.


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Table 2. Anti-HCV NS3 specific IgG subtypes generated in mice by immunization with various formulations of recombinant HCV NS3 proteins

Sera were collected 4 weeks after immunization. The IgG subtype was determined by ELISA with subtype-specific rat anti-mouse secondary antibodies. IgG1 or IgG2a concentration (ng ml–1) was calibrated against a standard curve using purified mouse IgG1{kappa} or IgG2a{kappa} as standards, respectively, and expressed as geometric means. Preferential Th1 response in mice immunized with various formulated recombinant HCV NS3 proteins indicating by specific IgG subtypes of anti-HCV NS3.

 
In mice immunized with liposome-rNS3, IgG1 production was almost the same as in naked rNS3-immunized mice, but IgG2a production was sevenfold that in mice immunized with rNS3 alone. The IgG1 : IgG2a ratio was 1·0, indicating that, compared with free-protein immunization, cationic liposome-mediated protein immunization had the tendency to improve the Th1/Th2 balance in favour of the Th1 pathway (P=0·002).

In mice immunized with the mixture of rNS3 and CpG, IgG1 production was only about one-fifth of that produced in the mice immunized with rHCV alone, while IgG2a production was twofold. The IgG1 : IgG2a ratio was 0·76. This result suggests that CpG ODN, like cationic liposomes, has the potential to improve the Th1/Th2 balance induced by protein immunization (P=0·003).

Although cationic liposomes or CpG ODN improved the Th1/Th2 balance, both failed to enhance the immune response induced by protein immunization (Fig. 1, Table 2). In contrast, in mice immunized with liposome-rNS3-CpG, the production of IgG1 and IgG2a was over 250- and 3000-fold higher, respectively, than in mice immunized with rNS3 alone and the ratio of IgG1 : IgG2a was reduced to 0·43. Thus, compared with free-rNS3 immunization, the isotype of IgG in liposome-rNS3-CpG-immunized mice was switched from IgG1 to IgG2a (P<0·001), and the immune response was markedly enhanced.

As a control, nucleotides C and G in CpG ODN were replaced with nucleotides G and C in GpC ODN. In mice immunized with liposome-NS3-GpC, IgG1 production was 220-fold that in mice immunized with rNS3 alone, but lower than in mice immunized with liposome-NS3-CpG. Similarly, IgG2a production was 180-fold of that in mice immunized with rNS3 alone, but only one-twentieth of that in mice immunized with liposome-rNS3-CpG. The ratio of IgG1 : IgG2a was 8·6. Compared with free-rNS3 protein, liposome-rNS3-GpC induced stronger immune responses, but unlike liposome-rNS3-CpG, the IgG isotope in liposome-rNS3-GpC immunized mice was still predominantly IgG1 and the immune response favoured the Th2 pathway.

Analysis based upon individual mice provided similar results (Table 3). In mice immunized with free-rNS3 protein, 10 out of 10 were IgG1 predominant, demonstrating again that the immune responses induced by protein immunization were of the Th2 type. In liposome-rNS3-immunized mice, 5 out of 10 were IgG1 dominant, 4 out of 10 were IgG2a dominant and 1 out of 10 was a non-responder. In mice immunized with the mixture of rNS3 and CpG, 1 out of 5 was IgG1 dominant, 2 out of 5 were IgG2a dominant and 2 out of 5 were non-responders. Thus, both liposome-rNS3 and a mixture of rNS3 and CpG ODN have some ability to switch IgG isotope from IgG1 to IgG2a, but compared to free protein immunization, the differences were not significant (P=0·097 and 0·179, respectively). The effects of the liposome-rNS3 or rNS3+CpG were also not optimal because of several non-responders. In contrast, mice immunized with liposomes encapsulating recombinant protein and CpG (liposome-rNS3-CpG), all were responders and 9 out of 10 were IgG2a dominant. The proportion of IgG2a dominant mice was significantly higher than free-protein-immunized mice (P<0·001) and immunization with liposome-rNS3-CpG almost uniformly induced Th1 type immune responses. In the control experiment, 4 out of 5 mice immunized with liposome-rNS3-GpC were IgG1 dominant and 1 out of 5 was IgG2a dominant. This was not significantly different from free-protein-immunized mice, but was significantly different from liposome-rNS3-CpG-immunized mice (P<0·033), indicating that the switch of the immune response by immune-modulating oligonucleotides is sequence dependent.


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Table 3. Switch of anti-HCV NS3-specific IgG subtype induced by different recombinant HCV NS3 formulations based on the analysis of individual mice

Sera were collected 4 weeks after immunization. The IgG subtype was determined by ELISA. The IgG1 or IgG2a concentration (ng ml–1) was calibrated against a standard curve using purified mouse IgG1{kappa} or IgG2a{kappa} as standards, respectively. (IgG1 : IgG2a ratio >1 is considered as IgG1 dominant and <1 is considered IgG2a dominant.)

 
Cytokine profiles in mice immunized with recombinant HCV NS3 in various formulations
Using the ELISPOT technique, cells secreting either Th1 cytokines, IFN-{gamma} and IL-2, or Th2 type cytokines, IL-4 and IL-5, were detected 5 days after stimulation with recombinant HCV/NS3 proteins. The frequency of spot-forming cells (FSFC) was expressed as the spot number per 400 000 cells. As shown in Fig. 2(A), the FSFC of IFN-{gamma} in liposome-NS3-CpG-immunized mice was significantly higher than that of the mice immunized with liposome-NS3 (P=0·034). The FSFC secreting IL-2 (Fig. 2B) in mice immunized with liposome-NS3-CpG was significantly higher than that in mice immunized with either liposome-NS3 (P<0·0001) or with free-rNS3 (P<0·0001). There was no statistically significant difference of FSFC of IL-2 between liposome-NS3-immunized mice and free-NS3-immunized mice (P=0·45).



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Fig. 2. (A) Numbers of IFN-{gamma}-, (B) IL-2-, (C) IL-4- and (D) IL-5-spot-forming cells (SFC) in response to 5 days stimulation with recombinant HCV NS3 protein (3 µg ml–1) from mice immunized with recombinant HCV NS3 protein in different formulations. The mice were immunized by direct i.m. injections. The numbers of SFC were obtained from 4x105 spleen cells. The results showed that liposome-CpG-NS3 induced the strongest Th1 immune response. The number of the sample in each group was rNS3, 8; liposome-NS3, 10; rNS3+CpG, 10; liposome-NS3-CpG, 10.

 
The FSFC of Th2 cytokine, IL-4, of mice immunized with either liposome-NS3-CpG or liposome-NS3 was lower than that of mice immunized with naked rNS3 (P<0·1 and 0·01, respectively) (Fig. 2C). Another Th2 cytokine, IL-5, also showed a higher FCSC in free-rNS3-immunized mice as compared with either of the liposome formulations (Fig. 2D).

The spleens from mice immunized with a mixture of rNS3 and CpG ODN did not induce any significant number of SFC for the cytokines studied in this experiment.

Mice immunized with either liposome-NS3-CpG or liposome-NS3 produced 1·4-fold more cells secreting IFN-{gamma} than were secreting IL-4. In contrast, in mice immunized with free-NS3, the cells secreting IFN-{gamma} were only one-sixteenth of the cells secreting IL-4, indicating that liposomes encapsulating NS3 alone or with CpG have the ability to switch the immune response against HCV NS3 from the Th2 to the Th1 pathway.

The FSFC for IL-12 in all immunized mice is shown in Fig. 3. The FSFC for IL-12 in mice immunized with liposome-NS3-CpG was significantly higher than in mice immunized with all other formulations (versus liposome-NS3, P<0·001; mixture of CpG and NS3, P<0·001; free-NS3, P<0·01). Thus, IL-12 secretion was also induced by cationic liposomes encapsulating CpG ODN immunization.



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Fig. 3. IL-12-related spot-forming cells in response to stimulation with recombinant HCV NS3 protein (3 µg ml–1) in mice immunized with recombinant HCV NS3 protein in various formulations. The results indicate that immunization with cationic liposomes co-encapsulating recombinant HCV NS3 protein and CpG ODN can induce a much stronger non-specific IL-12 secretion than other formulations of NS3, including liposome-NS3, mixture of NS3 and CpG ODN as well as free-NS3. The number of the sample in each group was the same as Fig. 2.

 

   DISCUSSION
Top
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
A specific Th1 immune response against HCV NS3 is very important in overcoming HCV infection, so the development of a vaccine directed against HCV NS3 has attracted considerable interest (Encke et al., 1998; Cho et al., 1999; Gorden et al., 2000; Lazdina et al., 2001; Wedemeyer et al., 2001; Brinster et al., 2001, 2002; Arribillaga et al., 2002; Frelin et al., 2003; Simon et al., 2003; Jiao et al., 2003, 2004). CpG ODN is an excellent immune adjuvant in various murine disease models and can drive Th1 immune responses (Chu et al., 1997; Weiner et al., 1997; Zimmermann et al., 1998; Moldoveanu et al., 1998; Davis et al., 1998; Brazolot-Millan et al., 1998). Several studies indicated that the adjuvant activities of CpG ODN are enhanced by its close physical association with the immunizing antigens (Weiner et al., 1997; Sun et al., 1998a). It was also demonstrated that the direct association of CpG ODN to a protein antigen, either via biotin–avidin linkage or covalent linkage, could enhance further the immune responses (Klinman et al., 1999; Shirota et al., 2000; Tighe et al., 2000). Liposomes could be used as an alternative method to co-deliver CpG ODN and antigens. It has been shown that liposomes co-encapsulating CpG ODN with the selected antigen could enhance the immune responses against T-independent antigen (Li et al., 2002). Previous studies in this laboratory also showed that cationic liposomes encapsulating DNA immunization enhanced and modulated the immune responses against HCV NS3 (Jiao et al., 2003). This study expands this earlier work to examine the effect of CpG and cationic lipid encapsulation on the immunogenecity of recombinant NS3. Immune modulation of NS3 could enhance either primary immunization with this recombinant protein or secondary immunization in a prime-boost strategy involving DNA vaccination in the initial stage. We showed that liposomes co-encapsulating rNS3 and CpG ODN strongly enhanced both humoral and cellular immune responses against HCV NS3. Compared with free rHCV NS3, rHCV NS3 encapsulated with liposome alone or rHCV NS3 mixed with CpG ODN alone, liposome-rNS3-CpG induced a significantly greater Th1-biased immunity as evidenced by the IgG1 : IgG2a ratio and Th1 cytokine production.

Liposomes protect the entrapped protein and CpG ODN from extracellular degradation. They also protect the proteins from neutralization by circulating antibodies and maintain the sustained release of the liposome-associated protein at the injection site (depot effect). In our experiments, the onset of anti-HCV/NS3 IgG in the mice immunized with any of three formulations containing liposomes encapsulating rNS3 proteins was 1 week later than in the mice immunized with the free form of NS3 proteins. This suggested that liposome encapsulation could delay the release of the entrapped contents, thus prolonging the time of stimulation.

The immunostimulatory activity of CpG ODN requires cellular uptake by endocytosis (Krieg, 2000) following their binding to a cell receptor belonging to the Toll-like receptor family, TLR9 (Hemmi et al., 2000). It has also been reported that CpG can guide tagged antigen specifically to dendritic cells (Shirota et al., 2001). CpG ODN carries net negative electric charges and has a strong electrostatic attraction to the membrane surface of cationic liposomes. It is inevitable for the binding of CpG to the outer surface during the formation of liposomes co-encapsulating rNS3 and CpG ODN, which provides a mechanism for liposome-rNS3-CpG specifically binding to dendritic cells.

In our previous study, we found that multiple plasmids encapsulated in cationic liposomes, with or without specific inserts, induced strong IL-12 secretion in immunized mice (Jiao et al., 2003). It has been reported that cationic lipid–DNA complexes possess potent antitumor effects related to innate immune responses following intravenous administration (Dow et al., 1999; Whitmore et al., 1999; Lanuti et al., 2000). The cationic liposomes by themselves or the plasmid DNA alone were without apparent immune stimulatory activity at the low doses employed in these studies. It has been postulated that enhancement of IL-12 secretion is due in part to the increased purine content of plasmids, which are from bacteria and enriched in CpG motifs relative to eukaryotic DNA (Klinman et al., 1996; Pisetsky, 1996; Krieg, 1996; Sun et al., 1998b). Our results show that the liposome-NS3-CpG complex induced significantly higher IL-12 secretion than other formulations, probably attributable to the CpG motif. IL-12 plays an important role in the differentiation of Th0 cells to Th1 cells at the site of antigen encounter (Kourilsky & Truffa-Bachi, 2001); these Th0 cells differentiate to Th1 cells under the stimulation of IL-12 and are induced to secret IFN-{gamma} in the presence of antigen.

The combination of antigen, CpG and cationic liposome encapsulation appears very effective in the murine model, fulfilling the goal of directing strong humoral and cell-mediated immune responses along the Th1 pathway. Although this appears to be a viable approach in the mouse model, one must caution that murine immunization is not always fully predictive of immune responses in higher primates. Thus, this vaccine strategy must be tested further in the chimpanzee, the only reliable non-human primate model for the study of HCV infectivity and protectivity. It will be necessary to demonstrate in the chimpanzee not only that CpG and cationic liposome encapsulation induce similar strong Th1 immune responses, but also that the increased quantity of liposomes required to deliver sufficient antigen for immunization of larger subjects is safe and well tolerated.

In summary, we have demonstrated that cationic liposomes co-encapsulating recombinant HCV NS3 together with CpG ODN can induce strong immune responses against HCV NS3 and that this formulation has the ability to switch the immune response from the Th2 to the Th1 pathway. The ability to switch the immune response pathway depends on both the immunomodulatory effects of CpG oligonuleotides and the ability of liposomes to co-deliver the antigen and immunomodulator to the same cells. We propose that the advantage of cationic liposomes is that their cationic outer surface can bind larger numbers of CpG oligonucleotides through electrostatic interaction and that the bound CpG in turn promotes the capture of the cationic liposome by APCs. We also suggest that cationic liposomes co-encapsulating recombinant HCV NS3 and CpG ODN induce IL-12 secretion, necessary for the differentiation of Th0 cells to Th1 cells. We conclude that the co-delivery of recombinant HCV NS3 protein and CpG ODN is a viable HCV vaccine strategy that should be the tested further in the chimpanzee model either as the primary immunogen or as a protein-boost following a liposome-encapsulated DNA prime. At least, this strategy could be used as a model and basis to develop an effective HCV vaccine in the future.


   REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 11 December 2003; accepted 3 February 2004.



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