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
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ABSTRACT |
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INTRODUCTION |
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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-
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 12511259 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 biotinavidin 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 liposomeDNA 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.
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METHODS |
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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 dehydrationrehydration 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 lipidDNA 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 ml1 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 (Kirkegaard & Perry Laboratories) and strepavidinhorseradish 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 ml1) was calibrated against a standard curve using purified mouse IgG1
or IgG2a
(2·441250 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--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 FicollPaque, the concentration adjusted to 4x106 cells ml1, and then the cells were cultured with rNS3 protein at 3 µg ml1 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-
(BD Pharmingen)] at a concentration of 10 µg ml1 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 ml1 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 MannWhitney 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.
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RESULTS |
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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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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- than were secreting IL-4. In contrast, in mice immunized with free-NS3, the cells secreting IFN-
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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DISCUSSION |
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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 lipidDNA 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-
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.
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Received 11 December 2003;
accepted 3 February 2004.
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