Comparison of Antibody-and Cell-Mediated Immune Responses After

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topes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did ... strong cell-mediated response but a weak humoral re- sponse (7 ...



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VIRAL IMMUNOLOGY Volume 18, Number 4, 2005 © Mary Ann Liebert, Inc. Pp. 637–648

Comparison of Antibody- and Cell-Mediated Immune Responses After Intramuscular Hepatitis C Immunizations of BALB/c Mice M. GHORBANI,1 T. NASS,1 A. AZIZI,1 C. SOARE,1 S. AUCOIN,1 A. GIULIVI,2 D.E. ANDERSON,3 and F. DIAZ-MITOMA1

ABSTRACT Current treatments for hepatitis C infection have limited efficacy, and there is no vaccine available. The goal of this study was to compare the immune response to several immunization combinations against hepatitis C virus (HCV). Six groups of mice were immunized at weeks 0, 4, and 8 with different combinations of a candidate HCV vaccine consisting of 100 g recombinant HCV core/E1/E2 (rHCV) DNA plasmid and/or 25 g rHCV polyprotein and 50 L Montanide ISA51. Four weeks after the last injection, all groups of mice were sacrificed and blood samples and spleens were collected for measuring the levels of specific HCV antibodies (total IgG, IgG1, and IgG2a). Cell proliferation and intracellular interferon- were also measured. Among the groups of immunized mice, only the mice immunized with rHCV DNA plasmid, rHCV polyprotein, and montanide (group D) and mice immunized with rHCV polyprotein and montanide (group F) demonstrated a significant increase in the total IgG titer after immunization. IgG1 was the predominant antibody detected in both groups D and F. No IgG2a was detected in any of the groups. Proliferation assays demonstrated that splenocytes from group D and group C (rHCV DNA primed/rHCV polyprotein boost) developed significant anti-HCV proliferative responses. The combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high antibody titer with a predominance of IgG1 antibodies and recognized the major neutralization epitopes in HVR1. In contrast, group C did not show an increase in anti-HCV antibodies, but did show a proliferative response.



EPATITIS C VIRUS (HCV) is a positive, singlestranded 9.5-kb RNA virus that encodes a single polyprotein that is processed by cellular and viral proteases. The viral genome encodes three structural proteins and seven regulatory or nonstructural proteins.

There are six strains of HCV (genotypes), which are classified by number and letter (e.g., HCV 1a, HCV 2b). Genotypes 1a and 1b are the most common genotypes, affecting approximately 70–80% of infected individuals in the United States (6), 66% in China (31), and 58% in Canada (2). More than 170 million individuals worldwide are infected with HCV. Those infected develop chronic


of Virology, Children’s Hospital of Eastern Ontario and University of Ottawa, Ottawa, Ontario, Canada. of Blood Borne Pathogens, Health Canada, Ottawa, Ontario, Canada. 3Neurology Associates, School of Medicine, Harvard University, Boston, Massachusetts. 2Division




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638 HCV infection or non-A, non-B hepatitis (8), and a portion will develop cirrhosis, liver failure, or hepatocellular carcinoma (14,34). Current attempts to treat HCV are limited to the use of interferon- (IFN-) in combination with ribavirin (33). This treatment clears HCV RNA in 50% of cases (32,40,41), necessitating the development of a novel treatment strategy. An effective preventive or therapeutic HCV vaccine will likely require the simultaneous induction of robust humoral and cellular immune responses. However, there are many obstacles in the development of an HCV vaccine. Research efforts are limited by a lack of readily available animal models. The only reliable model is the chimpanzee. Its use is restricted, however, by high cost and by the fact that the chimpanzee is an endangered species. In addition, viral variability is a complicating factor; and although only six HCV genotypes exist, multiple quasispecies may evolve after infection. Despite these obstacles, efforts to develop an effective vaccine continue. Use of purified recombinant proteins (17) and naked DNA–based compounds (18,51,53) seem to be the most promising approaches for the development of an HCV vaccine candidate. DNA vaccines are particularly interesting in that they express antigens intracellularly and induce effective cellular immune responses (58). The HCV core protein (20 kDa) has well-defined human B cell, T helper cell, and CTL epitopes. Immunization with an HCV core protein–based DNA vaccine appears to induce a strong class I–binding peptide delayed type hypersensitivity (DTH) response in mice (1). Because of its conserved nature across viral genotypes, the core protein is a promising candidate antigen for vaccine development, as it can induce a broad immune response. Plasmids encoding core protein are known to induce a strong cell-mediated response but a weak humoral response (7,28). Core protein is a nucleocapsid protein and thus is not secreted from cells. As such, it is a poor inducer of antibodies, which limits its use in vaccine de-

TABLE 1. Group A B C D E F

velopment. However, the immunogenecity of this protein can increase by using either a stronger adjuvant or different routes and forms of immunization. Other structural proteins include the envelope 1 (E1 33 kDa) and envelope 2 (E2 72 kDa), which contain the ligand for the cellular receptor, CD81, and the human scavenger receptor class B type I (3,10,19,45). An effective antibody response to viral envelope proteins may be important in protecting against infection by preventing virus from binding to its cellular receptor. The Hypervariable Region 1 (HVR1) is a 22-aa sequence that is the main recognition site for epitopes that induce neutralizing antibodies (13). The immunization of chimpanzees with a DNA vaccine encoding E2 protein suggested that it was capable of inducing anti-HCV immune responses that could potentially prevent the progression of disease (15,16). DNA immunization has also yielded disappointing results in some models (23). It seems that DNA alone may not be an efficient immunogen for the induction of humoral immune responses (43). Prime boost immunization, consisting of DNA priming followed by boosting with protein or with a viral vector encoding the recombinant viral product, has been used successfully in several disease models including hepatitis B and C, human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and malaria (18,21,30,46). In general, priming with DNA and boosting with protein or a viral vector encoding the recombinant viral product induces good humoral and cellular immunity. In light of these findings in our study, we compared a DNA prime/protein and adjuvant boost immunization strategies to one involving the combined use of DNA, protein, and adjuvant for both priming and boosting (Table 1). The adjuvant of choice in this study was montanide ISA-51, a mineral oil–based adjuvant from Seppic Co. (Fairfield, NJ) that can enhance the protective efficacy of a subunit vaccine (24,27). Our studies performed in BALB/c mice demonstrated that priming and boosting with a combination of DNA,





Boost twice

Empty pVAX1 rHCV DNA alone rHCV DNA alone Combined vaccine consisting of rHCV DNA,rHCV polyprotein, and montanide rHCV DNA and montanide rHCV polyprotein and montanide

Empty pVAX1 rHCV DNA alone rHCV polyprotein and montanide Combined vaccine consisting of rHCV DNA, rHCV polyprotein, and the montanide rHCV DNA and montanide rHCV polyprotein and montanide

aBALB/C mice 6–8 weeks old were vaccinated at weeks 0,4, and 8. Plasma was taken 1 day before vaccination for antibody titer analysis by enzyme-linked immunoassay. rHCV, recombinant hepatitis C virus core/E1/E2.



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VACCINE DEVELOPMENTS FOR HEPATITIS C protein, and adjuvant induced a stronger systemic humoral response than priming with DNA followed by boosting with protein and adjuvant. Specifically, the recombinant HCV core/E1/E2 (rHCV) DNA plasmid prime, followed by the rHCV polyprotein and montanide boost regimen, failed to induce a high HCV antibody titer (group C mice). However, the combination of an rHCV DNA plasmid, rHCV polyprotein, and montanide induced a high HCV-specific antibody titer with a predominance of IgG1 (group D mice), suggesting that this immunization strategy may be effective in protecting against HCV infection.

MATERIALS AND METHODS Animals. Male BALB/c were purchased from Charles River Canada (St. Constant, PQ, Canada) at 6–8 weeks of age. The mice were divided into six groups of seven animals each and were placed in appropriate cages in an animal care facility. An independent animal care ethics review board approved the animal study protocol. Mice were immunized intramuscularly (i.m.) in the quadriceps muscle with 100 L of immunogens at weeks 0, 4, and 8. Four weeks after the last injection, all groups of mice were sacrificed and the plasma samples and spleens were collected. Before to each immunization, blood was collected from the femoral vein or directly from the heart and placed in tubes containing heparin. After centrifugation, the plasma was collected and stored at 80°C for further testing. Spleens were collected in ice-cold phosphate-buffered saline (PBS; pH 7.4), mechanically disrupted with a sterile plastic pipette, and washed through a 100-m nylon cell strainer (Becton Dickinson, Franklin Lake, NJ). The isolated cells were then washed with PBS, re-strained, and re-suspended at a concentration of 2  106 cells/mL in Eagle’s minimal essential medium (IMDM) supplemented with 10% fetal calf serum (FCS), 100 U/mL penicillin, 100 g/mL gentamycin, 10 mmol/L HEPES, and 2 mmol/L glutamine. Cells were cultured immediately or frozen in a solution of 90% FCS/10% DMSO and stored in liquid nitrogen until used. Construction of plasmid DNA. Total RNA was extracted from the plasma of a patient infected with HCV genotype 1b. The RNA was used as a template to amplify Core, E1, and E2 genes. The HCV fragment containing Core, E1, and truncated E2 genes encoded for amino acid residues 1-683 (2049 nucleotides) was constructed by reverse transcription–polymerase chain reaction (RT-PCR) using forward primer 5 ACC ATG AGC ACG AAT CCT AAA CCTC 3 and reverse primer 5 TGG TAG GGT TGT GAA GGA ACA CG 3. The amplified fragment was cloned into the EcoR1 sites of pCR 2.1 vector using the TOPO-TA cloning kit (Invitrogen, Burlington, ON).

639 The nucleotide sequence was verified by DNA sequencing using the University of Ottawa DNA sequencing facility. White colonies containing the insert were picked and grown in LB medium (30 g tryptone, 15 g yeast extract, 15 g NaCl, ph 7.4) for 18 h, subjected to plasmid purification, digested with EcoRI restriction enzyme, and electrophoresed using a 1% agarose gel. A 2.1-kb band corresponding to the core/E1/E2 HCV genes was excised from the gel and extracted using a gel extraction kit (Qiagen, Mississauga, ON, Canada). The DNA fragment was then subcloned into pVAX-1 plasmid (Invitrogen, Burlington, ON, Canada), which was used for transformation of the DH5- cell line (Gibco-BRL, Burlington, ON, Canada). The transformed cells were then incubated on LB-agar plates and the colonies were picked the next day and grown in LB medium. The cells were pelleted by centrifugation and subjected to plasmid purification followed by digestion with several restriction enzymes. The cloned insert with the correct orientation was subjected to sequence analysis for further confirmation of its HCV origin. Bacteria containing the recombinant plasmid were incubated in 500 mL LB medium. The plasmid DNA was purified using a Qiagen Endofree Plasmid Mega Kit (Burlington, ON, Canada) and used to immunize mice. Expression of recombinant HCV polyprotein. To express and purify the recombinant HCV core/E1/E2 polyprotein, the TOPO-TA HCVcore/E1/E2 construct was subcloned into the pEF6/Myc-His expression vector (Invitrogen Burlington, ON); this vector contains six histidine residues and would ultimately allow purification of the HCV polyprotein by immobilized metal affinity chromatography (Clontech Talon Metal Affinity Resin Kit, Palo Alto, CA). The recombinant plasmid containing the correctly oriented insert was transfected into DH5 cells, amplified, and purified using the Endofree purification kit (Qiagen) as previously described. Chinese hamster ovary (CHO) cells were transiently transfected with the recombinant pEF6/Myc-His vector containing the core/E1/E2 insert. Transfection was performed by 2 electroporation shocks at 1.4–1.6 KV using an electrophoration apparatus (BTX Inc., San Diego, CA). The transfected cells were incubated in IMDM (Sigma-Aldrich, St. Louis, MO) containing 10% FCS (Life Technologies Laboratories, Grand Island, NY) and 50 g/mL penicillin-gentamicin. At 65 h after transfection the cells were harvested, lysed in lysis buffer (25 mmol/L Tris base, 2.5 mmol/L mercaptoethanol, and 1% Triton-X100), sonicated, and subjected to protein purification using the Talon affinity resin kit as described before. The purity of the protein was checked by mass spectrophotometery, and protein with 85% purity was used for immunization. Human sera. Human sera were obtained from healthy adult volunteers and HCV-positive individuals after ap-



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640 proval of the protocol by the Ethics Review Committee of the Children’s Hospital of Eastern Ontario (Ottawa, ON, Canada). HCV-positive patients were previously tested by the IMX System ELISA kit (Abbott, Weisbaden, Germany) for the presence of HCV-specific antibodies. HCV-seropositive patients who also had a positive RNA test in sera were tested for the specific genotype by the Inno-Lipa immunoassay. One of these sera was used as a template for the amplification of genotype 1b for the purpose of cloning and expression of the viral structural proteins, as previously described. Several genotype-specific sera were used to determine whether human antibodies were able to recognize the immunogens used in this study. O.D. values at least two standard deviations above the mean O.D. for the HCV-negative sera were considered positive for the presence of cross reactive antibodies to genotype 1b. Immunization. Mice were immunized at weeks 0, 4 and 8 via intramuscular (IM) injection in the quadriceps major with 100 g of the recombinant HCV core/E1/E2 DNA vaccine and/or 25 g recombinant HCV core/ E1/E2 polyprotein in PBS solution and 50 L Montanide ISA-51 (Seppic Inc., Fairfield, NJ) as an adjuvant; the total vaccine volume was 100 L. Six groups of mice were immunized as follows: group A (the negative control group) was immunized with pVAX1 expression plasmid not containing the HCV recombinant polyprotein gene insert; group B was immunized with the rHCV DNA plasmid; group C was primed with rHCV DNA plasmid and boosted twice with rHCV polyprotein and adjuvant; group D was primed with rHCV DNA plasmid, rHCV polyprotein and adjuvant and boosted twice with the same preparation; group E was primed with rHCV DNA plasmid and adjuvant and boosted twice with the same preparation; group F was primed with rHCV polyprotein and adjuvant and boosted twice with the same preparation (Table 1). Immunofluorecent microscopy. CHO cells expressing CoreE1E2 protein were grown on chamber slides for 5 h, washed twice with PBS and fixed with ice-cold methanol at 20°C for 10 min. Cells were washed with PBS 3 times for 10 min each at room temperature and were permeabilized with acetone for 15 min at 4°C. Cells were washed again 3 times for 10 min, blocked with 3% bovine serum albumin (BSA) in PBS for 1 h, and incubated with rabbit anti-CoreE1E2 antibody (1:100) in blocking buffer for 1 h at room temperature. Cells were washed 3 times with PBS and incubated with FITC-conjugated goat anti-rabbit (BioRad, Hercules, CA) diluted 1:500 in blocking buffer for 1 h at room temperature. Cells were then washed 3–4 times for 10 min each with PBS, mounted with mounting media, covered with cover slips, and sealed with nail polish.

GHORBANI ET AL. ELISA measurements to determine concentrations of antibodies specific for recombinant core/E1/E2 polyprotein and HVR1. Serum levels of hepatitis C–specific antibodies were measured using the HCV recombinant core/E1/E2 polyprotein as a capture molecule and a mouse-specific monoclonal antibody–horseradish peroxidase (HRP) conjugate detection system. EIA/RIA Strip Well TM plates (Corning CoStar Inc., New York, NY) were coated for 4 h at 37°C with 20 g/mL recombinant core/E1/E2 polyprotein dissolved in sterile distilled/deionized water and blocked overnight at 4°C with 1% BSA (SigmaAldrich, St. Louis, MO) in PBS. Dilutions of sera were incubated for 2 h at 37°C and antibodies were detected with a 1/1000 dilution in 1% BSA/PBS of the required goat anti-species–specific HRP conjugate (IgG H+L: Jackson Immunoresearch Laboratories, West Grove, PA; IgG1, IgG2a: Serotec, Oxford, UK). After each incubation, the plates were washed six times with PBS/0.05% Tween-20 (Sigma-Aldrich). O-phenylenediamine dihydrochloride (Sigma-Aldrich) and hydrogen peroxide were used to develop the color reaction. The OD was read at 490 nm after the reaction was stopped with 1 N HCL. An IgG2a monoclonal antibody specific for core protein aa 1-120 (Clone 0126, Biogenesis Ltd., Poole, England) and hepatitis C–negative or pre-immune sera were run in parallel, with all samples tested as negative control. Optical density (OD) values at least 2 standard deviations above the mean OD of the pre-immunization sera were considered positive for an HCV-antibody response. Cell proliferation and cytokine release assays. After the third vaccination, mice were anesthetized with 50 L Somnotal (MTC Pharmaceuticals, Cambridge, ON, Canada), sacrificed, and blood and spleens were collected. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by centrifugation on a density gradient and were washed and cultured in IMDM culture medium containing 100 U/ml penicillin, 100 mg/mL streptomycin and 10% heat-inactivated FCS. Spleen cells were harvested by rubbing the spleen over a Falcon Cell Strainer (100 m Nylon). Splenocytes were washed twice with PBS and cultured in triplicate using 96-well, round-bottom plates at 106 cells/well in 200 L RPMI #1640 (Gibco-BRL, Burlington, ON, Canada) containing 10% FCS, 50 mol/L -mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin. Cells were stimulated with recombinant core/E1/E2 polyprotein at a concentration of 10 g/mL. After 72 h, 100 L of supernatant was collected from each well for enzymelinked immunoassay (ELISA) analysis of interferon- (IFN-) and interleukin-5 (IL-5) (Pharmingen Inc, San Diego, CA) on standard ELISA plates (Costar, Corning Incorporated, Corning, NY); this volume was replaced



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VACCINE DEVELOPMENTS FOR HEPATITIS C with the appropriate culture media containing [3H]thymidine (1 Ci/well). Cells were incubated for an additional 18 h, and [3H]thymidine incorporation into DNA was measured after harvesting cells onto glass fiber filtermats (Wallac Oy, Turku, Finland) using a Wallace Microbeta  counter (model 1450; Wallac Oy, Turku, Finland). Results are expressed as [3H]thymidine incorporation count per minute (CPM). Western blot analysis. Purified protein extract was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to High bond-C Nitrocellulose membrane (Amersham Life Science, Piscataway, NJ). Incubation with primary mouse monoclonal anti-core antibody (Biogenesis, England) was followed by an anti-mouse horseradish peroxidase (HRP) conjugated secondary antibody (Bio-Rad, Missisauga, ON, Canada). Bound antibodies were detected by exposure to enhanced chemiluminescence reagent (ECL+, Amersham Biosciences) and analyzed by Kodak X-ray film. The immunoblot was then incubated with stripping buffer (100 mmol/L Tris-HCl, 100 mmol/L mercaptoethanol, 2% SDS, pH 6.8) for 30 min at 50°C. The stripped blot was once incubated with HCV-positive human sera followed by incubation with goat anti-human HRP-conjugated antibody and detected with electrochemiluminescence (ECL) detector. For the second time, the stripped blot was incubated with monoclonal mouse anti-His tag-HRP–conjugated antibody (BD Bioscience) and detected by ECL. Statistical analysis. To compare differences among groups, a one-way analysis of variance with the New-

FIG. 1. Immunofluorescence analysis of recombinant HCV core/E1/E2 polyprotein expression in mammalian cells. CHO cells were transfected by electrophoration with 50 g of pVAX1 plasmid containing the genes encoding HCV core/ E1/E2 proteins. After 48 h, CHO cells were trypsinized, centrifuged and incubated overnight on slides. Cells were fixed with either acetone or isopropanol and analyzed by immunofluorescense microscopy with a monoclonal antibody directed against HCV core protein. (A) CHO cells transfected with pVAX1 plasmid containing the recombinant HCV Core/E1/E2 insert. (B) CHO cells transfected with empty pVAX1 plasmid. (C) Western blot analysis of purified coreE1E2 protein expressed in CHO cells. Anti-His antibody was used to detect the purified protein (Lane 1). To confirm the protein specificity, the membrane was stripped once and re-blotted with mouse monoclonal antibody against HCV core protein (Lane 2), and re-stripped and blotted again with HCV positive Human pooled sera (Lane 3). In all cases, a 110 kd band corresponding to coreE1E2 protein was detected. The smaller band is corresponding to only E1E2 protein since the core protein is probably chopped of from the polyprotein by cell proteases. See text for abbreviations.

man-Keuls post-test was used. Values of p  0.05 were considered to be significant.

RESULTS Expression of recombinant HCV Core/E1/E2 polyprotein. After transfection of CHO cells with the pVAXHCV construct, the intracellular expression of the recombinant viral polyprotein consisting of the core, E1,






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and truncated E2 proteins was assessed by immunofluorescent staining using a monoclonal antibody against the core protein (Fig. 1). The purified recombinant HCV polyprotein cross-reacted with antisera from patients infected with diverse genotypes. However, the 3a and 4h genotype titers (both 1/900) were not as high as the titers observed for genotypes 1a and 1b (1/2700 and 1/8100, respectively). The induction of anti-HCV antibodies by immunization. The status of seroconversion was determined for each group of immunized mice (Fig. 2). Three of six groups (groups C, D, and F) showed seroconversion in response to the hepatitis C immunogens. However, only three of seven mice seroconverted in group C (the group primed with DNA and boosted with polyprotein and adjuvant); the increase in antibody was only marginal for this group. In contrast, all seven mice in group D (the FIG. 3. Immunogenicity of various HCV immunogen combinations as determined by ELISA analysis of total IgG. Six groups of mice were immunized with various HCV immunogen preparations. Mice were immunized three times on days 0, 30, and 60. On day 75, serum samples were taken, serially diluted, and tested for reactivity with the recombinant HCV core/E1/E2 polyprotein. Sera taken from mice prior to immunization were used as a baseline (Group G -). The murine groups were immunized as follows: immunization with empty pVAX1 (Group A -); immunization with rHCV DNA alone (Group B -*); priming with rHCV DNA and boosting with rHCV polyprotein and montanide (Group C -); priming and boosting with the combined vaccine consisting of rHCV DNA, rHCV polyprotein and montanide (Group D -); priming and boosting with rHCV DNA and montanide (Group E -); priming and boosting with rHCV polyprotein and montanide (Group F -). See text for abbreviations.

FIG. 2. ELISA analysis to measure total IgG antibodies induced by immunization with various HCV immunogen combinations. Briefly, ELISA plates were coated with 50 L of 20 g/mL purified coreE1E2 polyprotein and incubated with serial dilution of mice sera and detected with goat anti-mouse antiserum conjugated with HRP as described in the material and methods. The bars from A to F represent the mouse groups: immunized with empty pVAX1 (group A); primed and boosted twice with rHCV DNA alone (group B); primed with rHCV DNA and boosted twice with rHCV polyprotein and montanide (group C); primed and boosted with the combined vaccine consisting of rHCV DNA, rHCV polyprotein and montanide (group D); primed and boosted with rHCV DNA and montanide (group E); primed and boosted with rHCV polyprotein and montanide (group F). For each group, the fraction of mice exhibiting at least twice the optical density of the pre-immunized controls is shown above the bar for that immunization regimen. This ELISA was performed in mouse sera diluted 1/100. *p  0.05. See text for abbreviations.

group that was both primed and boosted with the combination of DNA, recombinant core/E1/E2 proteins and montanide) seroconverted and developed significant antiHCV antibody levels. In comparison, four of seven mice in group F (the group that was primed and boosted with polyprotein and adjuvant) seroconverted. Only groups D and F demonstrated a significant increase in the total IgG after the third immunization (p  0.01 and p  0.05 respectively). Similarly, groups D and F had significantly elevated total IgG anti-HCV titers after the third immunization (group D  1:2700; group F  1:900) (Fig. 3). All other groups had a total IgG titer of anti-HCV antibodies of 1:300. IgG1 was the predominant antibody detected in group D with an end-point titer of 1:900 (Fig. 4). In comparison, groups C and F had IgG1 titers of 1:300. No significant levels of IgG2a were detected in any of the immunized groups (data not shown). Antibodies induced by HCV immunization recognize the HVR1 neutralizing epitope. To determine the



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level of both IL-5 and IFN- significantly (p  0.05) compared to unstimulated control cells (Fig. 7A and 7B). Similar results were obtained from mice in group E when primed and boosted with HCV DNA and adjuvant. Although vaccination with protein and adjuvant in group F increased the level of IFN-, albeit to a lesser extent than in group C and E, it did not have any effect on IL-5 production in this group of immunized mice. The IFN- and IL-5 production in all other group of mice was either undetectable or not significant (data not shown).


FIG. 4. Induction of IgG1 antibodies in mice immunized with various HCV immunogen combinations. EIA/RIA Strip well TM plates were coated for 4 h at 37°C with 20 g/mL recombinant core/E1/E2 polyprotein dissolved in sterile distilled/deionized water and blocked overnight at 4°C with 1% BSA in PBS. Dilutions of sera were incubated for 2 h at 37°C and antibodies were detected with a 1/1000 dilution in 1% BSA/PBS of goat anti mouse IgG1 conjugated with HRP. The pattern of immunization, symbols, and colors are same as in Fig. 3. See text for abbreviations.

specificity of the antibodies induced by the various immunization regimens, pre-immunization sera and serially diluted post-immunization sera were tested for their ability to bind to HVR1 neutralizing epitope (Fig. 5). Only groups C and D showed significant but low antibody response to HVR1 antigen compared to the pre-immunization sera from the same group of mice, p  0.05. Other experimental groups showed no significant response to HVR1 neutralizing epitope (data not shown). Proliferation assays. The proliferative responses of cultured splenocytes induced by recombinant HCV core/E1/E2 polyprotein are shown in Figure 6. Group D, the group of mice primed and boosted with the combined vaccine consisting of rHCV DNA, rHCV polyprotein, and montanide, demonstrated a marked proliferative response to the antigen. Group C, the group of mice primed with DNA and boosted with protein plus adjuvant, also showed a fairly high proliferative response; but this response was significantly (p  0.05) lower than that in mice immunized with combined vaccine. None of the other groups showed any proliferation in response to the antigen. Serum cytokines. Each vaccine regimen induced a different pattern of cytokine release in the supernatant of proliferative PBMCs from immunized mice. Priming with HCV DNA and boosting with recombinant HCV CoreE1E2 protein and adjuvant in group C increased the

In this study we used different immunogen formulations to determine the modulation of the antibody response to viral structural antigens. A plasmid encoding the nucleocapsid core protein, the E1 envelope protein, a truncated form of the E2 envelope protein, and various combinations of the recombinant core/E1/E2 polyprotein and the adjuvant montanide were studied. The humoral response to the candidate HCV vaccine was characterized by a relatively high level of hepatitis C–specific IgG antibodies after the third immunization (Fig. 2). This combination of DNA, protein, and adjuvant to both prime and boost induced HCV seroconversion in 100% of the immunized mice (group D) (Fig. 2), and also resulted in the highest titer of antibody to HCV compared to the

FIG. 5. Antibodies induced by various HCV immunization combinations recognize the HVRI neutralizing epitope. EIA/RIA Strip wells coated with 20 g/mL recombinant core/E1/E2 polyprotein were incubated with a serial dilutions of mice sera and detected by goat anti mouse HVRI neutralizing epitope antibody as described in the material and methods section. Other experimental groups showed no significant response. See text for abbreviations.



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FIG. 6. Splenocyte proliferation response following immunization with various HCV immunogen combinations. Murine spleen cells were harvested 2 weeks after the last immunization, washed with PBS and cultured in triplicate using 96-well round-bottom plates at 106 cells/well in 200 L RPMI containing 10% FCS, 50 mol/L -mercaptoethanol and penicillin/streptomycin. Cells were stimulated with recombinant HCV core/E1/E2 polyprotein at a concentration of 10 g/mL. After 72 h, 100 L of supernatant was collected from each well for detection of various cytokines and this volume was replaced with RPMI containing [3H]thymidine (1 Ci/well). Cells were incubated for an additional 18 h and [3H]thymidine incorporation into DNA was measured after harvesting cells onto glass fiber filtermats. See text for abbreviations.

other vaccinated murine groups (Fig. 3). The combination of protein and adjuvant to prime and boost also induced a good antibody response in four of seven of immunized mice (57%) in group F (Fig. 2). In comparison, only 42% of mice seroconverted in the DNA prime/protein and montanide-boost group (group C); the anti-HCV IgG antibody titers for this group were relatively low at 1:300 (Fig. 3). IgG1 was the predominant antibody detected in the responder BALB/c mice (Fig. 4). Antibody responses have been demonstrated in several viral systems after DNA immunization of mice and primates. Wolff et al. (1990) demonstrated that foreign genes could be expressed in murine muscle after DNA immunization. Since then, many studies have shown that antibody responses to viral genes could be induced by DNA immunization (18,22,23,38,46). However, in instances in which DNA immunization failed to induce an antibody response (12), it is postulated that its main effect is the induction of CTL responses in the context of MHC class I antigen presentation. Antibodies against the hepatitis C viral nucleocapsid are among the first antibodies detected upon human infection (11,35). Major et al. (32) were able to induce antibodies against the core protein only when it was fused to hepatitis B surface antigen but not when administered

GHORBANI ET AL. as a DNA vaccine encoding only the core protein. We were able to demonstrate a robust cellular response to the recombinant HCV polyprotein in a lymphocyte proliferation assay. Mice primed and boosted with the combined DNA, protein, and adjuvant vaccine (group D) had higher T-cell proliferation and higher antibody titers to the HCV polyprotein than the DNA prime/protein and adjuvant boost group (group C). There was a direct correlation between antibody titer and intensity of lymphocyte proliferation. Similar anti-HVR1 antibody titers were observed in both groups. In contrast, the highest proliferative response was observed in the group of mice primed and boosted with the combined vaccine (group D). The HCV core protein forms the nucleocapsid of the HCV virion, has a well-conserved sequence, and contains several B and T cell epitopes (47,51). Priming mice with synthetic peptides corresponding to amino acids 121–140 of this protein, followed by a booster dose of total core protein, resulted in a 64-fold higher antibody titer than without peptide priming (7). Another study also indicated that the 16-residue synthetic peptide (HCV 129–144) is highly recognized by murine and human CTL with a prevalent HLA class I molecule (11,48,49).

FIG. 7. Cytokine measurement in supernatant of cultured splenocyte. (A) IL-5 measurement in supernatant of proliferating splenocytes. Cells were stimulated with 20 g of recombinant CoreE1E2 protein in IMDM media for 48–72 h. Only IMDM was added to control wells containing splenocytes. (B) IFN- measurement in unstimulated and stimulated splenocytes. *p  0.05. See text for abbreviations.



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VACCINE DEVELOPMENTS FOR HEPATITIS C Intramuscular administration may result in an increased concentration of immunogens in lymph nodes, enhancing helper T-cell responses by increasing antigen presentation by dendritic cells (13). It may also result in the systemic distribution of the immunogen, resulting in its entrapment in the reticuloendothelial system of different organs such as the spleen and liver, allowing increased exposure to B cells. ELISA of the supernatants of cultured PBMC demonstrated increased levels of IFN-, a Th1 cytokine, and of IL-5, a Th2 cytokine. However, the predominant immune response in BALB/c mice was a Th2 response as suggested by the predominance of IgG1 after immunization. This result was as expected, as BALB/c mice have a bias toward this type of immune response (36,52). In the cytokine release assay there was elevation of IFN- in the groups (C and E) that received DNA vaccine, which expectedly induces cellular immunity (Fig. 7A and 7B). However, there was only a small antibody response in these two groups of mice, which confirms the possibility that the plasmid mixed with the protein-based vaccine acts essentially as an adjuvant, based on the CpG motif contained within the plasmid. The lack of Th1 cytokine release in group D is counter-balanced by a high antibody response in mice that receiving recombinant protein in all injections (group D). We were unable to detect a prominent CTL response in experiments involving intracellular staining of CD8 T cells (data not shown). The lack of a CTL response may be explained by the finding of Webby et al. (55) that a biological competition for the same pathogen may occur between the Th1driven CTL response and the Th2-driven antibody response to the same pathogen may occur. Furthermore, Palmowski et al. (37) demonstrated that prime-boost immunization might induce strong CTL responses to some epitopes, which may inhibit the ability of the immune system response to others. In fact, a strong CTL response to one epitope may inhibit the response to others. The HCV E2 envelope glycoprotein contains a neutralizing epitope, hypervariable region 1 (HVR1), which can allow mutant viruses to escape the host immune system because of its variability (26, 56). Several studies have shown that the E2 protein is a critical immunogen given its ability to induce protection against HCV infection (5,13,54). Vaccination of chimpanzees with recombinant HCV E1/E2 proteins has been shown to induce in vivo protection against HCV, concomitant with anti-E2 antibody production (9). It has also been demonstrated that the antibodies present in immunized chimpanzees could prevent infection when incubated in vitro with virus before infection challenge (15,29). Moreover, the antibodies present in protected chimpanzees could inhibit the binding of E2 protein to human cells (17,44). Other observations have indicated that the HVR1 of E2 protein, which contains CTL epitopes, might play a role in HCV

protection (15,17,22,26,29). However, the protection is specific to the vaccine HCV strain, and thus protection is not achieved against wild-type viral strains. We used a truncated form of the E2 protein because it has been shown to induce neutralizing antibodies and are more efficient in inducing a robust immune response than the complete protein sequence (22). We were able to demonstrate the presence of antibodies to the HVR1 region, suggesting that these antibodies could recognize the neutralizing epitope of the E2 protein. The DNA, protein, and adjuvant combination closely resembles the antigen presentation that occurs upon natural infection with hepatitis C and therefore should induce a more robust immune response. Accordingly, the proliferative response to the immunogens was highest in the DNA/protein/adjuvant prime and boost group. The cell proliferation results concord with the humoral response, which was also robust in the combination vaccine prime-boost group. Our results are similar to a recent murine study of a candidate dengue vaccine consisting of a combination of DNA and recombinant protein; this vaccine induced one of the highest antibody titers compared to DNA priming and recombinant protein boosting (39,50,53). However, this study also demonstrated a high titer of antibody in mice immunized with DNA alone. Other studies have demonstrated the capacity of DNA priming and recombinant protein boosting to induce antibody responses (4,25). Raya et al. (42) successfully used montanide in a phase I HIV vaccine clinical trial in which a good humoral immune response was induced. These humoral responses may be dependent on the type of antigens, route of administration, or adjuvants. However, more balanced immune responses might be necessary to confer effective protection against HCV involving other immunomodulatory proteins such as interferon (20) to augment the cellular immune responses to DNA vaccination against HCV antigens. In an attempt to confirm the HVR1 epitope experiments, we are currently developing an assay to determine the titers of neutralizing antibody in samples of mice immunized with the combined vaccine. In addition, we are analyzing the Th2 cellular immune response to this vaccine preparation and assessing the role of adjuvants in the immune response to hepatitis C antigens.

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Address reprint requests to: Dr. Francisco Diaz-Mitoma Children’s Hospital of Eastern Ontario 401 Smyth Road Ottawa, ON, K1H 8L1, Canada E-mail: [email protected] Received February 19, 2005; accepted August 29, 2005.

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