Development of Th1 and Th2 Populations and the Nature of Immune Responses to Hepatitis B Virus DNA Vaccines Can Be Modulated by Codelivery of Various Cytokine Genes

Construction of expression vectors

The plasmid vector pS encoding HBV major envelope proteins was constructed previously (33). This eukaryotic expression vector was modified from plasmid pcDNA3 (Invitrogen, San Diego, CA) containing the CMV early promoter/enhancer sequence and the polyadenylation and 3′ splicing signals from bovine growth hormone. The cDNA of murine IFN-γ, IL-4, and GM-CSF were obtained by reverse transcription and PCR amplification of RNA derived from about 1 × 10⁸ mouse (C3H/HeN) splenocytes stimulated for 4 h, 8 h, and 24 h, respectively, with 5 ng/ml of PMA (Sigma Chemical Co., St. Louis, MO) and 5 μg/ml of Con A (Sigma) in culture medium. The fragment containing the murine IL-2 coding sequence was generated by PCR from plasmid pmut-1 (ATCC 37553; American Type Culture Collection (ATCC), Rockville, MD). The upstream PCR primer for each cytokine gene contains a BamHI site, and the downstream primer contains an EcoRI site. PCR products of IL-2, IL-4, GM-CSF, and IFN-γ were digested with BamHI and EcoRI, gel purified, and inserted between the BamHI and EcoRI sites of pcDNA3 to generate pIL-2, pIL-4, pGM, and pIFN-γ, respectively. The fragments containing the p35 and p40 coding sequences of murine IL-12 were generated by PCR from plasmids BLpSV35 and BLpSV40 (kindly provided by Michale T. Lotze, University of Pittsburgh, Pittsburgh, PA), respectively, and cloned into a bicistronic plasmid, pTCAE (33), under the control of discrete CMV promoters. Plasmid DNA was purified from transformed Erichia coli strain DH5α by Qiagen Plasmid Giga Kits (Qiagen, Hilden, Germany) according to the manufacturer’s instructions and stored at −70°C as pellets. The DNA was reconstituted in sterile saline at a concentration of 1 mg/ml for experimental use.

Cell transfection and cytokine gene expression

The expression of cytokine genes by the plasmid DNA was performed in a transient transfection assay using Lipofectamine (Life Technologies, Gaithersburg, MD) as specified by the manufacturer. Briefly, C2C12 mouse myoblasts (1 × 10⁵ cells; ATCC 1772) were cultured in DMEM plus 10% FCS (DMEM-10) in a six-well tissue culture plate until the cells reached approximately 50 to 80% confluence. Three micrograms of plasmid DNA was mixed with 20 μl of Lipofectamine in 200 μl of OPTI-MEM medium (Life Technologies) at room temperature. Following a 20-min incubation, the DNA-liposome complexes were diluted in 800 μl of OPTI-MEM and slowly added to cells, which had been prewashed twice with 5 ml of OPTI-MEM. After a 16-h incubation, the DNA-liposome complexes were removed, 2 ml of DMEM-10 was added to each well, and incubation was continued for another 48 h. The supernatant of each well was collected and stored at −80°C for cytokine analysis.

Cytokine ELISAs and proliferation assays

The cytokine activity present in the supernatant of plasmid DNA-transfected cells was assayed for its ability to support the proliferation of appropriate responsive cells: IL-2 and IL-4 were tested on HT-2 cells (36), GM-CSF was measured on NFS-60 cells (37), and IL-12 was determined on activated lymphoblasts. The HT-2 and NFS-60 cells were grown in RPMI 1640 containing 10% FCS (RPMI 10) supplemented with 1% supernatant of transfectoma cell lines Id-IL-2 (38) and Id-MoGM (39), which produce Id-cytokine fusion proteins containing murine IL-2 and GM-CSF activity, respectively. To perform the proliferation assay, samples were added in triplicate to 96-well plates in RPMI 10 with 5,000 HT-2 or NFS-60 cells to a total volume of 0.1 ml and incubated in a humidified incubator for 16 to 24 h at 37°C and 5% CO₂. IL-12 proliferation assay was performed as previously described (40). Briefly, the Con A-activated lymphoblasts were prepared by culturing C57BL/6 splenocytes in TCM medium (Celos Laboratory, Hopkins, MN) containing 2% FCS, 20 ng/ml of human rIL-2 (Endogen Inc., Cambridge, MA) and 2 μg/ml of Con A (Sigma) at a density of 2 × 10⁶ cells/ml for 2 to 3 days. The lymphoblast cells were then harvested and cultured at 50,000 cells/well with test samples in TCM plus 5% FCS for 24 h. After incubation, 1 μCi of [³H]thymidine (Amersham, Arlington Heights, IL) was added to each well in 50 μl of growth medium, and cells were harvested 4 to 6 h later using a FilterMate (Packard, Meriden, CT) automatic cell harvester; the incorporated radioactivity was determined using TopCount microplate scintillation counter (Packard). The concentration of cytokines in the samples was determined from the standard curves generated using recombinant murine IL-2, IL-4, GM-CSF, or IL-12, all of which were purchased from PharMingen (San Diego, CA).

ELISA detection systems (PharMingen) were also used to screen for the presence of IFN-γ, IL-2, and IL-4 cytokines in test samples. The capture Abs for murine IFN-γ, IL-2, and IL-4 were R46A2 (rat IgG1), JES6-1A12 (rat IgG2a), and BVD4-1D11 (rat IgG2b), respectively. The detection biotinylated Abs for IFN-γ, IL-2, and IL-4 were XMG1.2 (rat IgG1), JES6-5H4 (rat IgG2b), and BVD6-24G2 (rat IgG1), respectively. Briefly, microtiter plates were coated with 50 μl of anti-cytokine capture mAb at the concentration of 2 μg/ml overnight at 4°C. The plates were washed twice with PBS/Tween and blocked with 200 μl per well of 10% bovine calf serum in PBS for 2 h at room temperature. Then, the plates were washed twice and incubated with duplicates of serially diluted samples and standards overnight at 4°C. One hundred microliters of the biotinylated anti-cytokine mAb at a concentration of 2 μg/ml was added to each well and incubated at room temperature for 1 h. The plates were then washed six times, 100 μl of avidin-phosphatase (1:500; PharMingen) was added, and the mixture was incubated at room temperature for 30 min. Following multiple final washings, the color was developed with p-nitrophenyl phosphate (Sigma) as the substrate, and absorbance at 405 nm was measured using an ELISA plate reader. The concentration of cytokines in the samples was determined from the standard curve.

Immunization of mice

Female BALB/c (H-2^d) and C57BL/6 (H-2^b) mice were obtained form the Laboratory Animal Facility, Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan. All mice were immunized at 6 to 8 wk of age as described previously (33). In brief, groups of three to five mice were anesthetized and injected i.m. in the left hind thigh muscle with a mixture of 100 μg of pS and 100 μg of pcDNA3, pIL-2, pIL-4, pGM, pIFN-γ, or pIL-12. Mice immunized with 200 μg of pcDNA3 served as negative controls. In some experiments, mice were immunized with 100 μg of pS in the left thigh and 100 μg of each cytokine vectors or the control plasmid in the right thigh. For protein immunization controls, mice were given s.c. injections in the base of the tail with 4 μg of yeast-derived rHBsAg (41) in CFA (Life Technologies, Grand Island, NY).

Ab assay

Serum samples were collected by tail bleeding at different times, beginning 1 wk after immunization, and analyzed for the presence of HBsAg-specific Abs. Microtiter plates were coated with 5 μg/ml of yeast-derived rHBsAg. After incubation with 200 μl of 5% powdered milk in PBS on each well for 1 h to prevent nonspecific binding, 50 μl of serial dilutions of test sera was added to each well and incubated overnight at 4°C. After the samples were washed with PBS, bound proteins were detected with horseradish peroxidase-conjugated goat anti-mouse IgM (1:2000, Cappel-Organon Teknika, Veedijk Belgium) or anti-mouse IgG (1:2000; Cappel). Color was generated by adding 2,2′-azino-bis(ethylbenzthiazoline sulfonic acid), and absorbance at 492 nm was measured on an ELISA reader. For measurement of IgG1 and IgG2a anti-HBs isotypes, biotin-conjugated rat anti-mouse IgG1 (1:500, PharMingen) and rat anti-mouse IgG2a (1:500, PharMingen) were used as detectors. Avidin-alkaline phosphatase (1:1000, PharMingen) was then added. Color was developed with the addition of p-nitrophenyl phosphate (Sigma), and absorbance at 405 nm was measured. Concentrations of total IgG anti-HBs Abs in serum samples were estimated from the standard curve generated by a HBsAg-specific mAb (H25B10, ATCC CRL-8017) and expressed as μg/ml for IgG. For measurement of IgM, IgG1, and IgG2a Abs, readings were referenced to a standard serum pooled from four mice given i.p. injections of 2 μg of rHBsAg with CFA and bled 4 wk after immunization. The standard curves were generated using the pooled anti-HBs sera, and results were expressed as arbitrary units per milliliter (U/ml; 1 U = 50% maximum OD).

Flow cytometric analysis

The populations of splenocytes derived from immunized animals were analyzed by flow cytometer. Splenocytes were stained with FITC-conjugated anti-mouse CD4 or anti-mouse CD8α, combined with phycoerythrin-conjugated anti-Thy1.2; all Abs were purchased from PharMingen. Stained cells were analyzed on a FACSCallibur (Becton Dickinson, Mountain View, CA).

Assays for lymphocyte proliferation and cytokine secretion

To determine whether HBsAg-specific lymphoproliferative responses were induced in immunized aimals, spleens were removed 2 wk after immunization to make single-cell suspensions. CD4⁻-depleted and CD8⁻-depleted immune splenocytes were prepared by immunomagnetic depletion of CD4⁺ and CD8⁺ lymphocytes, respectively, and replaced with the same number of purified CD4⁺ or CD8⁺ cells isolated from naive mice. The immunomagnetic depletion or enrichment of CD4⁺ and CD8⁺ lymphocytes was performed by a magnetic activated cell sorter (MACS; Miltenyi Biotec, Bergisch Gladbach, Germany), using microbeads conjugated with rat anti-mouse CD4 and rat anti-mouse CD8 mAbs (Miltenyi Biotec), respectively, according to the manufacturer’s instructions. To perform the lymphoproliferative assay, 100 μl of 2 × 10⁶/ml unfractionated, CD4⁻-depleted, or CD8⁻-depleted splenocytes in complete RPMI 10 were added to each well in 96-well flat-bottom plates. Stimulated wells received purified rHBsAg at a concentration of 30, 10, or 1 μg/ml; transferrin (120 μg/ml, Sigma) served as a negative control Ag and Con A (5 μg/ml, Sigma) as a positive mitogenic control. Control wells received cells only. Cells in all the wells were cultured in a total volume of 200 μl of medium. After 4 days in culture, the cells were pulsed with [³H]thymidine (1 μCi/well) for 18 h and harvested with FilterMate (Packard) and the incorporated radioactivity was determined by TopCount (Packard). The stimulation index was calculated as the mean cpm of the stimulated wells divided by the mean cpm of the control wells.

To measure cytokine secretion, splenocytes were cultured as described above with the same panel of Ags or mitogen over the same range of concentrations with the exception that after 3 days in culture, cell-free supernatants were harvested and assayed immediately or stored at −80°C. These supernatant were screened for the presence of IFN-γ, IL-2, and IL-4 using ELISA detection systems as described in the previous section.

Cytotoxicity assays

BALB/c mice were immunized i.m. with 100 μg of pS and 100 μg of each of the cytokine vectors or the control plasmid. Spleens were removed 3 wk after immunization to measure the CTL activity. CD4⁻-depleted and CD8⁻-depleted immune splenocytes were prepared as described in the previous section. Single-cell suspensions of unfractionated, CD4⁻-depleted or CD8⁻-depleted splenocytes were treated with 5 ml per spleen of ACK lysis butter (0.15 M NH₄Cl, 1 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2) for 5 min at room temperature to remove RBC. HBsAg-expressing P815 cells, a murine mastocytoma line (H-2^d, DBA/2 mice), and CT26 cells, a murine colon carcinoma (H-2^d, BALB/c mice), were generated by permanent transfection with 20 μg of plasmid pS, selected for G418-resistant clones, and screened for the expression of HBsAg by ELISA and immunostaining. One of the HBsAg-expressing clone from each transfected cell line was selected and designated as P815/S and CT26/S, respectively. To perform the cytotoxic assay, responder splenocytes (4 × 10⁶ per well) were first stimulated with irradiated (8000 rad) P815/S or CT26/S transfectants (4 × 10⁶ per well) in RPMI 10 and 20 U/ml human rIL-2 (Genzyme Corp., Cambridge, MA) in 24-well plates for 4 days at 37°C. A chromium release assay was employed to measure the ability of in vitro-stimulated responder cells to lyse P815/S and CT26/S as well as the nontransfected parental cells. Target cells (1 × 10⁶ in 0.1 ml RPMI 10) were labeled with 0.1 mCi radiolabeled sodium chromate (Amersham) in 0.1 ml normal saline for 2 h at 37°C, washed three times with RPMI 10, and resuspended at a concentration of 5 × 10⁴ per ml in RPMI 10. A threefold serial dilution of 100 μl of stimulated responder splenocytes (starting from 5 × 10⁵ cells) was added to individual wells containing 100 μl of labeled target cells (5000 cells). In some experiments, the anti-CD4 mAb (GK1.5, ATCC TIB-207) or anti-CD8 mAb (53-6.72, ATCC TIB-105) was added to the culture medium during the cytotoxic assay at a concentration of 10 μg/ml. After a 6-h incubation at 37°C, 100 μl of culture supernatant was collected for gamma radiation counting. The percent specific lysis was calculated as [(experimental release − spontaneous release)]/(total release − spontaneous release)] × 100. Spontaneous release represents the amount of radioactivity released from target cells without the addition of effector cells. Total release represents the amount of radioactivity released following lysis of target cells after the addition of Triton X-100 to 1.0%.

In vivo tumor protection

Three weeks after injection of plasmid pS and various cytokine genes, individual groups of mice were challenged s.c. in the left lateral flank with 1 × 10⁵ CT26 cells or the HBsAg gene-transfected CT26/S cells. Tumor growth was measured every second or third day.

Construction and expression of cytokine vectors

The presence of certain cytokines during antigenic stimulation has been shown to be of decisive importance in directing the development of naive CD4⁺ cells into Th1 or Th2 effectors and thus may determine the outcome of many infectious and autoimmune diseases. DNA vaccination is a recently developed vaccine technology and has been reported as able to induce humoral and cellular immunities in many different disease models. To study whether the phenotype of immune responses to a DNA vaccine can be altered by cytokines, a previously described plasmid vector pS (33), which encodes HBsAg, was used as a model system. Since local expression of cytokines at the site of immune interaction is important to mediate its function, we hypothesized that codelivery of a cytokine vector with the HBV DNA vaccine might achieve this purpose. The cytokine-producing vectors pIL-2, pIL-4, pGM, pIFN-γ, and pIL-12 encoding murine IL-2, IL-4, GM-CSF, IFN-γ, and IL-12, respectively, were constructed as described in Materials and Methods. To test whether the various cytokine vectors can produce biologically active cytokines, mouse C2C12 myoblasts were transiently transfected with each of the vectors. Two days after transfection, the amount of cytokines present in the culture supernatant was analyzed by ELISA (for IFN-γ) or proliferation assays using appropriate responsive cells (HT-2 cells for IL-2 and IL-4; NFS-60 cells for GM-CSF and mitogen-activated lymphoblast for IL-12). Plasmids pIL-2, pIL-4, pGM, pIFN-γ, and pIL-12 produced 45.8 ± 4.8, 77.6 ± 6.8, 48.4 ± 6.5, 11.3 ± 0.3, and 17.6 ± 5.3 ng/ml of cytokine proteins, respectively, each with its particular biologic activity.

Codelivery of cytokine vectors enhances T cell proliferative responses to pS DNA vaccination

The enhancing effect of various cytokines on T cells was then examined. Groups of C57BL/6 mice were given i.m. injections of pS alone, or a mixture of pS plus a cytokine vector or the parental vector pcDNA3. Mice that received pcDNA3 served as negative controls. Mice immunized with rHBsAg were also included as protein immunization controls. At 2 wk after immunization, splenocytes were examined for proliferation in response to specific Ag stimulation. Both plasmid DNA and rHBsAg immunization elicited significant proliferative responses over a range of concentrations (1–30 μg/ml; Table I⇓). Coinjection of plasmids expressing IL-2, GM-CSF, IFN-γ, or IL-12 with pS enhanced the cellular proliferation by two- to threefold, while plasmid encoding IL-4 gave a less significant increase in the proliferative response (Table I⇓). The enhancement in cellular proliferation by cytokine vectors cannot be attributed simply to the intrinsic adjuvant effect of plasmid DNA as previously reported (42), since splenic lymphocytes derived from mice inoculated with pS plus pcDNA3 proliferated at similar levels as those immunized with pS alone. Mice vaccinated with the control pcDNA3 vector did not respond to HBsAg, and all mice failed to respond to transferrin included as a control Ag, indicating that the observed T cell proliferation was HBsAg specific. We also found that the adjuvant effect conveyed by the cytokine vector was only observed when the DNA vaccine was coinjected with the cytokine vector. Mice immunized with pS on the left thigh and cytokine cDNA vectors on the right thigh did not enhance the HBsAg-specific proliferative response (data not shown), indicating that the presence of cytokines at the site of immune interaction was important for this enhancement.

The splenic lymphocyte populations of immunized animals were evaluated by flow cytometric analysis. Codelivery of cytokine genes did not change the proportions of CD4⁺ and CD8⁺ Th cells in the plasmid DNA-vaccinated animals. The frequency of CD4⁺ lymphocytes in mice coinjected with pS and pIL-2, pIL-4, pGM, pIFN-γ, or pIL-12 was 32.7, 31.9, 31.1, 27.9, and 33.6%, respectively, which was comparable with that from mice immunized with pS plus pcDNA3 (30.8%) or pcDNA3 alone (30.9%). The frequency of CD8⁺ lymphocytes was also similar among groups of mice injected with or without cytokine vectors: 13.3% (pS + pIL-2), 14.2% (pS + pIL-4), 11.7% (pS + pGM), 12.0% (pS + pIFN-γ), and 13.6% (pS + pIL-12) vs 11.0% (pS + pcDNA3) and 12.7% (pcDNA3). To determine which lymphocyte population was responsive to HBsAg, spleen cells showing proliferative responses were further tested by immunomagnetic depletion of CD4⁺ or CD8⁺ lymphocytes. In all immunized groups, depletion of CD4⁺ cells significantly decreased the HBsAg-specific proliferative response, whereas depletion of CD8⁺ cells had little effect on this response (Fig. 1⇓), indicating that the bulk of the proliferative responses could be accounted for by CD4⁺ cells.

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FIGURE 1.

HBsAg-specific proliferative responses are mediated by CD4⁺ Th cells. Animals were treated as described in the legend to Table I⇑. The preparation of CD4⁻-depleted and CD8⁻-depleted splenocytes is detailed in Materials and Methods. Values are presented as the mean stimulation index for triplicate wells ± SD.

The magnitude of Ab responses to HBV DNA vaccination is altered by codelivery of various cytokine genes

To establish whether the humoral responses to the HBV DNA vaccine can be affected by simultaneous expression of cytokine genes, groups of five mice were given i.m. injections of a mixture of pS and various cytokine vectors. Mice receiving 4 μg of rHBsAg were also included as a comparison. Serum from each mouse was obtained at week 4 and week 8 following DNA or protein injection for analysis of IgM and IgG anti-HBs Ab responses, respectively. As shown in Figure 2⇓, both plasmid pS and rHBsAg elicited significant titers of HBsAg-specific IgM and IgG Abs. Coinjection of IL-2, IL-4, GM-CSF, IL-12, or IFN-γ genes with pS all resulted in much stronger IgM anti-HBs Ab compared with mice receiving pS plus the parental vector (Fig. 2⇓A). The IgG anti-HBs Ab were enhanced two- to threefold in mice that had received pS together with pIL-2, pIL-4, pGM, or pIL-12 compared with those immunized with pS plus the parental vector (Fig. 2⇓B). In contrast, we found that mice given injections of plasmids pS and pIFN-γ showed decreased IgG anti-HBs titers compared with the control mice. We also performed kinetic studies on the DNA vaccine-induced humoral responses and found that the adjuvant effect conveyed by the cytokine vector was observed only at the early time intervals, from week 4 through 20, after DNA immunization (data now shown). The influence was subsequently decreased, and by week 35, the difference in anti-HBs titers between groups immunized with and without cytokine coexpression became negligible. Mice that were given injections of pS and the cytokine vector on different thighs showed no alterations in the anti-HBs Ab titers (data not shown), indicating the importance of colocalization of cytokines with Ag to exert their adjuvant effects.

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FIGURE 2.

Effect of coinoculation with plasmids expressing various cytokines on HBsAg-specific Ab responses. Groups of five C57BL/6 mice were given i.m. injections of a mixture of 100 μg of pS plus 100 μg of various cytokine vectors or the control vector pcDNA3. For protein immunization controls, mice were injected s.c. with 4 μg of rHBsAg in CFA. Sera were collected at week 4 and 8 following vaccination for analysis of IgM and IgG anti-HBs Ab, respectively. The amounts of IgM and IgG anti-HBs Abs were measured from standard curves generated from the respective serially diluted control Abs and expressed as U/ml and μg/ml, respectively. The data are presented as mean ± SD for five animals per time point from one representative experiment of three performed.

Differentiation of CD4⁺ Th cell into Th1 or Th2 phenotype can be altered by codelivery of various cytokine vectors

It is known that the subsets of Th cells can be distinguished by the pattern of cytokines that they produce. Th1 cells produce IFN-γ and IL-2, and Th2 cells produce IL-4, IL-5, IL-10, and IL-13 (27, 43, 44, 45). To study the effect of cytokine coexpression on the development of Th cells induced by DNA vaccination, cytokines profiles released from HBsAg-stimulated splenocytes were compared among groups of mice immunized with pS with or without cytokine gene coinjection. Spleen cells from animals immunized with plasmid pS plus pcDNA3 or rHBsAg produced both Th1 (IFN-γ, IL-2) and Th2 (IL-4) cytokines (Fig. 3⇓). Coexpression of IL-12 or IFN-γ with the HBV DNA vaccine resulted in a significant increase in Th1 cytokine production (Fig. 3⇓, A and B); IFN-γ levels were increased six- to sevenfold, and IL-2 levels were increased about fivefold. This was accompanied with a complete shutoff of IL-4 production (Fig. 3⇓C). In contrast, cells from mice coimmunized with pS and pIL-4 produced much more IL-4 and much less IL-2 and IFN-γ as compared with cells from the control mice, indicating a bias toward the Th2 immunity. Coinjection of plasmids pIL-2 or pGM enhanced development of T cells that produce IL-2 and IFN-γ in response to challenge with a specific Ag, while cells that produce IL-4 were not significantly affected. These results indicate that the specific Th cell populations induced by a DNA vaccine can be altered by local expression of cytokines. Coexpression of IL-12 or IFN-γ promotes Th1 and down-regulates Th2 development, coexpression of IL-4 promotes Th2 and down-regulates Th1 development, and coexpression of IL-2 or GM-CSF largely enhance Th1 development and to a lesser extent Th2 development.

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FIGURE 3.

Effect of codelivery of various cytokine vectors on HBsAg-specific Th1/Th2 development in immunized animals. Animals were treated as described in the legend to Table I⇑. At 2 wk after immunization, pooled splenocytes from each group were stimulated with 30 μg/ml of HBsAg. Culture supernatants obtained 3 days after stimulation were collected for quantitation of IFN-γ, IL-2, and IL-4 by ELISA.

IgG isotypes to HBV DNA vaccination can be modulated by coinjection of various cytokine genes

The patterns of Ab isotypes produced in response to immunization are reliable indicators of the types of cytokines produced in vivo. IgG2a is produced as a consequence of Th1 cell activation and IFN-γ secretion, whereas IL-4 enhances the production of IgG1 and suppresses IgG2a (46, 47). We therefore measured anti-HBs IgG isotypes in the sera of mice treated with pS and the plasmid encoding various cytokines at week 8 after immunization, the time point at which peak Ab titers were achieved in vaccinated animals. As shown in Figure 4⇓, coexpression of IL-12 or IFN-γ resulted in a dramatic increase of anti-HBs IgG2a Abs, whereas titers of HBsAg-specific IgG1 Ab were decreased, indicating enhancement of Th1 and suppression of Th2 cell function in immunized animals. In contrast, mice treated with plasmids pS and pIL-4 showed an inhibition of anti-HBs IgG2a Ab, while the production of IgG1 Ab was increased, indicating suppression of Th1 and promotion of Th2 cell function in vivo. Animals coinjected with plasmids encoding IL-2 or GM-CSF showed a significant, five- to sevenfold, increase of anti-HBs IgG2a Ab, and a much lower increase of IgG1 Ab, indicating enhancement of Th1 cell function but no significant change in Th2 cells. We also analyzed serum samples from mice given injections of pS and the cytokine vectors at different thighs and found that the levels of IgG1 to IgG2a isotypes were not altered (data not shown).

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FIGURE 4.

Effect of codelivery of various cytokine vectors on IgG isotypes of anti-HBs Ab. Animals were treated as described in the legend to Figure 2⇑. Sera collected 8 wk after immunization were used to assay for the presence of IgG1 and IgG2a anti-HBs Abs. Concentrations of IgG1 and IgG2a anti-HBs Abs were measured from a standard curve generated from a serially diluted control Ab and expressed as U/ml. Data represent the mean titers ± SD of five animals per time point from one representative experiment of three performed.

CTL activities induced by HBV DNA vaccination are affected by codelivery of cytokine genes

It is well known that cellular immunity is highly dependent upon the types of cytokines produced by Th cells. Thus, we tested whether the CTL activity induced by HBV DNA vaccine was influenced by coexpression of cytokines. Splenocytes from mice vaccinated with plasmids pS with or without a cytokine vector and restimulated in vitro were analyzed for their ability to lyse syngeneic cells expressing a transfected HBsAg gene (P815/S). Coinjection of pIL-2, pGM, pIFN-γ, or pIL-12 augmented HBsAg-specific CTL activity, in which enhancement by pIFN-γ or pIL-12 was consistently greater than that mediated by pIL-2 or pGM (Fig. 5⇓A). In contrast, coinjection of pIL-4 substantially decreased the CTL activity as compared with that induced by pS without cytokine coexpression (Fig. 5⇓A). The CTL activity was HBsAg specific, since mice immunized with pcDNA3 alone did not induce any detectable HBsAg-specific lysis and no CTL activity was observed in any of the groups of mice when parental P815 cells were used as target cells (data not shown). The effector cells that lysed HBsAg-expressing transfectants in vitro were eliminated by depletion of CD8⁺ lymphocytes but not by depletion of CD4⁺ lymphocytes (Fig. 5⇓A). The different cytokine effect on the HBV DNA vaccine-induced CTL activity was further confirmed by assays using another HBsAg-transfected target cells, CT26/S, which produced more HBsAg than P815/S and was found to be more sensitive to the CTL assay. As shown in Figure 5⇓B, HBsAg-specific cytolysis was enhanced by coinjection of the IL-12 gene but suppressed by the IL-4 gene (Fig. 5⇓B). This cytolytic activity was blocked by an anti-CD8 mAb but not by an anti-CD4 mAb (Fig. 5⇓B). Taken together, these results indicate that the CTL activity primed by a HBV DNA vaccine in vivo is mediated by cells expressing CD4⁻CD8⁺ surface phenotype and the CTL activity can be enhanced or suppressed depending on the cytokine gene coexpressed.

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FIGURE 5.

Effect of codelivery of various cytokine vectors on HBsAg-specific CTL activities. Groups of BALB/c mice were treated with plasmid pS plus a cytokine vector or a control vector as described in the legend to Table I⇑. Three weeks after immunization, splenocytes were collected, cocultured for 4 days with irradiated P815/S transfectants, and used as effectors in the CTL assay. A, The specific cytolytic reactivity of unfractionated, CD4⁻-depleted, or CD8⁻-depleted effectors was tested on P815/S cells. B, The cytolytic reactivity of unfractionated effectors was tested on CT26/S cells in the presence of 10 μg/ml of anti-CD4 or anti-CD8 mAbs. The E:T ratio is 100:1 in all of these assays. Values of specific lysis are from one representative experiment of three performed and are presented as mean specific lysis of triplicate cultures ± SD.

In vivo tumor immunity induced by HBV DNA vaccination is enhanced by coinjection IL-12 gene but suppressed by coinjection of IL-4 gene

To determine the effect of phenotype changes of immune responses on protective efficacy in vivo, the ability of the pS DNA vaccine in combination with IL-12 or IL-4 gene to immunoprotect against transplantation of syngeneic HBsAg-transfected tumor cells was examined. Groups of three to five mice were vaccinated once with 100 μg of pS plus 100 μg of pcDNA3 or vectors encoding IL-4 or IL-12. Mice that received pcDNA3 alone served as a negative control group. Three weeks after vaccination, all mice were inoculated with 1 × 10⁵ HBsAg-expressing CT26/S or the parental CT26 tumor cells. All mice that received pcDNA3 and hence were not immune to HBsAg developed rapidly growing CT26 or CT26/S tumors within 2 wk after transplantation (Fig. 6⇓, A and E). Four of five mice immunized with pS + pcDNA3 and challenged with CT26/S showed an inhibition of tumor growth (Fig. 6⇓B). The protective efficacy was dramatically increased when the IL-12 gene was coinjected with plasmid pS; tumor growth was significantly suppressed, and two of five mice remained tumor free up to 60 days following tumor challenge (Fig. 6⇓D). The tumor immunity is HBsAg specific, since mice immunized with pS + pcDNA3 (Fig. 6⇓F) or pS + pIL-12 (Fig. 6⇓H) and challenged with the parental CT26 cells showed little protection. In contrast, vaccination with pS + pIL-4 offered little if any protection against CT26/S (Fig. 6⇓C) or CT26 (Fig. 6⇓G) challenge. These results demonstrate that the difference in phenotype of immune responses stimulated by DNA vaccination under influence of various cytokines has a significant effect on protective efficacy against tumor challenge.

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FIGURE 6.

Effect of codelivery of various cytokine vectors on growth of CT26/S and CT26 cells. BALB/c mice were immunized with: 200 μg of control plasmid pcDNA3 (A, E); 100 μg of pS + 100 μg of pcDNA3 (B, F); 100 μg of pS + 100 μg of pIL-4 (C, G); or 100 μg of pS + 100 μg of pIL-12 (D, H). Three weeks after immunization, 1 × 10⁵ CT26/S cells (A–D) or 1 × 10⁵ CT26 cells (E–H) were transplanted s.c. into all mice. Growth of the tumor was measured every second or third day. The tumor volume from each animal at various time intervals after transplantation is plotted.