Female C57BL/6 mice at the age of 6-8 wk were purchased from Shanghai Sippr-Bk Company and allowed to acclimate for at least 2 wk prior to use. The mice were housed in laminar airflow cages with free access to food and water ad libitum. Analyses of specimens from those animals revealed no evidence of exposure to viral, bacterial or parasitic pathogens during these studies.

The HBcAg-expression plasmid pHBc144 was constructed by inserting a gene encoding hepatitis B core antigen N’ end 144 amino acid into a vector (pcDNA3, Invitrogen), driven by the cytomegalovirus (CMV) immediate early promoter. The secreted HBcAg could be detected in the supernatant of cell culture transiently transfected with pHBc144[6]. Anti-HBc could be detected in the serum of Balb/c mice, which were injected with pHBc144 DNA plasmid.

The HBV core Ag peptides were synthesized and generously provided by Dr. Rafi Ahmed (Emory University, GA)

PBMCs were isolated from peripheral blood of healthy volunteers by density gradient centrifugation over Ficoll. Fresh whole blood was drawn, heparinized and gently mixed with one volume of RPMI1640. The diluted blood was layered over Ficoll and centrifuged at 800 g, for 20 min at 4°C. PBMC were collected from the interface between the plasma and the density gradient solution. After being washed in PBS, PBMCs were resuspended in RPMI 1640 supplemented with 5 mg/mL LPS, 200 mmol/L L-guatmine, 10% FCS, 20 mmol/L Hepes, 100 U/mL penicillin, and 100 μg/mL streptomycin, at a final cell concentration of 5 ×10⁶/mL. Then the PBMCs were cultured in 24-well plates for 6 h.

RNA extraction as well as RT-PCR analysis were performed as previously described[7]. Briefly, total RNA was purified using Trizol (Invitrogen Life Technologies) following the manufacturer’s instructions. A 4 μg aliquot of total cellular RNA was reverse transcribed using random hexanucleotide primers and the Superscript II preamplification system (Invitrogen Life Technologies). One-tenth of the cDNA obtained was amplified in a 20-pl reaction, using 1 U of Pwo DNA Polymerase Expand^TM High Fidelity PCR System (Boehringer Mannheim) and the reagents provided with the preamplification system. Thirty-five cycles of PCR amplification were performed at 94°C for 2 min, at 60°C for 1 min and at 72°C for 1 min; followed by a final extension at 72°C for 7 min. Ten microliters of the reaction products was analyzed on 1% agarose gel. The primer sequences are sense: 5’-GCGGAATTCATGAGAATTTCGAAACCACATTTGAG-3’; antisense: 5’-GCGCTCGAGAATCAATTGCAATCAAGAAGTGT-3’. The amplified cDNA was ultimately digested with EcoR I and Xho I and cloned into pcDNA3 and pET2c. The sequences of the IL-15 DNA insert were verified by sequencing with the dideoxy chain termination method (Bioasia Co., Shanghai). Sequences were analyzed by Vector NTI Suite software package (InfoMax).

Escherichia coli strain BL21(DE3) (Novagen) was transformed by the pET2c-IL-15 plasmid. The detailed protocol for production of fusion proteins was as follows. E.coli cells were grown to absorbance 0.6 at 600 nm, then expression was induced with 0.4 mmol/L isopropyl-D-thiogalac-toside for 4 h at 37°C. The bacterial cells were harvested, the cell pellet was suspended in 50 mL MTPBS lysis buffer (150 mmol/L NaCl, 16 mmol/L NaH₂PO₄, 4 mmol/L Na₂HPO₄, pH 7.3, 1% TritonX-100) per liter of culture volume. The cells were sonicated three times, 10 s each time (Sinifer cell disrupter B-30; Branson Sonic Power), and centrifuged at 15 000 g for 10 min at 4°C. The supernatant was collected for SDS-PAGE and Western blotting.

E.coli cells were cultured for an indicated period of time, lysed by adding 2X Laemmli SDS sample buffer and boiled for 5 min in a water bath. The proteins were resolved by 15% SDS-PAGE and transferred to a polyvinylidene difluoride membrane (Immun-blot PVDF; Bio-Rad). Non-specific binding sites were blocked by incubating the blot in blocking buffer (5% nonfat dry milk in wash buffer) for 1 h at room temperature. The blot was washed three times (5 min each time) with wash buffer (0.5% Tween 20, 200 mmol/L NaCl, and 50 mmol/L Tris, pH 7.5), and incubated with the appropriate primary Ab overnight at 4°C. The blot was then washed three times (5 min each time) and incubated with the corresponding secondary Ab-HRP conjugate for 1 h at room temperature. The blot was washed three times again (5 min each time), and proteins were detected.

For CTLL-2 bioassays, COS cells were transfected with 30 μg of IL-15 expression constructs following a standard calcium-phosphate-mediated transfection protocol[7]. For these studies roughly 2-3 × 10⁵ adherent COS cells/well were transfected in six-well 35-mm plates. After 24 h, the medium was replaced with fresh complete DMEM, supernatants were harvested 72 h after the transfection for assay.

Aliquots (100 μL) of the supernatant and serial two-fold dilution were added to 96-well microtiter plates containing 1 × 10⁵ CTLL-2 cells in a final volume of 200 μL. The cultures were incubated for 18 h at 37°C and pulse-labeled for another 6 h with 0.5 μCi [³H] thymidine. Thymidine incorporation was quantified by liquid scintillation spectrometry[8] (BD). A standard curve was plotted from proliferation induced by standard murine rIL-2.

Plasmids were amplified in Escherichia coli and plasmid DNA was purified by using anion exchange column (Endofree plasmid purification kit, Qiagen, Chatworth, Calif) according to the manufacturer’s instructions. The plasmid DNA was dissolved in sterile physiological saline, and the final concentration was 1 μg/mL.

C57BL/6（H-2^b）mice were vaccinated at the age of 6-8 wk by injection of DNA in 0.9% saline (w/v), into the quadriceps of each hind limb as previously described[9]. Injection volume was 50 μL per leg and total DNA dose was 50 μg per leg. On d 30, all mice were booster-immunized with the same DNA. On d 0, 8, 14, 30, 90 after priming and d 90 after boostering, blood samples were collected from each mouse by orbital bleeding (Figure 1). Serum was separated from each blood sample and used for measurement of the anti-HBcAb titer. On the other hand, mouse blood was obtained using heparin as an anticoagulant. The blood was diluted with RPMI 1640. PBMCs were obtained by density gradient centrifugation over HISTOPAQUE-1083 (Sigma), washed three times with RPMI 1640 and counted afterward. The spleens were removed for the measurement of cytokine production.

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Figure 1 Immunization schedule of pHBc144 and pIL-15 plasmid.

Anti-HBc antibody was titrated with ELISA kit (Shanghai Infectious Diseases’ Hospital). Each serum sample was made in a series of three-fold dilution. Goat anti-mouse IgG-HRP conjugate (The Binding Site Limited Co, England) was used as the secondary antibody and O-pheylenediamine (OPD) was used as a substrate. The plates were read with Titerek Multiskan MCC/340 at A₄₅₀.The wells with an absorbance of ≥ 0.1 (above background, 0.020) were scored as positive.

For intracellular cytokine detection of IFN-γ, PBMCs and single suspension of splenocytes were plated in a 96-well plate (Costar) at a density of 1 × 10⁶ cells/well in a final volume of 200 μL. Cells were stimulated with peptides for 5 h. Brefeldin A (final concentration 2 μg/mL) was added to the cultures during stimulation. At the end of the incubation, the cells were washed twice and stained for 30 min at 4°C with phycoerythrin (PE)-conjugated anti-mouse CD8 (BD Bioscience) in FACS buffer [2% FCS, 0.1% sodium azide in phosphate buffered saline (PBS)]. Cells were collected, washed and incubated in permeabilization buffer for 20 min on ice. The permeabilized cells were washed and stained with fluorescein isothiocyanate (FITC)-conjugated anti-mouse IFN-γ (BD PharMingen, San Diego, CA) for 30 min. Cells were washed, fixed with 2% (w/v) paraformaldehyde, and acquired with a FACSCalibur flow cytometer (Becton Dickinson, Boston, MA). Forward and side scatters were collected on a linear scale, while the PE and FITC signals were collected on a 4-decade log scale. Overlaps of emission spectra were electronically compensated. The debris were eliminated using the threshold on forward scatter and 100 000 events were acquired with CellQuest software (BD, Biosciences).

Statistical analyses of data were performed using SPSS 10.0 for windows (SPSS, Inc., Chicago, IL).

PBMCs were isolated from peripheral blood of healthy volunteers by density gradient centrifugation over Ficoll. A total of 5 × 10⁵ PBMCs were transferred into 24-well plates and cultured for 6 h with 5 μg/mL LPS. RNA extraction and RT-PCR were performed (Figure 2). The complete human IL-15 cDNA (GenBank accession No. U14407) was 518 bp long and was comprised of 146 bp signal peptide (IL-15sp) and a IL-15 mature peptide gene sequence (IL-15mp).

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Figure 2 Electrophoretogram of PCR products. The human IL-15 coding sequence (GenBank U14407) was RT-PCR amplified from human PBL mRNA stimulated with LPS.

Amplified cDNA was ultimately digested with EcoR I and Xho I and cloned into pcDNA3. The sequences of IL-15 DNA insert were confirmed and this plasmid was named pIL-15.

For generation of the IL-15 prokaryotic expression construct, the IL-15 mp coding sequence was PCR amplified from pIL-15. The resulting fragments were digested with EcoR I and Xho I and cloned into pET2c named pET-IL-15 (data not shown) .

Escherichia coli strain BL21(DE3) (Novagen) was transformed by the pET-IL-15 plasmid. After induction by IPTG and temperature, the supernatant of bacteria lysates was taken for SDS-PAGE and Western blotting. IL-15 fragments could be expressed in pET-32c vector and its molecular weight was M_r 26 000 (Figure 3).

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Figure 3 SDS-PAGE and Western blotting of recombinant polypeptides. M: polypeptide molecular marker; lane 1: BL21; lane 2: pET32c ; lane 3: pET-15 uninduced ; lane 4: pET-15 induced.

The IL-15 was cloned into the pcDNA3 vector with CMV promoter (Invitrogen) and transfected into COS cells. Forty-eight hours after transfection, the COS supernatants were collected and subjected to CTLL-2 proliferation assay. CTLL-2 cell ³H-TdR cpm was related to the dilution fold of the COS supernatant. When the dilution fold of transfection supernatant increased, the incorporation of ³H-TdR cpm decreased. The incorporation ³H-TdR cpm of pIL-15-transfected COS supernatant was higher than that of vector-tansfected COS supernatant (Figure 4). So we could detect IL-15 activity in the supernatants of COS cells transfected with pIL-15.

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Figure 4 IL-15 bioactivity in supernatants of cells transfected with pIL-15 expression plasmid. COS cells were transfected with pIL-15 (▲) expression constructs or vector pcDNA3 (×), and the supernatants were collected 72 h after transfection. The supernatants were tested by CTLL-2 bioassay to measure IL-15 bioactivity. IL-2 (5 × 10³ U/ mL) was used as a positive control.

Both primary and memory antigen-experienced CD8⁺ T cells rapidly upregulated cytokine production after encountering cells displaying their cognate peptide ligand in association with a major histocompatibility complex class I molecule. We used ICCS to identify cytokine-producing HBc-specific CD8⁺ memory T cells directly ex vivo after DNA inoculation prior to this study, but it was unclear how quickly these responses developed when IL-15 as a gene adjuvant was coinjected with HBc DNA vaccine. Therefore, we used ICCS to directly enumerate antigen-specific effector CD8⁺ T cells in the spleens of mice at various time points after DNA immunization (pHBc144 and/or pIL-15). The average percentage of peptide-specific CD8⁺ T cells detected in three mice analyzed at each time point for each mode of immunization is shown in Figure 5A.

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Figure 5 Kinetics of antigen-specific CD8⁺ T-cell response in spleen (A) and PBMCs (B) following DNA immunization. Mice were immunized with pHBc144 plasmid DNA or vector DNA intramuscularly. At various time points, splenocytes and PBMCs were harvested, stimulated for 5 h with specific peptide, double stained with anti-CD8a-PE and anti-IFN-γ-APC. A total of 1 × 10⁵ events were acquired by a flow cytometer. The indicated values on the lines of the figure (plotted on a log₁₀ scale) reflect the percentage of peptide-specific CD8⁺ T-cells to total CD8⁺ T-cells in the spleens or PBMCs (about three mice/group at each time point). The cut-off value was 0.1%.

Seven days after-immunization, antigen specific CD8⁺ T cells were present at a frequency of less than 0.1% in all of the vaccinated mice (both pHBc144 + pcDNA3 group and pHBc144 + pIL-15 groups), and therefore accurate analysis of IFN-γ production was not possible (Figure 5A). By d 14, 2.64% ± 1.07% of the HBcAg specific CD8⁺ T cells primed by pHBc144 + pIL-15 DNA vaccination secreted IFN-γ, and at 30 d after-immunization, 0.39% ± 0.21% of the antigen-specific cells were positive. A similar transition was observed in CD8⁺ T cells primed by pHBc144 + pcDNA3 DNA vaccination (2.26% ± 2.50% on d 14 and 0.42% ± 0.06% on d 30). Thus, the cytokine expression patterns of HBcAg-specific T cells were similar regardless of the pIL-15 immunization.

These proportions decreased gradually, by d 90 post-priming, 0.48% ± 0.36% of the antigen-specific CD8⁺ T cells induced by pHBc144 in pIL-15 co-immunized group secreted IFN-γ, while 0.16% ± 0.04% of the antigen-specific CD8⁺ T cells in pHBc144 and pcDNA3 co-immunized group were IFN-γ⁺CD8⁺ T cells (Figure 6).

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Figure 6 Percentages of HBc specific CD8⁺ T cells in the spleen after primary (A) and secondary immunization (B) and in PBMCs after primary (C) and secondary immunization (D) detected by ICCs directly ex vivo at various time points. C57BL/6 mice were immunized with pHBc144 DNA or pIL-15 DNA and boosted at d 30 later. The splenocytes and harvested at various time points. Then the cells were cultured for 5 h in the absence or presence of the HBcAg peptide and double stained with anti-CD8a-PE and anti-IFN-γ-APC. CD8⁺IFN-γ⁺cells are enclosed in ovals, and the numbers in the upper right quadrants represent the percentage of CD8⁺IFN-γ⁺ cells to total CD8⁺ T cells. The data from each group at each time point represent mean ± SD (n = 3). Naïve mice were used as negative controls. The cut-off value was 0.1%.

After being boosted, the HBc Ag specific T cells expanded rapidly, and by d 90 post-boost the specific memory cells could still be detected, sustaining a higher level than that for the first response. On d 90 after boosting, 0.28% ± 0.04% of the antigen-specific CD8⁺ T cells induced by pHBc144 in pIL-15 co-immunized group secreted IFN-γ, while 0.11% ± 0.04% of the antigen-specific CD8⁺ T cells in pHBc144 and pcDNA3 co-immunized group were IFN-γ⁺ CD8⁺ T cells (Figures 6A and B ).

For examining the kinetics and distribution of antigen specific CD8⁺ T-cells in the non-lymphoid tissue induced by DNA vaccination, we separated the lymphocytes from blood. The kinetics of HBcAg-specific CD8⁺ T cells in the circulation stimulated with pHBc144 showed a similar pattern with that of the spleen (Figure 5). However the number of HBcAg-specific CD8⁺ T cells in PBMCs was lower than that in the spleen at the same time point. Eight days after DNA vaccination, HBcAg-specific CD8⁺ T cells were detectable. By d 14 the number of IFN-γ⁺ CD8⁺ T cells in PBMCs reached the peak and then dropped gradually. These proportions decreased gradually until d 90 post-priming, 0.17% ± 0.05% of the antigen-specific CD8⁺ T cells induced by pHBc144 in pIL-15 co-immunized group secreted IFN-γ, while 0.22% ± 0.01% antigen-specific CD8⁺ T cells in pHBc144 and pcDNA3 co-immunized group were IFN-γ⁺ CD8⁺ T cells.

On d 90, after boosting, 0.20% ± 0.13% of the antigen-specific CD8⁺ T cells induced by pHBc144 and pIL-15 co-immunized group secreted IFN-γ, while 0.12% ± 0.13% antigen-specific CD8⁺ T cells in pHBc144 and pcDNA3 co-immunized group were IFN-γ⁺ CD8⁺ T cells (Figure 5B, Figure 6 C and D ). The antigen-specific memory cells still could be detected directly ex vivo in the circulation on d 90 post-boost (Figure 5B, Figure 6D).

At the same time point, we checked the total CD4⁺ and CD8⁺ cell percentage (Table 1), IL-15 had no big effect on the ratio of CD4⁺ and CD8⁺.

The HBcAg specific humoral immune response induced by pHBc144 was detected by ELISA for anti-HBc antibody in serum. The plates were read with Titerek Multiskan MCC/340 at A₄₅₀.The wells with an absorbance of ≥0.1 (above background, 0.020) were scored as positive.

Four weeks after the first pHBc144 immunization, all mice underwent anti-HBc seroconversion. The mean titer of anti-HBc IgG in pHBc144 + pIL-15 group reached 2^9.81 ± 2^0.9 on d 30. After being boosted, the mice produced a higher titer of anti-HBc IgG (2^9.81 ± 2^0.9) than that of the primary response, and anti-HBc maintained at that level (2^9.81 ± 2^0.9) on d 90. The titer of anti-HBc IgG2a in pHBc144 + pIL-15 group (2^9.81 ± 2^0.9) was slightly higher than that of pHBc144 + pcDNA3 group (2^8.22 ± 2^0.9), but there was no significant difference (P > 0.05).