The present study was conducted in the Department of Microbiology and Immunology of the Medical College of the Jinan University (Guangzhou, China). The Ethics Committee of The First Affiliated Hospital of the Jinan University (Guangzhou, China) approved the animal procedures and the experimental protocol.

Based on the CDS sequence of the preS1-preS2-S gene (GI:157091234), designed the primers 5′-CAGCTAGCATGGGAGGTTGGTCTTCCAAA-3′ upstream) (NheI) (Takara Bio Inc., Otsu, Japan) and 5′-GGCGGAAGCTTAATGTATACCCAAAGAC-3′ (downstream) (HindIII) with appropriate restriction endonuclease sites. Hepatitis B DNA extraction was obtained from the hepatitis B surface antigen-positive serum (The First Affiliated Hospital of the Jinan University) using phenol/chloroform extraction methods and used as a template to amplify HBV preS1 preS2 S region. The coding sequences of these GM-CSF fragments were synthesized using PCR from pORF-GM-CSF using specific primers, upstream: 5′-CCAAGCTTGGTGGCGGTGGAAGCGGCGGTGGCGGAAGCGGCGGTGGCGGCAGCTGGCTGCAGAGCCTGCT-3′ (HindIII and Linker), downstream: 5′-CGGAATTCTCACTCCTGGACTGGCTC-3′ (EcoRI), and cloned into pVAX1 using the standard cloning techniques. PCR and restriction endonuclease assay were used to screen and identify positive clones. DNA sequencing analysis (Sangon Biological Engineering and Technology and Service Company, Shanghai, China) of the recombinant pVAX1-L-GM identified the sucessful constructions of recombinant plasmid pVAX1-L-GM.

L-02 cells were digested with 0.25% trypsin and diluted to 2×10⁵ cells/ml and plated to 6-well plates with 2 ml medium per well. Then, when cells were 70–80% confluent, 4 μg purified plasmid were transfected into the prepared cells using 8 μl Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA).

Non-transfected cells were considered as the blank, while pVAX1-transfected cells as the negative comparison. Immunocytochemical staining was performed according to the manufacturer’s instructions (Boster Biological Technology, Ltd., Wuhan, China), and mouse anti-HBsAg antibody was used as the primary antibody.

Western blot analysis of fusion proteins was performed according to the standard procedure. The purified protein was separated on 12% SDS-PAGE and transferred to nitrocellulose membrane. Anti-HBsAg mAb antibody (Beijing Biosynthesis Biotechnology Co., Beijing, China) at a dilution of 1:1000 or Anti-GM-CSF mAb was used as the primary antibody to detect the presence of protein. Blots were developed using the ECL (Thermo Fisher Scientific, Inc., Rockford, IL, USA) method with HRP-labeled rabbit anti-mouse IgG at a dilution of 1:5000.

Double-antibody sandwich ELISA (DAS-ELISA) was used to detect the GM-CSF protein level according to the manufacturer’s instructions (R&D Inc., Minneapolis, MN, USA). The results were presented as the mean ± standard deviation (SD), and statistically significant differences between values were analyzed using the SPSS 13.0 software. P<0.05 was considered to indicate a statistically significant difference.

Female BALB/c mice (n=30; 6–8 weeks old) were purchased from the Experimental Animal Center of the Jinan University (Guangzhou, China) and divided equally into 3 groups (n=10/group). Mice were immunized intramuscularly individually with pVAX1-L-GM, pVAX1 and pVAX1HBsAg. The mice were then injected with a dose of 100 μg/100 μl plasmid pVAX1-L-GM, pVAX1 and pVAX1HBsAg, 3 times every second week. Blood was obtained from the tail each week after immunization. The spleens of each mouse in the vaccinated groups were removed aseptically at week 13 after the first immunization.

After the first-immunization, serum was collected every week using the tail vein bleeding method. Absorbance at 450 nm was measured in a microplate reader, according to the manufacturer’s instruction of the Mouse HBsAb ELISA kit (Wuhan EIAab Science Co., Ltd., Wuhan, China). Levels of serum antibody in immunized mice were monitored for 12 weeks.

Lymphocyte proliferation of immunized mice was measured by MTT assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]. At week 13 following the first immunization, mice were sacrificed, their spleens were aseptically removed and splenocytes were prepared as single-cell suspensions. The cells were cultured in triplicate using 96-well round-bottom plates at 2×10⁷ cells per well. RPMI-1640 (Gibco, Paisley, UK) supplemented with 10% fetal calf serum (FCS) was added to each well, and stimulated with HBsAg at a final concentration of 10 μg/ml. Lymphocytes stimulated with the medium alone were used as the negative control. The cells were incubated for 48 h at 37°C in a humid atmosphere of 5% CO₂, then 20 ml of MTT (5 mg/ml) (Sigma-Aldrich, Shanghai, China) was added to each well. Following additional incubation for 4 h, the supernatant was carefully aspirated and 200 μl of dimethylsulfoxide (DMSO) was added into each well and absorbance of the soluble formazan was measured at 570 nm using an automatic micro-plate reader (Bio-Rad, Hercules, CA, USA).

Splenocytes obtained from mice at week 13, following the first immunization were cultured in 24-well plates with complete culture RPMI-1640 medium [with 10% FBS, 50 μM 2-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, 100 units of penicillin per ml and 100 μg of streptomycin per ml]. Complete culture RPMI-1640 medium was used containing 5 μg/ml Concanavalin A (Dingguo Biotech, Beijing, China) and 10 U/ml of IL-2 (Pepro Tech, London, UK) to culture splenocytes in vitro for 2 days as the effector. The stimulator cells, harvested from naive mice, were pulsed with the final concentration of 20 μg/ml of HBV-specific peptide for 4 h at 37°C in 5% CO₂, and were then treated with 80 μg/ml mitomycin C for another 2 h. The cells were washed extensively with RPMI-1640 medium. The effector cells (4×10⁷ cells) were incubated with stimulator cells at an effector-stimulator ratio of 10:1 for 7 days in culture medium containing 10 U/ml recombinant IL-2 (Peprotech, Rocky Hill, NJ, USA) at 37°C in 5% CO₂. The target cells were prepared by P815 cells (mouse mastocytoma cell line, Shanghai Institute of Biochemistry and Cell Biology of the Chinese Academy of Sciences) pulsed with HBV-specific peptide for 4 h at 37°C in 5% CO₂. The cytotoxic activity was tested by non-radioactive LDH release assay. The assays were performed in triplicate with 1×10⁴ target cells/well incubated with effector cells at various effector cell/target cell (E:T) ratios of 100:1, 50:1, 25:1 and 12.5:1 in 96-well round-bottom plates, according to the Non-Radioactive Cytotoxicity Assay Kit (Promega, Madison, WI, USA). The absorbance values from the supernatants were recorded at 490 nm using an ELISA microplate reader.

At week 13 following immunization, the mice were sacrificed and their spleens were removed aseptically. Phosphate-buffered saline (PBS) buffer (0.1 mmol/l) was used to wash the spleen cells and cell suspension was collected. The CD4⁺/CD8⁺ detection kit (Beckman Coulter, Inc., Brea, CA, USA) required the volume of 100 μl of each sample intake, in order to detect the number of CD4⁺, CD8⁺ molecules on the surface of spleen T cells using the Epics XL flow cytometry (Beckman Coulter, Miami, FL, USA).

The splenocytes of immunized mice were cultured following the same procedure in the proliferation assays for 72 h. Following incubation, the supernatant from each well was removed for evaluation of secreted IFN-γ and IL-2 levels using ELISA. The concentrations of IFN-γ and IL-2 in the culture supernatant were measured using murine cytokine ELISA kits (R&D Systems, Minneapolis, MN, USA). The limit of the detection was 2 pg/ml.

Measurement data show the mean ± SD. The statistical software SPSS was used to perform statistical analysis. Differences between groups were analyzed using stochastic analysis of variance. P<0.05 was considered to indicate a statistically significant difference.

To determine whether or not the recombinant plasmid pVAX1-L-GM was expressed in vitro, L-02 cells were transiently transfected with pVAX1-L-GM or pVAX1 and their expression at a protein level was detected using western blot analysis and ELISA assay, respectively. Western blot analysis indicated a protein level of ∼64 kDa, as expected (Figs. 1 and 2). Expression values of GM-CSF proteins in the pVAX1 control group were 1.382±0.081 pg/ml and 1.382±0.081 pg/ml (mean ± SD, n=3). While the expression values of the GM-CSF proteins of the pVAX1-L-GM-transfected group were 153.073±4.20 and 193.124±3.943 pg/ml, compared to the negative control groups (cell control and pVAX1), the results were considered statistically significant (P<0.05) (Fig. 3).

To assess the effect of the pVAX1-L-GM DNA vaccine on the humoral responses in mice, blood samples were collected using the tail vein bleeding method each week following the first immunization, and the sera were isolated. The presence of anti-HBsAg-specific antibodies in sera was analyzed by ELISA. Specific antibody response was detectable in the pVAX1-L-GM and pVAX1HBsAg groups (Fig. 4). Statistical analysis of antibody levels of pVAX1-L-GM and pVAX-1HBsAg groups was performed and an enhanced antibody response was observed in the pVAX1-L-GM group, and the difference was statistically significant (P<0.05). The pVAX1-L-GM group produced antibody 2 weeks earlier than the control plasmid pVAX1 and pVAX1HBsAg groups.

To determine whether or not the pVAX1-L-GM DNA vaccine influenced cell-mediated immunity, a single-cell suspension of lymphocytes was prepared from immunized mice at week 13 after immunization. As shown in Fig. 5, mice immunized with pVAX1-L-GM elicited the highest level of splenocyte T-cell proliferation compared to the pVAX1HBsAg and pVAX1 groups (P<0.05).

In the protection against or eradication of viruses or other intracellular pathogens, specific cytotoxic responses have been previously demonstrated to be a key factor. To analyze the ability of recombinant pVAX1-L-GM to enhance the HBsAg-specific CTL response, splenic cells, derived from the immunized mice 13 weeks after immunization, were restimulated specifically by naive mice splenocytes pulsed with HBsAg-specific peptides in vitro for 7 days. P815 cells pulsed with HBsAg-specific peptides were used as target cells. The cytotoxic activity was tested using non-radioactive LDH release assay and the specific lysis rates are shown in Fig. 6. HBsAg-specific CTL was detectable in the mice immunized with the HBV DNA vaccine pVAX1-L-GM plasmids compared to the pVAX1HBsAg or pVAX1 groups at the E/T ratio of 100:1 (P<0.05). The specific CTL activities increased significantly in the pVAX1-L-GM and pVAX1HBsAg groups compared to the pVAX1 group, while the strongest CTL response was detected at the E/T ratio of 100:1 (P<0.01). The results demonstrated that cellular immunity was markedly enhanced by pVAX1-L-GM DNA and pVAX1HBsAg DNA vaccine plasmids.

To evaluate the subsets of T cells, total T cells were isolated at week 13 following immunization and re-stimulated in culture with HBsAg. These cells were then analyzed using FACS with a gate set on CD4⁺ and CD8⁺ T cells. As shown in Fig. 7, the number of CD4⁺ and CD8⁺ molecules on the surface of spleen T cell produced by the pVAX1-L-GM group was higher compared to the pVAX1HBsAg group, and the difference was statistically significant (P<0.05), indicating that the GM-CSF gene enhanced cell immune function.

We quantified the production of the cytokines IFN-γ and IL-2 released from splenocytes from immunized mice re-stimulated with HBsAg in vitro. Mice immunized with DNA vaccine pVAX1-L-GM elicited a significant enhancement of IFN-γ and IL-2 production, even significantly higher compared to the pVAX1HBsAg group (P<0.05) (Fig. 8). This finding suggested that this DNA vaccine resulted in stronger Th1-type cell immune response.