ODN (BW006) was provided by Yunnan Wosen Biotechnology Company. BW006 was synthesized with the sequence of 5'-tcgacgttcgtcgttcgtcgttc-3' and followed by sulphurization. Lipopolysaccharide (LPS) level in BW6 was less than 0.5 ng/mg by Limulus assay.
The awd2 subtype HBsAg was expressed in Hansenular polymorpha yeast and produced by Yunnan Wosen Biotechnology Company. The antigen had a concentration of 0.236 mg/ml and purity of greater than 99.0% by high-performance liquid chromatography (HPLC) and silver staining. LPS level in the antigen was less than 10 EU/ml by Limulus assay.
The awd2 HB vaccine was made with alum adjuvant and provided by Yunnan Wosen Biocenology Company. The concentrations of HBsAg and alum were 24 μg/ml and 0.5 mg/ml respectively.
Reagent for the detection of cytokine expression, anti-HBs and leucocyte surface molecules
The reagents for cytokine detection were provided by Panomics Company and used per protocol of each cytokine. The Anti-HBs was detected with automated chemiluminescent microparticle immunoassay, Abbott ARCHITECT® anti-HBs. The reagents for the detection of leucocyte surface molecules were provided by BioLegend Company.
Evaluation of cytokine mRNA
Splenocytes were isolated from 6- to 8-week-old female BALB/c mouse (H-2d, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China). Single cell suspensions (1 × 106/ml) were prepared with NycoPrep ™ 1.077A (AXIS-SHIELD PoC AS, Oslo, Norway) and suspended in 1640 medium (RPMI 1640, Hyclone) with penicillin-streptomycin (final concentrations of 100 U/ml and 100 μg/ml respectively) (Sigma, Irvine, U.K.). 0.1 ml of the single cell suspension and 0.1 ml BW006 (final concentration of 5 μg/ml) were added to round-bottom 96 wells microtiter plates and cultured at 37°C with 5% CO2. The mRNA expression of IL-2, IL-4, IL-5, IL-10, IL-12a, IL-12b and IFN-r were analyzed at 1, 3, 6 and 12 hours respectively, each in triplicate.
Evaluation of in vivo HBsAb production
100 6- to 8-week-old female BALB/c mice (H-2d, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were evenly divided into 10 groups and respectively received a single intramuscular injection (i.m.) into left tibialis anterior muscles, of 0.1 ml solution containing 50 μg alum, 20 μg BW006, 2 μg recombinant HBsAg, 2 μg recombinant HBsAg with 1.25 μg BW006, 2 μg recombinant HBsAg with 5 μg BW006, 2 μg recombinant HBsAg with 20 μg BW006, 2 μg recombinant HB vaccine, 2 μg recombinant HB vaccine with 1.25 μg BW006, 2 μg recombinant HB vaccine with 5 μg BW006 and 2 μg recombinant HB vaccine with 20 μg BW006. The duplicate aliquots of plasma were collected weekly from 1-4 weeks, one for HBsAb testing and the other one for HBsAb titration if it was HBsAb positive. The antibodies against HBsAg were detected and quantified in triplicate for each specimen with Abbott ARCHITECT® anti-HBs reagent as described above. The average value of the triplicate equal to or greater than 10 mIU/ml was considered as positive HBsAb conversion. The numbers of serum HBsAb conversion and titer of each positive serum were calculated.
Detection of leucocyte surface molecules CD80 and CD86
Sixteen 6- to 8-week-old female BALB/c mice (H-2d, National Institute for the Control of Pharmaceutical and Biological Products, Beijing, China) were evenly divided into 4 groups and given a single subcutaneous injection of 0.1 ml solution containing 4 μg recombinant HBsAg, 20 μg BW006, 4 μg recombinant HBsAg with 20 μg BW006, or NS, respectively. The mice of all four groups were euthanized 12 hours after the injection and dendritic cells were separated by auto-MACs kit (Miltenyibiotec Com.) per manufacturer's instructions. Single cell suspensions (1 × 106/ml) were prepared with NycoPrep ™ 1.077A (AXIS-SHIELD PoC AS, Oslo, Norway). Cell concentration of dendritic cells were adjusted to 1 × 105/ml and co-culvated with rat-anti mouse APC-CD11c, CD80-FITC and CD86-PE antibodies from BioLegend company at room temperature for 20 minutes. The cells were centrifuged at 400 g for 10 minutes and re-suspended with 500 μl of 10% paraformaldehyde. The expression levels of the surface molecules CD80 and CD86 were detected and quantified by flow cytometry.
Statistical analysis was performed by SAS version 9.2 (SAS Institute Inc., Cary, North Carolina, USA). Categorical variables were compared by Chi's square test. If 50% of cells had expected counts less than 5, Fisher's Exact test results would be used. Continuous variables were expressed as mean with standard deviation and compared by Student's t-test or Mann Whitney U test as appropriate. All statistical tests were two-sided, and p values less than 0.05 were considered statistically significant.