Preparation and immunogenicity of a Pichia pastoris-derived hepatitis B vaccine containing preS1, preS2 and S epitopes / 生物工程学报

The preparation process and immunogenicity of a novel hepatitis B vaccine containing preS1, preS2 and S epitopes were investigated in this study. A Pichia pastoris stain GS115-SS1S2 harbouring two chimeric HBsAg gene constructs, SS1 and SS2 was cultivated by high-density fermentation. 300-600 mg/L of the expression level was achieved through 48-72 h methanol induction. SSIS2 antigen was extracted and purified by silica adsorption, HIC and SEC to 99% purity from the harvested cells. 82 mg purified antigen could be achieved from one liter of fermentation culture. The immunogenicity of the purified antigen was evaluated in NIH mice. Three groups of female NIH mice, 14-16 g in weight, were injected once intraperitoneally with 2.5, 0.625, 0.156 microg of each of the two vaccines: SS1S2 or a commercially available S vaccine. Part of the mice were bled in 30 days after injection to compare the ED50 of the two vaccines. For the SSIS2 vaccine, the ED50 is 0.46, 0.29 and 0.84 microg respectively for the preS1, preS2 and S antigens. For the S vaccine, the ED50 is 0.99 microg for the S antigen. Another part of the mice were bleed in 7 or 14 days to detect preS1, preS2 and S antibodies. Higher ratios of mice were seroconverted for preS1 and preS2 antibodies as compared to the S antibody in these two time points. These results suggest that the SS1S2 vaccine may be more immunogenic than the conventional S vaccines.

Simultaneous expression of modified hepatitis B surface antigen fusion polypeptides containing preS1, preS2 epitopes in Pichia pastoris / 生物工程学报

At present time, the widely used hepatitis B virus( HBV) vaccines consist of only the small hepatitis B surface antigen expressed in yeast or CHO cells. The frequency of non-responders to these vaccines has increased the demand for a more immunogenic vaccine. Some studies have suggested that the addition of preS region to the vaccine will improve its efficacy. However, the large protein (L) containing the whole preS region can not be effectively expressed in vitro. To overcome this problem, two chimeric contructs, SS1, surface gene containing preS1 region at C-terminus and SS2, surface gene containing preS2 region at C-terminus, were constructed and effectively expressed in our previous studies. Here we further constructed an expression vector containing both SS1 and SS2 expression cassettes by separation and ligation the SS2 cassette to a linearized SS1 expression vector pAO815-SS1. The recombinant vector was transformed into Pichia pastoris GS115 by electroporation. A high-level expression strain (GS115-SS1S2) was established by primary screening for His+ transformants and further analysis for induction products. ELISA results demonstrated that the expressed protein had S, preS1 and preS2 antigenicities simultaneously. Western blotting showed that the product can bind to all of the three antibodies, anti-S, anti-preS1 and anti-preS2. The expression protein was present in the form of particles of 20-35 nm diameter and the yield of recombinant particles reached 300-600 mg/L by fermentation. The SS1 and SS2 polypeptides kept intact in purified particles, suggesting that the stability of preS region has been significantly improved.