Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses.

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg+alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-gamma and IL-4, but neither IFN-gamma nor IL-4 was detected in the HBsAg+alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.

[Expression of ORF2 protein of HEV genotype IV in Hansenula polymorpha].

Hepatitis E, an acute infectious disease transmitted via the fecal-oral route, is caused by hepatitis E virus. However, no effective treatment currently exists for hepatitis E, and the only epidemic control approach is vaccination. But so for there are no commercial vaccine for hepatitis E available in the world. To find a new expression system to develop recombinant hepatitis E vaccine, in this study the expression system of methylotrophic yeast Hansenula polymorpha was used to express the gene encoding amino acid 112 - 607 of the open reading frame 2 (ORF2) of hepatitis E virus (HEV) genotype IV. In order to achieve high expression level, the coding sequence was optimized according to codon usage bias of Hansenula polymorpha and synthesized through overlapping PCR. Subsequently the gene was subcloned into the multi-copy expression vectors of Hansenula polymorpha, which include pDGXHP1.0 (MOX promotor), pDGXHP2.0 (MOX promotor) and pDGXHP2.1 ( FMD promotor). The series of one-copy and multi-copy recombinant plasmids were transformed into ATCC26012(Ura3-) by electroporation. The transformants were cultured in selection media MDL and screened for the existence of foreign gene by PCR. Then the strains were induced in MM media and the expression products were detected by SDS-PAGE, ELISA and Western blot assays to select the high-level expression strains. The result of SDS-PAGE showed that the HEV ORF2 expression product was accumulated up to 12% of total cellular protein and its molecular weight is 56kD. The expression product showed high immunoreactivity detected by ELISA and the highest titer is 1:2048. The result of Western blot demonstrated that the expression product could be specifically recognized by the polyclonal antibody against HEV. The successful expression of HEV ORF2 protein in Hansenula polymorpha provides foundation for the further development of recombinant subunit vaccine against hepatitis E.