Fermentation, purification and immunogenicity evaluation of hepatitis E virus-like particles expressed in Hansenula polymorpha

Abstract:

To develop a new recombinant hepatitis E vaccine, we used Hansenula polymorpha expression system to express recombinant hepatitis E virus-like particles (HEV VLPs), to construct a recombinant engineered strain HP/HEV2.3. The fermentation conditions and purification process were studied next. The first working seed lots were fermented in liquid culture, and the fermentation products were collected, then crushed, clarified, purified by ultrafiltration, silica gel adsorbed and desorbed, concentrated by ultrafiltration, purified by liquid chromatography and sterilized by filtration. The purity reached 99% with a yield of 33%. Electron microscopy analysis revealed that both the purified recombinant HEV VLPs from HP/HEV2.3 and natural hepatitis E virus particles appear identical of being 32 nm. The resulting DNA sequence obtained from VLPs is identical to the published HEV sequence. The SDS-PAGE analysis has revealed that the protein molecular weight of the HEV VLPs is 56 kDa, and the expression product HEV VLPs were accumulated up to 26% of total cellular protein. The expression level is 1.0 g/L. Western blotting, enzyme-linked immunosorbent assay (ELISA) results of the protein and ED50 of the vaccine showed that the HEV VLPs have good antigenicity and immunogenicity. In summary, the recombinant HEV VLPs from Hansenula polymorpha can be used in the manufacture of a new genetically engineered vaccine against hepatitis E.