Chronic hepatitis B (SHBs antigenemia for more than 6 months) develops in up to 5–10% of infected healthy adults and up to
90% of newborns.18,29 After persisting hepatitis B infection cirrhosis of the liver eventually develops. Even without preceding cirrhosis, the
development of hepatocellular carcinoma (HCC) is possible.31 It has been estimated that about 0.5% of all people infected with HBV develop HBV-associated HCC 20–40 years after infection.32 Thus, about 25% of chronic HBV carriers, i.e., up to 1 million people per year, die of cirrhosis or HCC as a consequence
of hepatitis B.19
Recombinant HBV Vaccines
In many countries, the first licensed vaccines against HBV became available at the beginning of the 1980s. Because there is
no possibility for propagation of the virus in vitro, this vaccine was produced by harvesting and purifying HBsAg from the serum of chronic carriers. While they are effective
and safe, serum-derived vaccines are expensive and in relatively short supply because of shortage of human carrier plasma
that meets the requirements for vaccine production. Since 1986, recombinant hepatitis B vaccines have been used as more practical
alternatives with the HBsAg produced in yeast. Immunization with HBsAg results in antibody production against the HBsAg, which
protects against an infection with HBV. Newborns of chronically HBV-infected mothers receive a combined active and passive
immunization. All commercially available recombinant vaccines produced in yeasts so far are based on small surface antigens
inserted into yeast-derived membranes (Figure 1).
Currently, there are two recombinant hepatitis B vaccines that are approved by the FDA and available for use. Both are S-antigen
vaccines produced in the yeast S. cerevisiae. Additionally, P. pastoris and H. polymorpha-based vaccines have been launched (Table 1) .
Table 1. Commercially available yeast-derived vaccines (selection)
The construction of recombinant H. polymorpha strains generally follows a standard approach similar to that described for S. cerevisiae,33 encompassing construction of the expression cassette for HBsAg contained on a plasmid vector, transformation of H. polymorpha, and isolation and characterization of recombinant strains. The construction of such H. polymorpha strains uses vectors inserting an S-antigen coding sequence into an expression cassette harboring either an FMD promoter
or a MOX promoter for expression control (in the case of P. pastoris, the AOX1 promoter).7 Additionally, the vectors contain genetic elements required for plasmid propagation and selection in E. coli, and those for selection in the yeast host. FMD promoter-controlled production strains have been engineered for Hepavax-Gene
and AgB, and MOX-promoter controlled strains for have been engineered for ButaNG. The engineered strain contains up to 60
copies of the functional expression cassette for HBsAg mitotically stable integrated into the genome.
The inherent characteristics of the two promoters, both derived from genes of the methanol utilization pathway levels provide
both high productivity and an attractive upstream process design that can be controlled by additions of appropriate amounts
of glycerol and methanol to the culture medium.7 In H. polymorpha, the recombinant viral surface antigen is found to be assembled into yeast-derived lipid membranes similar to the situation
in other yeasts, forming 22 nm, 1.17–1.20 g cm–3 particles. Previous studies have indicated that this lipoprotein particle structure is essential for the antigenicity of
the HBsAg.34 The H. polymorpha-based platform with its methanol pathway promoters and the inclusion of methanol in a fermentation process provides an especially
efficient process for a balanced co-production of both vaccine components because membrane proliferation in general is associated
with methanol induction.
Product-containing cells are usually generated by a two fermenter cascade, consisting of a 5-L seed fermenter used to inoculate
the 50-L main fermenter. The whole fermentation process, starting from a single vial from the working cell bank, yields a
biomass of more than 10 g dry cell weight per liter in 55 hours. The production fermentation is carried out in synthetic medium
feeding glycerol in the first phase, and a mixture of glycerol and methanol in the second phase.7