Recombinant Vaccine Production in Yeast
Human HBV can be grouped into eight genotypes, which differ by at least 8%.
ABSTRACT
Yeasts are distinguished by a growing track record as expression platforms for the production of pharmaceuticals. Commercially available, yeast-derived, recombinant pharmaceuticals include, among others, insulin, the anti-coagulant hirudin, interferon-alpha-2a, and various vaccines against the hepatitis B virus and papillomavirus infections. The vaccines are produced in either baker's yeast (Saccharomyces cerevisiae), or the methylotrophic species Hansenula polymorpha and Pichia pastoris. In this article, we focus on a production process for hepatitis B vaccines in methylotrophs. Methylotrophs provide highly balanced production of both the membrane and the protein component of a recombinant viral particle. A brief outlook is given for the development of yeast strains designed for the production of other vaccine candidates.
The advent of gene technology has provided new and powerful methods for the safe, efficient production of pharmaceuticals, with the bacterium Escherichia coli, mammalian cells, and various yeasts as preferred platforms for the production of such recombinant compounds.1 Early examples include human growth hormone2 and insulin3 produced in recombinant strains of E. coli. Among the most important available recombinant pharmaceuticals are yeast-derived vaccines against papillomavirus4 and hepatitis B infections. Hepatitis B vaccines are based on particles containing the hepatitis B surface antigen (HBsAg) inserted into host-derived membranes.5–7 The success of current vaccination programs against hepatitis B is a result of the development of effective, yeast-derived recombinant hepatitis B surface proteins. Initially, the production of such vaccines was restricted to baker's yeast, S. cerevisiae, but with improvements in biotechnological methods, alternative yeast expression systems have been identified and developed. In particular, the methylotrophic yeast H. polymorpha8–14 has been found to exhibit many superior expression characteristics, and is currently being used in the production of several vaccines against different subtypes of the hepatitis B virus.7,15
In this article, we briefly describe the hepatitis B virus, its subtypes, and the disease it causes. Subsequently, recombinant protein production is discussed, focusing in particular on the application of the H. polymorpha expression system. We describe how a heterologous H. polymorpha strain expressing HBsAg is constructed and how efficient vaccine production is developed based on such recombinant strains.
Finally, we will provide an outlook for alternative vaccine strategies and the development of alternative vaccine candidates.
Virus and Disease Characteristics
Hepatitis B virus (HBV) was identified as the causative agent of serum hepatitis in the 1970s16 after B. Blumberg discovered the Australia antigen.17 Blumberg first recognized this antigen as a serum protein specific for aborigines in Australia. It was only later that the infectious nature of the Australia antigen was identified; it turned out to be the surface protein of HBV that is secreted into the bloodstream of infected patients in large excess over viral particles.18 HBV was found to be endemic in many parts of the world, with more than 2 billion people having had contact with the virus and more than 350 million chronic carriers of the virus.19
Human HBV can be grouped into eight genotypes (A–H) which differ by at least 8%.20–22 Genotype F, which is found in Brazil, Colombia, and Polynesia is the most divergent genotype20,23 and is grouped into genotypes F1 and F2.24 The genome of hepadnaviruses codes for four groups of proteins on the minus strand, namely (1) the core protein (HBcAg) which forms the nucleocapsid, and a shorter form (HBeAg), which is secreted by the host cell; (2) the three hepatitis B surface antigen (HBsAg) proteins of different size having different initiation sites of transcription but share the sequence of the small surface protein (SHBs) of 24 kDa at their carboxy-terminus which is the most abundant representative of the HBsAg proteins; minority proteins are the middle HBs of 32 kDa and the large surface protein (LHBs) of 39 kDa (3) the DNA polymerase, which is also a reverse transcriptase with a primer function and an RNAse H domain; and (4) a protein X (HBx) with unknown function for the virus and a plethora of reported properties in vitro.
The hepadnaviridae are round, enveloped viruses of 42–52 nm in diameter25 which appear as 45 nm in negative staining. The viral genome is packed, together with the viral polymerase and a cellular kinase, into a capsid with a diameter of 34 nm.26 In the serum of chronic carriers, the viral surface protein is found as DNA-free spherical or filamentous particles in excess over infectious virions.27
The hepatitis B virus is taken up by an unknown mechanism or receptor by the hepatocytes. Somewhere in the cytoplasm, the viral envelope is removed and the free nucleocapsid moves to the nuclear pores28 where the HBV DNA-genome leaves the capsid and translocates into the nucleus. In the nucleus, the short plus strand is completed (by the attached viral DNA polymerase), leading to a covalently closed circular double stranded DNA (cccDNA). This cccDNA serves as template for transcription of viral RNAs in the nucleus by the cellular RNA-polymerase II. The different mRNA species are exported from the nucleus into the cytoplasm, where translation occurs. Synthesis of the HBsAg takes place at the endoplasmic reticulum (ER) and the HBsAg is anchored in the ER-membrane. The core protein and the viral polymerase are translated by free ribosomes in the cytoplasm from the largest mRNA (3.5 kb). These two proteins form a complex with their mRNA whereby the core protein encapsidates the viral pre-genome in the cytoplasm. The encapsidated viral RNA is reverse-transcribed into the complete DNA minus strand by the viral polymerase and synthesis of the incomplete plus strand occurs. Then the viral capsid is enveloped at the ER with the HBs-containing membrane, and finally, the infectious virus is secreted.29,30 Subtypes of HBV are determined by different epitopes on particles formed by SHBs and sometimes are distinguished by single amino acid exchanges in the HBsAg sequence. According to the Paris workshop on HBV surface antigen subtypes, eight serotypes exist (adr, ayr, ayw1, ayw2, ayw3, ayw4, adw2, and adw4).
Figure. 1
The hepatitis B virus is transmitted parenterally by infected blood or blood products, through mucosal routes, during organ transplantation, or perinatally during birth. The virus mainly infects liver tissue. Hepatitis B virus infections can be transient or chronic. About 90–95% of infected adults recover completely after apparent or inapparent hepatitis and are regarded as cured. Damage to hepatocytes infected by HBV is not caused by the virus itself—HBV per se is not cytopathic—but by the host immune response.
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
The vaccine particles are harvested and cells are disrupted by a sequence of ion exchange, ultra filtration, and gel filtration steps.7 The purified HBsAg particles are formulated by adsorption to an aluminum hydroxide adjuvant and addition of a preservative. A single adult dose containing 10 or 20 g of rHBsAg may be administered in three single injections at 0, 1, and 6 months.
Outlook
In more recent developments, yeast-based HepB vaccines are modified by inclusion of alternative adjuvants, among others RC-529, a non-toxic lipid A mimetic35 or synthetic oligodeoxynucleotides containing immunostimulatory CpG motifs36 to reduce the administration schedule or to improve seroprotection. Other developments include other antigens such as large surface antigen or core protein sequences which aim to reduce the number of nonresponders or to develop a therapeutic vaccine.37 Several combination vaccines are already on the market or under development that include yeast-derived HepB particles (Table 1).
The characteristics of yeasts as safe and efficient production platforms have resulted in the development of other recombinant vaccines. An S. cerevisiae-based papillomavirus vaccine was launched in 2006 (Table 1).4 Ongoing development include, among others, vaccines against hepatitis C in H. polymorpha (Granovski, pers. commun.) or against malaria38 or Clostridium botulinum neurotoxin39 in P. pastoris.
GEORG MELMER, PHD, is the co-founder and CEO, GERD GELLISSEN, PHD, is the chief scientific officer, both at PharmedArtis GmbH, Germany, +49.241.6085.13270, gerd.gellissen@pharmedartis.deGOTTHARD KUNZE, PHD, leads a group active in yeast biotechnology at the IPK-Gatersleben, Germany.
References
1. Melmer G. Biopharmaceuticals and the industrial environment. Gellissen G, editor. In: Production of recombinant proteins. Weinheim, Germany: Wiley VCH;2005, pp. 319–359.
2. Goeddel D, et al. Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone. Nature. 1979a;281:544–8.
3. Goeddel D, Kleid DG, Bolivar F. Expression in E. coli of chemically synthesized genes for human insulin. Proc Natl Acad Sci USA. 1979b; 76:106–110.
4. Cutts FT, et al. Human papillovirus and HPV vaccines: a review. Bull World Health Org. 2007; 719–726.
5. Harford N, Cabezon T, Coiau B, Delisse A-M, Rutgers T, De Wilde M. Construction and characterization of a Saccharomyces cerevisiae strain (RIT4376) expressing hepatitis B surface antigen. Postgrad Med J. 1987;63:65–70.
6. Schaefer S, Piontek M, Ahn S-J, Papendieck A, Janowicz ZA, Timmermans I, Gellissen G. Recombinant hepatitis B vaccines—disease characterization and vaccine production. Gellissen G, editor. In: Hansenula polymorpha—biology and applications. Weinheim, Germany: Wiley VCH;2002, pp. 185–210.
7. Brocke P, et al. Recombinant hepatitis B vaccines: disease characterization and vaccine production. Gellissen G, editors. In: Production of recombinant proteins. Weinheim, Germany: Wiley VCH;2000, pp. 319–359.
8. Gleeson M, Ortori S, Sudbery P. Transformation of the methylotrophic yeast Hansenula polymorpha. J Gen Microbiol. 1986; 132:3459–3465.
9. Roggenkamp R, Hansen H, Eckart M, Janowicz Z, Hollenberg C. Transformation of the methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet. 1986;202:302–308.
10. Gellissen G, et al. Die methylotrophe Hefe als Expressionssystem für heterologe Proteine BioEngin. 1990;5:20–26.
11. Hollenberg CP, Gellissen G. Gene expression in methylotrophic yeasts. Curr Opin Biotechnol. 1997;8:554–60.
12. Gellissen G, Hollenberg CP. Application of yeasts in gene expression studies: a comparison of Saccharomyces cerevisiae, Hansenula polymorpha and Kluyveromyces lactis—a review. Gene. 1997;190:87–97.
13. Gellissen G, Hollenberg CP. Hansenula. Robinson R, Batt CA, Patel PD, editors. In: Encyclopedia of Food Microbiology. Vol. 2. San Diego, CA: Academic Press;1997, pp. 976–982.
14. Gellissen G. In: Hansenula polymorpha—biology and applications. Weinheim, Germany: Wiley VCH;2002.
15. Gellissen G, Melber K. Methylotrophic yeast Hansenula polymorpha as production organism for recombinant pharmaceuticals. Arzneim Forsch/Drug Res. 1996;46:943–8.
16. Dane DS, Cameron CH, Briggs M. Virus like particles in serum of patients with Australia-antigen-associated hapatitis. Lancet. 1970;1:695–698.
17. Blumberg BS, Sutnick AI, London WT. Hepatitis and leukemia: their relation to Australia antigen. Bull. NY Acad Med. 1968;1566–1586.
18. Mahoney FJ. Update on Diagnosis, Management, and Prevention of Hepatitis B Virus Infection. Clin Microbiol Rev. 1999;12:351–366.
19. Zuckerman JN, Zuckerman AJ. Current topics in hepatitis B. J Infect. 2000;41:130–136.
20. Norder H, Couroucé AM, Magnius LO. Complete genomes, phylogenetic relatedness, and structural proteins of six strains of the hepatitis B virus, four of which represent two new genotypes. Virology. 1994;489–503.
21. Stuyver L, De Gendt S, Van Geyt C, Zoulim F, Fried M, Schinazi RF, Rossau R. A new genotype of hepatitis B virus: complete genome and phylogenetic relatedness. J Gen Virol. 2000;81:67–74.
22. Arauz-Ruiz P, Norder H, Robertson BH, Magnius LO. Genotype H: a new Amerindian genotype of hepatitis B virus revealed in Central America. J Gen Virol. 2002;83:2059–73.
23. Naumann H, Schaefer S, Yoshida CF, Gaspar AM, Re R, Gerlich WH. Identification of a new hepatitis B virus HBV genotype from Brazil that expresses HBV surface antigen subtype adw4. J Gen Virol. 1993;74:1627–32.
24. Norder H, Arauz-Ruiz P, Blitz L, Pujol FH, Echevarria JM, Magnius L. The T1858 variant predisposing for the pre-core stop mutation correlates with one of two major genotype F hepatitis B virus clades. J Gen Virol. 2003;84:2083–7.
25. Jursch CA. Grö enbestimmung von Viren in menschlichen Blutplasma durch Ausschlu chromatographie. 2000; Universität of Gie en, Germany.
26. Crowther RA, Kiselev NA, Bottcher B, Berriman JA, Borisova GP, Ose V. Three-dimensional structure of hepatitis B virus core particles determined by electron cryomicroscopy. Cell. 1994;77:943–950.
27. Heermann KH, Gerlich WH. Surface Proteins of Hepatitis B Viruses. MacLachlan A, editor. In: Molecular Biology of the Hapatitis B Virus. Boca Raton: FLCRC Press;1991, pp. 109–143.
28. Kann M, Bischof A, Gerlich W. In vitro model for the nuclear transport of the hepadnavirus genome. J Virol. 1997;71:1310–6.
29. Kann M, Gerlich WH. Hepatitis B. Mahy BWJ, Collier L, editors. In: Virology. London: Arnold;1998, pp. 745–773.
30. Nassal M, Schaller H. Hepatitis B virus replication—an update. J Viral Hepatitis. 1996;3:217–226.
31. Brechot C, et al. Impact of HBV, HCV, and GBV C/HGV on hepatocellular carcinomas in Europe: results of a European concerted action. J Hepatol. 1998;29:173–83.
32. Buendia MA, Paterlini P, Tiollais P, Brechot C. Hepatocellular carcinoma: molecular aspects. Zuckerman AJ, Thomas CT, editors. In: Viral Hepatitis. Livingstone, London: Churchill;1998, pp. 179–200.
33. Kang HA, Gellissen G. Hansenula polymorpha. Gellissen G, editor. In: Production of recombinant proteins. Weinheim, Germany: Wiley VCH;2005, pp. 319–359.
34. Rutgers T, et al. Expression of different forms of hepatitis B virus envelope proteins in yeast. Zuckerman A, editor. In: Viral Hepatitis and Liver Disease. New York: A. R. Liss;1988, pp. 304–308.
35. Dupont J, et al. A controlled clinical trial comparing nthe safety and immunogenicity of new adjunvanted hepatitis B vaccine with a standard hepatitis B vaccine. Vaccine. 2006;24:7167–74.
36. Cooper CL, et al. CPG7909 improves hepatitis B virus vaccine seroprotection in antiretroviral-treated HIV-infected adults. AIDS. 2005;19:1473–79.
37. Lobaina Y, Palenzuela D, Pichardo D, Muzio V, Guillén G, Aguilar JC. Immunological charcaterization of two hepatitis B core antigen variants and their immunoenhancing effect on co-delivered hepatitis B surface antigen. Mol Immunol. 2005;42;289–294.
38. Zhang Q, Xue X, Qu L, Pan W. Construction and evaluation of a multistage combination vaccine against malaria. Vaccine. 2007;25:2112–9.
39. Webb RP, Smith TJ, Wright PM, Montgomery VA, Meagher MM, Smith LA. Protection with recombinant Clostridium botulinum C1 and D binding domain subunit (Hc) vaccines against C and D neurotoxins. Vaccine. 2007:25:4273–82.
