ORIGINAL PAPER S. A. C. Jorge · C. Hera · A. M. M. Spina R. C. Moreira · J. R. R. Pinho · C. F. M. Menck Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector Received: 25 February 1996 / Received revision: 21 June 1996 / Accepted: 15 July 1996 Abstract The hepatitis B virus surface antigen (HBsAg) gene, under control of the inducible mouse meta- llothionein I gene promoter, was inserted in an expres- sion vector based on the Epstein-Barr virus (EBV). This vector was introduced into human cells by DNA trans- fection and clones were selected for their resistance to hygromycin B. The recombinant EBV vector replicates efficiently as an episome in human cells and approxi- mately six copies per cell were found in one clone of hygromycin-B-resistant cells. These cells produce high levels of HBsAg in the presence of metals. The protein is mainly found in the cell medium, suggesting that the HBsAg is secreted from the cells. Introduction Hepatitis B virus (HBV) is a major aetiological agent of worldwide human diseases, including hepatitis, cirrhosis and hepatocellular carcinoma. There are approximately 3 × 10 8 HBV carriers in the world at serious risk of de- veloping chronic liver disease and, possibly, primary li- ver cancer (Hoofnagle 1990). The major viral envelope protein, HBV surface anti- gen (HBsAg), induces a protective immune response during infection (Tiollais et al. 1981). Since antibodies specific for HBsAg neutralize HBV, this antigen re- presents the major component of the currently licensed HBV vaccines (Hilleman et al. 1983; McAleer et al. 1984; Stephenne 1988). The first vaccine approved for human use consisted of HBsAg purified from the plasma of chronically infected individuals. Although this subunit vaccine was efficient, the relatively high cost of its pre- paration, the limited availability of suitable chronic HBV carriers as donors and the concern over other pathogens present in human plasma have led to the use of recombinant DNA technology to express HBsAg in hosts such as bacteria, yeast and insect and mammalian cells. The large-scale expression of the S protein is the basis for the production of a less expensive vaccine against HBV. A yeast-derived recombinant HBsAg has been commercially available since 1987. This system consists exclusively of the expression of the S gene, which codes for HBsAg (McAleer et al. 1984; Valenzuela et al. 1985; Miyanohara et al. 1983). Several other systems have been described, employing cultured mammalian cells carrying the S gene (Wang et al. 1983; Michel et al. 1985; Yoneyama et al. 1988). In contrast to yeast systems, HBsAg is normally secreted as a component of 22-nm particles from mammalian cells, allowing easy protein purification (Tiollais et al. 1981). Particularly high levels of HBsAg were expressed in rodent cells when a system in which the S gene is highly amplified was employed (Michel et al. 1985). This is the basis of a commercially available vaccine (Tron et al. 1989). We were interested in measuring the efficiency of vectors based on the Epstein-Barr virus (EBV) to sup- port and express the S gene. These vectors replicate episomally in human cells (1–100 copies/cell) and they can be used as carriers for expression of heterologous genes (Yates et al. 1985; James et al. 1989). The S gene, under the control of the mouse metallothionein I gene promoter (mMT-I), was inserted in an EBV vector carrying the hygromycin-B-resistance gene. The expres- sion of HBsAg in cells harbouring the vector was ana- lysed. Appl Microbiol Biotechnol (1996) 46: 533 – 537  Springer-Verlag 1996 S. A. C. Jorge · C. Hera 1 · C. F. M. Menck (&) Depto. de Biologia, Instituto de Biocie ˆncias, USP, CP 11461, Sa ˜o Paulo, SP- 05422-970, Brazil. e-mail: cfmmenck@usp.br A. M. M. Spina · R. C. Moreira · J. R. R. Pinho Instituto Adolfo Lutz, Av. Dr. Arnaldo 335, Sa ˜o Paulo, Brazil C. F. M. Menck Depto. de Microbiologia, Instituto de Cie ˆncias Biome ´dicas, USP, Sa ˜o Paulo, Brazil Present address: 1 Universidad de Cordoba, Depto. de Gene ´tica, Fac. de Cie ˆncias, Av. San Alberto Magno s/n, 14071 Cordoba, Spain