Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated.

The DNA sequence coding for hepatitis B virus surface antigen (HBsAg) was placed under control of the repressible acid phosphatase promoter of the yeast Saccharomyces cerevisiae in a plasmid capable ofautonomous replication in both yeast and Escherichia coli Yeast transformed by this plasmid synthesized up to 5 x 105 molecules per cell of immunologically active HBsAg polypeptide in phosphate-free medium. The HBsAg polypeptides produced in the yeast cells were assembled into 20-to 22-nm spherical or oval particles and were immunogenic.Hepatitis B virus (HBV) causes serious human liver disease, including hepatoma. Infection by this virus is a worldwide health problem, and a considerable number of people, particularly in Southeast Asia, the Middle East, and some areas of Africa, suffer from transient or chronic HBV infection. The infectious agent has been identified as a 40-to 50-nm spherical particle, called the Dane particle, that is detected in patients' blood. The Dane particle contains a 3.2-kilobase (kb) circular DNA that has a single-stranded gap (1, 2).Intensive efforts have been made to understand the structural and behaviorial characteristics ofthis virus in order to control the disease and to produce vaccines. However, such efforts have been hampered seriously by the fact that HBV replicates only in human and chimpanzee livers.The viral genome has been converted to double-stranded form by filling-in and has been cloned and propagated in Escherichia coli (3-6). Analyses of the cloned HBV genome have brought new insights into the structure and function ofthis viral genome. At the same time, it was expected that vaccines might be produced in E. coli cells by allowing expression ofthe cloned surface antigen (HBsAg) gene. Despite many efforts, however, production of vaccines in E. coli has not succeeded because, in E. coli cells, the HBsAg gene product seems to be either unstable or to cause effects deleterious to the host, or both. In the present study, we report a yeast system that allows expression of the HBsAg gene.One ofthe yeast acid phosphatases [APase; orthophosphoricmonoester phosphohydrolase (acid optimum), EC 3.1.3.2] is an exocellular 60-kilodalton polypeptide designated P-60; the expression of the gene for P-60 is controlled by the level of inorganic phosphate (7-12). We took this gene, freed its promoter from the coding sequence of the polypeptide, and joined the promoter with the HBsAg gene in such a way that free, nonfusion polypeptides of HBsAg were made. The promoterHBsAg gene complex was inserted into a shuttle vector that replicates in both yeast and E. coli and then was used to transform yeast. The transformants produce a large quantity of HBsAg polypeptide in low P1 medium, and the HBsAg is assembled into spherical or oval immunogenic particles. MATERIALS AND METHODSStrains and Media. Yeast strain AH22 (a leu2 his4 cani cir') (13)

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Two recombinant plasmids were constructed that allow expression of the hepatitis B core (HBc) antigen gene in the yeast Saccharomyces cerevisiae under the control of the repressible acid phosphatase promoter. One plasmid was designed to produce polypeptide I, which consists of 183 amino acids, and the other plasmid was designed to produce polypeptide II, which has an additional 29-amino-acid sequence at the amino terminus of polypeptide I. The viral genome may code for either one or both of these two polypeptides, depending upon the selection of initiation codons. Both polypeptides produced in yeast cells reacted with anti-HBc antibody and were assembled into spherical particles approximately 27 nm in diameter. Particles made of polypeptide I were stable, whereas those made of polypeptide II readily dissociated when exposed to high salt levels. The antigenicity of the HBc (as defined by its reactivity to anti-HBc antibody in the reversed passive hemagglutination assay) disappeared as the particle dissociated, leaving materials that sedimented slowly and that reacted to anti-hepatitis B e antibody. These observations strongly suggest that native viral cores are mostly (if not all) made of polypeptide I, because it is reasonably stable, and that the N-terminal portion of this polypeptide has some, but not a profound, influence on the assembly of polypeptides into particles.