The worldwide importance of human hepatitis B virus infection and the toll it takes in chronic liver disease, cirrhosis and hepatocarcinoma, make it imperative that a vaccine be developed for worldwide application. Human hepatitis B vaccines are presently prepared using hepatitis B surface antigen (HBsAg) that is purified from the plasma of human carriers of hepatitis B virus infection. The preparation of hepatitis B vaccine from a human source is restricted by the available supply of infected human plasma and by the need to apply stringent processes that purify the antigen and render it free of infectious hepatitis B virus and other possible living agents that might be present in the plasma. Joint efforts between our laboratories and those of Drs W. Rutter and B. Hall led to the preparation of vectors carrying the DNA sequence for HBsAg and antigen expression in the yeast Saccharomyces cerevisiae. Here we describe the development of hepatitis B vaccine of yeast cell origin. HBsAg of subtype adw was produced in recombinant yeast cell culture, and the purified antigen in alum formulation stimulated production of antibody in mice, grivet monkeys and chimpanzees. Vaccinated chimpanzees were totally protected when challenged intravenously with either homologous or heterologous subtype adr and ayw virus of human serum source. This is the first example of a vaccine produced from recombinant cells which is effective against a human viral infection.

Hepatitis B surface antigen (HBsAg) has been extracted from yeast cells that produce HBsAg. These cells contain the gene for surface antigen carried on a plasmid that replicates in the cells. Analysis of the yeast-derived HBsAg by sucrose gradient centrifugation and by polyacrylamide gel electrophoresis shows that the antigen that is initially released from yeast cells is a high molecular weight aggregate of the fundamental Mr 25,000 subunit. Unlike HBsAg derived from human plasma, the yeast antigen is held together by noncovalent interactions and can be dissociated in 2% NaDodSO4 without the use of reducing agents. During in vitro purification of the yeast antigen, some disulfide bonds form spontaneously between the antigen subunits, resulting in a particle composed of a mixture of monomers and disulfide-bonded dimers. Treatment with 3 M thiocyanate converts the 20-nm particles into a fully disulfide-bonded form that is not disrupted in NaDodSO4 unless a reducing agent is added. This disulfidebonded particle resembles the naturally occurring, plasmaderived surface antigen particle, and the in vitro formed particle has been used to prepare a vaccine for humans against hepatitis B virus infection.

A population of procollagen molecules has been isolated from the culture medium of a clonal line of calf dermatosparactic cells and shown to have the amino-acid composition, physical properties, and molecular structure consistent with collagen precursors. Although this procollagen population shares immunologic determinants with the procollagen obtained from dermatosparactic skin, it differs from the latter in aminoacid composition, in subunit properties, and by its content of both amino and carboxyl terminal non-collagen peptide appendages. We propose that the cell culture procollagen contains earlier biosynthetic forms of dermatosparactic procollagen.We have recently found that clonal lines of dermal cells from dermatosparactic calves produce two general types of procollagen in cell culture; one can be converted to al and a2 components and the other to only al components (1). These procollagen types are separable by ion-exchange chromatography and probably originate from separate genomes. We have isolated and partially characterized these procollagens and find them to contain a population of molecules which have unique and distinct properties. In this report we describe some of the unusual features of that procollagen population which can be converted to al and a2 components (1). MATERIALS AND METHODSClonal cell lines were maintained as previously described (1) and were grown in roller bottles. Procollagen was labeled with either [14C]proline or [3H]proline and was purified from the culture medium by a three step method (Church et al., un-published). Briefly, this method consists of an initial precipitation of procollagen by 20% (NH4)2SO4, followed by dissolving the precipitate in potassium phosphate, pH 7.6, ionic strength 0.4. Insoluble proteins were removed by centrifugation and the procollagen was then precipitated by the slow addition of ethanol to 18%, at 4°. This precipitate was harvested and then mixed with DEAE-cellulose which had been equilibrated with 0.01 M Trist HCI, pH 7.4, containing 2 M urea.The mixture was applied to the top of a DEAE-cellulose column (2.5 X 20 cm), equilibrated with the same buffer, and eluted as previously described (1).High speed sedimentation equilibrium was conducted using interference optics and the data were analyzed by statistical methods (2). Optical rotary dispersion was done in a Cary 60 spectropolarimeter and viscometry was carried out in a semimicro Ubbelhode viscometer.Standard procedures were used for: digestion by purified bacterial collagenase in the presence of N-ethyl maleimide (3); electrophoresis in polyacrylamide gels containing Na dodecyl sulfate and dithioerythritol (4); protein hydrolysis by ptoluene sulfonic acid (5); reduction and alkylation by dithioerythritol and iodoacetamide, respectively, in 8 M urea, TrisHCl, pH 8.0 (6); isoelectric focusing on a micro scale in 7 M urea (7); precipitation of segment long spacing (SLS) forms of collagen and procollagen by ATP, followed by negative staining by phosphotungstic acid (8).Immune sera, directed...

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