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.

Studies of human hepatitis A (infectious hepatitis) have been severely hampered by the lack of a simple assay for specific antibody against hepatitis A virus.In 1973, we reported (1) the development of a specific serum neutralization test for human hepatitis A antibody using the CR326 virus isolated from a case of human hepatitis. The test, though highly specific, is carried out in marmosets and is too laborious and costly for routine purpose. More recently, we developed (2) a specific complement-fixation (CF) test for hepatitis A antibody employing, as antigen, liver extract of a marmoset infected with the CR326 virus. The test provided a workable in vitro assay for serodiagnostic and seroepidemiologic investigations of hepatitis A in man.The present report describes the development of yet another test for hepatitis A antibody. This is an immune adherence (IA) assay employing, as for the CF tests, extract of liver of marmosets infected with CR326 virus. Data relating to the development of IA antibody against the virus in the course of hepatitis A infection and to the seroepidemiology of the disease are given. Additionally, data relative to the content of hepatitis A antibody in commercial human immune globulin and in various subhuman primate species are presented.Materials and Methods. Virus. The CR326 strain (3) of human hepatitis A virus isolated in marmosets from a case of hepatitis A in Costa Rica was used. Preparation of hepatitis A immune adherence ( I A ) test antigen. The antigen was prepared from the liver of a marmoset (Saguinus mystax) infected intravenously with CR326 virus.The liver was taken 27 days after virus inoculation, at a time when the serum enzymes (SGOT and SICD) were elevated. The liver was perfused with phosphate buffered saline (PBS) at pH 7.2, minced with scissors, and ground with sterile alundum in a mortar to give a final 10% suspension by weight in PBS. The extract was clarified by low speed centrifugation, applied to cesium chloride gradients (4), and fractions in the density range of 1.32-1.36 were collected. The fractions were dialyzed against PBS and were used as antigen in the IA assays. Control antigen was similarly prepared from liver of an uninfected marmoset. Immune adherence (IA) test. The procedure was similar to that of Mayumi et al. (5) for assay of hepatitis B antigen and antibody. The sera were not heated. The commercial human immune globulins were diluted 1:lO and absorbed with an equal volume of 25% kaolin before they were assayed for antibody content. The antigens were standardized in grid titrations in which serial dilutions of antigen were assayed with serial dilutions of human hepatitis A convalescent serum. Four units of viral antigen were employed in the tests and the control antigen was used at identical dilution. Complement-jixation (CF) and serum neutralization tests. The techniques for the CF tests for hepatitis A antibody for hepatitis B surface antigen (HB,Ag or Australia antigen), and for neutralizing antibody were described previously (1, 2).Patients' ...

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