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The 22 nm spherical form of hepatitis B surface antigen was purified from the serum or plasma of chronic carriers of the antigen. Antigens of subtypes ayw and adr were individually prepared by isopycnic banding in cesium chloride followed by rate zonal separation in sucrose. Each preparation was stabilized with human serum albumin, and aliquots were inactivated with 1:2000 formalin at 37 C for 96 hours. The potency and immunogenicity of each preparation were determined: both antigenicity and immunogenicity were retained by the preparations following purification and inactivation. Seronegative chimpanzees were vaccinated with the antigen preparations. None of the vaccinated chimpanzees developed evidence of infection with hepatitis B virus during the follow-up period. Twenty-four weeks after vaccination vaccinated and control chimpanzees were inoculated with live hepatitis B virus. Control chimpanzees developed hepatitis associated with HBS Ag seven and nine weeks following challenge. In contrast, none of the chimpanzees vaccinated with HBS Ag developed HBS Ag or hepatitis. Thus, hepatitis B vaccine appeared to be safe and efficacious when tested in chimpanzees.