William J. McAleer

Overview of clinical studies with hepatitis B vaccine made by recombinant DNA

The Merck, Sharp and Dohme hepatitis B vaccine formulated from HBsAg produced by a recombinant strain of Saccharomyces cerevisiae has proven to be highly immunogenic and safe. A 10 micrograms dose of the vaccine produced an anti-HBs response of greater than or equal to 10 IU/l in 91% or more of healthy adults who completed the three-dose regimen. Children responded well to all levels of vaccine antigen utilised but developed maximum anti-HBs titres with 5 micrograms doses. The age of the vaccine recipient affected responsiveness. Younger adults (20-29 years) responded more rapidly and with higher anti-HBs titres than did older adults (greater than or equal to 50 years). Children responded faster and with higher anti-HBs levels than younger adults. Clinical reactions reported after vaccination were mild and transient.

Specific Immune Adherence Assay for Human Hepatitis A Antibody. Application to Diagnostic and Epidemiologic Investigations

Summary A specific immune adherence (IA) test for hepatitis A antibody in human serum was described employing liver extract of marmosets infected with CR326 strain human hepatitis A virus. Persons with hepatitis A, but not hepatitis B, developed hepatitis A IA antibody soon after onset of the acute illness and this persisted thereafter. There was very close agreement in the tests for human hepatitis A immune adherence, complement fixing (CF) and neutralizing antibodies. IA antibodies appeared to develop somewhat later than CF or neutralizing antibody. A limited epidemiologic study of a family outbreak of hepatitis A and B in Costa Rica showed simultaneous occurrence of the two diseases and was supportive of the concept that susceptible persons in a country with high hepatitis A prevalence generally acquire their infections at an early age and are immune thereafter. Most persons of high socioeconomic level in an area of low hepatitis A incidence may proceed to adulthood without experience with hepatitis A. Persons of low socioeconomic level, however, such as commercial blood bank donors and prisoners, show high incidence of hepatitis A antibody. Hepatitis IA and CF antibodies persisted in human subjects for at least 7 hr after hepatitis A virus infection. Captive chimpanzees and grivet and rhesus monkeys, not given hepatitis A virus, showed evidence of previous experience with human hepatitis A or an antigenically related virus based on tests for hepatitis A antibody. Other subhuman primates, rodents, and swine, not given hepatitis A virus, were without hepatitis A antibody. The IA test provides an excellent tool for diagnostic and epidemiologic investigations of hepatitis A and should be of considerable value to detect hepatitis A virus in attempts to propagate the virus in cell culture. There was considerable difference in hepatitis A IA antibody content of different lots of commercial human immune globulin, though the majority titered 1:4000 or 1:8000.

Production and immunological analysis of recombinant hepatitis B vaccine

The synthesis of the hepatitis B surface antigen (HBsAG) in cells of Saccharomyces cerevisiae and its subsequent isolation, purification and analysis is described. The final, purified HBsAg particle exhibits close structural and biochemical similarities to particles derived from the plasma of chronically infected humans. Particles of yeast and human origin have been found, by chimpanzee efficacy studies and by various in vitro analyses, to be immunologically equivalent. The antigenic expression of a determinant-specific epitopes, as measured by antibody binding to synthetic peptides, has also been shown to be equivalent.

Physical, chemical, and morphologic dimensions of human hepatitis. A virus strain CR326

Summary CR326 human hepatitis A virus purified by isopycnic banding from infected marmoset sera was shown to consist of 27 nm spherical particles on electron microscopic examination. The particles were identified as hepatitis A virus by tests for infectivity and by specific neutralization of infectivity with convalescent human hepatitis A serum. Also, identical 27 nm viruses in liver extracts gave specific reactions with hepatitis A antisera when tested by immune electron microscopy. The buoyant density of the virus in CsCl was 1.34 and it was heat (60°), ether and acid stable but was destroyed by heat (100°), formalin (1:4000) and ultraviolet irradiation. Electron microscopic studies of sections of infected marmoset liver showed intracytoplasmic virus particles, usually in vesicles. Presumptive findings for RNA, together with the intracytoplasmic location of the virus, indicated the virus to be of RNA-type. The attributes of the virus indicate it is closely related to the entero-virus family and not to hepatitis B virus. The authors are grateful to F. Banker, P. Giesa, L. Hoover, W. Fisher, M. Johnston, C. Dennis and R. Roehm for excellent technical assistance. Drs. A. Tytell and A. Klink gave important technical advice.

Studies in chimpanzees of live, attenuated hepatitis A vaccine candidates.

Abstract Human hepatitis A virus was attenuated in virulence for chimpanzees by passage in FRhK6 and human diploid lung fibroblast cell cultures. A number of variants were developed by passage in cell cultures which showed different levels of virulence/attenuation for chimpanzees. These results were compared to those obtained with marmosets and reported previously. In general, most variants behaved similarly in the two animal types. Two chimpanzees which gave vaccine-like responses following inoculation with HAV cell culture variants were challenged with virulent HAV. Both animals were immune to HAV infection. These findings provide further evidence for the feasibility of developing live, attenuated vaccines against human hepatitis A.

Progress toward a Live, Attenuated Human Hepatitis A Vaccine

Abstract Human hepatitis A virus was attenuated in virulence for marmosets by passage in FRhK6 and human diploid lung fibroblast cell cultures. A number of variants were produced that showed different levels of virulence/attenuation for marmosets. Marmosets that had attenuated virus-like responses following injection of variant virus were challenged with virulent virus; all were solidly immune to infection. Antibody stimulated by the vaccine equated with protection. These findings show that human hepatitis A virus can be attenuated and show the feasibility for eventual development of a live, attenuated hepatitis A vaccine for human use.

Stability on storage at various temperatures of live measles, mumps and rubella virus vaccines in new stabilizer.

Dried live measles, mumps and rubella virus vaccines prepared in a new stabilizing medium were tested for loss of potency, according to time, at a range of temperatures from −20°C to 54–56°C. All vaccines proved remarkably stable and predictive values for retention of adequate potency for periods of time at a range of temperatures are presented. The quality of the current vaccine should go far toward assuring potency under adverse conditions of handling in developed countries and in handling outside the cold chain in emerging parts of the world.

Summary of worldwide clinical experience with H-B-Vax® (B, MSD)

Summary The hepatitis B vaccine produced by MSD consists of highly purified HBsAg which is adsorbed on to aluminium hydroxide adjuvant. The vaccine produces an anti-HBs response in 90 per cent or more of healthy infants, children and adults who receive the recommended three-dose regimen. These antibodies persist for at least 36 months in most individuals studied. Age is the major factor determining responsiveness to the vaccine. Children show a brisk, high-level response and require only 10 μg of HBsAg per dose. Younger adults also respond rapidly to 20 μg doses of HBsAg. Older adults, especially those over 60, may show a sluggish response. Immunocompromised individuals, such as renal dialysis patients, also show reduced responsiveness to the vaccine. Vaccine-induced antibodies have been shown to protect the vaccinee against acute hepatitis B, asymptomatic infection and chronic antigenaemia. Monovalent type ad vaccine has been shown to protect against type ay disease. Studies to measure protection when vaccine is administered post-exposure are in progress.

Haemophilus influenzae Type b Polysaccharide-Protein Conjugate Vaccine

Abstract An Haemophilus influenzae type b capsular polysaccharide-protein conjugate has been prepared. The polysaccharide was coupled to the serotype II protein of group B meningococcus through the spacer 6-aminocaproic acid using cyanogen bromide and water soluble carbodiimide. The conjugate can be shown to be reproducible and is stable and highly immunogenic in mice and African green monkeys. Clinical evaluation of this conjugate in children 3 months to 4 years of age showed that it elicited an antibody titer to the polysaccharide moiety greater than 1000 ng/ml in children 8 months of age or older.

The preparation and safety of hepatitis B vaccine

Preparation of hepatitis B vaccine in our laboratories consists of a series of steps that include initial concentration of surface antigen by ammonium sulphate precipitation, followed by isopycnic banding and rate zonal centrifugation in a K-II centrifuge. The partially purified antigen concentrate is digested with pepsin at pH 2 and the antigen is unfolded in 8 M urea solution followed by renaturation. After gel filtration, the antigen is treated with formalin in I :4000 dilution, adsorbed on to alum, and preserved with thimerosal. The final product contains essentially pure hepatitis B surface antigen. The process relies both on physical elimination of infectious virus particles and treatment with highly viral-destructive reagents in the pepsin (pH 2), urea and formalin steps. The process is known to be highly destructive of all known viruses tested and to include procedures that are known to be highly destructive of representatives of all known groups of animal viral agents. The three-step process in inactivation provides a fail-safe system for establishing safety of the product. Tests in more than 20000 persons, who are under surveillance, have shown no untoward effect and have confirmed the safety of the product.