An affordable multideterminant plasma-derived hepatitis B virus vaccine.

A new vaccine is reported which contains hepatitis B virus (HBV) e antigen (HBeAg) and pre-S determinants, in addition to highly purified HBV surface antigen (HBsAg). The rationale for this approach depends on the following data indicating that anti-HBe and antibody to the core antigen (anti-HBc), and antibody to pre-S determinants may play an active role in preventing HBV infection: (1) active immunization of chimpanzees with HBeAg(s) devoid of detectable HBsAg protected against subsequent challenge with HBV; (2) passive immunization of chimpanzees with an anti-HBe/anti-HBc intravenous immunoglobulin devoid of anti-HBs significantly delayed and appeared to attenuate HBV infection following subsequent challenge with HBV; (3) antibody to pre-S determinants appears to be able to neutralize infective HBV. The purification procedure used for the production of the New York Blood Center vaccine was designed to accomplish a high degree of purification with minimal use of complex equipment. This may facilitate eventual utilization of the vaccine on a mass scale for prevention of the HBV carrier state in high-prevalence regions of the world. The procedure uses polyethylene glycol (PEG) precipitations and hydroxylapatite adsorption steps, followed by only a single isopycnic separation in a zonal rotor, to achieve a vaccine which is substantially free of serum proteins and detectable HBV DNA yet contains HBsAg, HBeAg and pre-S determinants. The original vaccine was inactivated by Tween 80 and formalin. Four lots passed chimpanzee safety tests; two of these have been tested in clinical trials. Recently an improved vaccine has been developed in which two heat inactivation steps are used instead of formalin inactivation. This has resulted in further improvement in yields of antigenicity and immunogenicity, and provides an additional margin of safety.

A new hepatitis B vaccine containing HBeAg in addition to HBsAg.

A new vaccine is reported which contains HBeAg in addition to highly purified HBsAg. The rationale for this approach depends on the following data indicating that anti-HBe/anti-HBc may play an active role in prevention of HBV infection; (1) active immunization of chimpanzees with HBeAg(s) devoid of detectable HBsAg protected against subsequent challenge with HBV; (2) passive immunization of chimpanzees with an anti-HBe/anti-HBc intravenous immunoglobulin devoid of anti-HBs significantly delayed and appeared to attenuate HBV infection following subsequent challenge with HBV. The purification procedure utilized for production of the NYBC vaccine was designed to accomplish a high degree of purification with minimal use of complex equipment. This may facilitate eventual utilization of the vaccine on a mass scale for prevention of the HBV carrier state in high prevalence regions of the world. This procedure uses PEG precipitations and hydroxylapatite adsorption steps, followed by only a single isopycnic separation in a zonal rotor, to achieve a vaccine which is substantially free of serum proteins and detectable HBV DNA yet contains immunogenic quantities of HBsAg and HBeAg. The vaccine is inactivated by Tween 80 and formalin. Four lots have passed chimpanzee safety tests; two of them have been tested in clinical trials.

Immunogenicity of HBeAg in the New York Blood Center hepatitis B vaccine.

A new hepatitis B vaccine has been developed which contains both HBsAg and HBeAg since evidence suggests that anti-HBe may have useful biologic activities. It was thus important to determine whether HBeAg is immunogenic in this type of a preparation. We now report data indicating that highly purified HBsAg particles, derived from HBeAg containing plasma, release a proportion of their contained cryptic HBeAg following the detergent treatment used in the preparation of this vaccine. Both the soluble and the residual particle associated HBeAg are immunogenic in guinea pigs. There was no detectable anti-HBe response in animals injected with similar HBsAg particles which had not been exposed to detergent.

[Hepatitis B vaccine. Assessment of vaccines obtained from human plasma]. / Hepatitis B-Vakzine Eine Bewertung der aus Humanplasma gewonnenen Impfstoffe.

This review addresses three questions: Why should HBV vaccines be used? Who should receive them? How should they be made and given? It is suggested that the major function of HBV vaccine, in addition to preventing acute hepatitis in high-risk groups, is prevention of chronic HBV infections (i.e., the HBV carrier state) particularly in high-prevalence regions, with their attendant eventual morbidity and mortality due to cirrhosis and hepatocellular carcinoma. Target populations for vaccine use, subject to vaccine availability, are defined in accordance with this concept. Existing approaches to vaccine manufacture are compared and discussed; all appear to provide safe and immunogenic vaccines. However, it is too early to draw conclusions as to their relative immunogenicity, efficacy, and economy.

Cryptic association of e antigen with different morphologic forms of hepatitis B surface antigen.

When highly purified HBsAg particles, separated by rate zonal centrifugation into populations differing in predominant size, were tested for HBeAg, the e1 specificity was detected preferentially in association with particle fractions containing large filaments and Dane particles. These results were obtained both by agar gel diffusion and by radioimmunoassay for e antigen. The e antigen activity present in these fractions was potentiated by prior treatment of particles with Tween 80, suggesting cryptic localization of e1 specificity within or under the outer membrane. The HBeAg released by detergent treatment from a purified preparation composed predominantly of small-particle forms of HBsAg was separated by electrofocusing into a peak of nonparticulate e antigen in the pH range of 5.7--6.0. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed three major polypeptides in this preparation with approximate molecular weights of 25,000, 55,000, and 70,000. Furthermore, two additional peaks of e antigen activity were detected which migrated in association with HBsAg particles at isoelectric points of 4.4 and 5.5--5.6. The major portion of e antigen remained in association with particles after further purification by rate zonal centrifugation.

Association of normal serum protein antigens with chimpanzee hepatitis B surface antigen particles.

HBsAg/adw was purified from 2.6 liters of pooled plasma from a single chimpanzee carrier by polyethylene glycol (PEG) precipitation followed by isopycnic and rate zonal centrifugation. The different morphological populatilons of HBsAg separated in the final rate zonal centrifugation step were combined into seven pools: two fractions rich in filaments and Dane particles, two pools composed of filaments and 20--28-nm spheres, and three fractions containing mostly 20--28-nm spheres. The purified preparations of HBsAg analyzed for normal serum protein contaminants revealed albumin and traces of IgG. The same samples analyzed after Tween-80 treatment, revealed enhanced quantitites of the previous two contaminants, and in addition, transferrin, traces of alpha2-macroglobulin, IgM, and complement (C3/C3c). The residual contaminants were mostly removed by further purification and fractionation after detergent treatment using zone convection electrofocusing and rate zonal centrifugation. Our findings indicate that conventional purification techniques will not provide preparations of HBsAg free of traces of serum protein contaminants. Many of these are released only by detergent treatments and subsequent purification. It is not yet clear whether detergents release these contaminants from the interior of the particles or from firm association or incorporation within the membranes.

Heterogeneity of hepatitis B surface antigen-associated particles isolated from chimpanzee plasma.

Hepatitis B surface antigen (HBsAg) was purified from approximately 8 liters of pooled plasma from a carrier chimpanzee. Precipitation of HBsAg with polyethylene glycol resulted in more than 20-fold purification, with about 80% recovery of antigenic activity. The sample was separated by further purification and fractionation into three populations of HBsAg-associated particles by column chromatography on hydroxylapatite: the first contained short filaments and 22- to 28-nm spheres, the second was composed of larger filaments and variable-sized spheres, and the third contained mostly 16- to 22-nm spherical particles. A large volume of the polyethylene glycol precipitate passed through hydroxyl-apatite twice yielded over 650 mg of partially purified HBsAg. A pooled preparation of purified HBsAg was separated by zone-convection electrofocusing into five peaks of antigenic activity within the pH range of 4.7 to 5.7.

Large-scale purification of hepatitis B surface antigen.

Hepatitis B surface antigen was concentrated and purified from plasma by two simple steps of purification. In the first step the antigen was purified 24-fold by polyethylene glycol precipitation. An additional 10-fold purification was achieved by batchwise adsorption to hydroxylapatite and subsequent elution with 0.02 M sodium phosphate buffer.

[Application of a reversed passive hemagglutination test to the detection of HBs antigen]. / Technique d'hémagglutination passive inverse appliquée à la détection de l'antigène HBs

In this report we present an evaluation of the sensitivity, specificity and ability to detect HBs Ag carriers of a new reversed passive hemagglutination test, using immunochemically purified chimpanzee anti HBs bound to stabilized human erythrocytes. The method was shown to have a sensitivity equal (within one two fold dilution) to that of the Ausria I 125 ratio immuno assay, and in a double blind comparison detected essentially the same number of Hbs Ag containing specimens among volunteer blood donors. The method therefore provides an economical method for the third generation testing of blood donors. The methodology which has been described incorporates a definitive specificity test in which serum drawn before and after immunization of chimpanzees with purified HBs Ag is compared for its ability to neutralize the hemagglutination reaction. The use of serum from the same animal for this purpose avoids the theoretical possibility that antiglobulin antibodies directed at subclass determinants such as Gm of Inv could be differentially inhibited due to possible subclass differences in the blocking sera employed. A reliable test for specificity of HBs Ag screening results is essential to avoid false notification of donors that they are carriers of hepatitis B virus.

A new reversed passive hemagglutination test for detection of HBsAg.

A new reversed passive hemagglutination test for HBsAg, termed Raphadex B, has been developed using immunochemically purified chimpanzee anti-HBs bound to stabilized human erythrocytes. The test has been found to have equivalent sensitivity to the Ausria 125I radioimmunoassay, and detected a similar number of HBsAg-containing specimens in screening of volunteer blood donors. This method offers an economical approach to third generation methodology for hepatitis B screening of blood donors.