Polypeptide micelles with relative molecular weights of 25,000 (p25) and 30,000 (gp30) daltons were prepared from native 22-nm hepatitis B surface antigen (HBsAg) particles. This p25/gp30 complex was alum-adsorbed, and three dosage levels (20 micrograms, 4 micrograms, and 0.8 micrograms) were administered at 0, 1, and 6 months to 51 human volunteers. Local and systemic reactions were clinically insignificant, and all vaccinees seroconverted, regardless of dose. As anticipated, antibody responses diminished as the dosage was reduced. Seroconversion rates and geometric mean antibody levels for the 20 micrograms dosage group were significantly better than those observed with a commercial vaccine and were comparable to those achieved after immunization with 40 micrograms of the intact 22-nm particles used to prepare the polypeptides. By 2 weeks, an anti-HBs response was elicited in 80% of the group receiving 20 micrograms of the polypeptide vaccine. This rapid response to immunization may be particularly beneficial for postexposure prophylaxis where the early development of immunity is advantageous.
The morphological forms of hepatitis B antigen (HBsAg) present in an HBsAg-positive plasma (15–25 nm spheres, filaments 20–22 nm in diameter and 80–300 nm in length, and 42 nm Dane particles) were isolated and characterized biophysically. The HBsAg in this plasma was fractionated into five distinct populations, containing predominately 15–19 nm spheres, 20–22 nm spheres, 20 × 300 nm filaments, a mixture containing 23–25 nm spheres and rare filaments, and 42 nm Dane particles, respectively. These populations were labeled with 125I and electrofocused in a pH ampholyte gradient. Each population had a distinct isoelectric pH with values ranging from 3.65 to 4.70. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of each of the above labeled populations revealed the presence of 7, 10, 9, 5, and 9 polypeptides, respectively. The population containing predominately 23–25 nm spheres failed to react with antibody specific for HBsAg and probably was composed of inner cores of the Dane particles.
Hepatitis B core antigen (HBcAg) was purified from Dane particles and from infected hepatocytes. An identical isoelectric pH of 4.0 was determined for labeled preparations of both Dane-derived and liver-derived HBcAg. Unlabeled liver-derived HBcAg demonstrated a lower isoelectric pH of 3.7. Molecular weight determinations by Sepharose 4B column chromatography revealed that liver-derived HBcAg had a molecular weight of 8.5–9.0 × 106 daltons. The sedimentation coefficient of both Dane- and liver-derived HBcAg was found to be 124S. PAGE revealed that iodinated HBcAg derived from Dane particles was very similar in polypeptide structure to HBcAg derived from infected liver tissue. Twelve polypeptides were resolved from Dane core particles, and seven to nine were resolved from liver core particles. Several of the polypeptides in both preparations co-migrated with iodinated hepatitis B surface antigen (HBsAg). However, three polypeptides (mol. wt. 88,000, 79,000 and 59,000) were found in both Dane- and liver-derived HBcAg but not in HBsAg, which suggests that these polypeptides are HBcAg-specific. Endogenous DNA polymerase activity was observed in both Dane- and liver-derived core particles.
Anti-idiotype reagents that recognize a common idiotype associated with antibody to hepatitis B surface antigen (anti-HBs) were used to induce anti-HBs in mice. The anti-idiotype-induced anti-HBs was found to recognize the group-specific a determinant of hepatitis B surface antigen and to express an interspecies idiotype. These findings suggest that anti-idiotypes may be useful as vaccines or vaccine primers.
The purification is described of liver-derived hepatitis B core antigen (HBe Ag) from human hepatocytes demonstrating only intranuclear particles by electron microscopy. The purified preparation contained 5 × 10u particles per ml. The particles were mono-dispersed and relatively free of background material. DNA-dependent polymerase activity was present and had greater activity than an equal number of cores derived from Dane particles isolated from plasma. Specificity of the polymerase reaction was confirmed by precipitation of the activity with specific anti-HBc antiserum. A proportion of the liver-derived core particles was nonreactive for DNA polymerase activity. The polymerase-positive population of particles had a larger size than the polymerase-negative population of liver-derived cores as evidenced by gel filtration in Sepharose 4B.
The receptors for polymerized (P) human (Hu) serum albumin (SA) present on intact hepatitis B surface antigen (HBsAg) have been shown to be associated with the major HBsAg polypeptides, as well as HBsAg produced in vitro by a hepatoma cell line. These observations demonstrate that the albumin receptors are virus-specific. Two laboratories [Neurdth and Strick, 1979; Imai et al, 19791 recently reported that the 22-nm particles representing purified preparations of hepatitis B surface antigen (HBsAg) contain a receptor for polymerized (P) human (Hu) serum albumin (SA). Imai et a1 [I9791 showed that this receptor was associated with HBsAg derived from serum positive for e antigen (HBeAg) and that the receptor was protein in nature. More recently, O'Neill [1979] reported that the binding of HBsAg to P-HuSA was temperature-dependent, because binding decreased as the temperature was raised above room temperature. In addition, he reported that this interaction did not appear to depend on the antigenic subtype of HBsAg. In the present study we have examined two of the major polypeptides derived from purified HBsAg for the presence of P-HuSA receptors. In addition, we have tested whether these receptors are associated with HBsAg particles produced by a hepatoma cell line [Alexander et al, 19781. P22 and P68, respectively, were derived from purified preparations of HBsAg. The HBsAg complement-fixing titer of the original plasma pool from which the HBsAg Two polypeptides with molecular weights of 22,000 and 68,000 daltons, termed
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