Jacinta Skelly

Analysis of Hepatitis B Surface Antigen Components Solubilized with Triton X-100

Three glycoproteins of intact hepatitis B surface antigen (HBsAg) with mol. wt. of 32 000, 30 000 and 28 000 respectively were identified by reaction with 125I-concanavalin A (Con A) after separation by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen was effectively disrupted with Triton X-100 to produce a structure with a sedimentation coefficient of 3.9S. Affinity chromatography of disrupted HBsAg using concanavalin A-Sepharose 4B (Con A-Sepharose) resulted in two fractions. The first contained material which did not bind to the lectin and consisted of a single polypeptide of mol. wt. 64 000. Further studies revealed this component to be serologically identical to serum albumin and to lack any affinity for antibody to HBsAg. A comparison of the tryptic peptide map of this polypeptide with that of purified serum albumin demonstrated identical amino-acid sequences. The second fraction contained material which bound to Con A and contained two polypeptides with mol. wt. of 28000 and 23000 respectively. HBsAg reactivity was associated with this fraction. This procedure allows the prepartion of HBsAg sub-units in milligram quantities for further immunological studies.

The labelling of galactose residues in hepatitis B surface antigen glycoprotein.

The 20 to 25 nm particles of hepatitis B surface antigen were purified from the serum of a carrier chimpanzee. Five major polypeptide species were revealed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Treatment of the particles with neuraminidase (EC 3.2.1.18) and galactose oxidase (EC 1.1.3.9) followed by reduction with tritiated sodium borohydride labelled galactose residues in a single glycoprotein with an apparent mol. wt. of 28 000. The glycoprotein was not labelled when neuraminidase treatment was omitted, indicating that the galactose residues are in subterminal positions in the oligosaccharide chains. There was no significant incorporation of radiolabel into lipid. The serological activity of the antigen, as measured by a competitive double-antibody radioimmunoprecipitation assay, was not altered by the labelling procedure nor by exposure to neuraminidase alone.

Nondetection of Infectious Hepatitis B Virus in a Human Hepatoma Cell Line Producing Hepatitis B Surface Antigen

The PLC/PRF/5 human hepatoma cell line producing hepatitis B surface antigen (HBsAg) was studied to determine whether infectious hepatitis B virus (HBV) was also being produced. 2 chimpanzees with no previous exposure to HBV and no serologic markers of past or active HBV infection were inoculated intravenously with 50 ml of either tissue culture supernatant fluid (357 ng/ml HBsAg) or a suspension of cells disrupted by repeated freeze-thaw cycles (57 ng/ml HBsAg). No evidence of HBV infection was detected in either chimpanzee during 6 months of evaluation. This study suggests that the expression of a portion of the HBV genome, when a portion or all of that genome has been incorporated into a host cell, can result in the production of HBsAg without infectious HBV. If it becomes possible to produce a similar expression of this portion of the genome by itself in nonmalignant cells, HBsAg without HBV may be produced in vitro for use in hepatitis B vaccines.

The use of markers in immune electron microscopy.

Immune electron microscopy (IEM) cannot be used successfully for structures that do not have recognisable morphology. However, at least some of these structures or components are related antigenically to recognisable antigens or viruses. We have therefore mixed unknown antigens with known markers and looked for the presence of mixed aggregates. The present study examined a low molecular weight subunit of rotavirus and a micellar form of hepatitis B surface antigen. In both cases mixed immune aggregates were found showing that the unknown components had antigens in common with the established virus or antigen.

Formaldehyde treatment of hepatitis B micelle vaccine.

Abstract Treatment with formaldehyde, under conditions generally used for viral inactivation, is a suitable method for inactivation during the preparation of hepatitis B polypeptide micelle vaccine either before disruption of the 22 nm particles with Triton X-100 or after preparation of the micelles.

Induction of cell-mediated immunity to HBsAg polypeptides.

Adult male guinea pigs were immunized with hepatitis B virus polypeptides prepared by Triton X-100 solubilization of purified 22-nm hepatitis B surface antigen (HBsAg) particles. A virus-specific subunit containing both the 28,000 molecular weight glycoprotein and the 23,000 molecular weight protein stimulated both cell-mediated and humoral immunity. A whole-blood-cell transformation assay additionally showed that the 64,000 molecular weight component of HBsAg, previously shown to contain host-specific antigens, also stimulated a cellular response to purified intact HBsAg particles, suggesting the additional presence of virus-specific material in this fraction. Immunization of animals with the 28,000--23,000 molecular weight polypeptides subsequently prepared in micelle form enhanced the demonstration of a cell-mediated reaction after only a single dose of immunogen. The results provide further evidence as to the suitability of HBsAg polypeptides prepared by Triton X-100 solubilization for hepatitis B prophylaxis.