Hepatitis B surface antigen in pregnant women

Screening of HBsAg in 289 randomly selected normal pregnant women was done with counterelectrophoresis. Ten percent were found to be HBsAg positive. All of the 289 cord blood samples were HBsAg negative. Babies born to the HBsAg positive mothers were followed 2-8 months (average 3.5 months) after delivery and 37.5% were positive for HBsAg. Accordingly, the vertical transmission of HBsAg was postulated to occur during labor or after birth.     

Hepatitis B virus surface antigen binds to apolipoprotein H.

We have previously demonstrated that a plasma membrane-enriched fraction isolated from human liver is capable of binding recombinant hepatitis B surface antigen (rHBsAg) (P. Pontisso, M. A. Petit, M. Bankowski, and M. E. Peeples, J. Virol. 63:1981-1988, 1989). In this study we have separated the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and used a ligand-blotting technique to identify a 46-kDa rHBsAg-binding protein. This protein could be removed from the membranes with a weakly acidic buffer, implying that it is peripherally bound. Examination of human serum revealed that the 46-kDa binding protein is a serum protein. Isolation of plasma lipoproteins revealed that the binding protein is in part associated with chylomicrons and high-density lipoproteins, both of which are targeted to the hepatocyte during the normal course of lipid metabolism. The binding protein was identified as apolipoprotein H (apo H), also known as beta 2-glycoprotein I, on the basis of copurification of the rHBsAg-binding activity with the apo H protein and the ability of cDNA-expressed apo H to bind rHBsAg. Serum-derived HBsAg also binds to apo H, indicating that binding is not unique to rHBsAg. Binding is saturable, requires only the small S protein of rHBsAg, and is inhibited by excess rHBsAg, antibodies to HBsAg, and antibodies to apo H. The binding activity of apo H is destroyed upon reduction, indicating that 1 or more of its 22 disulfide bonds are required for interaction with rHBsAg. The possibility that an interaction between hepatitis B virus particles and lipoprotein particles may facilitate entry of the virus into hepatocytes is discussed.     

Hepatitis B surface antigen assembles in a post-ER, pre-Golgi compartment.

Expression of hepatitis B surface antigen (HBsAg), the major envelope protein of the virus, in the absence of other viral proteins leads to its secretion as oligomers in the form of disk-like or tubular lipoprotein particles. The observation that these lipoprotein particles are heavily disulphide crosslinked is paradoxical since HBsAg assembly is classically believed to occur in the ER, and hence in the presence of high levels of protein disulphide isomerase (PDI) which should resolve these higher intermolecular crosslinks. Indeed, incubation of mature, highly disulphide crosslinked HBsAg with recombinant PDI causes the disassembly of HBsAg to dimers. We have used antibodies against resident ER proteins in double immunofluorescence studies to study the stages of the conversion of the HBsAg from individual protein subunits to the secreted, crosslinked, oligomer. We show that HBsAg is rapidly sorted to a post-ER, pre-Golgi compartment which excludes PDI and other major soluble resident ER proteins although it overlaps with the distribution of rab2, an established marker of an intermediate compartment. Kinetic studies showed that disulphide-linked HBsAg dimers began to form during a short (2 min) pulse, increased in concentration to peak at 60 min, and then decreased as the dimers were crosslinked to form higher oligomers. These higher oligomers are the latest identifiable intracellular form of HBsAg before its secretion (t 1/2 = 2 h). Brefeldin A treatment does not alter the localization of HBsAg in this PDI excluding compartment, however, it blocks the formation of new oligomers causing the accumulation of dimeric HBsAg. Hence this oligomerization must occur in a pre-Golgi compartment. These data support a model in which rapid dimer formation, catalyzed by PDI, occurs in the ER, and is followed by transport of dimers to a pre-Golgi compartment where the absence of PDI and a different lumenal environment allow the assembly process to be completed.     

Hepatitis B surface antigen and its subtypes in an institution for the mentally retarded.

The prevalence of hepatitis B surface antigen (HBsAg) in 155 patients with Down''s syndrome (DS) and 209 with other types of mental retardation (OMR) at Huronia Regional Centre, Orillia, Ontario was 34.8 and 5.3%, respectively. There was no significant difference in prevalence between males and females in either group of patients. In 75 matched pairs (DS-OMR) the HBsAg prevalence was 45% in DS and 8.3% in OMR males; in females 40% of those with DS were HBsAg-positive, whereas all the OMR residents were negative. The prevalence of HBsAg in both DS and OMR groups was higher in those admitted in early childhood and in those who had resided in the institution for more than 10 years. In all 54 HBsAg-positive DS patients the antigen subtype was ad. Among the 11 HBsAg-positive OMR patients the subtype was ad in 10 cases and ay in 1.     

Hepatitis B virus DNA and e antigen in serum from blood donors in the United Kingdom positive for hepatitis B surface antigen.

Serum samples from 214 blood donors in the United Kingdom who were carriers of hepatitis B surface antigen (HBsAg) were examined for hepatitis B virus deoxyribonucleic acid (DNA) by DNA:DNA hybridisation and for hepatitis B e antigen (HBeAg) and its antibody. One fifth of the donors carried infectious virus in their circulation. The presence of hepatitis B virus DNA correlated well with that of HBeAg, although hepatitis B virus DNA was found in five serum samples that were negative for HBeAg. It is concluded that analysis of serum samples for hepatitis B virus DNA by hybridisation should be the method of choice for determining whether carriers of HBsAg are infectious.     

Hepatitis B surface antigen: an unusual secreted protein initially synthesized as a transmembrane polypeptide.

Hepatitis B surface antigen (HBsAg), the major coat protein of hepatitis B virus, is also secreted from cells as a subviral particle, without concomitant cleavage of N-terminal amino acid sequences. We examined this unusual export process in a cell-free system and showed that the initial product of HBsAg biosynthesis is an integral transmembrane protein, with most or all of its C-terminal half on the lumenal side of the endoplasmic reticulum membrane. To study the nature of its topogenic signals, we synthesized fusion proteins between HBsAg and the nonsecreted protein alpha-globin. Fusion proteins in which approximately 100 amino acids of globin preceded all HBsAg sequences were successfully translocated in vitro; the same domain as in the wild-type HBsAg was transported into the vesicle lumen. Fusions in which the entire globin domain was C terminal were able to translocate both the C-terminal region of HBsAg and its attached globin domain. Thus, uncleaved signal sequences in p24s function to direct portions of the molecule across the membrane and are able to perform this function even when positioned in an internal protein domain.     

Hepatitis B surface antigen polypeptides: artifactual bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis caused by aggregation.

Hepatitis B surface antigen, subtype ad, was purified and studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Two major bands with molecular weights of 23,500 and 27,500 and several weaker bands with higher molecular weights were observed. When the low-molecular-weight bands and the group of high-molecular-weight bands were excised from the gel, eluted, and reelectrophoresed, neither the low-molecular-weight bands nor the high-molecular-weight bands ever appeared alone, but both high- and low-molecular-weight bands always appeared. It was concluded that the apparently high-molecular-weight bands represented aggregates of the two small polypeptides whose monomers formed the major bands. The preparation thus contained only two polypeptides.     

Hepatitis B surface antigen. Role of lipids in maintaining the structural and antigenic properties of protein components.

Most of the lipid components of hepatitis B surface antigen (HBsAg) can be removed by treatment with the non-ionic non-denaturing detergent beta-D-octyl glucoside (OG) followed by centrifugation through caesium chloride linear density gradients (density 1.15-1.32 g/ml). The conformational changes induced by the elimination of lipids decreased the helical content of HBsAg proteins from 52 to 28% as indicated by c.d. techniques. Measurements of the extent of quenching of protein fluorescence by iodide showed that half of the tryptophan residues which are buried in the native structure of HBsAg particles are brought close to the surface of the molecule by such conformational changes. The antigenic activity, as measured by binding to polyclonal antibodies, was decreased upon removal of lipids. Moreover, the six different antigenic sites recognized by our panel of monoclonal antibodies decreased their capacity to bind to the corresponding antibody when lipids were removed. However, the extent of this decrease differed for the different antibodies. Thus the apparent dependence of antibody binding on the lipid content seemed to indicate a greater involvement of the lipid-protein interaction for some of the epitopes than for others.     

Hepatitis B surface antigen immunopurification using a plant-derived specific antibody produced in large scale

This paper provides an evaluation of a plant-derived HBsAg-specific antibody in the immunopurification of the recombinant HBsAg for vaccine purposes. This plant-derived antibody was obtained from different batches of 100-200kg of tobacco leaves and coupled to Sepharose CL-4B with high efficiency. The plant-derived antibody immunoaffinity matrix purification behavior (elution capacity, antigen purity, purification cycles, and ligand leakage) was comparable to that of its mouse-derived monoclonal antibody homolog. This result supports the feasibility of using this plant-derived antibody for the immunopurification of the Hepatitis B surface antigen for human use, opening a new possibility to overcome the constrain of monoclonal antibody production in mice.     

Secondary sex ratio variation in the Cayo Santiago macaque population

Secondary sex ratios (SSR) were calculated from 1,385 offspring delivered by 372 females in the Cayo Santiago population of free-ranging rhesus monkeys (Macaca mulatta) from 1976 through 1984. The SSR for the entire colony ranged from 0.86 to 1.46 males per female (combined total: 1.08), but no significant difference was observed (P > .05). SSR values were compared among the troops for each year. The SSR differed significantly among the six social groups (P < .05) only in 1978. The annual SSR of each troop was compared over 9 years. Significant variation was found only in group O. The annual SSR was significantly skewed (P < .05, males > females) for three troops in 3 separate years. The SSR did not vary according to troop rank. No significant difference was found among the 17 matrilines of the population, but comparison of matrilines within each social group revealed a significant difference in the SSR (P < .02) for the three matrilines in group I. This was due to the significantly skewed SSR (P = .0080, females > males) of the DM genealogy in that troop. SSR values were not related to matrilineal rank. Individual dominance rank did not bias the SSR. Complete reproductive histories for 266 females showed no evidence of significantly skewed SSR values. Age-related effects on the SSR were examined by using cross-sectional and cohort-based analyses. The SSR did not vary significantly (P > .05) with maternal age, but it was significantly skewed (P < .05) toward males at the ages of 5 and 9 years. Parity had no significant effect (P > .05) on SSR values. Wide variation occurred in the SSR of the Cayo Santiago population. Rank-related adjustment of the SSR at the level of the troop, matriline, or individual, as reported in short-term studies of other primate social groups, may reflect normal annual variation in the SSR evident only from longitudinal observations of large multigroup primate populations.     

Hepatitis B surface antigen expression in NT-1 cells of tobacco using different expression cassettes

Nicotiana tabacum 1 (NT-1) cells were transformed with four different expression cassettes of hepatitis B surface antigen (HBsAg). The transformed nature of the cells was confirmed by polymerase chain reaction (PCR). The expression levels were assayed by enzyme linked immunosorbent assay (ELISA). The expressivities varied among the different cassettes and the maximum expression of 16.6 ng g⁻¹(f.m.) of cells was noted in pEFEHER transformed cells. Salicylic acid (100 μM) treatment resulted in 1.8 fold increase of expression in pEFEHBS transformed cells. The effect of different concentrations of kanamycin and geneticin was studied on the growth of transformed cells and HBsAg expression. The cell growth was optimum at lower concentrations of the antibiotics, and the maximum expression was noted at 200 mg dm⁻³ of kanamycin.     

Hepatitis B virus core antigen: enhancement of its production in Escherichia coli,and interaction of the core particles with the viral surface antigen

The core antigen (HBcAg) of hepatitis B Virus (HBV) can be expressed in Escherichia coil where it assembles into icosahedral particles containing 240 or 180 subunits. Analysis of the two kinds of particles by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) showed that a substantial proportion of their subunits were smaller than the full-length HBcAg monomer and of variable size, but all had the same N-terminal sequence showing that the smaller species were heterogeneous in their arginine-rich C-terminal regions. Around 50% of these arginine residues are encoded by the triplet AGA which is rare in E. coli. Supplementation of the level of AGA tRNA in the cell by transformation with plasmids expressing the T4 AGA tRNA gene significantly enhanced the yield of HBcAg. Fusion phage carrying a ligand specific for HBcAg showed no significant difference in the affinity for the two sizes of HBcAg particles, but in similar reactions in solution HBV surface antigen exhibited differential affinities for the same two HBcAg preparations.