HBsAg Particles Research Articles

Cell Culture Derived Recombinant HBsAg is Highly Immunogenic and Protects Chimpanzees from Infection with Hepatitis B Virus

Recombinant hepatitis B surface antigen (rHBsAg) particles, secreted from Chinese hamster ovary (CHO) cells stably transfected with the S gene of HBV, are indistinguishable antigenically from HBsAg particles derived from the plasma of patients chronically infected with hepatitis B virus (HBV). The recombinant vaccine containing CHO cell derived rHBsAg is more immunogenic in chimpanzees than previously reported vaccines of either plasma derived HBsAg or yeast derived rHBsAg based on the kinetics of antibody production and the maximum titers elicited. In addition, chimpanzees vaccinated with CHO cell–culture derived vaccine have a strong cellular immune response to rHBsAg as measured by cell proliferation assays. Animals vaccinated with cell culture derived rHBsAg (ad subtype) are protected from infection with both the ad and ay subtypes of HBV.

Hepatitis B virus pre-S gene-encoded antigenic specificity and anti-pre-S antibody: Relationship between anti-pre-S response and recovery

A solid-phase radioimmunoassay involving specific antibody was developed for determination of the pre-S gene-encoded epitopes of hepatitis B virus and anti-pre-S antibody in sera of hepatitis B patients. The reaction for pre-S determinants associated with HBsAg was quantitatively inhibited by soluble, polymerized human serum albumin, and the lower limit of the assay was about 1.6 ng of HBsAg per ml. Continuous expression of pre-S-coded antigenic sites on HBsAg particles in chronic hepatitis B patients seropositive for HBeAg or anti-HBe shows that these determinants may be considered as a marker of chronicity during hepatitis B virus infection. The anti-pre-S antibody was determined by inhibition of the reaction for pre-S determinants. This antibody, different from anti-HBs, was detected during HBsAg antigenemia in patients recovering from acute type B hepatitis, before anti-HBs response. Kinetics of synthesis of anti-pre-S antibody in the course of acute type B hepatitis, followed by elimination of HBsAg and recovery, suggest the possible role of this antibody in the immunological clearance of infective hepatitis B virus particles.

Antibody to the receptor for polymerized human serum albumin in acute and persistent infection with hepatitis B virus

The antibody against the receptor for polymerized human serum albumin was determined by radioimmunoassay. The method involved the inhibition by the test serum, absorbed with HBsAg particles without the receptor, on the binding of polymerized human serum albumin to HBsAg particles with the receptor fixed on a solid support. The amount of polymerized human serum albumin captured by the receptor on HBsAg was then determined by the radiolabeled monoclonal antibody directed to an epitope specific for polymerized human serum albumin. In acute infection, the antibody to the receptor for polymerized human serum albumin appeared in the early recovery phase while HBs antigenemia and elevated transaminase levels were still present, preceding the antibody to HBsAg (anti-HBs). The antibody was detected in 4 (1%) of 358 sera from asymptomatic carriers of HBsAg containing antibody to HBeAg, and in none of 67 sera containing HBeAg. Although the antibody was found in as many as 111 (74%) of 150 sera from blood donors who had presumably acquired anti-HBs after natural infection, it was not detected in any sera from 77 recipients of hepatitis B vaccine who had seroconverted for anti-HBs. On the basis of these observations, the determination of antibody to the receptor for polymerized human serum albumin helps in further understanding the immunity to hepatitis B virus.

Antigen Engineering in Yeast: Synthesis and Assembly of Hybrid Hepatitis B Surface Antigen-Herpes Simplex 1 gD Particles

A new approach for antigen presentation and polyvalent vaccines is tested. The idea is to recombine the gene of a second immunogen with that of hepatitis B surface antigen so that the second immunogen is assembled into, and presented on the surface of the HBsAg particle. We describe here the synthesis and assembly in yeast of a model hybrid particle containing both, hepatitis B virus and herpes simplex virus surface antigens.

Detection of antibodies directed against the pre-S gene product of hepatitis B virus: Relationship between anti-pre-S response and recovery

A haemagglutination test and a radioimmunoassay were developed for determination of the pre-S-gene-encoded antigenic specificity of hepatitis B virus. The reaction for pre-S-coded determinants was specifically inhibited by the antibody produced in patients recovering from acute type B hepatitis. Kinetics of synthesis of anti-pre-S antibody in the course of acute type hepatitis followed by elimination of HBsAg and recovery suggest the important role of this antibody in immunological neutralization and clearance of infective HBV particles. The possible protective role of anti-pre-S antibody and the use of pre-S-determinant-positive HBsAg particles in HBV vaccines should be considered.

Sensitive ELISA for IgG and IgM anti-albumin autoantibodies not influenced by HBsAG-associated polyalbumin receptors

An enzyme immunoassay for the detection of IgG and IgM anti-polymerized albumin autoantibodies (AAA) is described. It was found that polyalbumin receptors on HBsAg particles interfere in the detection of IgG AAA when polymerized human albumin (pHSA), but not polymerized bovine albumin (pBSA), is used as coating antigen. Polyalbumin receptors do not appear to interfere in the detection of IgM AAA, with either pHSA or pBSA as coating antigen. All normal sera showed evidence of AAA, of both IgG and IgM classes. Levels of IgG and IgM AAA in sera from most type A and type B acute hepatitis patients were above the range of normal controls. ELISA detection of AAA distinct from HBsAg reactivity can help in understanding the role of these autoantibodies in HBV infection.

Binding activity of HBsAg particles from chronic HBsAg carriers to polystyrene beads coated with polymerized human serum albumin: diagnostic relevance of the assay

The binding activity of HBsAg particles to polystyrene beads coated with polymerized human serum albumin (pHSA) was studied by radioimmunoassay in 48 patients with chronic HBsAg carrier state. The pHSA assay was positive in all 16 HBeAg-positive patients and in 22 HBeAg-negative HBsAg carriers with chronic hepatitis. Asymptomatic, "healthy" HBsAg carriers did not react in the pHSA assay. Mean binding activity was significantly higher in the HBeAg-positive group (P/N ratio 39.3) than in HBeAg-negative carriers with chronic hepatitis in various stages (P/N ratio 19.2). Fractionation of five representative HBeAg-positive sera by density gradient ultracentrifugation in cesium chloride yielded three peaks of HBsAg particles at 1.28, 1.22 and. 1.18 g/ml. The first HBsAg peak contained Dane particles and exhibited strong reactivity in pHSA assay. The second and third peaks, both consisting of 22 nm particles, reacted differently in pHSA assay. While about half of the HBsAg particles in the second peak were bound, reaction in the third HBsAg peak was predominantly negative. Intrahepatic HBsAg was detectable with the immunofluorescence technique in 19 of 22 HBsAg carriers. HBcAg was found in seven of 10 HBeAg-positive cases and in three of 16 HBeAg-negative patients with chronic hepatitis. The diagnostic value of pHSA assay might be seen in differentiation between HBeAg-negative chronic HBsAg carriers with liver disease or "healthy" carrier state.

A transformed Vero cell line stably producing the hepatitis B virus surface antigen

We have constructed plasmids in which transcription of the surface antigen gene of the human hepatitis B virus (HBsAg) is under the control of the SV40 early promoter. These plasmids have been used to analyze the expression of the HBsAg gene, both in mouse cells and in African green monkey kidney (Vero) cells. We have established a Vero cell line continuously expressing HBsAg that is indistinguishable from authentic 22 nm HBsAg particles. Post-transcriptional modification of HBsAg mRNA also appears to occur normally in the monkey cells. These cells might be useful as a source of antigen for the preparation of an antihepatitis vaccine.

Detection of serum blocking factors and antibodies to the albumin receptor on HBsAG particles in healthy persons and patients with liver diseases.

An enzyme-linked immunosorbent assay (ELISA) for the detection of serum blocking factors (BF), or antibodies to the albumin receptor on HBsAg particles, was developed, and its clinical usefulness was examined in healthy persons and patients with liver diseases. Thirteen of 80 anti-HBs-positive female (16.3%) had BF, but all 25 male anti-HBs-positive, 41 female and 32 male anti-HBs-negative subjects were negative for BF. The activity of BF in BF-positive cases was not associated with the positive reciprocal hemagglutination titer of anti-HBs. For a neutralization test of BF, the BFs from 5 cases were absorbed with IgG-immunobeads. It was determined that these IgG-BFs were antibodies to the albumin receptors on HBsAg particles. No significance between positive-BF and abnormal S-GPT levels was recognized. These results suggest that the present test for the detection of BF, or anti-albumin receptor antibody, different from anti-HBs, might be useful for diagnosis of hepatitis B and as a marker for HB virus.

Simplified single radial immunodiffusion detection of albumin receptors on HBsAG particles and its significance regarding sero-changes of HBeAG in hepatitis-B patients.

A simplified single radial immunodiffusion (SRID) method to detect HBsAg particles albumin receptor activity in regard to polymerized human serum albumin (pHSA) (binding-activity of pHSA = pHSA-BA or albumin receptor activity) was developed and its significance in HBeAg-positive hepatitis B patients after a one year follow-up study was discussed. The pHSA-BA determined by the simplified SRID method was classified into 5 grades; +, +/- and -, and the results of this grading of test sera agreed well with those obtained by the ELISA quantitative assay. According to the results of one year follow-up study in sero-changes of HBeAg in 35 HBeAg-positive cases, 7 cases with pHSA-BA did not show HBeAg sero-change, but of 16 cases with pHSA-BA and 12 cases with (+) pHSA-BA 3 (18.8%) and 7 (58.3%) cases showed HBeAg sero-changes, respectively. These results suggest that the present simplified SRID detection of albumin receptor on HBsAg particles might be a useful method for the laboratory investigation of hepatitis B patients.

An antibody which precipitates Dane particles in acute hepatitis type B: relation to receptor sites which bind polymerized human serum albumin on virus particles.

An antibody, which is distinct from the HBsAg- reacts with antigenic sites on Dane particles- HBcAg and HBeAg, was studied by radioimmunoprecipitation of radioactive intact hepatitis B virions in sera obtained early in the course of acute hepatitis type B. The antibody, previously termed anti-Dane particle (anti-DP) antibody, was reactive with Dane particles and HBsAg particles obtained from HBeAg-positive sera but not with HBsAg particles from anti-HBe containing sera. The expression on virus particles of the evoking antigen correlated with levels of binding sites for polymerized human serum albumin (pHSA) as detected by solid-phase radioimmunoassay. In acute hepatitis B sera, levels of anti-DP antibody activity showed inverse correlation with expression of pHSA receptors on circulating virus particles, although the two reactivities were not mutually exclusive. In inhibition experiments, pHSA blocked precipitation of Dane particles by anti-DP positive sera, while native human albumin and polymerized bovine albumin had no effect. The inhibition by pHSA of the anti-DP reaction appeared specific since identical concentrations of pHSA did not interfere with precipitation of virus particles by anti-HBs. Affinity chromatography studies with anti-DP insolubilized on Sepharose 4B columns showed selective binding to the gel of radioactive Dane particles; 125I-HBsAg was not reactive. The binding of Dane particles to anti-DP columns was completely inhibited when virus particles were applied to the gel in pHSA; pretreatment of the column with pHSA did not affect the reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Albumin receptors on HBsAG particles and subsequent sero-conversion designate HBeAG-positive patients among chronic active liver disease patients.

An enzyme-linked immunosorbent assay (ELISA) for the purpose of detecting the albumin receptor on hepatitis B virus surface antigen (HBsAg) particles was developed. Patient sera with moderate to high receptor values demonstrated significant correlations with serum DNA-polymerase activity (p less than 0.005), but not with HBeAg titer. Within one year of the study of 47 HBeAg-positive patients, only in the group of 12 patients with the moderately high values and 9 with low values, did 2 (16.7%) and 6 cases (66.7%) sero-convert, respectively. These results suggest that the albumin receptor might be a useful marker of HBsAg-positive patients.

AVOIDANCE OF PERINATAL TRANSMISSION OF HEPATITIS B VIRUS: IS PASSIVE IMMUNISATION ALWAYS NECESSARY?

Screening of 8918 pregnant women revealed that 107 (1·2%) were HBsAg-positive. 92 of them (50 of German and 42 of predominantly Asian origin) have already delivered and were followed up for 12 months together with their infants. 14 infants of HBeAg-positive (or anti-HBe-negative) carrier mothers received a single dose of hepatitis B immunoglobulin (HBIG) immediately after birth, while 60 infants of anti-HBe-positive mothers were not immunised. 57 of the 60 "unprotected" children remained seronegative for HBsAg and HBeAg. 3 children showed HBs-antigenaemia immediately after birth; 2 of these lost HBsAg and developed anti-HBs after 6 and 9 months. The third lost HBsAg after 4 months without an antibody response developing. This suggests that the HBsAg particle only was transmitted rather than the whole virus. 12 of the 14 infants born to HBeAg-positive, or anti-HBe-negative carrier mothers were protected with one single high dose of HBIG. 2 hed HBs-antigenaemia, 1 had HBeAg as well. These data show that in a West European population the risk of perinatally acquired HBV infection in the infants of anti-HBe-positive carrier motehrs is small, and that passive immunisation of this group is not necessarily indicated.

Receptors for polymerized human serum albumin on hepatitis B virus particles detected by radioimmunoassay: Changes in receptor activity in serum during acute and chronic infection

Receptor sites that bind polymerized human serum albumin on hepatitis B virus (HBV) particles were evaluated in purified virus particle preparations and in HBsAg positive sera, by using solid phase radioimmunoassay. The receptor, found in previous reports to be species-specific, was inhibited neither by native human serum albumin, nor by anti-human immunoglobulins or anti-C1q sera, indicating that these host components are not involved in the reaction. Differential receptor expression on various HBsAg particles was evaluated by relating levels of HBsAg to those of polyalbumin binding activity. This ratio was lower in HBsAg particles purified from hepatitis Be antigen (HBeAg) positive serum (mean ratio: 3.5) than in those obtained from anti-HBe positive serum (mean ratio: 20.1), suggesting that HBeAg/anti-HBe status influences the expression of the receptor on virus particles. Accordingly, during acute hepatitis type B receptor activity was high in sera obtained at the onset, but rapidly decreased afterwards, particularly at the time of anti-HBe seroconversion. A similar behavior of the receptor was observed in cases of chronic HBV infection, where mean receptor counts by radioimmunoassay were 3825±4422 (mean cpm ±SD) in HBeAg positive cases and 868±1196 cpm in anti-HBe positive cases ( P<0.01). Seven HBsAg chronic carriers, all initially HBeAg positive, were followed up over a period of 1 to 4 yr and in all of them a progressive decrease in receptor activity was noted, independently of HBeAg persistence. These results indicate that the expression of the receptor for polymerized human albumin on HBV particles is an early event in the natural course of acute and chronic HBV infection, being followed by a progressive reduction in receptor activity, that parallels the reduction in virus replication, often independently of HBsAg serum levels.

Presence of receptors for polymerized albumin in HBsAg-containing hepatocytes and hepatoma cell line.

Recent evidence suggests that hepatitis B virions (HBV) and HBsAg particles contain receptors for polymerized human serum albumin (pHSA). We studied, by immunohistochemical techniques, the relationship between HBsAg and pHSA receptors in liver tissue from eight patients with chronic HBV infection and in a human hepatocellular carcinoma cell line (PLC/PRF/5) producing HBsAg. Both parallel sections and double fluorescent antibody staining of liver tissue demonstrated that only HBsAg-containing hepatocytes expressed pHSA receptors. The receptors could not be demonstrated in eight HBsAg negative livers. Sequential studies of PLC/PRF/5 cells revealed that pHSA and HBsAg emerged simultaneously in the cytoplasm, on the cell surface, and in the supernatant culture media. These findings indicate that pHSA receptors are closely associated with HBsAg during its synthesis and secretion by hepatocytes and suggest that the receptors are HBV-specific.

Long-term Follow-up of Chronic Hepatitis Patients with HBsAg, HBeAg and Dane Particle Associated DIMA Polymerase in Serum

Nine patients with chronic HBsAg-positive hepatitis and persistence of HBeAg and/or core-associated DNA polymerase activity in serum for at least 1 year were followed up regularly with liver function tests for 2--7 years after onset of illness. HBsAg was present in all patients during an average follow-up period of 5 years. Seven of the 9 patients had persistently abnormal liver function tests. Persistence of HBeAg and/or Dane particle associated DNA polymerase in serum for more than 1 year after onset of illness thus seems to indicate a further long period of HBsAg present in serum and impaired liver function.

Tubular forms of hepatitis b surface antigen bind with the nucleus of 22‐nm spherical hbsag particles

Spherical hepatitis B surface antigen particles (HbsAg) of 22-nm diameter were treated with sodium dodecylsulphate in the absence of reducing agents, and their nuclei were exposed. Factors that interact with the nucleus of HbsAg were detected in the serum of HBsAg carriers who were seropositive for hepatitis B e antigen and identified as tubular forms of HBsAg. The other categories of hepatitis B antigen, Dane particles, and 22-nm spherical HBsAg did not bind with the nucleus of HBsAg. When tubular forms of HBsAg had been treated with a proteolytic enzyme, they lost the reactivity to bind with the nucleus of HBsAg. On the basis of the results obtained, tubular forms of HBsAg bear the receptor of protein nature for the nucleus of 22-nm spherical HBsAg. The receptor allows rapid determination of tubular forms by a haemagglutination method for the evaluation of their clinical and epidemiological implications.

Demonstration and Partial Characterization of 22-nm HBsAg and Dane Particles of Subtype HBsAg/ady

The present paper describes the demonstration of d, y, w, and r HBsAg determinants in one serum. It was shown that there are two populations of HBsAg particles: HBsAg/ad and HBsAg/ady. All complete Dane particles were of subtype HBsAg/ady. Further characterization of HBsAg/ady particles did not reveal morphologic differences when they were compared with HBsAg/ad and HBsAg/ay particles. An HBsAg/ady phenotype may be the result of a double infection with hepatitis B viruses or exchanges of DNA sequences that determine HBsAg/ay and HBsAg/ad to form a new genotype.

Polypeptide profile of HB SAg excreted by a human hepatoma cell line

In this study the polypeptide composition of HB sAg particles secreted by a human hepatoma cell line (Alexander) was analysed. Five major components were identified with molecular weights of 22,000, 28,000, 33,000, 38,000, and 49,000. The two smaller species (MW 22,000 and 28,000) were found to be the major components. The polypeptide profile is similar to that described for HB sAg derived from sera of acute hepatitis patients and of chronic carriers. Data are also presented suggesting that the various HB sAg polypeptide components are aggregates and glycosylated derivatives of the 22,000 MW polypeptide subunit.

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.