[Pattern of distribution of pre S and S coded viral envelope proteins of hepatitis B virus on the molecular level in serum and liver tissue of chronic HBsAg carriers].
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Adult↗
Biomedical subjects
W H Gerlich
Publications and source records attributed to W H Gerlich.
Occurrence of pre-S1 antigen in viremic and nonviremic carriers of hepatitis B surface antigen.
The proteins of viral envelope, encoded by the pre-S1 region of HBV-DNA, were measured quantitatively with enzyme immunoassay using monoclonal antibodies directed to pre-S1 epitope and correlated with the expression of pre-S2 region encoded epitope and other HBV markers. In acute HBV infection, both pre-S encoded proteins were detected in sera along with markers of viral replication and disappeared shortly before complete virus clearance while high HBsAg titers were still present. Pre-S1 antigen was present in most (95.5%) symptomatic and asymptomatic chronic HBsAg carriers. There was no correlation between the presence of pre-S1 and HBeAg or HBV-DNA in serum: 73% of sera with pre-S1 determinants were anti-HBe positive, and only 25.4% were positive for HBV-DNA. Most pre-S1 activity in sera of viremic carriers was detected in fractions of sucrose gradient containing subviral 22-nm particles, and much less in those containing infectious virions. In asymptomatic, nonviremic HBsAg carriers, pre-S1 was located only on subviral 22-nm forms. Pre-S1 positive particles had no accessible pre-S2 epitope, which is recognized specifically by monoclonal anti-pre-S2 (F124) antibody. These results show that the synthesis of the large protein of HBV envelope may occur also in the absence of active viral replication, and in these cases pre-S1 encoded sequences are on subviral particles of HBsAg. Therefore, pre-S1 is not a serologic marker of infectious virus. Disappearance of pre-S1 epitopes on HBsAg occurs only before complete clearance of the virus, and this may have potential prognostic relevance.
Acute Disease↗
Formation of transmembraneous hepatitis B e-antigen by cotranslational in vitro processing of the viral precore protein.
The gene encoding the major core protein P22c of hepatitis B virus is preceded by a precore sequence. Expression of the core gene with the precore in Escherichia coli results in a membrane protein of HBe antigenicity. Expression in mammalian cells generates secreted HBeAg. To study the biosynthetic pathway of HBeAg and the function of precore in this process, we translated mRNAs for core proteins with and without precore using reticulocyte lysates and microsomal vesicles. The precore sequence was cleaved cotranslationally as a signal peptide, probably at alanine 19. The processed product P23e was partially translocated to the lumen of the microsomes. The arginine-rich carboxy-terminal domain of P23e was however not translocated and susceptible to trypsin. Clusters of positive-charged amino acids seem to act as a novel type of translocation stop signal. Trypsin generated a P16e which no longer had a transmembraneous configuration. The findings may explain the biosynthesis and potential function of HBeAg in hepatitis B virus-infected hepatocytes.
Amino Acid Sequence↗
[Approaches to developing an optimal vaccine against hepatitis B viruses].
Using recombinant DNA methods, many different approaches may be followed to optimize immunoprophylaxis against hepatitis B viruses. Most obviously a future vaccine should contain besides the known major surface protein two further envelope proteins which have recently been identified. All three envelope proteins should be present on the same particle in natural proportion and conformation. Such a vaccine may induce a more reliable and more durable immune protection even in difficult cases. The protective potential of the viral core protein, in particular of HBeAg, ought to be studied further experimentally. Possibly, the core proteins may be helpful in an immune therapy of already infected persons.
Antigen-Presenting Cells↗
Cell type specific expression of pre S 1 antigen and secretion of hepatitis B virus surface antigen. Brief Report.
Production of the three hepatitis B surface (HBs) proteins was studied in a hepatoma cell line (PLC/PRF/5) and two HBs antigen secreting cell lines (HeLa and mouse L-cells), which had been transfected by a viral genome isolated by molecular cloning from PLC/PRF/5 chromosomal DNA. The DNA used for transfection contains the HBs-specific promoters and the enhancer which regulate the expression of HBs genes in the transfected cell lines. All three cell lines expressed well the small and middle HBs protein, but the larger pre S 1 containing protein was barely detectable in the L-cell. In vivo growth of the transfected HeLa cell as nude mouse tumour increased pre S 1 expression and suppressed secretion of HBsAg.
Animals↗
Pre-S encoded surface proteins in relation to the major viral surface antigen in acute hepatitis B virus infection.
The role of pre-S encoded viral surface proteins in acute hepatitis B virus infection is still poorly understood. Binding sites for polymerized human serum albumin have been found to be encoded by the pre-s2 region of the hepatitis B virus genome. Recently, murine monoclonal antibodies against pre-s1 and pre-s2 encoded hepatitis B virus gene products were generated and used for their specific detection in serum. In sera from patients with acute hepatitis B, pre-s1 and pre-s2 antigen occurred in 16 of 20 and 15 of 20 patients, respectively. In the initial stage of the disease, pre-S gene products correlated with binding sites for polymerized human serum albumin, but not with hepatitis B surface antigen. Subsequently, pre-s1 and pre-s2 antigens were cleared from the serum of patients with acute hepatitis B before binding sites for polymerized human serum albumin and hepatitis B surface antigen. Possibly, the early clearance of pre-S markers can be of prognostic value in acute hepatitis B. The mechanisms of the early clearance of the pre-S antigens in acute hepatitis B remain to be elucidated. However, elimination by immunologic mechanisms appears likely.
Acute Disease↗
Immunogenicity of the gene S and Pre-S domains in hepatitis B virions and HBsAg filaments.
The three morphological forms (20-nm particles, filaments, virions) of hepatitis B surface antigen (HBsAg) were isolated from serum of chronic virus carriers or from transfected cell lines. BALB/c mice and guinea pigs were immunized with the antigens and the antibody responses against the three antigenic domains of the viral envelope were assayed. The proportion of pre-S1, pre-S2 and gene S antibodies was similar to the molar proportion of the domains in the immunogens. The major gene S and pre-S1 epitopes were conformational, and the major epitopes of the pre-S2 domain were sequential. The immunogenicity of natural and recombinant antigens was identical. The proportion of subtype-specific antibodies was high. The results suggest that recombinant HBsAg filaments containing both subtypes ad and ay may be optimal hepatitis B vaccines.
Animals↗
Inhibition of albumin binding to hepatitis B virions by monoclonal antibody to the preS2 domain of the viral envelope.
The binding of polyalbumin to hepatitis B virus (HBV)-associated envelope epitopes has been studied by means of a radioimmunoprecipitation technique. HBV particles were purified from the sera of chronic hepatitis B surface antigen (HBsAg) carriers and labelled through the endogenous HBV-DNA polymerase reaction. Human albumin, polymerized through glutaraldehyde cross-linking, was able to precipitate (100%) labelled HBV at concentrations of 31.2 and 62.5 micrograms/ml, in contrast to monomeric albumin (HSA). This event was further confirmed by immune electron microscopy. The addition of anti-HSA to the mixture HBV plus polyalbumin gave a 100% precipitation in a wide dilution range (15.6-500 micrograms/ml). The binding of polyalbumin (31.2 micrograms/ml) to virions was strongly inhibited (up to 98%) when preincubating with antibody to a glycosylation-dependent preS2 epitope on HBV. The same was accounted (up to 99%) for polyvalent IgG anti-HBs. However, antibodies to the group 'a' and subtype 'd' determinants, as well as anti-preS1 region antibodies, inhibited weakly polyalbumin binding to HBV. The binding site of the inhibitory antibody overlaps probably with neutralizing epitopes. Our findings support the hypothesis that albumin binding plays an important role in the viral life cycle.
Antibodies, Monoclonal↗
Cell-type dependent expression and secretion of hepatitis B virus pre-S1 surface antigen.
The human hepatoma cell PLC/PRF/5 contains cloned genomes of hepatitis B virus, one of which was transfected to mouse cells (LTK-) or to human carcinoma cells (HeLa). Expression of the viral surface proteins was measured using monoclonal antibodies. The large protein with the pre-S1 domain was best expressed in HeLa cells and least in LTK- cells. In vivo growth of parental hepatoma cells or transfected HeLa cells increased expression of large, and decreased expression of small protein. Secretion of large protein required an excess of small protein. The secreted HBsAg from transfected HeLa cells contained filaments. Since the virus contains a defined proportion of all three proteins, a special regulatory situation is necessary for maturation of its envelope and its secretion in vivo.
Animals↗
Proteins of hepatitis delta virus.
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Animals↗
Assay of hepatitis B virus genome titers in sera of infected subjects.
A method for quantitative standardization of the DNA hybridization assay for hepatitis B virus (HBV) DNA protein complex in serum is described. This method was used to determine the titer of HBV DNA in various groups of subjects with HB surface antigen (HBsAg) in order to ascertain its accuracy as an index of infectivity. The method's detection limit was 10(5) genome equivalents or 0.3 pg DNA per ml. Titers of 5 X 10(7) to 5 X 10(8) genome equivalents per ml were found to be typical for persistent massive viremia, which occurred more frequently in symptomatic (30 of 48) than in asymptomatic (24 of 72) carriers positive for HBe antigen (HBeAg). Moderate viremia (10(5)-5 X 10(7)) was usually found in patients eliminating the virus from the blood. Patients with resolving acute hepatitis B were frequently positive at the onset (18 of 26) with moderate titers, but became negative within several weeks. In 11 patients who developed chronic hepatitis B, titers increased until typical massive viremia was evident. Whereas healthy HBsAg carriers with anti-HBe always had negative genome titers (144 of 144), symptomatic carriers with anti-HBe often had moderate genome titers (9 of 30). It is recommended that genome titers be monitored in HBeAg-positive and in symptomatic anti-HBe positive virus carriers in order to distinguish between virus carriers with high (greater than 5 X 10(7)), moderate (10(5) -5 X 10(7)) and low (less than 10(5)) infectivity.
Acute Disease↗
Precore sequence of hepatitis B virus inducing e antigen and membrane association of the viral core protein.
Hepatitis B virus (HBV) DNA contains a precore (pre-c) sequence of 29 codons with unknown function upstream of its gene for the major core protein. Its significance was studied by expression of core proteins with and without pre-c in Escherichia coli. Core protein without pre-c, P22c, assembled spontaneously to core particles and formed core antigen. It had the same size and antigenicity as core particles from infected liver. Core protein with pre-c, P25e, instead formed membrane-associated e antigen (HBeAg). The data suggest that pre-c functions as a signal peptide for the attachment of core protein P25e to cellular membranes. This hypothesis can explain the not yet understood relation between viremia and HbeAg and the protective role of anti-HBe antibody.
Amino Acid Sequence↗
Cutoff levels of immunoglobulin M antibody against viral core antigen for differentiation of acute, chronic, and past hepatitis B virus infections.
The titer of antibody against core antigen of hepatitis B virus in the immunoglobulin M class (IgM anti-HBc) was determined by an IgM capture assay of reduced sensitivity (30 arbitrary units). The distribution of titers among 235 acute hepatitis patients who were hepatitis B surface antigen (HBsAg) positive suggested that 600 U forms a lower cutoff value for acute hepatitis B. Clinically apparent cases of acute hepatitis with high IgM anti-HBc and without HBsAg were rare (2.6%). Acute, non-B hepatitis in HBsAg carriers was more frequent (9.4%). In chronic hepatitis B, 39% of 174 biopsy-proven cases had moderate titers of 30 to 600 U, whereas healthy HBsAg carriers were rarely (4/84) positive. In mild or inapparent infections without HBsAg, titers were between 50 and 400 U. Thus, sufficiently accurate and sensitive quantitation of IgM anti-HBc allows for differentiation of acute and nonacute hepatitis B virus infection in acute hepatitis, partial differentiation between clinically symptomatic and asymptomatic chronic infections, and identification of recent subclinical infections.
Acute Disease↗
Hepatitis delta virus: protein composition of delta antigen and its hepatitis B virus-derived envelope.
Hepatitis delta virus (HDV)-associated particles were purified from the serum of an experimentally infected chimpanzee by size chromatography and by density centrifugation. Hepatitis delta antigen (HDAg) was detected after mild detergent treatment at a column elution volume corresponding to 36-nm particles and banded at a density of 1.25 g/ml. The serum had an estimated titer of 10(9) to 10(10) HDV-associated particles and had only a 10-fold excess of hepatitis B surface antigen (HBsAg) not associated with HDAg. Therefore, HDV appears to be much more efficiently packed and secreted than is its helper virus, hepatitis B virus (HBV), which is usually accompanied by a 1,000-fold excess of HBsAg. The protein compositions of the HDAg-containing particles were analyzed by immunoblotting with HDAg-, HBsAg-, and hepatitis B core antigen-specific antisera and monoclonal antibodies to HBV surface gene products. The HBsAg envelope of HDAg contained approximately 95% P24/GP27s, 5% GP33/36s, and 1% P39/GP42s proteins. This protein composition was more similar to that of the 22-nm particles of HBsAg than to that of complete HBV. The significant amount of GP33/36s suggests that the HBsAg component of the HDV-associated particle carries the albumin receptor. Two proteins of 27 and 29 kilodaltons which specifically bound antibody to HDAg but not HBV-specific antibodies were detected in the interior of the 36-nm particle. Since these proteins were structural components of HDAg and were most likely coded for by HDV, they were designated P27d and P29d.
Animals↗
[Significance of genetic technics for the diagnosis and prevention of hepatitis B virus infections].
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Carcinoma, Hepatocellular↗
Direct radioimmunoassay of antibody against hepatitis B core antigen using 32P-labelled core particles.
A direct radioimmune assay for antibody against hepatitis B core antigen (HBcAg) was developed. Flexible microtiter plates were coated with HBcAg, incubated with test samples, and thereafter with 32P-labelled HBcAg. Labelling was achieved by endogenous protein kinase of the core particles. This assay was tenfold more sensitive than a conventional inhibition assay employing enzyme-labelled anti-HBc as reagent. The radioimmunoassay detected a large number of positive persons (88/200) in a population with a high prevalence of blood-transmitted hepatitis infections (medical staff, liver and dialysis patients, contact persons, implicated blood donors) which were not detected by the inhibition assay. Such results were rare in healthy blood donors. The weak anti-HBc activity, which was detected only by direct radioimmunoassay, co-purified with IgG and was inhibited by addition of HBcAg to the serum. The activity may be due to a very limited hepatitis B infection or, what is more likely, to cross-reacting antibodies against unknown antigen(s). The factor detected only by direct radioimmune assay appears to be related to viral hepatitis. For detection of anti-HBc as a marker for hepatitis B virus it is, however, preferable to use less sensitive assays.
Hepatitis B Antibodies↗
Large surface proteins of hepatitis B virus containing the pre-s sequence.
The sequence of hepatitis B virus DNA contains an open reading frame which codes for a not-yet-identified protein of at least 389 amino acids. Only the products starting at the third (GP33/GP36) or the fourth (P24/GP27) initiation signal have been characterized as components of the viral surface antigen. We found a larger protein, P39, and its glycosylated form, GP42, in hepatitis B virus particles and viral surface antigen filaments. Immunological cross-reactions showed that P39/GP42 is partially homologous to P24/GP27 and GP33/GP36. The unique portion of its sequence bound monoclonal antibodies which had been induced by immunization with hepatitis B virus particles. Proteolytic cleavage patterns and subtype-specific size differences suggested that the sequence of P39 starts with the first initiation signal of the open reading frame. Its amino-terminal part (pre-s coded) is exposed at the viral surface and, probably, is highly immunogenic. A model is presented of how the open reading frame for the viral envelope leads to defined amounts of three different proteins.
Antibodies, Monoclonal↗
Topology and immunoreactivity of capsid proteins in hepatitis A virus.
Hepatitis A virus (HAV) was propagated in a hepatoma cell line and complete viral particles with a density of 1.34 g/ml were purified from cell extracts. The topography of the viral proteins (VPs) was studied by surface labelling with 125I and a solid-phase oxidant. The order of labelling intensity in complete particles was VP1 much greater than VP3 greater than VP2; labelling of VP4 was undetectable. When the particles were denatured with sodium dodecyl sulfate at 100 degrees C before iodination, the labelling efficiency was 6 times higher and the order of labelling intensity was VP3 greater than VP2 greater than VP1. After denaturation, the viral proteins no longer reacted with human anti-HAV antibody. The results suggest that (i) as with other picornaviruses, HAV exposes an essential part of VP1 at its surface whereas VP3 and especially VP2 are more hidden; (ii) naturally immunized individuals do not form detectable amounts of antibodies against the denatured capsid proteins. The apparent molecular weights of the VPs were 33000, 29000 and 28000 daltons.
Capsid↗