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.     

A lack of direct role of hepatitis B virus in the activation of ras and c-myc oncogenes in human hepatocellular carcinogenesis

This study was performed to determine the relationship of the activation of ras and c-myc oncogenes in human hepatocellular carcinoma to the hepatitis B virus gene expression or the presence of hepatitis B virus DNA/RNA at the cellular level. This was done using immunocytochemical analysis with two different antibodies on serial sections. In addition, immunocytochemical assay for the detection of ras p21 or c-myc protein was performed in combination with in situ hybridization for hepatitis B virus DNA/RNA using 35S-labeled hepatitis B virus DNA as a probe. Investigation of a total of 14 paired human hepatocellular carcinoma and adjacent nontumorous hepatic tissues revealed enhanced expression of ras p21 in one human hepatocellular carcinoma whereas c-myc protein was found in one paired human hepatocellular carcinoma and nontumorous tissue of the same patient. Only a small proportion of human hepatocellular carcinoma cells or hepatocytes among a large number of cells on a given section showed enhanced expression, and the distribution of the oncogene product-expressing cells was focal. However, the cells overexpressing these oncogenes did not show hepatitis B surface antigen in the serial sections. Furthermore, the combined immunocytochemical and in situ hybridization assays revealed that human hepatocellular carcinoma cells overexpressing ras p21 did not show hepatitis B virus DNA/RNA, whereas some human hepatocellular carcinoma cells and nontumorous hepatocytes located away from the foci of oncogene-expressing cells gave positive signals. These findings suggest that continued expression of HBsAg or the presence of hepatitis B virus DNA/RNA in a given human hepatocellular carcinoma cell id not necessary for enhanced expression of ras or c-myc proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hepatitis B virus integration in hepatitis B virus-related hepatocellular carcinoma in childhood.

In Taiwan, hepatocellular carcinoma is one of the major malignancies in children between 5 and 14 yr of age. We studied the status of hepatitis B virus DNA in the hepatocellular carcinoma and nontumorous liver tissues of eight children with positive serum HBsAg and maternal HBsAg. The hepatocellular carcinoma tissues from five of the eight children showed integration of hepatitis B virus DNA into host cellular DNA sequences. A pattern of single-site integration in four children and a multiple-site integration pattern in one child were demonstrated. In the remaining three children, hepatitis B virus DNA could not be demonstrated in the tumor tissues. Using subgenomic fragments of the hepatitis B virus genome as probes, we found that the X gene fragment and the surface antigen gene fragment were the most conserved sequences. The single-site integration of hepatitis B virus DNA in childhood hepatocellular carcinoma may have hit the critical region, resulting in insertional mutagenesis and early development of hepatocellular carcinoma. With a short incubation period and less exposure to environmental carcinogens during early life, childhood hepatocellular carcinoma may provide a good model to study the carcinogenic potential of hepatitis B virus.     

Antibodies to translation products of the pre-S1 and pre-S2 regions of the envelope gene of hepatitis B virus in fulminant hepatitis B

Sera from 11 patients with fulminant hepatitis B were tested for antibodies to translation products of the pre-S1 and pre-S2 regions of hepatitis B virus of IgM, IgA and IgG classes, as well as of IgA1, IgA2 and SIgA, with solid-phase enzyme immunoassays using native viral polypeptides. Antibodies to pre-S1 region product of IgM and/or IgA class were detected invariably in six patients who still had detectable hepatitis B surface antigen in serum at the time of clinical presentation. The remaining five patients who had lost HBsAg at presentation had antibodies to pre-S region products of various immunoglobulin classes in higher titers. The five patients with fulminant hepatitis without HBsAg had higher levels of IgA antibodies to pre-S region products than the seven patients with nonfulminant acute hepatitis B who had lost HBsAg: IgA antibody to pre-S1 region product (75.6 +/- 63.8 vs. 2.9 +/- 3.2, p less than 0.01) and IgA antibody to pre-S2 region product (28.9 +/- 25.3 vs. 4.2 +/- 6.9, p less than 0.01). IgA antibodies to pre-S1 and pre-S2 region products were invariably polymeric in fulminant hepatitis B. These findings are compatible with the hypothesis that a heightened humoral antibody response to pre-S1 and pre-S2 region products occurs early during the course of fulminant hepatitis B, participating in severe hepatic injury and early clearance of virus characteristic of this disease.     

Detection of hepatitis B virus DNA in hepatocellular carcinoma: analysis by hybridization with subgenomic DNA fragments

We have previously reported an analysis of DNA extracted from 31 primary liver tumors where, in 25 cases, we found chromosomal integration of hepatitis B virus DNA sequences. We describe here an investigation of the extent of the viral genome at each integration site in 15 of the hepatitis B virus DNA-positive tumors using subgenomic fragments of the viral genome as probes. Probes were roughly equivalent to the pre-S region, the surface antigen gene, the region containing the enhancer, the x gene and the core antigen gene. We found the core antigen gene to be that most underrepresented in the tumors and speculate that, since cells which express core antigen in the infected liver may be targeted for lysis by the immune system, modifications of the integrated viral DNA which prevent core antigen expression may be selected. Conversely, the region of the genome present in the greatest number of integrations was the surface antigen gene and, because it is known that the major surface antigen promoter is active in the integrated state, we find promoter insertion an attractive hypothesis to explain oncogenesis by hepatitis B virus.     

Expression of immunogenic epitopes of hepatitis B surface antigen with hybrid flagellin proteins by a vaccine strain of Salmonella.

A nonvirulent Salmonella dublin flagellin-negative, aromatic-dependent live vaccine strain has been used to express hepatitis B virus surface antigen epitopes in an immunogenic form. The envelope proteins of the virion are encoded by the S gene, which contains the pre-S1, pre-S2, and S coding regions. Synthetic oligonucleotides corresponding to amino acid residues S-(122-137) and pre-S2-(120-145) were inserted in-frame into the hypervariable region of a cloned Salmonella flagellin gene, and the recombinant plasmids were introduced into a flagellin-negative aroA mutant live vaccine strain of S. dublin, SL5928. The flagellin gene was expressed in bacteria carrying the plasmids as detected by immunoblotting with anti-flagellin (H1-d) serum. Both the S and pre-S2 epitopes were detected in bacteria carrying the relevant plasmid by immunoblotting with anti-HBs (antibody to hepatitis B virus surface antigen) and anti-peptide antisera. Animals immunized intramuscularly or orally with the live recombinant bacteria developed antibodies specific to these hepatitis B virus epitopes as detected by ELISA.     

Antibody to the hepatitis B virus receptor for polymerized albumin in acute infection and in hepatitis B vaccine recipients

A receptor for polymerized human serum albumin is encoded by the pre-S region of the hepatitis B virus genome and may mediate attachment of the virion to hepatocytes. To investigate antibody response to the virus receptor we studied sera and their IgG fractions for inhibitory activity on hemagglutination of polyalbumin-coated red cells by virus particles containing the pre-S polypeptide. By this method antibody to the receptor was detected in serum in a goat immunized with pre-S containing particles, with no relation to levels of antibody to hepatitis B surface antigen, and in the sera of 33% and 83%, respectively, of acute hepatitis B patients studied during the early phase of illness and during convalescence. In contrast, antibody to the receptor was not detected in serum in any of the 47 subjects immunized with a commercial, plasma-derived, hepatitis B vaccine. These results demonstrate that natural acute infection with hepatitis B virus leads to production of antibody to the virus receptor for polyalbumin, while such antibody response is absent after immunization with currently licensed hepatitis B vaccines.     

A frameshift mutation in the pre-S region of the human hepatitis B virus genome allows production of surface antigen particles but eliminates binding to polymerized albumin.

The coding region for the major polypeptide (p24S) of hepatitis B surface antigen (HBsAg) is preceded by an in-phase open reading frame termed pre-S. The coding potential of the pre-S region was examined in mouse L cells transformed with cloned hepatitis B virus DNA. Such cells produce three HBsAg-related polypeptides of Mr 24,000, 27,000, and 35,000 organized into complex particles of 22 nm diameter. These HBsAg particles bind to polymerized human albumin, but not to polyalbumins of several other species. In contrast, cells transformed with hepatitis B virus DNA bearing a frameshift mutation near the 3' end of the pre-S region secrete immunoreactive HBsAg particles containing only the 24,000 and 27,000 Mr species. These mutant particles, which lack the 35,000 Mr species, are unable to bind polymerized human albumin. These studies indicate that the pre-S region encodes the 35,000 Mr species, that this product accounts for the known polyalbumin-binding activity of HBsAg but is not required for assembly and secretion of HBsAg 22-nm particles, and that the major polypeptide of HBsAg is not derived primarily by cleavage of larger precursors encoded by the pre-S region.     

Large hepatitis B surface antigen polypeptides of Dane particles with the receptor for polymerized human serum albumin

Large hepatitis B surface antigen polypeptides with apparent molecular sizes of 39,000 and 43,000 daltons (P39 and P43) were liberated from a purified preparation of Dane particles of subtype adr. They were tested for reactivity with monoclonal antibodies raised against three synthetic oligopeptides representing fundamental sequences of the pre-S region in deoxyribonucleic acid of hepatitis B virus (subtype adr), as well as with monoclonal antibody against the major surface antigen polypeptide (P22) coded for by the S gene. Both P39 and its glycosylated form P43 bound to all four monoclonal antibodies, thereby indicating that they were coded for by the sequence of 1200 nucleotides, from the second ATG codon in the pre-S region to the stop codon of the S gene. Both P39 and P43 bound to polymerized human and chimpanzee albumins, but not to polymerized albumin from species without susceptibility to hepatitis B virus. Due to their presence in Dane particles and the expression of a polyalbumin receptor, the immune responses against P39 and P43 may have significance in infection with hepatitis B virus and its immunoprophylaxis.     

Vaccine engineering: enhancement of immunogenicity of synthetic peptide vaccines related to hepatitis in chemically defined models consisting of T- and B-cell epitopes.

We report the development of two models for synthetic hepatitis B vaccines. The models were based on the multiple antigen peptide (MAP) system and contained the relevant B- and T-cell epitopes without any macromolecular carrier. Two peptides, representing the a determinant of the S region (S protein) of hepatitis B surface antigen, a dominant serotype of hepatitis B virus infection found in humans, and residues 12-26 of the pre-S(2) region of the middle protein were incorporated as either monoepitope or diepitope MAP models. Immunizations of outbred rabbits with the monoepitope MAP that contains the pre-S(2) antigen resulted in high-titered antibody response to the middle protein, but the other monoepitope, containing only the a-determinant peptide antigen, resulted in poor immune responses to either the peptide antigens or to the S protein. The diepitope MAPs containing both the a and the pre-S(2) determinants produced high-titer antibodies reactive to the a-synthetic peptide and the S protein, as well as to the middle proteins. Thus, our results show that the diepitope MAP models eliminate the need for a protein carrier and that the pre-S(2) peptide determinant serves as a T-helper cell epitope that enhances the immune response of the S region and overcomes the poor immunogenicity encountered with a single epitope of the S region.     

Hepatocellular carcinoma in Richardson's ground squirrels (Spermophilus richardsonii): evidence for association with hepatitis B-like virus infection.

During studies of seasonal obesity, a high frequency of hepatic neoplasms was observed in Richardson's ground squirrels. Of 12 Richardson's ground squirrels examined thoroughly, 7 had mild or moderate degrees of chronic portal hepatitis and 6 (50%) had hepatocellular carcinoma. Serological tests for hepadnavirus surface antigen, anti-core antibody and virion DNA that recognize the ground squirrel hepatitis virus of California ground squirrels (Spermophilus beecheyi) were uniformly negative. Southern blot analyses of EcoRI digests of liver cell DNA demonstrated 3.2 kb fragments that hybridized with a ground squirrel hepatitis virus-specific probe in nontumorous liver tissue from 6 of 10 ground squirrels and in hepatocellular carcinoma specimens from 2 of 5 squirrels indicating infection with a hepadnavirus related to ground squirrel hepatitis virus. Failure, however, to detect serum antibody to ground squirrel hepatitis core antigen suggested probable antigenic differences between the ground squirrel hepatitis virus of California ground squirrels and the putative Richardson's ground squirrel agent. Further studies are required to fully characterize the hepadnavirus of Richardson's ground squirrels and to determine its relationship to hepatocarcinogenesis in this species.