Rearrangement of the surface antigen gene of hepatitis B virus integrated in the human hepatoma cell lines.

The rearrangement of integrated HBV DNA sequences in three different hepatoma cell lines, huH-1, huH-2, KG-55-T from Japanese patients, were studied by blot hybridization using whole HBV genome or a HBsAg or HBcAg DNA as a probe. The characteristic existence of multiple integration sites of HBV DNA sequences in each HindIII-restricted hepatoma cell DNA was revealed by the HBV genome probe. Detection of the isolated HBsAg gene in the HindIII fragment indicates that the integration of HBV DNA was not always related to the maintenance of the whole viral genome, and that movement of the HBsAg gene to another location occurred by rearrangement. On the other hand, the presence of the HBV DNA sequence without the intact HBcAg gene was shown in some of the HindIII fragments, when the HBcAg gene, probe was used, but a HindIII fragment, containing only the HBcAg gene, was not detected so far. The absence of the intact HBcAg gene suggests that the viral genome may lose a part of the HBcAg gene in the process of integration. This is consistent with recent findings of Ogston et al. (1982) that in Woodchuck hepatocellular carcinoma viral sequences are extensively rearranged.     

Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line.

The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.     

Integration pattern of hepatitis B virus DNA sequences in human hepatoma cell lines.

Four human hepatoma cell lines established from primary hepatocellular carcinomas were examined for the presence of hepatitis B virus DNA sequences. Reassociation kinetic analysis indicated that the cell lines HEp-3B 217, HEp-3B 14, HEp-3B F1, and PLC/PRF/5 contained two, one, one, and four genome equivalents per cell, respectively. Southern blot hybridization analysis demonstrated that hepatitis B virus DNA was integrated into the cellular DNAs of these cell lines. Further liquid hybridization studies with 32P-labeled HincII restriction fragments of hepatitis B virus DNA established that DNA sequences from all regions of the HBV genome were represented in the integrated viral sequences. Although the three HEp-3B cell lines were derived from the same tumor, they differed significantly in their patterns of integration of hepatitis B virus DNA, the number of copies of viral DNA per cell, and their ability to produce the virus-coded surface antigen.     

Insulin suppresses hepatitis B surface antigen expression in human hepatoma cells

The human hepatoma Hep3B cells contain integrated hepatitis B viral genome and continually secret hepatitis B surface antigen (HBsAg). The production of HBsAg (but not alpha-fetoprotein) was suppressed by addition of low concentrations (0.1-1 nM) of insulin into serum-free medium. In addition, the suppression of HBsAg production by insulin was paralleled with the decrease in HBsAg mRNA abundance. Insulin also cause a rapid rate of disappearance of HBsAg mRNA (t 1/2, 2 h) in Hep3B cells. The Hep3B cells carry specific receptor with high affinity for insulin (Kd = 1.8 nM). The receptor showed an insulin-dependent protein tyrosine kinase activity. The half-maximal insulin concentration for the activation of the receptor kinase was about 5 nM. Only very high concentrations of insulin-like growth factor I and human proinsulin can compete for the insulin receptor binding and suppress HBsAg production, this suggests that insulin may act through its receptor binding to suppress HBsAg expression in human hepatoma Hep3B cells.     

Structure and expression of human hepatitis B virus surface antigen

In view of the growing occurrence rate of the virus-induced hepatitis B and also of the special role played by this particular virus (HBV) in the application of recombinant genetic techniques to the study of complex biological systems, an attempt was made to survey the available evidence concerning the widely investigated and practically the most important part of the viral genome, viz. the gene coding for the surface antigen (HBsAg) and the protein itself. The possible antigenic structure of the protein was investigated using data on the primary structure of 11 cloned HBsAg gene variants and on the synthesis of peptides simulating its immunological properties. Special emphasis was placed on quantitative assessment of antigenicity and immunogenicity. Expression of the gene in homologous systems was studied using cultures of eukaryotic tissues: both as part of HBV nucleotide sequences incorporated into the chromosome and as part of extrachromosomal DNA. The latest findings on HBsAg gene expression in yeast and bacteria are reviewed.     

Overcoming T cell tolerance to the hepatitis B virus surface antigen in hepatitis B virus-transgenic mice

The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.     

Expression of the hepatitis B virus surface,core and E antigen genes by stable rat and mouse cell lines

A recombinant plasmid containing four tandem head-to-tail copies of the HBV⁴ genome has been constructed and introduced into thymidine kinase-deficient mouse and rat fibroblast cells by co-transformation with the thymidine kinase gene from Herpes simplex virus as a selectable marker. Several mouse cell lines that synthesize HBsAg and four lines (three mouse and one rat) that synthesize both HBsAg and HBeAg were isolated. The rat line and at least one of the L cell lines that synthesizes HBeAg also produce small quantities of HBcAg. The rat line has many partial and 18 to 20 complete copies of the HBV genome integrated into high molecular weight DNA, whereas the L cells that produce HBeAg have only two to three complete copies of HBV genome. The kinetics of synthesis of HBsAg and HBeAg by the rat line are similar to that for HBsAg synthesis by two human hepatoma lines, and the HBsAg secreted by this line has similar biophysical characteristics to that found in human serum. The results suggest that the configuration of the recombinant molecule used may be an important prerequisite for the expression of HBcAg and HBeAg, and we speculate that the biogenesis of their messenger RNA may proceed via a precursor that is greater than the length of the HBV genome.     

Antigenicity and immunogenicity of novel chimeric hepatitis B surface antigen particles with exposed hepatitis C virus epitopes

The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127-128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.     

Major polypeptide of duck hepatitis B surface antigen particles

The 40- to 50-nm pleomorphic particles found in the sera of domestic Pekin ducks infected with duck hepatitis B virus were purified by rate zonal and isopycnic centrifugation. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic polypeptide analysis of these particles, called duck hepatitis B surface antigen particles, revealed the major component to be a single 17,500-dalton polypeptide. This result is in contrast to polypeptide analyses of the surface antigens of related mammalian viruses, including hepatitis B, in which a major doublet of polypeptides is seen with molecular weights ranging from 23,000 to 29,000. Tryptic maps of 17,500-dalton polypeptide resembled that of the major non-glycosylated polypeptide of the adw subtype of hepatitis B surface antigen. A serological assay for antibody to the purified duck virus particles is also described.     

Vaccine against human hepatitis B virus prepared from antigen derived from human hepatoma cells in culture

Vaccine against human hepatitis B was prepared using antigen derived from hepatitis B carrier hepatoma cells grown in the interstices of a Diaflo hollow filter unit. Hepatitis B surface antigen (HBsAg) produced by these cells was purified by immune affinity chromatography, digestion with DNase and pepsin, and Sephadex G-150 separation. The Formalin-treated antigen was formulated in 20-micrograms dose on alum adjuvant with thimerosal added as a preservative. This cell culture vaccine was as potent as human plasma-derived vaccine as measured in a mouse potency assay. The vaccine proved safe in tests in chimpanzees and in human subjects who were in late stages of cancer of the central nervous system and who were receiving therapy for their condition. None of five subjects who received the vaccine developed untoward clinical reactions. Two of the subjects who received all three doses of vaccine developed antibody against HBsAg. Three persons, two given only the primary doses and one who was given all three doses but was lost to follow-up, demonstrated no response. The slow and relatively low antibody responses to the vaccine were similar to those in other immunosuppressed persons who were given vaccine of human plasma origin.     

Expression of the hepatitis B virus surface antigen gene by rat hepatocyte X mouse hepatoma hybrid cells

The expression of the S gene of hepatitis B virus has been studied in the somatic hybrid cells resulting from the fusion between rat hepatocytes in primary culture and cells of the mouse hepatoma line BWTG3, and in the parental line BWTG3. The DNA of the S gene inserted into the plasmids pAC Tk+ and pNY4 has been co-transfected into these cells with a plasmid DNA bearing a resistance gene to aminoglycoside. The level of expression of the S gene among the co-transfected resistant clones was estimated by radioimmunoassay. The results show that a high number of the co-transfected cellular hybrid clones express the S gene, whereas it is found, by contrast, that the S gene is poorly expressed in the mouse hepatoma cells. The level of expression of the S gene (as the amount of HBs Ag synthesized) is high in the hybrid clones and the synthesis of the HBs antigen is stable in time. These observations suggest for the first time in cell cultures in vitro, the role which is probably played by the normal hepatocyte genome in the expression of the S gene of HBV.     

Targeting of polyplex to human hepatic cells by bio-nanocapsules, hepatitis B virus surface antigen L protein particles

We have previously demonstrated that lipoplex, a complex of cationic liposomes and DNA, could be targeted to human hepatic cells in vitro and in vivo by conjugation with bio-nanocapsules (BNCs) comprising hepatitis B virus (HBV) surface antigen L protein particles. Because the BNC-lipoplex complexes were endowed with the human hepatic cell-specific infection machinery from HBV, the complexes showed excellent specific transfection efficiency in human hepatic cells. In this study, we have found that polyplex (a complex of polyethyleneimine (PEI) and DNA) could form stable complexes with BNCs spontaneously. The diameter and ζ-potential of BNC-polyplex complexes are about 240 nm and +3.54 mV, respectively, which make them more suitable for in vivo use than polyplex alone. BNC-polyplex complexes with an N/P ratio (the molar ratio of the amine group of PEI to the phosphate group of DNA) of 40 showed excellent transfection efficiency in human hepatic cells. When acidification of endosomes was inhibited by bafilomycin A1, the complexes showed higher transfection efficiency than polyplex itself, strongly suggesting that the complexes escaped from endosomes by both fusogenic activity of BNCs and proton sponge activity of polyplex. Furthermore, the cytotoxicity is comparable to that of polyplex of the same N/P value. Thus, BNC-polyplex complexes would be a promising gene delivery carrier for human liver-specific gene therapy.     

Circulating hepatitis B surface antigen particles carry hepatocellular microRNAs

Hepatitis B virus (HBV) produces high quantities of subviral surface antigen particles (HBsAg) which circulate in the blood outnumbering virions of about 1\10^3–6 times. In individuals coinfected with the defective hepatitis Delta virus (HDV) the small HDV-RNA-genome and Delta antigen circulate as ribonucleoprotein complexes within HBsAg subviral particles. We addressed the question whether subviral HBsAg particles may carry in the same way cellular microRNAs (miRNAs) which are released into the bloodstream within different subcellular forms such as exosomes and microvescicles. Circulating HBsAg particles were isolated from sera of 11 HBsAg carriers by selective immunoprecipitation with monoclonal anti-HBs-IgG, total RNA was extracted and human miRNAs were screened by TaqMan real-time quantitative PCR Arrays. Thirty-nine human miRNAs were found to be significantly associated with the immunoprecipitated HBsAg, as determined by both comparative DDCT analysis and non-parametric tests (Mann-Whitney, p<0.05) with respect to controls. Moreover immunoprecipitated HBsAg particles contained Ago2 protein that could be revealed in ELISA only after 0.5% NP40. HBsAg associated miRNAs were liver-specific (most frequent = miR-27a, miR-30b, miR-122, miR-126 and miR-145) as well as immune regulatory (most frequent = miR-106b and miR-223). Computationally predicted target genes of HBsAg-associated miRNAs highlighted molecular pathways dealing with host-pathogenThe finding that HBsAg particles carry selective pools of hepatocellular miRNAs opens new avenues of research to disentangle the complex interactions between host and HBV and provides a non invasive tool to study the physiopathology of liver epigenetics.     

A cell surface protein that binds avian hepatitis B virus particles.

We have identified a 180-kDa cellular glycoprotein (gp180) that binds with high affinity to duck hepatitis B virus (DHBV) particles. The protein was detected by coprecipitating labeled duck hepatocyte proteins with virions or recombinant DHBV envelope proteins, using nonneutralizing monoclonal antibodies to the virion envelope. Binding of gp180 requires only the pre-S region of the viral large envelope protein, since recombinant fusion proteins bearing only this region efficiently coprecipitate gp180. The DHBV-gp180 interaction is blocked by two independent neutralizing monoclonal antibodies. The protein is found on both internal and surface membranes of the cell, and the species distribution of gp180 binding activity mirrors the known host range of DHBV infection. Functional gp180 is expressed in a wide variety of tissues in susceptible ducks.