C型肝炎ウイルス関連肝細胞癌の早期発症における潜在性B型肝炎ウイルス感染の影響

久留米大学2013年

Cloning and structural analyses of hepatitis B virus DNAs, subtype adr.

Entire genomes of hepatitis B virus (subtype adr) have been cloned. The nucleotide sequence data were compared with other sequences of HBV genome including: adw [Valenzuela et al. (1981) in Animal Virus Genetics. Fields et al. eds. Academic Press, Inc., NY. pp. 57-70], ayw [Galibert et al. (1979) Nature, 281, 646-650], and adyw [Pasek et al. (1979) Nature 282, 575-579]. Four open coding frames for polypeptides larger than 6,000 dalton were found to be conserved and were highly compressed by overlapping with each other in one strand (L-strand). Sites of initiation of the S gene and termination of the P gene were not conserved. No conserved coding frame was found on the opposite strand (S strand). Amino acid sequences of six surface antigen (HBsAg) peptides, including subtypes adr, adw, and ayw, are deduced from the DNA sequences, and the substitution of amino acid residues which are consistent with the change of subtypes are demonstrated

Integration of hepatitis B virus DNA in hepatocellular carcinoma tissue

Hepatitis B virus (HBV) infection and subsequent viral DNA integration have been suggested to play an important role in the development of human hepatocellular carcinoma (HCC). Ten HBV-related HCC samples from South Africa and China were analyzed by Southern blot hybridisation with genomic and subgenomic HBV DNA probes. Nine tumours contained integrated HBV DNA; four had a single integrant and the rest more than one. Genomic DNAs from three single-integrant- positive tumours and two samples with a simple pattern of viral DNA integration were further molecularly cloned, and integrated HBV DNA and the flanking cellular sequences of the clone LAI la were analyzed in detail by restriction mapping and DNA sequencing. Clone LAI la unfortunately contained only the upstream virus-host junction. An attempt was made to modify the polymerase chain reaction (PCR) technique to amplify the downstream virus-host sequences without success. In summary, the single HBV integrant of tumour A1 is more than 2.7 kb in length and contains all regions of HBV genome. The viral DNA integration occurs in an intron or some non-transcribed cellular sequences and one of the virus-host junctions lies at nt 1881 only 47 bp downstream of HBV DR1 (nt 1824-1834), sequences around which are known to be hotspots for integration. The feature of an in-phase stop codon, TAG, in the pre-C region of the integrated HBV DNA has also been observed, although there is no evidence for its association with HCC. Further, the single integrant contains certain cis-acting elements including the promoters for pre-S and surface and the X gene, an enhancer and the viral polyadenylation signal, which are essential for virus gene expression. Thus, cellular sequence(s) may have been placed under the control of these viral cis-acting sequences, leading to altered cellular gene expression and a step to liver oncogenesis. This supports the hypothesis that HBV may contribute to hepatocarcinogenesis through insertional mutagenesis

Expression of hepatitis B virus chimeric proteins in prokaryotic and eukaryotic systems

The particulate form of the core antigen of hepatitis B virus (HBcAg), is highly immunogenic. It has been used as a carrier molecule, for expression and presentation of heterologous viral epitopes on the surface of hybrid core particles, in immunogenicity studies. The aim of this project was to produce a hybrid antigen comprising HBcAg and an immunogenic epitope of human cytomegalovirus (HCMV). A direct comparison was made of amino and carboxyl terminal fusions, by investigating the influence of position of the foreign epitope on antigenicity, immunogenicity and hybrid core particle formation. A part of the HCMV genome, encoding a neutralizing glycoprotein epitope gp58, was inserted at the amino terminus or fused to the truncated carboxyl terminus of HBcAg in separate constructs and expressed in a prokaryotic system (E.coli). At the same time, in order to express the same fusion proteins in a eukaryotic system, the baculovirus expression vector system (BEVS) was selected and as an initial control two recombinant baculoviruses, containing genes encoding HBcAg and hepatitis B surface antigen were isolated by dot blot hybridization. It was realized that work in BEVS would require more time than previously expected therefore further work was only carried out on the prokaryotic system. The HBcAg carboxyl terminal fusion (HBC 3 - 1 4 4 -HCMV) was expressed in high yields in E.coli and assembled into core like particles resembling native HBcAg. A similar fusion in the amino terminus of HBcAg (HCMV-HBC1-183) could not be purified or characterized immunologically, although it formed core like particles. HBc3-144-HCMV displayed HBc antigenicity but HCMV antigenicity could not be detected. Following immunization of rabbits with HBC3-144-HCMV, a high level of anti-HBc specific antibody was produced along with HCMV/gp58 specific antibody. The data presented here provide evidence that the HCMV/gp58 region can be used as a candidate immunogen for an HCMV subunit vaccine and that HBcAg can effectively present this foreign epitope joined to its carboxyl terminus, to the immune system