<b>OBJECTIVEb>To investigate the presentation of a neutralization epitope-containing peptide antigen of hepatitis E virus (HEV) on chimeric virus-like particles (VLPs) of hepatitis B surface antigen (HBsAg).

<b>METHODSb>The gene fragment corresponding to amino acids (aa) 551-607 (HEnAg) of HEV capsid protein, which contains the only neutralization epitope identified to date, was fused via a synthetic glycine linker in frame with the gene of HBsAg. The resulted fusion gene was then integrated through transformation into the genome of Pichia pastoris under the control of a methanol-induced alcohol oxidase 1 (AOX1) promoter and expressed intracellularly. The expression products in the soluble cell extracts were characterized by Western blot, ELISA, CsCl density gradient analysis, and electron microscopic visualization.

<b>RESULTSb>The novel fusion protein incorporating HBsAg and the neutralization epitope-containing HEnAg was expressed successfully in Pichia pastoris with an expected molecular weight of approximately 32 kD. It was found to possess the ability to assemble into chimeric HBV/HEV VLPs with immunological physical and morphological characteristics akin to HBsAg particles. Not only did the chimeric VLPs show high activity levels in a HBsAg particle-specific ELISA but they were also strongly immunoreactive with hepatitis E (HE) positive human serum in a HEV specific ELISA, indicating that HEnAg peptide fragments were exposed on VLP surfaces and would be expected to be readily accessible by cells and molecules of the immune system. Similarity between chimeric VLPs to highly immunogenic HBsAg particles may confer good immunogenicity on surface-displayed HEnAg.

<b>CONCLUSIONb>The chimeric HBV/HEV VLPs produced in this study may have potential to be a recombinant HBV/HEV bivalent vaccine candidate.

Epitopes ; Genetic Engineering ; Hepatitis Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B Surface Antigens ; biosynthesis ; genetics ; immunology ; Hepatitis B virus ; genetics ; immunology ; Hepatitis E virus ; genetics ; immunology ; Pichia ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; Vaccines, Synthetic

In order to find out the distribution of Genotype of those people infected with HBV (hepatitis B virus) from north Guangxi and the relationship between different immune status of HBV infected people and their genotypes, the HBV infected people are classified into three types according to immune tolerance, immune clearance ( response) and immune incompetence (residues). 150 cases from each type, a total of 450 cases are chosen to be tested with real time fluorescence quantitative PCR assay for detection of HBV infection in three kinds of different immune state of the HBV genotype. In the 450 cases, 323 cases belong to type B, 94 cases belong to type B, 23 cases belong to mixed type B+C and 10 cases belong to none B and none C type. Type B are the majority in all the three HBV immune status, made up to 70%, 78%, 67.33% of each type. The different immune state genotype proportion difference don't have statistical significance; immune state and genotypic correlation isn't statistically significant; type B HBV-DNA load is higher than that of type C, groups of persons aged 30 years or older with type C are significantly higher than that of < 30 years of age, the difference was statistically significant; among the genotypes of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBil) positive rate showed no significant difference between male and female; there was no significant difference in genotype distribution. The results show that, in North Guangxi HBV genotypes B, C accounts for the proportion, a small amount of B+C hybrid, occasionally fails to type HBV infection; among immune tolerance, immune clearance (response) and immune incompetence (residues) type B are in majority in these three kinds of immune state, chronic HBV infection immunity with the HBV genotype correlations were not statistically significant.
Adult ; Aged ; China ; Female ; Genotype ; Hepatitis Antibodies ; immunology ; Hepatitis B ; immunology ; virology ; Hepatitis B virus ; classification ; genetics ; immunology ; isolation & purification ; Humans ; Male ; Middle Aged ; Young Adult

<b>OBJECTIVEb>To investigate whether the hepatitis B virus (HBV) has quasispecies character by studying nucleotide sequence polymorphism and mutation features of HBV PreC/C gene region, and preliminaryly explore the heterogeneity of HBV quasispecies.

<b>METHODSb>The serum sample was obtained from a patient with chronic hepatitis B, and the whole HBV PreC/C gene region was amplified by PCR and cloned. Thirty-four clones that contained HBV PreC/C gene fragments were sequenced.

<b>RESULTSb>There were 28 kinds of different nucleotide sequences in 34 clones, and the nucleotide sequences diversity ranged from 0.2% to 2.1%. The mutation points were almost distributed in the whole region, but there wasn't mutation at PreC region nt.1 896 point in all sequences.

<b>CONCLUSIONb>Hepatitis B virus has complex quasispecies character in the patients with chronic hepatitis B.

Adult ; Hepatitis B Core Antigens ; genetics ; immunology ; Hepatitis B virus ; classification ; genetics ; isolation & purification ; Hepatitis B, Chronic ; virology ; Humans ; Male ; Mutation ; Promoter Regions, Genetic ; genetics ; Sequence Analysis, DNA

DNA, Viral ; blood ; Female ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; blood ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Humans ; Male ; Polymerase Chain Reaction ; methods

BACKGROUNDS/AIMS: Occult HBV infection is characterized by the presence of HBV infection with undetectable HBsAg. This study was carried out to find out the frequency of HBV infection in HBsAg- negative patients. METHODS: Fifty-six HBsAg-negative patients including 17 anti-HCV positive patients were evaluated. Patients were grouped according to their serological status; group A (anti-HBc+, anti-HBs-, n=16), B (anti-HBc+, anti-HBs+, n=26), and C (anti-HBc-, anti-HBs+/-, n=14). DNA was extracted from frozen liver biopsy specimen, and HBV DNA level was measured with real-time PCR. RESULTS: Overall frequency of detectable intrahepatic HBV DNA was 34% (19/56). The frequency was 56% (9/16) in group A, 31% (8/26) in group B and 14% (2/14) in group C (P=0.01). Intrahepatic HBV DNA levels were as follows; 2,010 +/- 6,660 copies/mg in group A, 6,180 +/- 29,530 copies/mg in group B and 350 +/- 1,220 copies/mg in group C. The frequency of occult HBV infection was not increased in anti-HCV positive patients. CONCLUSIONS: Intrahepatic HBV DNA is frequently detected in anti-HBc positive, HBsAg-negative patients, although the concentration is low.
Middle Aged ; Male ; Liver/*virology ; Humans ; Hepatitis B virus/*genetics/isolation & purification ; Hepatitis B Surface Antigens/*analysis ; Hepatitis B/*diagnosis/immunology/virology ; Female ; DNA, Viral/*analysis ; Aged ; Adult

<b>OBJECTIVEb>To investigate the phenotypes and functions of peripheral blood monocyte derived dendritic cells (DC) of chronic hepatitis B (CHB) patients with different HBV DNA loads.

<b>METHODSb>Twenty-eight CHB patients were included in this study. All patients were treated with nucleoside analogues (lamivudine or LdT or adefovir) for 24 weeks. Peripheral blood HBV DNA loads and liver biopsies were assessed before and after the treatment. The patients were divided into two groups according to their peripheral blood HBV DNA loads: a high-load group with HBV DNA loads higher than 10(5) copies/ml, and a low-load group with HBV DNA loads lower than 10(3) copies/ml. Ten healthy people were included as controls. Peripheral blood DC of each subject was enriched. The phenotypes of DC were subjected to flow cytometric analysis. The lymphocyte allo-stimulatory capacity of DC was evaluated through MTT assay. IL-10 and IL-12 production were quantified by ELISA.

<b>RESULTSb>DC proliferated successfully when stimulated by cytokines in vitro; however, DC of the CHB patients proliferated much slower than those of the healthy controls. The expression of DC surface molecules such as HLA-DR, CD86, CD80 and CD83 had a positive rate of over 80% in the normal population. However in our CHB patients they showed lower than normal expressions, especially the HLA-DR, CD86, CD80 and CD83, but the differences were not significant between the two groups with different virus loads. The stimulatory capacity of the DC in mixed lymphocyte reaction showed no difference between the two groups of patients, but both were lower than that of the healthy controls. The production of IL-12 and IL-10 also decreased significantly in the patients.

<b>CONCLUSIONSb>Peripheral DC of CHB patients have some defects in their phenotypes and their stimulatory capacity. The changes in phenotypes and down-regulation of the functions are not relevant to peripheral HBV DNA loads of the patients.

Adult ; Dendritic Cells ; immunology ; metabolism ; Female ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; blood ; immunology ; virology ; Humans ; Male ; Middle Aged ; Phenotype ; Viral Load ; Young Adult

Biomarkers ; blood ; DNA, Viral ; blood ; Female ; Hepatitis B ; genetics ; immunology ; prevention & control ; Hepatitis B Antibodies ; biosynthesis ; Hepatitis B Core Antigens ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B Vaccines ; immunology ; Hepatitis B e Antigens ; blood ; immunology ; Hepatitis B virus ; isolation & purification ; Humans ; Male ; Vaccination

The Pichia pastoris strain GS115-PreS could produce a high expression level of full-length PreS protein that secreted to the supernatant after methanol induction in the fermentation. The Western blot analysis showed a single band with expected molecular mass of 48kD and that the major component of the particles was the full-length PreS protein (PreS1 + PreS2 + S) and small envelope protein (S) of 48 and 28 kD, respectively. Electron microscopy image showed PreS particles with 30 nm in diameter. The supernatants of the fermentation were desalted and concentrated. Purified PreS protein was obtained by DEAE-SFF anion exchange column chromatography and the PreS particles were obtained by ultracentrifugation and sucrose density gradient. The ELISA assay results proved that both full-length PreS protein and particles showed high immunogenicity and specificity. P/N ratio further demonstrated that the immunogenicity of the particles is higher than the full-length PreS protein.
Hepatitis B Surface Antigens ; biosynthesis ; genetics ; Hepatitis B virus ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Protein Precursors ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification ; Viral Envelope Proteins ; biosynthesis ; genetics

Adolescent ; Adult ; Antigen-Antibody Complex ; blood ; Antiviral Agents ; therapeutic use ; Child ; DNA, Viral ; blood ; Female ; Hepatitis B e Antigens ; blood ; immunology ; Hepatitis B virus ; genetics ; isolation & purification ; Hepatitis B, Chronic ; drug therapy ; immunology ; Humans ; Lamivudine ; therapeutic use ; Male ; Middle Aged

Human hepatitis B virus surface antigen (HBsAg) binding protein(SBP) shows a specific binding ability to HBV surface antigen HBsAg. Previous work proved an ability of SBP to enhance the immune response of HBsAg vaccine. To investigate the function and mechanism of this protein, we constructed SBP-expression strains with Pichia pastoris expression system. We screened these strains and have got an expression strain with high protein expression quantity. Fermentation product was collected and purified to gain a large amount of purified protein. Identification of purified SBP with SDS-PAGE, High performance liquid chromatography, Western blotting and mass spectrometry suggested that the protein was highly purified and with a good integrity. ELISA test of purified SBP showed a significant binding ability to HBsAg, suggesting a good protein activity. This work offers a solid foundation to the research of SBP function and mechanism of immune enhancement.
Binding Sites ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Humans ; Pichia ; genetics ; metabolism ; Receptors, Virus ; biosynthesis ; genetics ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; isolation & purification