BACKGROUNDTo determine the antigenicity of recombinant hepatitis E virus ORF2 (rHEV ORF2) protein expressed in Pichia pastoris (P. pastoris).

METHODSBy using the rHEV ORF2 protein from E.coli as control, an indirect ELISA was adopted to identify the sensitivity, specificity and stability of rHEV ORF2 protein from P. pastoris in detection of HEV IgM and IgG antibody in sera from patients with hepatitis E. The reactivity of the rHEV ORF2 against 5 HEV ORF2 monoclonal antibodies (McAbs) was also tested.

RESULTSThe minimum concentration of coated antigen with which HEV IgG could be detected was 12.5 ng/ml, while the highest serum dilution to detect both IgM and IgG antibodies against HEV was 1:5 120. No cross-reaction was found with sera from patients with any other types of hepatitis. The 37 degree C acceleration test showed that the rORF2 was highly stable within 12 months at 4 degrees C. The 5 HEV ORF2 McAbs showed better reaction with the rORF2 from P. pastoris, especially that 4B2, 2E2, whose reaction against the rORF2 were 125 and 25 times respectively higher than that of rORF2 from E.Coli.

CONCLUSIONThere may be more extensive conformational epitopes in the rHEV ORF2 from P. pastoris. The excellent antigenicity, sensitivity and stability suggest that it can be served as a new candidate antigen for the development of diagnostic reagents of hepatitis E.

Gene Expression ; Hepatitis Antibodies ; blood ; Hepatitis Antigens ; genetics ; immunology ; Hepatitis E ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Pichia ; genetics ; metabolism ; Recombinant Proteins ; genetics ; immunology ; Viral Proteins ; genetics ; immunology

OBJECTIVETo screen proteins in hepatocytes interacting with HBeAg transactivated protein (HBeAgTP) with yeast-two hybrid technique for investigating the biological functions of HBeAgTP.

METHODSSuppression subtractive hybridization (SSH) and bioinformatics techniques were used for screening and cloning of the target genes transactivated by HBeAg. The HBeAgTP gene was amplified by polymerase chain reaction (PCR) and HBeAgTP bait plasmid was constructed with yeast-two hybrid system 3, and then transformed into yeast AH109. The transformed yeast mated with yeast Y187 containing liver cDNA library plasmid in 2 x YPDA medium. Diploid yeast was plated on synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) and synthetic dropout nutrient medium (SD/-Trp-Leu-His-Ade) containing X-gal for selecting two times and screening. After extracting and sequencing of plasmid from blue colonies, the results were analyzed by bioinformatics.

RESULTSHBeAgTP gene was successfully cloned and expressed in yeast cells. Fifteen genes in twenty-four positive colonies were obtained using yeast-two hybrid technique.

CONCLUSIONHBeAgTP conjugated protein genes were successfully cloned, along with the genes involved in transcription and translation of proteins, immunoloregulation, materials and energy metabolism in vivo.

Hepatitis B e Antigens ; genetics ; metabolism ; Hepatitis B virus ; immunology ; Hepatocytes ; immunology ; metabolism ; Humans ; Protein Interaction Mapping ; Protein Precursors ; genetics ; metabolism ; Two-Hybrid System Techniques ; Yeasts ; genetics

An E. coli expressed recombinant antigen NE2 was reported to aggregate into homo-oligomer, and can induce protective antibodies on rhesus monkey, but its immunogenicty was much weak after being purified. In this study, three N-terminal extension mutant of NE2 were expressed in E. coli, one of which named HEV 239 was found to aggregate into particle. HEV 239 antigen had good reactivity with sera of hepatitis E patients. The reactivity of HEV 239 against neutralization monoclonal antibody 8C11 was similar as NE2 antigen, while the reactivity of it against another neutralization monoclonal antibody 8H3 is much better than NE2 antigen, which indicated better antigenicity of HEV 239 than NE2. The diameter of purified HEV 239 particulate antigen was between 15 nm to 30 nm. The ED50 of immunization of HEV 239 particle adsorbed by aluminum adjuvant to BALB/c mice was between 0.08 microg to 0.25 microg. In contrast, the seraconversion rate of mice immunized by NE2 antigen adsorbed by aluminium adjuvant was only 25% on 60 microg vaccination. These results suggested that HEV 239 antigen particle has better immunogenicity as well as antigenicity than those of NE2 antigen, so it is a better vaccine candidate against HEV.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Genetic Vectors ; genetics ; Hepatitis Antigens ; immunology ; Hepatitis E virus ; genetics ; immunology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Nucleocapsid Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Proteins ; biosynthesis ; genetics ; immunology ; Viral Hepatitis Vaccines ; biosynthesis ; immunology

Replication of the hepatitis B virus is suppressed by deficiency of the X protein. Although several molecules that block cellular targets of X protein reduce the production of hepatitis B virus progeny, the effect of a specific inhibitor of X protein on viral replication has not been investigated. To block X protein specifically, we adopted an intracellular expression approach using H7 single chain variable fragment (H7scFv), an antibody fragment against X protein. We previously demonstrated that cytoplasmic expression of H7scFv inhibits X protein-induced tumorigenicity and transactivation. In this study, intracellular H7scFv expression inhibits reporter gene transactivation but not viral replication determined by endogenous hepatitis B virus polymerase activity assay and real-time PCR. Our findings imply that intracellular expression of antibody fragment against X protein may not be an alternative therapeutic modality for inhibition of hepatitis B virus replication.
Virus Replication/*drug effects ; Trans-Activators/*antagonists & inhibitors/immunology ; Immunoglobulin Variable Region/genetics/metabolism/*pharmacology ; Hepatitis B virus/*drug effects/physiology ; Hepatitis B e Antigens/metabolism ; Cell Line

OBJECTIVEo study the replication of hepatitis B virus (HBV) in HepG2 cells infected with Ad-1.2 HBV.

METHODSHepG2 cells were transfected with adenovirus containing 1.2 copies of HBV DNA. The expression of HBV antigens were detected in the culture medium by means of enzyme-linked immunosorbent assay (ELISA), and the covalently closed circular DNA (cccDNA) in the cells was extracted with plasmid extraction kit and detected by real-time PCR with selective primer after treatment with mung bean nuclease.

RESULTSHBsAg, HBeAg and HBV cccDNA were all detected in HepG2 cells after tranfection with Ad-1.2 HBV. HBV cccDNA was detected 1 day after the infection, reaching the peak level 4 days after infection.

CONCLUSIONAd-1.2 HBV-infected cells can serve as the model for screening and evaluation of antiviral agents.

Adenoviridae ; genetics ; Calibration ; Cell Line, Tumor ; DNA, Complementary ; genetics ; metabolism ; DNA, Viral ; genetics ; metabolism ; Hepatitis B Surface Antigens ; metabolism ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; metabolism ; physiology ; Humans ; Polymerase Chain Reaction ; Time Factors ; Transfection ; Virus Replication

OBJECTIVETo develop a simple method for genotyping of hepatitis E virus (HEV) and to investigate HEV genotype distribution in Nanjing area.

METHODSTwenty-seven full HEV sequences currently-available in GenBank were analyzed with MegAlign and MapDraw programs of DNA STAR software. Degenerate primers were designed and applied to amplify a fragment in HEV ORF1 region. HEV genotypes were determined by the size of the PCR products and by single restriction endonuclease analysis.

RESULTSThe PCR products of HEV genotype 1 and 2 were 275 bp and 269 bp in size. Distinctively, the PCR products of genotype 3 and 4 were 317 bp and 314 bp in size. Moreover, the PCR products of genotype 1 could be digested by Nae 1, but the products of genotype 2 could not. Distinctively, the PCR products of HEV genotype 3 could be digested by Not 1, but the products of genotype 4 could not. Six HEV reference strains standing for different HEV genotypes were clustered into their own types as predicted. Within 43 HEV IgM-positive clinical specimens collected in Nanjing, 19 were HEV PCR-positive and identified as genotype 4.

CONCLUSIONA simple method of PCR combined with single restriction endonuclease analysis is developed for HEV genotyping. This assay allows rapid identification of a large number of HEV isolates directly from clinical specimens. Among patients with hepatitis E in Nanjing, most were infected with HEV genotype 4.

DNA Restriction Enzymes ; metabolism ; DNA, Complementary ; genetics ; metabolism ; Deoxyribonucleases, Type II Site-Specific ; metabolism ; Genotype ; Hepatitis E ; blood ; genetics ; immunology ; Hepatitis E virus ; genetics ; Humans ; Polymerase Chain Reaction ; methods ; RNA, Viral ; genetics ; Reverse Transcriptase Polymerase Chain Reaction

We recently developed a mouse model of hepatitis B virus (HBV) chronic infection by intravenous (i.v.) injection with rAAV8-1. 3HBV to C57BL/6 mice. To define the responses of different mouse strains after injection with rAAV8-1. 3HBV, we intravenously injected rAAV8-1. 3HBV at doses of 4 x10(9) (Viral genome,vg), 4 x 10(10) vg and 4 x 10(11) vg to C57BL/6 and BALB/c mice,respectively, and determined the levels of serum HBV antigen and antibody by ELISA,serum viral DNA by real-time PCR,and HBcAg expression in liver by immunohistochemical staining. For C57BL/6 mouse strain with injection of rAAV8-1. 3HBV at three doses, 100% of the mice carried HBV for more than 8 months. The levels of serum HBsAg and HBeAg, serum viral DNA and HBcAg-positive hepatocytes increased in a rAAV8-1. 3HBV dose-dependent manner. For C57BL/6 mice injected with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg,over 40% of hepatocytes expressed HBcAg and serum viral DNA reached over 10(5) IU/mL. No HBV antibody was detected in sera of C57BL/6 mice. For BALB/c mice with injection of rAAV8-1. 3HBV at three doses, serum HBeAg, serum viral DNA and HBcAg-positive hepatocytes persisted for more than 8 months, but serum HBsAg declined remarkably at 2 weeks after injection. The levels of serum HBeAg and HBcAg-positive hepatocytes in BALB/c mice increased in a rAAV8-1. 3HBV dose-dependent manner. Injection with rAAV8-1. 3HBV at the dose of 4 x 10(11) vg resulted in over 50% of BALB/c mice hepatocytes expressing HBcAg. Serum anti-HBsAg were detected in BALB/c mice with rAAV8-1. 3HBV injection at the dose of 4 x10 (10) vg. In conclusion, both C57BL/6 and BALB/c strains can be developed to chronic HBV infection mouse models by i. v. injection with rAAV8-1. 3HBV at doses of 4 x10(9) - 4 x 10(11) vg and the levels of HBV replication increase in a rAAV8-1. 3HBV dose-dependent manner. In contrast to C57BL/6 strain, the BALB/c mice carry out humoral immunity to HBsAg, but fail to mediate HBV clearance.
Animals ; Dependovirus ; genetics ; metabolism ; Disease Models, Animal ; Genetic Vectors ; genetics ; metabolism ; Hepatitis B ; immunology ; virology ; Hepatitis B Antibodies ; immunology ; Hepatitis B Surface Antigens ; immunology ; Hepatitis B e Antigens ; immunology ; Hepatitis B virus ; genetics ; immunology ; physiology ; Hepatocytes ; immunology ; virology ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Inbred C57BL ; Transduction, Genetic ; Virus Replication

Antibodies, Monoclonal ; chemistry ; immunology ; Antigens, Viral ; chemistry ; genetics ; immunology ; Binding Sites ; Capsid Proteins ; chemistry ; genetics ; immunology ; Epitopes ; chemistry ; genetics ; immunology ; Escherichia coli ; genetics ; metabolism ; Gene Expression ; Hepatitis E ; immunology ; prevention & control ; virology ; Hepatitis E virus ; chemistry ; immunology ; Humans ; Molecular Docking Simulation ; Mutagenesis, Site-Directed ; Peptide Mapping ; Protein Binding ; Recombinant Proteins ; chemistry ; genetics ; immunology ; Viral Hepatitis Vaccines ; administration & dosage ; biosynthesis

Hepatitis E is an acute hepatitis casused by hepatitis E virus (HEV) in developing countries, where it occurs as cases sporadic and in epidemics form. The causative agent, hepatitis E virus, is transmitted primarily by the fecal-oral route. HEV is icosahedron non-enveloped virus, and its genome is a single-stranded, positive-sense, 3'-polyadenylated RNA about 7.5 kb in length. It contains three open reading frames (ORFs). Of which ORF1 codes for a polyprotein of 1693 amino acids and contain domains homologous to a viral methyltransferase, a papainlike cysteine protease, an RNA helicasre, and an RNA-dependent RNA polymerase, besides the most hypervariable region of the HEV genome. And ORF3 codes for a 123-amino-acide-long polypeptide with unknown function. While the major viral capsid protein (pORF2, ORF2 codes) of 660 amino acid was showed to contain the protective epitope. The bacterially expressed polypeptide disignated as NE2 has been proved to be a protective antige. And the anti-NE2 monoclonal antibodies (mAb) was screend, two of these mAbs 8C11 and 8H3 were showed to be against separate conformational neutralization epitope of hepatitis E virus (HEV). And these two mAb were used to screen for binding peptides from a 7-peptides phage display library. After four rounds of panning, tweenty-one positive monoclonal phages (11 for 8C11, and 10 for 8H3) were selected and the inserted fragments were sequenced. The DNA sequence coding for the obtained dominant peptide 8C11 (N'-His-Pro-Thr-Leu-Leu-Arg-Ile-C', named 8C11A) and 8H3 (N'-Ser-Ile-Leu-Pro-Tyr-Pro-Tyr-C', named 8H3A) were then synthesized and cloned to insert between amino acid 78 to 83 of hepatitis B core antigen (HBcAg), then expressed in E. coli. The recombinant proteins aggregate into homodimer or polymer on SDS-PAGE, and could bind with mAb 8C11 and 8H3 in Western blotting. Respectively, the recombinant protein C8C11A showed to be dimer mainly, which can bind with mAb 8C11. The monomer and dimer of C8H3A are in the same amount on SDS-PAGE, but only the dimer could bind with mAb 8H3 on Western blotting. The renatured recombinant proteins were all showed to aggregate into virus like particles which were similar as HBcAg on transmission electron micrograph. The dominant peptide 8H3A (N'-Ser-Ile-Leu-Pro-Tyr-ProTyr-C') that selected out by mAb 8H3 was further chemo-synthesized, and its binding activity was confirmed by BIAcore biosensor. The result showed that this 7-peptide can bind with mAb 8H3 in a big Ka and Kd form, which means the binding is not stable. These results implicated that conformational dependent neutralization epitope could be partially modeled by short peptide, which provided a feasible route for subunit vaccine development.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; immunology ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Epitopes ; chemistry ; genetics ; immunology ; metabolism ; Hepatitis B Core Antigens ; genetics ; metabolism ; Hepatitis E virus ; genetics ; immunology ; metabolism ; Mice ; Microscopy, Electron ; Molecular Sequence Data ; Peptide Library ; Peptides ; chemistry ; genetics ; immunology ; metabolism ; Recombinant Proteins ; genetics ; immunology ; metabolism ; ultrastructure ; Sequence Homology, Amino Acid ; Viral Proteins ; chemistry ; genetics ; immunology ; metabolism ; ultrastructure

BACKGROUNDSome hepatitis B extracellular antigen (HBeAg)-positive chronic hepatitis B (CHB) patients in their immune active phase can clear the virus spontaneously and enter into an inactive hepatitis B virus (HBV) carrier state, indicating a benign prognosis. In this study, the association between cytokine-inducible SRC homology 2 domain protein (CISH) gene polymorphisms at -292 (rs414171) and the spontaneous clearance of HBV in HBeAg-positive CHB patients in immune the active phase was investigated.

METHODSSeventy HBeAg-positive CHB patients in the immune active phase were followed up for 76 weeks without antiviral therapy. The alanine transaminase, aspartate transaminase, HBV DNA, HBeAg and hepatitis B extracellular antibody levels were tested regularly. At week 76, 27 patients were classified into group A (HBV DNA level below 2 104 IU/ml and the value of HBeAg declined below 10% of the baseline at week 76), and 43 patients were classified into group B (HBV DNA level higher than 2×10(4) IU/ml or the value of HBeAg did not decline substantially at week 76). CISH (rs414171) polymorphisms were also tested using the iPLEX system.

RESULTSThe HBV DNA levels at week 12 were significantly greater in group B compared with group A (group A: (6.87±1.40) log10IU/ml; group B: (7.61±1.38) log10IU/ml, P = 0.034) and the HBeAg values were greater in group B at week 28 compared with group A (P = 0.001). The differences in HBV DNA and HBeAg values increased between the groups over time. Sixteen patients in group A and 11 in group B were genotype AA. Those with genotype AT or TT included 11 in group A and 31 in group B (AA vs. AT and TT, odds ratio 4.10 (95% confidence interval: 1.462-11.491), P = 0.006).

CONCLUSIONCISH gene polymorphisms at -292 (rs414171) are associated with HBV clearance in HBeAg-positive CHB patients in the immune active phase, and AA is a favorable genotype for this effect.

Adolescent ; Adult ; Aged ; Female ; Genotype ; Hepatitis B e Antigens ; metabolism ; Hepatitis B virus ; genetics ; immunology ; Hepatitis B, Chronic ; immunology ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; genetics ; Suppressor of Cytokine Signaling Proteins ; genetics ; Young Adult