In this study, three expression vectors encoding unmodified glycoproteins E1 and E2 from H77 (1a), Hebei (1b) and JFH1 (2a) strains were constructed to form pVRC-H77-E1E2, pVRC-HeBei-E1E2 and pVRC-JFH1-E1E2 expressing constructs. The protein expression was confirmed by immunofluorescene assay(IFA) and Western blot. The Lentiviral vector has the ability to package the cellular membrane into pseudo-particles. The plasmid expressing HCV E1-E2 glycoproteins in native form was co-transfected into 293FT cells with a lentiviral packaging plasmid (pHR'CMV delta R8.2)and a self-inactivated (SIN) transfer plasmid (pCS-CG) containing a reporter EGFP gene to produce infectious HCV pseudo-particles(pp). Flow cytometry assays showed that the HCVpp could infect Huh7 and Huh7-CD81, and the infectivity in Huh7-CD81 was about 2-3 times higher than that in Huh7 cells. Meanwhile, HCVpp could neither infect non-liver cells, for example, the 293 cells, nor HepG2 cell . Titration of HCVpp by p24 ELISA assay or infection assay showed that this HCVpp may contain 5-25 ng/mL p24 or 10(4)-10(5) TU (transducing unit)/ ml. An in vitro HCV neutralizing assays based on HCVpp (1a, 1b, 2a) were then established using AP33, a monoclone antibody with cross-neutralizing ability to different HCV strains. The neutralizing ability of the antibodies from HCV infected patients was further studied with this HCVpp system. In summary, three kinds of HCVpp (1a, 1b, 2a subtype) were successfully developed; In vitro HCV neutralizing assays based on HCVpp and SIN lentiviral system were established. This system paves a way for characterization of early steps of HCV infection (host tropisms, receptor binding, membrane fusion, et al. ) or screening anti-HCV drugs (such as inhibitor to virus entry). This system can be further applied to assess the human immune responses in HCV patients or evaluate HCV vaccine candidates.
Hepacivirus ; immunology ; Hepatitis C Antibodies ; immunology ; Hepatitis C, Chronic ; immunology ; Humans ; Neutralization Tests ; Viral Envelope Proteins ; immunology ; Virion ; immunology

To evaluate the clinical feasibility of the antibody titer against a chimeric polypeptide (named Core 518), in which a domain of Core and NS3 of hepatitis C virus (HCV) was fused, ELISA was performed in a total of 76 serum samples. Each serum was serially diluted using two-fold dilution method with distilled water into 10 concentrations. They were all positive for second generation anti-HCV assay (HCV EIA II; Abbott Laboratories). Genotyping RT-PCR, quantitative competitive RT-PCR, and RIBA (Lucky Confirm; LG Biotech) were also assayed. Anti-Core 518 antibody was detected in x 12800 or higher dilutions of sera from 35 of 43 chronic hepatitis C (81.4%) and nine of 16 hepatocellular carcinoma sera (56.3%), one of four cirrhosis (25%), 0 of four acute hepatitis C, and one of nine healthy isolated anti-HCV-positive subjects (p=0.0000). The anti-Core 518 antibody titers were well correlated with the presence of HCV RNA in serum (p=0.002). The anti-Core 518 antibody titers decreased significantly in nine of ten responders to IFN-alpha treatment. Monitoring anti-Core 518 titers may be helpful not only for differentiating the status of HCV infection among patients with various type C viral liver diseases, but also for predicting responses to IFN-alpha treatment.
Adult ; Aged ; Female ; Genotype ; Hepatitis C/immunology* ; Hepatitis C/drug therapy* ; Hepatitis C/diagnosis ; Hepatitis C/blood ; Hepatitis C Antibodies/immunology* ; Hepatitis C Antibodies/blood ; Hepatitis C Antigens/immunology* ; Hepatitis C-Like Viruses/immunology* ; Hepatitis C-Like Viruses/genetics ; Human ; Immunoblotting ; Interferon Alfa-2a/therapeutic use* ; Male ; Middle Age ; RNA, Viral/blood ; Recombinant Fusion Proteins/immunology ; Viral Core Proteins/immunology* ; Viral Nonstructural Proteins/immunology*

In order to optimize the fabrication of SiO2 tubes immobilized with antibody for hepatitis C virus antigen (HCAg) detection, we formed the activated amino on the surface of SiO2 tubes by using the activation of aminosilane. Then we immobilized the hepatitis C virus (HCV) monoclonal antibody on the surface of SiO2 tubes by using glutaraldehyde as a chemical cross-linker, followed by detecting HCAg. Sequence tests showed that when the SiO2 tubes were treated in 10% (V/V) aminosilane solution and 3% (V/V) glutaraldehyde solution for 3 hours and 2 hours, respectively, the HCV monoclonal antibody had high immobilization efficiency and low nonspecificity, and the HCAg was detected to 1 ng/mL. This experiment can provide principle and experimental data for establishment of HCAg magnetic immunoassay system.
Antibodies, Immobilized ; immunology ; Antibodies, Monoclonal ; chemistry ; immunology ; Hepatitis C Antibodies ; chemistry ; immunology ; Hepatitis C Antigens ; analysis ; immunology ; Humans ; Silicon Dioxide ; chemistry

BACKGROUNDTo study the antibody against hepatitis C virus first envelope (HCV-E1) protein in the sera from patients with HCV and to evaluate the application of HCV-E1 antigen in detection of HCV antibody.

METHODSPurified E1 engineering protein was used as antigen to develop an ELISA for detecting E1 antibody in 80 national reference sera, 821 blood donors' sera and l20 sera from clinical patients with hepatitis.

RESULTSAnti-HCV E1 was positive in 70% (28/40) and negative in 100% (40/40) of 80 national reference sera, and 1.9% (16/821) was positive in blood of the sera donors' and 68% (492/720) positive in sera of patients with hepatitis. Most anti-HCV E1 positive sera were positive for core, NS 3 and NS 5A, but only a few sera were positive for E1 antigen. Of the sera from 218 clinical patients, 813 blood donors and 848 normal people that were anti-HCV negative tested by commercial anti HCV ELISA kit, 1.4%, 1.1% and 0.9% were anti-HCV E1 positive, respectively. Investigation of seroconversion on three patients showed that anti-E1 was first detectable.

CONCLUSIONSDetection of anti-HCV E1 by engineered E1 protein is sensitive and specific. The prevalence and early presence of E1 antibody in HCV infected patients reflect the active status of the disease to a certain extent. Detection of the antibody is useful in clinical diagnosis.

Enzyme-Linked Immunosorbent Assay ; Hepatitis C ; diagnosis ; Hepatitis C Antibodies ; blood ; Humans ; Viral Structural Proteins ; immunology

Hepatitis C ; diagnosis ; Hepatitis C Antibodies ; blood ; Humans ; Serologic Tests ; Viral Envelope Proteins ; immunology

OBJECTIVETo detect humoral immune response against different function regions of hepatitis C virus (HCV) in chronic patients, and further to investigate the correlativity between anti-HCV antibody titers and HCV RNA concentration.

METHODSUsing recombinant dominate epitope antigens, e.g. HCV Core, NS3, NS4, NS5 and chimeric HVR1, a set of ELISA test reagents was formulated. Then, titers of antibodies against HCV different regions and the RNA concentration of HCV in chronic patient sera were detected by ELISA and quantitative RT-PCR technique, respectively.

RESULTSGreat differences have been noted in antibody titers and positive rate of different HCV function regions in chronic patients. Antibodies against HCV Core and HVR1 have the highest positive rate, then NS3, NS4, and NS5 in sequence.

CONCLUSIONThe titer of antibodies against different regions of HCV in chronic patients has good correlation with HCV RNA concentration.

Hepatitis C Antibodies ; blood ; Hepatitis C, Chronic ; immunology ; virology ; Humans ; RNA, Viral ; blood

Animals ; Antigens, CD ; immunology ; Cytokines ; metabolism ; Hepacivirus ; immunology ; Hepatitis C ; immunology ; prevention & control ; virology ; Hepatitis C Antibodies ; biosynthesis ; immunology ; Hepatitis C Antigens ; immunology ; Humans ; Killer Cells, Natural ; immunology ; T-Lymphocytes ; immunology ; metabolism ; T-Lymphocytes, Regulatory ; immunology ; metabolism ; Viral Proteins ; immunology

Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.
Animals ; Antibodies, Monoclonal ; immunology ; Enzyme-Linked Immunosorbent Assay ; Epitopes ; Hepatitis Antibodies ; immunology ; Hepatitis E virus ; immunology ; Mice ; Mice, Inbred BALB C ; Viral Proteins ; immunology

BACKGROUNDTo study preparation of polyvalent DNA vaccine and the control of multiple gene expression.

METHODSA bicistronic vector pcDNA3.0BA was constructed from pcDNA3.0. HCV PC154 gene and HBV preS2S gene were inserted into this vector to form bicistronic expression construct pcDNA3.0BAPC154S2S and monocistronic expression construct pcDNA3.0BAPC154 or pcDNA3.0BAS2S. These plasmids were transiently expressed in COS-7 cells and injected into muscles of BALB/c mice.

RESULTSpcDNA3.0BA contains two cistronic units, which can co-express two kinds of genes, with the first immunogen gene and the second gene serving as additional immunogen or as modulator for the immune responses. HBV surface Ag and HCV core Ag were coexpressed in vitro. The antibody responses and lymphoproliferation to antigens were similar between bicistronic and monocistronic expression construct in mice.

CONCLUSIONpcDNA3.0BA is a novel vector, which can coexpress two proteins and elicit polyvalent immune responses.

Animals ; COS Cells ; Cercopithecus aethiops ; DNA, Recombinant ; immunology ; Gene Expression ; Hepatitis B ; blood ; immunology ; Hepatitis B Antibodies ; blood ; Hepatitis B Surface Antigens ; genetics ; immunology ; Hepatitis C ; blood ; immunology ; Hepatitis C Antibodies ; blood ; Hepatitis C Antigens ; genetics ; immunology ; Immunization ; methods ; Mice ; Mice, Inbred BALB C ; Plasmids ; genetics ; Vaccines, DNA ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

OBJECTIVETo observe the immunogenicity of combined hepatitis A and B vaccine (HAB).

METHODSThe combined HAB vaccine was prepared and different concentrations of HAB were administered on mice in week 0, 4 and 24, and then we tested the antibodies to both hepatitis A virus and B virus. After the first injection, we tested the hepatitis A antigen-induced and hepatitis B surface antigen-induced stimulation indices in spleen monocyte as well as changes of CD4+ and CD8+ cell numbers.

RESULTSThe serum antibody positive rates were 100% in all three groups, and the antibody induced by HAB vaccine were earlier than by monovalent vaccine. The hepatitis A antibody and hepatitis B surface antibody titers after the combined vaccine inoculation were not significantly higher than those after the monovalent vaccine inoculation. On the other hand, after the first injection of the combined vaccine, the hepatitis A antigen-induced and hepatitis B surface antigen-induced stimulation indices in spleen monocyte were detected. The numbers of CD4+ and CD8+ cells increased.

CONCLUSIONSHAB vaccine has reliable immunogenicity.

Animals ; CD4-CD8 Ratio ; Hepatitis A ; prevention & control ; Hepatitis A Antibodies ; blood ; Hepatitis A Vaccines ; immunology ; Hepatitis B ; prevention & control ; Hepatitis B Antibodies ; blood ; Hepatitis B Vaccines ; immunology ; Leukocytes, Mononuclear ; immunology ; Mice ; Mice, Inbred BALB C ; Random Allocation ; Vaccination ; Vaccines, Combined ; immunology