Animals ; Hepacivirus ; genetics ; Hepatitis C ; immunology ; prevention & control ; therapy ; virology ; Humans ; Mutation ; Viral Hepatitis Vaccines ; immunology

Cloning, Molecular ; Escherichia coli ; genetics ; Hepacivirus ; genetics ; Humans ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; immunology ; Viral Nonstructural Proteins ; biosynthesis ; genetics ; immunology

Hepatitis C virus (HCV) core protein is considered to be an attractive candidate for development of protective HCV vaccines. However, this protein may attenuate the induction of systemic immune responses due to its immunomodulatory properties. In this study, we constructed a HCV core gene-containing eukaryotic expression plamid pCI-C, and an in vivo-inducible prokaryotic expression plasmid pZW-C, and transformed the recombinant plasmids into an attenuated Salmonella typhimurium aroA strain SL7207. The resulting bacterial strains SL7207/pCI-C and SL7207/pZW-C were used to orally immunize BALB/c mice, and the immune responses specific to HCV core protein were assessed. Immunization with the recombinant bacteria SL7207/pCI-C led to a persistent drop in percentage of CD3 CD4 T cells, and induced a weak anti-core IgG production. Splenocytes from SL7207/pCI-C immunized mice developed a relatively weak proliferation response and inferior cytotoxic activity compared to those from the mice immunized with bacteria SL7207/pZW-C. Boost immunization with SL7207/ pCI-C yielded limited improvement in immune strength, while the boost with bacteria SL7207/pZW-C significantly enhanced the immune response. These results suggest that de novo synthesis of native HCV core protein may blunt the induction of immune responses. Attenuated S. typhimurium carrying HCV core protein could efficiently activate systemic cellular and humoral responses, and may be a promising strategy for the development of core-based HCV vaccines.
Animals ; Hepacivirus ; genetics ; immunology ; Mice ; Plasmids ; genetics ; Salmonella typhimurium ; genetics ; metabolism ; Vaccines, DNA ; immunology ; Viral Core Proteins ; genetics ; immunology ; Viral Hepatitis Vaccines ; genetics ; immunology

OBJECTIVETo clone 1b type of HCV NS3-4b Gene and express in HEK 293 cells, lay the foundation for further study of the HCV NS3-4b recombinant adeno-associated virus vaccine and its dendritic cell vaccine.

METHODSHCV 1b patients' serum was collected, and full length NS3-4b segment was amplified by RT-PCR and cloned into adeno-associated virus' expression vector pAAV. CMV. EGFP in order to express in HEK 293 cells. At last, it was validated whether express or not by Western Blot.

RESULTSThe 1b type gene NS3-4b were amplified and consistent to the expected size (2838 bp), the recombinant plasmid has been confirmed its successful restructured by double enzyme and sequencing, at last, Western Blot map can see objective protein expression after it transfect HEK 293 cells.

CONCLUSIONThe adeno-associsted virus recombination HCV NS3-4b plasmid have successfully constructed and it can express in eukaryotic cells.

Dependovirus ; genetics ; Genetic Vectors ; HEK293 Cells ; Hepacivirus ; genetics ; Humans ; Plasmids ; Vaccines, Synthetic ; immunology ; Viral Nonstructural Proteins ; genetics ; Viral Vaccines ; immunology

OBJECTIVEObtain the hepatitis C virus high purified subunit fusion protein and detect its immunogenicity.

METHODSWith the vector of pET-11d, fusion protein was expressed in Escherichia coli BL21 (DE3) after induced by IPTG. The protein was then purified by DEAE negative ion exchange chromatography and Ni2+ affinity chromatography. Western Blot analysis was used to detect the antigenicity of the fusion protein. At the same time, the sera were collected and prepared from the immunized experimental animals in order to investigate the immunogenicity of the protein by EIA.

RESULTSHigh purified hepatitis C virus subunit fusion protein was obtained successfully. The EIA indicated that the fusion protein could elicit specific antibodies in the animals with very high titers.

CONCLUSIONThe hepatitis C virus subunit fusion protein expressed in prokaryotic system was proved to have strong immunogenicity. It could provide some helpful and useful information to the hepatitis C virus prophylactic and therapeutic vaccine development.

Animals ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Hepacivirus ; genetics ; metabolism ; Immunoassay ; Rabbits ; Recombinant Fusion Proteins ; genetics ; immunology ; metabolism ; Viral Proteins ; genetics ; immunology ; metabolism

OBJECTIVETo evaluate the first and second assay kits currently used in blood centers for screening HCV infected blood, and to provide basis for a better match of the two assay kits.

METHODSUsing the newly developed multi-recombinant-HCV-antigen supplementary assay kit, the authors evaluated concurrently the specificity and sensitivity of two domestic and one imported anti-HCV detection kits.

RESULTSDiscrepancy in specificity and sensitivity existed among the two domestic HCV kits, and overall quality was slightly below that of leading or main stream imported HCV kit.

CONCLUSIONThe newly developed multi-recombinant-HCV-antigen supplementary assay kit is useful in the evaluation of HCV antibody detection kit currently in use. It provides qualified assessing kit to capture antibodies against various HCV antigens. The present paper provided guidance for selecting a better match of the two screening kits and improved screening efficiency.

Blood Donors ; Evaluation Studies as Topic ; Hepacivirus ; genetics ; immunology ; Hepatitis C Antibodies ; blood ; immunology ; Hepatitis C Antigens ; genetics ; immunology ; Humans ; Reagent Kits, Diagnostic ; standards ; Sensitivity and Specificity

The aim was to develop a single multiple-epitope fusion antigen which incorporates all of the major immunodominant epitopes from the six functional regions of the HCV genome. A nucleic acid sequence consisting of viral core, E1, E2, NS3, NS4, and NS5 regions was constructed and inserted into the Promega Pinpoint Xa-1 T vector for inducing expression. The protein was expressed in JM109 (DE3) as a fusion protein with a 13 kD biotinlated tag to be used for detection and affinity purification. Immunogenicity and biotinylated tag of the fusion protein were detected by Western blot analysis with positive anti-HCV serum and streptavidin alkaline phosphatase. After purified by Promega SoftLink Soft Release Avidin Resin, the protein was pre-coated on microwell and detected with anti-core, anti-NS3, anti-NS4 and anti-NS5 positive sera by EIA, respectively. The results indicated that the recombinant soluble protein was expressed and labelled with biotin successfully, it reacted with anti-HCV positive serum, and exposed all of the major immunogenic epitopes chosen. In conclusion, this recombinant antigen may be used to design an double antigen sandwich anti-HCV immunoassay.
Antigens, Viral ; genetics ; immunology ; Biotinylation ; Cloning, Molecular ; Enzyme-Linked Immunosorbent Assay ; Hepacivirus ; immunology ; Hepatitis C Antibodies ; blood ; Humans ; Immunodominant Epitopes ; Recombinant Fusion Proteins ; genetics ; immunology

Natural killer (NK) cells play an important role in innate immunity, especially in the response to viral infections, such as hepatitis C virus (HCV). Killer cell immunoglobulin-like receptors (KIRs) are the primary receptors of NK cells that mediate innate immunity. KIRs are also involved in acquired immunity, because some KIRs are expressed on the surface of certain subsets of T cells. In this study, the frequency of KIR genes, HLA-C allotypes, and combinations of KIR genes with their HLA-C ligands were evaluated in two different groups of the Korean population: controls and patients with chronic HCV infection. The study population consisted of 147 Korean patients with chronic HCV infection. The frequency of KIR2DS2 in patients with chronic HCV infection was 9.5% which was significantly lower than 19.5% of the control (P < 0.01). However, there were no significant differences in the frequency of other KIR genes, HLA-C allotypes or different combinations of KIR genes with their HLA-C ligands. This study can contribute to the further prospective study with a larger scale, suggesting the assumption that KIR2DS2 might aid in HCV clearance by enhancing both the innate and acquired immune responses of people in Korea.
Adult ; Aged ; Female ; Genes, MHC Class I ; Genotype ; HLA-C Antigens/genetics ; Hepacivirus/immunology ; Hepatitis C, Chronic/*genetics/immunology ; Humans ; Killer Cells, Natural/immunology/virology ; Male ; Middle Aged ; Receptors, KIR/*genetics/immunology ; Republic of Korea ; T-Lymphocyte Subsets/immunology

Amino Acid Sequence ; Epitopes, T-Lymphocyte ; immunology ; Forecasting ; HLA Antigens ; immunology ; Hepacivirus ; genetics ; immunology ; Hepatitis C ; immunology ; virology ; Hepatitis C Antigens ; immunology ; Humans ; T-Lymphocytes, Cytotoxic ; immunology ; virology ; Viral Hepatitis Vaccines ; immunology

OBJECTIVETo investigate the in vivo functional roles of the La autoantigen (La), the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein (hVAP-33), and the subunit gamma of the human eukaryotic initiation factors 2B (eIF2Bgamma) as co-infection factors supporting chronic infection with hepatitis C virus (HCV).

METHODSSmall interfering (si)RNAs were designed against the HCV internal ribosome entry site (IRES) and transfected into Huh7 cells chronically infected with the HCV pseudovirus (designated as Huh7-HCV cells). The IRES siRNA producing the most effective silencing was selected for further analysis by fluorescence quantitative polymerase chain reaction (qPCR). siRNAs designed against La, hVAP-33, and eIF2Bgamma and the IRES-specific siRNA were then transfected, respectively or in various combinations, into the Huh7-HCV cell line for 48 h. The delta CT values were calculated and used to compare the HCV inhibitive efficacies of the siRNAs in isolation or in combination. Western blotting analysis was used to compare the quantity of core protein expression in each group.

RESULTSThe four gene-specific siRNAs, in isolation or in combination, caused inhibition of HCV replication and gene and protein expressions to varying degrees. The combination of La + IRES siRNAs produced the strongest inhibition of HCV core antigen expression. The combinations of hVAP-33 + IRES siRNAs and eIF2Bgamma + IRES siRNAs produced stronger inhibitions of HCV replication and gene and protein expressions than either hVAP-33 siRNA or eIF2Bgamma siRNA alone.

CONCLUSIONLa, hVAP-33, and eIF2Bgamma act as co-infection factors of HCV chronic infection in vivo. HCV replication and gene and protein expression can be inhibited significantly by RNA interference of these co-infection factors and/or HCV IRES.

Autoantigens ; genetics ; Cell Line ; Eukaryotic Initiation Factor-2B ; genetics ; Hepacivirus ; immunology ; physiology ; Humans ; RNA, Small Interfering ; genetics ; Ribonucleoproteins ; genetics ; Vesicular Transport Proteins ; genetics ; Virus Replication