Synthetic gene for the hepatitis C virus nucleocapsid protein.

A synthetic gene encoding the hepatitis C virus (HCV) nucleocapsid protein was constructed and expressed in E. coli. To synthesize this gene, we developed a new method that results in the enzymatic synthesis of long polydeoxyribonucleotides from oligodeoxyribonucleotides. The method, designated as the 'Exchangeable Template Reaction' (ETR), uses oligonucleotides as templates for DNA polymerase. A special mechanism was designed to exchange the templates during the polymerase reaction. The mechanism relies on the formation of a single-stranded 3'-protrusion at the 'growing point' of the elongating DNA such that it can be subsequently annealed, in a sequence-specific manner, with the next synthetic oligonucleotide. When annealed to the 3'-protrusion, the added oligonucleotide becomes a template for DNA polymerase, and the protruding 3'-end of the double-stranded DNA is used as the primer. The HCV nucleocapsid gene was assembled with DNA ligase from three fragments synthesized by ETR. The data verify that this method is efficient. The main advantage of ETR is the ability to combine more than two oligonucleotides in one tube together with polymerase and an enzymatic activity that produces a 3'-protrusion (e.g., BstXI) rather than the sequential addition of each component. The data demonstrate that as many as five oligonucleotides can be used simultaneously, resulting in a synthesized DNA fragment of designed sequence. The synthetic gene expressed in E. coli produced a 27 kDa protein that specifically interacted with antibodies from sera obtained from HCV-infected individuals.     

Expression of hepatitis C virus E2 ectodomain in E. coli and its application in the detection of anti-E2 antibodies in human sera

The second envelope glycoprotein (E2) of hepatitis C virus has been shown to bind human target cells and has become a major target for the development of anti-HCV vaccines. Anti-E2 antibodies have been suggested to be of clinical significance in diagnosis, treatment and prognosis of hepatitis C. However, large-scale expression and purification of E2 proteins in mammalian cells is difficult. As an alternative, E2 fragment (aa 385-730) with a four-amino-acid mutation (aa 568-571 PCNI to RVTS) was expressed as hexa-histidine-tagged full length protein [E2N730(m)] in E. coli and purified to over 85% purity. Purified E2N730(m) was specifically recognized by homologous hepatitis C patient serum in Western blot, suggesting that it displayed E2~specific antigenicity. Rabbit antiserum raised against E2N730(m) recognized E2 glyco-proteins expressed in mammalian cells in Western blot. Purified E2N730(m) was used to detect anti-E2 antibodies in human sera and showed better specificity and sensitivity than previousl     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and 1-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed in E. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCl density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBeAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B14d     

Immunological evaluation of Escherichia coli-derived hepatitis C virus second envelope protein (E2) variants.

Two variants of the hepatitis C virus (HCV) E2 envelope protein, lacking the C-terminal domain and comprising amino acids 458-650 (E2A) and 382-605 (E2C), respectively, were efficiently produced in BL21 (DE3) Escherichia coli cells. E2A and E2C were used to immunize mice. The E2C variant induced the maximal mean antibody titer. Anti-E2C mouse sera reacted mainly with E2 synthetic peptides covering the 70 amino acid N-terminal region of the E2 protein. Moreover, a panel of anti-HCV positive human sera recognized only the E2C protein (28.2%) and the synthetic peptide covering the HVR-1 of the E2 protein (23.1%). These data indicate the existence of an immunologically relevant region in the HVR-1 of the HCV E2 protein.     

Purification and application of bacterially expressed chimeric protein E1E2 of hepatitis C virus

E1 and E2 glycoproteins are structural components of hepatitis C virus (HCV) virion. They are involved in cellular receptors interaction, neutralising antibodies elicitation, and viral morphogenesis. They are considered as major candidates for anti-HCV vaccine. In this report, we first expressed tandem E1E2 as well as C-terminally truncated E1 fragment and C-terminally truncated E2 fragment, respectively, in Escherichia coli cells and the proteins were purified to homogenesis. All the purified proteins can react specifically with patient sera. Both purified chimeric protein E1E2 and protein E2 can interact with a putative cellular receptor CD81, while purified protein E1 cannot interact with CD81. The sera of rabbit immunized with the E1E2 inhibited the binding of E2 protein to the major extracellular loop of human CD81 and reacted with both proteins E1 and E2, respectively. Anti-E1 and E2 antibodies can be generated simultaneously in the rabbit immunized with the E1E2, and the titers of antibodies were 63 or 56% higher than the titers induced by E1 or E2 alone, respectively. The results suggest that E1 and E2 can enhance their immunogenicity each other in chimeric protein E1E2 and the E. coli-derived chimeric protein E1E2 and corresponding antisera can be used as an useful tools in anti-HCV vaccine research.     

Detection of antibody to hepatitis C E2/NS1 protein in patients with type C hepatitis.

Putative E2/NS1 sequence of hepatitis C virus was expressed in E. coli as a fusion protein with maltose binding protein. Approximately 80 kDa protein was obtained containing 38 kDa E2/NS1 protein. The antibody to this protein was detectable in the same serum from which the sequence was amplified. It was also detectable in none of 7 acute hepatitis, in 2 of 12 chronic persistent hepatitis, in 3 of 25 chronic active hepatitis, and in 2 of 4 cirrhosis. It was detectable in none of 10 normal subjects. In 3 cases who were positive for the antibody before the interferon treatment, it became undetectable after the treatment. Thus, it seems that the antibody is not a neutralizing antibody and is related to active viral replication.     

Expression of processed envelope protein of hepatitis C virus in mammalian and insect cells.

The putative envelope protein of hepatitis C virus (HCV) was expressed in insect cells by using a baculovirus expression vector and in monkey COS cells under the control of exogenous promoters. The expressed envelope proteins, identified by immunoblot analysis using sera from patients with chronic HCV infection, were a series of glycoproteins of 35 to 24 kDa (gp35-24) in insect cells and a single species of glycoprotein of 35 kDa (gp35) in monkey cells. The size difference of these proteins was due to the different degrees of glycosylation. The envelope proteins expressed in these cells were produced by common specific cleavage from the precursor protein, and cleavage positions of the envelope protein were mapped at about amino acids 190 and 380. The gp35-24 proteins expressed in insect cells were used for detection of antibody against HCV envelope protein in patient sera. The results showed that (i) the antibody is detected in 2 to 17% of various patients with hepatitis C, (ii) three patients were apparently cured after acquiring the antienvelope antibody, and (iii) in sera of patients with more than a 20-year history of infection, the antibody sometimes coexisted with HCV. These results suggest that the antienvelope antibody is neutralizing only in limited number of patients with hepatitis C.     

Suitability of yeast- and Escherichia coli-expressed hepatitis B virus core antigen derivatives for detection of anti-HBc antibodies in human sera

Antibody to hepatitis B virus core antigen (anti-HBc) is one of the most important serological markers during hepatitis B virus (HBV) infection. The quality of the hepatitis B virus core antigen (HBcAg; diagnostic antigen) is crucial to the accuracy of anti-HBc detection. In an attempt to explore the suitability of recombinant HBcAg (rHBcAg) for diagnostic purposes, HBcAg was expressed in Escherichia coli (E. coli) and Pichia pastoris (P. pastoris) and evaluated for the detection of anti-HBc. The expression level of the recombinant protein satisfied the criteria for large-scale biologic production. P. pastoris- and E. coli-derived rHBcAg were purified with gel filtration followed by sucrose gradient (reagents A and C) or with a monoclonal anti-HBc antibody binding (reagents B and D) and were utilized to detect anti-HBc in competitive inhibition enzyme-linked immunosorbent assay (ELISA) format. The ELISA using P. pastoris-derived rHBcAg had a higher specificity and sensitivity than that using E.coli-derived rHBcAg to detect the anti-HBc standard panel. Serum specimens were collected from HBV-infected patients and healthy individuals (voluntary blood donors). Anti-HBc was detected in those specimens using P. pastoris- and E. coli-derived rHBcAg. The positive rate of anti-HBc detection in HBV-infected patients' sera was 100% with reagents A and B, 96.4% with reagent C, and 93.6% with reagent D. The negative rate in healthy control sera was 100% with reagents A and B, 97.0% with reagent C, and 99.7% with reagent D. These data indicate that P. pastoris-derived rHBcAg is superior to E.coli-derived rHBcAg for the detection of anti-HBc using the diagnostic ELISA.     

Cloning and expression of cDNAs from enterically-transmitted non-A, non-B hepatitis virus.

The fragment gene of enterically-transmitted non-A, non-B hepatitis virus (ET-NANBHV) was cloned as a cDNA and inserted into an expression vector pUEX2. The recombinant protein was expressed in Escherichia coli HB101 as a fusion protein with beta-galactosidase (beta-Gal). The fusion protein reacted with the sera of infected cynomolgus monkeys and of patients from Myanmar. This reaction was highly related with ET-NANBHV infection, and obviously demonstrates in that the recombinant protein can be used for the detection of ET-NANBHV infection.     

Artificial mosaic protein containing antigenic epitopes of hepatitis E virus.

A synthetic gene encoding an artificial polypeptide composed of antigenic epitopes of the hepatitis E virus (HEV) proteins was constructed from short oligodeoxyribonucleotides by using PCR. The polypeptide comprises a mosaic of three antigenically active dominant regions from the protein encoded by open reading frame 2 (ORF2), one antigenically active region from the protein encoded by ORF3 of the Burmese HEV strain, and one antigenically active region from the protein encoded by ORF3 of the Mexican HEV strain. The mosaic protein was expressed in Escherichia coli as a chimera with glutathione S-transferase or beta-galactosidase. Guinea pig sera containing antibodies to the corresponding HEV synthetic peptides were used to demonstrate by Western immunoblot analysis and enzyme immunoassay the presence and accessibility of all HEV-specific antigenic epitopes introduced into the mosaic protein. Both the glutathione S-transferase and beta-galactosidase hybrid proteins were analyzed by using a panel of human anti-HEV-positive and -negative sera. The data obtained strongly indicate a diagnostic potential for the mosaic protein.     

Expression of processed core protein of hepatitis C virus in mammalian cells.

A structural protein of hepatitis C virus (HCV) was expressed in monkey COS cells under the control of an exogenous promoter, and a protein of 22 kDa was identified by immunoblot analysis. This protein (p22), which was produced by processing in COS cells, reacted specifically to sera of chronic hepatitis C patients, and its coding region was mapped at the most amino-terminal part of the HCV polyprotein. These results suggested that the p22 protein is the nucleocapsid (core) protein of HCV. Moreover, the assay detecting antibody to p22 was found to be useful for early diagnosis of HCV infection.     

Expression and characterization of hepatitis C virus core protein fused to hepatitis B virus core antigen

Recombinant plasmids were constructed by fusing the gene fragments encoding the full-length (1-191aa) and the truncated (1-40aa and l-69aa) HCV core proteins (HCc) respectively to the core gene of HBV at the position of amino acid 144 and expressed inE. coli. The products were analyzed by ELISA, Western blotting as well as the immunization of the mice. The results showed that those fusion proteins (B144C191, B144C69, B144C40) possessed the dual antigenicity and immunogenicity of both hepatitis B virus core antigen (HBcAg) and hepatitis C virus core protein (HCc). Analysis by electron microscopy and CsCI density gradient ultra-centrifugation revealed that similar to the HBcAg itself, all fusion proteins were able to form particles. Comparison of the antigenicity and immunogenicity of those fusion proteins showed that the length of HCc gene fused to HBcAg had no much effect on the antigenicity and immunogenicity of HBcAg, however, B144C69 and B144C40 induced higher titres antibodies against HCc than B144C191. Using those fusion proteins, ELISA for screening of antibodies against both HBV and HCV in human sera was also established.     

Expression of structural proteins of hepatitis C virus (HCV) in mammalian cells

ＨＣＶｉｓｔｈｅｍａｊｏｒｃａｕｓｅｏｆｐｏｓｔｔｒａｎｓｆｕｓｉｏｎｎｏｎＡ,ｎｏｎＢｈｅｐａｔｉｔｉｓ[1].Ａｂｏｕｔ50%ｏｆｔｈｅｉｎｆｅｃｔｉｏｎｓｗｉｌｌｄｅｖｅｌｏｐｉｎｔｏｃｈｒｏｎｉｃｈｅｐａｔｉｔｉｓａｎｄａｍｏｎｇｔｈｅｍａｂｏｕｔ20%ｗｉｌｌｒｅｓｕｌｔｉｎｌｉｖｅｒｃｉｒｒｈｏｓｉｓａｎｄｈｅｐａｔｏｃｅｌｌｕｌａｒｃａｒｃｉｎｏｍａ[2].ＢｅｃａｕｓｅｔｈｅｔｉｔｅｒｏｆＨＣＶｐａｒｔｉｃｌｅｉｎｐａｔｉｅｎｔ’ｓｂｌｏｏｄｉｓｅｘｔｒｅｍｅｌｙｌｏｗ,ａｎｄｔｈｅｒｅｉｓｎｏ…     

A weak neutralizing antibody response to hepatitis C virus envelope glycoprotein enhances virus infection

We have completed a phase 1 safety and immunogenicity trial with hepatitis C virus (HCV) envelope glycoproteins, E1 and E2, with MF59 adjuvant as a candidate vaccine. Neutralizing activity to HCV genotype 1a was detected in approximately 25% of the vaccinee sera. In this study, we evaluated vaccinee sera from poor responders as a potential source of antibody dependent enhancement (ADE) of HCV infection. Sera with poor neutralizing activity enhanced cell culture grown HCV genotype 1a or 2a, and surrogate VSV/HCV pseudotype infection titer, in a dilution dependent manner. Surrogate pseudotypes generated from individual HCV glycoproteins suggested that antibody to the E2 glycoprotein; but not the E1 glycoprotein, was the principle target for enhancing infection. Antibody specific to FcRII expressed on the hepatic cell surface or to the Fc portion of Ig blocked enhancement of HCV infection by vaccinee sera. Together, the results from in vitro studies suggested that enhancement of viral infectivity may occur in the absence of a strong antibody response to HCV envelope glycoproteins.     

Prevalence of GB virus C/hepatitis G virus in Hungary

In 1995 a new flavivirus, GB virus C/hepatitis G virus (GBV-C/HGV), was discovered. The aim of this study was to determine the prevalence of the virus in healthy persons and hepatitis patients in Hungary. The sera of 408 healthy persons older than 60 years were tested for the presence of GBV-C/HGV antibodies, and 113 were positive (28%). Eight of the 71 healthy persons younger than 60 years and twenty of the 51 sera (39%) taken from patients suffering from hepatitis of unknown origin proved to be positive for GBV-C/HGV antibodies. Ten of the 124 sera (8%) of healthy persons and 36 of the 247 sera (14.6%) of hepatitis patients proved to be positive for GBV-C/HGV RNA. Eleven PCR products were sequenced, and the sequences were found to be different from each other and from the previously published ones. However, three sequences taken from the same patient at different times were identical. These results show that GBV-C/HGV is present in Hungary and cannot be considered rare.     

Type-specific antibodies to hepatitis C virus in sera from patients with various hematologic diseases

The reactivity of 100 sera taken from patients with different blood diseases and donors with respect to synthesized peptides in the variable area of protein NS4 of hepatitis C virus was studied. The presence of type-specific antibodies in the blood sera of patients with hepatitis C was shown. Two antigenic determinant corresponding to 1683-1705 and 1711-1732 amino acid residues in the protein area under study were detected. In hematological patients undergoing frequent blood transfusions mixed infection with different types of hepatitis C virus was registered; these types could be reliably determined with the use of synthetic peptides. The serotype determined with the use of peptides corresponded to the type of the circulating virus.     

Substrate requirements of hepatitis C virus serine proteinase for intermolecular polypeptide cleavage in Escherichia coli.

Using as substrates a series of chimeric proteins containing various fragments of the hepatitis C virus precursor polyprotein between Escherichia coli maltose binding protein and dihydrofolate reductase, we analyzed the substrate requirements of hepatitis C viral serine proteinase (Cpro-2) for intermolecular polypeptide cleavage in E. coli. Cpro-2-dependent substrate cleavage was observed in E. coli cells simultaneously transformed with expression plasmids for the Cpro-2 molecule and substrate protein. The cleavage sites were estimated by determining the amino (N)-terminal amino acid sequences of dihydrofolate reductase-fused processed products purified partially by affinity chromatography from the lysates, indicating that cleavage occurred at sites identical to those observed in eukaryotic cells. Mutation analysis using the chimeric substrate indicated that the presence of cysteine and small uncharged residues at positions P1 and P1', respectively, of the putative cleavage site is necessary for cleavage and that acidic residues in the region upstream of the cleavage site are required for efficient cleavage.     

Capsid assembly and involved function analysis of twelve core protein mutants of duck hepatitis B virus.

The roles of different regions of the duck hepatitis B virus (DHBV) core protein on viral capsid assembly and related functions were examined. Twelve deletion and insertion mutations which covered 80% of the DHBV C open reading frame were constructed and expressed in Escherichia coli. The N-terminal region (amino acids 3 to 66) of DHBV core protein was important for its tertiary structure and function in E. coli. The expressed core mutants without this region apparently inhibited E. coli growth. The results of transmission electron microscopy of E. coli thin sections, capsid agarose gel, and sucrose gradient sedimentation demonstrated that a few DHBV core mutants with insertion in the N terminus and deletion in the C terminus retained the ability to form core-like particles in E. coli. However, other mutations in most of N-terminal and central regions strongly inhibited the self-assembly ability of DHBV core protein in E. coli. In addition, the mutant with a C-terminal region deletion (amino acids 181 to 228) lost most of the nucleic acid-binding activity of the DHBV core protein.     

Viroporin activity of murine hepatitis virus E protein

The viroporin activity of the E protein from murine hepatitis virus (MHV), a member of the coronaviruses, was analyzed. Viroporins are a growing family of viral proteins able to enhance membrane permeability, promoting virus budding. Initially, the MHV E gene was inducibly expressed in Escherichia coli cells, leading to the arrest of bacterial growth, cell lysis and permeabilization to different compounds. Thus, exit of labeled nucleotides from E. coli cells to the cytoplasm was apparent upon expression of MHV E. In addition, enhanced entry of the antibiotic hygromycin B occurred at levels comparable to those observed with the viroporin 6K from Sindbis virus. Mammalian cells are also readily permeabilized by the expression of MHV E protein. Finally, brefeldin A powerfully blocks the viroporin activity of the E protein in BHK cells, suggesting that an intact vesicular system is necessary for this coronavirus to permeabilize mammalian cells.