Expression of Hepatitis C Virus Core and E2 antigenic recombinant proteins and their use for development of diagnostic assays.

BACKGROUND: Early diagnosis of HCV infection is based on detection of antibodies against HCV proteins using recombinant viral antigens. The present study was designed to select, clone and express the antigenic regions of Core and E2 genes from local HCV-3a genotype and to utilize the antigenic recombinant proteins (Core & E2) to develop highly sensitive, specific and economical diagnostic assays for detection of HCV infection. METHODS: The antigenic sites were determined within Core and E2 genes and were then cloned in pET-28a expression vector. The right orientation of the desired inserted fragments of Core and E2 were confirmed via sequencing prior to expression and were then transformed in BL21 (DE3) pLysS strains of E. coli and induced with 0.5mM Isopropyl-b-D-thiogalactopyranoside (IPTG) for the production of antigenic recombinant proteins. The produced truncated antigens were then purified by Nickel affinity chromatography and were confirmed by western blotting, immunoblotting and enzyme-linked immunosorbent assay (ELISA). RESULTS: The expressed Core and E2 recombinant antigens were used to develop immunoblotting assay for the detection of anti-HCV antibodies in sera. With immunoblotting, a total of 93-HCV infected sera and 35-HCV negative individuals were tested for the presence of anti-HCV antibodies to the Core and E2 antigens. Recombinant antigen showed 100% reactivity against HCV infected sera, with no cross reactivity against HCV-negative sera. The immunoblot assay mixture of recombinant antigens (Core+E2) showed a strong reaction intensity in the test area (TA) as compared to the individual truncated Core and E2 recombinant antigens. In the in-house ELISA assay, mixed Core and E2 recombinant antigens showed 100% reactivity against a standardized panel of 150-HCV-positive sera and non reactivity against a standardized panel of 150 HCV-negative sera while also being non reactive to sera positive for other viral infections. The antigenic recombinant antigens also were tested for the 30 sera of known genotypes. The antigens did not detect antibodies to genotype-3a, but detected antibodies to all genotypes and did not discriminate them genotype wise. A panel of 175 of HCV-suspected serum samples was subjected to comparative analysis with our in-house ELISA assay and with commercial HCV screening assays. After subjecting the results to the formulas for determining the quality parameters, immunoblot assay had 100% sensitivity and specificity, while the ELISA assay had 100% sensitivity and 98.8% specificity as compared to commercially available assays. CONCLUSION: This study indicates that a mixture of Core and E2 antigens are potentially valuable antigens and there is the possibility of developing serological assays for monitoring HCV infection.

[NS3AG expression in chronic hepatitis C: relationship with clinical histological activity and HCV genotypes].

AIM: we have analyzed the peculiarities of NS3Ag expression in hepatocytes and dependence of necroinflam mation on the NS3Ag level and on its localization in hepatocytes in patients with different genotypes of HCV MATERIALS AND METHODS: 31 patients with chronic hepatitis C were examined, 14 (45%) of them with HCV geno type 1b and 17 (55%) with genotype 3a. NS3Ag was detected in the liver tissue by immunohistochemistry ("Novocastra" kit, UK). Routine laboratory analyses were performed. Staging and grading were assessed according to the Knodell score and a modified method, and sclerosis index was determined according to the Metavir scale. RESULTS: the level of NS3Ag in the cytoplasm of hepatocytes was 140 fold higher than in the nucleus. Different patterns of NS3Ag localization in the cytoplasm were observed: in 19 (61%) patients it was detected peripherally near the plasma membrane, whereas in 12 (39%) patients it was diffusely distributed in the cytoplasm. Necroinflammation was more pronounced in the case of peripheral near-membrane NS3Ag localization. The fatty an hydropic degeneration and small cell dysplasia of hepatocytes were more significant in high NS3Ag concentration. No correlation was revealed between the intracytoplasmic NS3Ag localization and the genotype of HCV. However, biochemical activity and the severity of inflammation and fibrosis in all zones of the acinus were highe in patients with HCV genotype 1b, in spite of the lower viral load compared with the patients with genotype 3a CONCLUSIONS: in chronic hepatitis C, cytoplasmatic expression of NS3Ag prevailed over nuclear expression. Sever ity of necroinflammation was higher in patients with peripheral near-membrane NS3Ag localization than in those with uniform diffuse localization. The degree of parenchymatous damage and small cells dysplasia of he patocytes depended on the level of NS3Ag in hepatocytes. In patients with HCV genotype 1b, biochemical an histologic indicators of activity were higher, while NS3Ag concentration was lower compared to genotype 3a.

The HCV ARFP/F/core+1 protein: production and functional analysis of an unconventional viral product.

Hepatitis C virus (HCV) is an enveloped positive-strand RNA virus of the Flaviviridae family. It has a genome of about 9,600 nucleotides encoding a large polyprotein (about 3,000 amino acids) that is processed by cellular and viral proteases into at least 10 structural and nonstructural viral proteins. A novel HCV protein has also been identified by our laboratory and others. This protein--known as ARFP (alternative reading frame protein), F (for frameshift) or core+1 (to indicate the position) protein--is synthesized by an open reading frame overlapping the core gene at nucleotide +1 (core+1 ORF). However, almost 10 years after its discovery, we still know little of the biological role of the ARFP/F/core+1 protein. Abolishing core+1 protein production has no affect on HCV replication in cell culture or uPA-SCID mice, suggesting that core+1 protein is probably not important for the HCV reproductive cycle. However, the detection of specific anti-core+1 antibodies and T-cell responses in HCV-infected patients, as reported by many independent laboratories, provides strong evidence that this protein is produced in vivo. Furthermore, analyses of the HCV sequences isolated from patients with hepatocellular carcinoma and in vitro studies have provided strong preliminary evidence to suggest that core+1 protein plays a role in advanced liver disease and liver cancer. The available in vitro data also suggest that certain core function proteins may depend on production of the core+1 protein. We describe here the discovery of the various forms of the core+1 protein and what is currently known about the mechanisms of their production and their biochemical and functional properties. We also provide a detailed summary of the results of patient-based research.

HBx or HCV core gene expression in HepG2 human liver cells results in a survival benefit against oxidative stress with possible implications for HCC development.

Hepatitis virus replication in the liver is often accompanied by inflammation resulting in the formation of reactive oxygen species (ROS) and nitric oxide (NO) and these may induce cell death. We investigated whether the expression of HBx or HCV core protein in HepG2 cells has an influence on the sensitivity of these cells for oxidative radicals. Our previous study, using the inducible HBV model of HepAD38, revealed that oxidative-stress-related genes are upregulated by virus replication. In the present study, we examined the intracellular pro-oxidant status with dichlorofluorescein (DCF) in HepG2 cell lines transfected with HBx, HbsAg and HCV core. Baseline intracellular oxidative levels were not different in the cell lines expressing viral proteins as compared to control. However, when these cells were exposed to H(2)O(2), the viral protein expressing cells, especially those expressing HBx, showed a reduced level of ROS. This suggests that HBx and HCV core transfected cells can convert H(2)O(2) to less reactive compounds at a higher rate than the control cells. When HBx or HCV core expressing cells were exposed to peroxynitrite (a highly reactive product formed under physiological conditions through interaction of superoxide (O(2)(-)) with NO) these cells were less sensitive to induction of cell death. In addition, these cell lines were less prone to cell death when exposed to H(2)O(2) directly. In conclusion, HBx and HCV core expression in HepG2 cells leads to a survival benefit under oxidative stress which in vivo can be induced during inflammation.

Expression and processing of hepatitis C virus structural proteins in Pichia pastoris yeast.

Development of heterologous systems to produce useful HCV vaccine candidates is an important part of HCV research. In this study different HCV structural region variants were designed to express the first 120 aa, 176 aa, 339 aa, and 650 aa of HCV polyprotein, and aa 384 to 521, or aa 384-605 or aa 384-746 of HCV E2 protein fused to the leader sequence of sucrose invertase 2 allowing the secretion of recombinant E2 proteins. Low expression levels were observed for HCV core protein (HCcAg) variants expressing the first 120 aa and 176 aa (HCcAg.120 and HCcAg.176, respectively). Higher expression levels were observed when HCcAg was expressed as a polypeptide with either E1 or E1 and E2 proteins. In addition, HCcAg was processed to produce two antigenic bands with 21 and 23kDa (P21 and P23, respectively) when expressed as a polypeptide with HCV E1 and E2 proteins. Results also suggest E1 processing in the context of HCcAg.E1.E2 polyprotein. On the other hand, E2.521, E2.605, and E2.680 were efficiently excreted to the culture medium. However, the entire E2.746 variant predominantly localized in the insoluble fraction of ruptured cells. Results suggest that the hydrophobic C-terminal E2 region from aa 681 to 746 is critical for intracellular retention of recombinant E2.746 protein in Pichia pastoris cells. Endo H or PNGase F treatment suggests that E2.746 was modified with high-mannose type oligosaccharides in P. pastoris. These data justify the usefulness of P. pastoris expression system to express HCV structural viral proteins which may be useful targets for HCV vaccine candidates.

Heterologous production of five Hepatitis C virus-derived antigens in three Saccharomyces cerevisiae host strains.

In this study, the production of recombinant Hepatitis C virus (HCV) derived proteins from transformed Saccharomyces cerevisiae yeast cells is reported. Three different yeast strains (GRF18U, BY4743-4A and CENPK 113-5D) have been transformed for the intracellular expression of five antigens of different dimensions (from 32.8 to 85.2 kDa), all derived from the non-structural (NS) region of different HCV viruses' genotypes and posed under the control of a glycolytic promoter. The putative trans-membrane domains contained in four antigens seem responsible of their accumulation as protein aggregates. Good productions of the smaller and of the bigger antigens (50 and 30 mgl(-1), respectively) have been observed in simple flask batch cultures. Productions are strongly dependent from the genetic background of the yeast host and from the cellular localization of the antigen, while they appear independent from the growth rate of the transformed hosts. For every recombinant antigen tested, the highest production levels were achieved with the CENPK 113-5D-host strain, while the GRF18U strain shows symptoms of a heavily stressed phenotype.

[Acceleration of mixed lymphocyte reaction by HCV C-Fc gene-transferred murine dendritic cells].

AIM: To observe the metergasis of murine dendritic cells (DCs) transfected with HCV C-Fc gene through electroporation. METHODS: Mononucleocytes isolated from murine bone marrow were co-cultured with rmGM-CSF and rm-IL-4 for 7 days. Morphological characteristics of the cultured cells were observed under scan electron-microscope (SEM) and the expression of DEC205 on the cells was detected by FACS. DCs derived from the culture were transfected with plasmids containing HCV C-Fc gene. HCV C-Fc level in the transfected cells was detected by indirect immunofluorescence assay. MLR was studied with DCs and T cells. RESULTS: Following 7-day culture, a large number of cells with typical characteristics of DC were observed. The HCV C-Fc level in the transfected DCs was higher. MLR was stimulated markedly by DCs transfected with HCV C-Fc gene in comparison with the control group. CONCLUSION: A large number of DCs could be generated from murine bone marrow mononucleocyte cultures supplemented with GM-CSF and IL-4 for 1 week. The function of DCs transfected with pcDNA3HCV C-Fc was enhanced in MLR.

Characterization of secreted and intracellular forms of a truncated hepatitis C virus E2 protein expressed by a recombinant herpes simplex virus.

A replication-defective herpes simplex virus type 1 (HSV-1) recombinant lacking the glycoprotein H (gH)-encoding gene and expressing a truncated form of the hepatitis C (HCV) E2 glycoprotein (E2-661) was constructed and characterized. We show here that cells infected with the HSV/HCV recombinant virus efficiently express the HCV E2-661 protein. Most importantly, cellular and secreted E2-661 protein were both readily detected by the E2-conformational mAb H53 and despite the high expression levels, only limited amounts of misfolded aggregates were detected in either the cellular or secreted fractions. Furthermore, cell-associated and secreted E2-661 protein bound to the major extracellular loop (MEL) of CD81 in a concentration-dependent manner and both were highly reactive with sera from HCV-infected patients. Finally, BALB/c mice immunized intraperitoneally with the recombinant HSV/HCV virus induced high levels of anti-E2 antibodies. Analysis of the induced immunoglobulin G (IgG) isotypes showed high levels of IgG2a while the levels of the IgG1 isotype were significantly lower, suggesting a Th1-type of response. We conclude that the HSV-1 recombinant virus represents a promising tool for production of non-aggregated, immunologically active forms of the E2-661 protein and might have potential applications in vaccine development.

[Expression of hepatitis C virus envelope proteins with a recombinant baculovirus expression system].

OBJECTIVE: To acquire stable expression of envelope proteins of hepatitis C virus in insect host cells and use the expressed envelope proteins for detecting the serums of patients with hepatitis C. METHODS: The envelope gene of HCV H strain was amplified by PCR and inserted in baculovirus vector BacPAK8, and then recombined with linear BacPAK6 DNA in insect cells. The recombinant baculoviruses were selected by the plaque assay. The insect cells were infected by the recombinant baculoviruses that contained the target gene produced E1, E2 proteins, which were characterized with the immunoblot assay and the immunofluorescence and were used to determine 35 serum samples of patients with hepatitis C. RESULTS: The expressed E1, E2 proteins showed that the relative molecular mass of E1 is about 21 x 10(3) and 33 x 10(3), and that of E2 is about 60 x 10(3). Detection of immunofluorescence indicated that E1, E2 proteins are localized in the cytoplasm of the infected cells. Four of the 35 serums responded to expressed E1; one of them was found to recognize E2 protein. Three of 9 serums which were HCV RNA positive by PCR testing got united to E1, E2. CONCLUSION: The HCV envelope protein can be expressed stably in the insect cells. Expressed E proteins could be used in the serologic analysis of the patients' serums.

A highly sensitive and specific enzyme-linked immunosorbent assay of antibodies to hepatitis C virus.

In this study, a 178 amino acids long portion of the hepatitis C virus (HCV) core gene was cloned, sequenced, expressed in Escherichia coli, and purified. The resulting antigen (C178) was tested with human sera enzyme-linked immunosorbent assay (ELISA) in order to assess its ability to diagnose HCV. It was shown by ELISA that 92% of the patients sera, diagnosed previously by a 3rd generation enzyme immunoassay (EIA) as HCV-positive, had antibodies against the C178 antigen. This antigen gave no false positive results when tested with anti-HCV-negative sera.

[Preparation of hepatitis C virus nonstructural region antigens and detection of their relative antibodies].

OBJECTIVE: To investigate the relationship between HCV NS3 and the pathogenesis of HCV and research a method to assess the therapeutic effect of interferon (lEN) in clinic. METHODS: In a retrospective research, we used HCV-NS3 antigens of different terminals expressed in E. coli which contained the recombinant plasmids involving amino terminal region (N') or carboxyl terminal region (C') of HCV-NS3, to detect the antibodies by Western blot in sera of 63 hepatitis C patients who were treated with IFN. RESULTS: It proved that antibodies against HCV-NS3 N' and/or HCV NS3 C' terminals in hepatitis C patients may predict the treatment result of IFN, especially in the patients with chronic hepatitis C. CONCLUSIONS: The detection of antibodies against HCV-NS3 N' and/or C' terminals may be used for evaluation of IFN treatment, especially in chronic hepatitis C ones.

Characterization of the humoral and cellular immune responses against hepatitis C virus core induced by DNA-based immunization.

Hepatitis C Virus (HCV) causes most cases of posttransfusion hepatitis. Chronic HCV infection is highly related to chronic hepatitis, cirrhosis and hepatocellular carcinoma. Current therapies are only minimally effective and no vaccine has been developed. DNA-based immunization could be of prophylactic and therapeutic value for HCV infection. By intramuscular inoculation in BALB/c mice with an HCV recombinant plasmid pCI-HCV-C, we found significant levels of IgM antibody, but no significant IgG rise. After boost the immunized mice with recombinant HCV-core protein (cp1-10; 1-164aa), the anticore IgG, verified by Western-blotting, rose rapidly, which was two weeks earlier than that with control plasmid. Spleen cells from pCI-HCV-C immunized mice gave higher proliferation index (PI) than control (P < 0.05). The PI of cp1-10 boosted mice was even higher. Proliferation blocking assay with mAb proved the responding cell to be of CD4+ CD8- phenotype, supporting specific priming of T helper cells. A 51Cr-releasing CTL assay specific for HCV-core was developed, and a specific CTL response against HCV-core was demonstrated in both pCI-HCV-C immunized mice and mice boosted with cp1-10. Strong cytotoxic activity against peptide-pulsed p815 cells (H-2d), but not EL-4 cells (H-2b), suggested MHC class I restriction of the CTL activity. Blocking of CTL with mAb proved the effector cells to be of CD4- CD8+. Three CTL epitopes in HCV-core protein were demonstrated. We failed to detect CTL when immunized only with core protein. The results suggested that vaccination with HCV-core derived DNA sequences could be an effective method to induce humoral and cellular immune responses to HCV.

Targeting of hepatitis C virus core protein for MHC I or MHC II presentation does not enhance induction of immune responses to DNA vaccination.

We analyzed different vaccine approaches aimed at enhancing CD4(+)- and CD8(+)-dependent responses against hepatitis C virus (HCV) core antigen. Specific DNA vectors expressing various forms of the core in fusion with the ubiquitin or the lysosome-associated membrane protein (LAMP) were generated. These expressed the full-length wildtype core; the full-length core expressed as a covalent fusion with the ubiquitin; the full-length core expressed as a noncovalent fusion with the ubiquitin and containing a N-stabilizing or N-destabilizing residue; and the full-length core expressed as a fusion with the LAMP sequence. In vitro expression levels of the different plasmids differed by as much as tenfold. After injection into mice, none of the plasmids yielded a detectable antibody response, whereas core-specific cytotoxic T-lymphocyte (CTL) activity could be observed with all plasmids as long as 21 weeks postimmunization. No increase in CTL activity (ranging from 7% to 34% specific lysis) was observed with the ubiquitin-fusion-expressed core antigens compared with the wildtype core. The lowest CTL activity (< 5% specific lysis) was observed with the LAMP fusion. This vector was nonetheless unable to induce a detectable proliferative response. Screening of 10 different putative CTL peptide epitopes failed to reveal newly targeted epitopes when the core-fusion plasmids were used compared with the wildtype core-expressing plasmid. These data underline the difficulty in optimizing anti-core cellular immune response using molecular targeting strategies in DNA-based vaccination.

Presence of effector CD8+ T cells in hepatitis C virus-exposed healthy seronegative donors.

CTL responses against multiple hepatitis C virus (HCV) epitopes were detected in 7 of 29 (24.1%) healthy family members (HFM) persistently exposed to chronically HCV-infected patients (HCV-HFM). These precursor CTL were at very low or undetectable frequencies, as determined by limiting dilution analysis. However, when HCV-specific effector CD8+ T cells, freshly isolated from PBMC of HCV-HFM, were assessed by a sensitive enzyme-linked immunospot assay, their frequencies were severalfold higher than those of precursor CTL. These results indicate that the two assays detect two functionally distinct T cell populations and that the effector cells are not assayed by the 51Cr-release assay. Furthermore, the combination of cell depletion and enzyme-linked immunospot analyses showed that the effector cells were confined into a CD8+ CD45RO+ CD28- population. The persistence of effector CD8+ T cells specific for both the structural and nonstructural viral proteins in uninfected HCV-HFM, suggest that: 1) an immunological memory is established upon a subclinical infection without any evidence of hepatitis, in a large cohort of HCV-exposed individuals; 2) because these cells required neither restimulation nor the addition of particular cytokines in vitro for differentiating in effectors, they should be capable of prompt HCV-specific effector function in vivo, possibly providing antiviral protection; and 3) the maintenance of effector T cell responses may be sustained by persisting low-level stimulation induced by inapparent infections.

[Expression of a HCV multi-epitopes antigen gene and study on its immunogenicity].

Due to the hypervariable character of hepatitis C virus (HCV), 5 conserved T and/or B cell epitopes from core, envelope, NS3 and NS5 protein of HCV were chosen to form a 270 bp multi-epitopes antigen gene. The gene was clone into a fusion vector pWR450-1 to express a beta-galactosidase-HCV hybrid protein GZ-PCX. The purified GZ-PCX protein was specifically recognized by human anti-HCV antibodies. These results show that the HCV hybrid multi-epitopes antigen has excellent immunogenicity, which might be able to be used as an effective diagnosis agent and to provide protectivity to any genotype of HCV which might partly solve the problems in the researches of HCV vaccines.

Hepatitis C virus-specific CTL responses in PBMC from chimpanzees with chronic hepatitis C: determination of CTL and CTL precursor frequencies using a recombinant canarypox virus (ALVAC).

The aim of this study was to evaluate HCV-cytotoxic T lymphocyte response from PBMC in bulk CTL assays and in CTL precursor analyses using in vitro stimulation with canarypox virus (ALVAC) expressing HCV-capsid/E1/E2/NS2/NS3 antigens. Canarypox virus is naturally host-range restricted and does not replicate or cause cytopathology on mammalian cells. PBMC were obtained from four chimpanzees with chronic hepatitis C infection and one uninfected chimpanzee. CTL from bulk culture of PBMC and CTL precursor frequencies were found in three of the four chronically infected chimpanzees using ALVAC in vitro stimulation. No CTL response was detected in PBMC from the uninfected chimpanzee. The precursor frequencies of CTL specific for capsid, NS2 and NS3 proteins ranged between 1/2663 and 1/27202. No correlation was observed between percent cytolysis in bulk culture and CTL precursor frequencies. This method may prove useful in assessing the correlation between HCV-CTL response and virological or histological status.

[Inhibition of hepatitis C virus by antisense oligodeoxynucleotide in vitro].

OBJECTIVE: To study inhibitory effect of antisense oligodeoxynucleotide (asODN) on HCV in vitro. METHODS: The H9 cells transfected by pCD-HCV, a recombinant HCV containing total HCV structural gene, were treated with two 15-mers phosphorothioate (PS) ODNs complementary (PS-ASON) and homologous (PS-ODN) to HCV core genomic region, which were labeled with digoxin (DIG). Spot blot hybridization was carried out. Treated by the two ODNs, rPS-ODN (a 15-mers PS ODN of random sequence) or PS-ASON were modified with two liposomes (DOTAP and Lipofectin) and calcium phosphate precipitation respectively. With a half-ration, the variation of level of HCV mRNA and HCV antigen expression was observed by RT-PCR and dot ELISA. 3H-TdR adding test was done to observe PS-ASON cytotoxicity. RESULTS: PS-ODN and PS-ASON were detected in the H9 cells. The target gene was hybridized to PS-ASON and PS-ODN labeled with DIG. PS-ASON cut down level of HCV mRNA and HCV antigen expression obviously. However, PS-ODN and rPS-ODN did not influence the level of the both. The time-dependent and dose-dependent inhibition of PS-ASON was observed. In contrast to free PS-ASON, both of liposomal PS-ASON showed more highly effective inhibition, but calcium phosphate precipitation-PS-ASON complex did not. The results showed PS-ASON did not influence the H9 cells growth at 10 mumol/L. CONCLUSION: PS-ASON complementary to HCV core gene is asODN and exerts antisense-inhibitory effect on the level of HCV translation obviously, but not on the level of HCV replication and transcription.

Fusion expression of green fluorescent protein and HCV capsid antigene in Escherichia coli cells.

A chimeric gene of the green fluorescent protein (GFP) and hepatitis C virus (HCV) core antigene were constructed and expressed in E. coli cells. The expressed fusion protein was examined by Dot-ELISA and Western blot and the three antigenic determinants were detected. The GFP-Core fusion protein showed not only the striking green fluorescence under natural light but also the HCV antigenic activity. A new method of immunological diagnosis is greatly anticipated in the light of this fusion protein which can be seen as the HCV antigen tagged with the green fluorescent protein.