Article

Expression and immune response to HCV core DNA based vaccine constructs

Authors:
  • Somahlution, United States, Jupiter FL
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Abstract

Hepatitis C virus (HCV) is a major worldwide cause of acute and chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The development of vaccines against HCV have been complicated by the high variability of the envelope region, and it is likely that the cellular immune responses to viral structural proteins may be important for eradicating persistent viral infection. Recently, it was reported that the injection into muscle cells of plasmids encoding viral genes resulted in the generation of strong cellular immune responses. We constructed vectors that express the highly conserved HCV core gene. In this regard, the pHCV 2-2 construct contained the entire HCV core region and pHCV 4-2 contained both the 5' noncoding region and the core gene. Cellular expression of HCV core protein was assessed following transfection into human and murine cell lines, and higher intracellular levels of the 21-kd core protein were observed with pHCV 2-2. These HCV core DNA constructs were used to immunize BALB/c mice and produced low-level anti-HCV core humoral immune responses. To assess cytotoxic T-lymphocyte (CTL) activity generated in vivo, a cloned syngeneic SP2/O myeloma cell line constitutively expressing HCV core protein was established and inoculated into BALB/c mice to produce growth of plasmacytomas. Strong CTL activity was generated because the tumor size and weight in pHCV 2-2-immunized mice were remarkably reduced compared with mice injected with mock DNA. Spontaneous CTL activity was also exhibited by splenocytes in an in vitro cytotoxicity assay. These investigations demonstrate that plasmid constructs expressing HCV core protein generate strong CTL activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents.

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... It was noteworthy that all animals developed detectable Ab responses after three immunizations. In this regard, these nonstructural proteins are far better Ags to stimulate humoral immune responses compared with previous studies by us using the HCV core structural protein (22,23). Similar to the findings of HCV core, the humoral immune response to the NS3 protein was weak; therefore, it may be necessary to activate APCs by the coadministration of cytokine-expressing plasmids such as IL-2 and granulocyte macrophage CSF to achieve optimal humoral and cellular immune responses, (23,24). ...
... In contrast to the data presented here, DNA immunization using a construct encoding for the HCV core structural protein produced less vigorous cellular and humoral immune responses (22,23,25). The envelope region has great sequence diversity among the various genotypes and may not be a good target region because of immunoselection of viral variants known to occur during natural viral infection (22,25,26). ...
... In contrast to the data presented here, DNA immunization using a construct encoding for the HCV core structural protein produced less vigorous cellular and humoral immune responses (22,23,25). The envelope region has great sequence diversity among the various genotypes and may not be a good target region because of immunoselection of viral variants known to occur during natural viral infection (22,25,26). The NS3 gene encodes for a serine protease that cleaves the viral polyprotein precursor posttranslationally at several junctions and also serves as the viral helicase. ...
Article
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Exposure to hepatitis C virus (HCV) is associated with a high prevalence of persistent viral infection and the development of chronic liver disease and hepatocellular carcinoma. Recovery from acute infection may depend upon the generation of broad-based cellular immune responses to viral structural and nonstructural proteins. We used the DNA-based immunization approach in BALB/c mice to determine whether the HCV nonstructural proteins NS3, NS4, and NS5 will induce Ab responses, CD4+ Th cell proliferation, and cytokine release in response to stimulation by recombinant proteins as well as generate CD8+ CTL activity both in vitro and in vivo. We found that the nonstructural proteins were particularly good immunogens and produced cellular immune responses when administered as a DNA construct. Indeed, a tumor model was established following inoculation of syngenic SP2/0 cells stably transfected with NS5. We observed protection against tumor formation and growth only in mice immunized with the NS5-encoding DNA construct, establishing the generation of significant CTL activity in vivo by this technique. The results indicate that genetic immunization may define the cellular immune response of the host to HCV nonstructural proteins and is a promising approach for vaccine development.
... However, this nucleocapsid protein is not secreted from the cell, and therefore, has been shown to be weakly immunogenic at the B and Th cell level in mice (K. Tokushige and J. Wands, unpublished observations) (14). This relatively low degree of antigenicity may limit the generation of a broad based protective immune response. ...
... This cDNA fragment was inserted into a plasmid expression vector (pAp031) containing a Rous sarcoma virus enhancer element and driven by a CMV promoter (Apollon, Malvern, PA). This construct was designated pHCV2-2 (14). ...
... The human hepatocellular carcinoma (HUH-7), rhabdomyosarcoma (RD), and murine myoblast (G8) cell lines were transfected transiently with expression constructs to assess intracellular levels of HCV core protein and amounts of IL-2, IL-4, and GM-CSF secreted into the culture medium. The SP2/0 syngeneic BALB/c mouse myeloma-derived cell line was used to generate target cells to measure CTL activity in vitro, as described (14). All cell lines were obtained from the American Type Culture Collection (Rockville, MD). ...
Article
Full-text available
Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for irradication of infection. DNA-based immunization is a promising approach to generate HCV-specific immune responses. Previous studies of DNA-based immunizations in mice using an HCV core DNA expression plasmid (pHCV2-2) demonstrated an efficient CTL response against HCV core epitopes; however, the humoral and Th cell proliferative responses were found to be weak. To enhance the immunogenicity of this nonsecreted viral structural protein at the B and T cell level, we coimmunized mice with pHCV2-2 and DNA expression constructs encoding for mouse IL-2, IL-4, and granulocyte-macrophage CSF proteins. Under these experimental conditions, a seroconversion frequency to anti-HCV core increased from 40 to 80% in immunized mice. The CD4+ inflammatory T cell proliferative responses as well as CD8+ CTL activity to HCV core protein were enhanced substantially after coimmunization with the IL-2 and granulocyte-macrophage CSF DNA expression constructs. In contrast, coimmunization with an IL-4-producing construct induced differentiation of Th cells toward a Th0 subtype and suppressed HCV core-specific CTL activity. Taken together, these studies emphasize that generation of antiviral immune responses using DNA-based immunization may be modified by local cytokine production at the site of Ag presentation.
... Globally, an estimated 170 million people are chronically infected with hepatitis C virus (HCV), and 3 to 4 million persons are newly infected each year [1,2]. The human immune system has difficulties in clearing the virus in either the acute, or chronic phase of the infection with up to 40% of patients progressing to cirrhosis and liver fail- ure3456 . ...
... In mice, potent experimentally induced anti-core immune response conferred partial protection against challenge with core expressing recombinant vaccinia virus [40]. However, despite high immunogenicity in the natural infection, core does not perform well as an immunogen, specifically if introduced as naked DNA [2,414243 . Attempts to enhance core immunogenicity by targeting HCV core protein to specific cellular compartments [44], co-immunization with cytokine expressing plasmids [2,41], adjuvants as CpG [45], or truncated core gene versions [46] had limited or no suc- cess. ...
... However, despite high immunogenicity in the natural infection, core does not perform well as an immunogen, specifically if introduced as naked DNA [2,414243 . Attempts to enhance core immunogenicity by targeting HCV core protein to specific cellular compartments [44], co-immunization with cytokine expressing plasmids [2,41], adjuvants as CpG [45], or truncated core gene versions [46] had limited or no suc- cess. Prime-boost strategies have been used to increase immune responses to a number of DNA vaccines. ...
Article
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Hepatitis C core protein is an attractive target for HCV vaccine aimed to exterminate HCV infected cells. However, although highly immunogenic in natural infection, core appears to have low immunogenicity in experimental settings. We aimed to design an HCV vaccine prototype based on core, and devise immunization regimens that would lead to potent anti-core immune responses which circumvent the immunogenicity limitations earlier observed. Plasmids encoding core with no translation initiation signal (pCMVcore); with Kozak sequence (pCMVcoreKozak); and with HCV IRES (pCMVcoreIRES) were designed and expressed in a variety of eukaryotic cells. Polyproteins corresponding to HCV 1b amino acids (aa) 1-98 and 1-173 were expressed in E. coli. C57BL/6 mice were immunized with four 25-microg doses of pCMVcoreKozak, or pCMV (I). BALB/c mice were immunized with 100 microg of either pCMVcore, or pCMVcoreKozak, or pCMVcoreIRES, or empty pCMV (II). Lastly, BALB/c mice were immunized with 20 microg of core aa 1-98 in prime and boost, or with 100 microg of pCMVcoreKozak in prime and 20 microg of core aa 1-98 in boost (III). Antibody response, [3H]-T-incorporation, and cytokine secretion by core/core peptide-stimulated splenocytes were assessed after each immunization. Plasmids differed in core-expression capacity: mouse fibroblasts transfected with pCMVcore, pCMVcoreIRES and pCMVcoreKozak expressed 0.22 +/- 0.18, 0.83 +/- 0.5, and 13 +/- 5 ng core per cell, respectively. Single immunization with highly expressing pCMVcoreKozak induced specific IFN-gamma and IL-2, and weak antibody response. Single immunization with plasmids directing low levels of core expression induced similar levels of cytokines, strong T-cell proliferation (pCMVcoreIRES), and antibodies in titer 103(pCMVcore). Boosting with pCMVcoreKozak induced low antibody response, core-specific T-cell proliferation and IFN-gamma secretion that subsided after the 3rd plasmid injection. The latter also led to a decrease in specific IL-2 secretion. The best was the heterologous pCMVcoreKozak prime/protein boost regiment that generated mixed Th1/Th2-cellular response with core-specific antibodies in titer >or= 3 x 10(3). Thus, administration of highly expressed HCV core gene, as one large dose or repeated injections of smaller doses, may suppress core-specific immune response. Instead, the latter is induced by a heterologous DNA prime/protein boost regiment that circumvents the negative effects of intracellular core expression.
... Chronic alcoholics have a high incidence of hepatitis C virus (HCV) infection (Schiff and Ozden 2003; Oshita et al., 1994; Siu et al., 2009) which may be due, in part, to the action of alcohol on the cellular immune response to epitopes that reside on viral structural and nonstructural proteins. Studies have been performed in animal models where genetic immunization has been employed to generate viral specific antibody and CD4+ and CD8+ responses to HCV core protein (Tokushige et al., 1996; Geissler et al., 1997a; Encke et al., 1998; Stylianou and Saklatvala, 1998). Low level expression of HCV proteins encoded by such plasmids either in muscle or fibroblasts following DNA inoculation led to activation of dendritic cells (DCs) at the site of immunization or in distant draining lymph nodes to subsequently prime anti-viral immune responses. ...
... Chronic alcohol feeding had a substantial suppressive effect on the generation of viral antigen specific CD4+ and CD8+ cellular immune activity when HCV core was employed as the immunogen. Of interest, this inhibitory effect of alcohol could be partially, or completely, reversed by co-immunization with an interleukin-2 (IL-2) or granulocyte macrophage colony-stimulating factor (GM- CSF) expression plasmid (Encke et al., 1998; Tokushige et al., 1996; Stylianou and Saklatvala, 1998; Geissler et al., 1997a; Encke and Wands, 2000; Geissler et al., 1999). Such experimental results are consistent with the concept that DNA based immunization was enhanced by cytokine production at the site of antigen presentation, and that DCs could be an important cellular target during chronic alcohol consumption (Encke et al., 1998; Tokushige et al., 1996; Stylianou and Saklatvala, 1998; Geissler et al., 1997a; Encke and Wands, 2000; Geissler et al., 1999). ...
... Of interest, this inhibitory effect of alcohol could be partially, or completely, reversed by co-immunization with an interleukin-2 (IL-2) or granulocyte macrophage colony-stimulating factor (GM- CSF) expression plasmid (Encke et al., 1998; Tokushige et al., 1996; Stylianou and Saklatvala, 1998; Geissler et al., 1997a; Encke and Wands, 2000; Geissler et al., 1999). Such experimental results are consistent with the concept that DNA based immunization was enhanced by cytokine production at the site of antigen presentation, and that DCs could be an important cellular target during chronic alcohol consumption (Encke et al., 1998; Tokushige et al., 1996; Stylianou and Saklatvala, 1998; Geissler et al., 1997a; Encke and Wands, 2000; Geissler et al., 1999). Similar results were obtained using a plasmid encoding for the HCV NS5 non-structural protein as well (Encke et al., 1998; Rehermann and Nascimbeni, 2005). ...
Article
Hepatitis C virus infection affects 170 million people worldwide, and the majority of individuals exposed to HCV develop chronic hepatitis leading to progressive liver damage, cirrhosis, and hepatocellular cancer. The natural history of HCV infection is influenced by genetic and environmental factors of which chronic alcohol use is an independent risk factor for cirrhosis in HCV-infected individuals. Both the hepatitis C virus and alcohol damage the liver and result in immune alterations contributing to both decreased viral clearance and liver injury. This review will capture the major components of the interactions between alcohol and HCV infection to provide better understanding for the molecular basis of the dangerous combination of alcohol use and HCV infection. Common targets of HCV and alcohol involve innate immune recognition and dendritic cells, the critical cell type in antigen presentation and antiviral immunity. In addition, both alcohol and HCV affect intracellular processes critical for hepatocyte and immune cell functions including mitochondrial and proteasomal activation. Finally, both chronic alcohol use and hepatitis C virus infection increase the risk of hepatocellular cancer. The common molecular mechanisms underlying the pathological interactions between alcohol and HCV include the modulation of cytokine production, lipopolysaccharide (LPS)-TLR4 signaling, and reactive oxygen species (ROS) production. LPS-induced chronic inflammation is not only a major cause of progressive liver injury and fibrosis, but it can also contribute to modification of the tissue environment and stem cells to promote hepatocellular cancer development. Alteration of these processes by alcohol and HCV produces an environment of impaired antiviral immune response, greater hepatocellular injury, and activation of cell proliferation and dedifferentiation.
... Recent reports show that DNA-immunization in mice using both the structural and/or nonstructural HCV proteins can generate humoral and cellular immune responses [266][267][268][269][270][271][272][273][274] . The use of DNA-plasmids encoding the nonstructural proteins 268, 270-272 seems to generate more potent humoral and cellular immune responses compared to responses induced by the structural proteins 266,267,275 . ...
... Recent reports show that DNA-immunization in mice using both the structural and/or nonstructural HCV proteins can generate humoral and cellular immune responses [266][267][268][269][270][271][272][273][274] . The use of DNA-plasmids encoding the nonstructural proteins 268, 270-272 seems to generate more potent humoral and cellular immune responses compared to responses induced by the structural proteins 266,267,275 . A major problem with genetic immunization against HCV has been the rather limited immunogenicity. ...
... Strategies used to overcome the high level of genetic variability include the use of conserved HCV proteins such as the core protein as antigen (Tokushige et al., 1996), Several researchers have attempted to overcome the problem by selection of synthetic HVR epitopes that are cross reactive with large numbers of patient sera and antibodies (termed mimotopes). ...
Thesis
p>For this project, a real-time PCR-based assay was developed for detection and quantitation of HCV RNA using the TaqMan 5' nuclease assay and the Applied Biosystems PRISM 7700 Sequence Detection System. TaqMan primers and probes were designed to amplify part of the 5' untranslated region and detected HCV genotypes 1 - 5 with equal efficiency. An HCV standard RNA was developed and used to generate standard curves for quantitation. An internal control RNA was produced and used to monitor efficiency of RNA extraction reverse transcription and amplification. The performance of the assay was evaluated using the World Health Organisation International Standard for HCV RNA. The assay quantified the International Standard to within a mean of 27% from the published load and had a limit of detection of 50 international units/ml. The assay had a wide dynamic range (50 - 10<sup>8</sup> copies/ml) and had intra and inter-assay coefficients of variation of 23.8% and 29.4%, respectively. The assay was compared with the Quantiplex HCV RNA 2.0 assay (Bayer) and viral loads given by the two assays correlated strongly. The quantitative assay was used to study HCV load in 53 symptomatic and 57 asymptomatic chronically infected individuals. No difference in load was found between the two populations. When the groups were combined, a weak correlation was found between viral load and the level of liver fibrosis. Longitudinal variation in load was also studied in 19 asymptomatic patients. Of these, eight showed an increase in load, ten showed a decrease and one showed no overall change in load. Viral load varied by <11og<sub>10</sub> copies/ml in the majority of patients (68%). HCV loads were also studied in eight HCV-positive patients treated with a combination of pegylated interferon and ribavirin (Roche). End of treatment virological and biochemical responses rates were 87.5% and 78%, respectively and the overall end of treatment response rate was 75%. Highest rates of decline in viral load were seen in patients infected with genotype 3 virus. The assay was also used to quantify HCV RNA in cultured peripheral blood mononuclear cells from HCV-positive patients. The cells were treated with immunomodulatory compounds (cyclosporin A, hydrocortisone, phytohaemagglutinin) and HCV RNA levels measured. HCV RNA levels decreased in all treated and untreated cells over the study period (seven to 14 days). The addition of the immunomodulatory compounds had no detectable effects on virus levels.</p
... 2000). DNA immunization can induce cell-mediated immune responses against structural and nonstructural (NS) HCV genes in mice (Bocher W O, 2001; Tokushige K, 1996). The HCV NS3 gene has been considered as a possible vaccine target (Bartenschlager R, 1993; Pang P S, 2002). ...
Article
Full-text available
Hepatitis C virus (HCV) chronic infection is a worldwide health problem, and numerous efforts have been invested to develop novel vaccines. An efficient vaccine requires broad immune response induction against viral proteins. To achieve this goal, we constructed a DNA vaccine expressing nonstructural 3 (NS3) gene (pcDNA3.1-HCV-NS3) and assessed the immune response in C57BL/6 mice. In this study, the NS3 gene was amplified with a nested-reverse transcriptase-polymerase chain reaction (RT-PCR) method using sera of HCV-infected patients with genotype 1a. The resulting NS3 gene was subcloned into a pcDNA3.1 eukaryotic expression vector, and gene expression was detected by western blot. The resultant DNA vaccine was co-administered with interleukin-12 (IL-12) as an adjuvant to female C57BL/6 mice. After the final immunizations, lymphocyte proliferation, cytotoxicity, and cytokine levels were assessed to measure immune responses. Our data suggest that co-administration of HCV NS3 DNA vaccine with IL-12 induces production of significant levels of both IL-4 and interferon (IFN)-γ (p<0.05). Cytotoxicity and lymphocyte proliferation responses of vaccinated mice were significantly increased compared to control (p<0.05). Collectively, our results demonstrated that co-administration of HCV NS3 and IL-12 displayed strong immunogenicity in a murine model.
... After stripping of the membrane using standard procedures and reprobing with Mab directed against the hepatitis B virus (HBV) core protein a specific band corresponding to the HBV core protein serving as a negative control could be visualized. A confluent 10 cm cell culture dish of Sp2/0 cells or SP2/19 cells that stably express HCV core protein additionally (31) were lysed in 1% Triton X 100 cell lysis buffer (30). Subsequently, 50 l of cell lysate was subjected to 15% SDS-PAGE. ...
Article
The monoclonal antibody C7-50 binds to the HCV core protein with high sensitivity and specificity. The coding sequences of the variable domains of the antibody were determined following cDNA cloning of the Fab and sFv fragments. Subsequently, intracellular expression and binding of these antibody fragments to the HCV core protein as a potential antiviral approach were studied. There was high specificity and sensitivity of binding of bacterially expressed, recombinant C7-50 Fab to HCV core as measured by EIA and immunoblot. For expression in mammalian cells, the C7-50 antibody was subcloned in the sFv format by the introduction of a (Gly(4)Ser)(3) linker spaced between light and heavy chains. Northern and Western blot analysis as well as confocal microscopy established the targeted expression of the C7-50 sFv antibody fragment in the endoplasmic reticulum of transfected cells. The colocalization and intracellular binding of the antibody fragment to HCV core protein was confirmed by immunoprecipitation and subsequent immunoblot analysis. This study demonstrates that gene delivery of cDNA coding sequences inducing intracellular expression of C7-50 antibody fragments leads to binding of the antibody fragment to the HCV core protein within the secretory compartment of transfected cells. Intracellular immunization represents a promising antiviral approach to interfere with the life cycle of HCV.
... Thus, a phase I clinical trial of an HCV vaccine has been completed in the United States (personal communication); the results of the trial and the initiation of a phase II trial are pending. DNA-based vaccines: DNA-based vaccines are an innovative technique in the treatment of chronic viral infections (68,69). An example of a model HBV DNA-based vaccine includes a plasmid containing a designed DNA gene fragment attached to a cytomegalovirus promoter intended for injection into muscle tissue. ...
Article
Full-text available
Hepatitis C virus (HCV) infects over 170 million people worldwide. While interferon is currently the most used single agent therapy, this drug may result in a sustained loss of virus from the blood in only up to 15% of patients; new options for treatment are needed. With the release of ribavirin in North America and Europe, a viral clearance rate or 'cure' may be attained in up to 40% of patients. Developing successful antiviral therapy that prevents or delays the development of cirrhosis, liver failure and liver cancer as well as decreasing the demand for liver transplantation are clearly identified goals. Unfortunately, there is no complete in vitro model of HCV replication or translation. Due to the lack of an animal or cell culture model of HCV infection, in vitro translation screening systems to identify inhibitors of HCV protein translation are being evaluated by a large number of biotechnology companies. With advancing computer technology, high throughput screening processes are now possible and can be joined to specific in vitro model testing systems. Along with examining some of the information known about HCV therapy and the HCV genome, the present review discusses potential targets for new therapies and identifies therapeutic agents that are nearing clinical application.
... It has been shown in a number of studies that injection of a DNA construct for the hepatitis C virus core protein generates corespecific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity (Major et al., 1995;Lagging et al., 1995;Tokushige et al., 1996;Inchauspe et al., 1997). Plasmids expressing different domains of the hepatitis C virus envelope E2 glycoprotein were injected in BALB/c mice intramuscularly or intraepidermally via a gene gun, and induced humoral immune responses were evaluated. ...
Article
Full-text available
Vaccines represent the most commonly employed immunologic intervention in medicine today. DNA vaccination or genetic immunization is a rapidly developing technology that offers new approaches for the prevention of disease. This method of vaccination provides a stable and long-lived source of the protein vaccine, and it is a simple, robust, and effective means of eliciting both antibody- and cell-mediated immune responses. Furthermore, DNA vaccines have a number of potential advantages such as they can address several diseases in one vaccine, they are cheap and easy to produce and have no special cold storage requirement because they are extremely stable. It has proven to be a generally applicable technology in various preclinical animal models of infectious and noninfectious diseases, and several DNA vaccines have now entered phase I/II, human clinical trials. There are several hurdles that need to be overcome on the road to the use of DNA vaccines widely. These include the technical challenges of improving delivery and/or potency so that low doses of DNA can achieve the efficacy of conventional vaccines.
... The advantage of this method is that it avoids dif®cult viral protein puri®cation, and also allows an immune response against viral structural proteins which may have less genotypic variation than envelope proteins. 57 The haemophilias ...
Article
Gene therapy for diseases of the gastrointestinal tract is an exciting prospect because of the fundamental cure that is potentially available. The gastrointestinal system, and especially the liver, is an area that will be central to the development of gene therapy. Techniques for gene replacement include homologous recombination and gene augmentation. For the treatment of cancer antisense strategy, pro-drug activation systems and gene immunotherapy are being investigated. Gene-carrying vectors divide into viral- and non-viral-based vectors, each with advantages and limitations. The accurate delivery of these vectors to sufficient numbers of target cells in vivo is still a major barrier to clinical use. Diseases that may be helped by gene therapy include: gastrointestinal malignancies, viral hepatitis, the haemophilias, hypercholesterolaemia, α1-antitrypsin deficiency, and metabolic diseases of the liver and cystic fibrosis. In this review we will outline the principles of gene therapy, delivery vectors under investigation, diseases that may benefit from this technology and some of the remaining problems to be overcome.
... Previous studies show that DNA-based rate of nonresponsiveness to the hepatitis B envelope vaccine immunization is a very effective method to induce humoral compared with nonalcoholic subjects. 37-40 The lower vaccine and especially cellular immune responses against different response rate in alcoholics was associated with diminished viral antigens including HBV. [21][22][23][24] That approach would allow CD4 / T-cell activation and suggests that the humoral imus to assess the effects of ethanol on cellular immune remune response to HBV envelope proteins was a T-cell-desponses to HBV in vivo, in the context of MHC class I and pendent event. Taken together, our studies provide direct II antigen presentation, in an attempt to explore the possible evidence that cellular immune responses to HBV envelope immunologic mechanism(s) relating to the induction of perproteins may be adversely influenced by chronic ethanol consistent viral infection. ...
Article
Full-text available
Hepatitis B virus (HBV) is common in alcoholics and may result in chronic infection. Persistence of HBV infection could be partially caused by the effects of ethanol on the cellular and humoral immune response to viral structural proteins. The DNA-based immunization approach was used to experimentally assess the effects of chronic ethanol feeding on immune responses directed against the middle envelope protein (MHBs) of HBV. Mice were fed an ethanol or isocaloric, pair-fed control liquid diet for 8 weeks, followed by immunization with a plasmid construct containing the pre-S2/S gene that encodes for MHBs. Chronic ethanol consumption marginally reduced the levels of the antibody to hepatitis B surface proteins (anti-HBs) generated by the DNA-based immunization approach. Initially, cytotoxic lymphocyte (CTL) activity was higher in ethanol-fed mice but progressively declined following the second and third immunizations as compared with control mice. In addition, CTL and CD4+ T helper (TH) cells responded poorly to increasing concentrations of envelope protein and peptides in vitro with respect to generation of CTL activity and proliferative responses. Finally, proliferating CD4+ T cells derived from ethanol-fed animals had substantial changes in the levels of cytokines secreted into the culture supernatants as compared with control mice. These studies show that chronic ethanol consumption substantially alters the cellular immune responses to a human viral structural protein, and that these effects may contribute to the persistence of viral infection.
... The purpose was to evaluate the performance of this new mode of vaccination applied to HCV envelope proteins, specifically E2, as well as to gain insights into the immunogenic nature of E2. DNA immunization with HCV genes has so far been described only in studies focused on the viral nucleocapsid (28,32,45). ...
Article
Full-text available
Plasmids expressing different domains of the hepatis C virus (HCV) envelope E2 glycoprotein from a genotype 1a isolate were constructed to compare the immunogenic potential of E2 in nucleic acid-based immunizations. One plasmid, pCIE2t, expressed a C-terminally truncated form of E2, while others, pS2.SE2A to pS2.SE2E, encoded the adjacent 60-amino-acid (aa) sequences of E2 (inserts A to E) expressed as a fusion with the hepatitis B virus surface antigen. BALB/c mice were given injections of the plasmids intramuscularly (i.m.) or intraepidermally (i.e.) via a gene gun (biolistic introduction), and induced humoral immune responses were evaluated. The i.e. injections resulted in higher seroconversion rates and antibody titers, up to 100-fold, than did the i.m. injections (P = 0.01 to 0.04). Three restricted immunogenic domains, E2A (aa 384 to 443), E2C (aa 504 to 555), and E2E (aa 609 to 674), that yielded antibody titers ranging from 1:59 to > 1:43,700 could be identified. Subtype 1a- and 1b-derived E2 antigens and synthetic peptides were used in Western blot and enzyme-linked immunosorbent assay analyses, which revealed that the cross-reactivity of the plasmid-induced antibodies was linked both to the type of antigen expressed and to the injection mode. Induced anti-E2 antibodies could immunoprecipitate noncovalent E1E2 complexes believed to exist on the surface of HCV virions. This study allowed us to identify restricted immunogenic domains within E2 and demonstrated that different routes of injection of HCV E2 plasmids can result in quantitatively and qualitatively different humoral immune responses.
... Many studies have been published on the development of DNA-based vaccines against HCV. It was reported that the injection into muscle cells of plasmids constructs expressing HCV core protein generate strong cytotoxic T-lymphocyte (CTL) activity, as assessed both in vivo and in vitro, and are promising candidates as antiviral agents (Tokushige et al., 1996; Gehring et al., 2004). The development of DNA base vaccine for our local setup will be valuable work and will be helpful in eradicating the whole nation from the blood born pathogen. ...
Article
Full-text available
Hepatitis C virus (HCV) is the major etiological agent of hepatitis. It infects 200 million people worldwide and 85% of them could develop chronic hepatitis, liver function failure or hepatocellular carcinoma. Hepatitis C is rapidly emerging as a major health problem in developing countries like Pakistan with prevalence rate of 10% and genotype 3a is the most prevalent. Here, approximately 80% of infections proceed to chronic infection and infected blood is the primary route of spread. In Pakistan, about 75% of patents do not receive standard anti HCV therapy (Interferon + Ribavirin) and of the 25% that do receive such treatment, the SVR rate is 60 -70%. This review is designed to cover the information about the status of HCV in Pakistan with major focus on its prevalence, genotypes, current diagnostic assays, available therapies and treatment outcomes. The present review further emphasizes the need to uncover exact HCV prevalence rate in the country, to develop diagnostic assays based on local genotype, to understand the interaction between HCV genotype 3a genes and cell line genes responsible HCV pathogenesis. In addition, this review discusses the need for the generation of infectious pseudo particle of HCV as a potential vaccine, to investigate DNA base vaccine, or siRNA-based anti HCV approaches for our local genotypes.
... Human gene therapy is defined as the introduction of new genetic material into the cells of an individual with the intention of producing a therapeutic benefit for the patient (4,7,90). Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management (4,7,35,51,83,94,96,99,137,138,151). Gene therapy for infectious diseases requires the introduction of genes designed to specifically block or inhibit the gene expression or function of gene products, such that the replication of the infectious agent is blocked or limited. ...
Chapter
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Human gene therapy is defined as the introduction of new genetic material into the cells of an individual with the intention of producing a therapeutic benefit for that individual. Gene therapy may be applied to any infectious disease whereby the infecting organism inserts its own genetic material into the genome of the host cell genome. Keywords: infectious diseases; human immunodeficiency virus; gene therapy
... In addition, infection with one genotype does not preclude the possibility of reinfection with the same or other genotypes [7]. The efforts of other researchers to induce immunity to HCV is encouraging [8][9][10][11][12][13][14][15][16]. In 1990, Wolff et al. [17] showed convincingly that plasmid DNA injected into mouse skeletal muscle in vivo could produce fully functional gene products for extended periods of time. ...
Article
DNA-based immunizations have been used to elicit cellular immunity to hepatitis C virus (HCV) proteins in mice. Mice were immunized by intramuscular or intradermal injections of plasmid DNA derived from a near-full-length HCV genotype 1b genomic clone (pRC/B2) or individual genomic clones. These immunizations induced cytotoxic T lymphocytes (CTLs), as revealed in standard chromium-release assays that used syngeneic peptide-pulsed or trans-fected target cells. These assays identified four CTL epitopes within the capsid, E1, and E2 regions of the polyprotein sequence of HCV genotype la that were cross-reactive with HCV genotype 1b. Additionally, CTLs derived from mice immunized with either NS3 or NS5 specifically lysed target cells sensitized to either the genotype 1a or 1b gene products. Nucleic acid immunizations also generated humoral immunity to HCV proteins, as detected by anti-HCV reactivity to NS3 and capsid in ELISAs and immunoblot assays.
... 脂质体法转染pGM-CSF于CHO细胞 [13] , 6孔组 细胞炎症反应过度级联放大以及树突状细胞功 能的改变等 [18][19][20][21][22] . 由于单用核心蛋白基因不能产 生有效的保护性抗体反应 [23][24][25][26] , 因此近年的研究 已有研究表明CD4 + T细胞在增强CTL杀伤 活性方面起着关键作用 [30,31] . 我们的研究表明 ...
Article
AIM: To study the effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) genetic adjuvant on immune response induced by plasmid DNA encoding the hepatitis C virus (HCV) core (C) protein. METHODS: The gene encoding the HCV C protein was amplified by PCR from HCV 1b genotype and inserted into the pUC119 vector. The HCV C gene was then subcloned into the pCMH6K eukaryotic vector, and the resulting plasmid was named pCMH6K/HCV-C. The recombinant vector was confirmed by restriction enzyme analysis and DNA sequencing, and transfected into China hamster ovary (CHO) cells with Lipofectamine 2000. Distribution of the HCV C protein in transfected CHO cells was detected by immunofluorescence. Balb/c mice were vaccinated with the recombinant plasmid with or without the GM-CSF gene. HCV C-specific antibody in serum was measured by ELISA. The changes in T lymphocyte subsets and levels of Th cell intracellular cytokines interferon-γ (IFN-γ) and interleukin-4 (IL-4) in splenic cell suspension from immunized mice were evaluated by flow cytometric analysis. CTL activity was assessed by LDH assay. RESULTS: Restrict enzyme digestion and DNA sequencing indicated that the recombinant pCMH6K/ HCV-C was successfully constructed. The expression of plasmid-encoded protein was mainly distributed in membrane and scarcely in cytoplasm of transfected CHO cells. The percentage of CD4+ T cells in spleen cells in the pCMH6K/HCV-C+pGM-CSF co-vaccination group was significantly higher than those in other groups (all P < 0.05). The percentage of CD8+ T cells showed no significant differences among each group (P > 0.05). CTL activity induced by GM-CSF DNA co-vaccination was significantly higher than that immunized with the same amount of other naked DNA (P < 0.05). The ratio of IFN-γ to IL-4 in spleen cells from GMCSF DNA co-vaccination group was significantly higher than those in other groups (all P < 0.05). CONCLUSION: GM-CSF DNA could enhance the immune stimulatory effects of HCV DNA vaccine and induce Th1-type immune response.
... [24] Extra to E2 protein, more conserved core protein have logically used to expand the immune response of the vaccine for including of the specific CTL epitopes. [25][26][27] NS3 protein as a targeted antigen in therapy-linked or spontaneously resolved HCV patients has recently considered and thus, as a potential target in vaccine development studies has been shown. [28,29] Polyepitope vaccine studies were recently described for HCV by application of epitopes derived from both structural and NS proteins. ...
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Background: Hepatitis C virus (HCV) infection is a serious public health threat worldwide. Cellular immune responses, especially cytotoxic T-lymphocytes (CTLs), play a critical role in immune response toward the HCV clearance. Since polytope vaccines have the ability to stimulate the cellular immunity, a recombinant fusion protein was developed in this study. Materials and methods: The designed fusion protein is composed of hepatitis B surface antigen (HBsAg), as an immunocarrier, fused to an HCV polytope sequence. The polytope containing five immunogenic epitopes of HCV was designed to induce specific CTL responses. The construct was cloned into the pET-28a, and its expression was investigated in BL21 (DE3), BL21 pLysS, BL21 pLysE, and BL21 AI Escherichia coli strains using 12% gel sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Finally, the identity of expressed fusion protein was confirmed by Western blotting using anti-His monoclonal antibody and affinity chromatography was applied to purify the expressed protein. Results: The accuracy of the construct was confirmed by restriction map analysis and sequencing. The transformation of the construct into the BL21 (DE3), pLysS, and pLysE E. coli strains did not lead to any expression. The fusion protein was found to be toxic for E. coli DE3. By applying two steps inhibition, the fusion protein was successfully expressed in BL21 (AI) E. coli strain. Conclusion: The HBsAg-polytope fusion protein expressed in this study can be further evaluated for its immunogenicity in animal models.
... Gene therapy is being investigated as an alternative treatment for a wide range of infectious diseases that are not amenable to standard clinical management. [50][51][52][53][54][55][56][57] Gene therapy for infectious diseases requires the introduction of genes designed to specifically block or inhibit the gene expression or function of gene products, such that the replication of the infectious agent is blocked or limited. In addition to this intracellular intervention, gene therapy may be used to intervene in the spread of the infectious agent at the extracellular level. ...
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Gene therapy can be broadly defined as the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Safe methods have been devised to do this, using several viral and non-viral vectors. Two main approaches emerged: in vivo modification and ex vivo modification. Retrovirus, adenovirus, adeno-associated virus are suitable for gene therapeutic approaches which are based on permanent expression of the therapeutic gene. Non-viral vectors are far less efficient than viral vectors, but they have advantages due to their low immunogenicity and their large capacity for therapeutic DNA. To improve the function of non-viral vectors, the addition of viral functions such as receptor mediated uptake and nuclear translocation of DNA may finally lead to the development of an artificial virus. Gene transfer protocols have been approved for human use in inherited diseases, cancers and acquired disorders. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.
Article
This chapter discusses the development of hepatitis C vaccines. Presently, numerous obstacles to hepatitis C vaccine development remain. The large degree of genetic and immunologic diversity of the virus may be the most difficult to overcome. However, in limited studies, it has been shown that hepatitis C virus (HCV) can be neutralized by antibody and that even with the lack of solid protection from infection; the vaccine-induced antibody can alter the natural history of infection toward a short-lasting, mild disease. The prevention of chronic infection in itself would be important as the most serious outcomes of HCV infections are the result of viral persistence. It is hoped that detailed studies of the immune response to HCV infections will provide insights to solving many of these problems. New technologies being applied to vaccine development may eventually lead to effective immunoprophylaxis and therapy.
Article
Development of a broad based cellular and humoral immune response to hepatitis C virus (HCV) structural proteins may be important for eradication of viral infection. In previous studies in mice we demonstrated that facilitated DNA-based immunization with an HCV core DNA-expression construct stimulated the generation of weak cytotoxic T lymphocyte (CTL), helper T cell (Th), and humoral immune responses against HCV core related epitopes. To enhance the immunogenicity of this non-secreted viral structural protein at both the B- and T-cell level, several chimeric HBV-HCV constructs were prepared which were designed to express and secrete HCV core protein along with various regions of the hepatitis B envelope protein. No secretion of the chimeric proteins into the culture supernatant was detected using sensitive radioimmunoassays. However, such chimeric proteins were capable of generating CD4+ inflammatory T cell and CD8+ CTL activity against both HBV and HCV components of the fusion proteins. It was determined that the proliferative activity of T cells as well as the humoral immune responses to HCV core protein were substantially enhanced by some chimeric fusion proteins as compared to the HCV core protein alone. The strength of the immune responses appeared directly related to the level of Th1 cytokines produced by CD4+ T cells obtained from immunized animals. Further characterization of the immune responses stimulated by these DNA constructs studied helped to define some of the most immunogenic regions of the chimeric proteins that they encode.
Chapter
DNA vaccination involves the direct injection of a suitable designed plasmid DNA molecule into a tissue such that the antigenic proteins encoded by the plasmid are expressed in the cells of the treated host. This extraordinarily simple approach has been translated into practice for many antigens from infectious agents, for putative tumor antigens, and for certain potential allergens. If the results currently obtained in animal models can be replicated with equal efficiency in humans and large animals, and if the justifiable concerns about safety can be resolved, then the DNA vaccination may become the basis for an entirely new generation of vaccines.
Chapter
Hepatitisviren sind eine heterogene Gruppe von Viren, deren hauptsächliche klinische Manifestation eine Hepatitis ist. Hepatitisviren wirken aber auch auf andere Organsysteme und beeinflussen die Embryo- und Fetogenese. Demgegenüber ist die Hepatitis bei hepatotropen Viren nicht das hauptsächliche Symptom. Bedeutung für das Ungeborene und Neugeborene haben die in Tabelle 1 aufgelisteten Hepatitisviren.
Article
Hepatitis C virus (HCV) is a positive-strand RNA virus that codes for at least six different proteins (Fig. 1). Its worldwide prevalence ranges from 0.5% to 10% and it is currently believed that more than 70% of HCV-infected patients will evolve towards a chronic carrier state with the risk, in more than 40%, of developing cirrhosis associated with an enhanced change of evolution towards hepatocellular carcinomas (HCC) [32]. In France, HCV infections have become the prevalent cause of HCC and it is estimated that more than 30,000 cases of HCC directly related to HCV infection will be accounted for by the year 2010 [3]. Community acquired infection is still common causing a significant amount of morbidity as well as important economic burdens [26]. In addition, the current lack of efficient antiviral treatment makes the development of a vaccine desirable.
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Genetic immunization is a potentially useful strategy to prevent or treat hepatitis B virus (HBV) infection. We have previously shown that HBV envelope proteins are highly immunogenic using this technique. The large envelope protein (LHBs), however, induced significantly weaker humoral and cellular immune responses when compared with the middle envelope protein (MHBs). We studied the effect of co-immunizations with cytokine DNA expression constructs encoding for interleukin (IL)-2 and (GM-CSF) on the immunogenicity of LHBs at the B-and T-cell level. Co-immunizations of mice with plasmids encoding for MHBs and IL-2 or GM-CSF increased anti-HBs responses, helper T-cell proliferative activity, and cytotoxic T lymphocyte (CTL) killing. In contrast, co-immunizations of plasmids encoding for LHBs and IL-2 or GM-CSF had no effect on humoral and cellular immune responses. LHBs did not inhibit the production or secretion of IL-2 and GM-CSF. In addition, IL-2, tumor necrosis factor alfa (TNF-alpha), and interferon gamma (IFN-gamma) had no suppressive effect on HBV envelope protein expression in vitro. Based on these data, MHBs, but not LHBs, genetic immunization can be augmented by IL-2 or GM-CSF cytokines.
Article
Development of a broad-based cellular immune response to hepatitis B viral structural proteins may be important for recovery from infection, and lack of such responses may lead to persistent viral infection and chronic liver disease. Strategies designed to enhance the hepatitis B virus (HBV)-specific immune response may be able to reduce persistent viral infection of the liver. The aim of this study was to induce HBV-specific cellular and humoral immune responses in mice using DNA-based immunizations with the large and middle envelope and nucleocapsid proteins. Antibodies to HBV structural proteins, T-helper-cell proliferation, and cytokine release and generation of cytotoxic T lymphocyte (CTL) activity were measured in vaccinated mice. Immunized mice developed high-titer antibodies against envelope and core proteins in serum. More importantly, 93% of the immunized mice produced strong inflammatory CD4+ T-cell and CD8+ CTL responses to viral proteins. This study shows that DNA-based vaccination will generate broad-based CTL activity as well as strong T-helper cell responses with the production of TH1-type cytokines to HBV structural proteins. Such constructs are promising candidates as antiviral agents, and these studies have defined some of the most immunogenic antigens for an immunotherapeutic approach of chronic HBV infection.
Article
DNA vaccines have been termed The Third Generation of Vaccines. The recent successful immunization of experimental animals against a range of infectious agents and several tumour models of disease with plasmid DNA testifies to the powerful nature of this revolutionary approach in vaccinology. Among numerous advantages, a major attraction of DNA vaccines over conventional vaccines is that they are able to induce protective cytotoxic T-cell responses as well as helper T-cell and humoral immunity. Here we review the current state of nucleic acid vaccines and cover a wide range of topics including delivery mechanisms, uptake and expression of plasmid DNA, and the types of immune responses generated. Further, we discuss safety issues, and document the use of nucleic acid vaccines against viral, bacterial and parasitic diseases, and cancer. The early potential promise of DNA vaccination has been fully substantiated with recent, exciting developments including the movement from testing DNA vaccines in laboratory models to non-human primates and initial human clinical trials. These advances and the emerging voluminous literature on DNA vaccines highlight the rapid progress that has been made in the DNA immunization field. It will be of considerable interest to see whether the progress and optimism currently prevailing can be maintained, and whether the approach can indeed fulfil the medical and commerical promise anticipated.
Article
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.
Thesis
DNA vaccines have become the spotlight in vaccinology in the past ten years as an alternative to conventional vaccines. In our study we characterized newly formulated non-ionic surfactant vesicles (niosomes) and assessed their potential as a delivery vehicle for DNA vaccines. Plasmid DNA was successfully entrapped into cationic niosomes using the dehydration-rehydration method. The DRV niosomes entrapping or complexing the plasmid DNA were shown to protect the latter from deoxyribonuclease attack as well as damage from probe sonication. Although niosome stability was composition dependent, overall all formulations were more stable in skeletal muscle extract than in plasma at 37°C. Niosomes were shown to offer significant protection to the incorporated plasmid DNA in the presence of plasma and skeletal muscle extract at 37°C. Tissue distribution with 125I-labelled DNA studies indicated that the plasmid DNA is recovered in the injection site as well as the local lymph nodes. Also expression of a plasmid DNA (pEGFP encoding the enhanced green fluorescent protein) after intramuscular injection occurred in the thigh muscle and local lymph nodes to a greater extent compared to the naked DNA. After immunization with cationic DRV niosomes entrapping the plasmid DNA results indicated significantly greater IgG antibody responses against the encoded antigen compared to naked plasmid DNA. The presence of DOPE was shown to be vital for better immune response. Furthermore, production of endogenous IL-4 and IFN-γ cytokines was also detected in the spleen. The cytokine levels were mainly significantly higher in animals injected with niosomal-DNA compared to those injected with naked DNA. It appears that niosome mediated DNA immunization is effective in inducing both humoral and/or cell-mediated immune response and, therefore, niosomes are promising vehicles for the delivery of DNA vaccines.
Article
Plasmid DNA-based immunization has been shown to be an effective means of vaccination in animal models. In this study, the immune responses to various hepatitis C virus structural protein antigens were evaluated using this technique. Six recombinant plasmids were constructed. These include, individually, the coding regions for the core protein (pC); E1 (pE1) and E2 (pE2); as well as core, E1, and E2 together (pCE1E2); E1 and E2 together (pE1E2); and finally an E2 construct from which the N-terminal hypervariable region had been deleted (pE2 deltaHVR). These plasmids were transfected into mammalian cells to test their protein expression and were injected into the quadriceps muscles of BALB/c mice to measure specific antibodies and cytotoxic T-lymphocyte responses. All the recombinant plasmids were shown to express specific antigens transiently in cells and elicited specific antibody responses to core, E1, and E2 in mice. Specific cytotoxic T lymphocyte responses were detected only in mice injected with plasmid constructs encoding the core. Genetic immunization can aid the development of hepatitis C virus vaccines by allowing for the rapid construction and evaluation of different expression plasmids as potential immunogens.
Article
The direct injection of a naked plasmid DNA vaccine encoding a foreign antigen results in plasmid uptake and protein expression leading to the induction of antigen-specific cellular and humoral immune responses. The ability of DNA vaccine-elicited immune responses to protect against viral and bacterial infections, parasites, cancers, and autoimmune diseases has been well documented in numerous animal models. Phase I human clinical trials have shown that experimental DNA vaccines are safe and well tolerated, however, these preliminary studies indicate that measures must be taken to improve vaccine immunogenicity. One approach to improve the immunogenicity of DNA vaccines is through the co-delivery of cytokine expression plasmids as genetic adjuvants. Studies in a variety of animal models clearly demonstrate that plasmid DNA-encoded immunomodulatory cytokines not only alter the magnitude and direction of the DNA vaccine-elicited immune response, but can also improve vaccine efficacy. These studies suggest that the use of immunomodulatory cytokines with plasmid DNA vaccines may allow clinicians to tailor the resulting immune response to more closely resemble the correlates of protection for a given pathogen.
Chapter
This chapter focuses on hepatitis C virus (HCV), causes viral hepatitis, called non-A, non-B (NANB) hepatitis that spread predominantly via transfusion of blood, blood products, and intravenous drug abuse. The high morbidity and mortality due to HCV infection has provided impetus for ongoing research into biology, epidemiology, pathogenicity, treatment, and prevention of HCV infection. The identification of the infectious agent responsible for NANB hepatitis was made possible only through the use of molecular cloning methods that allowed the isolation and sequencing of a portion of the viral genome. HCV consists of a single-stranded RNA genome of positive polarity surrounded by a nucleocapsid protein core enclosed in a lipid membrane or envelope. The RNA genome is approximately 9400-9500 nucleotides in length. Molecular biology provides several new approaches to the study of HCV replication, thereby providing means to screen and identify novel therapeutic compounds that interfere with the virus life cycle. With the availability of enhanced tests for detecting HCV nucleic acids or proteins directly, there is greater accuracy both in preventing transmission of HCV through the use of blood products, and in determining the activity of HCV in untreated patients and in patients being treated with antiviral therapy. Some recent research suggested that HCV is a curable disease, and that as new therapies and combinations of therapies are utilized, there may be further improvements in the rate of viral clearance.
Article
This review will focus on the prevalence of hepatitis c virus (HCV) infection in alcoholics with and without liver disease. Evidence will be presented to demonstrate that ethanol and chronic HCV infection synergistically accelerate liver injury. Some of the major postulated mechanisms responsible for disease progression include high rates of apoptosis, lipid peroxidation, and generation of free radicals and reactive oxygen species with reduced antioxidant capacity of the liver. Acquisition and persistence of HCV infection may be due to the adverse effects of ethanol on humoral and cellular immune responses to HCV. Dendritic cells (DC) appear to be one of the major targets for ethanol's action and DC dysfunction impairs the ability of the host to generate viral specific cluster of differentiation 4 (CD4+) and cluster of differentiation 8 (CD8+) immune responses. There is a relationship between increased alcohol intake and decreased response to interferon (IFN) therapy, which may be reversed by abstinence. Clinical studies are needed to optimize treatment responses in alcoholic patients with chronic HCV infection.
Article
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A multivalent vaccine candidate against hepatitis B virus (HBV) and hepatitis C virus (HCV) infections was constructed on the basis of HBV core (HBc) virus-like particles (VLPs) as carriers. Chimeric VLPs that carried a virus-neutralizing HBV pre-S1 epitope corresponding to amino acids (aa) 20 to 47 in the major immunodominant region (MIR) and a highly conserved N-terminal HCV core epitope corresponding to aa 1 to 60 at the C terminus of the truncated HBcΔ protein (N-terminal aa 1 to 144 of full-length HBc) were produced in Escherichia coli cells and examined for their antigenicity and immunogenicity. The presence of two different foreign epitopes within the HBc molecule did not interfere with its VLP-forming ability, with the HBV pre-S1 epitope exposed on the surface and the HCV core epitope buried within the VLPs. After immunization of BALB/c mice, specific T-cell activation by both foreign epitopes and a high-titer antibody response against the pre-S1 epitope were found, whereas an antibody response against the HBc carrier was notably suppressed. Both inserted epitopes also induced a specific cytotoxic-T-lymphocyte (CTL) response, as shown by the gamma interferon (IFN-γ) production profile.
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Hepatitis C virus (HCV) is responsible for no less than 71 million people chronically infected and is one of the most frequent indications for liver transplantation worldwide. Despite direct-acting antiviral therapies fuel optimism in controlling HCV infections, there are several obstacles regarding treatment accessibility and reinfection continues to remain a possibility. Indeed, the majority of new HCV infections in developed countries occur in people who inject drugs and are more plausible to get reinfected. To achieve global epidemic control of this virus the development of an effective prophylactic or therapeutic vaccine becomes a must. The coronavirus disease 19 (COVID-19) pandemic led to auspicious vaccine development against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) virus, which has renewed interest on fighting HCV epidemic with vaccination. The aim of this review is to highlight the current situation of HCV vaccine candidates designed to prevent and/or to reduce HCV infectious cases and their complications. We will emphasize on some of the crossroads encountered during vaccine development against this insidious virus, together with some key aspects of HCV immunology which have, so far, hampered the progress in this area. The main focus will be on nucleic acid-based as well as recombinant viral vector-based vaccine candidates as the most novel vaccine approaches, some of which have been recently and successfully employed for SARS-CoV-2 vaccines. Finally, some ideas will be presented on which methods to explore for the design of live-attenuated vaccines against HCV.
Article
Recent studies have raised the possibility that DNA-based vaccination may prove useful for generating virus-specific cytotoxic T-lymphocytes (CTL) responses. Recently, a plasmid containing the human elongation factor 1α(EF1-α) promoter, pEF321, was reported to be a versatile expression vector for gene expression in mammalian cells in vitro. In the present study, we assessed the capability of a novel plasmid, pEFCE1E2, encoding hepatitis C virus (HCV) structural proteins (core, E1 and E2) under the EF1-α promoter to generate CTL against HCV in vivo. BALB/c mice were immunized with the pEFCE1E2 but not with a plasmid possessing the same cDNA under the cytomegalovirus developed HCV-specific effector cells by a single immunization. These effector cells elicited by pEFCE1E2 immunization were CD8+ and major histocompatibility complex class I restricted. These studies provided evidence for the potential utility of the EF1-α promoter for development of DNA vaccines against HCV infections.
Article
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.
Article
We analyzed the humoral immune response elicited by hepatitis C virus (HCV) E2 protein expressed in vivo after injection of plasmid DNA into mice and rhesus macaques. Three plasmids were used for immunization: a plasmid containing the entire sequence of the E2 and p7 genes (pE2); a plasmid encoding a truncated form of the E2 protein targeted to the cell surface (pE2surf); a control plasmid (pDisplay) lacking an HCV insert. Each plasmid was injected intramuscularly into 5 mice and intraepidermally (via gene gun) into 5 mice. Immunization was repeated three times at three week intervals. Five macaques were injected intramuscularly (two with pE2, two with pE2surf and one with pDisplay) and immunization was repeated after 8 weeks. All mice immunized via gene gun with pE2 or pE2surf developed anti-E2. The animals immunized with pE2surf developed an earlier and stronger humoral immune response than those immunized with pE2. Only 2 of the mice injected by the intramuscular route, both immunized with pE2surf, developed detectable anti-E2. One of the two macaques immunized with pE2 and both macaques immunized with pE2surf developed anti-E2; the humoral immune response was much stronger in the animals immunized with pE2surf. Our results suggest that presentation of HCV E2 on the cell surface may increase its immunogenicity while preserving its ability to react with antibodies generated during a natural infection.
Chapter
From 1796, when Edward Jenner infected a young boy with cowpox in hopes of preventing smallpox, to the present day, vaccinations have changed the incidence of many diseases worldwide. The history of immunology actually predates Jenner to ancient China, India and Persia. The observation that recovery from a particular disease rendered people “immune” to a second episode of that same disease, led the Chinese to try to prevent smallpox by exposing uninfected people to pustule samples obtained from smallpox lesions. Jenner then improved the safety of vaccination by using a different, but similar, virus to vaccinate a young boy with cowpox rather than exposing him to the authentic smallpox virus. His observations that milkmaids who had developed cowpox lesions did not develop smallpox led Jenner to “vaccinate” twenty-three other people with cowpox and publish his findings in 1801 (1). This initial experiment serves as an important foundation of modern day immunology, and served to popularize the term “vaccine”, which is derived from the Latin word “vacca”, which means cow. Years later, Louis Pasteur, who had advanced the germ theory of infection, was able to prove that a disease could be prevented by exposure to “weakened” germs, which only caused harmless infections. His theories supported his development of the rabies vaccine.
Article
Hepatitis B virus (HBV) is common in alcoholics and may result in chronic infection. Persistence of HBV infection could be partially caused by the effects of ethanol on the cellular and humoral immune response to viral structural proteins. The DNA-based immunization approach was used to experimentally assess the effects of chronic ethanol feeding on immune responses directed against the middle envelope protein (MHBs) of HBV. Mice were fed an ethanol or isocaloric, pair-fed control liquid diet for 8 weeks, followed by immunization with a plasmid construct containing the pre-S2/S gene that encodes for MHBs. Chronic ethanol consumption marginally reduced the levels of the antibody to hepatitis B surface proteins (anti-HBs) generated by the DNA-based immunization approach. Initially, cytotoxic lymphocyte (CTL) activity was higher in ethanol-fed mice but progressively declined following the second and third immunizations as compared with control mice. In addition, CTL and CD4+ T helper (TH) cells responded poorly to increasing concentrations of envelope protein and peptides in vitro with respect to generation of CTL activity and proliferative responses. Finally, proliferating CD4+ T cells derived from ethanol-fed animals had substantial changes in the levels of cytokines secreted into the culture supernatants as compared with control mice. These studies show that chronic ethanol consumption substantially alters the cellular immune responses to a human viral structural protein, and that these effects may contribute to the persistence of viral infection. (Hepatology 1997 Sep;26(3):764-70)
Therapeutic vaccination is still a controversial subject regarding its potentially beneficial effects. It is often forgotten that therapeutic vaccination has been used for at least 40 years in modern medicine. Doctors treated allergies by administering the allergen by a different route and in a different form than when it causes the allergic reaction. The aim here was to modulate the direction of the host immune response from a ‘proallergic’ histamine releasing and T-helper 2-like response to an ‘antiallergic’ γIFN-producing T-helper 1-like response. Thus, the aim is to use the vaccine to deliver the antigen in a different way to modulate the host immune response. This is exactly the aim with a therapeutic vaccination of a chronic viral infection. A chronic viral infection has, depending on the virus, most probably modulated the host immune response to become less effective against the invading virus, thereby promoting viral persistence. Different viruses use different strategies to do this, and consequently, the therapeutic vaccine should be designed to remedy, correct, or at least partially modulate the host immune response in a more antiviral direction. There are no examples yet in human infections where a therapeutic vaccine has been found to undisputedly mediate a clearance of the infection. Numerous reports have suggested that the therapeutic vaccination did prime or reactivate immune responses, which may be regarded as more antiviral including γIFN-producing CD4+ T-helper cells and cytotoxic T lymphocytes. However, substantial hurdles need to be overcome before we have several therapeutically active vaccines, meaning that they indeed affect the disease progression and are commonly used in the clinic. These difficulties and problems are discussed in the present review.Section editor:Charles Craig – Clinical Virology Specialist
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Purpose Dry eye condition is an extrahepatic manifestation associated with chronic hepatitis C virus (HCV) infection. Since conjunctival inflammation can contribute to the dry eye condition, in the present study we analyzed the conjunctival inflammatory response to HCV core and NS3 proteins. Methods We used primary human conjunctival fibroblasts for our study. Cytokines were measured with enzyme-linked immunosorbent assay (ELISA). Toll-like receptor (TLR) and cell adhesion molecule gene expression patterns were analyzed with semiquantitative reverse transcription (RT)–PCR. Immunofluorescence staining was performed for the MyD88, nuclear factor-kappa B (NF-kB), and inducible nitric oxide synthase (iNOS) proteins. Nitric oxide (NO) was measured with the Griess assay; terminal deoxynucleotidyl transferase-mediated uridine 5′-triphosphate-biotin nick end labeling (TUNEL) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays were performed for apoptosis and cell viability, respectively. Results When exposed to the HCV core and NS3 proteins, the conjunctival fibroblasts secreted interleukin-8 (IL-8), IL-6, tumor necrosis factor-alpha (TNF-α), and IL-10 in a dose-dependent manner. Various TLRs were involved in the innate immune response via MyD88 signaling without NF-kB involvement. The gene expression of cell adhesion molecules such as CD44 and ICAM-1 was upregulated, and the cells secreted NO via iNOS. As the sum of these stress responses, the cells underwent apoptosis, which eventually lead to cell death. Conclusions HCV core and NS3 proteins induced conjunctival inflammation that may form the pathogenesis of dry eye condition.
Article
Chronic infections with hepatitis B (HBV) and hepatitis C (HCV) virus are worldwide problems often leading to the development of chronic liver disease and hepatocellular carcinoma. Genetic immunizations with DNA encoding for structural and nonstructural proteins of HCV and HBV in experimental mice generate a broad base CD4+ and CD8+ cellular immune response which may be required for viral clearance from the host. DNA based immunization is a promising antiviral approach for the development of therapeutic and prophylactic vaccine against HBV and HCV.
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The mechanism of initiation of translation on hepatitis C virus (HCV) RNA was investigated in vitro. HCV RNA was transcribed from the cDNA that corresponded to nucleotide positions 9 to 1772 of the genome by using phage T7 RNA polymerase. Both capped and uncapped RNAs thus transcribed were active as mRNAs in a cell-free protein synthesis system with lysates prepared from HeLa S3 cells or rabbit reticulocytes, and the translation products were detected by anti-gp35 antibodies. The data indicate that protein synthesis starts at the fourth AUG, which was the initiator AUG at position 333 of the HCV RNA used in this study. Efficiency of translation of the capped methylated RNA appeared to be similar to that of the capped unmethylated RNA. However, a capped methylated RNA showed a much higher activity as mRNA than did the capped unmethylated RNA in rabbit reticulocyte lysates when the RNA lacked a nucleotide sequence upstream of position 267. The results strongly suggest that HCV RNA carries an internal ribosome entry site (IRES). Artificial mono- and dicistronic mRNAs were prepared and used to identify the region that carried the IRES. The results indicate that the sequence between nucleotide positions 101 and 332 in the 5' untranslated region of HCV RNA plays an important role in efficient translation. Our data suggest that the IRES resides in this region of the RNA. Furthermore, an IRES in the group II HCV RNA was found to be more efficient than that in the group I HCV RNA.
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E2/nonstructural protein 1, the putative envelope glycoprotein (gp72) of HCV, possesses an N-terminal hypervariable (E2 HV) domain from amino acids 384 to 414 of unknown significance. The high degree of amino acid sequence variation in the E2 HV domain appears to be comparable to that observed in the human immunodeficiency virus type 1 gp120 V3 domain. This observation and the observation that the HCV E2 HV domain lacks conserved secondary structure imply that, like the V3 loop of human immunodeficiency virus 1 gp120, the N-terminal E2 region may encode protective epitopes that are subject to immune selection. Antibody-epitope binding studies revealed five isolate-specific linear epitopes located in the E2 HV region. These results suggest that the E2 HV domain is a target for the human immune response and that, in addition to the three major groups of HCV, defined by nucleotide and amino acid sequence identity among HCV isolates, E2 HV-specific subgroups also exist. Analysis of the partial or complete E2 sequences of two individuals indicated that E2 HV variants can either coexist simultaneously in a single individual or that a particular variant may predominate during different episodes of disease. In the latter situation, we found one individual who developed antibodies to a subregion of the E2 HV domain (amino acids 396-407) specific to a variant that was predominant during one major episode of hepatitis but who lacked detectable antibodies to the corresponding region of a second variant that was predominant during a later episode of disease. The data suggest that the variability in the E2 HV domain may result from immune selection. The findings of this report could impact vaccine strategies and drug therapy programs designed to control and eliminate HCV.
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We have determined the nucleotide sequence of the 5' noncoding (NC) region of the hepatitis C virus (HCV) genome in 44 isolates from around the world. We have identified several HCV isolates with significantly greater sequence heterogeneity than reported previously within the 5' NC region. The most distantly related isolates were only 90.1% identical. Nucleotide insertions were seen in three isolates. Analysis of the nucleotide sequence from 44 HCV isolates in this study combined with that of 37 isolates reported in the literature reveals that the 5' NC region of HCV consists of highly conserved domains interspersed with variable domains. The consensus sequence was identical to the prototype HCV sequence. Nucleotide variations were found in 45 (16%) of the 282 nucleotide positions analyzed and were primarily located in three domains of significant heterogeneity (positions -239 to -222, -167 to -118, and -100 to -72). Conversely, there were three highly conserved domains consisting of 18, 22, and 63 completely invariant nucleotides (positions -263 to -246, -199 to -178, and -65 to -3, respectively). Two nucleotide domains within the 5' NC region, conserved among all HCV isolates studied to date, shared statistically significant similarity with pestivirus 5' NC sequences, providing further evidence for a close evolutionary relationship between these two groups of viruses. Additional analysis revealed the presence of short open reading frames in all HCV isolates. Our sequence analysis of the 5' NC region of the HCV genome provides additional information about conserved elements within this region and suggests a possible functional role for the region in viral replication or gene expression. These data also have implications for selection of optimal primer sequences for the detection of HCV RNA by the PCR assay.
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Genomic RNA from the human prototype strain H of the hepatitis C virus (HCV-H) has been molecularly cloned and sequenced. The HCV-H sequence reported consists of 9416 nucleotides including the 5' and 3' untranslated regions. HCV-H shows 96% amino acid identity with the American isolate HCV-1 but only 84.9% with the Japanese isolates HCV-J and HCV-BK. In addition to the hypervariable region (region V) previously identified in the putative E2 domain, three other variable domains were identified: region V1 (putative E1), region V2 (putative E2), and region V3 (putative NS5). These regions appear rather conserved (86-100%) among the American isolates (HCV-1 and HC-J1) or among various Japanese isolates (HCV-J, HCV-BK, HCV-JH, and HC-J4) but show striking heterogeneity when the two subgroups are compared (42-87.5% amino acid difference). A structural similarity between the 5'-terminal hairpin structure of HCV and of poliovirus was observed. This study further suggests the existence of at least two genomic subtypes of HCV and confirms a distant relationship between HCV and pestiviruses.
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We have determined the nucleotide sequence at the extreme 5' and 3' termini of the hepatitis C virus (HCV) genome. Our analyses of these sequences show (i) the nucleotide sequence in the 5' untranslated region is highly conserved among HCV isolates of widely varying geographical origin, (ii) within this region, there are blocks of nucleotide sequence homology with pestiviruses but not with other viruses, (iii) the relative position of short open reading frames present in the same region of the HCV genome is similar to that of the pestiviral genome, (iv) RNAs truncated at the 5' and 3' ends are found, but the origin and functions of these RNAs are unknown, and (v) poly(A) tails appear to be present on 3' subgenomic RNAs. These data differentiate HCV from the flaviviruses and indicate a closer evolutionary relationship of HCV with the pestiviruses. However, HCV also appears to be substantially different from other known pestiviruses. These data are consistent with the assignment of HCV to a separate viral genus.
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Hepatitis C virus (HCV) is a major causative agent of parenterally transmitted non-A, non-B hepatitis. The genomic region encoding the virion-associated core protein is relatively conserved among HCV strains. To generate a DNA vaccine capable of expressing the HCV core protein, the genomic region encoding amino acid residues 1 to 191 of the HCV-1 strain was amplified and cloned into an eukaryotic expression vector. Intramuscular inoculation of recombinant plasmid DNA into BALB/c mice (H-2d) generated core-specific antibody responses, lymphoproliferative responses, and cytotoxic T-lymphocyte activity. Our results suggest that the HCV core polynucleotide warrants further investigation as a potential vaccine against HCV infection.
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Vectors expressing the first 58 amino acids of the hepatitis C virus (HCV) nucleocapsid alone or as a fusion protein with the middle (pre-S2 and S) or major (S) surface antigens of hepatitis B virus (HBV) were constructed. Intramuscular immunization of BALB/c mice with the chimeric constructs in the form of naked DNA elicited humoral responses to antigens from both viruses within 2 to 6 weeks postinjection. No anti-HCV responses were obtained in mice immunized with the vector expressing the HCV sequence in the nonfusion context. Sera from chimera-injected mice specifically recognized both HCV capsid and HBV surface antigens in enzyme-linked immunosorbent assay and immunoblot testing. Anti-HCV serum titers formed plateaus of approximately 1:3,000; these remained stable until the end of the study (18 weeks postinfection). Anti-HBV immune responses were found to be lower in the chimera-injected animals (< 200 mIU/ml) than in those immunized with the native HBV vector (> 2,000 mIU/ml). This is the first report of the use of DNA-based immunization for the generation of immune responses to an HCV protein. In addition, these findings show that it is possible to elicit responses to viral epitopes from two distinct viruses via DNA immunization with chimeric vectors.
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Hepatitis C virus (HCV) is a major cause of posttransfusion and community-acquired hepatitis, and a majority of individuals infected with this virus will subsequently develop chronic hepatitis. Characterization of the host immune response to this infection is an important first step that should facilitate the development of immunomodulatory agents and vaccines. Cellular immune responses, especially those mediated by cytotoxic T lymphocytes (CTL), are important in the control of many viral diseases. In this study, liver-infiltrating lymphocytes from persons with chronic HCV hepatitis were examined for evidence of HCV-specific CTL by using target cells infected with recombinant vaccinia viruses expressing the HCV core, E1, E2, and part of the NS2 proteins. Bulk expansion of liver-derived CD8+ lymphocytes resulted in the detection of HCV-specific CTL activity, whereas activity could not be found in CD8+ lymphocytes expanded from peripheral blood. Epitopes recognized by these CTL were defined by using CTL clones obtained by limiting dilution and target cells sensitized with synthetic HCV peptides. Four distinct HLA class I-restricted epitopes were identified, including two epitopes in the amino-terminal portion of the core protein. These studies provide evidence that the highly conserved core protein is a target for HCV-specific CTL and identify CTL epitopes within the more highly variable E2 envelope protein. Our studies also suggest that HCV-specific CTL are localized at the site of tissue injury in infected persons with chronic hepatitis. Identification of the epitopes recognized by HCV-specific CTL will facilitate exploration of their role in disease pathogenesis and may provide information useful in development of therapeutic interventions or vaccines.
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The HLA class I-restricted cytotoxic T lymphocyte (CTL) response is a major defense mechanism in viral infections. It has been suggested that the CTL response may contribute to viral clearance and liver cell injury during hepatitis C virus (HCV) infection. To test this hypothesis requires an understanding of the characteristics of HCV-specific cytotoxic effector cells and identification of the target antigens to which they respond. To begin this process we stimulated peripheral blood mononuclear cells (PBMC) from a group of HLA-A2 positive patients with chronic hepatitis C with a panel of 130 HCV-derived peptides containing the HLA-A2 binding motif. Effector cells were tested for their capacity to lyse HLA-A2-matched target cells that were either sensitized with peptide or infected with a vaccinia virus construct containing HCV sequences. Using this approach we have identified nine immunogenic peptides in HCV, three of which are derived from the putative core protein, three from the nonstructural (NS) 3 domain, two from NS4 and one from NS5. Selected responses were shown to be HLA-A2 restricted, mediated by CD8+ T cells and to recognize endogenously synthesized viral antigen. Unexpectedly, peptide-specific CTL responses could also be induced in sero-negative individuals, suggesting in vitro activation of naive CTL precursors. The precursor frequency of peptide-specific CTL was 10 to 100-fold higher in infected patients compared to uninfected controls, and the responses were greatly diminished by removal of CD45 RO+ (memory) T cells. Further quantitative studies are clearly required to establish whether a correlation exists between the HCV-specific CTL response and the clinical course of this disease. Definition of the molecular targets of the human CTL response to HCV creates this opportunity, and may also contribute to the development of a T cell-based HCV vaccine.
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This study shows that DNA vaccination can confer protection against a persistent viral infection by priming CD8+ cytotoxic T lymphocytes (CTL). Adult BALB/c (H-2d) mice were injected intramuscularly with a plasmid expressing the nucleoprotein (NP) gene of lymphocytic choriomeningitis virus (LCMV) under the control of the cytomegalovirus promoter. The LCMV NP contains the immunodominant CTL epitope (amino acids 118 to 126) recognized by mice of the H-2d haplotype. After three injections with 200 micrograms of NP DNA, the vaccinated mice were challenged with LCMV variants (clones 13 and 28b) that establish persistent infection in naive adult mice. Fifty percent of the DNA-vaccinated mice were protected, as evidenced by decreased levels of infectious virus in the blood and tissues, eventual clearance of viral antigen from all organs tested, the presence of an enhanced LCMV-specific CD8+ CTL response, and maintenance of memory CTL after clearance of virus infection. However, it should be noted that protection was seen in only half of the vaccinated mice, and we were unable to directly measure virus-specific immune responses in any of the DNA-vaccinated mice prior to LCMV challenge. Thus, at least in the system that we have used, gene immunization was a suboptimal method of inducing protective immunity and was several orders of magnitude less efficient than vaccination with live virus. In conclusion, our results show that DNA immunization works against a persistent viral infection but that efforts should be directed towards improving this novel method of vaccination.
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Genetic immunization is a simple method for producing polyclonal antibodies in mice. To test if this approach could be used for monoclonal antibody production, biolistic transfection was used to immunize a mouse. High levels of polyclonal antibodies against human growth hormone (hGH) were elicited following three inoculations with the gene for hGH. When hybridoma cells were created from the mouse's splenocytes, approximately 17% secreted antibodies vs. hGH. Of these, some recognized only native or denatured hGH, while most recognized both forms of the protein. These findings demonstrate the utility of genetic immunization as a method to produce monoclonal antibodies.
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The effect of sense and antisense oligodeoxynucleotides (ODNs) on hepatitis C virus (HCV) gene expression was studied to determine the role of the highly conserved 5'-untranslated region in the life cycle of the virus. It was found that antisense ODNs complementary to nucleotides (nt) 38-65, 134-175, and 312-339 in the 5' noncoding region and 341-377 in the core open reading frame efficiently blocked HCV RNA translation. Overlapping ODNs that differed by only several nucleotides showed substantially different inhibition of HCV RNA translation. Fine sequence specificity testing at nt positions 351-377 revealed that ODNs as small as a 12-mer (nt 351-363) retained a high degree (80%) of inhibitory activity compared to ODNs of longer sequences. These results suggest that there are three highly specific domains in the 5' noncoding region and a sequence immediately downstream of the HCV core initiation codon that may be critical for translation of HCV RNA. This study also provides an experimental approach for the selection of target HCV RNA sequences susceptible to antisense effects, as well as for definition of functional regions of the genome necessary for viral replication.
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We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
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Evidence suggests that cellular immunity to hepatitis C virus (HCV) core protein may be important in the pathogenesis of viral infection. Therefore, interferon gamma (IFN-γ) production by peripheral blood mononuclear cells (PBMC) derived from patients with chronic HCV infection (genotype lb) was examined. The cellular immune response was evaluated with a recombinant HCV core fusion protein derived from a patient with genotype lb. To identify the immunodominant epitopes, IFN-γ production in responders was also assessed with a panel of nine synthetic peptides that covered the entire core region. It was found that mononuclear cells from 24 (52%) of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate (14%, P < .05). More important, individuals who had received IFN-α treatment and went into clinical and virological remission had a higher response rate (75%, P < .05) compared with those with ongoing hepatitis whose treatment failed (31%). Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 141 to 160 was recognized in 9 patients. Interestingly, 7 of 8 patients bearing HLA DR 4 and w53 haplotypes recognized the peptide sequence 141 to 160. Thus, IFN-γ production of the mononuclear cell response appeared to be HLA DR restricted, and the responding cells were identified as CD4+ T cells. This study suggests the presence of immunodominant T cell epitopes within the HCV core protein in association with HLA DR phenotypes in patients with HCV-associated liver disease.
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Twenty-nine patients with chronic hepatitis C and 15 asymptomatic hepatitis C virus antibody–positive subjects who clinically recovered from hepatitis C virus infection were studied for their peripheral blood lymphomononuclear cell proliferative response to hepatitis C virus structural and nonstructural antigens (core, envelope, nonstructural 4 and nonstructural 5) expressed in yeast as superoxide dismutase fusion proteins, in an initial attempt to define some of the features of the virus-specific immune response. Hepatitis C virus core was the most immunogenic antigen for human leukocyte antigen class II–restricted T cells in both groups of patients studied, and the proliferative response to it was the most vigorous and the most frequently expressed in comparison with the other antigens tested. The specificity of the results was supported by the lack of response to hepatitis C virus antigens by healthy uninfected controls and confirmed by recognition of recombinant core proteins of different origin (yeast and baculovirus) by polyclonal T-cell lines produced by T-cell stimulation with yeast-derived core. Each of the antigens tested was able to induce significant although variable levels of proliferative response, indicating that all can be immunogenic at the T-cell level. Significant proliferative responses to core, nonstructural 4 and nonstructural 5 antigens were more frequently detected in subjects who were able to eradicate infection than in patients with chronic hepatitis C, although the difference was statistically not significant. No difference was observed between the two groups of patients with respect to the response to the putative envelope antigens. (Hepatology 1994;19:286–295).
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Cytotoxic T lymphocytes have been reported to be involved in the immune clearance of virus-infected cells and in the pathogenesis of viral infection. We studied the cytotoxic T lymphocyte response to the putative nucleocapsid protein of hepatitis C virus in patients with chronic hepatitis C. Cytotoxic T lymphocytes specific for hepatitis C virus nucleocapsid protein were generated from peripheral blood lymphocytes by means of repeated stimulation with a synthetic hepatitis C virus nucleocapsid protein peptide. The cytotoxic T lymphocytes were CD8 positive and recognized an epitope in hepatitis C virus nucleocapsid protein residues 81 to 100 in association with a human leukocyte antigen class I molecule, B44. The peptideinduced cytotoxic T lymphocytes recognized target cells synthesizing hepatitis C virus nucleocapsid protein endogenously, though less efficiently than peptide-pulsed target cells. The human leukocyte antigen B44–restricted cytotoxic T lymphocyte response was observed in three of five patients with chronic hepatitis C and a human leukocyte antigen B44 molecule but in neither of two hepatitis C virus–negative healthy individuals with human leukocyte antigen B44 molecules. The results demonstrate the presence of hepatitis C virus–specific cytotoxic T lymphocytes in the peripheral blood of patients with chronic hepatitis C and provide a strategy to study the role of cytotoxic T lymphocytes in the viral clearance and the pathogenesis of hepatitis C virus infection. (HEPATOLOGY 1993;18:1039-1044).
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Interferon-α therapy is of proven efficacy in chronic hepatitis C, but it is not universally effective and may be associated with intolerable side effects. Ribavirin is a nucleoside analog with a broad spectrum of antiviral action. We conducted an uncontrolled pilot study of ribavirin therapy in 13 patients with chronic hepatitis C. Ribavirin was given for 6 mo, in a dose that was increased, at 2-mo intervals, from 600 mg to 1,000 mg to 1,200 mg/day. Serum ALT levels gradually decreased in all 13 treated patients; the mean percentage of decrease was 67% (from 210 U/L [range = 109 to 593] to 63 U/L [range = 22 to 108 U/L]; p = 0.0006) after 6 mo of treatment. Serum aminotransferase levels fell to the normal range in four patients (31%). In the 3 to 6 mo after cessation of ribavirin therapy, serum aminotransferase activities gradually rose to near pretreatment levels in all but one patient. Therapy was associated with a significant decrease in the geometric mean titer of hepatitis C virus RNA in serum (1:1,981 vs. 1:199; p < 0.02) although no patients lost hepatitis C virus RNA from serum during therapy. No significant improvement was seen in liver histological appearance. Ribavirin therapy resulted in mild, reversible hemolysis; no patient exhibited symptomatic anemia. These findings suggest that ribavirin has a beneficial effect in patients with chronic hepatitis C, although further studies are needed to determine how ribavirin is best used. (HEPATOLOGY 1992;16:649–654.)
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Outcomes for patients with hepatocellular carcinoma (HCC) without cirrhosis and factors associated with disease progression remain unclear. The goals of this single-institution study were to define the outcomes for such patients, and to determine factors associated with survival and disease progression. This was a retrospective review of consecutive patients with HCC without cirrhosis who underwent hepatic resection between 1985 and 2003. Survival was estimated by the Kaplan-Meier method and risk factors were identified by Cox proportional hazards models. A total of 143 patients were enrolled, of whom 29·4 per cent had identifiable risk factors for chronic liver disease. Major resection (at least three segments) was undertaken in 63·6 per cent of patients. The operative mortality rate was 3·5 per cent. Median disease-free survival was 2·4 years. Multivariable analysis revealed presence of multiple tumours as the only independent predictor of tumour recurrence. Median overall survival was 3·3 years. Factors independently associated with decreased overall survival were multiple tumours, high histological grade, perioperative transfusion, male sex and age at least 66 years. Patients with HCC but without cirrhosis have acceptable outcomes after resection. Specific risk factors for the development of HCC in these patients have yet to be defined.
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Chronic liver disease develops in more than half of patients with post-transfusion hepatitis C, but little is known about the natural history of community-acquired hepatitis C. In 1985 and 1986 we identified adults with acute non-A, non-B hepatitis in four counties in the United States and followed them prospectively. We used three markers to detect hepatitis C virus (HCV) infection in stored samples of serum: antibody to HCV (anti-HCV) detected by second-generation serologic assays; HCV RNA detected by polymerase-chain-reaction assay; and antibody to HCV antigen (anti-HCVAg) detected by fluorescent-antibody-blocking assay. Of 130 patients with non-A, non-B hepatitis, 106 (82 percent) had HCV infection, 93 were positive for anti-HCV, and 13 were positive only for HCV RNA or anti-HCVAg. Chronic hepatitis developed in 60 (62 percent) of 97 HCV-infected patients followed for 9 to 48 months, with no relation to the risk factors for infection. Ten of the 30 patients who had liver biopsies had chronic active hepatitis. In samples collected 42 to 48 months after the onset of hepatitis, HCV RNA was detected in 12 of 13 tested patients with chronic hepatitis and in all 15 tested patients with hepatitis that had resolved. Anti-HCV persisted in all but two of the initially positive patients, for a rate of antibody loss of 0.6 per 100 person-years. Patients with community-acquired hepatitis C have a high rate of chronic hepatitis. HCV may be a major cause of chronic liver disease in the United States, and in most patients HCV infection seems to persist for at least several years, even in the absence of active liver disease.
Article
To determine whether chronic hepatitis C virus (HCV) infection is an independent risk factor for hepatocellular carcinoma and whether it increases the cirrhosis-related risk for hepatocellular carcinoma. Two pair-matched case-control studies. A referral-based hospital. In study I, 212 patients with hepatocellular carcinoma (197 of whom had known underlying cirrhosis) were compared with controls who had chronic nonhepatic diseases. In study II, the 197 patients with hepatocellular carcinoma and cirrhosis were compared with 197 pair-matched controls who had cirrhosis but not hepatocellular carcinoma. Levels of antibody to HCV (anti-HCV), hepatitis B surface antigen (HBsAg), and antibody to hepatitis B core antigen (anti-HBc) were assayed, and alcohol abuse was assessed by history. In study I, 151 patients (71%) with hepatocellular carcinoma were anti-HCV positive compared with 11 controls (5%) with chronic nonhepatic diseases (odds ratio, 42; 95% CI, 22 to 95). Multivariate analysis showed that anti-HCV was an independent risk factor for hepatocellular carcinoma (odds ratio, 69; CI, 15 to 308). The analysis also showed that HBsAg (odds ratio, 8.7; CI, 1.5 to 50) and anti-HBc (odds ratio, 4.2 (CI, 1.7 to 11) were risk factors for hepatocellular carcinoma. No statistically significant interaction was found between anti-HCV and the markers of HBV infection. In study II, 146 patients (74%) with hepatocellular carcinoma and cirrhosis were anti-HCV positive compared with 122 patients (62%) with cirrhosis alone (odds ratio, 1.8; CI, 1.1 to 2.8). Multivariate analysis confirmed that anti-HCV (odds ratio, 2.0; CI, 1.3 to 32) and HBsAg (odds ratio, 2.0; CI, 1.0 to 4.2) were independent risk factors for hepatocellular carcinoma. Hepatitis C virus infection is a risk factor for hepatocellular carcinoma, apparently by inducing cirrhosis and, to a lesser extent, by enhancing the risk in patients with cirrhosis. Hepatitis C virus infection acts independently of HBV infection (another risk factor) and of alcohol abuse, age, or gender.
Article
The RNA genomes of human hepatitis C virus (HCV) and the animal pestiviruses responsible for bovine viral diarrhea (BVDV) and hog cholera (HChV) have relatively lengthy 5′ nontranslated regions (5′NTRs) sharing short segments of conserved primary nucleotlde sequence. The functions of these 5′NTRs are poorly understood. By comparative sequence analysis and thermodynamic modeling of the 5′NTRs of multiple BVDV and HChV strains, we developed models of the secondary structures of these RNAs. These pestiviral 5′NTRs are highly conserved structurally, despite substantial differences in their primary nucleotlde sequences. The assignment of similar structures to conserved segments of primary nucleotide sequence present in the 5′NTR of HCV resulted in a model of the secondary structure of the HCV 5′NTR which was refined by determining sites at which synthetic HCV RNA was cleaved by double- and single-strand specific RNases. These studies indicate the existence of a large conserved stem-loop structure within the 3′ 200 bases of the 5′NTRs of both HCV and pestiviruses which corresponds to the ribosomal landing pad (Internal ribosomal entry site) of HCV. This structure shows little relatedness to the ribosomal landing pad of hepatitis A virus, suggesting that these functionally similar structures may have evolved independently.
Article
We previously identified two hypervariable regions [HVR1 (27 amino acids) and HVR2 (7 amino acids)] in the putative envelope glycoprotein (gp70) by comparison of the amino acid sequences of many isolates of the HCV-II genotype. To understand the functional features of these HVRs, using the polymerase chain reaction we analyzed the rate of actual sequence variability in the region including HVR1 and HVR2 of HCV isolated successively at intervals of several months from two patients with chronic C-type hepatitis. In both patients, the amino acid sequence of HVR1, but not HVR2, was found to change dramatically during the observation period (about one amino acid per month). However, no alteration of the amino acid sequence of HVR1 of HCV was observed in a patient in the acute phase of chronic hepatitis. Restriction digestion analysis of sequence diversity showed that a HCV genome with a newly introduced mutation in HVR1 often became the predominant population at the next time of examination. Alterations of amino acids in HVR1 occurred sequentially in the two patients in the chronic phase. These findings suggest that mutations in HVR1 are involved in the mechanism of persistent chronic HCV infection.
Article
Hepatitis C virus (HCV) is a major cause of post-transfusion and sporadic hepatitis worldwide, leading to chronic liver disease in at least 50% of infected individuals. The pathogenic mechanisms that result in chronic hepatitis are unknown. Lymphocytes are typically observed within the hepatic parenchyma, but the functional characteristics of these cells have not been defined. In this study, liver-infiltrating lymphocytes from two subjects with chronic HCV hepatitis were cloned at limiting dilution and tested for HCV-specific cytolytic activity using autologous target cells infected with vaccinia viruses expressing recombinant HCV Ag or sensitized with synthetic HCV peptides. In both subjects, HCV-specific, HLA class I-restricted CTL were identified that recognized epitopes in variable regions of either the envelope or nonstructural proteins. These results demonstrate the presence of HCV-specific CTL at the site of tissue damage in persons with chronic HCV hepatitis, and provide a means to evaluate the possible pathogenic role of these cells in HCV infection.
Article
Interferon-alpha therapy is of proven efficacy in chronic hepatitis C, but it is not universally effective and may be associated with intolerable side effects. Ribavirin is a nucleoside analog with a broad spectrum of antiviral action. We conducted an uncontrolled pilot study of ribavirin therapy in 13 patients with chronic hepatitis C. Ribavirin was given for 6 mo, in a dose that was increased, at 2-mo intervals, from 600 mg to 1,000 mg to 1,200 mg/day. Serum ALT levels gradually decreased in all 13 treated patients; the mean percentage of decrease was 67% (from 210 U/L [range = 109 to 593] to 63 U/L [range = 22 to 108 U/L]; p = 0.0006) after 6 mo of treatment. Serum aminotransferase levels fell to the normal range in four patients (31%). In the 3 to 6 mo after cessation of ribavirin therapy, serum aminotransferase activities gradually rose to near pretreatment levels in all but one patient. Therapy was associated with a significant decrease in the geometric mean titer of hepatitis C virus RNA in serum (1:1,981 vs. 1:199; p less than 0.02) although no patients lost hepatitis C virus RNA from serum during therapy. No significant improvement was seen in liver histological appearance. Ribavirin therapy resulted in mild, reversible hemolysis; no patient exhibited symptomatic anemia. These findings suggest that ribavirin has a beneficial effect in patients with chronic hepatitis C, although further studies are needed to determine how ribavirin is best used.
Article
To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.
Article
The complete nucleotide sequence of a hepatitis C virus derived from plasma of a human carrier in Japan was determined. The cDNA of the isolate (HC-J6) contained 9481 nucleotides and an additional T stretch of 30 to 108 nucleotides at the 3' end, and had one large open reading frame coding for a polyprotein of 3033 amino acids. It differed by 31.8 to 32.1% in the nucleotide sequence and by 27.4 to 27.7% in the amino acid sequence from an American isolate and two Japanese isolates previously reported. Among these four isolates, the 5' non-coding region of 329 to 341 nucleotides was well conserved (greater than 93% identity), whereas the 3' non-coding region of 39 to 45 nucleotides (T stretches not included) was more variable (greater than 30% identity). An excellent degree of conservation of the 5' non-coding region would reflect its pivotal role in replication, and primers deduced from this region could be applied for the sensitive and specific detection of viral RNA by polymerase chain reaction. Due to a high degree of similarity in the amino acid sequence of the putative core protein (greater than 90%), antigen probes deduced from it would be suitable for the serological diagnosis of HCV infection. Low sequence similarity in the putative envelope protein (greater than 53% identity), however, would have to be taken into account in considering the immunoprophylaxis of HCV infection.
Article
Hepatitis C virus (HCV) is a major causative agent of posttransfusion non-A, non-B hepatitis, which often develops into malignant chronic diseases, including liver cirrhosis and hepatocellular carcinoma. We have cloned from human carriers overlapping cDNAs (9,416 bp) covering the entire coding region of the HCV genome. The latter encodes a 3,010-amino-acid polyprotein. In addition, there are 332 and 54 bases of 5' and 3' noncoding sequences, respectively. Our HCV strain has a 77% nucleic acid identity to the HCV strain cloned by workers at Chiron Corporation. The hydrophobicity profile of the putative polyprotein is similar to those of flaviviruses, but it has limited amino acid homology to polyproteins of flaviviruses and other viruses, indicating that HCV is at most distantly related to flaviviruses.
Article
The nucleotide sequence of the RNA genome of the human hepatitis C virus (HCV) has been determined from overlapping cDNA clones. The sequence (9379 nucleotides) has a single large open reading frame that could encode a viral polyprotein precursor of 3011 amino acids. While there as little overall amino acid and nucleotide sequence homology with other viruses, the 5' HCV nucleotide sequence upstream of this large open reading frame has substantial similarity to the 5' termini of pestiviral genomes. The polyprotein also has significant sequence similarity to helicases encoded by animal pestiviruses, plant potyviruses, and human flaviviruses, and it contains sequence motifs widely conserved among viral replicases and trypsin-like proteases. A basic, presumed nucleocapsid domain is located at the N terminus upstream of a region containing numerous potential N-linked glycosylation sites. These HCV domains are located in the same relative position as observed in the pestiviruses and flaviviruses and the hydrophobic profiles of all three viral polyproteins are similar. These combined data indicate that HCV is an unusual virus that is most related to the pestiviruses. Significant genome diversity is apparent within the putative 5' structural gene region of different HCV isolates, suggesting the presence of closely related but distinct viral genotypes.
Article
A possible causative role for the recently discovered hepatitis C virus (HCV) in the development of hepatocellular carcinoma (HCC) was investigated by assay of sera from HCC patients in Japan for antibodies to a recombinant HCV antigen and to hepatitis B virus (HBV) antigens. Among the 253 HCC patients examined, 156 (61.7%) had no serum markers of either a previous or a current HBV infection (group I), 46 (18.2%) were negative for HBV surface antigen but positive for anti-HBV surface and/or anti-HBV core antibody, indicating the occurrence of a previous, transient HBV infection (group II), and 51 (20.2%) were chronically infected HBV carriers as evidenced by positivity for HBV surface antigen (group III). The prevalence of HCV antibody in group I (68.6%) and II (58.7%) patients was significantly higher than for group III (3.9%) or in 148 additional patients with other (non-HCC) cancers (10.1%) (P less than 0.01). Thus, there appears to be a strong association between HCV infection and the development of HCC, particularly in patients for which HBV infection cannot be implicated as a causative factor. The data also suggest an additional mode of transmission for HCV other than blood transfusion, since a history of blood transfusion was shown in only about 30% of the HCV antibody-positive HCC patients in groups I and II. A high prevalence of HCV antibody was also shown among patients with HCC whose disease was originally thought to be due to very high ethanol consumption.
Article
The nucleotide sequence of the Japanese type of hepatitis C virus (HCV-J) genome, consisting of 9413 nucleotides, was determined by analyses of cDNA clones from plasma specimens from Japanese patients with chronic hepatitis. HCV-J genome contains a long open reading frame that can encode a sequence of 3010 amino acid residues. Comparison of HCV-J with the American isolate of HCV showed 22.6% difference in nucleotide sequence and 15.1% difference in amino acid sequence. Thus HCV-J and the American isolate of HCV are probably different subtypes of HCV. The relationship of HCV-J with other animal RNA virus families and the putative organization of the HCV-J genome are discussed.
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A sensitive radioimmunoassay was used to detect antibodies to hepatitis C virus (HCV) in patients with hepatocellular carcinoma and chronic hepatitis. HCV antibodies (anti-HCV) were detected in 86 of 132 patients with hepatocellular carcinoma with no relation to the presence or absence of hepatitis B surface antigen (HBsAg). The prevalence of anti-HCV was also high in patients with diseases thought to predispose to hepatocellular carcinoma, such as non-A, non-B chronic hepatitis and cirrhosis (74%). In HBsAg-negative patients with hepatocellular carcinoma the prevalence of anti-HCV was lower than that in patients with non-A, non-B chronic hepatitis (16% vs 55%); the prevalence of serum antibodies to hepatitis B core antigen (anti-HBc), a marker of hepatitis B virus infection, was 70% and 28%, respectively. In HBsAg-negative patients with hepatocellular carcinoma, anti-HCV and anti-HBc occurred together nearly three times as often as in patients with chronic hepatitis (54% vs 19%). These data indicate that, in Italy, HCV is an important factor associated with hepatocellular carcinoma and non-A, non-B chronic hepatitis.
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We describe a simple calcium phosphate transfection protocol and neo marker vectors that achieve highly efficient transformation of mammalian cells. In this protocol, the calcium phosphate-DNA complex is formed gradually in the medium during incubation with cells and precipitates on the cells. The crucial factors for obtaining efficient transformation are the pH (6.95) of the buffer used for the calcium phosphate precipitation, the CO2 level (3%) during the incubation of the DNA with the cells, and the amount (20 to 30 micrograms) and the form (circular) of DNA. In sharp contrast to the results with circular DNA, linear DNA is almost inactive. Under these conditions, 50% of mouse L(A9) cells can be stably transformed with pcDneo, a simian virus 40-based neo (neomycin resistance) marker vector. The NIH3T3, C127, CV1, BHK, CHO, and HeLa cell lines were transformed at efficiencies of 10 to 50% with this vector and the neo marker-incorporated pcD vectors that were used for the construction and transduction of cDNA expression libraries as well as for the expression of cloned cDNA in mammalian cells.
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The natural history of chronic hepatitis C is just beginning to be clarified, with its more common course being an insidiously progressive liver disease that often remains clinically inconsequential for many years or even decades. Although chronic hepatitis C progresses histologically, the impact on the clinical well-being of the patient is less evident. Interferon is an effective therapy for this disease because of its antiviral effect on the cytopathic hepatitis C virus, lowering serum alanine aminotransferase (ALT) levels, rather than because of any immune modulatory mechanism. Unfortunately, interferon therapy does not permanently eradicate hepatitis C virus in the majority of patients, and relapse with return of the serum ALT level to the pretreatment range occurs in approximately 70% of responding patients. Other interferon-treated patients continue to be viremic and are not considered to be responders. In addition, not all patients with chronic hepatitis C require treatment. A systematic approach to patient evaluation is necessary to determine the need for treatment, assess treatment response, identify side effects of therapy, and assist in other clinical decisions.
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Evidence suggests that cellular immunity to hepatitis C virus (HCV) core protein may be important in the pathogenesis of viral infection. Therefore, interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) derived from patients with chronic HCV infection (genotype 1b) was examined. The cellular immune response was evaluated with a recombinant HCV core fusion protein derived from a patient with genotype 1b. To identify the immunodominant epitopes, IFN-gamma production in responders was also assessed with a panel of nine synthetic peptides that covered the entire core region. It was found that mononuclear cells from 24 (52%) of 46 patients with chronic liver disease responded to the core protein; asymptomatic HCV carriers demonstrated a lower response rate (14%, P < .05). More important, individuals who had received IFN-alpha treatment and went into clinical and virological remission had a higher response rate (75%, P < .05) compared with those with ongoing hepatitis whose treatment failed (31%). Of 25 patients whose mononuclear cells responded to HCV core protein, 18 had a significant response to one or more peptides; 12 patients reacted to a peptide mixture containing hydrophilic sequences. The core peptide amino acid sequence 141 to 160 was recognized in 9 patients. Interestingly, 7 of 8 patients bearing HLA DR 4 and w53 haplotypes recognized the peptide sequence 141 to 160. Thus, IFN-gamma production of the mononuclear cell response appeared to be HLA DR restricted, and the responding cells were identified as CD4+ T cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Article
Hepatitis C virus (HCV) is the major cause of transfusion-acquired non-A, non-B hepatitis. HCV is an enveloped positive-sense RNA virus which has been classified as a new genus in the flavivirus family. Like the other two genera in this family, the flaviviruses and the pestiviruses, HCV polypeptides appear to be produced by translation of a long open reading frame and subsequent proteolytic processing of this polyprotein. In this study, a cDNA clone encompassing the long open reading frame of the HCV H strain (3,011 amino acid residues) has been assembled and sequenced. This clone and various truncated derivatives were used in vaccinia virus transient-expression assays to map HCV-encoded polypeptides and to study HCV polyprotein processing. HCV polyproteins and cleavage products were identified by using convalescent human sera and a panel of region-specific polyclonal rabbit antisera. Similar results were obtained for several mammalian cell lines examined, including the human HepG2 hepatoma line. The data indicate that at least nine polypeptides are produced by cleavage of the HCV H strain polyprotein. Putative structural proteins, located in the N-terminal one-fourth of the polyprotein, include the capsid protein C (21 kDa) followed by two possible virion envelope proteins, E1 (31 kDa) and E2 (70 kDa), which are heavily modified by N-linked glycosylation. The remainder of the polyprotein probably encodes nonstructural proteins including NS2 (23 kDa), NS3 (70 kDa), NS4A (8 kDa), NS4B (27 kDa), NS5A (58 kDa), and NS5B (68 kDa). An 82- to 88-kDa glycoprotein which reacted with both E2 and NS2-specific HCV antisera was also identified (called E2-NS2). Preliminary results suggest that a fraction of E1 is associated with E2 and E2-NS2 via disulfide linkages.