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Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.

ABSTRACT: The genome of hepatitis C virus (HCV) consists of seven functional regions: the core, E1, E2/NS1, NS2, NS3, NS4, and NS5 regions. The U. S. Food and Drug Administration-licensed 2.0G immunoassay for the detection of anti-HCV uses proteins from the core, NS3, and NS4 regions (McHutchinson et al., Hepatology 15:19-25, 1992). The 3.0G enzyme-linked immunosorbent assay includes the protein from the NS5 region (Uyttendaele et al., Vox Sang. 66:122-129, 1994). The necessity of detecting antibodies to viral envelope proteins (E1 and E2) and to different genotype samples has been demonstrated previously (Chien et al., Lancet 342:933, 1993; Lok et al., Hepatology 18:497-502, 1993). In this study we have attempted to improve the sensitivity of the anti-HCV assay by developing a single multiple-epitope fusion antigen (MEFA; MEFA-6) which incorporates all of the major immunodominant epitopes from the seven functional regions of the HCV genome. A nucleic acid sequence consisting of proteins from the viral core, E1, E2, NS3, NS4, and NS5 regions and different subtype-specific regions of the NS4 region was constructed, cloned, and expressed in yeast. The epitopes present on this antigen can be detected by epitope-specific monoclonal and polyclonal antibodies. In a competition assay, the MEFA-6 protein competed with 83 to 96% of genotype-specific antibodies from HCV genotype-specific peptides. This recombinant antigen was subsequently used to design an anti-HCV chemiluminescent immunoassay. We designed our assay using a monoclonal anti-human immunoglobulin G antibody bound to the solid phase. Because MEFA-6 is fused with human superoxide dismutase (h-SOD), we used an anti-human superoxide dismutase, dimethyl acridinium ester-labeled monoclonal antibody for detection. Our results indicate that MEFA-6 exposes all of the major immunogenic epitopes. Its excellent sensitivity and specificity for the detection of clinical seroconversion are demonstrated by this assay.

SUBMITTER: Chien DY

PROVIDER: S-EPMC84783 | biostudies-literature | 1999 May

REPOSITORIES: biostudies-literature

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Project description:BACKGROUND:As direct acting antiviral (DAA) therapy is progressively rolled out for patients with hepatitis C virus (HCV) infection, careful scrutiny of HCV epidemiology, diagnostic testing, and access to care is crucial to underpin improvements in delivery of treatment, with the ultimate goal of elimination. METHODS:We retrospectively studied microbiology records from a large UK teaching hospital in order to compare the performance of HCV screening and diagnostic tests (antibody, antigen and HCV RNA detection). Having described a local cohort of adults with active HCV infection, we investigated the proportion who attended hospital appointments, were prescribed direct acting antiviral (DAA) therapy, and cleared HCV RNA following treatment. RESULTS:Over a total time period of 33 months between 2013 and 2016, we tested 38,509 individuals for HCV infection and confirmed a new diagnosis of active HCV infection (HCV-Ag + and/or HCV RNA+) in 353 (positive rate 0.9%). Our in-house HCV-Ab screening test had a positive predictive value of 87% compared to repeat HCV-Ab testing in a reference laboratory, highlighting the potential for false positives to arise using this test. HCV-Ag had 100% positive predictive value compared to detection of HCV RNA. There was a strong correlation between quantitative HCV-Ag and HCV RNA viral load (p < 0.0001). Among the cases of infection, genotype-1 and genotype-3 predominated, the median age was 37 years, 84% were male, and 36% were in prison. Hepatology review was provided in 39%, and 22% received treatment. Among those who received DAA therapy with 12 weeks of follow-up, 93% achieved a sustained virologic response (SVR12). CONCLUSIONS:HCV-Ag performs well as a diagnostic test compared to PCR for HCV RNA. Active HCV infection is over-represented among men and in the prison population. DAA therapy is successful in those who receive it, but a minority of patients with a diagnosis of HCV infection access clinical care. Enhanced efforts are required to provide linkage to clinical care within high risk populations.

Project description:UnlabelledA lack of immunocompetent-small-primate models has been an obstacle for developing hepatitis C virus (HCV) vaccines and affordable antiviral drugs. In this study, HCV/GB virus B (GBV-B) chimeric virus carrying the major nonstructural proteins NS2 to NS4A (HCV NS2 to -4A chimera) was produced and used to infect common marmosets, since HCV NS2 to NS4A proteins are critical proteases and major antigens. Seven marmosets were inoculated intrahepatically with HCV NS2 to -4A chimera RNA for primary infection or intravenously injected with chimera-containing serum for passage infection. Three animals used as controls were injected with phosphate-buffered saline (PBS) or GBV-B, respectively. Six of seven HCV NS2 to -4A chimera-infected marmosets exhibited consistent viremia and one showed transient viremia during the course of follow-up detection. All six infected animals with persistent circulating viremia presented characteristics typical of viral hepatitis, including viral RNA and proteins in hepatocytes and histopathological changes in liver tissue. Viremia was consistently detected for 5 to 54 weeks of follow-up. FK506 immunosuppression facilitated the establishment of persistent chimera infection in marmosets. An animal with chimera infection spontaneously cleared the virus in blood 7 weeks following the first inoculation, but viral-RNA persistence, low-level viral protein, and mild necroinflammation remained in liver tissue. The specific antibody and T-cell response to HCV NS3 in this viremia-resolved marmoset was boosted by rechallenging, but no viremia was detected during 57 weeks of follow-up. The chimera-infected marmosets described can be used as a suitable small-primate animal model for studying novel antiviral drugs and T-cell-based vaccines against HCV infection.ImportanceHCV infection causes approximately 70% of chronic hepatitis and is frequently associated with primary liver cancer globally. Chimpanzees have been used as a reliable primate model for HCV infection, but ethical considerations have restricted their utility in biomedical research. GB virus B (GBV-B) is a flavivirus related to HCV. It can infect common marmosets, a New World small primate, and induces viral hepatitis similar to HCV infection in humans. To minimize differences between GBV-B and HCV, we generated HCV NS2 to -4A/GBV-B chimeric viruses and established a chimera-infected marmoset model. HCV NS2 to -4A chimera-infected marmosets provide a small-animal model for evaluating novel antiviral drugs targeting HCV NS3-NS4A protease and T-cell-based HCV vaccines.

Project description:BACKGROUND: No recognized risk factors can be identified in 10-40% of hepatitis C virus (HCV)-infected patients suggesting that the modes of transmission involved could be underestimated or unidentified. Invasive diagnostic procedures, such as endoscopy, have been considered as a potential HCV transmission route; although the actual extent of transmission in endoscopy procedures remains controversial. Most reported HCV outbreaks related to nosocomial acquisition have been attributed to unsafe injection practices and use of multi-dose vials. Only a few cases of likely patient-to-patient HCV transmission via a contaminated colonoscope have been reported to date. Nosocomial HCV infection may have important medical and legal implications and, therefore, possible transmission routes should be investigated. In this study, a case of nosocomial transmission of HCV from a common source to two patients who underwent colonoscopy in an endoscopy unit is reported. RESULTS: A retrospective epidemiological search after detection of index cases revealed several potentially infective procedures: sample blood collection, use of a peripheral catheter, anesthesia and colonoscopy procedures. The epidemiological investigation showed breaches in colonoscope reprocessing and deficiencies in the recording of valuable tracing data. Direct sequences from the NS5B region were obtained to determine the extent of the outbreak and cloned sequences from the E1-E2 region were used to establish the relationships among intrapatient viral populations. Phylogenetic analyses of individual sequences from viral populations infecting the three patients involved in the outbreak confirmed the patient pointed out by the epidemiological search as the source of the outbreak. Furthermore, the sequential order in which the patients underwent colonoscopy correlates with viral genetic variability estimates. CONCLUSIONS: Patient-to-patient transmission of HCV could be demonstrated although the precise route of transmission remained unclear. Viral genetic variability is proposed as a useful tool for tracing HCV transmission, especially in recent transmissions.

Project description:A distinctive feature of HCV is that its life cycle depends on lipoprotein metabolism. Viral morphogenesis and secretion follow the very low-density lipoprotein (VLDL) biogenesis pathway and, consequently, infectious HCV in the serum is associated with triglyceride-rich lipoproteins (TRL). Lipoprotein lipase (LPL) hydrolyzes TRL within chylomicrons and VLDL but, independently of its catalytic activity, it has a bridging activity, mediating the hepatic uptake of chylomicrons and VLDL remnants. We previously showed that exogenously added LPL increases HCV binding to hepatoma cells by acting as a bridge between virus-associated lipoproteins and cell surface heparan sulfate, while simultaneously decreasing infection levels. We show here that LPL efficiently inhibits cell infection with two HCV strains produced in hepatoma cells or in primary human hepatocytes transplanted into uPA-SCID mice with fully functional human ApoB-lipoprotein profiles. Viruses produced in vitro or in vivo were separated on iodixanol gradients into low and higher density populations, and the infection of Huh 7.5 cells by both virus populations was inhibited by LPL. The effect of LPL depended on its enzymatic activity. However, the lipase inhibitor tetrahydrolipstatin restored only a minor part of HCV infectivity, suggesting an important role of the LPL bridging function in the inhibition of infection. We followed HCV cell entry by immunoelectron microscopy with anti-envelope and anti-core antibodies. These analyses demonstrated the internalization of virus particles into hepatoma cells and their presence in intracellular vesicles and associated with lipid droplets. In the presence of LPL, HCV was retained at the cell surface. We conclude that LPL efficiently inhibits HCV infection by acting on TRL associated with HCV particles through mechanisms involving its lipolytic function, but mostly its bridging function. These mechanisms lead to immobilization of the virus at the cell surface. HCV-associated lipoproteins may therefore be a promising target for the development of new therapeutic approaches.

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Use of a novel hepatitis C virus (HCV) major-epitope chimeric polypeptide for diagnosis of HCV infection.

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